Cystic Fibrosis Gene Therapy in the UK and Elsewhere

PubMed Central

Pytel, Kamila M.; Alton, Eric W.F.W.

2015-01-01

Abstract The cystic fibrosis transmembrane conductance regulator (CFTR) gene was identified in 1989. This opened the door for the development of cystic fibrosis (CF) gene therapy, which has been actively pursued for the last 20 years. Although 26 clinical trials involving approximately 450 patients have been carried out, the vast majority of these trials were short and included small numbers of patients; they were not designed to assess clinical benefit, but to establish safety and proof-of-concept for gene transfer using molecular end points such as the detection of recombinant mRNA or correction of the ion transport defect. The only currently published trial designed and powered to assess clinical efficacy (defined as improvement in lung function) administered AAV2-CFTR to the lungs of patients with CF. The U.K. Cystic Fibrosis Gene Therapy Consortium completed, in the autumn of 2014, the first nonviral gene therapy trial designed to answer whether repeated nonviral gene transfer (12 doses over 12 months) can lead to clinical benefit. The demonstration that the molecular defect in CFTR can be corrected with small-molecule drugs, and the success of gene therapy in other monogenic diseases, is boosting interest in CF gene therapy. Developments are discussed here. PMID:25838137

Control of the proinflammatory state in cystic fibrosis lung epithelial cells by genes from the TNF-alphaR/NFkappaB pathway.

PubMed Central

Eidelman, O.; Srivastava, M.; Zhang, J.; Leighton, X.; Murtie, J.; Jozwik, C.; Jacobson, K.; Weinstein, D. L.; Metcalf, E. L.; Pollard, H. B.

2001-01-01

BACKGROUND: Cystic fibrosis (CF) is the most common, lethal autosomal recessive disease affecting children in the United States and Europe. Extensive work is being performed to develop both gene and drug therapies. The principal mutation causing CF is in the CFTR gene ([Delta F508]CFTR). This mutation causes the mutant protein to traffic poorly to the plasma membrane, and degrades CFTR chloride channel activity. CPX, a candidate drug for CF, binds to mutant CFTR and corrects the trafficking deficit. CPX also activates mutant CFTR chloride channel activity. CF airways are phenotypically inundated by inflammatory signals, primarily contributed by sustained secretion of the proinflammatory cytokine interleukin 8 (IL-8) from mutant CFTR airway epithelial cells. IL-8 production is controlled by genes from the TNF-alphaR/NFkappaB pathway, and it is possible that the CF phenotype is due to dysfunction of genes from this pathway. In addition, because drug therapy with CPX and gene therapy with CFTR have the same common endpoint of raising the levels of CFTR, we have hypothesized that either approach should have a common genomic endpoint. MATERIALS AND METHODS: To test this hypothesis, we studied IL-8 secretion and global gene expression in IB-3 CF lung epithelial cells. The cells were treated by either gene therapy with wild-type CFTR, or by pharmacotherapy with the CFTR-surrogate drug CPX. CF cells, treated with either CFTR or CPX, were also exposed to Pseudomonas aeruginosa, a common chronic pathogen in CF patients. cDNA microarrays were used to assess global gene expression under the different conditions. A novel bioinformatic algorithm (GENESAVER) was developed to identify genes whose expression paralleled secretion of IL-8. RESULTS: We report here that IB3 CF cells secrete massive levels of IL-8. However, both gene therapy with CFTR and drug therapy with CPX substantially suppress IL-8 secretion. Nonetheless, both gene and drug therapy allow the CF cells to respond with physiologic secretion of IL-8 when the cells are exposed to P. aeruginosa. Thus, neither CFTR nor CPX acts as a nonspecific suppressor of IL-8 secretion from CF cells. Consistently, pharmacogenomic analysis indicates that CF cells treated with CPX greatly resemble CF cells treated with CFTR by gene therapy. Additionally, the same result obtains in the presence of P. aeruginosa. Classical hierarchical cluster analysis, based on similarity of global gene expression, also supports this conclusion. The GENESAVER algorithm, using the IL-8 secretion level as a physiologic variable, identifies a subset of genes from the TNF-alphaR/NFkappaB pathway that is expressed in phase with IL-8 secretion from CF epithelial cells. Certain other genes, previously known to be positively associated with CF, also fall into this category. Identified genes known to code for known inhibitors are expressed inversely, out of phase with IL-8 secretion. CONCLUSIONS: Wild-type CFTR and CPX both suppress proinflammatory IL-8 secretion from CF epithelial cells. The mechanism, as defined by pharmacogenomic analysis, involves identified genes from the TNF-alphaR/NFkappaB pathway. The close relationship between IL-8 secretion and genes from the TNF-alphaR/NFkappaB pathway suggests that molecular or pharmaceutical targeting of these novel genes may have strategic use in the development of new therapies for CF. From the perspective of global gene expression, both gene and drug therapy have similar genomic consequences. This is the first example showing equivalence of gene and drug therapy in CF, and suggests that a gene therapy-defined endpoint may prove to be a powerful paradigm for CF drug discovery. Finally, because the GENESAVER algorithm is capable of isolating disease-relevant genes in a hypothesis-driven manner without recourse to any a priori knowledge about the system, this new algorithm may also prove useful in applications to other genetic diseases. PMID:11591888

Effects of FVIII immunity on hepatocyte and hematopoietic stem cell–directed gene therapy of murine hemophilia A

PubMed Central

Lytle, Allison M; Brown, Harrison C; Paik, Na Yoon; Knight, Kristopher A; Wright, J Fraser; Spencer, H Trent; Doering, Christopher B

2016-01-01

Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV. PMID:26909355

Gene therapy: charting a future course--summary of a National Institutes of Health Workshop, April 12, 2013.

PubMed

O'Reilly, Marina; Federoff, Howard J; Fong, Yuman; Kohn, Donald B; Patterson, Amy P; Ahmed, Nabil; Asokan, Aravind; Boye, Shannon E; Crystal, Ronald G; De Oliveira, Satiro; Gargiulo, Linda; Harper, Scott Q; Ikeda, Yasuhiro; Jambou, Robert; Montgomery, Maureen; Prograis, Lawrence; Rosenthal, Eugene; Sterman, Daniel H; Vandenberghe, Luk H; Zoloth, Laurie; Abedi, Mehrdad; Adair, Jennifer; Adusumilli, Prasad S; Goins, William F; Gray, Jhanelle; Monahan, Paul; Popplewell, Leslie; Sena-Esteves, Miguel; Tannous, Bakhos; Weber, Thomas; Wierda, William; Gopal-Srivastava, Rashmi; McDonald, Cheryl L; Rosenblum, Daniel; Corrigan-Curay, Jacqueline

2014-06-01

Recently, the gene therapy field has begun to experience clinical successes in a number of different diseases using various approaches and vectors. The workshop Gene Therapy: Charting a Future Course, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities, brought together early and mid-career researchers to discuss the key scientific challenges and opportunities, ethical and communication issues, and NIH and foundation resources available to facilitate further clinical advances.

Gene Therapy: Charting a Future Course—Summary of a National Institutes of Health Workshop, April 12, 2013

PubMed Central

O'Reilly, Marina; Federoff, Howard J.; Fong, Yuman; Kohn, Donald B.; Patterson, Amy P.; Ahmed, Nabil; Asokan, Aravind; Boye, Shannon E.; Crystal, Ronald G.; De Oliveira, Satiro; Gargiulo, Linda; Harper, Scott Q.; Ikeda, Yasuhiro; Jambou, Robert; Montgomery, Maureen; Prograis, Lawrence; Rosenthal, Eugene; Sterman, Daniel H.; Vandenberghe, Luk H.; Zoloth, Laurie; Abedi, Mehrdad; Adair, Jennifer; Adusumilli, Prasad S.; Goins, William F.; Gray, Jhanelle; Monahan, Paul; Popplewell, Leslie; Sena-Esteves, Miguel; Tannous, Bakhos; Weber, Thomas; Wierda, William; Gopal-Srivastava, Rashmi; McDonald, Cheryl L.; Rosenblum, Daniel

2014-01-01

Abstract Recently, the gene therapy field has begun to experience clinical successes in a number of different diseases using various approaches and vectors. The workshop Gene Therapy: Charting a Future Course, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities, brought together early and mid-career researchers to discuss the key scientific challenges and opportunities, ethical and communication issues, and NIH and foundation resources available to facilitate further clinical advances. PMID:24773122

Advances in Gene Therapy for Hemophilia.

PubMed

Nathwani, Amit C; Davidoff, Andrew M; Tuddenham, Edward G D

2017-11-01

Gene therapy provides hope for a cure for patients with hemophilia by establishing continuous endogenous expression of factor VIII or factor IX following transfer of a functional gene copy to replace the hemophilic patient's own defective gene. Hemophilia may be considered a "low-hanging fruit" for gene therapy because a small increment in blood factor levels (≥2% of normal) significantly improves the bleeding tendency from severe to moderate, eliminating most spontaneous bleeds. After decades of research, the first trial to provide clear evidence of efficiency after gene transfer in patients with hemophilia B using adeno-associated virus vectors was reported by the authors' group in 2011. This has been followed by unprecedented activity in this area, with the commencement of seven new early-phase trials involving >55 patients with hemophilia A or hemophilia B. These studies have, in large part, generated promising clinical data that lay a strong foundation for gene therapy to move forward rapidly to market authorization. This review discusses the data from the authors' studies and emerging results from other gene therapy trials in both hemophilia A and B.

A Novel Combination of Thermal Ablation and Heat-Inducible Gene Therapy for Breast Cancer Treatment

DTIC Science & Technology

2008-04-01

focused ultrasound ( HIFU ) thermal ablation and HIFU -induced gene therapy represents a promising approach in improving the overall efficacy and quality...STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT High intensity focused ultrasound ...al., High intensity focused ultrasound -induced gene activation in solid tumors. Journal of the Acoustical Society of America, 2006. 120(1): p. 492

Characteristics of lentiviral vectors harboring the proximal promoter of the vav proto-oncogene: a weak and efficient promoter for gene therapy.

PubMed

Almarza, Elena; Río, Paula; Meza, Nestor W; Aldea, Montserrat; Agirre, Xabier; Guenechea, Guillermo; Segovia, José C; Bueren, Juan A

2007-08-01

Recent published data have shown the efficacy of gene therapy treatments of certain monogenic diseases. Risks of insertional oncogenesis, however, indicate the necessity of developing new vectors with weaker or cell-restricted promoters to minimize the trans-activation activity of integrated proviruses. We have inserted the proximal promoter of the vav proto-oncogene into self-inactivating lentiviral vectors (vav-LVs) and investigated the expression pattern and therapeutic efficacy of these vectors. Compared with other LVs frequently used in gene therapy, vav-LVs mediated a weak, though homogeneous and stable, expression in in vitro-cultured cells. Transplantation experiments using transduced mouse bone marrow and human CD34(+) cells confirmed the stable activity of the promoter in vivo. To investigate whether the weak activity of this promoter was compatible with a therapeutic effect, a LV expressing the Fanconi anemia A (FANCA) gene was constructed (vav-FANCA LV). Although this vector induced a low expression of FANCA, compared to the expression induced by a LV harboring the spleen focus-forming virus (SFFV) promoter, the two vectors corrected the phenotype of cells from a patient with FA-A with the same efficacy. We propose that self-inactivating vectors harboring weak promoters, such as the vav promoter, will improve the safety of gene therapy and will be of particular interest for the treatment of diseases where a high expression of the transgene is not required.

Nerve Growth Factor Gene Therapy Activates Neuronal Responses in Alzheimer’s Disease

PubMed Central

Tuszynski, Mark H.; Yang, Jennifer H.; Barba, David; U, H S.; Bakay, Roy; Pay, Mary M.; Masliah, Eliezer; Conner, James M.; Kobalka, Peter; Roy, Subhojit; Nagahara, Alan H.

2016-01-01

IMPORTANCE Alzheimer’s disease (AD) is the most common neurodegenerative disorder, and lacks effective disease modifying therapies. In 2001 we initiated a clinical trial of Nerve Growth Factor (NGF) gene therapy in AD, the first effort at gene delivery in an adult neurodegenerative disorder. This program aimed to determine whether a nervous system growth factor prevents or reduces cholinergic neuronal degeneration in AD patients. We present post-mortem findings in 10 subjects with survival times ranging from 1 to 10 years post-treatment. OBJECTIVE To determine whether degenerating neurons in AD retain an ability to respond to a nervous system growth factor delivered after disease onset. DESIGN, SETTING, AND PARTICIPANTS 10 patients with early AD underwent NGF gene therapy using either ex vivo or in vivo gene transfer. The brains of all eight patients in the first Phase 1 ex vivo trial and two patients in a subsequent Phase 1 in vivo trial were examined. MAIN OUTCOME MEASURES Brains were immunolabeled to evaluate in vivo gene expression, cholinergic neuronal responses to NGF, and activation of NGF-related cell signaling. In two cases, NGF protein levels were measured by ELISA. RESULTS Degenerating neurons in the AD brain respond to NGF. All patients exhibited a trophic response to NGF, in the form of axonal sprouting toward the NGF source. Comparing treated and non-treated sides of the brain in three patients that underwent unilateral gene transfer, cholinergic neuronal hypertrophy occurred on the NGF-treated side (P>0.05). Activation of cellular signaling and functional markers were present in two patients that underwent AAV2-mediated NGF gene transfer. Neurons exhibiting tau pathology as well as neurons free of tau expressed NGF, indicating that degenerating cells can be infected with therapeutic genes with resulting activation of cell signaling. No adverse pathological effects related to NGF were observed. CONCLUSIONS AND RELEVANCE These findings indicate that neurons of the degenerating brain retain the ability to respond to growth factors, with axonal sprouting, cell hypertrophy and activation of functional markers. NGF-induced sprouting persists over ten years. Growth factor therapy appears safe over extended time periods and merits continued testing as a means of treating neurodegenerative disorders. Trial Registration: NCT00087789 and NCT00017940 PMID:26302439

The Effect of Neonatal Gene Therapy on Skeletal Manifestations in Mucopolysaccharidosis VII Dogs after a Decade

PubMed Central

Xing, Elizabeth M.; Knox, Van W.; O'Donnell, Patricia A.; Sikura, Tracey; Liu, Yuli; Wu, Susan; Casal, Margret L.; Haskins, Mark E.; Ponder, Katherine P.

2013-01-01

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disease due to deficient activity of β-glucuronidase (GUSB), and results in glycosaminoglycan accumulation. Skeletal manifestations include bone dysplasia, degenerative joint disease, and growth retardation. One gene therapy approach for MPS VII involves neonatal intravenous injection of a gamma retroviral vector expressing GUSB, which results in stable expression in liver and secretion of enzyme into blood at levels predicted to be similar or higher to enzyme replacement therapy. The goal of this study was to evaluate the long-term effect of neonatal gene therapy on skeletal manifestations in MPS VII dogs. Treated MPS VII dogs could walk throughout their lives, while untreated MPS VII dogs could not stand beyond 6 months and were dead by 2 years. Luxation of the coxofemoral joint and the patella, dysplasia of the acetabulum and supracondylar ridge, deep erosions of the distal femur, and synovial hyperplasia were reduced, and the quality of articular bone was improved in treated dogs at 6 to 11 years of age compared with untreated MPS VII dogs at 2 years or less. However, treated dogs continued to have osteophyte formation, cartilage abnormalities, and an abnormal gait. Enzyme activity was found near synovial blood vessels, and there was 2% as much GUSB activity in synovial fluid as in serum. We conclude that neonatal gene therapy reduces skeletal abnormalities in MPS VII dogs, but clinically-relevant abnormalities remain. Enzyme replacement therapy will probably have similar limitations long-term. PMID:23628461

Clinical development of gene- and cell-based therapies: overview of the European landscape

PubMed Central

de Wilde, Sofieke; Guchelaar, Henk-Jan; Zandvliet, Maarten Laurens; Meij, Pauline

2016-01-01

In the last decade, many clinical trials with gene- and cell-based therapies were performed and increasing interest in the development was established by (national) authorities, academic developers, and commercial companies. However, until now only eight products have received marketing authorization (MA) approval. In this study, a comprehensive overview of the clinical development of gene- and cell-based therapies in Europe is presented, with a strong focus on product-technical aspects. Public data regarding clinical trials with gene- and cell-based therapies, obtained from the European Union (EU) clinical trial database (EudraCT) between 2004 and 2014 were analyzed, including product-technical variables as potential determinants affecting development. 198 unique gene and cell therapy products were identified, which were studied in 278 clinical trials, mostly in phase 1/2 trials and with cell therapies as major group. Furthermore, most products were manufactured from autologous starting material mostly manufactured from stem cells. The majority of the trials were sponsored by academia, whereas phase 3 trials mostly by large companies. Academia dominated early-stage development by mainly using bone marrow derived products and stem cells. Conversely, commercial sponsors were more actively pursuing in vivo gene therapy medicinal product development, and cell therapies derived from differentiated tissue in later-stage development. PMID:27990447

Gene Delivery in Neuro-Oncology.

PubMed

Dixit, Karan; Kumthekar, Priya

2017-09-02

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults with a dismal prognosis despite aggressive multimodal management thus novel treatments are urgently needed. Gene therapy is a versatile treatment strategy being investigated in multiple cancers including GBM. In gene therapy, a variety of vectors or "carriers" are used to deliver genes designed for different anti-tumoral effects. Gene delivery vehicles and approaches to treatment will be addressed in this review. The most commonly studied vectors are viral based, however, driven by advances in biomedical engineering, mesenchymal and neural stem cells, as well as multiple different types of nanoparticles have been developed to improve tumor tropism and also increase gene transfer into tumor cells. Different genes have been studied including suicide genes, which convert non-toxic prodrug into cytotoxic drug; immunomodulatory genes, which stimulate the immune system; and tumor suppressor genes which repair the defect that allow cells to divide unchecked. Gene therapy may be a promising treatment strategy in neuro-oncology as it is versatile and flexible due to the ability to tailor vectors and genes for specific therapeutic activity. Pre-clinical studies and clinical trials have demonstrated feasibility and safety of gene therapy; however, further studies are required to determine efficacy.

Genetic basis and gene therapy trials for thyroid cancer.

PubMed

Al-Humadi, Hussam; Zarros, Apostolos; Al-Saigh, Rafal; Liapi, Charis

2010-01-01

Gene therapy is regarded as one of the most promising novel therapeutic approaches for hopeless cases of thyroid cancer and those not responding to traditional treatment. In the last two decades, many studies have focused on the genetic factors behind the origin and the development of thyroid cancer, in order to investigate and shed more light on the molecular pathways implicated in different differentiated or undifferentiated types of thyroid tumors. We, herein, review the current data on the main genes that have been proven to (or thought to) be implicated in thyroid cancer etiology, and which are involved in several well-known signaling pathways (such as the mitogen-activated protein kinase and phosphatidylinositol-3-kinase/Akt pathways). Moreover, we review the results of the efforts made through multiple gene therapy trials, via several gene therapy approaches/strategies, on different thyroid carcinomas. Our review leads to the conclusion that future research efforts should seriously consider gene therapy for the treatment of thyroid cancer, and, thus, should: (a) shed more light on the molecular basis of thyroid cancer tumorigenesis, (b) focus on the development of novel gene therapy approaches that can achieve the required antitumoral efficacy with minimum normal tissue toxicity, as well as (c) perform more gene therapy clinical trials, in order to acquire more data on the efficacy of the examined approaches and to record the provoked adverse effects.

Genome-editing Technologies for Gene and Cell Therapy.

PubMed

Maeder, Morgan L; Gersbach, Charles A

2016-03-01

Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed.

Genome-editing Technologies for Gene and Cell Therapy

PubMed Central

Maeder, Morgan L; Gersbach, Charles A

2016-01-01

Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed. PMID:26755333

Active site mutant transgene confers tolerance to human β-glucuronidase without affecting the phenotype of MPS VII mice

PubMed Central

Sly, William S.; Vogler, Carole; Grubb, Jeffrey H.; Zhou, Mi; Jiang, Jinxing; Zhou, Xiao Yan; Tomatsu, Shunji; Bi, Yanhua; Snella, Elizabeth M.

2001-01-01

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is an autosomal recessive lysosomal storage disorder due to an inherited deficiency of β-glucuronidase. A naturally occurring mouse model for this disease was discovered at The Jackson Laboratory and shown to be due to homozygosity for a 1-bp deletion in exon 10 of the gus gene. The murine model MPS VII (gusmps/mps) has been very well characterized and used extensively to evaluate experimental strategies for lysosomal storage diseases, including bone marrow transplantation, enzyme replacement therapy, and gene therapy. To enhance the value of this model for enzyme and gene therapy, we produced a transgenic mouse expressing the human β-glucuronidase cDNA with an amino acid substitution at the active site nucleophile (E540A) and bred it onto the MPS VII (gusmps/mps) background. We demonstrate here that the mutant mice bearing the active site mutant human transgene retain the clinical, morphological, biochemical, and histopathological characteristics of the original MPS VII (gusmps/mps) mouse. However, they are now tolerant to immune challenge with human β-glucuronidase. This “tolerant MPS VII mouse model” should be useful for preclinical trials evaluating the effectiveness of enzyme and/or gene therapy with the human gene products likely to be administered to human patients with MPS VII. PMID:11226217

Lentiviral Gene Therapy Using Cellular Promoters Cures Type 1 Gaucher Disease in Mice

PubMed Central

Dahl, Maria; Doyle, Alexander; Olsson, Karin; Månsson, Jan-Eric; Marques, André R A; Mirzaian, Mina; Aerts, Johannes M; Ehinger, Mats; Rothe, Michael; Modlich, Ute; Schambach, Axel; Karlsson, Stefan

2015-01-01

Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease. PMID:25655314

Gene therapy and gastrointestinal cancer: concepts and clinical facts.

PubMed

Hauses, M; Schackert, H K

1999-10-01

Principles of the treatment of gastrointestinal cancer with gene therapy evolved from the advent of techniques in molecular biology, from increasing insights into the molecular basis of tumorigenesis and from the need to develop more efficient treatment modalities. Any gene therapy approach has to take two major tasks into consideration: the therapeutic gene has to be delivered into the target cell population with high efficiency, specificity and safety, and has to act in a way that provides a benefit to the patient. Data on 22 clinical trials on malignancies of the gastrointestinal tract are available. They utilize a variety of gene-delivery methods and target cell populations, and there is considerable variety among their strategies. Gene transfer is performed by injection of naked plasmid DNA and by use of DNA-liposome complexes and viral vectors. In some cases, the gene transfer is carried out ex vivo and the patients receive genetically modified cells, whereas other approaches deliver the vector to the target cell population in vivo. The theoretical concepts of gene therapy can be divided into three groups. One approach makes use of suicide genes comprising bacterial or viral genes that convert a nontoxic prodrug into a highly cytotoxic chemotherapeutic agent at the tumor site. This approach aims at higher therapeutic specificity and fewer side effects than with the systemic delivery of cytotoxic agents. The second strategy makes an attempt to invoke the immune system to destroy malignant cells. Different strategies, such as immunization with genetically modified tumor cells or transfer of new genes to T cells, are considered to have clinical benefits. The major advantage of these immunotherapeutic approaches is the systemic effect both on the primary tumor and on metastases. The third strategy evolved from the insight that cancer is a genetic disease caused by activation of oncogenes or inactivation of tumor-suppressor genes. Compensation of genetic defects by the downregulation of activated oncogenes or the restoration of tumor-suppressor-gene functions may be able to revert the malignant phenotype of cancer cells. Of the 22 gene-therapy trials, 17 trials focus on immunotherapy. Only two trials make use of suicide genes and, in three trials, a functional copy of the p53 tumor-suppressor gene was reintroduced into malignant cells. Modalities for gene transfer and the strategies underlying gene therapy will be discussed in the context of gastrointestinal malignancies and the potential benefits for patients.

Standards for gene therapy clinical trials based on pro-active risk assessment in a London NHS Teaching Hospital Trust.

PubMed

Bamford, K B; Wood, S; Shaw, R J

2005-02-01

Conducting gene therapy clinical trials with genetically modified organisms as the vectors presents unique safety and infection control issues. The area is governed by a range of legislation and guidelines, some unique to this field, as well as those pertinent to any area of clinical work. The relevant regulations covering gene therapy using genetically modified vectors are reviewed and illustrated with the approach taken by a large teaching hospital NHS Trust. Key elements were Trust-wide communication and involvement of staff in a pro-active approach to risk management, with specific emphasis on staff training and engagement, waste management, audit and record keeping. This process has led to the development of proposed standards for clinical trials involving genetically modified micro-organisms.

Mobile genetic elements and cancer. From mutations to gene therapy.

PubMed

Kozeretska, I A; Demydov, S V; Ostapchenko, L I

2011-12-01

In the present review, an association between cancer and the activity of the non-LTR retroelements L1, Alu, and SVA, as well as endogenous retroviruses, in the human genome, is analyzed. Data suggesting that transposons have been involved in embryogenesis and malignization processes, are presented. Events that lead to the activation of mobile elements in mammalian somatic cells, as well as the use of mobile elements in genetic screening and cancer gene therapy, are reviewed.

Gene therapy in an era of emerging treatment options for hemophilia B.

PubMed

Monahan, P E

2015-06-01

Factor IX deficiency (hemophilia B) is less common than factor VIII deficiency (hemophilia A), and innovations in therapy for hemophilia B have generally lagged behind those for hemophilia A. Recently, the first sustained correction of the hemophilia bleeding phenotype by clotting factor gene therapy has been described using recombinant adeno-associated virus (AAV) to deliver factor IX. Despite this success, many individuals with hemophilia B, including children, men with active hepatitis, and individuals who have pre-existing natural immunity to AAV, are not eligible for the current iteration of hemophilia B gene therapy. In addition, recent advances in recombinant factor IX protein engineering have led some hemophilia treaters to reconsider the urgency of genetic cure. Current clinical and preclinical approaches to advancing AAV-based and alternative approaches to factor IX gene therapy are considered in the context of current demographics and treatment of the hemophilia B population. © 2015 International Society on Thrombosis and Haemostasis.

Chromatin status of apoptosis genes correlates with sensitivity to chemo-, immune- and radiation therapy in colorectal cancer cell lines.

PubMed

Benard, Anne; Janssen, Connie M; van den Elsen, Peter J; van Eggermond, Marja C J A; Hoon, Dave S B; van de Velde, Cornelis J H; Kuppen, Peter J K

2014-12-01

The apoptosis pathway of programmed cell death is frequently deregulated in cancer. An intact apoptosis pathway is required for proper response to anti-cancer treatment. We investigated the chromatin status of key apoptosis genes in the apoptosis pathway in colorectal cancer cell lines in relation to apoptosis induced by chemo-, immune- or radiation therapy. Using chromatin immunoprecipitation (ChIP), we measured the presence of transcription-activating histone modifications H3Ac and H3K4me3 and silencing modifications H3K9me3 and H3K27me3 at the gene promoter regions of key apoptosis genes Bax, Bcl2, Caspase-9, Fas (CD95) and p53. Cell lines DLD1, SW620, Colo320, Caco2, Lovo and HT29 were treated with cisplatin, anti-Fas or radiation. The apoptotic response was measured by flow cytometry using propidium iodide and annexin V-FITC. The chromatin status of the apoptosis genes reflected the activation status of the intrinsic (Bax, Bcl2, Caspase-9 and p53) and extrinsic (Fas) pathways. An active intrinsic apoptotic pathway corresponded to sensitivity to cisplatin and radiation treatment of cell lines DLD1, SW620 and Colo320. An active Fas promoter corresponded to an active extrinsic apoptotic pathway in cell line DLD1. mRNA expression data correlated with the chromatin status of the apoptosis genes as measured by ChIP. In conclusion, the results presented in this study indicate that the balance between activating and silencing histone modifications, reflecting the chromatin status of apoptosis genes, can be used to predict the response of tumor cells to different anti-cancer therapies and could provide a novel target to sensitize tumors to obtain adequate treatment responses.

Newer Gene Editing Technologies toward HIV Gene Therapy

PubMed Central

Manjunath, N.; Yi, Guohua; Dang, Ying; Shankar, Premlata

2013-01-01

Despite the great success of highly active antiretroviral therapy (HAART) in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called “Berlin patient” who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy. PMID:24284874

E phage gene transfection associated to chemotherapeutic agents increases apoptosis in lung and colon cancer cells.

PubMed

Rama, Ana R; Prados, Jose; Melguizo, Consolacion; Alvarez, Pablo J; Ortiz, Raúl; Madeddu, Roberto; Aranega, Antonia

2011-01-01

The limited ability of conventional therapies to achieve the long-term survival of metastatic lung and colon cancer patients suggests the need for new treatment options. In this respect, genes encoding cytotoxic proteins have been proposed as a new strategy to enhance the activity of drugs, and combined therapies involving such genes and classical antitumoral drugs have been studied intensively. The E gene from phiX174 encodes a membrane protein with a toxic domain that leads to a decrease in tumour cell growth rates. Therefore, in order to improve the anti-tumour effects of currently used chemotherapeutic drugs on cancer cells, we investigated the association of the E suicide gene with these antineoplastic drugs. The E gene has antitumoral effects in both lung and colon cancer cells. In addition, expression of this gene induces ultrastructural changes in lung cancer transfected cells (A-549), although the significance of these changes remains unknown. The effect of combined therapy (gene and cytotoxic therapy) enhances the inhibition of tumour cell proliferation in comparison to single treatments. Indeed, our in vitro results indicate that an experimental therapeutic strategy based on this combination of E gene therapy and cytotoxic drugs may result in a new treatment strategy for patients with advanced lung and colon cancer.

Novel Biomarkers of Human GM1 Gangliosidosis Reflect the Clinical Efficacy of Gene Therapy in a Feline Model.

PubMed

Gray-Edwards, Heather L; Regier, Debra S; Shirley, Jamie L; Randle, Ashley N; Salibi, Nouha; Thomas, Sarah E; Latour, Yvonne L; Johnston, Jean; Golas, Gretchen; Maguire, Annie S; Taylor, Amanda R; Sorjonen, Donald C; McCurdy, Victoria J; Christopherson, Peter W; Bradbury, Allison M; Beyers, Ronald J; Johnson, Aime K; Brunson, Brandon L; Cox, Nancy R; Baker, Henry J; Denney, Thomas S; Sena-Esteves, Miguel; Tifft, Cynthia J; Martin, Douglas R

2017-04-05

GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats-either untreated or AAV treated for more than 5 years-and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment. Copyright © 2017 The American Society of Gene and Cell Therapy. All rights reserved.

Developing protocols for recombinant adeno-associated virus-mediated gene therapy in space.

PubMed

Ohi, S

2000-07-01

With the advent of the era of International Space Station (ISS) and Mars exploration, it is important more than ever to develop means to cure genetic and acquired diseases, which include cancer and AIDS, for these diseases hamper human activities. Thus, our ultimate goal is to develop protocols for gene therapy, which are suitable to humans on the earth as well as in space. Specifically, we are trying to cure the hemoglobinopathies, beta-thalassemia (Cooley's anemia) and sickle cell anemia, by gene therapy. These well-characterized molecular diseases serve as models for developing ex vivo gene therapy, which would apply to other disorders as well. For example, the procedure may become directly relevant to treating astronauts for space-anemia, immune suppression and bone marrow derived tumors, e.g. leukemia. The adeno-associated virus serotype 2 (AAV2) is a non-pathogenic human parvovirus with broad host-range and tissue specificity. Exploiting these characteristics we have been developing protocols for recombinant AAV2 (rAAV)-based gene therapy. With the rAAV constructs and hematopoietic stem cell (HSC) culture systems in hand, we are currently attempting to cure the mouse model of beta-thalassemia [C57BL/6- Hbbth/Hbbth, Hb(d-minor)] by HSC transplantation (HST) as well as by gene therapy. This paper describes the current status of our rAAV-gene therapy research.

RecA Inhibitors Potentiate Antibiotic Activity and Block Evolution of Antibiotic Resistance.

PubMed

Alam, Md Kausar; Alhhazmi, Areej; DeCoteau, John F; Luo, Yu; Geyer, C Ronald

2016-03-17

Antibiotic resistance arises from the maintenance of resistance mutations or genes acquired from the acquisition of adaptive de novo mutations or the transfer of resistance genes. Antibiotic resistance is acquired in response to antibiotic therapy by activating SOS-mediated DNA repair and mutagenesis and horizontal gene transfer pathways. Initiation of the SOS pathway promotes activation of RecA, inactivation of LexA repressor, and induction of SOS genes. Here, we have identified and characterized phthalocyanine tetrasulfonic acid RecA inhibitors that block antibiotic-induced activation of the SOS response. These inhibitors potentiate the activity of bactericidal antibiotics, including members of the quinolone, β-lactam, and aminoglycoside families in both Gram-negative and Gram-positive bacteria. They reduce the ability of bacteria to acquire antibiotic resistance mutations and to transfer mobile genetic elements conferring resistance. This study highlights the advantage of including RecA inhibitors in bactericidal antibiotic therapies and provides a new strategy for prolonging antibiotic shelf life. Copyright © 2016 Elsevier Ltd. All rights reserved.

Generation of genetic constructs that simultaneously express several shRNAs.

PubMed

Kretova, Olga V; Alembekov, Ildar R; Tchurikov, Nickolai A

2012-05-01

RNAi has potential as an antiviral gene therapy strategy. Cassette constructs simultaneously expressing several siRNAs could prove to be the most efficient technique in developing gene therapy approaches for highly mutable viruses such as HIV-1. Here we describe a rapid and cost-saving protocol to generate cassettes that simultaneously express three siRNAs for repression of HIV-1 and CCR5 transcripts. siRNA biological activity was tested in a non-viral system, and exhibited both efficiency and specificity. Our results suggest this protocol can be used to rapidly generate cassette constructs for antiviral gene therapy applications.

GeneXpert MTB/RIF Testing in the Management of Patients with Active Tuberculosis; A Real Life Experience from Saudi Arabia.

PubMed

Omrani, Ali S; Al-Otaibi, Mohammed F; Al-Ateah, Souad M; Al-Onazi, Fahad M; Baig, Kamran; El-Khizzi, Noura A; Albarrak, Ali M

2014-03-01

GeneXpert MTB/RIF is a real-time PCR assay with established diagnostic performance in pulmonary and extra-pulmonary forms of tuberculosis. The aim of this study was to assess the contribution of GeneXpert MTB/RIF assay to the management of patients with any form of active tuberculosis in a single large tertiary center in Saudi Arabia, with a special focus on the impact on time to start of antituberculous therapy compared with Ziehl-Neelsen (ZN) smears and mycobacterial cultures. Clinical, radiological and laboratory records for all patients who were commenced on antituberculous therapy between March 2011 and February 2013 were retrospectively reviewed. A total of 140 patients were included, 38.6% of which had pulmonary tuberculosis. GeneXpert MTB/RIF was requested for only 39.2% of patients and was the only reason for starting antituberculous therapy for only 12.1%. The median time to a positive GeneXpert MTB/RIF result was 0 days (IQR 3) compared with 0 day (IQR 1) for smear microscopy (P > 0.999) and 22 days (IQR 21) for mycobacterial cultures (P < 0.001). No patients discontinued antituberculous therapy because of a negative GeneXpert MTB/RIF result. In a setting wherein physicians are highly experienced in the diagnosis and treatment of tuberculosis, GeneXpert MTB/RIF was remarkably under-utilized and had only a limited impact on decisions related to starting or stopping antituberculous therapy. Cost-effectiveness and clinical utility of routine testing of all smear-negative clinical samples submitted for tuberculosis investigations by GeneXpert MTB/RIF warrant further study.

The effect of neonatal gene therapy on skeletal manifestations in mucopolysaccharidosis VII dogs after a decade.

PubMed

Xing, Elizabeth M; Knox, Van W; O'Donnell, Patricia A; Sikura, Tracey; Liu, Yuli; Wu, Susan; Casal, Margret L; Haskins, Mark E; Ponder, Katherine P

2013-06-01

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disease due to deficient activity of β-glucuronidase (GUSB), and results in glycosaminoglycan accumulation. Skeletal manifestations include bone dysplasia, degenerative joint disease, and growth retardation. One gene therapy approach for MPS VII involves neonatal intravenous injection of a gamma retroviral vector expressing GUSB, which results in stable expression in liver and secretion of enzyme into blood at levels predicted to be similar or higher to enzyme replacement therapy. The goal of this study was to evaluate the long-term effect of neonatal gene therapy on skeletal manifestations in MPS VII dogs. Treated MPS VII dogs could walk throughout their lives, while untreated MPS VII dogs could not stand beyond 6 months and were dead by 2 years. Luxation of the coxofemoral joint and the patella, dysplasia of the acetabulum and supracondylar ridge, deep erosions of the distal femur, and synovial hyperplasia were reduced, and the quality of articular bone was improved in treated dogs at 6 to 11 years of age compared with untreated MPS VII dogs at 2 years or less. However, treated dogs continued to have osteophyte formation, cartilage abnormalities, and an abnormal gait. Enzyme activity was found near synovial blood vessels, and there was 2% as much GUSB activity in synovial fluid as in serum. We conclude that neonatal gene therapy reduces skeletal abnormalities in MPS VII dogs, but clinically-relevant abnormalities remain. Enzyme replacement therapy will probably have similar limitations long-term. Copyright © 2013 Elsevier Inc. All rights reserved.

The use of genes for performance enhancement: doping or therapy?

PubMed

Oliveira, R S; Collares, T F; Smith, K R; Collares, T V; Seixas, F K

2011-12-01

Recent biotechnological advances have permitted the manipulation of genetic sequences to treat several diseases in a process called gene therapy. However, the advance of gene therapy has opened the door to the possibility of using genetic manipulation (GM) to enhance athletic performance. In such 'gene doping', exogenous genetic sequences are inserted into a specific tissue, altering cellular gene activity or leading to the expression of a protein product. The exogenous genes most likely to be utilized for gene doping include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor type 1 (IGF-1), myostatin antagonists, and endorphin. However, many other genes could also be used, such as those involved in glucose metabolic pathways. Because gene doping would be very difficult to detect, it is inherently very attractive for those involved in sports who are prepared to cheat. Moreover, the field of gene therapy is constantly and rapidly progressing, and this is likely to generate many new possibilities for gene doping. Thus, as part of the general fight against all forms of doping, it will be necessary to develop and continually improve means of detecting exogenous gene sequences (or their products) in athletes. Nevertheless, some bioethicists have argued for a liberal approach to gene doping.

A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma.

PubMed

Söling, Ariane; Theiss, Christian; Jungmichel, Stephanie; Rainov, Nikolai G

2004-08-04

BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.

Gene therapy for achromatopsia.

PubMed

Michalakis, Stylianos; Schön, Christian; Becirovic, Elvir; Biel, Martin

2017-03-01

The present review summarizes the current status of achromatopsia (ACHM) gene therapy-related research activities and provides an outlook for their clinical application. ACHM is an inherited eye disease characterized by a congenital absence of cone photoreceptor function. As a consequence, ACHM is associated with strongly impaired daylight vision, photophobia, nystagmus and a lack of color discrimination. Currently, six genes have been linked to ACHM. Up to 80% of the patients carry mutations in the genes CNGA3 and CNGB3 encoding the two subunits of the cone cyclic nucleotide-gated channel. Various animal models of the disease have been established and their characterization has helped to increase our understanding of the pathophysiology associated with ACHM. With the advent of adeno-associated virus vectors as valuable gene delivery tools for retinal photoreceptors, a number of promising gene supplementation therapy programs have been initiated. In recent years, huge progress has been made towards bringing a curative treatment for ACHM into clinics. The first clinical trials are ongoing or will be launched soon and are expected to contribute important data on the safety and efficacy of ACHM gene supplementation therapy. Copyright © 2017 John Wiley & Sons, Ltd.

Radiogenetic therapy: strategies to overcome tumor resistance.

PubMed

Marples, B; Greco, O; Joiner, M C; Scott, S D

2003-01-01

The aim of cancer gene therapy is to selectively kill malignant cells at the tumor site, by exploiting traits specific to cancer cells and/or solid tumors. Strategies that take advantage of biological features common to different tumor types are particularly promising, since they have wide clinical applicability. Much attention has focused on genetic methods that complement radiotherapy, the principal treatment modality, or that exploit hypoxia, the most ubiquitous characteristic of most solid cancers. The goal of this review is to highlight two promising gene therapy methods developed specifically to target the tumor volume that can be readily used in combination with radiotherapy. The first approach uses radiation-responsive gene promoters to control the selective expression of a suicide gene (e.g., herpes simplex virus thymidine kinase) to irradiated tissue only, leading to targeted cell killing in the presence of a prodrug (e.g., ganciclovir). The second method utilizes oxygen-dependent promoters to produce selective therapeutic gene expression and prodrug activation in hypoxic cells, which are refractive to conventional radiotherapy. Further refining of tumor targeting can be achieved by combining radiation and hypoxia responsive elements in chimeric promoters activated by either and dual stimuli. The in vitro and in vivo studies described in this review suggest that the combination of gene therapy and radiotherapy protocols has potential for use in cancer care, particularly in cases currently refractory to treatment as a result of inherent or hypoxia-mediated radioresistance.

Endogenous versus Exogenous Growth Factor Regulation of Articular Chondrocytes

PubMed Central

Shi, Shuiliang; Chan, Albert G.; Mercer, Scott; Eckert, George J.; Trippel, Stephen B.

2014-01-01

Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF-I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF-ß1 stimulated these reparative functions, while endogenous TGF-ß1 had little effect. Endogenous TGF-ß1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for articular cartilage repair may differ for different growth factors. In the case of IGF-I, gene therapy or protein therapy appear to be viable options. In contrast, TGF-ß1 gene therapy may be constrained by a limited ability of chondrocytes to convert latent complexes to an active form. PMID:24105960

Poly (amidoamine) (PAMAM) dendrimer mediated delivery of drug and pDNA/siRNA for cancer therapy.

PubMed

Li, Jun; Liang, Huamin; Liu, Jing; Wang, Ziyuan

2018-07-30

Poly (amidoamine) (PAMAM) dendrimers are well-defined, highly branched macromolecules with numerous active amine groups on the surface. Because of their unique properties, PAMAM dendrimers have steadily grown in popularity in drug delivery, gene therapy, medical imaging and diagnostic application. This review focuses on the recent developments on the application in PAMAM dendrimers as effective carriers for drug and gene (pDNA, siRNA) delivery in cancer therapy, including: a) PAMAM for anticancer drug delivery; b) PAMAM and gene therapy; c) PAMAM used in overcoming tumor multidrug resistance; d) PAMAM used for hybrid nanoparticles; and e) PAMAM linked or loaded in other nanoparticles. Copyright © 2018 Elsevier B.V. All rights reserved.

Tracking Progesterone Receptor-Mediated Actions in Breast Cancer

PubMed Central

Knutson, Todd P.; Lange, Carol A.

2014-01-01

Ovarian steroid hormones contribute to breast cancer initiation and progression primarily through the actions of their nuclear transcription factors, the estrogen receptor alpha (ERα) and progesterone receptors (PRs). These receptors are important drivers of the luminal A and B subtypes of breast cancer, where estrogen-blocking drugs have been effective endocrine therapies for patients with these tumors. However, many patients do not respond, or become resistant to treatment. When endocrine therapies fail, the luminal subtypes of breast cancer are more difficult to treat because these subtypes are among the most heterogeneous in terms of mutation diversity and gene expression profiles. Recent evidence suggests that progestin and PR actions may be important drivers of luminal breast cancers. Clinical trial data has demonstrated that hormone replacement therapy with progestins drives invasive breast cancer and results in greater mortality. PR transcriptional activity is dependent upon cross-talk with growth factor signaling pathways that alter PR phosphorylation, acetylation, or SUMOylation as mechanisms for regulating PR target gene selection required for increased cell proliferation and survival. Site-specific PR phosphorylation is the primary driver of gene-selective PR transcriptional activity. However, PR phosphorylation and heightened transcriptional activity is coupled to rapid PR protein degradation; the range of active PR detected in tumors is likely to be dynamic. Thus, PR target gene signatures may provide a more accurate means of tracking PR’s contribution to tumor progression rather than standard clinical protein-based (IHC) assays. Further development of antiprogestin therapies should be considered along side antiestrogens and aromatase inhibitors. PMID:24291072

Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

PubMed Central

Siner, Joshua I.; Samelson-Jones, Benjamin J.; Crudele, Julie M.; French, Robert A.; Lee, Benjamin J.; Zhou, Shanzhen; Merricks, Elizabeth; Raymer, Robin; Camire, Rodney M.; Arruda, Valder R.

2016-01-01

Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645–1648) in the design of B-domain–deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector–based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy. PMID:27734034

Comparison and evaluation of gene therapy and epigenetic approaches for wound healing.

PubMed

Cutroneo, K R; Chiu, J F

2000-01-01

During the past decade considerable evidence has mounted concerning the importance of growth factors in the wound healing process both for cell replication and for stimulating reparative cells to synthesize and secrete extracellular matrix components. During normal wound healing the growth factor concentration has to be maintained at a certain level. If the growth factor concentration is too low, normal healing fails to occur. Whereas if the growth factor concentration is too high due to either over-expression of the growth factor or too much growth factor being applied to the wound, aberrant wound healing will occur. One approach for controlling the amount of growth factor at the wound site during normal healing is through gene therapy and the titration of gene dosage. However if a narrow window exists between the beneficial therapeutic effect and toxic effects with increasing gene dosage, an agent may be necessary to give in combination with gene therapy to regulate the over-expression of growth factor. In addition to genetic approaches to regulate wound healing, epigenetic approaches also exist. Antisense oligodeoxynucleotides have been shown to regulate wound repair in certain model systems and to determine the protein(s) necessary for normal wound healing. A novel approach to regulate the activity of collagen genes, thereby affecting fibrosis, is to use a sense oligodeoxynucleotide having the same sequence of the cis element which regulates the promoter activity of a particular collagen gene. This exogenous oligodeoxynucleotide will compete with the cis element in the collagen gene for the trans-acting factor which regulates promoter activity. These epigenetic approaches afford the opportunity to regulate over-expression of growth factor and therefore preclude the potential toxic effects of gene therapy. Both genetic and epigenetic approaches for regulating the wound healing process, either normal or aberrant wound healing, have certain advantages and disadvantages which are discussed in the present article.

In silico Pathway Activation Network Decomposition Analysis (iPANDA) as a method for biomarker development.

PubMed

Ozerov, Ivan V; Lezhnina, Ksenia V; Izumchenko, Evgeny; Artemov, Artem V; Medintsev, Sergey; Vanhaelen, Quentin; Aliper, Alexander; Vijg, Jan; Osipov, Andreyan N; Labat, Ivan; West, Michael D; Buzdin, Anton; Cantor, Charles R; Nikolsky, Yuri; Borisov, Nikolay; Irincheeva, Irina; Khokhlovich, Edward; Sidransky, David; Camargo, Miguel Luiz; Zhavoronkov, Alex

2016-11-16

Signalling pathway activation analysis is a powerful approach for extracting biologically relevant features from large-scale transcriptomic and proteomic data. However, modern pathway-based methods often fail to provide stable pathway signatures of a specific phenotype or reliable disease biomarkers. In the present study, we introduce the in silico Pathway Activation Network Decomposition Analysis (iPANDA) as a scalable robust method for biomarker identification using gene expression data. The iPANDA method combines precalculated gene coexpression data with gene importance factors based on the degree of differential gene expression and pathway topology decomposition for obtaining pathway activation scores. Using Microarray Analysis Quality Control (MAQC) data sets and pretreatment data on Taxol-based neoadjuvant breast cancer therapy from multiple sources, we demonstrate that iPANDA provides significant noise reduction in transcriptomic data and identifies highly robust sets of biologically relevant pathway signatures. We successfully apply iPANDA for stratifying breast cancer patients according to their sensitivity to neoadjuvant therapy.

In silico Pathway Activation Network Decomposition Analysis (iPANDA) as a method for biomarker development

PubMed Central

Ozerov, Ivan V.; Lezhnina, Ksenia V.; Izumchenko, Evgeny; Artemov, Artem V.; Medintsev, Sergey; Vanhaelen, Quentin; Aliper, Alexander; Vijg, Jan; Osipov, Andreyan N.; Labat, Ivan; West, Michael D.; Buzdin, Anton; Cantor, Charles R.; Nikolsky, Yuri; Borisov, Nikolay; Irincheeva, Irina; Khokhlovich, Edward; Sidransky, David; Camargo, Miguel Luiz; Zhavoronkov, Alex

2016-01-01

Signalling pathway activation analysis is a powerful approach for extracting biologically relevant features from large-scale transcriptomic and proteomic data. However, modern pathway-based methods often fail to provide stable pathway signatures of a specific phenotype or reliable disease biomarkers. In the present study, we introduce the in silico Pathway Activation Network Decomposition Analysis (iPANDA) as a scalable robust method for biomarker identification using gene expression data. The iPANDA method combines precalculated gene coexpression data with gene importance factors based on the degree of differential gene expression and pathway topology decomposition for obtaining pathway activation scores. Using Microarray Analysis Quality Control (MAQC) data sets and pretreatment data on Taxol-based neoadjuvant breast cancer therapy from multiple sources, we demonstrate that iPANDA provides significant noise reduction in transcriptomic data and identifies highly robust sets of biologically relevant pathway signatures. We successfully apply iPANDA for stratifying breast cancer patients according to their sensitivity to neoadjuvant therapy. PMID:27848968

Applications of microscopy to genetic therapy of cystic fibrosis and other human diseases.

PubMed

Moninger, Thomas O; Nessler, Randy A; Moore, Kenneth C

2006-01-01

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.

Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue

NASA Astrophysics Data System (ADS)

Lee, Hyun Chul; Kim, Su-Jin; Kim, Kyung-Sup; Shin, Hang-Cheol; Yoon, Ji-Won

2000-11-01

A cure for diabetes has long been sought using several different approaches, including islet transplantation, regeneration of β cells and insulin gene therapy. However, permanent remission of type 1 diabetes has not yet been satisfactorily achieved. The development of type 1 diabetes results from the almost total destruction of insulin-producing pancreatic β cells by autoimmune responses specific to β cells. Standard insulin therapy may not maintain blood glucose concentrations within the relatively narrow range that occurs in the presence of normal pancreatic β cells. We used a recombinant adeno-associated virus (rAAV) that expresses a single-chain insulin analogue (SIA), which possesses biologically active insulin activity without enzymatic conversion, under the control of hepatocyte-specific L-type pyruvate kinase (LPK) promoter, which regulates SIA expression in response to blood glucose levels. Here we show that SIA produced from the gene construct rAAV-LPK-SIA caused remission of diabetes in streptozotocin-induced diabetic rats and autoimmune diabetic mice for a prolonged time without any apparent side effects. This new SIA gene therapy may have potential therapeutic value for the cure of autoimmune diabetes in humans.

Phospholamban mutants compete with wild type for SERCA binding in living cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gruber, Simon J.; Haydon, Suzanne; Thomas, David D., E-mail: ddt@umn.edu

2012-04-06

Highlights: Black-Right-Pointing-Pointer PLB phosphorylation in HEK cells increased FRET between YFP-PLB and CFP-SERCA. Black-Right-Pointing-Pointer Competition: Expressing loss-of-function PLB mutants in the system decreased FRET. Black-Right-Pointing-Pointer The FRET assay could screen potential therapeutic PLB mutants to activate SERCA. -- Abstract: We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca{sup 2+} cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCAmore » activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLB{sub M}) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLB{sub M} in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLB{sub M} and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLB{sub M} for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.« less

Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta 1-producing fibroblasts.

PubMed

Lee, K H; Song, S U; Hwang, T S; Yi, Y; Oh, I S; Lee, J Y; Choi, K B; Choi, M S; Kim, S J

2001-09-20

Transforming growth factor beta (TGF-beta) has been considered as a candidate for gene therapy of orthopedic diseases. The possible application of cell-mediated TGF-beta gene therapy as a new treatment regimen for degenerative arthritis was investigated. In this study, fibroblasts expressing active TGF-beta 1 were injected into the knee joints of rabbits with artificially made cartilage defects to evaluate the feasibility of this therapy for orthopedic diseases. Two to 3 weeks after the injection there was evidence of cartilage regeneration, and at 4 to 6 weeks the cartilage defect was completely filled with newly grown hyaline cartilage. Histological analyses of the regenerated cartilage suggested that it was well integrated with the adjacent normal cartilage at the sides of the defect and that the newly formed tissue was indeed hyaline cartilage. Our findings suggest that cell-mediated TGF-beta 1 gene therapy may be a novel treatment for orthopedic diseases in which hyaline cartilage damage has occurred.

Applications of Gene Editing Technologies to Cellular Therapies.

PubMed

Rein, Lindsay A M; Yang, Haeyoon; Chao, Nelson J

2018-03-27

Hematologic malignancies are characterized by genetic heterogeneity, making classic gene therapy with a goal of correcting 1 genetic defect ineffective in many of these diseases. Despite initial tribulations, gene therapy, as a field, has grown by leaps and bounds with the recent development of gene editing techniques including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR) sequences and CRISPR-associated protein-9 (Cas9) nuclease or CRISPR/Cas9. These novel technologies have been applied to efficiently and specifically modify genetic information in target and effector cells. In particular, CRISPR/Cas9 technology has been applied to various hematologic malignancies and has also been used to modify and improve chimeric antigen receptor-modified T cells for the purpose of providing effective cellular therapies. Although gene editing is in its infancy in malignant hematologic diseases, there is much room for growth and application in the future. Copyright © 2018 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Advanced therapies for the treatment of hemophilia: future perspectives.

PubMed

Liras, Antonio; Segovia, Cristina; Gabán, Aline S

2012-12-13

Monogenic diseases are ideal candidates for treatment by the emerging advanced therapies, which are capable of correcting alterations in protein expression that result from genetic mutation. In hemophilia A and B such alterations affect the activity of coagulation factors VIII and IX, respectively, and are responsible for the development of the disease. Advanced therapies may involve the replacement of a deficient gene by a healthy gene so that it generates a certain functional, structural or transport protein (gene therapy); the incorporation of a full array of healthy genes and proteins through perfusion or transplantation of healthy cells (cell therapy); or tissue transplantation and formation of healthy organs (tissue engineering). For their part, induced pluripotent stem cells have recently been shown to also play a significant role in the fields of cell therapy and tissue engineering. Hemophilia is optimally suited for advanced therapies owing to the fact that, as a monogenic condition, it does not require very high expression levels of a coagulation factor to reach moderate disease status. As a result, significant progress has been possible with respect to these kinds of strategies, especially in the fields of gene therapy (by using viral and non-viral vectors) and cell therapy (by means of several types of target cells). Thus, although still considered a rare disorder, hemophilia is now recognized as a condition amenable to gene therapy, which can be administered in the form of lentiviral and adeno-associated vectors applied to adult stem cells, autologous fibroblasts, platelets and hematopoietic stem cells; by means of non-viral vectors; or through the repair of mutations by chimeric oligonucleotides. In hemophilia, cell therapy approaches have been based mainly on transplantation of healthy cells (adult stem cells or induced pluripotent cell-derived progenitor cells) in order to restore alterations in coagulation factor expression.

Gene therapy clinical trials worldwide to 2017: An update.

PubMed

Ginn, Samantha L; Amaya, Anais K; Alexander, Ian E; Edelstein, Michael; Abedi, Mohammad R

2018-03-25

To date, almost 2600 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical activity from trial databases, official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our November 2017 update, we have entries on 2597 trials undertaken in 38 countries. We have analysed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and the genes that have been transferred. Details of the analyses presented, and our searchable database are available via The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at: http://www.wiley.co.uk/genmed/clinical. We also provide an overview of the progress being made in gene therapy clinical trials around the world, and discuss key trends since the previous review, namely the use of chimeric antigen receptor T cells for the treatment of cancer and advancements in genome editing technologies, which have the potential to transform the field moving forward. Copyright © 2018 John Wiley & Sons, Ltd.

Cellular innate immunity and restriction of viral infection: implications for lentiviral gene therapy in human hematopoietic cells.

PubMed

Kajaste-Rudnitski, Anna; Naldini, Luigi

2015-04-01

Hematopoietic gene therapy has tremendous potential to treat human disease. Nevertheless, for gene therapy to be efficacious, effective gene transfer into target cells must be reached without inducing detrimental effects on their biological properties. This remains a great challenge for the field as high vector doses and prolonged ex vivo culture conditions are still required to reach significant transduction levels of clinically relevant human hematopoietic stem and progenitor cells (HSPCs), while other potential target cells such as primary macrophages can hardly be transduced. The reasons behind poor permissiveness of primary human hematopoietic cells to gene transfer partly reside in the retroviral origin of lentiviral vectors (LVs). In particular, host antiviral factors referred to as restriction factors targeting the retroviral life cycle can hamper LV transduction efficiency. Furthermore, LVs may activate innate immune sensors not only in differentiated hematopoietic cells but also in HSPCs, with potential consequences on transduction efficiency as well as their biological properties. Therefore, better understanding of the vector-host interactions in the context of hematopoietic gene transfer is important for the development of safer and more efficient gene therapy strategies. In this review, we briefly summarize the current knowledge regarding innate immune recognition of lentiviruses in primary human hematopoietic cells as well as discuss its relevance for LV-based ex vivo gene therapy approaches.

Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

PubMed Central

Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

2015-01-01

Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

Recent trends in the gene therapy of β-thalassemia

PubMed Central

Finotti, Alessia; Breda, Laura; Lederer, Carsten W; Bianchi, Nicoletta; Zuccato, Cristina; Kleanthous, Marina; Rivella, Stefano; Gambari, Roberto

2015-01-01

The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases. PMID:25737641

Production of recombinant streptokinase from Streptococcus pyogenes isolate and its potential for thrombolytic therapy.

PubMed

Assiri, Abdullah S; El-Gamal, Basiouny A; Hafez, Elsayed E; Haidara, Mohamed A

2014-12-01

To produce an effective recombinant streptokinase (rSK) from pathogenic Streptococcus pyogenes isolate in yeast, and evaluate its potential for thrombolytic therapy. This study was conducted from November 2012 to December 2013 at King Khalid University, Abha, Kingdom of Saudi Arabia (KSA). Throat swabs collected from 45 pharyngitis patients in Asser Central Hospital, Abha, KSA were used to isolate Streptococcus pyogenes. The bacterial DNA was used for amplification of the streptokinase gene (1200 bp). The gene was cloned and in vitro transcribed in an eukaryotic expression vector that was transformed into yeast Pichia pastoris SMD1168, and the rSK protein was purified and tested for its thrombolytic activity. The Streptococcus pyogenes strain was isolated and its DNA nucleotide sequence revealed similarity to other Streptococcus pyogenes in the Gene bank. Sequencing of the amplified gene based on DNA nucleotide sequence revealed a SK gene closely related to other SK genes in the Gene bank. However, based on deduced amino acids sequence, the gene formed a separate cluster different from clusters formed by other examined genes, suggesting a new bacterial isolate and accordingly a new gene. The purified protein showed 82% clot lysis compared to a commercial SK (81%) at an enzyme concentration of 2000 U/ml. The present yeast rSK showed similar thrombolytic activity in vitro as that of a commercial SK, suggesting its potential for thrombolytic therapy and large scale production. 

Combining Cytotoxic and Immune-Mediated Gene Therapy to Treat Brain Tumors

PubMed Central

Curtin, James F.; King, Gwendalyn D.; Candolfi, Marianela; Greeno, Remy B.; Kroeger, Kurt M.; Lowenstein, Pedro R.; Castro, Maria G.

2006-01-01

Glioblastoma (GBM) is a type of intracranial brain tumor, for which there is no cure. In spite of advances in surgery, chemotherapy and radiotherapy, patients die within a year of diagnosis. Therefore, there is a critical need to develop novel therapeutic approaches for this disease. Gene therapy, which is the use of genes or other nucleic acids as drugs, is a powerful new treatment strategy which can be developed to treat GBM. Several treatment modalities are amenable for gene therapy implementation, e.g. conditional cytotoxic approaches, targeted delivery of toxins into the tumor mass, immune stimulatory strategies, and these will all be the focus of this review. Both conditional cytotoxicity and targeted toxin mediated tumor death, are aimed at eliminating an established tumor mass and preventing further growth. Tumors employ several defensive strategies that suppress and inhibit anti-tumor immune responses. A better understanding of the mechanisms involved in eliciting anti-tumor immune responses has identified promising targets for immunotherapy. Immunotherapy is designed to aid the immune system to recognize and destroy tumor cells in order to eliminate the tumor burden. Also, immune-therapeutic strategies have the added advantage that an activated immune system has the capability of recognizing tumor cells at distant sites from the primary tumor, therefore targeting metastasis distant from the primary tumor locale. Pre-clinical models and clinical trials have demonstrated that in spite of their location within the central nervous system (CNS), a tissue described as ‘immune privileged’, brain tumors can be effectively targeted by the activated immune system following various immunotherapeutic strategies. This review will highlight recent advances in brain tumor immunotherapy, with particular emphasis on advances made using gene therapy strategies, as well as reviewing other novel therapies that can be used in combination with immunotherapy. Another important aspect of implementing gene therapy in the clinical arena is to be able to image the targeting of the therapeutics to the tumors, treatment effectiveness and progression of disease. We have therefore reviewed the most exciting non-invasive, in vivo imaging techniques which can be used in combination with gene therapy to monitor therapeutic efficacy over time. PMID:16248789

Robust, synergistic regulation of human gene expression using TALE activators.

PubMed

Maeder, Morgan L; Linder, Samantha J; Reyon, Deepak; Angstman, James F; Fu, Yanfang; Sander, Jeffry D; Joung, J Keith

2013-03-01

Artificial activators designed using transcription activator-like effector (TALE) technology have broad utility, but previous studies suggest that these monomeric proteins often exhibit low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy, and guide design of monomeric TALE-based fusion proteins.

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A.

PubMed

Du, Lily M; Nurden, Paquita; Nurden, Alan T; Nichols, Timothy C; Bellinger, Dwight A; Jensen, Eric S; Haberichter, Sandra L; Merricks, Elizabeth; Raymer, Robin A; Fang, Juan; Koukouritaki, Sevasti B; Jacobi, Paula M; Hawkins, Troy B; Cornetta, Kenneth; Shi, Qizhen; Wilcox, David A

2013-01-01

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A.

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

PubMed Central

Du, Lily M.; Nurden, Paquita; Nurden, Alan T.; Nichols, Timothy C.; Bellinger, Dwight A.; Jensen, Eric S.; Haberichter, Sandra L.; Merricks, Elizabeth; Raymer, Robin A.; Fang, Juan; Koukouritaki, Sevasti B.; Jacobi, Paula M.; Hawkins, Troy B.; Cornetta, Kenneth; Shi, Qizhen; Wilcox, David A.

2013-01-01

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A. PMID:24253479

Fight fire with fire: Gene therapy strategies to cure HIV.

PubMed

Huyghe, Jon; Magdalena, Sips; Vandekerckhove, Linos

2017-08-01

Human Immunodeficiency Virus (HIV) to date remains one of the most notorious viruses mankind has ever faced. Despite enormous investments in HIV research for more than 30 years an effective cure for HIV has been elusive. Areas covered: Combination antiretroviral therapy (cART) suppresses active viral replication, but is not able to eliminate the virus completely due to stable integration of HIV inside the host genome of infected cells and the establishment of a latent reservoir, that is insensitive to cART. Nevertheless, this latent HIV reservoir is fully capable to refuel viral replication when treatment is stopped, creating a major obstacle towards a cure for HIV. Several gene therapy approaches ranging from the generation of HIV resistant CD4 + T cells to the eradication of HIV infected cells by immune cell engineering are currently under pre-clinical and clinical investigation and may present a promising road to a cure. In this review, we focus on the status and the prospects of gene therapy strategies to cure/eradicate HIV. Expert commentary: Recent advances in gene therapy for oncology and infectious diseases indicate that gene therapy may be a feasible and very potent cure strategy, and therefore a potential game changer in the search for an effective HIV cure.

The human visual cortex responds to gene therapy–mediated recovery of retinal function

PubMed Central

Ashtari, Manzar; Cyckowski, Laura L.; Monroe, Justin F.; Marshall, Kathleen A.; Chung, Daniel C.; Auricchio, Alberto; Simonelli, Francesca; Leroy, Bart P.; Maguire, Albert M.; Shindler, Kenneth S.; Bennett, Jean

2011-01-01

Leber congenital amaurosis (LCA) is a rare degenerative eye disease, linked to mutations in at least 14 genes. A recent gene therapy trial in patients with LCA2, who have mutations in RPE65, demonstrated that subretinal injection of an adeno-associated virus (AAV) carrying the normal cDNA of that gene (AAV2-hRPE65v2) could markedly improve vision. However, it remains unclear how the visual cortex responds to recovery of retinal function after prolonged sensory deprivation. Here, 3 of the gene therapy trial subjects, treated at ages 8, 9, and 35 years, underwent functional MRI within 2 years of unilateral injection of AAV2-hRPE65v2. All subjects showed increased cortical activation in response to high- and medium-contrast stimuli after exposure to the treated compared with the untreated eye. Furthermore, we observed a correlation between the visual field maps and the distribution of cortical activations for the treated eyes. These data suggest that despite severe and long-term visual impairment, treated LCA2 patients have intact and responsive visual pathways. In addition, these data suggest that gene therapy resulted in not only sustained and improved visual ability, but also enhanced contrast sensitivity. PMID:21606598

Gene and cell therapy for pancreatic cancer.

PubMed

Singh, Hans Martin; Ungerechts, Guy; Tsimberidou, Apostolia M

2015-04-01

The clinical outcomes of patients with pancreatic cancer are poor, and the limited success of classical chemotherapy underscores the need for new, targeted approaches for this disease. The delivery of genetic material to cells allows for a variety of therapeutic concepts. Engineered agents based on synthetic biology are under clinical investigation in various cancers, including pancreatic cancer. This review focuses on Phase I - III clinical trials of gene and cell therapy for pancreatic cancer and on future implications of recent translational research. Trials available in the US National Library of Medicine (www.clinicaltrials.gov) until February 2014 were reviewed and relevant published results of preclinical and clinical studies were retrieved from www.pubmed.gov . In pancreatic cancer, gene and cell therapies are feasible and may have synergistic antitumor activity with standard treatment and/or immunotherapy. Challenges are related to application safety, manufacturing costs, and a new spectrum of adverse events. Further studies are needed to evaluate available agents in carefully designed protocols and combination regimens. Enabling personalized cancer therapy, insights from molecular diagnostic technologies will guide the development and selection of new gene-based drugs. The evolving preclinical and clinical data on gene-based therapies can lay the foundation for future avenues improving patient care in pancreatic cancer.

Current status of gene therapy for brain tumors

PubMed Central

MURPHY, ANDREA M.; RABKIN, SAMUEL D.

2013-01-01

Glioblastoma (GBM) is the most common and deadliest primary brain tumor in adults, with current treatments having limited impact on disease progression. Therefore the development of alternative treatment options is greatly needed. Gene therapy is a treatment strategy that relies on the delivery of genetic material, usually transgenes or viruses, into cells for therapeutic purposes, and has been applied to GBM with increasing promise. We have included selectively replication-competent oncolytic viruses within this strategy, although the virus acts directly as a complex biologic anti-tumor agent rather than as a classic gene delivery vehicle. GBM is a good candidate for gene therapy because tumors remain locally within the brain and only rarely metastasize to other tissues; the majority of cells in the brain are post-mitotic, which allows for specific targeting of dividing tumor cells; and tumors can often be accessed neurosurgically for administration of therapy. Delivery vehicles used for brain tumors include nonreplicating viral vectors, normal adult stem/progenitor cells, and oncolytic viruses. The therapeutic transgenes or viruses are typically cytotoxic or express prodrug activating suicide genes to kill glioma cells, immunostimulatory to induce or amplify anti-tumor immune responses, and/or modify the tumor microenvironment such as blocking angiogenesis. This review describes current preclinical and clinical gene therapy strategies for the treatment of glioma. PMID:23246627

Mammalian synthetic biology: emerging medical applications

PubMed Central

Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M.; Krams, Rob

2015-01-01

In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes. PMID:25808341

Small RNA-induced INTS6 gene up-regulation suppresses castration-resistant prostate cancer cells by regulating β-catenin signaling.

PubMed

Chen, Hong; Shen, Hai-Xiang; Lin, Yi-Wei; Mao, Ye-Qing; Liu, Ben; Xie, Li-Ping

2018-06-12

Small RNAs play an important role in gene regulatory networks. The gene suppressive effect of small RNAs was previously the dominant focus of studies, but during the recent decade, small RNA-induced gene activation has been reported and has become a notable gene manipulation technique. In this study, a putative tumor suppressor, INTS6, was activated by introducing a promoter-targeted small RNA (dsRNA-915) into castration-resistant prostate cancer (CRPC) cells. Unique dynamics associated with the gene upregulation phenomenon was observed. Following gene activation, cell proliferation and motility were suppressed in vitro. Downregulation of Wnt/β-catenin signaling was observed during the activation period, and the impairment of β-catenin degradation reversed the tumor suppressor effects of INTS6. These results suggest the potential application of small activating RNAs in targeted gene therapy for CRPC.

The therapeutic application of CRISPR/Cas9 technologies for HIV

PubMed Central

Saayman, Sheena; Ali, Stuart A.; Morris, Kevin V.; Weinberg, Marc S.

2015-01-01

Introduction The use of antiretroviral therapy (ART) has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing system. Areas covered CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs (sgRNAs) can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease deficient Cas9 fused to transcription activating domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus. Expert opinion The diversity of the CRISPR/Cas9 technologies hold great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV. PMID:25865334

Oncosuppressive suicide gene virotherapy "PVH1-yCD/5-FC" for pancreatic peritoneal carcinomatosis treatment: NFκB and Akt/PI3K involvement.

PubMed

Réjiba, Soukaina; Bigand, Christelle; Parmentier, Celine; Masmoudi, Ahmed; Hajri, Amor

2013-01-01

Peritoneal carcinomatosis is common in advanced pancreatic cancer. Despite current standard treatment, patients with this disease until recently were considered incurable. Cancer gene therapy using oncolytic viruses have generated much interest over the past few years. Here, we investigated a new gene directed enzyme prodrug therapy (GDEPT) approach for an oncosuppressive virotherapy strategy using parvovirus H1 (PV-H1) which preferentially replicates and kills malignant cells. Although, PV-H1 is not potent enough to destroy tumors, it represents an attractive vector for cancer gene therapy. We therefore sought to determine whether the suicide gene/prodrug system, yCD/5-FC could be rationally combined to PV-H1 augmenting its intrinsic oncolytic activity for pancreatic cancer prevention and treatment. We showed that the engineered recombinant parvovirus rPVH1-yCD with 5-FC treatment increased significantly the intrinsic cytotoxic effect and resulted in potent induction of apoptosis and tumor growth inhibition in chemosensitive and chemoresistant cells. Additionally, the suicide gene-expressing PV-H1 infection reduced significantly the constitutive activities of NFκB and Akt/PI3K. Combination of their pharmacological inhibitors (MG132 and LY294002) with rPVH1-yCD/5-FC resulted in substantial increase of antitumor activity. In vivo, high and sustained expression of NS1 and yCD was observed in the disseminated tumor nodules and absent in normal tissues. Treatment of mice bearing intraperitoneal pancreatic carcinomatosis with rPVH1-yCD/5-FC resulted in a drastic inhibition of tumor cell spreading and subsequent increase in long-term survival. Together, the presented data show the improved oncolytic activity of wPV-H1 by yCD/5-FC and thus provides valuable effective and promising virotherapy strategy for prevention of tumor recurrence and treatment. In the light of this study, the suicide gene parvovirotherapy approach represents a new weapon in the war against pancreatic cancer. Moreover, these preliminary accomplishments are opening new field for future development of new combined targeted therapies to have a meaningful impact on advanced cancer.

Oncosuppressive Suicide Gene Virotherapy “PVH1-yCD/5-FC” for Pancreatic Peritoneal Carcinomatosis Treatment: NFκB and Akt/PI3K Involvement

PubMed Central

Réjiba, Soukaina; Bigand, Christelle; Parmentier, Celine; Masmoudi, Ahmed; Hajri, Amor

2013-01-01

Peritoneal carcinomatosis is common in advanced pancreatic cancer. Despite current standard treatment, patients with this disease until recently were considered incurable. Cancer gene therapy using oncolytic viruses have generated much interest over the past few years. Here, we investigated a new gene directed enzyme prodrug therapy (GDEPT) approach for an oncosuppressive virotherapy strategy using parvovirus H1 (PV-H1) which preferentially replicates and kills malignant cells. Although, PV-H1 is not potent enough to destroy tumors, it represents an attractive vector for cancer gene therapy. We therefore sought to determine whether the suicide gene/prodrug system, yCD/5-FC could be rationally combined to PV-H1 augmenting its intrinsic oncolytic activity for pancreatic cancer prevention and treatment. We showed that the engineered recombinant parvovirus rPVH1-yCD with 5-FC treatment increased significantly the intrinsic cytotoxic effect and resulted in potent induction of apoptosis and tumor growth inhibition in chemosensitive and chemoresistant cells. Additionally, the suicide gene-expressing PV-H1 infection reduced significantly the constitutive activities of NFκB and Akt/PI3K. Combination of their pharmacological inhibitors (MG132 and LY294002) with rPVH1-yCD/5-FC resulted in substantial increase of antitumor activity. In vivo, high and sustained expression of NS1 and yCD was observed in the disseminated tumor nodules and absent in normal tissues. Treatment of mice bearing intraperitoneal pancreatic carcinomatosis with rPVH1-yCD/5-FC resulted in a drastic inhibition of tumor cell spreading and subsequent increase in long-term survival. Together, the presented data show the improved oncolytic activity of wPV-H1 by yCD/5-FC and thus provides valuable effective and promising virotherapy strategy for prevention of tumor recurrence and treatment. In the light of this study, the suicide gene parvovirotherapy approach represents a new weapon in the war against pancreatic cancer. Moreover, these preliminary accomplishments are opening new field for future development of new combined targeted therapies to have a meaningful impact on advanced cancer. PMID:23967078

Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy.

PubMed

Zonari, Erika; Desantis, Giacomo; Petrillo, Carolina; Boccalatte, Francesco E; Lidonnici, Maria Rosa; Kajaste-Rudnitski, Anna; Aiuti, Alessandro; Ferrari, Giuliana; Naldini, Luigi; Gentner, Bernhard

2017-04-11

Ex vivo gene therapy based on CD34 + hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34 + cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E 2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34 + CD38 - cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs.

PubMed

Callan, Mary Beth; Haskins, Mark E; Wang, Ping; Zhou, Shangzhen; High, Katherine A; Arruda, Valder R

2016-01-01

Severe hemophilia A (HA) is an inherited bleeding disorder characterized by <1% of residual factor VIII (FVIII) clotting activity. The disease affects several mammals including dogs, and, like humans, is associated with high morbidity and mortality. In gene therapy using adeno-associated viral (AAV) vectors, the canine model has been one of the best predictors of the therapeutic dose tested in clinical trials for hemophilia B (factor IX deficiency) and other genetic diseases, such as congenital blindness. Here we report our experience with liver gene therapy with AAV-FVIII in two outbred, privately owned dogs with severe HA that resulted in sustained expression of 1-2% of normal FVIII levels and prevented 90% of expected bleeding episodes. A Thr62Met mutation in the F8 gene was identified in one dog. These data recapitulate the improvement of the disease phenotype in research animals, and in humans, with AAV liver gene therapy for hemophilia B. Our experience is a novel example of the benefits of a relevant preclinical canine model to facilitate both translational studies in humans and improved welfare of privately owned dogs.

Gastric Carcinogenesis and Underlying Molecular Mechanisms: Helicobacter pylori and Novel Targeted Therapy

PubMed Central

Nishizawa, Toshihiro

2015-01-01

The oxygen-derived free radicals that are released from activated neutrophils are one of the cytotoxic factors of Helicobacter pylori-induced gastric mucosal injury. Increased cytidine deaminase activity in H. pylori-infected gastric tissues promotes the accumulation of various mutations and might promote gastric carcinogenesis. Cytotoxin-associated gene A (CagA) is delivered into gastric epithelial cells via bacterial type IV secretion system, and it causes inflammation and activation of oncogenic pathways. H. pylori infection induces epigenetic transformations, such as aberrant promoter methylation in tumor-suppressor genes. Aberrant expression of microRNAs is also reportedly linked to gastric tumorogenesis. Moreover, recent advances in molecular targeting therapies provided a new interesting weapon to treat advanced gastric cancer through anti-human epidermal growth factor receptor 2 (HER-2) therapies. This updated review article highlights possible mechanisms of gastric carcinogenesis including H. pylori-associated factors. PMID:25945346

Genetic therapies against HIV

PubMed Central

Rossi, John J; June, Carl H; Kohn, Donald B

2015-01-01

Highly active antiretroviral therapy prolongs the life of HIV-infected individuals, but it requires lifelong treatment and results in cumulative toxicities and viral-escape mutants. Gene therapy offers the promise of preventing progressive HIV infection by sustained interference with viral replication in the absence of chronic chemotherapy. Gene-targeting strategies are being developed with RNA-based agents, such as ribozymes, antisense, RNA aptamers and small interfering RNA, and protein-based agents, such as the mutant HIV Rev protein M10, fusion inhibitors and zinc-finger nucleases. Recent advances in T-cell–based strategies include gene-modified HIV-resistant T cells, lentiviral gene delivery, CD8+ T cells, T bodies and engineered T-cell receptors. HIV-resistant hematopoietic stem cells have the potential to protect all cell types susceptible to HIV infection. The emergence of viral resistance can be addressed by therapies that use combinations of genetic agents and that inhibit both viral and host targets. Many of these strategies are being tested in ongoing and planned clinical trials. PMID:18066041

Molecular Diagnostics of Copper-Transporting Protein Mutations Allows Early Onset Individual Therapy of Menkes Disease.

PubMed

Králík, L; Flachsová, E; Hansíková, H; Saudek, V; Zeman, J; Martásek, P

2017-01-01

Menkes disease is a severe X-linked recessive disorder caused by a defect in the ATP7A gene, which encodes a membrane copper-transporting ATPase. Deficient activity of the ATP7A protein results in decreased intestinal absorption of copper, low copper level in serum and defective distribution of copper in tissues. The clinical symptoms are caused by decreased activities of copper-dependent enzymes and include neurodegeneration, connective tissue disorders, arterial changes and hair abnormalities. Without therapy, the disease is fatal in early infancy. Rapid diagnosis of Menkes disease and early start of copper therapy is critical for the effectiveness of treatment. We report a molecular biology-based strategy that allows early diagnosis of copper transport defects and implementation of individual therapies before the full development of pathological symptoms. Low serum copper and decreased activity of copperdependent mitochondrial cytochrome c oxidase in isolated platelets found in three patients indicated a possibility of functional defects in copper-transporting proteins, especially in the ATPA7 protein, a copper- transporting P-type ATPase. Rapid mutational screening of the ATP7A gene using high-resolution melting analysis of DNA indicated presence of mutations in the patients. Molecular investigation for mutations in the ATP7A gene revealed three nonsense mutations: c.2170C>T (p.Gln724Ter); c.3745G>T (p.Glu1249Ter); and c.3862C>T (p.Gln1288Ter). The mutation c.3745G>T (p.Glu1249Ter) has not been identified previously. Molecular analysis of the ATOX1 gene as a possible modulating factor of Menkes disease did not reveal presence of pathogenic mutations. Molecular diagnostics allowed early onset of individual therapies, adequate genetic counselling and prenatal diagnosis in the affected families.

Gene set analysis of purine and pyrimidine antimetabolites cancer therapies.

PubMed

Fridley, Brooke L; Batzler, Anthony; Li, Liang; Li, Fang; Matimba, Alice; Jenkins, Gregory D; Ji, Yuan; Wang, Liewei; Weinshilboum, Richard M

2011-11-01

Responses to therapies, either with regard to toxicities or efficacy, are expected to involve complex relationships of gene products within the same molecular pathway or functional gene set. Therefore, pathways or gene sets, as opposed to single genes, may better reflect the true underlying biology and may be more appropriate units for analysis of pharmacogenomic studies. Application of such methods to pharmacogenomic studies may enable the detection of more subtle effects of multiple genes in the same pathway that may be missed by assessing each gene individually. A gene set analysis of 3821 gene sets is presented assessing the association between basal messenger RNA expression and drug cytotoxicity using ethnically defined human lymphoblastoid cell lines for two classes of drugs: pyrimidines [gemcitabine (dFdC) and arabinoside] and purines [6-thioguanine and 6-mercaptopurine]. The gene set nucleoside-diphosphatase activity was found to be significantly associated with both dFdC and arabinoside, whereas gene set γ-aminobutyric acid catabolic process was associated with dFdC and 6-thioguanine. These gene sets were significantly associated with the phenotype even after adjusting for multiple testing. In addition, five associated gene sets were found in common between the pyrimidines and two gene sets for the purines (3',5'-cyclic-AMP phosphodiesterase activity and γ-aminobutyric acid catabolic process) with a P value of less than 0.0001. Functional validation was attempted with four genes each in gene sets for thiopurine and pyrimidine antimetabolites. All four genes selected from the pyrimidine gene sets (PSME3, CANT1, ENTPD6, ADRM1) were validated, but only one (PDE4D) was validated for the thiopurine gene sets. In summary, results from the gene set analysis of pyrimidine and purine therapies, used often in the treatment of various cancers, provide novel insight into the relationship between genomic variation and drug response.

Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR-Cas9 when targeting the human CCR5 gene.

PubMed

Nerys-Junior, Arildo; Braga-Dias, Luciene P; Pezzuto, Paula; Cotta-de-Almeida, Vinícius; Tanuri, Amilcar

2018-01-01

The human C-C chemokine receptor type-5 (CCR5) is the major transmembrane co-receptor that mediates HIV-1 entry into target CD4+ cells. Gene therapy to knock-out the CCR5 gene has shown encouraging results in providing a functional cure for HIV-1 infection. In gene therapy strategies, the initial region of the CCR5 gene is a hotspot for producing functional gene knock-out. Such target gene editing can be done using programmable endonucleases such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). These two gene editing approaches are the most modern and effective tools for precise gene modification. However, little is known of potential differences in the efficiencies of TALEN and CRISPR-Cas9 for editing the beginning of the CCR5 gene. To examine which of these two methods is best for gene therapy, we compared the patterns and amount of editing at the beginning of the CCR5 gene using TALEN and CRISPR-Cas9 followed by DNA sequencing. This comparison revealed that CRISPR-Cas9 mediated the sorting of cells that contained 4.8 times more gene editing than TALEN+ transfected cells.

Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR-Cas9 when targeting the human CCR5 gene

PubMed Central

Nerys-Junior, Arildo; Braga-Dias, Luciene P.; Pezzuto, Paula; Cotta-de-Almeida, Vinícius; Tanuri, Amilcar

2018-01-01

Abstract The human C-C chemokine receptor type-5 (CCR5) is the major transmembrane co-receptor that mediates HIV-1 entry into target CD4+ cells. Gene therapy to knock-out the CCR5 gene has shown encouraging results in providing a functional cure for HIV-1 infection. In gene therapy strategies, the initial region of the CCR5 gene is a hotspot for producing functional gene knock-out. Such target gene editing can be done using programmable endonucleases such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). These two gene editing approaches are the most modern and effective tools for precise gene modification. However, little is known of potential differences in the efficiencies of TALEN and CRISPR-Cas9 for editing the beginning of the CCR5 gene. To examine which of these two methods is best for gene therapy, we compared the patterns and amount of editing at the beginning of the CCR5 gene using TALEN and CRISPR-Cas9 followed by DNA sequencing. This comparison revealed that CRISPR-Cas9 mediated the sorting of cells that contained 4.8 times more gene editing than TALEN+ transfected cells. PMID:29583154

Production of MPS VII mouse (Gustm(hE540A·mE536A)Sly) doubly tolerant to human and mouse β-glucuronidase

PubMed Central

Tomatsu, Shunji; Orii, Koji O.; Vogler, Carole; Grubb, Jeffrey H.; Snella, Elizabeth M.; Gutierrez, Monica; Dieter, Tatiana; Holden, Christopher C.; Sukegawa, Kazuko; Orii, Tadao; Kondo, Naomi; Sly, William S.

2006-01-01

Mucopolysaccharidosis VII (MPS VII, Sly syndrome) is an autosomal recessive lysosomal storage disease caused by β-glucuronidase (GUS) deficiency. A naturally occurring mouse model of that disease has been very useful for studying experimental approaches to therapy. However, immune responses can complicate evaluation of the long-term benefits of enzyme replacement or gene therapy delivered to adult MPS VII mice. To make this model useful for studying the long-term effectiveness and side effects of experimental therapies delivered to adult mice, we developed a new MPS VII mouse model, which is tolerant to both human and murine GUS. To achieve this, we used homologous recombination to introduce simultaneously a human cDNA transgene expressing inactive human GUS into intron 9 of the murine Gus gene and a targeted active site mutation (E536A) into the adjacent exon 10. When the heterozygote products of germline transmission were bred to homozygosity, the homozygous mice expressed no GUS enzyme activity but expressed inactive human GUS protein highly and were tolerant to immune challenge with human enzyme. Expression of the mutant murine Gus gene was reduced to about 10% of normal levels, but the inactive murine GUS enzyme also conferred tolerance to murine GUS. This MPS VII mouse model should be useful to evaluate therapeutic responses in adult mice receiving repetitive doses of enzyme or mice receiving gene therapy as adults. Heterozygotes expressed only 9.5–26% of wild-type levels of murine GUS instead of the expected 50%, indicating a dominant-negative effect of the mutant enzyme monomers on the activity of GUS tetramers in different tissues. Corrective gene therapy in this model should provide high enough levels of expression of normal GUS monomers to overcome the dominant negative effect of mutant monomers on newly synthesized GUS tetramers in most tissues. PMID:12700165

Viral delivery of genome-modifying proteins for cellular reprogramming.

PubMed

Mikkelsen, Jacob Giehm

2018-06-18

Following the successful development of virus-based gene vehicles for genetic therapies, exploitation of viruses as carriers of genetic tools for cellular reprogramming and genome editing should be right up the street. However, whereas persistent, potentially life-long gene expression is the main goal of conventional genetic therapies, tools and bits for genome engineering should ideally be short-lived and active only for a limited time. Although viral vector systems have already been adapted for potent genome editing both in vitro and in vivo, regulatable gene expression systems or self-limiting expression circuits need to be implemented limiting exposure of chromatin to genome-modifying enzymes. As an alternative approach, emerging virus-based protein delivery technologies support direct protein delivery, providing a short, robust boost of enzymatic activity in transduced cells. Is this potentially the perfect way of shipping loads of cargo to many recipients and still maintain short-term activity? Copyright © 2018 Elsevier Ltd. All rights reserved.

Personalizing gene therapy in gastric cancer.

PubMed

Vogiatzi, P; Cassone, M; Claudio, P P

2006-11-01

Gene therapy was proposed many decades ago as a more straightforward and definitive way of curing human diseases, but only recently technical advancements and improved knowledge have allowed its active development as a broad and promising research field. After the first successes in the cure of genetic and infectious diseases, it has been actively investigated as a means to decrease the burden and suffering generated by cancer. The field of gastric cancer is witnessing an impressive flourishing of studies testing the possibilities and actual efficacy of the many different strategies employed in gene therapy, and overall results seem to be two-sided: while original ideas and innovative protocols are providing extremely interesting contributions with great potential, more advanced-phase studies concluded so far have fallen short of expectations regarding efficacy, although invariably demonstrating little or no toxicity. An overview of the major efforts in this field is provided here, and a critical discussion is presented on the single strategies undertaken and on the overall balance between potentiality and pitfalls. Copyright 2006 Prous Science. All rights reserved.

An oncolytic adenovirus regulated by a radiation-inducible promoter selectively mediates hSulf-1 gene expression and mutually reinforces antitumor activity of I131-metuximab in hepatocellular carcinoma.

PubMed

Zhang, Yan; Fang, Lin; Zhang, Quan'an; Zheng, Qin; Tong, Jinlong; Fu, Xiaohui; Jiang, Xiaoqing; Su, Changqing; Zheng, Junnian

2013-06-01

Gene therapy and antibody approaches are crucial auxiliary strategies for hepatocellular carcinoma (HCC) treatment. Previously, we established a survivin promoter-regulated oncolytic adenovirus that has inhibitory effect on HCC growth. The human sulfatase-1 (hSulf-1) gene can suppress the growth factor signaling pathways, then inhibit the proliferation of cancer cells and enhance cellular sensitivity to radiotherapy and chemotherapy. I(131)-metuximab (I(131)-mab) is a monoclonal anti-HCC antibody that conjugated to I(131) and specifically recognizes the HAb18G/CD147 antigen on HCC cells. To integrate the oncolytic adenovirus-based gene therapy and the I(131)-mab-based radioimmunotherapy, this study combined the CArG element of early growth response-l (Egr-l) gene with the survivin promoter to construct a radiation-inducible enhanced promoter, which was used to recombine a radiation-inducible oncolytic adenovirus as hSulf-1 gene vector. When I(131)-mab was incorporated into the treatment regimen, not only could the antibody produce radioimmunotherapeutic effect, but the I(131) radiation was able to further boost adenoviral proliferation. We demonstrated that the CArG-enhanced survivin promoter markedly improved the proliferative activity of the oncolytic adenovirus in HCC cells, thereby augmenting hSulf-1 expression and inducing cancer cell apoptosis. This novel strategy that involved multiple, synergistic mechanisms, including oncolytic therapy, gene therapy and radioimmunotherapy, was demonstrated to exert an excellent anti-cancer outcome, which will be a promising approach in HCC treatment. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Ewing's Sarcoma: Development of RNA Interference-Based Therapy for Advanced Disease

PubMed Central

Simmons, Olivia; Maples, Phillip B.; Senzer, Neil; Nemunaitis, John

2012-01-01

Ewing's sarcoma tumors are associated with chromosomal translocation between the EWS gene and the ETS transcription factor gene. These unique target sequences provide opportunity for RNA interference(i)-based therapy. A summary of RNAi mechanism and therapeutically designed products including siRNA, shRNA and bi-shRNA are described. Comparison is made between each of these approaches. Systemic RNAi-based therapy, however, requires protected delivery to the Ewing's sarcoma tumor site for activity. Delivery systems which have been most effective in preclinical and clinical testing are reviewed, followed by preclinical assessment of various silencing strategies with demonstration of effectiveness to EWS/FLI-1 target sequences. It is concluded that RNAi-based therapeutics may have testable and achievable activity in management of Ewing's sarcoma. PMID:22523703

Breast Cancer Gene Therapy: Development of Novel Non-Invasive Magnetic Resonance Assay to Optimize Efficacy

DTIC Science & Technology

2007-05-01

inhalation-mouse). HFB had been proposed as a veterinary anesthetic and has been used in many species including ponies, sheep, cats, dogs , rats and...anesthetic and has been used in many species including ponies, sheep, cats, dogs , rats and mice, but was abandoned due to its flammability [265...gene activity detection- specifically, using fluorinated substrates of β-galactosidase to reveal gene activity. Our prototype molecule PFONPG (p

Androgen receptor (AR) cistrome in prostate differentiation and cancer progression.

PubMed

Wang, Fengtian; Koul, Hari K

2017-01-01

Despite the progress in development of better AR-targeted therapies for prostate cancer (PCa), there is no curative therapy for castration-resistant prostate cancer (CRPC). Therapeutic resistance in PCa can be characterized in two broad categories of AR therapy resistance: the first and most prevalent one involves restoration of AR activity despite AR targeted therapy, and the second one involves tumor progression despite blockade of AR activity. As such AR remains the most attractive drug target for CRPC. Despite its oncogenic role, AR signaling also contributes to the maturation and differentiation of prostate luminal cells during development. Recent evidence suggests that AR cistrome is altered in advanced PCa. Alteration in AR may result from AR amplification, alternative splicing, mutations, post-translational modification of AR, and altered expression of AR co-factors. We reasoned that such alterations would result in the transcription of disparate AR target genes and as such may contribute to the emergence of castration-resistance. In the present study, we evaluated the expression of genes associated with canonical or non-canonical AR cistrome in relationship with PCa progression and prostate development by analyzing publicly available datasets. We discovered a transcription switch from canonical AR cistrome target genes to the non-canonical AR cistrome target genes during PCa progression. Using Gene Set Enrichment Analysis (GSEA), we discovered that canonical AR cistrome target genes are enriched in indolent PCa patients and the loss of canonical AR cistrome is associated with tumor metastasis and poor clinical outcome. Analysis of the datasets involving prostate development, revealed that canonical AR cistrome target genes are significantly enriched in prostate luminal cells and can distinguish luminal cells from basal cells, suggesting a pivotal role for canonical AR cistrome driven genes in prostate development. These data suggest that the expression of canonical AR cistrome related genes play an important role in maintaining the prostate luminal cell identity and might restrict the lineage plasticity observed in lethal PCa. Understanding the molecular mechanisms that dictate AR cistrome may lead to development of new therapeutic strategies aimed at restoring canonical AR cistrome, rewiring the oncogenic AR signaling and overcome resistance to AR targeted therapies.

Gene expression in cerebral ischemia: a new approach for neuroprotection.

PubMed

Millán, Mónica; Arenillas, Juan

2006-01-01

Cerebral ischemia is one of the strongest stimuli for gene induction in the brain. Hundreds of genes have been found to be induced by brain ischemia. Many genes are involved in neurodestructive functions such as excitotoxicity, inflammatory response and neuronal apoptosis. However, cerebral ischemia is also a powerful reformatting and reprogramming stimulus for the brain through neuroprotective gene expression. Several genes may participate in both cellular responses. Thus, isolation of candidate genes for neuroprotection strategies and interpretation of expression changes have been proven difficult. Nevertheless, many studies are being carried out to improve the knowledge of the gene activation and protein expression following ischemic stroke, as well as in the development of new therapies that modify biochemical, molecular and genetic changes underlying cerebral ischemia. Owing to the complexity of the process involving numerous critical genes expressed differentially in time, space and concentration, ongoing therapeutic efforts should be based on multiple interventions at different levels. By modification of the acute gene expression induced by ischemia or the apoptotic gene program, gene therapy is a promising treatment but is still in a very experimental phase. Some hurdles will have to be overcome before these therapies can be introduced into human clinical stroke trials. Copyright 2006 S. Karger AG, Basel.

Metatranscriptome Analysis of the Vaginal Microbiota Reveals Potential Mechanisms for Protection against Metronidazole in Bacterial Vaginosis.

PubMed

Deng, Zhi-Luo; Gottschick, Cornelia; Bhuju, Sabin; Masur, Clarissa; Abels, Christoph; Wagner-Döbler, Irene

2018-06-27

Bacterial vaginosis (BV) is a prevalent multifactorial disease of women in their reproductive years characterized by a shift from the Lactobacillus species-dominated microbial community toward a taxonomically diverse anaerobic community. For unknown reasons, some women do not respond to therapy. In our recent clinical study, among 37 women diagnosed with BV, 31 were successfully treated with metronidazole, while 6 still had BV after treatment. To discover possible reasons for the lack of response in those patients, we performed a metatranscriptome analysis of their vaginal microbiota, comparing them to the patients who responded. Seven of 8 clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) genes of Gardnerella vaginalis were highly upregulated in nonresponding patients. Cas genes, in addition to protecting against phages, might be involved in DNA repair, thus mitigating the bactericidal effect of DNA-damaging agents such as metronidazole. In the second part of our study, we analyzed the vaginal metatranscriptomes of four patients over 3 months and showed high in vivo expression of genes for pore-forming toxins in L. iners and of genes encoding enzymes for the production of hydrogen peroxide and d-lactate in L. crispatus IMPORTANCE Bacterial vaginosis is a serious issue for women in their reproductive years. Although it can usually be cured by antibiotics, the recurrence rate is very high, and some women do not respond to antibiotic therapy. The reasons for that are not known. Therefore, we undertook a study to detect the activity of the complete microbiota in the vaginal fluid of women who responded to antibiotic therapy and compared it to the activity of the microbiota in women who did not respond. We found that one of the most important pathogens in bacterial vaginosis, Gardnerella vaginalis , has activated genes that can repair the DNA damage caused by the antibiotic in those women that do not respond to therapy. Suppressing these genes might be a possibility to improve the antibiotic therapy of bacterial vaginosis. Copyright © 2018 Deng et al.

Therapeutic gene editing: delivery and regulatory perspectives.

PubMed

Shim, Gayong; Kim, Dongyoon; Park, Gyu Thae; Jin, Hyerim; Suh, Soo-Kyung; Oh, Yu-Kyoung

2017-06-01

Gene-editing technology is an emerging therapeutic modality for manipulating the eukaryotic genome by using target-sequence-specific engineered nucleases. Because of the exceptional advantages that gene-editing technology offers in facilitating the accurate correction of sequences in a genome, gene editing-based therapy is being aggressively developed as a next-generation therapeutic approach to treat a wide range of diseases. However, strategies for precise engineering and delivery of gene-editing nucleases, including zinc finger nucleases, transcription activator-like effector nuclease, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated nuclease Cas9), present major obstacles to the development of gene-editing therapies, as with other gene-targeting therapeutics. Currently, viral and non-viral vectors are being studied for the delivery of these nucleases into cells in the form of DNA, mRNA, or proteins. Clinical trials are already ongoing, and in vivo studies are actively investigating the applicability of CRISPR/Cas9 techniques. However, the concept of correcting the genome poses major concerns from a regulatory perspective, especially in terms of safety. This review addresses current research trends and delivery strategies for gene editing-based therapeutics in non-clinical and clinical settings and considers the associated regulatory issues.

Therapeutic gene editing: delivery and regulatory perspectives

PubMed Central

Shim, Gayong; Kim, Dongyoon; Park, Gyu Thae; Jin, Hyerim; Suh, Soo-Kyung; Oh, Yu-Kyoung

2017-01-01

Gene-editing technology is an emerging therapeutic modality for manipulating the eukaryotic genome by using target-sequence-specific engineered nucleases. Because of the exceptional advantages that gene-editing technology offers in facilitating the accurate correction of sequences in a genome, gene editing-based therapy is being aggressively developed as a next-generation therapeutic approach to treat a wide range of diseases. However, strategies for precise engineering and delivery of gene-editing nucleases, including zinc finger nucleases, transcription activator-like effector nuclease, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated nuclease Cas9), present major obstacles to the development of gene-editing therapies, as with other gene-targeting therapeutics. Currently, viral and non-viral vectors are being studied for the delivery of these nucleases into cells in the form of DNA, mRNA, or proteins. Clinical trials are already ongoing, and in vivo studies are actively investigating the applicability of CRISPR/Cas9 techniques. However, the concept of correcting the genome poses major concerns from a regulatory perspective, especially in terms of safety. This review addresses current research trends and delivery strategies for gene editing-based therapeutics in non-clinical and clinical settings and considers the associated regulatory issues. PMID:28392568

Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

PubMed

Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L

2010-01-01

The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases: An NHLBI Resource for the Gene Therapy Community

PubMed Central

Skarlatos, Sonia I.

2012-01-01

Abstract The goals of the National Heart, Lung, and Blood Institute (NHLBI) Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases are to conduct gene transfer studies in monkeys to evaluate safety and efficiency; and to provide NHLBI-supported investigators with expertise, resources, and services to actively pursue gene transfer approaches in monkeys in their research programs. NHLBI-supported projects span investigators throughout the United States and have addressed novel approaches to gene delivery; “proof-of-principle”; assessed whether findings in small-animal models could be demonstrated in a primate species; or were conducted to enable new grant or IND submissions. The Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases successfully aids the gene therapy community in addressing regulatory barriers, and serves as an effective vehicle for advancing the field. PMID:22974119

Targeting Signal Transducers and Activators of Transcription-3 (STAT3) as a Novel Strategy in Sensitizing Breast Cancer to EGFR-Targeted Therapy

DTIC Science & Technology

2009-06-01

Osman, F. The human glutathione S-transferase P1 ( GSTP1 ) gene is transactivated by cyclic AMP (cAMP) via a cAMP response element (CRE) proximal to the...transcription start site. Chem-Biol. Interactions 133, 320-321, 2001. 4. Lo, H.-W. and Ali-Osman, F. Cyclic AMP mediated GSTP1 gene activation in...tumor cells involves the interaction of activated CREB-1 with the GSTP1 CRE: a novel mechanism of cellular GSTP1 gene regulation. Journal of Cellular

Expression of complement and pentraxin proteins in acute phase response elicited by tumor photodynamic therapy: the engagement of adrenal hormones.

PubMed

Merchant, Soroush; Huang, Naiyan; Korbelik, Mladen

2010-12-01

Treatment of solid tumors by photodynamic therapy (PDT) was recently shown to trigger a strong acute phase response. Using the mouse Lewis lung carcinoma (LLC) model, the present study examined complement and pentraxin proteins as PDT-induced acute phase reactants. The results show a distinct pattern of changes in the expression of genes encoding these proteins in the tumor, as well as host liver and spleen, following PDT mediated by photosensitizer Photofrin™. These changes were influenced by glucocorticoid hormones, as evidenced by transcriptional activation of glucocorticoid receptor and the upregulation of gene encoding this receptor. The expression of gene for glucocorticoid-induced zipper (GILZ) protein, whose activity is particularly susceptible to glucocorticoid regulation, was also changed in PDT-treated tumors. A direct demonstration that tumor PDT induces glucocorticoid hormone upregulation is provided by documenting elevated levels of serum corticosterone in mice bearing PDT-treated LLC tumors. Tumor response to PDT was negatively affected by blocking glucocorticoid receptor activity, which suggests that glucocorticoid hormones have a positive impact on the therapeutic outcome with this therapy. Copyright © 2010 Elsevier B.V. All rights reserved.

The natural dietary genistein boosts bacteriophage-mediated cancer cell killing by improving phage-targeted tumor cell transduction.

PubMed

Tsafa, Effrosyni; Al-Bahrani, Mariam; Bentayebi, Kaoutar; Przystal, Justyna; Suwan, Keittisak; Hajitou, Amin

2016-08-09

Gene therapy has long been regarded as a promising treatment for cancer. However, cancer gene therapy is still facing the challenge of targeting gene delivery vectors specifically to tumors when administered via clinically acceptable non-invasive systemic routes (i.e. intravenous). The bacteria virus, bacteriophage (phage), represents a new generation of promising vectors in systemic gene delivery since their targeting can be achieved through phage capsid display ligands, which enable them to home to specific tumor receptors without the need to ablate any native eukaryotic tropism. We have previously reported a tumor specific bacteriophage vector named adeno-associated virus/phage, or AAVP, in which gene expression is under a recombinant human rAAV2 virus genome targeted to tumors via a ligand-directed phage capsid. However, cancer gene therapy with this tumor-targeted vector achieved variable outcomes ranging from tumor regression to no effect in both experimental and natural preclinical models. Herein, we hypothesized that combining the natural dietary genistein, with proven anticancer activity, would improve bacteriophage anticancer safe therapy. We show that combination treatment with genistein and AAVP increased targeted cancer cell killing by AAVP carrying the gene for Herpes simplex virus thymidine kinase (HSVtk) in 2D tissue cultures and 3D tumor spheroids. We found this increased tumor cell killing was associated with enhanced AAVP-mediated gene expression. Next, we established that genistein protects AAVP against proteasome degradation and enhances vector genome accumulation in the nucleus. Combination of genistein and phage-guided virotherapy is a safe and promising strategy that should be considered in anticancer therapy with AAVP.

Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma.

PubMed

Galal El-Shemi, A; Mohammed Ashshi, A; Oh, E; Jung, B-K; Basalamah, M; Alsaegh, A; Yun, C-O

2018-01-01

Current treatments of hepatocellular carcinoma (HCC) are ineffective and unsatisfactory in many aspects. Cancer-targeting gene virotherapy using oncolytic adenoviruses (OAds) armed with anticancer genes has shown efficacy and safety in clinical trials. Nowadays, both inhibitor of growth 4 (ING4), as a multimodal tumor suppressor gene, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as a potent apoptosis-inducing gene, are experiencing a renaissance in cancer gene therapy. Herein we investigated the antitumor activity and safety of mono- and combined therapy with OAds armed with ING4 (Ad-ΔB/ING4) and TRAIL (Ad-ΔB/TRAIL) gene, respectively, on preclinical models of human HCC. OAd-mediated expression of ING4 or TRAIL transgene was confirmed. Ad-ΔB/TRAIL and/or Ad-ΔB/ING4 exhibited potent killing effect on human HCC cells (HuH7 and Hep3B) but not on normal liver cells. Most importantly, systemic therapy with Ad-ΔB/ING4 plus Ad-ΔB/TRAIL elicited more eradicative effect on an orthotopic mouse model of human HCC than their monotherapy, without causing obvious overlapping toxicity. Mechanistically, Ad-ΔB/ING4 and Ad-ΔB/TRAIL were remarkably cooperated to induce antitumor apoptosis and immune response, and to repress tumor angiogenesis. This is the first study showing that concomitant therapy with Ad-ΔB/ING4 and Ad-ΔB/TRAIL may provide a potential strategy for HCC therapy and merits further investigations to realize its possible clinical translation.

Alterations in mRNA profiles of trastuzumab‑resistant Her‑2‑positive breast cancer.

PubMed

Zhao, Bin; Zhao, Yang; Sun, Yan; Niu, Haitao; Sheng, Long; Huang, Dongfang; Li, Li

2018-05-07

Breast cancer is one of the most common malignancies in women. Neoadjuvant trastuzumab therapy improves the prognosis of certain Her‑2‑positive breast cancer patients, however around two‑thirds of patients with Her‑2‑positive breast cancer do not benefit from Her‑2‑targeted therapy. To investigate the key mechanisms in trastuzumab resistance, potential biomarkers for neoadjuvant trastuzumab sensitivity were investigated using the gene expression omnibus (GEO) database for mRNA microarray data of Her‑2‑positive breast cancer patients who received neoadjuvant trastuzumab therapy. GEO profiles of 22 patients with a complete response and 48 patients with a partial response were identified in the GSE22358, GSE62327 and GSE66305 datasets. A total of 2,376, 1,000 and 1,152 differentially expressed genes in GSE22358, GSE62327 and GSE66305 datasets were demonstrated, respectively, utilizing GEO2R software. Furthermore, enriched gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were analyzed using the Database for Annotation, Visualization and Integrated Discovery software. Subsequently, a protein‑protein interaction network was established using STRING software. The results demonstrated that low sex‑determining region Y‑box 11 and high Bcl‑2 expression may be employed as markers for neoadjuvant trastuzumab therapy for Her‑2‑positive breast cancer. More importantly, phosphoinositide 3‑kinase/Akt and angiogenesis pathways, which are known to be the key targets of trastuzumab, were activated at a lower level in the partial response patients, while the Wnt and estrogen receptor signaling pathways were activated in these patients. Therefore, combination therapy of trastuzumab and anti‑Wnt or hormone therapy may be a promising treatment modality and should be tested in further studies.

Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model

PubMed Central

Visigalli, Ilaria; Delai, Stefania; Politi, Letterio S.; Di Domenico, Carmela; Cerri, Federica; Mrak, Emanuela; D'Isa, Raffaele; Ungaro, Daniela; Stok, Merel; Sanvito, Francesca; Mariani, Elisabetta; Staszewsky, Lidia; Godi, Claudia; Russo, Ilaria; Cecere, Francesca; del Carro, Ubaldo; Rubinacci, Alessandro; Brambilla, Riccardo; Quattrini, Angelo; Di Natale, Paola; Ponder, Katherine; Naldini, Luigi

2010-01-01

Type I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of α-L-iduronidase, which results in glycosaminoglycan accumulation in tissues. Clinical manifestations include skeletal dysplasia, joint stiffness, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly, and mental retardation (the last being present exclusively in the severe Hurler variant). The available treatments, enzyme-replacement therapy and hematopoietic stem cell (HSC) transplantation, can ameliorate most disease manifestations, but their outcome on skeletal and brain disease could be further improved. We demonstrate here that HSC gene therapy, based on lentiviral vectors, completely corrects disease manifestations in the mouse model. Of note, the therapeutic benefit provided by gene therapy on critical MPS I manifestations, such as neurologic and skeletal disease, greatly exceeds that exerted by HSC transplantation, the standard of care treatment for Hurler patients. Interestingly, therapeutic efficacy of HSC gene therapy is strictly dependent on the achievement of supranormal enzyme activity in the hematopoietic system of transplanted mice, which allows enzyme delivery to the brain and skeleton for disease correction. Overall, our data provide evidence of an efficacious treatment for MPS I Hurler patients, warranting future development toward clinical testing. PMID:20847202

TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer

PubMed Central

Nyquist, Michael D.; Li, Yingming; Hwang, Tae Hyun; Manlove, Luke S.; Vessella, Robert L.; Silverstein, Kevin A. T.; Voytas, Daniel F.; Dehm, Scott M.

2013-01-01

Androgen receptor (AR) target genes direct development and survival of the prostate epithelial lineage, including prostate cancer (PCa). Thus, endocrine therapies that inhibit the AR ligand-binding domain (LBD) are effective in treating PCa. AR transcriptional reactivation is central to resistance, as evidenced by the efficacy of AR retargeting in castration-resistant PCa (CRPC) with next-generation endocrine therapies abiraterone and enzalutamide. However, resistance to abiraterone and enzalutamide limits this efficacy in most men, and PCa remains the second-leading cause of male cancer deaths. Here we show that AR gene rearrangements in CRPC tissues underlie a completely androgen-independent, yet AR-dependent, resistance mechanism. We discovered intragenic AR gene rearrangements in CRPC tissues, which we modeled using transcription activator-like effector nuclease (TALEN)-mediated genome engineering. This modeling revealed that these AR gene rearrangements blocked full-length AR synthesis, but promoted expression of truncated AR variant proteins lacking the AR ligand-binding domain. Furthermore, these AR variant proteins maintained the constitutive activity of the AR transcriptional program and a CRPC growth phenotype independent of full-length AR or androgens. These findings demonstrate that AR gene rearrangements are a unique resistance mechanism by which AR transcriptional activity can be uncoupled from endocrine regulation in CRPC. PMID:24101480

Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs

PubMed Central

Callan, Mary Beth; Haskins, Mark E.; Wang, Ping; Zhou, Shangzhen; High, Katherine A.; Arruda, Valder R.

2016-01-01

Severe hemophilia A (HA) is an inherited bleeding disorder characterized by <1% of residual factor VIII (FVIII) clotting activity. The disease affects several mammals including dogs, and, like humans, is associated with high morbidity and mortality. In gene therapy using adeno-associated viral (AAV) vectors, the canine model has been one of the best predictors of the therapeutic dose tested in clinical trials for hemophilia B (factor IX deficiency) and other genetic diseases, such as congenital blindness. Here we report our experience with liver gene therapy with AAV-FVIII in two outbred, privately owned dogs with severe HA that resulted in sustained expression of 1–2% of normal FVIII levels and prevented 90% of expected bleeding episodes. A Thr62Met mutation in the F8 gene was identified in one dog. These data recapitulate the improvement of the disease phenotype in research animals, and in humans, with AAV liver gene therapy for hemophilia B. Our experience is a novel example of the benefits of a relevant preclinical canine model to facilitate both translational studies in humans and improved welfare of privately owned dogs. PMID:27011017

Tumor-specific expression of shVEGF and suicide gene as a novel strategy for esophageal cancer therapy.

PubMed

Liu, Ting; Wu, Hai-Jun; Liang, Yu; Liang, Xu-Jun; Huang, Hui-Chao; Zhao, Yan-Zhong; Liao, Qing-Chuan; Chen, Ya-Qi; Leng, Ai-Min; Yuan, Wei-Jian; Zhang, Gui-Ying; Peng, Jie; Chen, Yong-Heng

2016-06-21

To develop a potent and safe gene therapy for esophageal cancer. An expression vector carrying fusion suicide gene (yCDglyTK) and shRNA against vascular endothelial growth factor (VEGF) was constructed and delivered into EC9706 esophageal cancer cells by calcium phosphate nanoparticles (CPNP). To achieve tumor selectivity, expression of the fusion suicide gene was driven by a tumor-specific human telomerase reverse transcriptase (hTERT) promoter. The biologic properties and therapeutic efficiency of the vector, in the presence of prodrug 5-fluorocytosine (5-FC), were evaluated in vitro and in vivo. Both in vitro and in vivo testing showed that the expression vector was efficiently introduced by CPNP into tumor cells, leading to cellular expression of yCDglyTK and decreased VEGF level. With exposure to 5-FC, it exhibited strong anti-tumor effects against esophageal cancer. Combination of VEGF shRNA with the fusion suicide gene demonstrated strong anti-tumor activity. The shVEGF-hTERT-yCDglyTK/5-FC system provided a novel approach for esophageal cancer-targeted gene therapy.

In vivo osteoprotegerin gene therapy preventing bone loss induced by periodontitis.

PubMed

Tang, H; Mattheos, N; Yao, Y; Jia, Y; Ma, L; Gong, P

2015-08-01

The objective of this study was to investigate the effects of osteoprotegerin (OPG) gene therapy on alveolar bone resorption caused by experimental periodontitis in rats, thus forming a foundation for potential clinical applications of OPG gene therapy in the treatment of periodontitis and peri-implantitis. To study the effects of OPG on alveolar bone protection, an experimental periodontitis model was used by placing a bacterial plaque retentive silk ligature in the gingival sulcus around the maxillary second molar tooth, injection of Porphyromonas gingivalis and high carbohydrate diet. A total of 30 Sprague-Dawley rats were randomly divided into three groups, with 10 rats in each group: group I (control) was treated with 10 μL normal saline injection; group II with 10 μL mock vector; and group III with 10 μL local OPG gene transfer by transfection with in vitro constructed pcDNA3.1-human OPG (pcDNA3.1-hOPG). A subperiosteal injection was done adjacent to the second molars on days 0, 7, 14 and 21. Four weeks later, all animals were killed and radiographic, histological and immunohistochemical examinations were performed. Statistical analysis included ANOVA and LSD-Bonferroni test. Group III (OPG gene therapy) had significantly enhanced (p < 0.05) integrated optical density of OPG, had significantly decreased alveolar bone resorption volume and active osteoclast number (p < 0.05) through descriptive histological examination when compared with the other two groups at week 4. Local recombinant OPG plasmid-mediated gene therapy suppresses osteoclastogenesis in vivo and inhibits alveolar bone height reduction caused by experimental periodontitis in rats. OPG gene therapy may be beneficial in preventing progressive periodontal bone loss. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Adiponectin gene therapy ameliorates high-fat, high-sucrose diet-induced metabolic perturbations in mice

PubMed Central

Kandasamy, A D; Sung, M M; Boisvenue, J J; Barr, A J; Dyck, J R B

2012-01-01

Background and Design: Adiponectin is an adipokine secreted primarily from adipose tissue that can influence circulating plasma glucose and lipid levels through multiple mechanisms involving a variety of organs. In humans, reduced plasma adiponectin levels induced by obesity are associated with insulin resistance and type 2 diabetes, suggesting that low adiponectin levels may contribute the pathogenesis of obesity-related insulin resistance. Methods and Results: The objective of the present study was to investigate whether gene therapy designed to elevate circulating adiponectin levels is a viable strategy for ameliorating insulin resistance in mice fed a high-fat, high-sucrose (HFHS) diet. Electroporation-mediated gene transfer of mouse adiponectin plasmid DNA into gastrocnemius muscle resulted in elevated serum levels of globular and high-molecular weight adiponectin compared with control mice treated with empty plasmid. In comparison to HFHS-fed mice receiving empty plasmid, mice receiving adiponectin gene therapy displayed significantly decreased weight gain following 13 weeks of HFHS diet associated with reduced fat accumulation, and exhibited increased oxygen consumption and locomotor activity as measured by indirect calorimetry, suggesting increased energy expenditure in these mice. Consistent with improved whole-body metabolism, mice receiving adiponectin gene therapy also had lower blood glucose and insulin levels, improved glucose tolerance and reduced hepatic gluconeogenesis compared with control mice. Furthermore, immunoblot analysis of livers from mice receiving adiponectin gene therapy showed an increase in insulin-stimulated phosphorylation of insulin signaling proteins. Conclusion: Based on these data, we conclude that adiponectin gene therapy ameliorates the metabolic abnormalities caused by feeding mice a HFHS diet and may be a potential therapeutic strategy to improve obesity-mediated impairments in insulin sensitivity. PMID:23446660

Cell Cycle Status of CD34+ Hemopoietic Stem Cells Determines Lentiviral Integration in Actively Transcribed and Development-related Genes

PubMed Central

Papanikolaou, Eleni; Paruzynski, Anna; Kasampalidis, Ioannis; Deichmann, Annette; Stamateris, Evangelos; Schmidt, Manfred; von Kalle, Christof; Anagnou, Nicholas P

2015-01-01

Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer may cause insertional mutagenesis. Although, such risks had been originally estimated as extremely low, several reports of leukemias or clonal dominance, have led to a re-evaluation of the mechanisms operating in insertional mutagenesis. Therefore, unraveling the mechanism of retroviral integration is mandatory toward safer gene therapy applications. In the present study, we undertook an experimental approach which enabled direct correlation of the cell cycle stage of the target cell with the integration profile of LVs. CD34+ cells arrested at different stages of cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate transcribed genes with integration sites. The results indicate that ~10% of integration events occurred in actively transcribed genes and that the cell cycle stage of target cells affects integration pattern. Specifically, use of thymine promoted a safer profile, since it significantly reduced integration within cell cycle-related genes, while we observed increased possibility for integration into genes related to development, and decreased possibility for integration within cell cycle and cancer-related genes, when transduction occurs during mitosis. PMID:25523760

Parkinson's Disease Gene Therapy: Success by Design Meets Failure by Efficacy

PubMed Central

Bartus, Raymond T; Weinberg, Marc S; Samulski, R. Jude

2014-01-01

Over the past decade, nine gene therapy clinical trials for Parkinson's disease (PD) have been initiated and completed. Starting with considerable optimism at the initiation of each trial, none of the programs has yet borne sufficiently robust clinical efficacy or found a clear path toward regulatory approval. Despite the immediately disappointing nature of the efficacy outcomes in these trials, the clinical data garnered from the individual studies nonetheless represent tangible and significant progress for the gene therapy field. Collectively, the clinical trials demonstrate that we have overcome the major safety hurdles previously suppressing central nervous system (CNS) gene therapy, for none produced any evidence of untoward risk or harm after administration of various vector-delivery systems. More importantly, these studies also demonstrated controlled, highly persistent generation of biologically active proteins targeted to structures deep in the human brain. Therefore, a renewed, focused emphasis must be placed on advancing clinical efficacy by improving clinical trial design, patient selection and outcome measures, developing more predictive animal models to support clinical testing, carefully performing retrospective analyses, and most importantly moving forward—beyond our past limits. PMID:24356252

Mammalian synthetic biology: emerging medical applications.

PubMed

Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M; Krams, Rob

2015-05-06

In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON-OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences

NASA Technical Reports Server (NTRS)

Li, X.; Eastman, E. M.; Schwartz, R. J.; Draghia-Akli, R.

1999-01-01

Relatively low levels of expression from naturally occurring promoters have limited the use of muscle as a gene therapy target. Myogenic restricted gene promoters display complex organization usually involving combinations of several myogenic regulatory elements. By random assembly of E-box, MEF-2, TEF-1, and SRE sites into synthetic promoter recombinant libraries, and screening of hundreds of individual clones for transcriptional activity in vitro and in vivo, several artificial promoters were isolated whose transcriptional potencies greatly exceed those of natural myogenic and viral gene promoters.

Insulin-like growth factor-I gene delivery to astrocytes reduces their inflammatory response to lipopolysaccharide

PubMed Central

2011-01-01

Background Insulin-like growth factor-I (IGF-I) exerts neuroprotective actions in the central nervous system that are mediated at least in part by control of activation of astrocytes. In this study we have assessed the efficacy of exogenous IGF-I and IGF-I gene therapy in reducing the inflammatory response of astrocytes from cerebral cortex. Methods An adenoviral vector harboring the rat IGF-I gene and a control adenoviral vector harboring a hybrid gene encoding the herpes simplex virus type 1 thymidine kinase fused to Aequorea victoria enhanced green fluorescent protein were used in this study. Primary astrocytes from mice cerebral cortex were incubated for 24 h or 72 h with vehicle, IGF-I, the IGF-I adenoviral vector, or control vector; and exposed to bacterial lipopolysaccharide to induce an inflammatory response. IGF-I levels were measured by radioimmunoassay. Levels of interleukin 6, tumor necrosis factor-α, interleukin-1β and toll-like receptor 4 mRNA were assessed by quantitative real-time polymerase chain reaction. Levels of IGF-I receptor and IGF binding proteins 2 and 3 were assessed by western blotting. The subcellular distribution of nuclear factor κB (p65) was assessed by immunocytochemistry. Statistical significance was assessed by one way analysis of variance followed by the Bonferroni pot hoc test. Results IGF-I gene therapy increased IGF-I levels without affecting IGF-I receptors or IGF binding proteins. Exogenous IGF-I, and IGF-I gene therapy, decreased expression of toll-like receptor 4 and counteracted the lipopolysaccharide-induced inflammatory response of astrocytes. In addition, IGF-I gene therapy decreased lipopolysaccharide-induced translocation of nuclear factor κB (p65) to the cell nucleus. Conclusion These findings demonstrate efficacy of exogenous IGF-I and of IGF-I gene therapy in reducing the inflammatory response of astrocytes. IGF-I gene therapy may represent a new approach to reduce inflammatory reactions in glial cells. PMID:21371294

Radiographic evaluation of bones and joints in mucopolysaccharidosis I and VII dogs after neonatal gene therapy.

PubMed

Herati, Ramin Sedaghat; Knox, Van W; O'Donnell, Patricia; D'Angelo, Marina; Haskins, Mark E; Ponder, Katherine P

2008-11-01

Mucopolysaccharidosis I (MPS I) and MPS VII are due to deficient activity of the glycosaminoglycan-degrading lysosomal enzymes alpha-L-iduronidase and beta-glucuronidase, respectively, and result in abnormal bones and joints. Here, the severity of skeletal disease in MPS I and MPS VII dogs and the effects of neonatal gene therapy were evaluated. For untreated MPS VII dogs, the lengths of the second cervical vertebrae (C2) and the femur were only 56% and 84% of normal, respectively, and bone dysplasia and articular erosions, and joint subluxation were severe. Previously, we reported that neonatal intravenous injection of a retroviral vector (RV) with the appropriate gene resulted in expression in liver and blood cells, and high serum enzyme activity. In this study, we demonstrate that C2 and femurs of RV-treated MPS VII dogs were longer at 82% and 101% of normal, respectively, and there were partial improvements of qualitative abnormalities. For untreated MPS I dogs, the lengths of C2 and femurs (91% and 96% of normal, respectively) were not significantly different from normal dogs. Qualitative changes in MPS I bones and joints were generally modest and were partially improved with RV treatment, although cervical spine disease was severe and was difficult to correct with gene therapy in both models. The greater severity of skeletal disease in MPS VII than in MPS I dogs may reflect accumulation of chondroitin sulfate in cartilage in MPS VII, or could relate to the specific mutations. Neonatal RV-mediated gene therapy ameliorates, but does not prevent, skeletal disease in MPS I and MPS VII dogs.

Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma.

PubMed

Kim, Yun-Hee; Kim, Kyung Tae; Lee, Sang-Jin; Hong, Seung-Hee; Moon, Ju Young; Yoon, Eun Kyung; Kim, Sukyoung; Kim, Eun Ok; Kang, Se Hun; Kim, Seok Ki; Choi, Sun Il; Goh, Sung Ho; Kim, Daehong; Lee, Seong-Wook; Ju, Mi Ha; Jeong, Jin Sook; Kim, In-Hoo

2016-01-01

Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy.

A Quantitative Comparison of Anti-HIV Gene Therapy Delivered to Hematopoietic Stem Cells versus CD4+ T Cells

PubMed Central

Savkovic, Borislav; Nichols, James; Birkett, Donald; Applegate, Tanya; Ledger, Scott; Symonds, Geoff; Murray, John M.

2014-01-01

Gene therapy represents an alternative and promising anti-HIV modality to highly active antiretroviral therapy. It involves the introduction of a protective gene into a cell, thereby conferring protection against HIV. While clinical trials to date have delivered gene therapy to CD4+T cells or to CD34+ hematopoietic stem cells (HSC), the relative benefits of each of these two cellular targets have not been conclusively determined. In the present analysis, we investigated the relative merits of delivering a dual construct (CCR5 entry inhibitor + C46 fusion inhibitor) to either CD4+T cells or to CD34+ HSC. Using mathematical modelling, we determined the impact of each scenario in terms of total CD4+T cell counts over a 10 year period, and also in terms of inhibition of CCR5 and CXCR4 tropic virus. Our modelling determined that therapy delivery to CD34+ HSC generally resulted in better outcomes than delivery to CD4+T cells. An early one-off therapy delivery to CD34+ HSC, assuming that 20% of CD34+ HSC in the bone marrow were gene-modified (G+), resulted in total CD4+T cell counts ≥180 cells/ µL in peripheral blood after 10 years. If the uninfected G+ CD4+T cells (in addition to exhibiting lower likelihood of becoming productively infected) also exhibited reduced levels of bystander apoptosis (92.5% reduction) over non gene-modified (G-) CD4+T cells, then total CD4+T cell counts of ≥350 cells/ µL were observed after 10 years, even if initially only 10% of CD34+ HSC in the bone marrow received the protective gene. Taken together our results indicate that: 1.) therapy delivery to CD34+ HSC will result in better outcomes than delivery to CD4+T cells, and 2.) a greater impact of gene therapy will be observed if G+ CD4+T cells exhibit reduced levels of bystander apoptosis over G- CD4+T cells. PMID:24945407

Prospects for personalized combination immunotherapy for solid tumors based on adoptive cell therapies and immune checkpoint blockade therapies.

PubMed

Kato, Daiki; Yaguchi, Tomonori; Iwata, Takashi; Morii, Kenji; Nakagawa, Takayuki; Nishimura, Ryohei; Kawakami, Yutaka

2017-01-01

  Immune checkpoint blockade (ICB) and adoptive cell therapies (ACT) with antigen-receptor gene-engineered T cells have been shown to be successful for a limited number of patients with solid tumors. Responders to ICB therapy typically have T cell-inflamed tumors. Thus, it is important to develop strategies that convert non-T cell-inflamed tumors to T cell-inflamed tumors. Although chimeric antigen receptor transduced T (CAR-T) cell therapy targeting hematological malignancies demonstrated durable clinical responses, the success of gene-engineered T cell therapies in solid tumors is hampered by a lack of unique antigens, antigen loss in cancer cells, and the immune-suppressive tumor microenvironment (TME) of solid tumors. However, gene-engineered T cells possess strong killing activity and cytokine production capacity, which can induce antigen spreading and modulate the TME of non-T cell-inflamed tumors seen in non-responders to ICB therapy. Immune responses against cancer are highly heterogeneous, not only between tumor types, but also within a patient or between different patients with the same type of cancer, indicating that personalized immunotherapy should be employed, based on the immune status of the individual patient. Here, we offer our perspective for personalized combination immunotherapy for solid tumors based on ACT and ICB therapies.

Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors.

PubMed

Thakore, Pratiksha I; Gersbach, Charles A

2016-01-01

Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.

Targeting gene therapy to cancer: a review.

PubMed

Dachs, G U; Dougherty, G J; Stratford, I J; Chaplin, D J

1997-01-01

In recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root. This is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide. This report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment. In order to specifically target malignant cells while at the same time sparing normal tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation. Although the efficient delivery of DNA to tumor sites remains a formidable task, progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods. In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be combined in the future for effective therapy. To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements. Another novel possibility that will be discussed is the regulation of therapeutic gene products by tumor-specific gene splicing. Gene expression could also be targeted at conditions specific to the tumor microenvironment, such as glucose deprivation and hypoxia. We have concentrated on hypoxia-targeted gene expression and this report will discuss our progress in detail. Chronic hypoxia occurs in tissue that is more than 100-200 microns away from a functional blood supply. In solid tumors hypoxia is widespread both because cancer cells are more prolific than the invading endothelial cells that make up the blood vessels and because the newly formed blood supply is disorganized. Measurements of oxygen partial pressure in patients' tumors showed a high percentage of severe hypoxia readings (less than 2.5 mmHg), readings not seen in normal tissue. This is a major problem in the treatment of cancer, because hypoxic cells are resistant to radiotherapy and often to chemotherapy. However, severe hypoxia is also a physiological condition specific to tumors, which makes it a potentially exploitable target. We have utilized hypoxia response elements (HRE) derived from the oxygen-regulated phosphoglycerate kinase gene to control gene expression in human tumor cells in vitro and in experimental tumors. The list of genes that have been considered for use in the treatment of cancer is extensive. It includes cytokines and costimulatory cell surface molecules intended to induce an effective systemic immune response against tumor antigens that would not otherwise develop. Other inventive strategies include the use of internally expressed antibodies to target oncogenic proteins (intrabodies) and the use of antisense technology (antisense oligonucleotides, antigenes, and ribozymes). This report will concentrate more on novel genes encoding prodrug activating enzymes, so-called suicide genes (Herpes simplex virus thymidine kinase, Escherichia coli nitroreductase, E. (ABSTRACT TRUNCATED)

A Dynamic Bronchial Airway Gene Expression Signature of Chronic Obstructive Pulmonary Disease and Lung Function Impairment

PubMed Central

Steiling, Katrina; van den Berge, Maarten; Hijazi, Kahkeshan; Florido, Roberta; Campbell, Joshua; Liu, Gang; Xiao, Ji; Zhang, Xiaohui; Duclos, Grant; Drizik, Eduard; Si, Huiqing; Perdomo, Catalina; Dumont, Charles; Coxson, Harvey O.; Alekseyev, Yuriy O.; Sin, Don; Pare, Peter; Hogg, James C.; McWilliams, Annette; Hiemstra, Pieter S.; Sterk, Peter J.; Timens, Wim; Chang, Jeffrey T.; Sebastiani, Paola; O’Connor, George T.; Bild, Andrea H.; Postma, Dirkje S.; Lam, Stephen

2013-01-01

Rationale: Molecular phenotyping of chronic obstructive pulmonary disease (COPD) has been impeded in part by the difficulty in obtaining lung tissue samples from individuals with impaired lung function. Objectives: We sought to determine whether COPD-associated processes are reflected in gene expression profiles of bronchial airway epithelial cells obtained by bronchoscopy. Methods: Gene expression profiling of bronchial brushings obtained from 238 current and former smokers with and without COPD was performed using Affymetrix Human Gene 1.0 ST Arrays. Measurements and Main Results: We identified 98 genes whose expression levels were associated with COPD status, FEV1% predicted, and FEV1/FVC. In silico analysis identified activating transcription factor 4 (ATF4) as a potential transcriptional regulator of genes with COPD-associated airway expression, and ATF4 overexpression in airway epithelial cells in vitro recapitulates COPD-associated gene expression changes. Genes with COPD-associated expression in the bronchial airway epithelium had similarly altered expression profiles in prior studies performed on small-airway epithelium and lung parenchyma, suggesting that transcriptomic alterations in the bronchial airway epithelium reflect molecular events found at more distal sites of disease activity. Many of the airway COPD-associated gene expression changes revert toward baseline after therapy with the inhaled corticosteroid fluticasone in independent cohorts. Conclusions: Our findings demonstrate a molecular field of injury throughout the bronchial airway of active and former smokers with COPD that may be driven in part by ATF4 and is modifiable with therapy. Bronchial airway epithelium may ultimately serve as a relatively accessible tissue in which to measure biomarkers of disease activity for guiding clinical management of COPD. PMID:23471465

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

PubMed

Kawashita, Yujo; Deb, Niloy J; Garg, Madhur K; Kabarriti, Rafi; Fan, Zuoheng; Alfieri, Alan A; Roy-Chowdhury, Jayanta; Guha, Chandan

2014-08-01

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and natural killer cells. The addition of Adeno-CD40L further improved the median survival of animals to 80 ± 15 days and this effect was abolished only when using anti-CD8 antibodies. Chromium-51 (51Cr) release assay showed cytotoxic T lymphocyte (CTL) activation, suggesting efficient dendritic cell (DC) activation with CTL activation after the treatment. Furthermore, when surviving mice were rechallenged with BNL-ETK cells on the foot pad, RT + HSV-TK/GCV + Flt3L + CD40L-treated mice developed a small tumor on day 56 but the tumor eventually disappeared after 105 days. Mice treated with RT + HSV-TK/GCV + Flt3L showed a slowed tumor growth curve compared with untreated mice. Therefore, combination therapy using Flt3L to induce DC proliferation and CD40L to enhance DC maturation holds great promise for immunomodulation of radiation therapy to enhance HCC tumor control and prevent progression of disease in patients with diffuse HCC.

Non-viral gene activated matrices for mesenchymal stem cells based tissue engineering of bone and cartilage.

PubMed

Raisin, Sophie; Belamie, Emmanuel; Morille, Marie

2016-10-01

Recent regenerative medicine and tissue engineering strategies for bone and cartilage repair have led to fascinating progress of translation from basic research to clinical applications. In this context, the use of gene therapy is increasingly being considered as an important therapeutic modality and regenerative technique. Indeed, in the last 20 years, nucleic acids (plasmid DNA, interferent RNA) have emerged as credible alternative or complement to proteins, which exhibited major issues including short half-life, loss of bioactivity in pathologic environment leading to high dose requirement and therefore high production costs. The relevance of gene therapy strategies in combination with a scaffold, following a so-called "Gene-Activated Matrix (GAM)" approach, is to achieve a direct, local and sustained delivery of nucleic acids from a scaffold to ensure efficient and durable cell transfection. Among interesting cells sources, Mesenchymal Stem Cells (MSC) are promising for a rational use in gene/cell therapy with more than 1700 clinical trials approved during the last decade. The aim of the present review article is to provide a comprehensive overview of recent and ongoing work in non-viral genetic engineering of MSC combined with scaffolds. More specifically, we will show how this inductive strategy can be applied to orient stem cells fate for bone and cartilage repair. Copyright © 2016 Elsevier Ltd. All rights reserved.

The natural dietary genistein boosts bacteriophage-mediated cancer cell killing by improving phage-targeted tumor cell transduction

PubMed Central

Tsafa, Effrosyni; Al-Bahrani, Mariam; Bentayebi, Kaoutar; Przystal, Justyna; Suwan, Keittisak; Hajitou, Amin

2016-01-01

Gene therapy has long been regarded as a promising treatment for cancer. However, cancer gene therapy is still facing the challenge of targeting gene delivery vectors specifically to tumors when administered via clinically acceptable non-invasive systemic routes (i.e. intravenous). The bacteria virus, bacteriophage (phage), represents a new generation of promising vectors in systemic gene delivery since their targeting can be achieved through phage capsid display ligands, which enable them to home to specific tumor receptors without the need to ablate any native eukaryotic tropism. We have previously reported a tumor specific bacteriophage vector named adeno-associated virus/phage, or AAVP, in which gene expression is under a recombinant human rAAV2 virus genome targeted to tumors via a ligand-directed phage capsid. However, cancer gene therapy with this tumor-targeted vector achieved variable outcomes ranging from tumor regression to no effect in both experimental and natural preclinical models. Herein, we hypothesized that combining the natural dietary genistein, with proven anticancer activity, would improve bacteriophage anticancer safe therapy. We show that combination treatment with genistein and AAVP increased targeted cancer cell killing by AAVP carrying the gene for Herpes simplex virus thymidine kinase (HSVtk) in 2D tissue cultures and 3D tumor spheroids. We found this increased tumor cell killing was associated with enhanced AAVP-mediated gene expression. Next, we established that genistein protects AAVP against proteasome degradation and enhances vector genome accumulation in the nucleus. Combination of genistein and phage-guided virotherapy is a safe and promising strategy that should be considered in anticancer therapy with AAVP. PMID:27437775

Arrhenius analysis of the relationship between hyperthermia and Hsp70 promoter activation: a comparison between ex vivo and in vivo data.

PubMed

Deckers, Roel; Debeissat, Christelle; Fortin, Pierre-Yves; Moonen, Chrit T W; Couillaud, Franck

2012-01-01

Tight regulation of gene expression in the region where therapy is necessary and for the duration required to achieve a therapeutic effect and to minimise systemic toxicity is very important for clinical applications of gene therapy. Hyperthermia in combination with a temperature sensitive heat shock protein (Hsp70) promoter presents a unique approach allowing non-invasive spatio-temporal control of transgene expression. In this study we investigated the in vivo and ex vivo relationship between temperature and duration of thermal stress with respect to the resulting gene expression using an Arrhenius analysis. A transgenic mouse expressing the luciferase reporter gene under the transcriptional control of a thermosensitive promoter was used to assure identical genotype for in vivo (mouse leg) and ex vivo (bone marrow mononuclear and embryonic fibroblast cells) studies. The mouse leg and cells were heated at different temperatures and different exposure times. Bioluminescence imaging and in vitro enzymatic assay were used to measure the resulting transgene expression. We showed that temperature-induced Hsp70 promoter activation was modulated by both temperature as well as duration of hyperthermia. The relationship between temperature and duration of hyperthermia and the resulting reporter gene expression can be modelled by an Arrhenius analysis for both in vivo as well as ex vivo. However, the increase in reporter gene expression after elevating the temperature of the thermal stress with 1°C is not comparable for in vivo and ex vivo situations. This information may be valuable for optimising clinical gene therapy protocols.

In vitro therapeutic effect of PDT combined with VEGF-A gene therapy

NASA Astrophysics Data System (ADS)

Lecaros, Rumwald Leo G.; Huang, Leaf; Hsu, Yih-Chih

2014-02-01

Vascular endothelial growth factor A (VEGF-A), commonly known as VEGF, is one of the primary factors that affect tumor angiogenesis. It was found to be expressed in cancer cell lines including oral squamous cell carcinoma. Photodynamic therapy (PDT) is a novel therapeutic modality to treat cancer by using a photosensitizer which is activated by a light source to produce reactive oxygen species and mediates oxygen-independent hypoxic conditions to tumor. Another emerging treatment to cure cancer is the use of interference RNA (e.g. siRNA) to silence a specific mRNA sequence. VEGF-A was found to be expressed in oral squamous cell carcinoma and overexpressed after 24 hour post-PDT by Western blot analysis. Cell viability was found to decrease at 25 nM of transfected VEGF-A siRNA. In vitro combined therapy of PDT and VEGF-A siRNA showed better response as compared with PDT and gene therapy alone. The results suggest that PDT combined with targeted gene therapy has a potential mean to achieve better therapeutic outcome.

Insights into molecular therapy of glioma: current challenges and next generation blueprint

PubMed Central

Rajesh, Y; Pal, Ipsita; Banik, Payel; Chakraborty, Sandipan; Borkar, Sachin A; Dey, Goutam; Mukherjee, Ahona; Mandal, Mahitosh

2017-01-01

Glioma accounts for the majority of human brain tumors. With prevailing treatment regimens, the patients have poor survival rates. In spite of current development in mainstream glioma therapy, a cure for glioma appears to be out of reach. The infiltrative nature of glioma and acquired resistance substancially restrict the therapeutic options. Better elucidation of the complicated pathobiology of glioma and proteogenomic characterization might eventually open novel avenues for the design of more sophisticated and effective combination regimens. This could be accomplished by individually tailoring progressive neuroimaging techniques, terminating DNA synthesis with prodrug-activating genes, silencing gliomagenesis genes (gene therapy), targeting miRNA oncogenic activity (miRNA-mRNA interaction), combining Hedgehog-Gli/Akt inhibitors with stem cell therapy, employing tumor lysates as antigen sources for efficient depletion of tumor-specific cancer stem cells by cytotoxic T lymphocytes (dendritic cell vaccination), adoptive transfer of chimeric antigen receptor-modified T cells, and combining immune checkpoint inhibitors with conventional therapeutic modalities. Thus, the present review captures the latest trends associated with the molecular mechanisms involved in glial tumorigenesis as well as the limitations of surgery, radiation and chemotherapy. In this article we also critically discuss the next generation molecular therapeutic strategies and their mechanisms for the successful treatment of glioma. PMID:28317871

[Molecular mechanisms of primary and secondary resistance, molecular-genetic features and characteristics of KIT/PDGFRA non-mutated GISTs].

PubMed

Kalfusová, Alena; Kodet, Roman

2017-01-01

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Most of them arise due to activating mutations in KIT (75 - 85 %) or PDGFRA (less than 10 %) genes. Identification of the activating mutations in KIT and PDGFRA genes, which code for receptor tyrosine kinases (RTKs), has improved the outcome of targeted therapy of metastatic, unresectable or recurrent GISTs. Primary and/or secondary resistance represents a significant problem in the targeted therapy by Imatinib mesylate (IM) in patients with GIST. An important mechanism of the secondary resistance is the evolvement of secondary mutations. Except for primary and secondary resistance, there is another problem of disease progression - a failure of tumor cells eradication even in the long term therapy of tyrosine kinase inhibitors. GISTs without mutations in KIT/PDGFRA genes constitute 10 - 15% GISTs in adults, and a majority (85 %) of pediatric GISTs. KIT/PDGFRA wild-type GISTs represent a heterogeneous group of tumors with several molecular-genetics and/or morphologic differences. KIT/PDGFRA wild-type GISTs are different in their molecular features, for example in mutations in the BRAF, KRAS, NF1 genes or defects of succinate dehydrogenase (SDH) subunits. KIT/PDGFRA wild-type GISTs are generally less sensitive to targeted therapy by tyrosine kinase inhibitors in comparison with KIT/PDGFRA mutated GISTs. Inhibitors of BRAF, PI3K (mTOR) or inhibitors of IGF1R and VEGFR receptors provide alternative therapeutic strategies.

Hematopoietic stem cell gene therapy for IFNγR1 deficiency protects mice from mycobacterial infections.

PubMed

Hetzel, Miriam; Mucci, Adele; Blank, Patrick; Nguyen, Ariane Hai Ha; Schiller, Jan; Halle, Olga; Kühnel, Mark-Philipp; Billig, Sandra; Meineke, Robert; Brand, Daniel; Herder, Vanessa; Baumgärtner, Wolfgang; Bange, Franz-Christoph; Goethe, Ralph; Jonigk, Danny; Förster, Reinhold; Gentner, Bernhard; Casanova, Jean-Laurent; Bustamante, Jacinta; Schambach, Axel; Kalinke, Ulrich; Lachmann, Nico

2018-02-01

Mendelian susceptibility to mycobacterial disease is a rare primary immunodeficiency characterized by severe infections caused by weakly virulent mycobacteria. Biallelic null mutations in genes encoding interferon gamma receptor 1 or 2 ( IFNGR1 or IFNGR2 ) result in a life-threatening disease phenotype in early childhood. Recombinant interferon γ (IFN-γ) therapy is inefficient, and hematopoietic stem cell transplantation has a poor prognosis. Thus, we developed a hematopoietic stem cell (HSC) gene therapy approach using lentiviral vectors that express Ifnγr1 either constitutively or myeloid specifically. Transduction of mouse Ifnγr1 -/- HSCs led to stable IFNγR1 expression on macrophages, which rescued their cellular responses to IFN-γ. As a consequence, genetically corrected HSC-derived macrophages were able to suppress T-cell activation and showed restored antimycobacterial activity against Mycobacterium avium and Mycobacterium bovis Bacille Calmette-Guérin (BCG) in vitro. Transplantation of genetically corrected HSCs into Ifnγr1 -/- mice before BCG infection prevented manifestations of severe BCG disease and maintained lung and spleen organ integrity, which was accompanied by a reduced mycobacterial burden in lung and spleen and a prolonged overall survival in animals that received a transplant. In summary, we demonstrate an HSC-based gene therapy approach for IFNγR1 deficiency, which protects mice from severe mycobacterial infections, thereby laying the foundation for a new therapeutic intervention in corresponding human patients. © 2018 by The American Society of Hematology.

Chinese medicine protein and peptide in gene and cell therapy.

PubMed

Feng, Yinglu; Yin, Zifei; Zhang, Daniel; Srivastava, Arun; Ling, Chen

2018-06-11

The success of gene and cell therapy in clinic during the past two decades as well as our expanding ability to manipulate these biomaterials are leading to new therapeutic options for a wide range of inherited and acquired diseases. Combining conventional therapies with this emerging field is a promising strategy to treat those previously-thought untreatable diseases. Traditional Chinese medicine (TCM) has evolved for thousands of years in China and still plays an important role in human health. As part of the active ingredients of TCM, proteins and peptides have attracted long-term enthusiasm of researchers. More recently, they have been utilized in gene and cell therapy, resulting in promising novel strategies to treat both cancer and non-cancer diseases. This manuscript presents a critical review on this field, accompanied with perspectives on the challenges and new directions for future research in this emerging frontier. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Simultaneous regulation of apoptotic gene silencing and angiogenic gene expression for myocardial infarction therapy: Single-carrier delivery of SHP-1 siRNA and VEGF-expressing pDNA.

PubMed

Kim, Dongkyu; Ku, Sook Hee; Kim, Hyosuk; Jeong, Ji Hoon; Lee, Minhyung; Kwon, Ick Chan; Choi, Donghoon; Kim, Sun Hwa

2016-12-10

Gene therapy is aimed at selectively knocking up or knocking down the target genes involved in the development of diseases. In many human diseases, dysregulation of disease-associated genes is occurred concurrently: some genes are abnormally turned up and some are turned down. In the field of non-viral gene therapy, plasmid DNA (pDNA) and small interfering RNA (siRNA) are suggested as representative regulation tools for activating and silencing the expression of genes of interest, representatively. Herein, we simultaneously loaded both siRNA (Src homology region 2 domain-containing tyrosine phosphatase-1 siRNA, siSHP-1) for anti-apoptosis and pDNA (hypoxia-inducible vascular endothelial growth factor expression vector, pHI-VEGF) for angiogenesis in a single polymeric nanocarrier and used to synergistically attenuate ischemia-reperfusion (IR)-induced myocardial infarction, which is mainly caused by dysregulating of cardiac apoptosis and angiogenesis. For dual-modality cardiac gene delivery, siSHP-1 and pHI-VEGF were sequentially incorporated into a stable nanocomplex by using deoxycholic acid-modified polyethylenimine (DA-PEI). The resulting DA-PEI/siSHP-1/pHI-VEGF complexes exhibited the high structural stability against polyanion competition and the improved resistance to digestion by nucleases. The cardiac administration of DA-PEI/siSHP-1/pHI-VEGF reduced cardiomyocyte apoptosis and enhanced cardiac microvessel formation, thereby reducing infarct size in rat ischemia-reperfusion model. The simultaneous anti-apoptotic and angiogenic gene therapies synergized the cardioprotective effects of each strategy; thus our dual-modal single-carrier gene delivery system can be considered as a promising candidate for treating ischemic heart diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Meta-analysis of human gene expression in response to Mycobacterium tuberculosis infection reveals potential therapeutic targets.

PubMed

Wang, Zhang; Arat, Seda; Magid-Slav, Michal; Brown, James R

2018-01-10

With the global emergence of multi-drug resistant strains of Mycobacterium tuberculosis, new strategies to treat tuberculosis are urgently needed such as therapeutics targeting potential human host factors. Here we performed a statistical meta-analysis of human gene expression in response to both latent and active pulmonary tuberculosis infections from nine published datasets. We found 1655 genes that were significantly differentially expressed during active tuberculosis infection. In contrast, no gene was significant for latent tuberculosis. Pathway enrichment analysis identified 90 significant canonical human pathways, including several pathways more commonly related to non-infectious diseases such as the LRRK2 pathway in Parkinson's disease, and PD-1/PD-L1 signaling pathway important for new immuno-oncology therapies. The analysis of human genome-wide association studies datasets revealed tuberculosis-associated genetic variants proximal to several genes in major histocompatibility complex for antigen presentation. We propose several new targets and drug-repurposing opportunities including intravenous immunoglobulin, ion-channel blockers and cancer immuno-therapeutics for development as combination therapeutics with anti-mycobacterial agents. Our meta-analysis provides novel insights into host genes and pathways important for tuberculosis and brings forth potential drug repurposing opportunities for host-directed therapies.

A Functional Genomic Meta-Analysis of Clinical Trials in Systemic Sclerosis: Toward Precision Medicine and Combination Therapy.

PubMed

Taroni, Jaclyn N; Martyanov, Viktor; Mahoney, J Matthew; Whitfield, Michael L

2017-05-01

Systemic sclerosis is an orphan, systemic autoimmune disease with no FDA-approved treatments. Its heterogeneity and rarity often result in underpowered clinical trials making the analysis and interpretation of associated molecular data challenging. We performed a meta-analysis of gene expression data from skin biopsies of patients with systemic sclerosis treated with five therapies: mycophenolate mofetil, rituximab, abatacept, nilotinib, and fresolimumab. A common clinical improvement criterion of -20% or -5 modified Rodnan skin score was applied to each study. We applied a machine learning approach that captured features beyond differential expression and was better at identifying targets of therapies than the differential expression alone. Regardless of treatment mechanism, abrogation of inflammatory pathways accompanied clinical improvement in multiple studies suggesting that high expression of immune-related genes indicates active and targetable disease. Our framework allowed us to compare different trials and ask if patients who failed one therapy would likely improve on a different therapy, based on changes in gene expression. Genes with high expression at baseline in fresolimumab nonimprovers were downregulated in mycophenolate mofetil improvers, suggesting that immunomodulatory or combination therapy may have benefitted these patients. This approach can be broadly applied to increase tissue specificity and sensitivity of differential expression results. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation.

PubMed

Moreno, Ana M; Fu, Xin; Zhu, Jie; Katrekar, Dhruva; Shih, Yu-Ru V; Marlett, John; Cabotaje, Jessica; Tat, Jasmine; Naughton, John; Lisowski, Leszek; Varghese, Shyni; Zhang, Kang; Mali, Prashant

2018-04-25

Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Precision genome editing in the CRISPR era.

PubMed

Salsman, Jayme; Dellaire, Graham

2017-04-01

With the introduction of precision genome editing using CRISPR-Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end-joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology-directed repair (HDR) using an homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations, or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR-Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR-Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.

Immunotherapy and gene therapy as novel treatments for cancer

PubMed Central

Rangel-Sosa, Martha Montserrat; Aguilar-Córdova, Estuardo

2017-01-01

Abstract The immune system interacts closely with tumors during the disease development and progression to metastasis. The complex communication between the immune system and the tumor cells can prevent or promote tumor growth. New therapeutic approaches harnessing protective immunological mechanisms have recently shown very promising results. This is performed by blocking inhibitory signals or by activating immunological effector cells directly. Immune checkpoint blockade with monoclonal antibodies directed against the inhibitory immune receptors CTLA-4 and PD-1 has emerged as a successful treatment approach for patients with advanced melanoma. Ipilimumab is an anti-CTLA-4 antibody which demonstrated good results when administered to patients with melanoma. Gene therapy has also shown promising results in clinical trials. Particularly, Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (TK) gene to tumor cells in combination with ganciclovir (GCV) may provide an effective suicide gene therapy for destruction of glioblastomas, prostate tumors and other neoplasias by recruiting tumor-infiltrating lymphocytes into the tumor. The development of new treatment strategies or combination of available innovative therapies to improve cell cytotoxic T lymphocytes trafficking into the tumor mass and the production of inhibitory molecules blocking tumor tissue immune-tolerance are crucial to improve the efficacy of cancer therapy. PMID:29213157

The search for biomarkers in the critically ill: a cautionary tale.

PubMed

Moran, John L; Solomon, Patricia J

2018-06-01

The search for biomarkers has been described as a dismal patchwork of fragmented research. We review biomarkers in sepsis in the critically ill in terms of conventional single circulating proteins. Despite sepsis biomarker publications trebling over the past 6 years, currently only one, procalcitonin, has materialised promise. We survey genomic biomarker initiatives, single nucleotide polymorphisms (SNPs) and gene signatures. Despite many SNP associations with sepsis susceptibility and a limited number of genome-wide association studies, the status of these associations is that of genomic signposts only. The standing of gene signatures in the paradigmatic discipline, breast cancer, is described. Uncertainties in the understanding of the sepsis process are documented - the dissociation between blood and tissue element activity, or compartmentalisation. The paradox of the active search for gene signatures to refine the sepsis phenotype and discover target subtypes for new therapies in the absence of such therapies is presented.

Antioxidant gene therapy against neuronal cell death

PubMed Central

Navarro-Yepes, Juliana; Zavala-Flores, Laura; Annadurai, Anandhan; Wang, Fang; Skotak, Maciej; Chandra, Namas; Li, Ming; Pappa, Aglaia; Martinez-Fong, Daniel; Razo, Luz Maria Del; Quintanilla-Vega, Betzabet; Franco, Rodrigo

2014-01-01

Oxidative stress is a common hallmark of neuronal cell death associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, as well as brain stroke/ischemia and traumatic brain injury. Increased accumulation of reactive species of both oxygen (ROS) and nitrogen (RNS) has been implicated in mitochondrial dysfunction, energy impairment, alterations in metal homeostasis and accumulation of aggregated proteins observed in neurodegenerative disorders, which lead to the activation/modulation of cell death mechanisms that include apoptotic, necrotic and autophagic pathways. Thus, the design of novel antioxidant strategies to selectively target oxidative stress and redox imbalance might represent important therapeutic approaches against neurological disorders. This work reviews the evidence demonstrating the ability of genetically encoded antioxidant systems to selectively counteract neuronal cell loss in neurodegenerative diseases and ischemic brain damage. Because gene therapy approaches to treat inherited and acquired disorders offer many unique advantages over conventional therapeutic approaches, we discussed basic research/clinical evidence and the potential of virus-mediated gene delivery techniques for antioxidant gene therapy. PMID:24333264

Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.

PubMed Central

Bandara, G; Mueller, G M; Galea-Lauri, J; Tindal, M H; Georgescu, H I; Suchanek, M K; Hung, G L; Glorioso, J C; Robbins, P D; Evans, C H

1993-01-01

Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy. Images Fig. 1 Fig. 2 PMID:8248169

Neonatal Systemic AAV Induces Tolerance to CNS Gene Therapy in MPS I Dogs and Nonhuman Primates

PubMed Central

Hinderer, Christian; Bell, Peter; Louboutin, Jean-Pierre; Zhu, Yanqing; Yu, Hongwei; Lin, Gloria; Choa, Ruth; Gurda, Brittney L; Bagel, Jessica; O'Donnell, Patricia; Sikora, Tracey; Ruane, Therese; Wang, Ping; Tarantal, Alice F; Casal, Margret L; Haskins, Mark E; Wilson, James M

2015-01-01

The potential host immune response to a nonself protein poses a fundamental challenge for gene therapies targeting recessive diseases. We demonstrate in both dogs and nonhuman primates that liver-directed gene transfer using an adeno-associated virus (AAV) vector in neonates induces a persistent state of immunological tolerance to the transgene product, substantially improving the efficacy of subsequent vector administration targeting the central nervous system (CNS). We applied this approach to a canine model of mucopolysaccharidosis type I (MPS I), a progressive neuropathic lysosomal storage disease caused by deficient activity of the enzyme α-l-iduronidase (IDUA). MPS I dogs treated systemically in the first week of life with a vector expressing canine IDUA did not develop antibodies against the enzyme and exhibited robust expression in the CNS upon intrathecal AAV delivery at 1 month of age, resulting in complete correction of brain storage lesions. Newborn rhesus monkeys treated systemically with AAV vector expressing human IDUA developed tolerance to the transgene, resulting in high cerebrospinal fluid (CSF) IDUA expression and no antibody induction after subsequent CNS gene therapy. These findings suggest that inducing tolerance to the transgene product during a critical period in immunological development can improve the efficacy and safety of gene therapy. PMID:26022732

Neonatal Systemic AAV Induces Tolerance to CNS Gene Therapy in MPS I Dogs and Nonhuman Primates.

PubMed

Hinderer, Christian; Bell, Peter; Louboutin, Jean-Pierre; Zhu, Yanqing; Yu, Hongwei; Lin, Gloria; Choa, Ruth; Gurda, Brittney L; Bagel, Jessica; O'Donnell, Patricia; Sikora, Tracey; Ruane, Therese; Wang, Ping; Tarantal, Alice F; Casal, Margret L; Haskins, Mark E; Wilson, James M

2015-08-01

The potential host immune response to a nonself protein poses a fundamental challenge for gene therapies targeting recessive diseases. We demonstrate in both dogs and nonhuman primates that liver-directed gene transfer using an adeno-associated virus (AAV) vector in neonates induces a persistent state of immunological tolerance to the transgene product, substantially improving the efficacy of subsequent vector administration targeting the central nervous system (CNS). We applied this approach to a canine model of mucopolysaccharidosis type I (MPS I), a progressive neuropathic lysosomal storage disease caused by deficient activity of the enzyme α-l-iduronidase (IDUA). MPS I dogs treated systemically in the first week of life with a vector expressing canine IDUA did not develop antibodies against the enzyme and exhibited robust expression in the CNS upon intrathecal AAV delivery at 1 month of age, resulting in complete correction of brain storage lesions. Newborn rhesus monkeys treated systemically with AAV vector expressing human IDUA developed tolerance to the transgene, resulting in high cerebrospinal fluid (CSF) IDUA expression and no antibody induction after subsequent CNS gene therapy. These findings suggest that inducing tolerance to the transgene product during a critical period in immunological development can improve the efficacy and safety of gene therapy.

Advances in treatment of achondroplasia and osteoarthritis.

PubMed

Klag, Kendra A; Horton, William A

2016-04-15

Achondroplasia (ACH) is the prototype and most common of the human chondrodysplasias. It results from gain-of-function mutations that exaggerate the signal output of the fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase that negatively regulates growth plate activity and linear bone growth. Several approaches to reduce FGFR3 signaling by blocking receptor activation or inhibiting downstream signals have been proposed. Five show promise in preclinical mouse studies. Two candidate therapies target the extracellular domain of FGFR3. The first is a decoy receptor that competes for activating ligands. The second is a synthetic blocking peptide that prevents ligands from binding and activating FGFR3. Two established drugs, statins and meclozine, improve growth of ACH mice. The strongest candidate therapy employs an analog of C-type natriuretic peptide (CNP), which antagonizes the mitogen-activated-protein (MAP) kinase pathway downstream of the FGFR3 receptor and may also act independently in the growth plate. Only the CNP analog has reached clinical trials. Preliminary results of Phase 2 studies show a substantial increase in growth rate of ACH children after six months of therapy with no serious adverse effects. A challenge for drug therapy in ACH is targeting agents to the avascular growth plate. The application of gene therapy in osteoarthritis offers insights because it faces similar technical obstacles. Major advances in gene therapy include the emergence of recombinant adeno-associated virus as the vector of choice, capsid engineering to target vectors to specific tissues, and development of methods to direct vectors to articular chondrocytes. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Anti-TNFα therapy for inflammatory bowel diseases is associated with Epstein-Barr virus lytic activation.

PubMed

Lapsia, Sameer; Koganti, Siva; Spadaro, Salvatore; Rajapakse, Ramona; Chawla, Anupama; Bhaduri-McIntosh, Sumita

2016-02-01

Anti-TNFα therapy, known to suppress T-cell immunity, is increasingly gaining popularity for treatment of autoimmune diseases including inflammatory bowel diseases (IBD). T-cell suppression increases the risk of B-cell EBV-lymphoproliferative diseases and lymphomas. Since EBV-lytic activation is essential for development of EBV-lymphomas and there have been reports of EBV-lymphomas in patients treated with anti-TNFα therapy, we investigated if patients treated with anti-TNFα antibodies demonstrate greater EBV-lytic activity in blood. Peripheral blood mononuclear cells from 10 IBD patients solely on anti-TNFα therapy compared to 3 control groups (10 IBD patients not on immunosuppressive therapy, 10 patients with abdominal pain but without IBD, and 10 healthy subjects) were examined for the percentage of T-cells, EBV load and EBV-lytic transcripts. Patients on anti-TNFα therapy had significantly fewer T-cells, greater EBV load, and increased levels of transcripts from EBV-lytic genes of all kinetic classes compared to controls. Furthermore, exposure of EBV-infected B-cell lines to anti-TNFα antibodies resulted in increased levels of BZLF1 mRNA; BZLF1 encodes for ZEBRA, the viral latency-to-lytic cycle switch. Thus, IBD patients treated with anti-TNFα antibodies have greater EBV loads likely due to enhanced EBV-lytic gene expression and anti-TNFα antibodies may be sufficient to activate the EBV lytic cycle. Findings from this pilot study lay the groundwork for additional scientific and clinical investigation into the effects of anti-TNFα therapy on the life cycle of EBV, a ubiquitous oncovirus that causes lymphomas in the setting of immunocompromise. © 2015 Wiley Periodicals, Inc.

Targeted Cancer Therapy: Vital Oncogenes and a New Molecular Genetic Paradigm for Cancer Initiation Progression and Treatment.

PubMed

Willis, Rudolph E

2016-09-14

It has been declared repeatedly that cancer is a result of molecular genetic abnormalities. However, there has been no working model describing the specific functional consequences of the deranged genomic processes that result in the initiation and propagation of the cancer process during carcinogenesis. We no longer need to question whether or not cancer arises as a result of a molecular genetic defect within the cancer cell. The legitimate questions are: how and why? This article reviews the preeminent data on cancer molecular genetics and subsequently proposes that the sentinel event in cancer initiation is the aberrant production of fused transcription activators with new molecular properties within normal tissue stem cells. This results in the production of vital oncogenes with dysfunctional gene activation transcription properties, which leads to dysfunctional gene regulation, the aberrant activation of transduction pathways, chromosomal breakage, activation of driver oncogenes, reactivation of stem cell transduction pathways and the activation of genes that result in the hallmarks of cancer. Furthermore, a novel holistic molecular genetic model of cancer initiation and progression is presented along with a new paradigm for the approach to personalized targeted cancer therapy, clinical monitoring and cancer diagnosis.

The Role of Gene Therapy in the Treatment of Retinal Diseases: A Review.

PubMed

Campa, C; Gallenga, C E; Bolletta, E; Perri, P

2017-01-01

Gene therapy represents the therapeutic delivery of nucleic acid polymers into patient cells with the aim of treating an underlying disease. Over the past 2 decades this new therapy has made substantial progress owing to better understanding of the pathobiologic basis of various diseases coupled with growth of gene transfer biotechnologies. The eye, in particular, represents a suitable target for such therapy due to the immune privilege provided by the blood-ocular barrier, the ability to directly visualize, access and locally treat the cells and the minimal amount of vector needed given the size of this organ. It is not surprising therefore that several clinical trials are now ongoing in this field. The purpose of this review was to provide an update on gene therapy for retinal diseases, discussing differences in treatment strategies, vector designs and surgical techniques. Research was performed on PubMed, ClinicalTrials.gov, and Home Genetic Reference. We additionally utilized the internet database for genetics of retinal diseases, the portal for rare diseases and orphan drugs and the NCBI database Online Mendelian Inheritance in Man. No restriction was applied on the language of publications. We present the available results of current active clinical trials for inherited retinal disease such as Leber's congenital amaurosis type 2, choroideremia, Stargardt disease, achromatopsia and juvenile X-linked retinoschisis. We also illustrate a new approach of this therapy for the treatment of much more common ocular diseases such as age-related macular degeneration and diabetic retinopathy. Gene therapy represents an emerging and promising therapeutic approach for the treatment not only of rare inherited retinal diseases but also much more common retinal pathologies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Dual systemic tumor targeting with ligand-directed phage and Grp78 promoter induces tumor regression.

PubMed

Kia, Azadeh; Przystal, Justyna M; Nianiaris, Nastasia; Mazarakis, Nicholas D; Mintz, Paul J; Hajitou, Amin

2012-12-01

The tumor-specific Grp78 promoter is overexpressed in aggressive tumors. Cancer patients would benefit greatly from application of this promoter in gene therapy and molecular imaging; however, clinical benefit is limited by lack of strategies to target the systemic delivery of Grp78-driven transgenes to tumors. This study aims to assess the systemic efficacy of Grp78-guided expression of therapeutic and imaging transgenes relative to the standard cytomegalovirus (CMV) promoter. Combination of ligand and Grp78 transcriptional targeting into a single vector would facilitate systemic applications of the Grp78 promoter. We generated a dual tumor-targeted phage containing the arginine-glycine-aspartic acid tumor homing ligand and Grp78 promoter. Next, we combined flow cytometry, Western blot analysis, bioluminescence imaging of luciferase, and HSVtk/ganciclovir gene therapy and compared efficacy to conventional phage carrying the CMV promoter in vitro and in vivo in subcutaneous models of rat and human glioblastoma. We show that double-targeted phage provides persistent transgene expression in vitro and in tumors in vivo after systemic administration compared with conventional phage. Next, we showed significant tumor killing in vivo using the HSVtk/ganciclovir gene therapy and found a systemic antitumor effect of Grp78-driven HSVtk against therapy-resistant tumors. Finally, we uncovered a novel mechanism of Grp78 promoter activation whereby HSVtk/ganciclovir therapy upregulates Grp78 and transgene expression via the conserved unfolded protein response signaling cascade. These data validate the potential of Grp78 promoter in systemic cancer gene therapy and report the efficacy of a dual tumor targeting phage that may prove useful for translation into gene therapy and molecular imaging applications.

Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yu, E-mail: xuyu1001@gmail.com; Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071; Liu, Zhengchun, E-mail: l135027@126.com

Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expressionmore » of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.« less

Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer

PubMed Central

Takahashi, Hiroshi; Hirai, Yukihiko; Migita, Makoto; Seino, Yoshihiko; Fukuda, Yuh; Sakuraba, Hitoshi; Kase, Ryoichi; Kobayashi, Toshihide; Hashimoto, Yasuhiro; Shimada, Takashi

2002-01-01

Fabry disease is a systemic disease caused by genetic deficiency of a lysosomal enzyme, α-galactosidase A (α-gal A), and is thought to be an important target for enzyme replacement therapy. We studied the feasibility of gene-mediated enzyme replacement for Fabry disease. The adeno-associated virus (AAV) vector containing the α-gal A gene was injected into the right quadriceps muscles of Fabry knockout mice. A time course study showed that α-gal A activity in plasma was increased to ≈25% of normal mice and that this elevated activity persisted for up to at least 30 weeks without development of anti-α-gal A antibodies. The α-gal A activity in various organs of treated Fabry mice remained 5–20% of those observed in normal mice. Accumulated globotriaosylceramide in these organs was completely cleared by 25 weeks after vector injection. Reduction of globotriaosylceramide levels was also confirmed by immunohistochemical and electronmicroscopic analyses. Echocardiographic examination of treated mice demonstrated structural improvement of cardiac hypertrophy 25 weeks after the treatment. AAV vector-mediated muscle-directed gene transfer provides an efficient and practical therapeutic approach for Fabry disease. PMID:12370426

Albumin based versatile multifunctional nanocarriers for cancer therapy: Fabrication, surface modification, multimodal therapeutics and imaging approaches.

PubMed

Kudarha, Ritu R; Sawant, Krutika K

2017-12-01

Albumin is a versatile protein used as a carrier system for cancer therapeutics. As a carrier it can provide tumor specificity, reduce drug related toxicity, maintain therapeutic concentration of the active moiety like drug, gene, peptide, protein etc. for long period of time and also reduce drug related toxicities. Apart from cancer therapy, it is also utilized in the imaging and multimodal therapy of cancer. This review highlights the important properties, structure and types of albumin based nanocarriers with regards to their use for cancer targeting. It also provides brief discussion on methods of preparation of these nanocarriers and their surface modification. Applications of albumin nanocarriers for cancer therapy, gene delivery, imaging, phototherapy and multimodal therapy have also been discussed. This review also provides brief discussion about albumin based marketed nano formulations and those under clinical trials. Copyright © 2017 Elsevier B.V. All rights reserved.

Radiographic Evaluation of Bones and Joints in Mucopolysaccharidosis I and VII Dogs After Neonatal Gene Therapy

PubMed Central

Herati, Ramin Sedaghat; Knox, Van W.; O’Donnell, Patricia; D’Angelo, Marina; Haskins, Mark E.; Ponder, Katherine P.

2009-01-01

Mucopolysaccharidosis I (MPS I) and MPS VII are due to deficient activity of the glycosaminoglycan-degrading lysosomal enzymes α-L-iduronidase and β-glucuronidase, respectively, and result in abnormal bones and joints. Here, the severity of skeletal disease in MPS I and MPS VII dogs and the effects of neonatal gene therapy were evaluated. For untreated MPS VII dogs, the lengths of the second cervical vertebrae (C2) and the femur were only 56% and 84% of normal, respectively, and bone dysplasia and articular erosions, and joint subluxation were severe. Previously, we reported that neonatal intravenous injection of a retroviral vector (RV) with the appropriate gene resulted in expression in liver and blood cells, and high serum enzyme activity. In this study, we demonstrate that C2 and femurs of RV-treated MPS VII dogs were longer at 82% and 101% of normal, respectively, and there were partial improvements of qualitative abnormalities. For untreated MPS I dogs, the lengths of C2 and femurs (91% and 96% of normal, respectively) were not significantly different from normal dogs. Qualitative changes in MPS I bones and joints were generally modest and were partially improved with RV treatment, although cervical spine disease was severe and was difficult to correct with gene therapy in both models. The greater severity of skeletal disease in MPS VII than in MPS I dogs may reflect accumulation of chondroitin sulfate in cartilage in MPS VII, or could relate to the specific mutations. Neonatal RV-mediated gene therapy ameliorates, but does not prevent, skeletal disease in MPS I and MPS VII dogs. PMID:18707908

Adenovirus-Mediated p202 Gene Transfer in Breast Cancer Gene Therapy

DTIC Science & Technology

2005-05-01

transcriptional regulation of genes important for cell cycle control, differentiation, and apoptosis (1, 3, 4). Our previous studies have shown that p202...leads to induction of p53 and activation of p53 target gene (e.g., p21 CIP 1). 10. The positive regulation of p53 by IFIXcd can be observed only in...cancers. Together, our data suggest that both Ad-p202 and IFIX may be further developed into efficient therapeutic agents for human cancer gene

Engineering HSV-1 vectors for gene therapy.

PubMed

Goins, William F; Huang, Shaohua; Cohen, Justus B; Glorioso, Joseph C

2014-01-01

Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications, and with the approval of Glybera (alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20: 1831-1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I-III human trials in patients with glioblastoma multiforme, a fatal form of brain cancer, and in malignant melanoma. In fact, T-VEC (talimogene laherparepvec, formerly known as OncoVex GM-CSF) displayed efficacy in a recent Phase III trial when compared to standard GM-CSF treatment alone (Andtbacka et al. J Clin Oncol 31: sLBA9008, 2013) and may soon become the second FDA-approved gene therapy product used in standard patient care. In addition to the replication-competent oncolytic HSV vectors like T-VEC, replication-defective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy, and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.

Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors

PubMed Central

Thakore, Pratiksha I.; Gersbach, Charles A.

2016-01-01

Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy. PMID:26443215

Adenosine Deaminase (ADA)-Deficient Severe Combined Immune Deficiency (SCID): Molecular Pathogenesis and Clinical Manifestations.

PubMed

Bradford, Kathryn L; Moretti, Federico A; Carbonaro-Sarracino, Denise A; Gaspar, Hubert B; Kohn, Donald B

2017-10-01

Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme of purine metabolism encoded by the Ada gene, is a cause of human severe combined immune deficiency (SCID). Numerous deleterious mutations occurring in the ADA gene have been found in patients with profound lymphopenia (T - B - NK - ), thus underscoring the importance of functional purine metabolism for the development of the immune defense. While untreated ADA SCID is a fatal disorder, there are multiple life-saving therapeutic modalities to restore ADA activity and reconstitute protective immunity, including enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) with autologous gene-corrected hematopoietic stem cells (HSC). We review the pathogenic mechanisms and clinical manifestations of ADA SCID.

Gene doping: an overview and current implications for athletes.

PubMed

van der Gronde, Toon; de Hon, Olivier; Haisma, Hidde J; Pieters, Toine

2013-07-01

The possibility of gene doping, defined as the transfer of nucleic acid sequences and/or the use of normal or genetically modified cells to enhance sport performance, is a real concern in sports medicine. The abuse of knowledge and techniques gained in the area of gene therapy is a form of doping, and is prohibited for competitive athletes. As yet there is no conclusive evidence that that gene doping has been practiced in sport. However, given that gene therapy techniques improve continuously, the likelihood of abuse will increase. A literature search was conducted to identify the most relevant proteins based on their current gene doping potential using articles from Pubmed, Scopus and Embase published between 2006 and 2011. The final list of selected proteins were erythropoietin, insulin-like growth factor, growth hormone, myostatin, vascular endothelial growth factor, fibroblast growth factor, endorphin and enkephalin, α actinin 3, peroxisome proliferator-activated receptor-delta (PPARδ) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C). We discuss these proteins with respect to their potential benefits, existing gene therapy experience in humans, potential risks, and chances of detection in current and future anti-doping controls. We have identified PPARδ and PEPCK-C as having high potential for abuse. But we expect that for efficiency reasons, there will be a preference for inserting gene target combinations rather than single gene doping products. This will also further complicate detection.

Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer.

PubMed

Lopes, Gabriel Lima; Vattimo, Edoardo Filippo de Queiroz; Castro Junior, Gilberto de

2015-01-01

Lung cancer is the leading cause of cancer-related deaths worldwide. Promising new therapies have recently emerged from the development of molecular targeted drugs; particularly promising are those blocking the signal transduction machinery of cancer cells. One of the most widely studied cell signaling pathways is that of EGFR, which leads to uncontrolled cell proliferation, increased cell angiogenesis, and greater cell invasiveness. Activating mutations in the EGFR gene (deletions in exon 19 and mutation L858R in exon 21), first described in 2004, have been detected in approximately 10% of all non-squamous non-small cell lung cancer (NSCLC) patients in Western countries and are the most important predictors of a response to EGFR tyrosine-kinase inhibitors (EGFR-TKIs). Studies of the EGFR-TKIs gefitinib, erlotinib, and afatinib, in comparison with platinum-based regimens, as first-line treatments in chemotherapy-naïve patients have shown that the EGFR-TKIs produce gains in progression-free survival and overall response rates, although only in patients whose tumors harbor activating mutations in the EGFR gene. Clinical trials have also shown EGFR-TKIs to be effective as second- and third-line therapies in advanced NSCLC. Here, we review the main aspects of EGFR pathway activation in NSCLC, underscore the importance of correctly identifying activating mutations in the EGFR gene, and discuss the main outcomes of EGFR-TKI treatment in NSCLC.

Non-invasive, neuron-specific gene therapy by focused ultrasound-induced blood-brain barrier opening in Parkinson's disease mouse model.

PubMed

Lin, Chung-Yin; Hsieh, Han-Yi; Chen, Chiung-Mei; Wu, Shang-Rung; Tsai, Chih-Hung; Huang, Chiung-Yin; Hua, Mu-Yi; Wei, Kuo-Chen; Yeh, Chih-Kuang; Liu, Hao-Li

2016-08-10

Focused ultrasound (FUS)-induced with microbubbles (MBs) is a promising technique for noninvasive opening of the blood-brain barrier (BBB) to allow targeted delivery of therapeutic substances into the brain and thus the noninvasive delivery of gene vectors for CNS treatment. We have previously demonstrated that a separated gene-carrying liposome and MBs administration plus FUS exposure can deliver genes into the brain, with the successful expression of the reporter gene and glial cell line-derived neurotrophic factor (GDNF) gene. In this study, we further modify the delivery system by conjugating gene-carrying liposomes with MBs to improve the GDNF gene-delivery efficiency, and to verify the possibility of using this system to perform treatment in the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animal disease model. FUS-BBB opening was verified by contrast-enhanced MRI, and GFP gene expression was verified via in vivo imaging system (IVIS). Western blots as well as enzyme-linked immunosorbent assay (ELISA) were conducted to measure protein expression, and immunohistochemistry (IHC) was conducted to test the Tyrosine hydroxylase (TH)-neuron distribution. Dopamine (DA) and its metabolites as well as dopamine active transporter (DAT) were quantitatively analyzed to show dopaminergic neuronal dopamine secretion/activity/metabolism. Motor performance was evaluated by rotarod test weekly. Results demonstrated that the LpDNA-MBs (gene-liposome-MBs) complexes successfully serve as gene carrier and BBB-opening catalyst, and outperformed the separated LpDNA/MBs administration both in terms of gene delivery and expression. TH-positive IHC and measurement of DA and its metabolites DOPAC and HVA confirmed improved neuronal function, and the proposed system also provided the best neuroprotective effect to retard the progression of motor-related behavioral abnormalities. Immunoblotting and histological staining further confirmed the expression of reporter genes in neuronal cells. This study suggests that FUS exposures with the administration of LpDNA-MBs complexes synergistically can serve as an effective gene therapy strategy for MPTP-animal treatment, and may have potential for further application to perform gene therapy for neurodegenerative disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Gene therapy in pancreatic cancer

PubMed Central

Liu, Si-Xue; Xia, Zhong-Sheng; Zhong, Ying-Qiang

2014-01-01

Pancreatic cancer (PC) is a highly lethal disease and notoriously difficult to treat. Only a small proportion of PC patients are eligible for surgical resection, whilst conventional chemoradiotherapy only has a modest effect with substantial toxicity. Gene therapy has become a new widely investigated therapeutic approach for PC. This article reviews the basic rationale, gene delivery methods, therapeutic targets and developments of laboratory research and clinical trials in gene therapy of PC by searching the literature published in English using the PubMed database and analyzing clinical trials registered on the Gene Therapy Clinical Trials Worldwide website (http://www. wiley.co.uk/genmed/ clinical). Viral vectors are main gene delivery tools in gene therapy of cancer, and especially, oncolytic virus shows brighter prospect due to its tumor-targeting property. Efficient therapeutic targets for gene therapy include tumor suppressor gene p53, mutant oncogene K-ras, anti-angiogenesis gene VEGFR, suicide gene HSK-TK, cytosine deaminase and cytochrome p450, multiple cytokine genes and so on. Combining different targets or combination strategies with traditional chemoradiotherapy may be a more effective approach to improve the efficacy of cancer gene therapy. Cancer gene therapy is not yet applied in clinical practice, but basic and clinical studies have demonstrated its safety and clinical benefits. Gene therapy will be a new and promising field for the treatment of PC. PMID:25309069

SFRP1 is a possible candidate for epigenetic therapy in non-small cell lung cancer.

PubMed

Taguchi, Y-H; Iwadate, Mitsuo; Umeyama, Hideaki

2016-08-12

Non-small cell lung cancer (NSCLC) remains a lethal disease despite many proposed treatments. Recent studies have indicated that epigenetic therapy, which targets epigenetic effects, might be a new therapeutic methodology for NSCLC. However, it is not clear which objects (e.g., genes) this treatment specifically targets. Secreted frizzled-related proteins (SFRPs) are promising candidates for epigenetic therapy in many cancers, but there have been no reports of SFRPs targeted by epigenetic therapy for NSCLC. This study performed a meta-analysis of reprogrammed NSCLC cell lines instead of the direct examination of epigenetic therapy treatment to identify epigenetic therapy targets. In addition, mRNA expression/promoter methylation profiles were processed by recently proposed principal component analysis based unsupervised feature extraction and categorical regression analysis based feature extraction. The Wnt/β-catenin signalling pathway was extensively enriched among 32 genes identified by feature extraction. Among the genes identified, SFRP1 was specifically indicated to target β-catenin, and thus might be targeted by epigenetic therapy in NSCLC cell lines. A histone deacetylase inhibitor might reactivate SFRP1 based upon the re-analysis of a public domain data set. Numerical computation validated the binding of SFRP1 to WNT1 to suppress Wnt signalling pathway activation in NSCLC. The meta-analysis of reprogrammed NSCLC cell lines identified SFRP1 as a promising target of epigenetic therapy for NSCLC.

Eliminating SCID row: new approaches to SCID.

PubMed

Kohn, Donald B

2014-12-05

Treatments for patients with SCID by hematopoietic stem cell transplantation (HSCT) have changed this otherwise lethal primary immune deficiency disorder into one with an increasingly good prognosis. SCID has been the paradigm disorder supporting many key advances in the field of HSCT, with first-in-human successes with matched sibling, haploidentical, and matched unrelated donor allogeneic transplantations. Nevertheless, the optimal approaches for HSCT are still being defined, including determining the optimal stem cell sources, the use and types of pretransplantation conditioning, and applications for SCID subtypes associated with radiosensitivity, for patients with active viral infections and for neonates. Alternatively, autologous transplantation after ex vivo gene correction (gene therapy) has been applied successfully to the treatment of adenosine deaminase-deficient SCID and X-linked SCID by vector-mediated gene addition. Gene therapy holds the prospect of avoiding risks of GVHD and would allow each patient to be their own donor. New approaches to gene therapy by gene correction in autologous HSCs using site-specific endonuclease-mediated homology-driven gene repair are under development. With newborn screening becoming more widely adopted to detect SCID patients before they develop complications, the prognosis for SCID is expected to improve further. This chapter reviews recent advances and ongoing controversies in allogeneic and autologous HSCT for SCID. © 2014 by The American Society of Hematology. All rights reserved.

Estrogenic effects of herbal medicines from Costa Rica used for the management of menopausal symptoms.

PubMed

Doyle, Brian J; Frasor, Jonna; Bellows, Lauren E; Locklear, Tracie D; Perez, Alice; Gomez-Laurito, Jorge; Mahady, Gail B

2009-01-01

Outcomes from the Women's Health Initiative have demonstrated adverse effects associated with hormone therapy and have prioritized the need to develop new alternative treatments for the management of menopause and osteoporosis. To this end, we have been investigating natural herbal medicines used by Costa Rican women to manage menopausal symptoms. Seventeen plant species were collected and extracted in Costa Rica. To establish possible mechanisms of action and to determine their potential future use for menopause or osteoporosis, we investigated the estrogenic activities of the herbal extracts in an estrogen-reporter gene estrogen receptor (ER) beta-Chemically Activated Luciferase Expression assay in U2-OS cells and in reporter and endogenous gene assays in MCF-7 cells. Six of the plant extracts bound to the ERs. Four of the six extracts stimulated reporter gene expression in the ER-beta-Chemically Activated Luciferase Expression assay. All six extracts modulated expression of endogenous genes in MCF-7 cells, with four extracts acting as estrogen agonists and two extracts, Pimenta dioica and Smilax domingensis, acting as partial agonist/antagonists by enhancing estradiol-stimulated pS2 mRNA expression but reducing estradiol-stimulated PR and PTGES mRNA expression. Both P. dioica and S. domingensis induced a 2ERE-luciferase reporter gene in transient transfected MCF-7 cells, which was inhibited by the ER antagonist ICI 182,780. This work presents a plausible mechanism of action for many of the herbal medicines used by Costa Rican women to treat menopausal symptoms. However, it further suggests that studies of safety and efficacy are needed before these herbs should be used as alternative therapies to hormone therapy.

Assessment of Radiation Induced Therapeutic Effect and Cytotoxicity in Cancer Patients Based on Transcriptomic Profiling.

PubMed

Karim, Sajjad; Mirza, Zeenat; Chaudhary, Adeel G; Abuzenadah, Adel M; Gari, Mamdooh; Al-Qahtani, Mohammed H

2016-02-19

Toxicity induced by radiation therapy is a curse for cancer patients undergoing treatment. It is imperative to understand and define an ideal condition where the positive effects notably outweigh the negative. We used a microarray meta-analysis approach to measure global gene-expression before and after radiation exposure. Bioinformatic tools were used for pathways, network, gene ontology and toxicity related studies. We found 429 differentially expressed genes at fold change >2 and p-value <0.05. The most significantly upregulated genes were synuclein alpha (SNCA), carbonic anhydrase I (CA1), X-linked Kx blood group (XK), glycophorin A and B (GYPA and GYPB), and hemogen (HEMGN), while downregulated ones were membrane-spanning 4-domains, subfamily A member 1 (MS4A1), immunoglobulin heavy constant mu (IGHM), chemokine (C-C motif) receptor 7 (CCR7), BTB and CNC homology 1 transcription factor 2 (BACH2), and B-cell CLL/lymphoma 11B (BCL11B). Pathway analysis revealed calcium-induced T lymphocyte apoptosis and the role of nuclear factor of activated T-cells (NFAT) in regulation of the immune response as the most inhibited pathways, while apoptosis signaling was significantly activated. Most of the normal biofunctions were significantly decreased while cell death and survival process were activated. Gene ontology enrichment analysis revealed the immune system process as the most overrepresented group under the biological process category. Toxicity function analysis identified liver, kidney and heart to be the most affected organs during and after radiation therapy. The identified biomarkers and alterations in molecular pathways induced by radiation therapy should be further investigated to reduce the cytotoxicity and development of fatigue.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Naumov, Inna; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; Kazanov, Dina

2012-01-15

Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1more » and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.« less

Ethical, social and public awareness issues in gene therapy EuropaBio.

PubMed

2002-01-01

EuropaBio, the European Association for Bio-industries, represents 40 corporate members operating world wide and 14 national associations (totaling up to 700 small- and medium-sized enterprises) involved in research, development, testing, manufacturing, sales, and distribution of biotechnology-derived products and services in the field of health cae, agriculture, food, and the environment. AGE is a group of researchers and university professors involved in high-level professional activities related to bioethics, and particularly interested in ethical issues related to the development and use of modern technology. It is essential that industry actively participates in, and contributes to, the social debate on emerging technologies. Therefore, EuropaBio presents herein its view on gene therapy and its responsible development and use.

Sustained secretion of anti-tumor necrosis factor α monoclonal antibody from ex vivo genetically engineered dermal tissue demonstrates therapeutic activity in mouse model of rheumatoid arthritis.

PubMed

Zafir-Lavie, Inbal; Miari, Reem; Sherbo, Shay; Krispel, Simi; Tal, Osnat; Liran, Atar; Shatil, Tamar; Badinter, Felix; Goltsman, Haim; Shapir, Nir; Benhar, Itai; Neil, Garry A; Panet, Amos

2017-08-01

Rheumatoid arthritis (RA) is a symmetric inflammatory polyarthritis associated with high concentrations of pro-inflammatory, cytokines including tumor necrosis factor (TNF)-α. Adalimumab is a monoclonal antibody (mAb) that binds TNF-α, and is widely used to treat RA. Despite its proven clinical efficacy, adalimumab and other therapeutic mAbs have disadvantages, including the requirement for repeated bolus injections and the appearance of treatment limiting anti-drug antibodies. To address these issues, we have developed an innovative ex vivo gene therapy approach, termed transduced autologous restorative gene therapy (TARGT), to produce and secrete adalimumab for the treatment of RA. Helper-dependent (HD) adenovirus vector containing adalimumab light and heavy chain coding sequences was used to transduce microdermal tissues and cells of human and mouse origin ex vivo, rendering sustained secretion of active adalimumab. The genetically engineered tissues were subsequently implanted in a mouse model of RA. Transduced human microdermal tissues implanted in SCID mice demonstrated 49 days of secretion of active adalimumab in the blood, at levels of tens of microgram per milliliter. In addition, transduced autologous dermal cells were implanted in the RA mouse model and demonstrated statistically significant amelioration in RA symptoms compared to naïve cell implantation and were similar to recombinant adalimumab bolus injections. The results of the present study report microdermal tissues engineered to secrete active adalimumab as a proof of concept for sustained secretion of antibody from the novel ex vivo gene therapy TARGT platform. This technology may now be applied to a range of antibodies for the therapy of other diseases. Copyright © 2017 John Wiley & Sons, Ltd.

Post-translational regulation of gene expression using the ATF4 oxygen-dependent degradation domain for hypoxia-specific gene therapy.

PubMed

Cho, Su Hee; Oh, Binna; Kim, Hyun Ah; Park, Jeong Hyun; Lee, Minhyung

2013-11-01

Solid tumors have hypoxic regions in their cores, due to low blood supply levels. Therefore, hypoxia-specific gene regulation systems have been developed for tumor-specific gene therapy. In this study, the oxygen-dependent degradation (ODD) domain on activating transcription factor-4 (ATF4) was evaluated for post-translational regulation of proteins. The ATF4 ODD cDNA was amplified by RT-PCR, and a luciferase plasmid containing the ATF4 ODD domain, pSV-Luc-ATF4-ODD, was constructed. Luciferase expression was induced under hypoxia by the ATF4 ODD domain in transfection assays into N2A neuroblastoma cells, C6 glioblastoma cells, and U87 glioblastoma cells. In the transfection assay with pSV-Luc-ATF4-ODD, RT-PCR results showed that the mRNA level did not change under hypoxia. This suggests that the induction of luciferase under hypoxia was mediated by post-translational regulation. A plasmid expressing thymidine kinase from herpes simplex virus (HSV-tk), pSV-HSVtk-ATF4-ODD, was constructed with the ATF4 ODD cDNA. The transfection assay with pSV-TK-ATF4-ODD showed that the ATF4 ODD domain induced HSV-tk expression under hypoxia and facilitated the death of C6 cells in the presence of ganciclovir (GCV). Furthermore, pSV-HSVtk-ATF4-ODD induced caspase-3 activity in the hypoxic cells. In conclusion, the ATF4 ODD may be useful for hypoxia-specific gene therapy by post-translational regulation of gene expression.

Mesenchymal stem cell-based NK4 gene therapy in nude mice bearing gastric cancer xenografts

PubMed Central

Zhu, Yin; Cheng, Ming; Yang, Zhen; Zeng, Chun-Yan; Chen, Jiang; Xie, Yong; Luo, Shi-Wen; Zhang, Kun-He; Zhou, Shu-Feng; Lu, Nong-Hua

2014-01-01

Mesenchymal stem cells (MSCs) have been recognized as promising delivery vehicles for gene therapy of tumors. Gastric cancer is the third leading cause of worldwide cancer mortality, and novel treatment modalities are urgently needed. NK4 is an antagonist of hepatocyte growth factor receptors (Met) which are often aberrantly activated in gastric cancer and thus represent a useful candidate for targeted therapies. This study investigated MSC-delivered NK4 gene therapy in nude mice bearing gastric cancer xenografts. MSCs were transduced with lentiviral vectors carrying NK4 complementary DNA or enhanced green fluorescent protein (GFP). Such transduction did not change the phenotype of MSCs. Gastric cancer xenografts were established in BALB/C nude mice, and the mice were treated with phosphate-buffered saline (PBS), MSCs-GFP, Lenti-NK4, or MSCs-NK4. The tropism of MSCs toward gastric cancer cells was determined by an in vitro migration assay using MKN45 cells, GES-1 cells and human fibroblasts and their presence in tumor xenografts. Tumor growth, tumor cell apoptosis and intratumoral microvessel density of tumor tissue were measured in nude mice bearing gastric cancer xenografts treated with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 via tail vein injection. The results showed that MSCs migrated preferably to gastric cancer cells in vitro. Systemic MSCs-NK4 injection significantly suppressed the growth of gastric cancer xenografts. MSCs-NK4 migrated and accumulated in tumor tissues after systemic injection. The microvessel density of tumor xenografts was decreased, and tumor cellular apoptosis was significantly induced in the mice treated with MSCs-NK4 compared to control mice. These findings demonstrate that MSC-based NK4 gene therapy can obviously inhibit the growth of gastric cancer xenografts, and MSCs are a better vehicle for NK4 gene therapy than lentiviral vectors. Further studies are warranted to explore the efficacy and safety of the MSC-based NK4 gene therapy in animals and cancer patients. PMID:25525335

Genes and Gene Therapy

MedlinePlus

... a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

Genome-wide target profiling of piggyBac and Tol2 in HEK 293: pros and cons for gene discovery and gene therapy

PubMed Central

2011-01-01

Background DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery. Results We have observed that (1) the Tol2 transposase (but not piggyBac) is highly sensitive to molecular engineering; (2) the piggyBac donor with only the 40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective transposition; and (3) a small amount of piggyBac transposases results in robust transposition suggesting the piggyBac transpospase is highly active. Performing genome-wide target profiling on data sets obtained by retrieving chromosomal targeting sequences from individual clones, we have identified several piggyBac and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear difference in targeting preferences in the human genome. Finally, we have observed that (5) only sites with a particular sequence context can be targeted by either piggyBac or Tol2. Conclusions The non-overlapping targeting preference of piggyBac and Tol2 makes them complementary research tools for manipulating mammalian genomes. PiggyBac is the most promising transposon-based vector system for achieving site-specific targeting of therapeutic genes due to the flexibility of its transposase for being molecularly engineered. Insights from this study will provide a basis for engineering piggyBac transposases to achieve site-specific therapeutic gene targeting. PMID:21447194

Gene Therapy in the Cornea: 2005-present

PubMed Central

Mohan, Rajiv R.; Tovey, Jonathan C.K.; Sharma, Ajay; Tandon, Ashish

2011-01-01

Successful restoration of vision in human patients with gene therapy affirmed its promise to cure ocular diseases and disorders. The efficacy of gene therapy is contingent upon vector and mode of therapeutic DNA introduction into targeted cells/tissues. The cornea is an ideal tissue for gene therapy due to its ease of access and relative immune-privilege. Considerable progress has been made in the field of corneal gene therapy in last 5 years. Several new gene transfer vectors, techniques and approaches have evolved. Although corneal gene therapy is still in its early stages of development, the potential of gene-based interventions to treat corneal abnormalities have begun to surface. Identification of next generation viral and nanoparticle vectors, characterization of delivered gene levels, localization, and duration in the cornea, and significant success in controlling corneal disorders, particularly fibrosis and angiogenesis, in experimental animal disease models, with no major side effects have propelled gene therapy a step closer towards establishing gene-based therapies for corneal blindness. Recently, researchers have assessed the delivery of therapeutic genes for corneal diseases and disorders due to trauma, infections, chemical, mechanical, and surgical injury, and/or abnormal wound healing. This review provides an update on the developments in gene therapy for corneal diseases and discusses the barriers that hinder its utilization for delivering genes in the cornea. PMID:21967960

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line

PubMed Central

Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

2017-01-01

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines. PMID:28810654

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line.

PubMed

Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

2017-08-01

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines.

Multifunctional cationic polyurethanes designed for non-viral cancer gene therapy.

PubMed

Cheng, Jian; Tang, Xin; Zhao, Jie; Shi, Ting; Zhao, Peng; Lin, Chao

2016-01-01

Nano-polyplexes from bioreducible cationic polymers have a massive promise for cancer gene therapy. However, the feasibility of cationic polyurethanes for non-viral gene therapy is so far not well studied. In this work, a linear cationic polyurethane containing disulfide bonds, urethane linkages and protonable tertiary amino groups was successfully generated by stepwise polycondensation reaction between 2,2'-dithiodiethanol bis(p-nitrophenyl carbonate) and 1,4-bis(3-aminopropyl)piperazine (BAP). We confirmed that the cationic polyurethane (denoted as PUBAP) displayed superior gene delivery properties to its cationic polyamide analogue, thus causing higher in vitro transfection efficiency in MCF-7 and SKOV-3 cells. Besides, further folate-PEGylation and hydrophobic deoxycholic acid (DCA) conjugation to amino-containing PUBAP can be conducted to afford multifunctional polyurethane gene delivery system. After optimization, folate-decorated nano-polyplexes from the PUBAP conjugated with 8 folate-PEG chains and 12 DCA residues exhibited superb colloidal stability under physiological conditions, and performed rapid uptake via folate receptor-mediated endocytosis, efficient intracellular gene release and nucleus translocation into SKOV-3 cells in vitro and in vivo. Importantly, PUBAP based polyplexes possess low cytotoxicity as a result of PUBAP biodegradability. Therefore, marked growth inhibition of SKOV-3 tumor xenografted in Balb/c nude mice was achieved with negligible side effects on the mouse health after intravenous administration of PUBAP based polyplexes with a therapeutic plasmid encoding for TNF-related apoptosis-inducing ligand. This work provides a new insight into biomedical application of bio-responsive polyurethanes for cancer therapy. In this study, we have confirmed that disulfide-based cationic polyurethane presents a new non-viral vector for gene transfer and cancer gene therapy. The significance of this work includes: (1) design and synthesis of a group of novel disulfide-based cationic polyurethane by non-isocyanate chemistry; (2) comparative study of transfection activity between cationic polyurethanes and cationic polyamides; (3) feasibility of bioreducible cationic polyurethanes for in vivo cancer gene therapy. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Decoy receptor 3 inhibits renal mononuclear leukocyte infiltration and apoptosis and prevents progression of IgA nephropathy in mice.

PubMed

Ka, Shuk-Man; Hsieh, Tai-Tzu; Lin, Shih-Hua; Yang, Sung-Sen; Wu, Chin-Chen; Sytwu, Huey-Kang; Chen, Ann

2011-12-01

The progression of IgA nephropathy (IgAN), the most frequent type of primary glomerulonephritis, is associated with high levels of mononuclear leukocyte infiltration into the kidney. These cells consist mainly of T cells and macrophages. Our previous study showed that a decoy receptor 3 (DCR3) gene therapy can prevent the development of a mouse autoimmune glomerulonephritis model by its potent immune modulating effects (Ka SM, Sytwu HK, Chang DM, Hsieh SL, Tsai PY, Chen A. J Am Soc Nephrol 18: 2473-2485, 2007). Here, we tested the hypothesis that DCR3 might prevent the progression of IgAN, an immune complex-mediated primary glomerulonephritis, by inhibiting T cell activation, renal T cell/macrophage infiltration, and protecting the kidney from apoptosis. We used a progressive IgAN (Prg-IgAN) model in B cell-deficient mice, because the mice are characterized by a dramatic proliferation of activated T cells systemically and progressive NF-κB activation in the kidney. We treated the animals with short-term gene therapy with DCR3 plasmids by hydrodynamics-based gene delivery. When the mice were euthanized on day 21, we found that, compared with empty vector-treated (disease control) Prg-IgAN mice, DCR3 gene therapy resulted in 1) systemic inhibition of T cell activation and proliferation; 2) lower serum levels of proinflammatory cytokines; 3) improved proteinuria, renal function, and renal pathology (inhibiting the development of marked glomerular proliferation, crescent formation, glomerulosclerosis, and interstitial inflammation); 5) suppression of T cell and macrophage infiltration into the periglomerular interstitium of the kidney; and 5) a reduction in apoptotic figures in the kidney. On the basis of these findings, DCR3 might be useful therapeutically in preventing the progression of IgAN.

Therapy of chronic myeloid leukemia: twilight of the imatinib era?

PubMed

Trela, Ewelina; Glowacki, Sylwester; Błasiak, Janusz

2014-01-01

Chronic myeloid leukemia (CML) results from the clonal expansion of pluripotent hematopoietic stem cells containing the active BCR/ABL fusion gene produced by a reciprocal translocation of the ABL1 gene to the BCR gene. The BCR/ABL protein displays a constitutive tyrosine kinase activity and confers on leukemic cells growth and proliferation advantage and resistance to apoptosis. Introduction of imatinib (IM) and other tyrosine kinase inhibitors (TKIs) has radically improved the outcome of patients with CML and some other diseases with BCR/ABL expression. However, a fraction of CML patients presents with resistance to this drug. Regardless of clinical profits of IM, there are several drawbacks associated with its use, including lack of eradication of the malignant clone and increasing relapse rate resulting from long-term therapy, resistance, and intolerance. Second and third generations of TKIs have been developed to break IM resistance. Clinical studies revealed that the introduction of second-generation TKIs has improved the overall survival of CML patients; however, some with specific mutations such as T315I remain resistant. Second-generation TKIs may completely replace imatinib in perspective CML therapy, and addition of third-generation inhibitors may overcome resistance induced by every form of point mutations.

Stereotactic delivery of a recombinant adenovirus into a C6 glioma cell line in a rat brain tumor model.

PubMed

Badie, B; Hunt, K; Economou, J S; Black, K L

1994-11-01

The dismal results of conventional therapy for primary malignant brain tumors has justified exploring gene therapy approaches for this disease. Transduction of animal brain tumor models in vivo has been reported previously with retroviruses and herpes viruses. Because adenoviruses have the advantage of transducing quiescent and actively dividing tumor cells, they may prove to be more effective in such therapy. We used a replication-deficient recombinant adenovirus bearing the Escherichia coli beta-galactosidase gene in a rat C6 glioma tumor model. Transduced cells were detected by X-5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to reveal beta-galactosidase activity. Initial experiments in vitro showed 50% and 90% transduction at vector titers of approximately 10(7) and 10(8) plaque-forming units/ml, respectively. Although no cytopathic effects were seen at 10(7) plaque-forming units/ml, more than 50% reduction in tumor cell growth was noted at 10(8) plaque-forming units/ml both in vitro and in vivo. Stereotactic delivery of the recombinant adenovirus into the frontal lobe of normal rat brains resulted in intense staining of all cell types, that is, neurons, astrocytes, and ependymal cells. Stereotactic injection into C6 glioma brain tumors in rats stained 25 to 30% of the tumor cells. We conclude that adenovirus vectors can be used to transfer genes to central nervous system tumors in vivo. Using stereotactic delivery, adenovirus vectors can transfer genes into the central nervous system intended for tumor therapy.

RNA Structure Design Improves Activity and Specificity of trans-Splicing-Triggered Cell Death in a Suicide Gene Therapy Approach.

PubMed

Poddar, Sushmita; Loh, Pei She; Ooi, Zi Hao; Osman, Farhana; Eul, Joachim; Patzel, Volker

2018-06-01

Spliceosome-mediated RNA trans-splicing enables correction or labeling of pre-mRNA, but therapeutic applications are hampered by issues related to the activity and target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure design to improve both on-target activity and specificity of tsRNA in a herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy approach targeting alpha fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC) or human papillomavirus type 16 (HPV-16) pre-mRNA. While unstructured, mismatched target binding domains significantly improved 3' exon replacement (3'ER), 5' exon replacement (5'ER) correlated with the thermodynamic stability of the tsRNA 3' end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA that harbors secondary target binding domains shielding alternative on-target and blinding off-target splicing events. Such rationally designed suicide RNAs efficiently triggered death of HPV-16-transduced or hepatoblastoma-derived human tissue culture cells without evidence for off-target cell killing. Highest cell death activities were observed with novel dual-targeting tsRNAs programmed for trans-splicing toward AFP and a second HCC pre-mRNA biomarker. Our observations suggest trans-splicing represents a promising approach to suicide gene therapy. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Creation of an In vivo cytosensor using engineered mesangial cells. Automatic sensing of glomerular inflammation controls transgene activity.

PubMed

Kitamura, M; Kawachi, H

1997-09-15

Automatic control over exogenous gene expression in response to the activity of disease is a crucial hurdle for gene transfer-based therapies. Towards achieving this goal, we created a "cytosensor" that perceives local inflammatory states and subsequently regulates foreign gene expression. alpha-Smooth muscle actin is known to be expressed in glomerular mesangial cells exclusively in pathologic situations. CArG box element, the crucial regulatory sequence of the alpha-smooth muscle actin promoter, was used as a sensor for glomerular inflammation. Rat mesangial cells were stably transfected with an expression plasmid that introduces a beta-galactosidase gene under the control of CArG box elements. In vitro, the established cells expressed beta-galactosidase exclusively after stimulation with serum. To examine whether the cells are able to automatically control transgene activity in vivo, serum-stimulated or unstimulated cells were transferred into normal rat glomeruli or glomeruli subjected to anti-Thy 1 glomerulonephritis. When stimulated cells were transferred into the normal glomeruli, beta-galactosidase expression was switched off in vivo within 3 d. In contrast, when unstimulated cells were transferred into the nephritic glomeruli, transgene expression was substantially induced. These data indicate the feasibility of using the CArG box element as a molecular sensor for glomerular injury. In the context of advanced forms of gene therapy, this approach provides a novel concept for automatic regulation of local transgene expression where the transgene is required to be activated during inflammation and deactivated when the inflammation has subsided.

Generation of a Hypomorphic Model of Propionic Acidemia Amenable to Gene Therapy Testing

PubMed Central

Guenzel, Adam J; Hofherr, Sean E; Hillestad, Matthew; Barry, Mary; Weaver, Eric; Venezia, Sarah; Kraus, Jan P; Matern, Dietrich; Barry, Michael A

2013-01-01

Propionic acidemia (PA) is a recessive genetic disease that results in an inability to metabolize certain amino acids and odd-chain fatty acids. Current treatment involves restricting consumption of these substrates or liver transplantation. Deletion of the Pcca gene in mice mimics the most severe forms of the human disease. Pcca− mice die within 36 hours of birth, making it difficult to test intravenous systemic therapies in them. We generated an adult hypomorphic model of PA in Pcca− mice using a transgene bearing an A138T mutant of the human PCCA protein. Pcca−/−(A138T) mice have 2% of wild-type PCC activity, survive to adulthood, and have elevations in propionyl-carnitine, methylcitrate, glycine, alanine, lysine, ammonia, and markers associated with cardiomyopathy similar to those in patients with PA. This adult model allowed gene therapy testing by intravenous injection with adenovirus serotype 5 (Ad5) and adeno-associated virus 2/8 (AAV8) vectors. Ad5-mediated more rapid increases in PCCA protein and propionyl-CoA carboxylase (PCC) activity in the liver than AAV8 and both vectors reduced propionylcarnitine and methylcitrate levels. Phenotypic correction was transient with first generation Ad whereas AAV8-mediated long-lasting effects. These data suggest that this PA model may be a useful platform for optimizing systemic intravenous therapies for PA. PMID:23648696

Conserved sequences in the current strains of HIV-1 subtype A in Russia are effectively targeted by artificial RNAi in vitro.

PubMed

Tchurikov, Nickolai A; Fedoseeva, Daria M; Gashnikova, Natalya M; Sosin, Dmitri V; Gorbacheva, Maria A; Alembekov, Ildar R; Chechetkin, Vladimir R; Kravatsky, Yuri V; Kretova, Olga V

2016-05-25

Highly active antiretroviral therapy has greatly reduced the morbidity and mortality of AIDS. However, many of the antiretroviral drugs are toxic with long-term use, and all currently used anti-HIV agents generate drug-resistant mutants. Therefore, there is a great need for new approaches to AIDS therapy. RNAi is a powerful means of inhibiting HIV-1 production in human cells. We propose to use RNAi for gene therapy of HIV/AIDS. Previously we identified a number of new biologically active siRNAs targeting several moderately conserved regions in HIV-1 transcripts. Here we analyze the heterogeneity of nucleotide sequences in three RNAi targets in sequences encoding the reverse transcriptase and integrase domains of current isolates of HIV-1 subtype A in Russia. These data were used to generate genetic constructs expressing short hairpin RNAs 28-30-bp in length that could be processed in cells into siRNAs. After transfection of the constructs we observed siRNAs that efficiently attacked the selected targets. We expect that targeting several viral genes important for HIV-1 reproduction will help overcome the problem of viral adaptation and will prevent the appearance of RNAi escape mutants in current virus strains, an important feature of gene therapy of HIV/AIDS. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of neonatal gene therapy on lumbar spine disease in mucopolysaccharidosis VII dogs

PubMed Central

Smith, Lachlan J; Martin, John T; O'Donnell, Patricia; Wang, Ping; Elliott, Dawn M; Haskins, Mark E; Ponder, Katherine P

2012-01-01

Mucopolysaccharidosis VII (MPS VII) is due to deficient β-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan, and dermatan sulfate glycosaminoglycans in various tissues including those of the spine. Associated spine disease can be due to abnormalities in the vertebrae, the intervertebral discs, or other spine tissues. The goal of this study was to determine if neonatal gene therapy could prevent lumbar spine disease in MPS VII dogs. MPS VII dogs were injected intravenously with a retroviral vector (RV) expressing canine GUSB at 2 to 3 days after birth, which resulted in transduction of hepatocytes that secreted GUSB into blood. Expression was stable for up to 11 years, and mean survival was increased from 0.4 years in untreated dogs to 6.1 years in treated dogs. Despite a profound positive clinical effect, 6-month-old RV-treated MPS VII dogs still had hypoplastic ventral epiphyses with reduced calcification in the lumbar spine, which resulted in a reduced stiffness and increased range of motion that was not improved relative to untreated MPS VII dogs. At six to 11 years of age, ventral vertebrae remained hypoplastic in RV-treated MPS VII dogs, and there was desiccation of the nucleus pulposus in some discs. Histochemical staining demonstrated that discs did not have detectable GUSB activity despite high serum GUSB activity, which is likely due to poor diffusion into this relatively avascular structure. Thus, neonatal gene therapy cannot prevent lumbar spine disease in MPS VII dogs, which predicts that enzyme replacement therapy (ERT) will similarly be relatively ineffective even if started at birth. PMID:22510705

Identification and characterization of HPV-independent cervical cancers.

PubMed

Banister, Carolyn E; Liu, Changlong; Pirisi, Lucia; Creek, Kim E; Buckhaults, Phillip J

2017-02-21

Human papillomavirus (HPV) initiates cervical cancer, and continuous expression of HPV oncogenes E6 and E7 is thought to be necessary to maintain malignant growth. Current therapies target proliferating cells, rather than specific pathways, and most experimental therapies specifically target E6/E7. We investigated the presence and expression of HPV in cervical cancer, to correlate HPV oncogene expression with clinical and molecular features of these tumors that may be relevant to new targeted therapies. While virtually all cervical cancers contained HPV DNA, and most expressed E6/E7 (HPV-active), a subset (8%) of HPV DNA-positive cervical cancers did not express HPV transcripts (HPV-inactive). HPV-inactive tumors occurred in older women (median 54 vs. 45 years, p = 0.02) and were associated with poorer survival (median 715 vs 3046 days, p = 0.0003). Gene expression profiles of HPV-active and -inactive tumors were distinct. HPV-active tumors expressed E2F target genes and increased AKT/MTOR signaling. HPV-inactive tumors had increased WNT/β-catenin and Sonic Hedgehog signaling. Substantial genome-wide differences in DNA methylation were observed. HPV-inactive tumors had a global decrease in DNA methylation; however, many promoter-associated CpGs were hypermethylated. Many inflammatory response genes showed promoter methylation and decreased expression. The somatic mutation landscapes were significantly different. HPV-active tumors carried few somatic mutations in driver genes, whereas HPV-inactive tumors were enriched for non-synonymous somatic mutations (p-value < 0.0000001) specifically targeting TP53, ARID, WNT, and PI3K pathways. The Cancer Genome Atlas (TCGA) cervical cancer data were analyzed. Many of the gene expression changes and somatic mutations found in HPV-inactive tumors alter pathways for which targeted therapeutics are available. Treatment strategies focused on WNT, PI3K, or TP53 mutations may be effective against HPV-inactive tumors and could improve survival for these cervical cancer patients.

Recombination-activating gene 1 (Rag1)-deficient mice with severe combined immunodeficiency treated with lentiviral gene therapy demonstrate autoimmune Omenn-like syndrome.

PubMed

van Til, Niek P; Sarwari, Roya; Visser, Trudi P; Hauer, Julia; Lagresle-Peyrou, Chantal; van der Velden, Guus; Malshetty, Vidyasagar; Cortes, Patricia; Jollet, Arnaud; Danos, Olivier; Cassani, Barbara; Zhang, Fang; Thrasher, Adrian J; Fontana, Elena; Poliani, Pietro L; Cavazzana, Marina; Verstegen, Monique M A; Villa, Anna; Wagemaker, Gerard

2014-04-01

Recombination-activating gene 1 (RAG1) deficiency results in severe combined immunodeficiency (SCID) caused by a complete lack of T and B lymphocytes. If untreated, patients succumb to recurrent infections. We sought to develop lentiviral gene therapy for RAG1-induced SCID and to test its safety. Constructs containing the viral spleen-focus-forming virus (SF), ubiquitous promoters, or cell type-restricted promoters driving sequence-optimized RAG1 were compared for efficacy and safety in sublethally preconditioned Rag1(-/-) mice undergoing transplantation with transduced bone marrow progenitors. Peripheral blood CD3(+) T-cell reconstitution was achieved with SF, ubiquitous promoters, and cell type-restricted promoters but 3- to 18-fold lower than that seen in wild-type mice, and with a compromised CD4(+)/CD8(+) ratio. Mitogen-mediated T-cell responses and T cell-dependent and T cell-independent B-cell responses were not restored, and T-cell receptor patterns were skewed. Reconstitution of mature peripheral blood B cells was approximately 20-fold less for the SF vector than in wild-type mice and often not detectable with the other promoters, and plasma immunoglobulin levels were abnormal. Two months after transplantation, gene therapy-treated mice had rashes with cellular tissue infiltrates, activated peripheral blood CD44(+)CD69(+) T cells, high plasma IgE levels, antibodies against double-stranded DNA, and increased B cell-activating factor levels. Only rather high SF vector copy numbers could boost T- and B-cell reconstitution, but mRNA expression levels during T- and B-cell progenitor stages consistently remained less than wild-type levels. These results underline that further development is required for improved expression to successfully treat patients with RAG1-induced SCID while maintaining low vector copy numbers and minimizing potential risks, including autoimmune reactions resembling Omenn syndrome. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Why commercialization of gene therapy stalled; examining the life cycles of gene therapy technologies.

PubMed

Ledley, F D; McNamee, L M; Uzdil, V; Morgan, I W

2014-02-01

This report examines the commercialization of gene therapy in the context of innovation theories that posit a relationship between the maturation of a technology through its life cycle and prospects for successful product development. We show that the field of gene therapy has matured steadily since the 1980s, with the congruent accumulation of >35 000 papers, >16 000 US patents, >1800 clinical trials and >$4.3 billion in capital investment in gene therapy companies. Gene therapy technologies comprise a series of dissimilar approaches for gene delivery, each of which has introduced a distinct product architecture. Using bibliometric methods, we quantify the maturation of each technology through a characteristic life cycle S-curve, from a Nascent stage, through a Growing stage of exponential advance, toward an Established stage and projected limit. Capital investment in gene therapy is shown to have occurred predominantly in Nascent stage technologies and to be negatively correlated with maturity. Gene therapy technologies are now achieving the level of maturity that innovation research and biotechnology experience suggest may be requisite for efficient product development. Asynchrony between the maturation of gene therapy technologies and capital investment in development-focused business models may have stalled the commercialization of gene therapy.

Gene therapy improves dental manifestations in hypophosphatasia model mice.

PubMed

Okawa, R; Iijima, O; Kishino, M; Okawa, H; Toyosawa, S; Sugano-Tajima, H; Shimada, T; Okada, T; Ozono, K; Ooshima, T; Nakano, K

2017-06-01

Hypophosphatasia is a rare inherited skeletal disorder characterized by defective bone mineralization and deficiency of tissue non-specific alkaline phosphatase (TNSALP) activity. The disease is caused by mutations in the liver/bone/kidney alkaline phosphatase gene (ALPL) encoding TNSALP. Early exfoliation of primary teeth owing to disturbed cementum formation, periodontal ligament weakness and alveolar bone resorption are major complications encountered in oral findings, and discovery of early loss of primary teeth in a dental examination often leads to early diagnosis of hypophosphatasia. Although there are no known fundamental treatments or effective dental approaches to prevent early exfoliation of primary teeth in affected patients, several possible treatments have recently been described, including gene therapy. Gene therapy has also been applied to TNSALP knockout mice (Alpl -/- ), which phenocopy the infantile form of hypophosphatasia, and improved their systemic condition. In the present study, we investigated whether gene therapy improved the dental condition of Alpl -/- mice. Following sublethal irradiation (4 Gy) at the age of 2 d, Alpl -/- mice underwent gene therapy using bone marrow cells transduced with a lentiviral vector expressing a bone-targeted form of TNSALP injected into the jugular vein (n = 3). Wild-type (Alpl +/+ ), heterozygous mice (Alpl +/- ) and Alpl -/- mice were analyzed at 9 d of age (n = 3 of each), while Alpl +/+ mice and treated or untreated Alpl -/- mice were analyzed at 1 mo of age (n = 3 of each), and Alpl +/- mice and Alpl -/- mice with gene therapy were analyzed at 3 mo of age (n = 3 of each). A single mandibular hemi-section obtained at 1 mo of age was analyzed using a small animal computed tomography machine to assess alveolar bone formation. Other mandibular hemi-sections obtained at 9 d, 1 mo and 3 mo of age were subjected to hematoxylin and eosin staining and immunohistochemical analysis of osteopontin, a marker of cementum. Immunohistochemical analysis of osteopontin, a marker of acellular cementum, revealed that Alpl -/- mice displayed impaired formation of cementum and alveolar bone, similar to the human dental phenotype. Cementum formation was clearly present in Alpl -/- mice that underwent gene therapy, but did not recover to the same level as that in wild-type (Alpl +/+ ) mice. Micro-computed tomography examination showed that gene therapy improved alveolar bone mineral density in Alpl -/- mice to a similar level to that in Alpl +/+ mice. Our results suggest that gene therapy can improve the general condition of Alpl -/- mice, and induce significant alveolar bone formation and moderate improvement of cementum formation, which may contribute to inhibition of early spontaneous tooth exfoliation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Rational Design of Iron Oxide Nanoparticles as Targeted Nanomedicines for Cancer Therapy

NASA Astrophysics Data System (ADS)

Kievit, Forrest M.

2011-07-01

Nanotechnology provides a flexible platform for the development of effective therapeutic nanomaterials that can interact specifically with a target in a biological system and provoke a desired biological response. Of the nanomaterials studied, superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as one of top candidates for cancer therapy due to their intrinsic superparamagnetism that enables non-invasive magnetic resonance imaging (MRI) and biodegradability favorable for in vivo application. This dissertation is aimed at development of SPION-based nanomedicines to overcome the current limitations in cancer therapy. These limitations include non-specificity of therapy which can harm healthy tissue, the difficulty in delivering nucleic acids for gene therapy, the formation of drug resistance, and the inability to detect and treat micrometastases. First, a SPION-based non-viral gene delivery vehicle was developed through functionalization of the SPION core with a co-polymer designed to provide stable binding of DNA and low toxicity which showed excellent gene delivery in vitro and in vivo. This SPION-based non-viral gene delivery vehicle was then activated with a targeting agent to improve gene delivery throughout a xenograft tumor of brain cancer. It was found that targeting did not promote the accumulation of SPIONs at the tumor site, but rather improved the distribution of SPIONs throughout the tumor so a higher proportion of cells received treatment. Next, the high surface area of SPIONs was utilized for loading large amounts of drug which was shown to overcome the multidrug resistance acquired by many cancer cells. Drug bound to SPIONs showed significantly higher multidrug resistant cell uptake as compared to free drug which translated into improved cell kill. Also, an antibody activated SPION was developed and was shown to be able to target micrometastases in a transgenic animal model of metastatic breast cancer. These SPION-based nanomedicines provide a platform for the future development of therapies that are hoped to overcome the current limitations in cancer therapy. Finally, a three-dimensional in vitro tumor tissue culture model was developed for mimicking the tumor microenvironment in which cultured cells showed higher malignancy than traditional two-dimensional and three-dimensional models. This in vitro model should provided researches with a better tool for testing novel nanomedicines in vitro before moving to the more costly in vivo experiments.

Intracoronary Cytoprotective Gene Therapy: A Study of VEGF-B167 in a Pre-Clinical Animal Model of Dilated Cardiomyopathy.

PubMed

Woitek, Felix; Zentilin, Lorena; Hoffman, Nicholas E; Powers, Jeffery C; Ottiger, Isabel; Parikh, Suraj; Kulczycki, Anna M; Hurst, Marykathryn; Ring, Nadja; Wang, Tao; Shaikh, Farah; Gross, Polina; Singh, Harinder; Kolpakov, Mikhail A; Linke, Axel; Houser, Steven R; Rizzo, Victor; Sabri, Abdelkarim; Madesh, Muniswamy; Giacca, Mauro; Recchia, Fabio A

2015-07-14

Vascular endothelial growth factor (VEGF)-B activates cytoprotective/antiapoptotic and minimally angiogenic mechanisms via VEGF receptors. Therefore, VEGF-B might be an ideal candidate for the treatment of dilated cardiomyopathy, which displays modest microvascular rarefaction and increased rate of apoptosis. This study evaluated VEGF-B gene therapy in a canine model of tachypacing-induced dilated cardiomyopathy. Chronically instrumented dogs underwent cardiac tachypacing for 28 days. Adeno-associated virus serotype 9 viral vectors carrying VEGF-B167 genes were infused intracoronarily at the beginning of the pacing protocol or during compensated heart failure. Moreover, we tested a novel VEGF-B167 transgene controlled by the atrial natriuretic factor promoter. Compared with control subjects, VEGF-B167 markedly preserved diastolic and contractile function and attenuated ventricular chamber remodeling, halting the progression from compensated to decompensated heart failure. Atrial natriuretic factor-VEGF-B167 expression was low in normally functioning hearts and stimulated by cardiac pacing; it thus functioned as an ideal therapeutic transgene, active only under pathological conditions. Our results, obtained with a standard technique of interventional cardiology in a clinically relevant animal model, support VEGF-B167 gene transfer as an affordable and effective new therapy for nonischemic heart failure. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Gene Therapy for Skin Diseases

PubMed Central

Gorell, Emily; Nguyen, Ngon; Lane, Alfred; Siprashvili, Zurab

2014-01-01

The skin possesses qualities that make it desirable for gene therapy, and studies have focused on gene therapy for multiple cutaneous diseases. Gene therapy uses a vector to introduce genetic material into cells to alter gene expression, negating a pathological process. This can be accomplished with a variety of viral vectors or nonviral administrations. Although results are promising, there are several potential pitfalls that must be addressed to improve the safety profile to make gene therapy widely available clinically. PMID:24692191

Genetically engineered suicide gene in mesenchymal stem cells using a Tet-On system for anaplastic thyroid cancer.

PubMed

Kalimuthu, Senthilkumar; Oh, Ji Min; Gangadaran, Prakash; Zhu, Liya; Lee, Ho Won; Jeon, Yong Hyun; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

2017-01-01

Anaplastic thyroid cancer (ATC) is the most aggressive malignancy of the thyroid, during which undifferentiated tumors arise from the thyroid follicular epithelium. ATC has a very poor prognosis due to its aggressive behavior and poor response to conventional therapies. Gene-directed enzyme/prodrug therapy using genetically engineered mesenchymal stromal cells (MSC) is a promising therapeutic strategy. The doxycycline (DOX)-controlled Tet inducible system is the most widely utilized regulatory system and could be a useful tool for therapeutic gene-based therapies. For example, use a synthetic "tetracycline-on" switch system to control the expression of the therapeutic gene thymidine kinase, which converts prodrugs to active drugs. The aim of this study was to develop therapeutic MSCs, harboring an inducible suicide gene, and to validate therapeutic gene expression using optical molecular imaging of ATC. We designed the Tet-On system using a retroviral vector expressing herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Mouse bone marrow-derived mesenchymal stromal cells (BM-MSC) were transduced using this system with (MSC-Tet-TK/Fluc2) or without (MSC-TK/Fluc) the Tet-On system. Transduced cells were screened and characterized. Engineered MSCs were co-cultured with ATC (CAL62/Rluc) cells in the presence of the prodrug ganciclovir (GCV) and stimulated with DOX. The efficiency of cell killing monitored by assessing Rluc (CAL62/Rluc) and Fluc (MSC-Tet-TK/Fluc and MSC-TK/Fluc) activities using IVIS imaging. Fluc activity increased in MSC-Tet-TK/Fluc cells in a dose dependent manner following DOX treatment (R2 = 0.95), whereas no signal was observed in untreated cells. eGFP could also be visualized after induction with DOX, and the HSV1-TK protein could be detected by western blotting. In MSC-TK/Fluc cells, the Fluc activity increased with increasing cell number (R2 = 0.98), and eGFP could be visualized by fluorescence microscopy. The Fluc activity and cell viability of MSC-Tet-TK/Fluc and MSC-TK/Fluc cells decreased significantly following GCV treatment. A bystander effect of the therapeutic cells confirmed in co-cultures of CAL62 cells, an anaplastic thyroid cancer cell line, with either MSC-Tet-TK/Fluc cells or MSC-TK/Fluc cells. The Rluc activity in MSC-Tet-TK/Fluc co-cultures, derived from the CAL62/Rluc cells, decreased significantly with GCV treatment of DOX treated cultures, whereas no significant changes were observed in untreated cultures. In addition, the Fluc activity of MSC-Tet-TK/Fluc cells also decreased significantly with DOX treatment whereas no signal was present in untreated cultures. A bystander effect also be demonstrated in co-cultures with MSC-TK/Fluc cells and CAL62/Rluc; both the Rluc activity and the Fluc activity were significantly decreased following GCV treatment. We have successfully developed a Tet-On system of gene-directed enzyme/prodrug delivery using MSCs. We confirmed the therapeutic bystander effect in CAL62/Rluc cells with respect to MSC-Tet-TK/Fluc and MSC-TK/Fluc cells after GCV treatment with and without DOX. Our results confirm the therapeutic efficiency of a suicide gene, with or without the Tet-On system, for ATC therapy. In addition, our findings provide an innovative therapeutic approach for using the Tet-On system to eradicate tumors by simple, repeated administration of MSC-Tet-TK/Fluc cells with DOX and GCV.

Gene therapy for carcinoma of the breast

PubMed Central

Stoff-Khalili, MA; Dall, P; Curiel, DT

2007-01-01

In view of the limited success of available treatment modalities for breast cancer, alternative and complementary strategies need to be developed. The delineation of the molecular basis of breast cancer provides the possibility of specific intervention by gene therapy through the introduction of genetic material for therapeutic purposes. In this regard, several gene therapy approaches for carcinoma of the breast have been developed. These approaches can be divided into six broad categories: (1) mutation compensation, (2) molecular chemotherapy, (3) proapoptotic gene therapy, (4) antiangiogenic gene therapy, (5) genetic immunopotentiation, and (6) genetic modulation of resistance/sensitivity. Clinical trials for breast cancer have been initiated to evaluate safety, toxicity, and efficacy. Combined modality therapy with gene therapy and chemotherapy or radiation therapy has shown promising results. It is expected that as new therapeutic targets and approaches are identified and advances in vector design are realized, gene therapy will play an increasing role in clinical breast cancer treatment. PMID:16410823

Nuclear Calcium Signaling Controls Expression of a Large Gene Pool: Identification of a Gene Program for Acquired Neuroprotection Induced by Synaptic Activity

PubMed Central

Zhang, Sheng-Jia; Zou, Ming; Lu, Li; Lau, David; Ditzel, Désirée A. W.; Delucinge-Vivier, Celine; Aso, Yoshinori; Descombes, Patrick; Bading, Hilmar

2009-01-01

Synaptic activity can boost neuroprotection through a mechanism that requires synapse-to-nucleus communication and calcium signals in the cell nucleus. Here we show that in hippocampal neurons nuclear calcium is one of the most potent signals in neuronal gene expression. The induction or repression of 185 neuronal activity-regulated genes is dependent upon nuclear calcium signaling. The nuclear calcium-regulated gene pool contains a genomic program that mediates synaptic activity-induced, acquired neuroprotection. The core set of neuroprotective genes consists of 9 principal components, termed Activity-regulated Inhibitor of Death (AID) genes, and includes Atf3, Btg2, GADD45β, GADD45γ, Inhibin β-A, Interferon activated gene 202B, Npas4, Nr4a1, and Serpinb2, which strongly promote survival of cultured hippocampal neurons. Several AID genes provide neuroprotection through a common process that renders mitochondria more resistant to cellular stress and toxic insults. Stereotaxic delivery of AID gene-expressing recombinant adeno-associated viruses to the hippocampus confers protection in vivo against seizure-induced brain damage. Thus, treatments that enhance nuclear calcium signaling or supplement AID genes represent novel therapies to combat neurodegenerative conditions and neuronal cell loss caused by synaptic dysfunction, which may be accompanied by a deregulation of calcium signal initiation and/or propagation to the cell nucleus. PMID:19680447

Improved animal models for testing gene therapy for atherosclerosis.

PubMed

Du, Liang; Zhang, Jingwan; De Meyer, Guido R Y; Flynn, Rowan; Dichek, David A

2014-04-01

Gene therapy delivered to the blood vessel wall could augment current therapies for atherosclerosis, including systemic drug therapy and stenting. However, identification of clinically useful vectors and effective therapeutic transgenes remains at the preclinical stage. Identification of effective vectors and transgenes would be accelerated by availability of animal models that allow practical and expeditious testing of vessel-wall-directed gene therapy. Such models would include humanlike lesions that develop rapidly in vessels that are amenable to efficient gene delivery. Moreover, because human atherosclerosis develops in normal vessels, gene therapy that prevents atherosclerosis is most logically tested in relatively normal arteries. Similarly, gene therapy that causes atherosclerosis regression requires gene delivery to an existing lesion. Here we report development of three new rabbit models for testing vessel-wall-directed gene therapy that either prevents or reverses atherosclerosis. Carotid artery intimal lesions in these new models develop within 2-7 months after initiation of a high-fat diet and are 20-80 times larger than lesions in a model we described previously. Individual models allow generation of lesions that are relatively rich in either macrophages or smooth muscle cells, permitting testing of gene therapy strategies targeted at either cell type. Two of the models include gene delivery to essentially normal arteries and will be useful for identifying strategies that prevent lesion development. The third model generates lesions rapidly in vector-naïve animals and can be used for testing gene therapy that promotes lesion regression. These models are optimized for testing helper-dependent adenovirus (HDAd)-mediated gene therapy; however, they could be easily adapted for testing of other vectors or of different types of molecular therapies, delivered directly to the blood vessel wall. Our data also supports the promise of HDAd to deliver long-term therapy from vascular endothelium without accelerating atherosclerotic disease.

CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.

PubMed

Wrona, Dominik; Siler, Ulrich; Reichenbach, Janine

2017-03-13

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47 phox -deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47 phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47 phox -subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47 phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47 phox -deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Gene therapy for prostate cancer: where are we now?

PubMed

Steiner, M S; Gingrich, J R

2000-10-01

The ability to recombine specifically and alter DNA sequences followed by techniques to transfer these sequences or even whole genes into normal and diseased cells has revolutionized medical research and ushered the clinicians of today into the age of gene therapy. We provide urologists a review of relevant background information, outline current treatment strategies and clinical trials, and delineate current challenges facing the field of gene therapy for advanced prostate cancer. We comprehensively reviewed the literature, including PubMed and recent abstract proceedings from national meetings, relevant to gene therapy and advanced prostate cancer. We selected for review literature representative of the principal scientific background for current gene therapy strategies and National Institutes of Health Recombinant DNA Advisory Committee approved clinical trials. Current prostate cancer gene therapy strategies include correcting aberrant gene expression, exploiting programmed cell death pathways, targeting critical cell biological functions, introducing toxic or cell lytic suicide genes, enhancing the immune system antitumor response and combining treatment with conventional cytotoxic chemotherapy or radiation therapy. Many challenges lie ahead for gene therapy, including improving DNA transfer efficiency to cells locally and at distant sites, enhancing levels of gene expression and overcoming immune responses that limit the time that genes are expressed. Nevertheless, despite these current challenges it is almost certain that gene therapy will be part of the urological armamentarium against prostate cancer in this century.

Gene Therapy Approaches For The Treatment Of Retinal Disorders

PubMed Central

Petit, Lolita; Punzo, Claudio

2016-01-01

There is an impelling need to develop effective therapeutic strategies for patients with retinal disorders. Gleaning from the large quantity of information gathered over the past two decades on the mechanisms governing degeneration of the retina, it is now possible to devise innovative therapies based on retinal gene transfer. Different gene-based approaches are under active investigation. They include strategies to correct the specific genetic defect in inherited retinal diseases, strategies to delay the onset of blindness independently of the disease-causing mutations and strategies to reactivate residual cells at late stages of the diseases. In this review, we discuss the status of application of these technologies, outlining the future therapeutic potential for many forms of retinal blinding diseases. PMID:27875674

Stem cell gene therapy for fanconi anemia: report from the 1st international Fanconi anemia gene therapy working group meeting.

PubMed

Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

2011-07-01

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA.

Seamless correction of the sickle cell disease mutation of the HBB gene in human induced pluripotent stem cells using TALENs.

PubMed

Sun, Ning; Zhao, Huimin

2014-05-01

Sickle cell disease (SCD) is the most common human genetic disease which is caused by a single mutation of human β-globin (HBB) gene. The lack of long-term treatment makes the development of reliable cell and gene therapies highly desirable. Disease-specific patient-derived human induced pluripotent stem cells (hiPSCs) have great potential for developing novel cell and gene therapies. With the disease-causing mutations corrected in situ, patient-derived hiPSCs can restore normal cell functions and serve as a renewable autologous cell source for the treatment of genetic disorders. Here we successfully utilized transcription activator-like effector nucleases (TALENs), a recently emerged novel genome editing tool, to correct the SCD mutation in patient-derived hiPSCs. The TALENs we have engineered are highly specific and generate minimal off-target effects. In combination with piggyBac transposon, TALEN-mediated gene targeting leaves no residual ectopic sequences at the site of correction and the corrected hiPSCs retain full pluripotency and a normal karyotype. Our study demonstrates an important first step of using TALENs for the treatment of genetic diseases such as SCD, which represents a significant advance toward hiPSC-based cell and gene therapies. © 2013 Wiley Periodicals, Inc.

HIV-derived vectors for gene therapy targeting dendritic cells.

PubMed

Rossetti, Maura; Cavarelli, Mariangela; Gregori, Silvia; Scarlatti, Gabriella

2013-01-01

Human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) have the potential to mediate stable therapeutic gene transfer. However, similarly to other viral vectors, their benefit is compromised by the induction of an immune response toward transgene-expressing cells that closely mimics antiviral immunity. LV share with the parental HIV the ability to activate dendritic cells (DC), while lack the peculiar ability of subverting DC functions, which is responsible for HIV immune escape. Understanding the interaction between LV and DC, with plasmacytoid and myeloid DC playing fundamental and distinct roles, has paved the way to novel approaches aimed at regulating transgene-specific immune responses. Thanks to the ability to target either DC subsets LV might be a powerful tool to induce immunity (i.e., gene therapy of cancer), cell death (i.e., in HIV/AIDS infection), or tolerance (i.e., gene therapy strategies for monogenic diseases). In this chapter, similarities and differences between the LV-mediated and HIV-mediated induction of immune responses, with specific focus on their interactions with DC, are discussed.

Gene therapy for ocular diseases meditated by ultrasound and microbubbles (Review)

PubMed Central

WAN, CAIFENG; LI, FENGHUA; LI, HONGLI

2015-01-01

The eye is an ideal target organ for gene therapy as it is easily accessible and immune-privileged. With the increasing insight into the underlying molecular mechanisms of ocular diseases, gene therapy has been proposed as an effective approach. Successful gene therapy depends on efficient gene transfer to targeted cells to prove stable and prolonged gene expression with minimal toxicity. At present, the main hindrance regarding the clinical application of gene therapy is not the lack of an ideal gene, but rather the lack of a safe and efficient method to selectively deliver genes to target cells and tissues. Ultrasound-targeted microbubble destruction (UTMD), with the advantages of high safety, repetitive applicability and tissue targeting, has become a potential strategy for gene- and drug delivery. When gene-loaded microbubbles are injected, UTMD is able to enhance the transport of the gene to the targeted cells. High-amplitude oscillations of microbubbles act as cavitation nuclei which can effectively focus ultrasound energy, produce oscillations and disruptions that increase the permeability of the cell membrane and create transient pores in the cell membrane. Thereby, the efficiency of gene therapy can be significantly improved. The UTMD-mediated gene delivery system has been widely used in pre-clinical studies to enhance gene expression in a site-specific manner in a variety of organs. With reasonable application, the effects of sonoporation can be spatially and temporally controlled to improve localized tissue deposition of gene complexes for ocular gene therapy applications. In addition, appropriately powered, focused ultrasound combined with microbubbles can induce a reversible disruption of the blood-retinal barrier with no significant side effects. The present review discusses the current status of gene therapy of ocular diseases as well as studies on gene therapy of ocular diseases meditated by UTMD. PMID:26151686

Activation of mTor Signaling by Gene Transduction to Induce Axon Regeneration in the Central Nervous System Following Neural Injury

DTIC Science & Technology

2015-03-01

terminus amino acids of amyloid precursor protein (cAPP). cAPP was found in our recent publication in Gene Therapy (2013) to be the most effective...Therapy. 2012;20:275-86 PubMed PMID: 22008911. 7. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing...NatRevMolCell Biol. 2011;12:21-35. 8. Morita T, Sobue K. Specification of neuronal polarity regulated by local translation of CRMP2 and Tau via the mTOR

Hypoxia-inducible tumour-specific promoters as a dual-targeting transcriptional regulation system for cancer gene therapy

PubMed Central

Javan, Bita; Shahbazi, Majid

2017-01-01

Transcriptional targeting is the best approach for specific gene therapy. Hypoxia is a common feature of the tumour microenvironment. Therefore, targeting gene expression in hypoxic cells by placing transgene under the control of a hypoxia-responsive promoter can be a good strategy for cancer-specific gene therapy. The hypoxia-inducible gene expression system has been investigated more in suicide gene therapy and it can also be of great help in knocking down cancer gene therapy with siRNAs. However, this system needs to be optimised to have maximum efficacy with minimum side effects in normal tissues. The combination of tissue-/tumour-specific promoters with HRE core sequences has been found to enhance the specificity and efficacy of this system. In this review, hypoxia-inducible gene expression system as well as gene therapy strategies targeting tumour hypoxia will be discussed. This review will also focus on hypoxia-inducible tumour-specific promoters as a dual-targeting transcriptional regulation systems developed for cancer-specific gene therapy. PMID:28798809

The nucleolus: an emerging target for cancer therapy.

PubMed

Hein, Nadine; Hannan, Katherine M; George, Amee J; Sanij, Elaine; Hannan, Ross D

2013-11-01

For over 100 years, pathologists have utilised an increase in size and number of nucleoli, the subnuclear site of ribosome synthesis, as a marker of aggressive tumours. Despite this, the contribution of the nucleolus and ribosomal RNA synthesis to cancer has been largely overlooked. This concept has recently changed with the demonstration that the nucleolus indirectly controls numerous other cellular functions, in particular, the cellular activity of the critical tumour suppressor protein, p53. Moreover, selective inhibition of ribosomal gene transcription in the nucleolus has been shown to be an effective therapeutic strategy to promote cancer-specific activation of p53. This article reviews the largely untapped potential of the nucleolus and ribosomal gene transcription as exciting new targets for cancer therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Influence of Cytochrome P450 Pharmacogenetics on Disposition of Common Antidepressant and Antipsychotic Medications

PubMed Central

van der Weide, Jan; Hinrichs, John WJ

2006-01-01

Since the identification of all the major drug-metabolising cytochrome P450 (CYP) enzymes and their major gene variants, pharmacogenetics has had a major impact on psychotherapeutic drug therapy. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is an important reason for drug therapy failure. Variability in CYP activity may be caused by various factors, including endogenous factors such as age, gender and morbidity as well as exogenous factors such as co-medication, food components and smoking habit. However, polymorphisms, present in most CYP genes, are responsible for a substantial part of this variability. Although CYP genotyping has been shown to predict the majority of aberrant phenotypes, it is currently rarely performed in clinical practice. PMID:16886044

A lentiviral vector with expression controlled by E2F-1: A potential tool for the study and treatment of proliferative diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, Bryan E.; Patricio, Juliana Rotelli; Program in Biotechnology, University of Sao Paulo

2006-10-06

We have constructed a lentiviral vector with expression limited to cells presenting active E2F-1 protein, a potential advantage for gene therapy of proliferative diseases. For the FE2FLW vector, the promoter region of the human E2F-1 gene was utilized to drive expression of luciferase cDNA, included as a reporter of viral expression. Primary, immortalized, and transformed cells were transduced with the FE2FLW vector and cell cycle alterations were induced with serum starvation/replacement, contact inhibition or drug treatment, revealing cell cycle-dependent changes in reporter activity. Forced E2F-1 expression, but not E2F-2 or E2F-3, increased reporter activity, indicating a major role for thismore » factor in controlling expression from the FE2FLW virus. We show the utility of this vector as a reporter of E2F-1 and proliferation-dependent cellular alterations upon cytotoxic/cytostatic treatment, such as the introduction of tumor suppressor genes. We propose that the FE2FLW vector may be a starting point for the development of gene therapy strategies for proliferative diseases, such as cancer or restinosis.« less

Republished review: Gene therapy for ocular diseases.

PubMed

Liu, Melissa M; Tuo, Jingsheng; Chan, Chi-Chao

2011-07-01

The eye is an easily accessible, highly compartmentalised and immune-privileged organ that offers unique advantages as a gene therapy target. Significant advancements have been made in understanding the genetic pathogenesis of ocular diseases, and gene replacement and gene silencing have been implicated as potentially efficacious therapies. Recent improvements have been made in the safety and specificity of vector-based ocular gene transfer methods. Proof-of-concept for vector-based gene therapies has also been established in several experimental models of human ocular diseases. After nearly two decades of ocular gene therapy research, preliminary successes are now being reported in phase 1 clinical trials for the treatment of Leber congenital amaurosis. This review describes current developments and future prospects for ocular gene therapy. Novel methods are being developed to enhance the performance and regulation of recombinant adeno-associated virus- and lentivirus-mediated ocular gene transfer. Gene therapy prospects have advanced for a variety of retinal disorders, including retinitis pigmentosa, retinoschisis, Stargardt disease and age-related macular degeneration. Advances have also been made using experimental models for non-retinal diseases, such as uveitis and glaucoma. These methodological advancements are critical for the implementation of additional gene-based therapies for human ocular diseases in the near future.

Gene therapy for ocular diseases.

PubMed

Liu, Melissa M; Tuo, Jingsheng; Chan, Chi-Chao

2011-05-01

The eye is an easily accessible, highly compartmentalised and immune-privileged organ that offers unique advantages as a gene therapy target. Significant advancements have been made in understanding the genetic pathogenesis of ocular diseases, and gene replacement and gene silencing have been implicated as potentially efficacious therapies. Recent improvements have been made in the safety and specificity of vector-based ocular gene transfer methods. Proof-of-concept for vector-based gene therapies has also been established in several experimental models of human ocular diseases. After nearly two decades of ocular gene therapy research, preliminary successes are now being reported in phase 1 clinical trials for the treatment of Leber congenital amaurosis. This review describes current developments and future prospects for ocular gene therapy. Novel methods are being developed to enhance the performance and regulation of recombinant adeno-associated virus- and lentivirus-mediated ocular gene transfer. Gene therapy prospects have advanced for a variety of retinal disorders, including retinitis pigmentosa, retinoschisis, Stargardt disease and age-related macular degeneration. Advances have also been made using experimental models for non-retinal diseases, such as uveitis and glaucoma. These methodological advancements are critical for the implementation of additional gene-based therapies for human ocular diseases in the near future.

Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer *

PubMed Central

Lopes, Gabriel Lima; Vattimo, Edoardo Filippo de Queiroz; de Castro, Gilberto

2015-01-01

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide. Promising new therapies have recently emerged from the development of molecular targeted drugs; particularly promising are those blocking the signal transduction machinery of cancer cells. One of the most widely studied cell signaling pathways is that of EGFR, which leads to uncontrolled cell proliferation, increased cell angiogenesis, and greater cell invasiveness. Activating mutations in the EGFR gene (deletions in exon 19 and mutation L858R in exon 21), first described in 2004, have been detected in approximately 10% of all non-squamous non-small cell lung cancer (NSCLC) patients in Western countries and are the most important predictors of a response to EGFR tyrosine-kinase inhibitors (EGFR-TKIs). Studies of the EGFR-TKIs gefitinib, erlotinib, and afatinib, in comparison with platinum-based regimens, as first-line treatments in chemotherapy-naïve patients have shown that the EGFR-TKIs produce gains in progression-free survival and overall response rates, although only in patients whose tumors harbor activating mutations in the EGFR gene. Clinical trials have also shown EGFR-TKIs to be effective as second- and third-line therapies in advanced NSCLC. Here, we review the main aspects of EGFR pathway activation in NSCLC, underscore the importance of correctly identifying activating mutations in the EGFR gene, and discuss the main outcomes of EGFR-TKI treatment in NSCLC. PMID:26398757

Evolving Complexity in Hemophilia Management.

PubMed

Croteau, Stacy E

2018-06-01

Rapid expansion of therapeutic options have increased the complexity of hemophilia care. Previously, on-demand therapy aimed to reduce morbidity and early mortality; however, now aggressive prophylaxis, particularly in children, encourages an active lifestyle. Accurate diagnosis, recognition of early threats to musculoskeletal health, and optimization of therapy are critical for both males and females affected by hemophilia. The diversity of emerging hemophilia therapies, from modified factor protein concentrates, to gene therapy, to nonfactor hemostatic strategies, provide an exciting opportunity to target unmet needs in the bleeding disorder community. Copyright © 2018 Elsevier Inc. All rights reserved.

Repetitive Transcranial Magnetic Stimulation Activates Specific Regions in Rat Brain

NASA Astrophysics Data System (ADS)

Ji, Ru-Rong; Schlaepfer, Thomas E.; Aizenman, Carlos D.; Epstein, Charles M.; Qiu, Dike; Huang, Justin C.; Rupp, Fabio

1998-12-01

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive technique to induce electric currents in the brain. Although rTMS is being evaluated as a possible alternative to electroconvulsive therapy for the treatment of refractory depression, little is known about the pattern of activation induced in the brain by rTMS. We have compared immediate early gene expression in rat brain after rTMS and electroconvulsive stimulation, a well-established animal model for electroconvulsive therapy. Our result shows that rTMS applied in conditions effective in animal models of depression induces different patterns of immediate-early gene expression than does electroconvulsive stimulation. In particular, rTMS evokes strong neural responses in the paraventricular nucleus of the thalamus (PVT) and in other regions involved in the regulation of circadian rhythms. The response in PVT is independent of the orientation of the stimulation probe relative to the head. Part of this response is likely because of direct activation, as repetitive magnetic stimulation also activates PVT neurons in brain slices.

Co-Targeting Prostate Cancer Epithelium and Bone Stroma by Human Osteonectin-Promoter-Mediated Suicide Gene Therapy Effectively Inhibits Androgen-Independent Prostate Cancer Growth.

PubMed

Sung, Shian-Ying; Chang, Junn-Liang; Chen, Kuan-Chou; Yeh, Shauh-Der; Liu, Yun-Ru; Su, Yen-Hao; Hsueh, Chia-Yen; Chung, Leland W K; Hsieh, Chia-Ling

2016-01-01

Stromal-epithelial interaction has been shown to promote local tumor growth and distant metastasis. We sought to create a promising gene therapy approach that co-targets cancer and its supporting stromal cells for combating castration-resistant prostate tumors. Herein, we demonstrated that human osteonectin is overexpressed in the prostate cancer epithelium and tumor stroma in comparison with their normal counterpart. We designed a novel human osteonectin promoter (hON-522E) containing positive transcriptional regulatory elements identified in both the promoter and exon 1 region of the human osteonectin gene. In vitro reporter assays revealed that the hON-522E promoter is highly active in androgen receptor negative and metastatic prostate cancer and bone stromal cells compared to androgen receptor-positive prostate cancer cells. Moreover, in vivo prostate-tumor-promoting activity of the hON-522E promoter was confirmed by intravenous administration of an adenoviral vector containing the hON-522E promoter-driven luciferase gene (Ad-522E-Luc) into mice bearing orthotopic human prostate tumor xenografts. In addition, an adenoviral vector with the hON-522E-promoter-driven herpes simplex virus thymidine kinase gene (Ad-522E-TK) was highly effective against the growth of androgen-independent human prostate cancer PC3M and bone stromal cell line in vitro and in pre-established PC3M tumors in vivo upon addition of the prodrug ganciclovir. Because of the heterogeneity of human prostate tumors, hON-522E promoter-mediated gene therapy has the potential for the treatment of hormone refractory and bone metastatic prostate cancers.

Oligonucleotide-based therapy for neurodegenerative diseases.

PubMed

Magen, Iddo; Hornstein, Eran

2014-10-10

Molecular genetics insight into the pathogenesis of several neurodegenerative diseases, such as Alzheimer׳s disease, Parkinson׳s disease, Huntington׳s disease and amyotrophic lateral sclerosis, encourages direct interference with the activity of neurotoxic genes or the molecular activation of neuroprotective pathways. Oligonucleotide-based therapies are recently emerging as an efficient strategy for drug development and these can be employed as new treatments of neurodegenerative states. Here we review advances in this field in recent years which suggest an encouraging assessment that oligonucleotide technologies for targeting of RNAs will enable the development of new therapies and will contribute to preservation of brain integrity. Copyright © 2014 Elsevier B.V. All rights reserved.

Current Status and Prospects of Gene Therapy for the Inner Ear

PubMed Central

Huang, Aji

2011-01-01

Abstract Inner ear diseases are common and often result in hearing disability. Sensorineural hearing loss is the main cause of hearing disability. So far, no effective treatment is available although some patients may benefit from a hearing aid equipped with a hearing amplifier or from cochlear implantation. Inner ear gene therapy has become an emerging field of study for the treatment of hearing disability. Numerous new discoveries and tremendous advances have been made in inner ear gene therapy including gene vectors, routes of administration, and therapeutic genes and targets. Gene therapy may become a treatment option for inner ear diseases in the near future. In this review, we summarize the current state of inner ear gene therapy including gene vectors, delivery routes, and therapeutic genes and targets by examining and analyzing publications on inner ear gene therapy from the literature and patent documents, and identify promising patents, novel techniques, and vital research projects. We also discuss the progress and prospects of inner ear gene therapy, the advances and shortcomings, with possible solutions in this field of research. PMID:21338273

[New possibilities will open up in human gene therapy].

PubMed

Portin, Petter

2016-01-01

Gene therapy is divided into somatic and germ line therapy. The latter involves reproductive cells or their stem cells, and its results are heritable. The effects of somatic gene therapy are generally restricted to a single tissue of the patient in question. Until now, all gene therapies in the world have belonged to the regime of somatic therapy, germ line therapy having been a theoretical possibility only. Very recently, however, a method has been developed which is applicable to germ line therapy as well. In addition to technical challenges, severe ethical problems are associated with germ line therapy, demanding opinion statement.

75 FR 66381 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-28

...] Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting AGENCY: Food and Drug...: Cellular, Tissue and Gene Therapies Advisory Committee. General Function of the Committee: To provide... Competent Retrovirus (RCR)/Lentivirus (RCL) in Retroviral and Lentiviral Vector Based Gene Therapy Products...

[Collaborative study on regulatory science for facilitating clinical development of gene therapy products for genetic diseases].

PubMed

Uchida, Eriko; Igarashi, Yuka; Sato, Yoji

2014-01-01

Gene therapy products are expected as innovative medicinal products for intractable diseases such as life-threatening genetic diseases and cancer. Recently, clinical developments by pharmaceutical companies are accelerated in Europe and the United States, and the first gene therapy product in advanced countries was approved for marketing authorization by the European Commission in 2012. On the other hand, more than 40 clinical studies for gene therapy have been completed or ongoing in Japan, most of them are conducted as clinical researches by academic institutes, and few clinical trials have been conducted for approval of gene therapy products. In order to promote the development of gene therapy products, revision of the current guideline and/or preparation of concept paper to address the evaluation of the quality and safety of gene therapy products are necessary and desired to clearly show what data should be submitted before First-in-Human clinical trials of novel gene therapy products. We started collaborative study with academia and regulatory agency to promote regulatory science toward clinical development of gene therapy products for genetic diseases based on lentivirus and adeno-associated virus vectors; National Center for Child Health and Development (NCCHD), Nippon Medical School and PMDA have been joined in the task force. At first, we are preparing pre-draft of the revision of the current gene therapy guidelines in this project.

Gene therapy for haemophilia.

PubMed

Sharma, Akshay; Easow Mathew, Manu; Sriganesh, Vasumathi; Neely, Jessica A; Kalipatnapu, Sasank

2014-11-14

Haemophilia is a genetic disorder which is characterized by spontaneous or provoked, often uncontrolled, bleeding into joints, muscles and other soft tissues. Current methods of treatment are expensive, challenging and involve regular administration of clotting factors. Gene therapy has recently been prompted as a curative treatment modality. To evaluate the safety and efficacy of gene therapy for treating people with haemophilia A or B. We searched the Cochrane Cystic Fibrosis & Genetic Disorders Group's Coagulopathies Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched the reference lists of relevant articles and reviews.Date of last search: 06 November 2014. Eligible trials included randomised or quasi-randomised clinical trials, including controlled clinical trials comparing gene therapy (with or without standard treatment) with standard treatment (factor replacement) or other 'curative' treatment such as stem cell transplantation individuals with haemophilia A or B of all ages who do not have inhibitors to factor VIII or IX. No trials of gene therapy for haemophilia were found. No trials of gene therapy for haemophilia were identified. No randomised or quasi-randomised clinical trials of gene therapy for haemophilia were identified. Thus, we are unable to determine the effects of gene therapy for haemophilia. Gene therapy for haemophilia is still in its nascent stages and there is a need for well-designed clinical trials to assess the long-term feasibility, success and risks of gene therapy for people with haemophilia.

Development of Gene Therapeutics for Head and Neck Cancer in China: From Bench to Bedside.

PubMed

Guo, Wei; Song, Hao

2018-02-01

Head and neck cancer represents the seventh most common cancer worldwide. Although multidisciplinary sequential treatments have been used, there is still an urgent need for new treatment approaches that can effectively improve the outcomes of patients with advanced stages of head and neck cancer. Gene therapy is a rapidly evolving field in cancer therapy that has been shown to improve the efficacy of antitumor treatment. China is at the forefront in clinical trials and practice of gene therapy. Chinese researchers have mainly focused on gene therapeutics based on oncolytic virus and recombinant adenovirus expressing p53, antiangiogenesis factor or herpes simplex virus-thymidine kinase. Currently, two gene therapy drugs, Gendicine and Oncorine, have been marketed in China, and a number of upcoming gene therapy agents are under development for the treatment of head and neck cancer. Most gene therapy agents have demonstrated excellent tolerance. However, the therapeutic effects need further improvement. With current innovations in tumor biology and knowledge, gene therapy has great potential as a safe and effective anticancer treatment. In recent years, new gene therapy agents with promising effects have been incorporated into clinical trials in China. Thus, gene therapy may become an important part of anticancer therapy and is expected to improve the therapeutic effect of head and neck cancers in the near future.

Stem Cell Gene Therapy for Fanconi Anemia: Report from the 1st International Fanconi Anemia Gene Therapy Working Group Meeting

PubMed Central

Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

2011-01-01

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA. PMID:21540837

Biological Gene Delivery Vehicles: Beyond Viral Vectors

PubMed Central

Seow, Yiqi; Wood, Matthew J

2009-01-01

Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or acquired diseases such as cancer, to vaccination, each with different requirements for gene delivery. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immunological features, which restrict gene therapy applications and hold back the potential for preventive gene therapy. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents which include bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts, and exosomes. Exploiting the natural properties of these biological entities for specific gene delivery applications will expand the repertoire of gene therapy vectors available for clinical use. Here, we review the prospects for nonviral biological delivery vehicles as gene therapy agents with focus on their unique evolved biological properties and respective limitations and potential applications. The potential of these nonviral biological entities to act as clinical gene therapy delivery vehicles has already been shown in clinical trials using bacteria-mediated gene transfer and with sufficient development, these entities will complement the established delivery techniques for gene therapy applications. PMID:19277019

Biological gene delivery vehicles: beyond viral vectors.

PubMed

Seow, Yiqi; Wood, Matthew J

2009-05-01

Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or acquired diseases such as cancer, to vaccination, each with different requirements for gene delivery. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immunological features, which restrict gene therapy applications and hold back the potential for preventive gene therapy. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents which include bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts, and exosomes. Exploiting the natural properties of these biological entities for specific gene delivery applications will expand the repertoire of gene therapy vectors available for clinical use. Here, we review the prospects for nonviral biological delivery vehicles as gene therapy agents with focus on their unique evolved biological properties and respective limitations and potential applications. The potential of these nonviral biological entities to act as clinical gene therapy delivery vehicles has already been shown in clinical trials using bacteria-mediated gene transfer and with sufficient development, these entities will complement the established delivery techniques for gene therapy applications.

cDNA microarray analysis of human keratinocytes cells of patients submitted to chemoradiotherapy and oral photobiomodulation therapy: pilot study.

PubMed

Antunes, Heliton S; Wajnberg, Gabriel; Pinho, Marcos B; Jorge, Natasha Andressa Nogueira; de Moraes, Joyce Luana Melo; Stefanoff, Claudio Gustavo; Herchenhorn, Daniel; Araújo, Carlos M M; Viégas, Celia Maria Pais; Rampini, Mariana P; Dias, Fernando L; de Araujo-Souza, Patricia Savio; Passetti, Fabio; Ferreira, Carlos G

2018-01-01

Oral mucositis is an acute toxicity that occurs in patients submitted to chemoradiotherapy to treat head and neck squamous cell carcinoma. In this study, we evaluated differences in gene expression in the keratinocytes of the oral mucosa of patients treated with photobiomodulation therapy and tried to associate the molecular mechanisms with clinical findings. From June 2009 to December 2010, 27 patients were included in a randomized double-blind pilot study. Buccal smears from 13 patients were obtained at days 1 and 10 of chemoradiotherapy, and overall gene expression of samples from both dates were analyzed by complementary DNA (cDNA) microarray. In addition, samples from other 14 patients were also collected at D1 and D10 of chemoradiotherapy for subsequent validation of cDNA microarray findings by qPCR. The expression array analysis identified 105 upregulated and 60 downregulated genes in our post-treatment samples when compared with controls. Among the upregulated genes with the highest fold change, it was interesting to observe the presence of genes related to keratinocyte differentiation. Among downregulated genes were observed genes related to cytotoxicity and immune response. The results indicate that genes known to be induced during differentiation of human epidermal keratinocytes were upregulated while genes associated with cytotoxicity and immune response were downregulated in the laser group. These results support previous clinical findings indicating that the lower incidence of oral mucositis associated with photobiomodulation therapy might be correlated to the activation of genes involved in keratinocyte differentiation.

75 FR 65640 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-26

...] Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting AGENCY: Food and Drug... closed to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee. General... Branch, Office of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and Research, FDA...

76 FR 22405 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2011-04-21

...] Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting AGENCY: Food and Drug...: Cellular, Tissue and Gene Therapies Advisory Committee. General Function of the Committee: To provide... June 29, 2011, the committee will discuss cellular and gene therapy products for the treatment of...

76 FR 81513 - Cellular, Tissue, and Gene Therapies Advisory Committee; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-28

...] Cellular, Tissue, and Gene Therapies Advisory Committee; Notice of Meeting AGENCY: Food and Drug... meeting will be closed to the public. Name of Committee: Cellular, Tissue, and Gene Therapies Advisory... programs in the Cellular and Tissue Branch, Office of Cellular, Tissue and Gene Therapies, Center for...

[Non-viral gene therapy approach for regenerative recovery of skin wounds in mammals].

PubMed

Efremov, A M; Dukhovlinov, I V; Dizhe, E B; Burov, S V; Leko, M V; Akif'ev, B N; Mogilenko, D A; Ivanov, I A; Perevozchikov, A P; Orlov, S V

2010-01-01

The rate and character of skin tissue regeneration after wounds, burns and other traumas depend on the cell proliferation within damaged area. Acceleration of healing by stimulation of cell proliferation and extracellular matrix synthesis is one of the most important tasks of modern medicine. There are gene therapy approaches to wound treatment consisting in the transfer of genes encoding mitogenic growth factors to wound area. The most important step in the development of gene therapy approaches is the design of gene delivery tools. In spite of high efficacy of viral vectors, the non-viral means have some preferences (low toxicity, low immunogenity, safety and the absence of backside effects). Among non-viral gene delivery tools, molecular conjugates are the most popular because of their efficacy, simplicity, and the capacity to the targeted gene transfer. In the present work we have developed two molecular conjugates--NLS-TSF7 and NLS-TSF12 consisting of the modified signal of nuclear localization of T-antigen of SV40 virus (cationic part) and the peptide ligands of mammalian transferrin receptor (ligand part). These conjugates bind to plasmid DNA with formation of polyelectrolytic complexes and are capable to deliver plasmid DNA into cells expressing transferrin receptors by receptor-mediated endocytosis. Transfer of the expression vector of luciferase gene in the complex with molecular conjugate NLS-TSF7 to murine surface tissues led to about 100 fold increasing of luciferase activity in comparison with the transfer of free expression vector. Treatment of slash wounds in mice with the complexes of expression vector of synthetic human gene encoding insulin-like growth factor 1 with molecular conjugates NLS-TSF7 led to acceleration of healing in comparison with mice treated with free expression vector. The results obtained confirm the high efficiency of the developed regenerative gene therapy approach for the treatment of damaged skin tissues in mammals.

American Society of Gene & Cell Therapy

MedlinePlus

... Learn More Close The American Society of Gene & Cell Therapy ASGCT is the primary membership organization for ... Official Journal of the American Society of Gene & Cell Therapy Molecular Therapy is the leading journal for ...

RPE65 gene therapy slows cone loss in Rpe65-deficient dogs.

PubMed

Mowat, F M; Breuwer, A R; Bartoe, J T; Annear, M J; Zhang, Z; Smith, A J; Bainbridge, J W B; Petersen-Jones, S M; Ali, R R

2013-05-01

Recent clinical trials of retinal pigment epithelium gene (RPE65) supplementation therapy in Leber congenital amaurosis type 2 patients have demonstrated improvements in rod and cone function, but it may be some years before the effects of therapy on photoreceptor survival become apparent. The Rpe65-deficient dog is a very useful pre-clinical model in which to test efficacy of therapies, because the dog has a retina with a high degree of similarity to that of humans. In this study, we evaluated the effect of RPE65 gene therapy on photoreceptor survival in order to predict the potential benefit and limitations of therapy in patients. We examined the retinas of Rpe65-deficient dogs after RPE65 gene therapy to evaluate the preservation of rods and cone photoreceptor subtypes. We found that gene therapy preserves both rods and cones. While the moderate loss of rods in the Rpe65-deficient dog retina is slowed by gene therapy, S-cones are lost extensively and gene therapy can prevent that loss, although only within the treated area. Although LM-cones are not lost extensively, cone opsin mislocalization indicates that they are stressed, and this can be partially reversed by gene therapy. Our results suggest that gene therapy may be able to slow cone degeneration in patients if intervention is sufficiently early and also that it is probably important to treat the macula in order to preserve central function.

Effects of blood-activating and stasis-removing drugs combined with VEGF gene transfer on angiogenesis in ischemic necrosis of the femoral head.

PubMed

Li, Jun-Hui; Wu, Ya-Ling; Ye, Jian-Hong; Ning, Ya-Gong; Yu, Hai-Ying; Peng, Zhong-Jie; Luan, Xiao-Wen

2009-09-01

To observe the promoting effects of blood-activating and stasis-removing Chinese drugs combined with vascular endothelial growth factor (VEGF) gene transfer on angiogenesis in ischemic necrosis of the femoral head. Forty Japanese giant-ear rabbits were randomly divided into a control group, a model group, a Chinese drug group, a gene group, and a combined group. After 8 weeks of treatment, the rate of VEGF positive cell expression in the synovium of the femoral head was measured using the immunohistochemical method, and the number of blood vessels in the femoral head was measured by digital subtraction angiography. The rate of VEGF positive cell expression in the model group was significantly lower than that in the Chinese drug group (P < 0.05) and very significantly lower than those in other groups (P < 0.01); but in the combined group it was significantly higher than in the Chinese drug group (P < 0.05). The differences in the number of blood vessels in area A between the model group and other groups were not statistically significant. However, in area B, the number of blood vessels significantly increased in the control group, the gene group and the combined group as compared with the model group (P < 0.05), and in the combined group the number of blood vessels was significantly more than in the gene group (P < 0.05); but in the Chinese drug group it was not significantly different than the model group (P > 0.05). Either the blood-activating and stasis-removing Chinese drugs or VEGF gene transfer can promote the angiogenesis and building of collateral circulation for femoral head ischemic necrosis, and the combined therapy with Chinese drugs or VEGF gene transfer may show a better therapeutic effect. The present study provides an experimental basis for clinical application of the combined therapy with the blood-activating and stasis-removing Chinese drugs and VEGF gene transfer.

Early gene expression profiles of patients with chronic hepatitis C treated with pegylated interferon-alfa and ribavirin.

PubMed

Younossi, Zobair M; Baranova, Ancha; Afendy, Arian; Collantes, Rochelle; Stepanova, Maria; Manyam, Ganiraju; Bakshi, Anita; Sigua, Christopher L; Chan, Joanne P; Iverson, Ayuko A; Santini, Christopher D; Chang, Sheng-Yung P

2009-03-01

Responsiveness to hepatitis C virus (HCV) therapy depends on viral and host factors. Our aim was to assess sustained virologic response (SVR)-associated early gene expression in patients with HCV receiving pegylated interferon-alpha2a (PEG-IFN-alpha2a) or PEG-IFN-alpha2b and ribavirin with the duration based on genotypes. Blood samples were collected into PAXgene tubes prior to treatment as well as 1, 7, 28, and 56 days after treatment. From the peripheral blood cells, total RNA was extracted, quantified, and used for one-step reverse transcription polymerase chain reaction to profile 154 messenger RNAs. Expression levels of messenger RNAs were normalized with six "housekeeping" genes and a reference RNA. Multiple regression and stepwise selection were performed to assess differences in gene expression at different time points, and predictive performance was evaluated for each model. A total of 68 patients were enrolled in the study and treated with combination therapy. The results of gene expression showed that SVR could be predicted by the gene expression of signal transducer and activator of transcription-6 (STAT-6) and suppressor of cytokine signaling-1 in the pretreatment samples. After 24 hours, SVR was predicted by the expression of interferon-dependent genes, and this dependence continued to be prominent throughout the treatment. Early gene expression during anti-HCV therapy may elucidate important molecular pathways that may be influencing the probability of achieving virologic response.

Curing genetic disease with gene therapy.

PubMed

Williams, David A

2014-01-01

Development of viral vectors that allow high efficiency gene transfer into mammalian cells in the early 1980s foresaw the treatment of severe monogenic diseases in humans. The application of gene transfer using viral vectors has been successful in diseases of the blood and immune systems, albeit with several curative studies also showing serious adverse events (SAEs). In children with X-linked severe combined immunodeficiency (SCID-X1), chronic granulomatous disease, and Wiskott-Aldrich syndrome, these SAEs were caused by inappropriate activation of oncogenes. Subsequent studies have defined the vector sequences responsible for these transforming events. Members of the Transatlantic Gene Therapy Consortium [TAGTC] have collaboratively developed new vectors that have proven safer in preclinical studies and used these vectors in new clinical trials in SCID-X1. These trials have shown evidence of early efficacy and preliminary integration analysis data from the SCID-X1 trial suggest an improved safety profile.

Curing Genetic Disease with Gene Therapy

PubMed Central

Williams, David A.

2014-01-01

Development of viral vectors that allow high efficiency gene transfer into mammalian cells in the early 1980s foresaw the treatment of severe monogenic diseases in humans. The application of gene transfer using viral vectors has been successful in diseases of the blood and immune systems, albeit with several curative studies also showing serious adverse events (SAEs). In children with X-linked severe combined immunodeficiency (SCID-X1), chronic granulomatous disease, and Wiskott-Aldrich syndrome, these SAEs were caused by inappropriate activation of oncogenes. Subsequent studies have defined the vector sequences responsible for these transforming events. Members of the Transatlantic Gene Therapy Consortium [TAGTC] have collaboratively developed new vectors that have proven safer in preclinical studies and used these vectors in new clinical trials in SCID-X1. These trials have shown evidence of early efficacy and preliminary integration analysis data from the SCID-X1 trial suggest an improved safety profile. PMID:25125725

Gene therapy and its implications in Periodontics

PubMed Central

Mahale, Swapna; Dani, Nitin; Ansari, Shumaila S.; Kale, Triveni

2009-01-01

Gene therapy is a field of Biomedicine. With the advent of gene therapy in dentistry, significant progress has been made in the control of periodontal diseases and reconstruction of dento-alveolar apparatus. Implementation in periodontics include: -As a mode of tissue engineering with three approaches: cell, protein-based and gene delivery approach. -Genetic approach to Biofilm Antibiotic Resistance. Future strategies of gene therapy in preventing periodontal diseases: -Enhances host defense mechanism against infection by transfecting host cells with an antimicrobial peptide protein-encoding gene. -Periodontal vaccination. Gene therapy is one of the recent entrants and its applications in the field of periodontics are reviewed in general here. PMID:20376232

Activation of caspase-3 noninvolved in the bystander effect of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system.

PubMed

Zhang, Zhihong; Lin, Juqiang; Chu, Jun; Ma, Yan; Zeng, Shaoqun; Luo, Qingming

2008-01-01

Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system.

Zalypsis has in vitro activity in acute myeloid blasts and leukemic progenitor cells through the induction of a DNA damage response

PubMed Central

Colado, Enrique; Paíno, Teresa; Maiso, Patricia; Ocio, Enrique M.; Chen, Xi; Álvarez-Fernández, Stela; Gutiérrez, Norma C.; Martín-Sánchez, Jesús; Flores-Montero, Juan; San Segundo, Laura; Garayoa, Mercedes; Fernández-Lázaro, Diego; Vidriales, Maria-Belen; Galmarini, Carlos M.; Avilés, Pablo; Cuevas, Carmen; Pandiella, Atanasio; San-Miguel, Jesús F.

2011-01-01

Background Although the majority of patients with acute myeloid leukemia initially respond to conventional chemotherapy, relapse is still the leading cause of death, probably because of the presence of leukemic stem cells that are insensitive to current therapies. We investigated the antileukemic activity and mechanism of action of zalypsis, a novel alkaloid of marine origin. Design and Methods The activity of zalypsis was studied in four acute myeloid leukemia cell lines and in freshly isolated blasts taken from patients with acute myeloid leukemia before they started therapy. Zalypsis-induced apoptosis of both malignant and normal cells was measured using flow cytometry techniques. Gene expression profiling and western blot studies were performed to assess the mechanism of action of the alkaloid. Results Zalypsis showed a very potent antileukemic activity in all the cell lines tested and potentiated the effect of conventional antileukemic drugs such as cytarabine, fludarabine and daunorubicin. Interestingly, zalypsis showed remarkable ex vivo potency, including activity against the most immature blast cells (CD34+ CD38− Lin−) which include leukemic stem cells. Zalypsis-induced apoptosis was the result of an important deregulation of genes involved in the recognition of double-strand DNA breaks, such as Fanconi anemia genes and BRCA1, but also genes implicated in the repair of double-strand DNA breaks, such as RAD51 and RAD54. These gene findings were confirmed by an increase in several proteins involved in the pathway (pCHK1, pCHK2 and pH2AX). Conclusions The potent and selective antileukemic effect of zalypsis on DNA damage response mechanisms observed in acute myeloid leukemia cell lines and in patients’ samples provides the rationale for the investigation of this compound in clinical trials. PMID:21330323

Gene expression profiling of breast cancer cell lines treated with proton and electron radiations.

PubMed

Bravatà, Valentina; Minafra, Luigi; Cammarata, Francesco Paolo; Pisciotta, Pietro; Lamia, Debora; Marchese, Valentina; Manti, Lorenzo; Cirrone, Giuseppe Ap; Gilardi, Maria Carla; Cuttone, Giacomo; Forte, Giusi Irma; Russo, Giorgio

2018-06-11

Technological advances in radiation therapy are evolving with the use of hadrons, such as protons, indicated for tumors where conventional radiotherapy does not give significant advantages or for tumors located in sensitive regions, which need the maximum of dose-saving of the surrounding healthy tissues. The genomic response to conventional and non conventional Linear Energy Transfer exposure is a poor investigated topic and became an issue of radiobiological interest. The aim of this work was to analyze and compare molecular responses in term of gene expression profiles, induced by electron and proton irradiation in breast cancer cell lines. We studied the gene expression profiling differences by cDNA microarray activated in response to electron and proton irradiation with different Linear Energy Transfer values, among three breast cell lines (the tumorigenic MCF7 and MDA-MB-231 and the non tumorigenic MCF10A), exposed to the same sub-lethal dose of 9 Gy. Gene expression profiling pathway analyses showed the activation of different signaling and molecular networks in a cell line and radiation type-dependent manner. MCF10A and MDA-MB-231 cell lines were found to induce factors and pathways involved in the immunological process control. Here we describe in a detailed way the gene expression profiling and pathways activated after electron and proton irradiation in breast cancer cells. Summarizing, although specific pathways are activated in a radiation type-dependent manner, each cell line activates overall similar molecular networks in response to both these two types of ionizing radiation. Advances in knowledge: In the era of personalized medicine and breast cancer target-directed intervention, we trust that this study could drive radiation therapy towards personalized treatments, evaluating possible combined treatments, based on the molecular characterization.

Effect of neonatal gene therapy on lumbar spine disease in mucopolysaccharidosis VII dogs.

PubMed

Smith, Lachlan J; Martin, John T; O'Donnell, Patricia; Wang, Ping; Elliott, Dawn M; Haskins, Mark E; Ponder, Katherine P

2012-09-01

Mucopolysaccharidosis VII (MPS VII) is due to deficient β-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan, and dermatan sulfate glycosaminoglycans in various tissues including those of the spine. Associated spine disease can be due to abnormalities in the vertebrae, the intervertebral disks, or other spine tissues. The goal of this study was to determine if neonatal gene therapy could prevent lumbar spine disease in MPS VII dogs. MPS VII dogs were injected intravenously with a retroviral vector (RV) expressing canine GUSB at 2 to 3 days after birth, which resulted in transduction of hepatocytes that secreted GUSB into blood. Expression was stable for up to 11 years, and mean survival was increased from 0.4 years in untreated dogs to 6.1 years in treated dogs. Despite a profound positive clinical effect, 6-month-old RV-treated MPS VII dogs still had hypoplastic ventral epiphyses with reduced calcification in the lumbar spine, which resulted in a reduced stiffness and increased range of motion that were not improved relative to untreated MPS VII dogs. At six to 11 years of age, ventral vertebrae remained hypoplastic in RV-treated MPS VII dogs, and there was desiccation of the nucleus pulposus in some disks. Histochemical staining demonstrated that disks did not have detectable GUSB activity despite high serum GUSB activity, which is likely due to poor diffusion into this relatively avascular structure. Thus, neonatal gene therapy cannot prevent lumbar spine disease in MPS VII dogs, which predicts that enzyme replacement therapy (ERT) will similarly be relatively ineffective even if started at birth. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular Approach to Targeted Therapy for Multiple Sclerosis.

PubMed

Sherbet, Gajanan V

2016-01-01

The development and evolution of targeted therapy to any disease require the identification of targets amenable to treatment of patients. Here the pathogenetic signalling systems involved in multiple sclerosis are scrutinised to locate nodes of deregulation and dysfunction in order to devise strategies of drug development for targeted intervention. Oliogoclonal bands (OCB) are isoelectric focusing profiles of immunoglobulins synthesised in the central nervous system. OCBs enable the diagnosis of multiple sclerosis with high sensitivity and specificity and are related to the course of the disease and progression. The OCB patterns can be linked with the expression of angiogenic molecular species. Angiogenic signalling which has also been implicated in demyelination provides the option of using angiogenesis inhibitors in disease control. The PI3K (phosphoinositide 3-kinase)/Akt axis has emerged with a key role in myelination with its demonstrable links with mTOR mediated transcription of downstream target genes. Inflammatory signals and innate and acquired immunity from the activation of NF-κB (nuclear factor κB) responsive genes are considered. NF-κB signalling could be implicated in myelination. The transcription factor STAT (signal transducers and activators of transcription) and the EBV (Epstein- Barr virus) transcription factor BZLF1 contributing significantly to the disease process are a major environmental factor linked to MS. EBV can activate TGF (transforming growth factor) and VEGF (vascular endothelial growth factor) signalling. EBV microRNAs are reviewed as signalling mediators of pathogenesis. Stem cell transplantation therapy has lately gained much credence, so the current status of mesenchymal and hematopoietic stem cell therapy is reviewed with emphasis on the differential expression immune-related genes and operation of signalling systems.

Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

NASA Astrophysics Data System (ADS)

Yuan, Chenyan; An, Yanli; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng

2014-08-01

Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression.

Orexin Gene Therapy Restores the Timing and Maintenance of Wakefulness in Narcoleptic Mice

PubMed Central

Kantor, Sandor; Mochizuki, Takatoshi; Lops, Stefan N.; Ko, Brian; Clain, Elizabeth; Clark, Erika; Yamamoto, Mihoko; Scammell, Thomas E.

2013-01-01

Study Objectives: Narcolepsy is caused by selective loss of the orexin/hypocretin-producing neurons of the hypothalamus. For patients with narcolepsy, chronic sleepiness is often the most disabling symptom, but current therapies rarely normalize alertness and do not address the underlying orexin deficiency. We hypothesized that the sleepiness of narcolepsy would substantially improve if orexin signaling were restored in specific brain regions at appropriate times of day. Design: We used gene therapy to restore orexin signaling in a mouse model of narcolepsy. In these Atx mice, expression of a toxic protein (ataxin-3) selectively kills the orexin neurons. Interventions: To induce ectopic expression of the orexin neuropeptides, we microinjected an adeno-associated viral vector coding for prepro-orexin plus a red fluorescence protein (AAV-orexin) into the mediobasal hypothalamus of Atx and wild-type mice. Control mice received an AAV coding only for red fluorescence protein. Two weeks later, we recorded sleep/wake behavior, locomotor activity, and body temperature and examined the patterns of orexin expression. Measurements and Results: Atx mice rescued with AAV-orexin produced long bouts of wakefulness and had a normal diurnal pattern of arousal, with the longest bouts of wake and the highest amounts of locomotor activity in the first hours of the night. In addition, AAV-orexin improved the timing of rapid eye movement sleep and the consolidation of nonrapid eye movement sleep in Atx mice. Conclusions: These substantial improvements in sleepiness and other symptoms of narcolepsy demonstrate the effectiveness of orexin gene therapy in a mouse model of narcolepsy. Additional work is needed to optimize this approach, but in time, AAV-orexin could become a useful therapeutic option for patients with narcolepsy. Citation: Kantor S; Mochizuki T; Lops SN; Ko B; Clain E; Clark E; Yamamoto M; Scammell TE. Orexin gene therapy restores the timing and maintenance of wakefulness in narcoleptic mice. SLEEP 2013;36(8):1129–1138. PMID:23904672

Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours.

PubMed

Buhles, Alexandra; Collins, Sara A; van Pijkeren, Jan P; Rajendran, Simon; Miles, Michelle; O'Sullivan, Gerald C; O'Hanlon, Deirdre M; Tangney, Mark

2009-03-09

The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metastasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate reduction in primary subcutaneous tumour growth. Overall, this study demonstrates the potential for Nk4 gene therapy of metastatic tumours, when delivered by AAV or non-viral methods.

Association of angiotensin-converting-enzyme gene polymorphism with the depressor response to mild exercise therapy in patients with mild to moderate essential hypertension.

PubMed

Zhang, B; Sakai, T; Miura, S; Kiyonaga, A; Tanaka, H; Shindo, M; Saku, K

2002-10-01

We studied the association of angiotensin I-converting enzyme (ACE) gene polymorphism with the depressor response to exercise therapy in 64 Japanese subjects with mild to moderate essential hypertension. Each subject performed 10 weeks of mild (lactate threshold intensity: approximately 50% maximum oxygen consumption) exercise therapy on a bicycle ergometer. Systolic blood pressure (SPB), diastolic blood pressure (DPB), and mean arterial pressure (MAP) were significantly decreased by exercise therapy in subjects with the ACE-II and ID genotypes but not in DD subjects. The time-by-genotype interaction effects were significant for DBP and MAP. According to a multiple logistic regression analysis, the age- and baseline plasma renin activity-adjusted relative risk (odds ratio) for the lack of a depressor response conferred by the D allele (assuming an additive effect) was 2.72 [95% confidence interval (CI), 1.07-6.91; p = 0.034]; for DD genotypes, as compared with the DI and II genotypes (assuming that the D allele is recessive), it was 11.7 (95% CI, 2.25-60.6; p = 0.003). ACE gene I/D polymorphism is associated with the depressor response of essential hypertensives to mild exercise therapy, which suggests that genetic features may underlie, at least in part, the heterogeneity of the depressor response in essential hypertensives to mild exercise therapy.

In vivo evaluation of a cancer therapy strategy combining HSV1716-mediated oncolysis with gene transfer and targeted radiotherapy.

PubMed

Sorensen, Annette; Mairs, Robert J; Braidwood, Lynne; Joyce, Craig; Conner, Joe; Pimlott, Sally; Brown, Moira; Boyd, Marie

2012-04-01

Oncolytic herpes viruses show promise for cancer treatment. However, it is unlikely that they will fulfill their therapeutic potential when used as monotherapies. An alternative strategy is to use these viruses not only as oncolytic agents but also as a delivery mechanism of therapeutic transgenes to enhance tumor cell killing. The herpes simplex virus 1 deletion mutant HSV1716 is a conditionally replicating oncolytic virus that selectively replicates in and lyses dividing tumor cells. It has a proven safety profile in clinical trials and has demonstrated efficacy as a gene-delivery vehicle. To enhance its therapeutic potential, we have engineered HSV1716 to convey the noradrenaline transporter (NAT) gene (HSV1716/NAT), whose expression endows infected cells with the capacity to accumulate the noradrenaline analog metaiodobenzylguanidine (MIBG). Thus, the NAT gene-infected cells are susceptible to targeted radiotherapy using radiolabeled (131)I-MIBG, a strategy that has already shown promise for combined targeted radiotherapy-gene therapy in cancer cells after plasmid-mediated transfection. We used HSV1716/NAT as a dual cell lysis-gene delivery vehicle for targeting the NAT transgene to human tumor xenografts in vivo. In tumor xenografts that did not express NAT, intratumoral or intravenous injection of HSV1716/NAT induced the capacity for active uptake of (131)I-MIBG. Administration of HSV1716/NAT and (131)I-MIBG resulted in decreased tumor growth and enhanced survival relative to injection of either agent alone. Efficacy was dependent on the scheduling of delivery of the 2 agents. These findings support a role for combination radiotherapy-gene therapy for cancer using HSV1716 expressing the NAT transgene and targeted radionuclide therapy.

Gene therapy for eye as regenerative medicine? Lessons from RPE65 gene therapy for Leber's Congenital Amaurosis.

PubMed

Rakoczy, Elizabeth P; Narfström, Kristina

2014-11-01

Recombinant virus mediated gene therapy of Leber's Congenital Amaurosis has provided a wide range of data on the utility of gene replacement therapy for recessive diseases. Studies to date demonstrate that gene therapy in the eye is safe and can result in long-term recovery of visual function, but they also highlight that further research is required to identify optimum intervention time-points, target populations and the compatibility of associate therapies. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Gene-environment interaction between adiponectin gene polymorphisms and environmental factors on the risk of diabetic retinopathy.

PubMed

Li, Yuan; Wu, Qun Hong; Jiao, Ming Li; Fan, Xiao Hong; Hu, Quan; Hao, Yan Hua; Liu, Ruo Hong; Zhang, Wei; Cui, Yu; Han, Li Yuan

2015-01-01

To evaluate whether the adiponectin gene is associated with diabetic retinopathy (DR) risk and interaction with environmental factors modifies the DR risk, and to investigate the relationship between serum adiponectin levels and DR. Four adiponectin polymorphisms were evaluated in 372 DR cases and 145 controls. Differences in environmental factors between cases and controls were evaluated by unconditional logistic regression analysis. The model-free multifactor dimensionality reduction method and traditional multiple regression models were applied to explore interactions between the polymorphisms and environmental factors. Using the Bonferroni method, we found no significant associations between four adiponectin polymorphisms and DR susceptibility. Multivariate logistic regression found that physical activity played a protective role in the progress of DR, whereas family history of diabetes (odds ratio 1.75) and insulin therapy (odds ratio 1.78) were associated with an increased risk for DR. The interaction between the C-11377 G (rs266729) polymorphism and insulin therapy might be associated with DR risk. Family history of diabetes combined with insulin therapy also increased the risk of DR. No adiponectin gene polymorphisms influenced the serum adiponectin levels. Serum adiponectin levels did not differ between the DR group and non-DR group. No significant association was identified between four adiponectin polymorphisms and DR susceptibility after stringent Bonferroni correction. The interaction between C-11377G (rs266729) polymorphism and insulin therapy, as well as the interaction between family history of diabetes and insulin therapy, might be associated with DR susceptibility.

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators

PubMed Central

Polstein, Lauren R.; Perez-Pinera, Pablo; Kocak, D. Dewran; Vockley, Christopher M.; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E.; Reddy, Timothy E.; Gersbach, Charles A.

2015-01-01

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. PMID:26025803

Brain-targeted stem cell gene therapy corrects mucopolysaccharidosis type II via multiple mechanisms.

PubMed

Gleitz, Hélène Fe; Liao, Ai Yin; Cook, James R; Rowlston, Samuel F; Forte, Gabriella Ma; D'Souza, Zelpha; O'Leary, Claire; Holley, Rebecca J; Bigger, Brian W

2018-06-08

The pediatric lysosomal storage disorder mucopolysaccharidosis type II is caused by mutations in IDS, resulting in accumulation of heparan and dermatan sulfate, causing severe neurodegeneration, skeletal disease, and cardiorespiratory disease. Most patients manifest with cognitive symptoms, which cannot be treated with enzyme replacement therapy, as native IDS does not cross the blood-brain barrier. We tested a brain-targeted hematopoietic stem cell gene therapy approach using lentiviral IDS fused to ApoEII (IDS.ApoEII) compared to a lentivirus expressing normal IDS or a normal bone marrow transplant. In mucopolysaccharidosis II mice, all treatments corrected peripheral disease, but only IDS.ApoEII mediated complete normalization of brain pathology and behavior, providing significantly enhanced correction compared to IDS. A normal bone marrow transplant achieved no brain correction. Whilst corrected macrophages traffic to the brain, secreting IDS/IDS.ApoEII enzyme for cross-correction, IDS.ApoEII was additionally more active in plasma and was taken up and transcytosed across brain endothelia significantly better than IDS via both heparan sulfate/ApoE-dependent receptors and mannose-6-phosphate receptors. Brain-targeted hematopoietic stem cell gene therapy provides a promising therapy for MPS II patients. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Clinical development of gene therapy needs a tailored approach: a regulatory perspective from the European Union.

PubMed

Narayanan, Gopalan; Cossu, Giulio; Galli, Maria Cristina; Flory, Egbert; Ovelgonne, Hans; Salmikangas, Paula; Schneider, Christian K; Trouvin, Jean-Hugues

2014-03-01

Gene therapy is a rapidly evolving field that needs an integrated approach, as acknowledged in the concept article on the revision of the guideline on gene transfer medicinal products. The first gene therapy application for marketing authorization was approved in the International Conference on Harmonisation (ICH) region in 2012, the product being Alipogene tiparvovec. The regulatory process for this product has been commented on extensively, highlighting the challenges posed by such a novel technology. Here, as current or previous members of the Committee for Advanced Therapies, we share our perspectives and views on gene therapy as a treatment modality based on current common understanding and regulatory experience of gene therapy products in the European Union to date. It is our view that a tailored approach is needed for a given gene therapy product in order to achieve successful marketing authorization.

Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease

PubMed Central

Jison, Maria L.; Munson, Peter J.; Barb, Jennifer J.; Suffredini, Anthony F.; Talwar, Shefali; Logun, Carolea; Raghavachari, Nalini; Beigel, John H.; Shelhamer, James H.; Danner, Robert L.; Gladwin, Mark T.

2016-01-01

In sickle cell disease, deoxygenation of intra-erythrocytic hemoglobin S leads to hemoglobin polymerization, erythrocyte rigidity, hemolysis, and microvascular occlusion. Ischemia-reperfusion injury, plasma hemoglobin-mediated nitric oxide consumption, and free radical generation activate systemic inflammatory responses. To characterize the role of circulating leukocytes in sickle cell pathogenesis we performed global transcriptional analysis of blood mononuclear cells from 27 patients in steady-state sickle cell disease (10 patients treated and 17 patients untreated with hydroxyurea) compared with 13 control subjects. We used gender-specific gene expression to validate human microarray experiments. Patients with sickle cell disease demonstrated differential gene expression of 112 genes involved in heme metabolism, cell-cycle regulation, antioxidant and stress responses, inflammation, and angiogenesis. Inducible heme oxygenase-1 and downstream proteins biliverdin reductase and p21, a cyclin-dependent kinase, were up-regulated, potentially contributing to phenotypic heterogeneity and absence of atherosclerosis in patients with sickle cell disease despite endothelial dysfunction and vascular inflammation. Hydroxyurea therapy did not significantly affect leukocyte gene expression, suggesting that such therapy has limited direct anti-inflammatory activity beyond leukoreduction. Global transcriptional analysis of circulating leukocytes highlights the intense oxidant and inflammatory nature of steady-state sickle cell disease and provides insight into the broad compensatory responses to vascular injury. PMID:15031206

Genetic correction of dystrophin deficiency and skeletal muscle remodeling in adult MDX mouse via transplantation of retroviral producer cells.

PubMed Central

Fassati, A; Wells, D J; Sgro Serpente, P A; Walsh, F S; Brown, S C; Strong, P N; Dickson, G

1997-01-01

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disease caused by mutations of the dystrophin gene. No effective therapy is available, but dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. We have developed a strategy for efficient in vivo gene transfer of dystrophin cDNA into regenerating skeletal muscle. Retroviral producer cells, which release a vector carrying the therapeutically active dystrophin minigene, were mitotically inactivated and transplanted in adult nude/mdx mice. Transplantation of 3 x 10(6) producer cells in a single site of the tibialis anterior muscle resulted in the transduction of between 5.5 and 18% total muscle fibers. The same procedure proved also feasible in immunocompetent mdx mice under short-term pharmacological immunosuppression. Minidystrophin expression was stable for up to 6 mo and led to alpha-sarcoglycan reexpression. Muscle stem cells could be transduced in vivo using this procedure. Transduced dystrophic skeletal muscle showed evidence of active remodeling reminiscent of the genetic normalization process which takes place in female DMD carriers. Overall, these results demonstrate that retroviral-mediated dystrophin gene transfer via transplantation of producer cells is a valid approach towards the long-term goal of gene therapy of DMD. PMID:9239410

Adenovirus-mediated suicide gene therapy under the control of Cox-2 promoter for colorectal cancer.

PubMed

Wang, Zhao-Xia; Bian, Hai-Bo; Yang, Jing-Song; De, Wei; Ji, Xiao-Hui

2009-08-01

Colorectal cancer is a most frequent type of gastrointestinal tract cancers. The prognosis of patients with colorectal cancer remains poor despite intensive interventions. Tumor specific promoter-directed gene therapy and adenoviral technology can be promising strategies for such advanced disease. This study was conducted to explore the possible therapeutic approach of Cox-2 promoter-directed suicide gene therapy with herpes simplex virus thymidine kinase (HSV-tk) in combination with adenoviral technology for advanced colorectal cancer. Firstly, the activity of Cox-2 promoter was assessed by dual luciferase and enhanced green fluorescent protein reporter gene assays in colorectal cancer cell lines and normal human intestinal epithelial cell line. Then, the expression of coxsackievirus and adenovirus receptor (CAR) was detected in colorectal cancer cell lines. The Cox-2 promoter-directed HSV-tk/ganciclovir (GCV) system mediated by adenovirus (Ad-Cp-TK) was developed (Ad-CMVp-TK, Ad-null and no Ad as controls). In vitro cytoxicity, colony formation and apoptosis assays were performed using Ad-Cp-TK. An animal study was carried out in which BALB/C nude mice bearing tumors were treated with Ad-Cp-TK and GCV treatments. Results showed that Cox-2 promoter possessed high transcriptional activity in a tumor-specific manner. All colorectal cancer cells were detected CAR-positive. In vitro cytotoxic and colony formation assays showed that colorectal cancer cells infected with Ad-Cp-TK became more sensitive to GCV but the sensitivity of normal cells infected with Ad-Cp-TK to GCV were not altered. Moreover, the Ad-Cp-TK system combined with GCV treatment could significantly induce apoptosis of colorectal cancer cells but not normal intestinal epithelial cells. Furthermore, this system also significantly inhibited the growth of subcutaneous tumors and prolonged survival of mice. Thus, adenovirus primary receptor was positive in colorectal cancer cells and adenovirus-mediated suicide gene therapy under the control of Cox-2 promoter could provide a promising treatment modality for advanced colorectal cancer with tumor specificity.

Tyrosine kinase receptor status in endometrial stromal sarcoma: an immunohistochemical and genetic-molecular analysis.

PubMed

Cossu-Rocca, Paolo; Contini, Marcella; Uras, Maria Gabriela; Muroni, Maria Rosaria; Pili, Francesca; Carru, Ciriaco; Bosincu, Luisanna; Massarelli, Giovannino; Nogales, Francisco F; De Miglio, Maria Rosaria

2012-11-01

Endometrial stromal sarcomas (ESS) are rare uterine malignant mesenchymal neoplasms, which are currently treated by surgery, as effective adjuvant therapies have not yet been established. Tyrosine kinase inhibitors have rarely been applied in ESS therapy, with few reports describing imatinib responsivity. The aim of this study was to analyze the status of different tyrosine kinase receptors in an ESS series, in order to evaluate their potential role as molecular targets. Immunohistochemistry was performed for EGFR, c-KIT, PDGFR-α, PDGFR-β, and ABL on 28 ESS. EGFR, PDGFR-α, and PDGFR-β gene expression was investigated by real-time polymerase chain reaction (qRT-PCR) on selected cases. "Hot-spot" mutations were screened for on EGFR, c-KIT, PDGFR-α, and PDGFR-β genes, by sequencing. All analysis was executed from formalin-fixed, paraffin-embedded specimens. Immunohistochemical overexpression of 2 or more tyrosine kinase receptors was observed in 18 of 28 tumors (64%), whereas only 5 tumors were consistently negative. Gene expression profiles were concordant with immunohistochemical overexpression in only 1 tumor, which displayed both high mRNA levels and specific immunoreactivity for PDGFR-α, and PDGFR-β. No activating mutations were found on the tumors included in the study. This study confirms that TKRs expression is frequently observed in ESS. Considering that the responsiveness to tyrosine kinase inhibitors is known to be related to the presence of specific activating mutations or gene over-expression, which are not detectable in ESS, TKRs immunohistochemical over-expression alone should not be considered as a reliable marker for targeted therapies in ESS. Specific post-translational abnormalities, responsible for activation of TKRs, should be further investigated.

75 FR 54351 - Cell and Gene Therapy Clinical Trials in Pediatric Populations; Public Workshop

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-07

...] Cell and Gene Therapy Clinical Trials in Pediatric Populations; Public Workshop AGENCY: Food and Drug... Biologics Evaluation and Research (CBER) is announcing a public workshop entitled ``Cell and Gene Therapy... regarding best practices related to cell and gene therapy clinical trials in pediatric populations, as well...

77 FR 65693 - Cellular, Tissue and Gene Therapies Advisory Committee; Amendment of Notice

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-30

...] Cellular, Tissue and Gene Therapies Advisory Committee; Amendment of Notice AGENCY: Food and Drug... notice of a meeting of the Cellular, Tissue and Gene Therapies Advisory Committee. This meeting was... announced that a meeting of the Cellular, Tissue and Gene Therapies Advisory Committee would be held on...

Genotoxicity of retroviral hematopoietic stem cell gene therapy

PubMed Central

Trobridge, Grant D

2012-01-01

Introduction Retroviral vectors have been developed for hematopoietic stem cell (HSC) gene therapy and have successfully cured X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy, and Wiskott-Aldrich syndrome. However, in HSC gene therapy clinical trials, genotoxicity mediated by integrated vector proviruses has led to clonal expansion, and in some cases frank leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity with the aim of developing safer vectors and safer gene therapy protocols. These genotoxicity studies are critical to advancing HSC gene therapy. Areas covered This review provides an introduction to the mechanisms of retroviral vector genotoxicity. It also covers advances over the last 20 years in designing safer gene therapy vectors, and in integration site analysis in clinical trials and large animal models. Mechanisms of retroviral-mediated genotoxicity, and the risk factors that contribute to clonal expansion and leukemia in HSC gene therapy are introduced. Expert opinion Continued research on virus–host interactions and next-generation vectors should further improve the safety of future HSC gene therapy vectors and protocols. PMID:21375467

Strategy of Cancer Targeting Gene-Viro-Therapy (CTGVT) a trend in both cancer gene therapy and cancer virotherapy.

PubMed

Liu, Xin-Yuan; Li, Hua-Guang; Zhang, Kang-Jian; Gu, Jin-Fa

2012-07-01

Cancer Targeting Gene-Viro-Therapy (CTGVT) and Gene Armed Oncolytic Virus Therapy (GAOVT) both are identical by inserting an antitumor gene into an oncolytic virus. This approach has gradually become a hot topic in cancer therapy, because that CTGVT (GAOVT) has much higher antitumor than that of either gene therapy alone or oncolytic virotherapy alone. We proposed the CTGVT strategy in 1999-2001, insisted it as a long term systematic approach to be examined over 10 years and have published 68 SCI papers some in good Journals. The CD gene armed oncolytic adenovirus therapy (GAOVT) for cancer treatment with potent antitumor effect was also named in our laboratory in 2003. Several modifications to CTGVT will be carried out by our group and will be introduced briefly in this paper. Most importantly, the modifications of CTGVT usually resulted in complete eradication of xenograft tumors in nude mice. In future best antitumor drugs may emerge from the modified CTGVT strategy and not from either gene therapy or virotherapy alone.

The function of dog models in developing gene therapy strategies for human health.

PubMed

Nowend, Keri L; Starr-Moss, Alison N; Murphy, Keith E

2011-08-01

The domestic dog is of great benefit to humankind, not only through companionship and working activities cultivated through domestication and selective breeding, but also as a model for biomedical research. Many single-gene traits have been well-characterized at the genomic level, and recent advances in whole-genome association studies will allow for better understanding of complex, multigenic hereditary diseases. Additionally, the dog serves as an invaluable large animal model for assessment of novel therapeutic agents. Thus, the dog has filled a crucial step in the translation of basic research to new treatment regimens for various human diseases. Four well-characterized diseases in canine models are discussed as they relate to other animal model availability, novel therapeutic approach, and extrapolation to human gene therapy trials.

Nanoparticles for cancer gene therapy: Recent advances, challenges, and strategies.

PubMed

Wang, Kui; Kievit, Forrest M; Zhang, Miqin

2016-12-01

Compared to conventional treatments, gene therapy offers a variety of advantages for cancer treatment including high potency and specificity, low off-target toxicity, and delivery of multiple genes that concurrently target cancer tumorigenesis, recurrence, and drug resistance. In the past decades, gene therapy has undergone remarkable progress, and is now poised to become a first line therapy for cancer. Among various gene delivery systems, nanoparticles have attracted much attention because of their desirable characteristics including low toxicity profiles, well-controlled and high gene delivery efficiency, and multi-functionalities. This review provides an overview on gene therapeutics and gene delivery technologies, and highlight recent advances, challenges and insights into the design and the utility of nanoparticles in gene therapy for cancer treatment. Copyright © 2016. Published by Elsevier Ltd.

Advances in gene therapy for heart failure.

PubMed

Fish, Kenneth M; Ishikawa, Kiyotake

2015-04-01

Chronic heart failure is expected to increase its social and economic burden as a consequence of improved survival in patients with acute cardiac events. Cardiac gene therapy holds significant promise in heart failure treatment for patients with currently very limited or no treatment options. The introduction of adeno-associated virus (AAV) gene vector changed the paradigm of cardiac gene therapy, and now it is the primary vector of choice for chronic heart failure gene therapy in clinical and preclinical studies. Recently, there has been significant progress towards clinical translation in this field spearheaded by AAV-1 mediated sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) gene therapy targeting chronic advanced heart failure patients. Meanwhile, several independent laboratories are reporting successful gene therapy approaches in clinically relevant large animal models of heart failure and some of these approaches are expected to enter clinical trials in the near future. This review will focus on gene therapy approaches targeting heart failure that is in clinical trials and those close to its initial clinical trial application.

Gene Therapy in Heart Failure.

PubMed

Fargnoli, Anthony S; Katz, Michael G; Bridges, Charles R; Hajjar, Roger J

2017-01-01

Heart failure is a significant burden to the global healthcare system and represents an underserved market for new pharmacologic strategies, especially therapies which can address root cause myocyte dysfunction. Modern drugs, surgeries, and state-of-the-art interventions are costly and do not improve survival outcome measures. Gene therapy is an attractive strategy, whereby selected gene targets and their associated regulatory mechanisms can be permanently managed therapeutically in a single treatment. This in theory could be sustainable for the patient's life. Despite the promise, however, gene therapy has numerous challenges that must be addressed together as a treatment plan comprising these key elements: myocyte physiologic target validation, gene target manipulation strategy, vector selection for the correct level of manipulation, and carefully utilizing an efficient delivery route that can be implemented in the clinic to efficiently transfer the therapy within safety limits. This chapter summarizes the key developments in cardiac gene therapy from the perspective of understanding each of these components of the treatment plan. The latest pharmacologic gene targets, gene therapy vectors, delivery routes, and strategies are reviewed.

Successful Combination of Sequential Gene Therapy and Rescue Allo-HSCT in Two Children with X-CGD - Importance of Timing.

PubMed

Siler, Ulrich; Paruzynski, Anna; Holtgreve-Grez, Heidi; Kuzmenko, Elena; Koehl, Ulrike; Renner, Eleonore D; Alhan, Canan; de Loosdrecht, Arjan A van; Schwäble, Joachim; Pfluger, Thomas; Tchinda, Joelle; Schmugge, Markus; Jauch, Anna; Naundorf, Sonja; Kühlcke, Klaus; Notheis, Gundula; Güngor, Tayfun; Kalle, Christof V; Schmidt, Manfred; Grez, Manuel; Seger, Reinhard; Reichenbach, Janine

2015-01-01

We report on a series of sequential events leading to long-term survival and cure of pediatric X-linked chronic granulomatous disease (X-CGD) patients after gamma-retroviral gene therapy (GT) and rescue HSCT. Due to therapyrefractory life-threatening infections requiring hematopoietic stem cell transplantation (HSCT) but absence of HLAidentical donors, we treated 2 boys with X-CGD by GT. Following GT both children completely resolved invasive Aspergillus nidulans infections. However, one child developed dual insertional activation of ecotropic viral integration site 1 (EVI1) and signal transducer and activator of transcription 3 (STAT3) genes, leading to myelodysplastic syndrome (MDS) with monosomy 7. Despite resistance to mismatched allo-HSCT with standard myeloablative conditioning, secondary intensified rescue allo-HSCT resulted in 100 % donor chimerism and disappearance of MDS. The other child did not develop MDS despite expansion of a clone with a single insertion in the myelodysplasia syndrome 1 (MDS1) gene and was cured by early standard allo-HSCT. The slowly developing dominance of clones harboring integrations in MDS1-EVI1 may guide clinical intervention strategies, i.e. early rescue allo-HSCT, prior to malignant transformation. GT was essential for both children to survive and to clear therapy-refractory infections, and future GT with safer lentiviral self-inactivated (SIN) vectors may offer a therapeutic alternative for X-CGD patients suffering from life-threatening infections and lacking HLA-identical HSC donors.

Gene therapy for haemophilia.

PubMed

Sharma, Akshay; Easow Mathew, Manu; Sriganesh, Vasumathi; Reiss, Ulrike M

2016-12-20

Haemophilia is a genetic disorder characterized by spontaneous or provoked, often uncontrolled, bleeding into joints, muscles and other soft tissues. Current methods of treatment are expensive, challenging and involve regular administration of clotting factors. Gene therapy has recently been prompted as a curative treatment modality. This is an update of a published Cochrane Review. To evaluate the safety and efficacy of gene therapy for treating people with haemophilia A or B. We searched the Cochrane Cystic Fibrosis & Genetic Disorders Group's Coagulopathies Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched the reference lists of relevant articles and reviews.Date of last search: 18 August 2016. Eligible trials include randomised or quasi-randomised clinical trials, including controlled clinical trials comparing gene therapy (with or without standard treatment) with standard treatment (factor replacement) or other 'curative' treatment such as stem cell transplantation for individuals with haemophilia A or B of all ages who do not have inhibitors to factor VIII or IX. No trials of gene therapy for haemophilia were found. No trials of gene therapy for haemophilia were identified. No randomised or quasi-randomised clinical trials of gene therapy for haemophilia were identified. Thus, we are unable to determine the safety and efficacy of gene therapy for haemophilia. Gene therapy for haemophilia is still in its nascent stages and there is a need for well-designed clinical trials to assess the long-term feasibility, success and risks of gene therapy for people with haemophilia.

Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation.

PubMed

Nomikou, N; Feichtinger, G A; Saha, S; Nuernberger, S; Heimel, P; Redl, H; McHale, A P

2018-01-01

Gene-activated matrix (GAM)-based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here, an advanced ultrasound-responsive GAM is described containing target cells that facilitates matrix-assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound-responsive GAMs consisting of fibrin/collagen hybrid-matrices containing microbubbles, bone morphogenetic protein BMP2/7 coexpression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immunohistochemical analysis for osteocalcin expression, MAS-stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7 days after treatment with ultrasound. At day 30 post-treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X-ray microcomputed tomography and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could be exploited to facilitate ex vivo gene transfer with subsequent surgical implantation or alternatively provide a minimally invasive means of stimulating in situ transgene delivery for osteoinductive gene-based therapies. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Insulated Foamy Viral Vectors

PubMed Central

Browning, Diana L.; Collins, Casey P.; Hocum, Jonah D.; Leap, David J.; Rae, Dustin T.; Trobridge, Grant D.

2016-01-01

Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34+ cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy. PMID:26715244

Ferritin heavy chain as a molecular imaging reporter gene in glioma xenografts.

PubMed

Cheng, Sen; Mi, Ruifang; Xu, Yu; Jin, Guishan; Zhang, Junwen; Zhou, Yiqiang; Chen, Zhengguang; Liu, Fusheng

2017-06-01

The development of glioma therapy in clinical practice (e.g., gene therapy) calls for efficiently visualizing and tracking glioma cells in vivo. Human ferritin heavy chain is a novel gene reporter in magnetic resonance imaging. This study proposes hFTH as a reporter gene for MR molecular imaging in glioma xenografts. Rat C6 glioma cells were infected by packaged lentivirus carrying hFTH and EGFP genes and obtained by fluorescence-activated cell sorting. The iron-loaded ability was analyzed by the total iron reagent kit. Glioma nude mouse models were established subcutaneously and intracranially. Then, in vivo tumor bioluminescence was performed via the IVIS spectrum imaging system. The MR imaging analysis was analyzed on a 7T animal MRI scanner. Finally, the expression of hFTH was analyzed by western blotting and histological analysis. Stable glioma cells carrying hFTH and EGFP reporter genes were successfully obtained. The intracellular iron concentration was increased without impairing the cell proliferation rate. Glioma cells overexpressing hFTH showed significantly decreased signal intensity on T 2 -weighted MRI both in vitro and in vivo. EGFP fluorescent imaging could also be detected in the subcutaneous and intracranial glioma xenografts. Moreover, the expression of the transferritin receptor was significantly increased in glioma cells carrying the hFTH reporter gene. Our study illustrated that hFTH generated cellular MR imaging contrast efficiently in glioma via regulating the expression of transferritin receptor. This might be a useful reporter gene in cell tracking and MR molecular imaging for glioma diagnosis, gene therapy and tumor metastasis.

HER2 activating mutations are targets for colorectal cancer treatment.

PubMed

Kavuri, Shyam M; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M; Migliardi, Giorgia; Searleman, Adam C; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A; Bertotti, Andrea; Bose, Ron

2015-08-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer. ©2015 American Association for Cancer Research.

Overcoming resistance to single-agent therapy for oncogenic BRAF gene fusions via combinatorial targeting of MAPK and PI3K/mTOR signaling pathways

PubMed Central

Jain, Payal; Silva, Amanda; Han, Harry J.; Lang, Shih-Shan; Zhu, Yuankun; Boucher, Katie; Smith, Tiffany E.; Vakil, Aesha; Diviney, Patrick; Choudhari, Namrata; Raman, Pichai; Busch, Christine M.; Delaney, Tim; Yang, Xiaodong; Olow, Aleksandra K.; Mueller, Sabine; Haas-Kogan, Daphne; Fox, Elizabeth; Storm, Phillip B.; Resnick, Adam C.; Waanders, Angela J.

2017-01-01

Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs. PMID:29156677

Hypoxia-activated chemotherapeutic TH-302 enhances the effects of VEGF-A inhibition and radiation on sarcomas.

PubMed

Yoon, C; Lee, H-J; Park, D J; Lee, Y-J; Tap, W D; Eisinger-Mathason, T S K; Hart, C P; Choy, E; Simon, M C; Yoon, S S

2015-06-30

Human sarcomas with a poor response to vascular endothelial growth factor-A (VEGF-A) inhibition and radiation therapy (RT) have upregulation of hypoxia-inducible factor 1α (HIF-1α) and HIF-1α target genes. This study examines the addition of the hypoxia-activated chemotherapy TH-302 to VEGF-A inhibition and RT (a.k.a. trimodality therapy). Trimodality therapy was examined in two xenograft models and in vitro in tumour endothelial cells and sarcoma cell lines. In both mouse models, VEGF-A inhibition and radiation showed greater efficacy than either therapy alone in slowing sarcoma growth. When TH-302 was added, this trimodality therapy completely blocked tumour growth with tumours remaining dormant for over 3 months after cessation of therapy. Trimodality therapy caused 2.6- to 6.2-fold more endothelial cell-specific apoptosis than bimodality therapies, and microvessel density and HIF-1α activity were reduced to 11-13% and 13-20% of control, respectively. When trimodality therapy was examined in vitro, increases in DNA damage and apoptosis were much more pronounced in tumour endothelial cells compared with that in sarcoma cells, especially under hypoxia. The combination of TH-302, VEGF-A inhibition, and RT is highly effective in preclinical models of sarcoma and is associated with increased DNA damage and apoptosis in endothelial cells and decreased HIF-1α activity.

Combined antitumor activity of the nitroreductase/CB1954 suicide gene system and γ-rays in HeLa cells in vitro

PubMed Central

Teng, Geling; Ju, Yuanrong; Yang, Yepeng; Hua, Hu; Chi, Jingyu; Mu, Xiuan

2016-01-01

Escherichia coli nitroreductase (NTR) may convert the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent, which may lead to DNA crosslinks and the apoptosis of cancer cells. NTR/CB1954 has been demonstrated to be an effective gene therapy in cancer cells. The present study examined whether the NTR/CB1954 suicide gene system had cytotoxic effects on HeLa cells and may improve the radiosensitivity of HeLa cells to γ-rays. It was observed that the NTR/CB1954 suicide gene system exerted marked cytotoxic effects on HeLa cells. The combined therapeutic effects of NTR/CB1954 and γ-rays on HeLa cells demonstrated a synergistic effect. CB1954 at concentrations of 12.5 and 25 µmol/l increased the sensitization enhancement ratio of HeLa cells to 1.54 and 1.66, respectively. Therefore, when compared with monotherapy, the combined therapy of NTR/CB1954 and γ-rays may increase the apoptotic rate and enhance the radiosensitivity of HeLa cells. The combined therapy of γ-ray radiation and the NTR/CB1954 suicide gene system may be a novel and potent therapeutic method for the treatment of cervical carcinoma. PMID:27840931

Combined antitumor activity of the nitroreductase/CB1954 suicide gene system and γ-rays in HeLa cells in vitro.

PubMed

Teng, Geling; Ju, Yuanrong; Yang, Yepeng; Hua, Hu; Chi, Jingyu; Mu, Xiuan

2016-12-01

Escherichia coli nitroreductase (NTR) may convert the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent, which may lead to DNA crosslinks and the apoptosis of cancer cells. NTR/CB1954 has been demonstrated to be an effective gene therapy in cancer cells. The present study examined whether the NTR/CB1954 suicide gene system had cytotoxic effects on HeLa cells and may improve the radiosensitivity of HeLa cells to γ‑rays. It was observed that the NTR/CB1954 suicide gene system exerted marked cytotoxic effects on HeLa cells. The combined therapeutic effects of NTR/CB1954 and γ‑rays on HeLa cells demonstrated a synergistic effect. CB1954 at concentrations of 12.5 and 25 µmol/l increased the sensitization enhancement ratio of HeLa cells to 1.54 and 1.66, respectively. Therefore, when compared with monotherapy, the combined therapy of NTR/CB1954 and γ‑rays may increase the apoptotic rate and enhance the radiosensitivity of HeLa cells. The combined therapy of γ‑ray radiation and the NTR/CB1954 suicide gene system may be a novel and potent therapeutic method for the treatment of cervical carcinoma.

Gene therapy in plastic surgery.

PubMed

Tepper, Oren M; Mehrara, Babak J

2002-02-01

Recent developments in gene therapy have shown promise in the treatment of soft-tissue repair, bone formation, nerve regeneration, and cranial suture development. This special topic article reviews commonly used methods of gene therapy and discusses their various advantages and disadvantages. In addition, an overview of new developments in gene therapy as they relate to plastic surgery is provided.

Alterations of immune response of non-small cell lung cancer with Azacytidine

PubMed Central

Easwaran, Hariharan; Mohammad, Helai P.; Vendetti, Frank; VanCriekinge, Wim; DeMeyer, Tim; Du, Zhengzong; Parsana, Princy; Rodgers, Kristen; Yen, Ray-Whay; Zahnow, Cynthia A.; Taube, Janis M.; Brahmer, Julie R.; Tykodi, Scott S.; Easton, Keith; Carvajal, Richard D.; Jones, Peter A.; Laird, Peter W.; Weisenberger, Daniel J.; Tsai, Salina; Juergens, Rosalyn A.; Topalian, Suzanne L.; Rudin, Charles M.; Brock, Malcolm V.; Pardoll, Drew; Baylin, Stephen B.

2013-01-01

Innovative therapies are needed for advanced Non-Small Cell Lung Cancer (NSCLC). We have undertaken a genomics based, hypothesis driving, approach to query an emerging potential that epigenetic therapy may sensitize to immune checkpoint therapy targeting PD-L1/PD-1 interaction. NSCLC cell lines were treated with the DNA hypomethylating agent azacytidine (AZA – Vidaza) and genes and pathways altered were mapped by genome-wide expression and DNA methylation analyses. AZA-induced pathways were analyzed in The Cancer Genome Atlas (TCGA) project by mapping the derived gene signatures in hundreds of lung adeno (LUAD) and squamous cell carcinoma (LUSC) samples. AZA up-regulates genes and pathways related to both innate and adaptive immunity and genes related to immune evasion in a several NSCLC lines. DNA hypermethylation and low expression of IRF7, an interferon transcription factor, tracks with this signature particularly in LUSC. In concert with these events, AZA up-regulates PD-L1 transcripts and protein, a key ligand-mediator of immune tolerance. Analysis of TCGA samples demonstrates that a significant proportion of primary NSCLC have low expression of AZA-induced immune genes, including PD-L1. We hypothesize that epigenetic therapy combined with blockade of immune checkpoints – in particular the PD-1/PD-L1 pathway – may augment response of NSCLC by shifting the balance between immune activation and immune inhibition, particularly in a subset of NSCLC with low expression of these pathways. Our studies define a biomarker strategy for response in a recently initiated trial to examine the potential of epigenetic therapy to sensitize patients with NSCLC to PD-1 immune checkpoint blockade. PMID:24162015

Vectors for Inhaled Gene Therapy in Lung Cancer. Application for Nano Oncology and Safety of Bio Nanotechnology

PubMed Central

Zarogouldis, Paul; Karamanos, Nikos K.; Porpodis, Konstantinos; Domvri, Kalliopi; Huang, Haidong; Hohenforst-Schimdt, Wolfgang; Goldberg, Eugene P.; Zarogoulidis, Konstantinos

2012-01-01

Novel aerosol therapeutic modalities have been investigated for lung cancer. Inhaled gene therapy has presented safety and effectiveness previously in cystic fibrosis. However, safety concerns have been raised regarding the safety of non-viral vectors for inhaled gene therapy in lung cancer, and therefore small steps have been made towards this multifunctional treatment modality. During the last decade, numerous new nanocomplexes have been created and investigated as a safe gene delivery nano-vehicle. These formulations are multifunctional; they can be used as either local therapy or carrier for an effective inhaled gene therapy for lung cancer. Herein, we present current and future perspectives of nanocomplexes for inhaled gene therapy treatment in lung cancer. PMID:23109824

All-in-one processing of heterogeneous human cell grafts for gene and cell therapy.

PubMed

Lukianova-Hleb, Ekaterina Y; Yvon, Eric S; Shpall, Elizabeth J; Lapotko, Dmitri O

2016-01-01

Current cell processing technologies for gene and cell therapies are often slow, expensive, labor intensive and are compromised by high cell losses and poor selectivity thus limiting the efficacy and availability of clinical cell therapies. We employ cell-specific on-demand mechanical intracellular impact from laser pulse-activated plasmonic nanobubbles (PNB) to process heterogeneous human cell grafts ex vivo with dual simultaneous functionality, the high cell type specificity, efficacy and processing rate for transfection of target CD3+ cells and elimination of subsets of unwanted CD25+ cells. The developed bulk flow PNB system selectively processed human cells at a rate of up to 100 million cell/minute, providing simultaneous transfection of CD3+ cells with the therapeutic gene (FKBP12(V36)-p30Caspase9) with the efficacy of 77% and viability 95% (versus 12 and 60%, respectively, for standard electroporation) and elimination of CD25+ cells with 99% efficacy. PNB flow technology can unite and replace several methodologies in an all-in-one universal ex vivo simultaneous procedure to precisely and rapidly prepare a cell graft for therapy. PNB's can process various cell systems including cord blood, stem cells, and bone marrow.

TALEN-mediated functional correction of human iPSC-derived macrophages in context of hereditary pulmonary alveolar proteinosis.

PubMed

Kuhn, Alexandra; Ackermann, Mania; Mussolino, Claudio; Cathomen, Toni; Lachmann, Nico; Moritz, Thomas

2017-11-09

Hereditary pulmonary alveolar proteinosis (herPAP) constitutes a rare, life threatening lung disease characterized by the inability of alveolar macrophages to clear the alveolar airspaces from surfactant phospholipids. On a molecular level, the disorder is defined by a defect in the CSF2RA gene coding for the GM-CSF receptor alpha-chain (CD116). As therapeutic options are limited, we currently pursue a cell and gene therapy approach aiming for the intrapulmonary transplantation of gene-corrected macrophages derived from herPAP-specific induced pluripotent stem cells (herPAP-iPSC) employing transcriptional activator-like effector nucleases (TALENs). Targeted insertion of a codon-optimized CSF2RA-cDNA driven by the hybrid cytomegalovirus (CMV) early enhancer/chicken beta actin (CAG) promoter into the AAVS1 locus resulted in robust expression of the CSF2RA gene in gene-edited herPAP-iPSCs as well as thereof derived macrophages. These macrophages displayed typical morphology, surface phenotype, phagocytic and secretory activity, as well as functional CSF2RA expression verified by STAT5 phosphorylation and GM-CSF uptake studies. Thus, our study provides a proof-of-concept, that TALEN-mediated integration of the CSF2RA gene into the AAVS1 safe harbor locus in patient-specific iPSCs represents an efficient strategy to generate functionally corrected monocytes/macrophages, which in the future may serve as a source for an autologous cell-based gene therapy for the treatment of herPAP.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

PubMed Central

Migita, M; Medin, J A; Pawliuk, R; Jacobson, S; Nagle, J W; Anderson, S; Amiri, M; Humphries, R K; Karlsson, S

1995-01-01

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected. Images Fig. 2 Fig. 3 PMID:8618847

Complete TCRα gene locus control region activity in T cells derived in vitro from embryonic stem cells

PubMed Central

Lahiji, Armin; Kučerová-Levisohn, Martina; Lovett, Jordana; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D.

2013-01-01

Locus Control Regions (LCR) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin’s gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. Herein we report manifestation of all key features of mouse T cell receptor (TCR)-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC). High level, copy number-related TCRα LCR-linked reporter gene expression levels are cell type-restricted in this system, and upregulated during the expected stage transition of T cell development. We further report that de novo introduction of TCRα LCR linked transgenes into existing T cell lines yields incomplete LCR activity. Together, these data indicate that establishing full TCRα LCR activity requires critical molecular events occurring prior to final T-lineage determination. This study additionally validates a novel, tractable and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering. PMID:23720809

Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

PubMed

Nance, Michael E; Duan, Dongsheng

2015-12-01

Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

Integration of High-Risk Human Papillomavirus into Cellular Cancer-Related Genes in Head and Neck Cancer Cell Lines

PubMed Central

Walline, Heather M; Komarck, Christine M; McHugh, Jonathan B; Tang, Alice L; Owen, John H; Teh, Bin T; McKean, Erin; Glover, Thomas; Graham, Martin P; Prince, Mark E; Chepeha, Douglas B; Chinn, Steven B; Ferris, Robert L; Gollin, Susanne M; Hoffmann, Thomas K; Bier, Henning; Brakenhoff, Ruud; Bradford, Carol R; Carey, Thomas E

2017-01-01

Background HPV-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. This study evaluates viral oncogene expression and viral integration sites in HPV16 and HPV18-positive squamous carcinoma cell lines. Methods E6-E7 alternate transcripts were assessed by RT-PCR. Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. Results All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 HNSCC lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. Conclusions HPV integration into cancer-related genes occurred in 7/9 HPV-positive cell lines and of these six were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. PMID:28236344

T-cell receptor gene therapy: critical parameters for clinical success.

PubMed

Linnemann, Carsten; Schumacher, Ton N M; Bendle, Gavin M

2011-09-01

T-cell receptor (TCR) gene therapy aims to induce immune reactivity against tumors by introducing genes encoding a tumor-reactive TCR into patient T cells. This approach has been extensively tested in preclinical mouse models, and initial clinical trials have demonstrated the feasibility and potential of TCR gene therapy as a cancer treatment. However, data obtained from preclinical and clinical studies suggest that both the therapeutic efficacy and the safety of TCR gene therapy can be and needs to be further enhanced. This review highlights those strategies that can be followed to develop TCR gene therapy into a clinically relevant treatment option for cancer patients.

Enhanced EJ Cell Killing of (125)I Radiation by Combining with Cytosine Deaminase Gene Therapy Regulated by Synthetic Radio-Responsive Promoter.

PubMed

Li, Ling; Zhang, Chun-li; Kang, Lei; Wang, Rong-Fu; Yan, Ping; Zhao, Qian; Yin, Lei; Guo, Feng-qin

2015-10-01

To investigate the enhancing effect of radionuclide therapy by the therapeutic gene placed under the control of radio-responsive promoter. The recombinant lentivirus E8-codA-GFP, including a synthetic radiation-sensitive promoter E8, cytosine deaminase (CD) gene, and green fluorescent protein gene, was constructed. The gene expression activated by (125)I radiation was assessed by observation of green fluorescence. The ability of converting 5-fluorocytosine (5-FC) to 5-fluorourial (5-FU) by CD enzyme was assessed by high-performance liquid chromatography. The viability of the infected cells exposed to (125)I in the presence of 5-FC was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the infected cells exposed to (125)I alone served as negative control and 5-FU as positive control. The recombinant lentiviral vector was constructed successfully. On exposure of infected cells to (125)I, green fluorescence can be observed and 5-FU can be detected. MTT assay showed that the survival rate for infected cells treated with (125)I was lower compared with the (125)I control group, but higher than the positive control group. The synthetic promoter E8 can induce the expression of downstream CD gene under (125)I radiation, and the tumor killing effect of (125)I can be enhanced by combining CD gene therapy with radiosensitive promoter.

Converting cancer genes into killer genes.

PubMed Central

Da Costa, L T; Jen, J; He, T C; Chan, T A; Kinzler, K W; Vogelstein, B

1996-01-01

Over the past decade, it has become clear that tumorigenesis is driven by alterations in genes that control cell growth or cell death. Theoretically, the proteins encoded by these genes provide excellent targets for new therapeutic agents. Here, we describe a gene therapy approach to specifically kill tumor cells expressing such oncoproteins. In outline, the target oncoprotein binds to exogenously introduced gene products, resulting in transcriptional activation of a toxic gene. As an example, we show that this approach can be used to specifically kill cells overexpressing a mutant p53 gene in cell culture. The strategy may be generally applicable to neoplastic diseases in which the underlying patterns of genetic alterations or abnormal gene expression are known. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8633039

Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma: an innovative approach for cancer therapy.

PubMed Central

Huber, B E; Richards, C A; Krenitsky, T A

1991-01-01

An approach involving retroviral-mediated gene therapy for the treatment of neoplastic disease is described. This therapeutic approach is called "virus-directed enzyme/prodrug therapy" (VDEPT). The VDEPT approach exploits the transcriptional differences between normal and neoplastic cells to achieve selective killing of neoplastic cells. We now describe development of the VDEPT approach for the treatment of hepatocellular carcinoma. Replication-defective, amphotrophic retroviruses were constructed containing a chimeric varicella-zoster virus thymidine kinase (VZV TK) gene that is transcriptionally regulated by either the hepatoma-associated alpha-fetoprotein or liver-associated albumin transcriptional regulatory sequences. Subsequent to retroviral infection, expression of VZV TK was limited to either alpha-fetoprotein- or albumin-positive cells, respectively. VZV TK metabolically activated the nontoxic prodrug 6-methoxypurine arabinonucleoside (araM), ultimately leading to the formation of the cytotoxic anabolite adenine arabinonucleoside triphosphate (araATP). Cells that selectively expressed VZV TK became selectively sensitive to araM due to the VZV TK-dependent anabolism of araM to araATP. Hence, these retroviral-delivered chimeric genes generated tissue-specific expression of VZV TK, tissue-specific anabolism of araM to araATP, and tissue-specific cytotoxicity due to araM exposure. By utilizing such retroviral vectors, araM was anabolized to araATP in hepatoma cells, producing a selective cytotoxic effect. Images PMID:1654555

Gene therapy for sickle cell disease.

PubMed

Olowoyeye, Abiola; Okwundu, Charles I

2016-11-14

Sickle cell disease encompasses a group of genetic disorders characterized by the presence of at least one hemoglobin S (Hb S) allele, and a second abnormal allele that could allow abnormal hemoglobin polymerisation leading to a symptomatic disorder.Autosomal recessive disorders (such as sickle cell disease) are good candidates for gene therapy because a normal phenotype can be restored in diseased cells with only a single normal copy of the mutant gene. This is an update of a previously published Cochrane Review. The objectives of this review are:to determine whether gene therapy can improve survival and prevent symptoms and complications associated with sickle cell disease;to examine the risks of gene therapy against the potential long-term gain for people with sickle cell disease. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Haemoglobinopathies Trials Register, which comprises of references identified from comprehensive electronic database searches and searching relevant journals and abstract books of conference proceedings.Date of the most recent search of the Group's Haemoglobinopathies Trials Register: 15 August 2016. All randomised or quasi-randomised clinical trials (including any relevant phase 1, 2 or 3 trials) of gene therapy for all individuals with sickle cell disease, regardless of age or setting. No trials of gene therapy for sickle cell disease were found. No trials of gene therapy for sickle cell disease were reported. No randomised or quasi-randomised clinical trials of gene therapy for sickle cell disease were reported. Thus, no objective conclusions or recommendations in practice can be made on gene therapy for sickle cell disease. This systematic review has identified the need for well-designed, randomised controlled trials to assess the benefits and risks of gene therapy for sickle cell disease.

Gene therapy for sickle cell disease.

PubMed

Olowoyeye, Abiola; Okwundu, Charles I

2014-10-10

Sickle cell disease encompasses a group of genetic disorders characterized by the presence of at least one hemoglobin S (Hb S) allele, and a second abnormal allele that could allow abnormal hemoglobin polymerisation leading to a symptomatic disorder.Autosomal recessive disorders (such as sickle cell disease) are good candidates for gene therapy because a normal phenotype can be restored in diseased cells with only a single normal copy of the mutant gene. The objectives of this review are:- to determine whether gene therapy can improve survival and prevent symptoms and complications associated with sickle cell disease;- to examine the risks of gene therapy against the potential long-term gain for people with sickle cell disease. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Haemoglobinopathies Trials Register, which comprises of references identified from comprehensive electronic database searches and searching relevant journals and abstract books of conference proceedings.Date of the most recent search of the Group's Haemoglobinopathies Trials Register: 21 July 2014. All randomised or quasi-randomised clinical trials (including any relevant phase 1, 2 or 3 trials) of gene therapy for all individuals with sickle cell disease, regardless of age or setting. No trials of gene therapy for sickle cell disease were found. No trials of gene therapy for sickle cell disease were reported. No randomised or quasi-randomised clinical trials of gene therapy for sickle cell disease were reported. Thus, no objective conclusions or recommendations in practice can be made on gene therapy for sickle cell disease. This systematic review has identified the need for well-designed, randomised controlled trials to assess the benefits and risks of gene therapy for sickle cell disease.

Duchenne Muscular Dystrophy Gene Therapy in the Canine Model

PubMed Central

2015-01-01

Abstract Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disease caused by dystrophin deficiency. Gene therapy has significantly improved the outcome of dystrophin-deficient mice. Yet, clinical translation has not resulted in the expected benefits in human patients. This translational gap is largely because of the insufficient modeling of DMD in mice. Specifically, mice lacking dystrophin show minimum dystrophic symptoms, and they do not respond to the gene therapy vector in the same way as human patients do. Further, the size of a mouse is hundredfolds smaller than a boy, making it impossible to scale-up gene therapy in a mouse model. None of these limitations exist in the canine DMD (cDMD) model. For this reason, cDMD dogs have been considered a highly valuable platform to test experimental DMD gene therapy. Over the last three decades, a variety of gene therapy approaches have been evaluated in cDMD dogs using a number of nonviral and viral vectors. These studies have provided critical insight for the development of an effective gene therapy protocol in human patients. This review discusses the history, current status, and future directions of the DMD gene therapy in the canine model. PMID:25710459

[Clinical and genetic analysis for two children with congenital disturbance of glycosylation with PMM2 gene mutations].

PubMed

Ren, Changhong; Fang, Fang; Huang, Yu; Cheng, Hua; Dai, Lifang

2015-12-01

To analyze the clinical and PMM2 gene mutation features of congenital disturbance of glycosylation caused by PMM2 gene mutation (PMM2-CDG, previously known as CDG 1a). The clinical data of two Chinese patients who were clinically diagnosed as PMM2-CDG at neurology department of Beijing Children's Hospital in 2012 were retrospectively collected. The gene mutations were identified by Sanger sequencing. Both patients were female, aged 1 year and 1 month and 8 months respectively. The main clinical features of the two cases were developmental delay after birth, chronic diarrhea and metabolic acidosis, associated with elevated serum transaminases, and decreased antithrombin III activity. Physical examination showed esotropia, inverted nipples, and abnormal subcutaneous fat pads. The cranial MRI showed cerebellar atrophy. Both cases were treated with occupational therapy, physical therapy and speech therapy. The development was gradually improved but also delayed as compared with normal peers during follow-up for more than 3 years. Genetic analysis showed that patient 1 was compound heterozygous for c. 422G>A(p.Arg141His), which was reported for known pathogenic mutation, and c. 669C>A(p.Asp223Glu), was a new mutation. The patient 2 showed compound heterozygous mutation for c. 634A>G (p.Met212Val)and c. 713G>C(p.Arg238Pro), which were both new mutations. PMM2-CDG is a rare metabolic disease, and the diagnosis should be considered in a child with developmental delay, elevated serum transaminases, decreased antithrombin III activity, inverted nipples, abnormal subcutaneous fat pads, esotropia, and cerebellar atrophy on MRI. It can be confirmed by PMM2 gene analysis.

Glutathione-responsive nano-transporter-mediated siRNA delivery: silencing the mRNA expression of Ras.

PubMed

Doss, C George Priya; Debottam, S; Debajyoti, C

2013-06-01

Gene therapy through antisense technology via intracellular delivery of a gene-silencing element is a promising approach to treat critical diseases like cancers. Ras acts as molecular switch, considered as one of the proto-oncogenes whose modification or mutation may promote tumor formation. The recent trends of nano-carrier-based drug delivery have gained superiority and proved to be 100 times more potent in drug delivery compared to standard therapies. The nano-based drug delivery has provided the basis of achieving successful target-specific drug delivery. Glutathione (GSH) is considered as one of the best and ubiquitous internal stimulus for swift destabilization of nano-transporters inside cells to accomplish proficient intracellular drug release. This concept has given a new hope to oncologists of modifying the existing drugs to be delivered to their desired destination. RNA interference is a primary tool in functional genomics to selectively silence messenger RNA (mRNA) expression, which can be exploited quickly to develop novel drugs against lethal disease target. Silencing of mRNA molecules using siRNA has also come of age to become one of the latest weapons developed in the concept of gene therapy. However, this strategy has severely failed to achieve target specificity especially to a tumor cell. In this context, we have proposed the incorporation of an antisense siRNA packed inside a GSH-responsive nano-transporter to be delivered specifically to a tumor cell against the sense mRNA of the Ras protein. It will limit the Ras-mediated activation of other proteins and transcription factors. Thus, it will knock down several differential gene expressions being regulated by Ras-activated pathways like enzyme-linked receptor kinase pathway. Henceforth, gene silencing technology through nano-drug delivery can be combined as a single weapon to terminate malignancy.

Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

PubMed

Rohmer, Stanimira; Mainka, Astrid; Knippertz, Ilka; Hesse, Andrea; Nettelbeck, Dirk M

2008-04-01

Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication-deficient or more recently by conditionally replication-competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat-regulated transcription control as a promising tool for this purpose. We investigated the feasibility of a short fragment of the human hsp70B' promoter, with and without upstream insulator elements, for the regulation of transgene expression by replication-deficient or oncolytic adenoviruses. Two novel adenoviral vectors with an insulated hsp70B' promoter were developed and showed stringent heat-inducible gene expression with induction ratios up to 8000-fold. In contrast, regulation of gene expression from the hsp70B' promoter without insulation was suboptimal. In replication-competent/oncolytic adenoviruses regulation of the hsp70B' promoter was lost specifically during late replication in permissive cells and could not be restored by the insulators. We developed novel adenovirus gene transfer vectors that feature improved and stringent regulation of transgene expression from the hsp70B' promoter using promoter insulation. These vectors have potential for gene therapy applications that benefit from external modulation of therapeutic gene expression or for combination therapy with hyperthermia. Furthermore, our study reveals that vector replication can deregulate inserted cellular promoters, an observation which is of relevance for the development of replication-competent/oncolytic gene transfer vectors. (c) 2008 John Wiley & Sons, Ltd.

Inhibitory effect of Survivin promoter-regulated oncolytic adenovirus carrying P53 gene against gallbladder cancer.

PubMed

Liu, Chen; Sun, Bin; An, Ni; Tan, Weifeng; Cao, Lu; Luo, Xiangji; Yu, Yong; Feng, Feiling; Li, Bin; Wu, Mengchao; Su, Changqing; Jiang, Xiaoqing

2011-12-01

Gene therapy has become an important strategy for treatment of malignancies, but problems remains concerning the low gene transferring efficiency, poor transgene expression and limited targeting specific tumors, which have greatly hampered the clinical application of tumor gene therapy. Gallbladder cancer is characterized by rapid progress, poor prognosis, and aberrantly high expression of Survivin. In the present study, we used a human tumor-specific Survivin promoter-regulated oncolytic adenovirus vector carrying P53 gene, whose anti-cancer effect has been widely confirmed, to construct a wide spectrum, specific, safe, effective gene-viral therapy system, AdSurp-P53. Examining expression of enhanced green fluorecent protein (EGFP), E1A and the target gene P53 in the oncolytic adenovirus system validated that Survivin promoter-regulated oncolytic adenovirus had high proliferation activity and high P53 expression in Survivin-positive gallbladder cancer cells. Our in vitro cytotoxicity experiment demonstrated that AdSurp-P53 possessed a stronger cytotoxic effect against gallbladder cancer cells and hepatic cancer cells. The survival rate of EH-GB1 cells was lower than 40% after infection of AdSurp-P53 at multiplicity of infection (MOI) = 1 pfu/cell, while the rate was higher than 90% after infection of Ad-P53 at the same MOI, demonstrating that AdSurp-P53 has a potent cytotoxicity against EH-GB1 cells. The tumor growth was greatly inhibited in nude mice bearing EH-GB1 xenografts when the total dose of AdSurp-P53 was 1 × 10(9) pfu, and terminal dUTP nick end-labeling (TUNEL) revealed that the apoptotic rate of cancer cells was (33.4 ± 8.4)%. This oncolytic adenovirus system overcomes the long-standing shortcomings of gene therapy: poor transgene expression and targeting of only specific tumors, with its therapeutic effect better than the traditional Ad-P53 therapy regimen already on market; our system might be used for patients with advanced gallbladder cancer and other cancers, who are not sensitive to chemotherapy, radiotherapy, or who lost their chance for surgical treatment. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A snapshot of gene therapy in Latin America.

PubMed

Linden, Rafael; Matte, Ursula

2014-03-01

Gene therapy attempts the insertion and expression of exogenous genetic material in cells for therapeutic purposes. Conceived in the 1960s, gene therapy reached its first clinical trial at the end of the 1980s and by December 2013 around 600 genuine open clinical trials of gene therapy were registered at NIH Clinical Trials Database. Here, we summarize the current efforts towards the development of gene therapy in Latin America. Our survey shows that the number of scientists involved in the development of gene therapy and DNA vaccines in Latin America is still very low. Higher levels of investment in this technology are necessary to boost the advancement of innovation and intellectual property in this field in a way that would ease both the social and financial burden of various medical conditions in Latin America.

Type 1 Diabetes Mellitus Donor Mesenchymal Stromal Cells Exhibit Comparable Potency to Healthy Controls In Vitro.

PubMed

Davies, Lindsay C; Alm, Jessica J; Heldring, Nina; Moll, Guido; Gavin, Caroline; Batsis, Ioannis; Qian, Hong; Sigvardsson, Mikael; Nilsson, Bo; Kyllonen, Lauri E; Salmela, Kaija T; Carlsson, Per-Ola; Korsgren, Olle; Le Blanc, Katarina

2016-11-01

: Bone marrow mesenchymal stromal cells (BM-MSCs) have been characterized and used in many clinical studies based on their immunomodulatory and regenerative properties. We have recently reported the benefit of autologous MSC systemic therapy in the treatment of type 1 diabetes mellitus (T1D). Compared with allogeneic cells, use of autologous products reduces the risk of eliciting undesired complications in the recipient, including rejection, immunization, and transmission of viruses and prions; however, comparable potency of autologous cells is required for this treatment approach to remain feasible. To date, no analysis has been reported that phenotypically and functionally characterizes MSCs derived from newly diagnosed and late-stage T1D donors in vitro with respect to their suitability for systemic immunotherapy. In this study, we used gene array in combination with functional in vitro assays to address these questions. MSCs from T1D donors and healthy controls were expanded from BM aspirates. BM mononuclear cell counts and growth kinetics were comparable between the groups, with equivalent colony-forming unit-fibroblast capacity. Gene microarrays demonstrated differential gene expression between healthy and late-stage T1D donors in relation to cytokine secretion, immunomodulatory activity, and wound healing potential. Despite transcriptional differences, T1D MSCs did not demonstrate a significant difference from healthy controls in immunosuppressive activity, migratory capacity, or hemocompatibility. We conclude that despite differential gene expression, expanded MSCs from T1D donors are phenotypically and functionally similar to healthy control MSCs with regard to their immunomodulatory and migratory potential, indicating their suitability for use in autologous systemic therapy. The potential for mesenchymal stromal cells (MSCs) as a cell-based therapy in the treatment of immunologic disorders has been well established. Recent studies reported the clinical potential for autologous MSCs as a systemic therapy in the treatment of type I diabetes mellitus (T1D). The current study compared the genotypic and phenotypic profiles of bone marrow-derived MSCs from T1D and healthy donors as autologous (compared with allogeneic) therapy provides distinct advantages, such as reduced risk of immune reaction and transmission of infectious agents. The findings of the current study demonstrate that despite moderate differences in T1D MSCs at the gene level, these cells can be expanded in culture to an extent corresponding to that of MSCs derived from healthy donors. No functional difference in terms of immunosuppressive activity, blood compatibility, or migratory capacity was evident between the groups. The study findings also show that autologous MSC therapy holds promise as a T1D treatment and should be evaluated further in clinical trials. ©AlphaMed Press.

Gene therapy of the brain: the trans-vascular approach.

PubMed

Schlachetzki, Felix; Zhang, Yun; Boado, Ruben J; Pardridge, William M

2004-04-27

Many chronic neurologic diseases do not respond to small molecule therapeutics, and have no effective long-term therapy. Gene therapy offers the promise of an effective cure for both genetic and acquired brain disease. However, the limiting problem in brain gene therapy is delivery to brain followed by regulation of the expression of the transgene. Present day gene vectors do not cross the blood-brain barrier (BBB). Consequently, brain gene therapy requires craniotomy and the local injection of a viral gene vector. However, there are few brain disorders that can be effectively treated with local injection. Most applications of gene therapy require global expression in the brain of the exogenous gene, and this can only be achieved with a noninvasive delivery through the BBB--the trans-vascular route to brain. An additional consideration is the potential toxicity of all viral and nonviral approaches, which may either integrate into the host genome and cause insertional mutagenesis or cause inflammation in the brain. Nonviral, noninvasive gene therapy of the brain is now possible with the development of a new approach to targeting therapeutic genes to the brain following an IV administration. This approach utilizes genetically engineered molecular Trojan horses, which ferry the gene across the BBB and into neurons. Global and reversible expression of therapeutic genes in the human brain without surgery and without viral vectors is now possible.

Targeted enzyme prodrug therapies.

PubMed

Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

2010-09-01

The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

Progresses towards safe and efficient gene therapy vectors.

PubMed

Chira, Sergiu; Jackson, Carlo S; Oprea, Iulian; Ozturk, Ferhat; Pepper, Michael S; Diaconu, Iulia; Braicu, Cornelia; Raduly, Lajos-Zsolt; Calin, George A; Berindan-Neagoe, Ioana

2015-10-13

The emergence of genetic engineering at the beginning of the 1970's opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.

Progresses towards safe and efficient gene therapy vectors

PubMed Central

Chira, Sergiu; Jackson, Carlo S.; Oprea, Iulian; Ozturk, Ferhat; Pepper, Michael S.; Diaconu, Iulia; Braicu, Cornelia; Raduly, Lajos-Zsolt; Calin, George A.; Berindan-Neagoe, Ioana

2015-01-01

The emergence of genetic engineering at the beginning of the 1970′s opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors. PMID:26362400

[Current strategies in the treatment of renal-cell cancer: targeted therapies].

PubMed

Trigo, José Manuel; Bellmunt, Joaquim

2008-03-22

Renal-cell carcinoma represents 95% of all renal tumours. The Von Hippel-Lindau (VHL) tumor-suppressor gene is mutated or silenced in most clear cell renal carcinomas. pVHL loss results in the stabilization of the heterodimeric transcription factor hypoxia-inducible factor (HIF) and enhanced transactivation of HIF target genes. HIF itself has been difficult to inhibit with drug-like molecules although a number of agents that indirectly inhibit HIF, including mTOR (mammalian target of rapamycin) inhibitors, have been identified. Moreover, a number of drugs have been developed that target HIF-responsive gene products, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), implicated in tumor angiogenesis. Many of these targeted therapies, especially sunitinib, have demonstrated significant activity in kidney cancer clinical trials and represent a substantive advance in the treatment of this disease.

Radiation Gene-expression Signatures in Primary Breast Cancer Cells.

PubMed

Minafra, Luigi; Bravatà, Valentina; Cammarata, Francesco P; Russo, Giorgio; Gilardi, Maria C; Forte, Giusi I

2018-05-01

In breast cancer (BC) care, radiation therapy (RT) is an efficient treatment to control localized tumor. Radiobiological research is needed to understand molecular differences that affect radiosensitivity of different tumor subtypes and the response variability. The aim of this study was to analyze gene expression profiling (GEP) in primary BC cells following irradiation with doses of 9 Gy and 23 Gy delivered by intraoperative electron radiation therapy (IOERT) in order to define gene signatures of response to high doses of ionizing radiation. We performed GEP by cDNA microarrays and evaluated cell survival after IOERT treatment in primary BC cell cultures. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to validate candidate genes. We showed, for the first time, a 4-gene and a 6-gene signature, as new molecular biomarkers, in two primary BC cell cultures after exposure at 9 Gy and 23 Gy respectively, for which we observed a significantly high survival rate. Gene signatures activated by different doses of ionizing radiation may predict response to RT and contribute to defining a personalized biological-driven treatment plan. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Twenty Years of European Union Support to Gene Therapy and Gene Transfer.

PubMed

Gancberg, David

2017-11-01

For 20 years and throughout its research programmes, the European Union has supported the entire innovation chain for gene transfer and gene therapy. The fruits of this investment are ripening as gene therapy products are reaching the European market and as clinical trials are demonstrating the safety of this approach to treat previously untreatable diseases.

B-Cell Maturation Antigen, A Proliferation-Inducing Ligand, and B-Cell Activating Factor Are Candidate Mediators of Spinal Cord Injury-Induced Autoimmunity

PubMed Central

Saltzman, Jonah W.; Battaglino, Ricardo A.; Salles, Loise; Jha, Prateek; Sudhakar, Supreetha; Garshick, Eric; Stott, Helen L.; Zafonte, Ross

2013-01-01

Abstract Autoimmunity is thought to contribute to poor neurological outcomes after spinal cord injury (SCI). There are few mechanism-based therapies, however, designed to reduce tissue damage and neurotoxicity after SCI because the molecular and cellular bases for SCI-induced autoimmunity are not completely understood. Recent groundbreaking studies in rodents indicate that B cells are responsible for SCI-induced autoimmunity. This novel paradigm, if confirmed in humans, could aid in the design of neuroprotective immunotherapies. The aim of this study was to investigate the molecular signaling pathways and mechanisms by which autoimmunity is induced after SCI, with the goal of identifying potential targets in therapies designed to reduce tissue damage and inflammation in the chronic phase of SCI. To that end, we performed an exploratory microarray analysis of peripheral blood mononuclear cells to identify differentially expressed genes in chronic SCI. We identified a gene network associated with lymphoid tissue structure and development that was composed of 29 distinct molecules and five protein complexes, including two cytokines, a proliferation-inducing ligand (APRIL) and B-cell–activating factor (BAFF), and one receptor, B-cell maturation antigen (BMCA) involved in B cell development, proliferation, activation, and survival. Real-time polymerase chain reaction analysis from ribonucleic acid samples confirmed upregulation of these three genes in SCI. To our knowledge, this is the first report that peripheral blood mononuclear cells produce increased levels of BAFF and APRIL in chronic SCI. This finding provides evidence of systemic regulation of SCI-autoimmunity via APRIL and BAFF mediated activation of B cells through BMCA and points toward these molecules as potential targets of therapies designed to reduce neuroinflammation after SCI. PMID:23088438

Positron emission tomography and gene therapy: basic concepts and experimental approaches for in vivo gene expression imaging.

PubMed

Peñuelas, Iván; Boán, JoséF; Martí-Climent, Josep M; Sangro, Bruno; Mazzolini, Guillermo; Prieto, Jesús; Richter, José A

2004-01-01

More than two decades of intense research have allowed gene therapy to move from the laboratory to the clinical setting, where its use for the treatment of human pathologies has been considerably increased in the last years. However, many crucial questions remain to be solved in this challenging field. In vivo imaging with positron emission tomography (PET) by combination of the appropriate PET reporter gene and PET reporter probe could provide invaluable qualitative and quantitative information to answer multiple unsolved questions about gene therapy. PET imaging could be used to define parameters not available by other techniques that are of substantial interest not only for the proper understanding of the gene therapy process, but also for its future development and clinical application in humans. This review focuses on the molecular biology basis of gene therapy and molecular imaging, describing the fundamentals of in vivo gene expression imaging by PET, and the application of PET to gene therapy, as a technology that can be used in many different ways. It could be applied to avoid invasive procedures for gene therapy monitoring; accurately diagnose the pathology for better planning of the most adequate therapeutic approach; as treatment evaluation to image the functional effects of gene therapy at the biochemical level; as a quantitative noninvasive way to monitor the location, magnitude and persistence of gene expression over time; and would also help to a better understanding of vector biology and pharmacology devoted to the development of safer and more efficient vectors.

Advances in the Engineering of the Gene Editing Enzymes and the Genomes: Understanding and Handling the Off-Target Effects of CRISPR/Cas9.

PubMed

Yin, Yufang; Wang, Qian; Xiao, Li; Wang, Fengjiao; Song, Zhuo; Zhou, Cuilan; Liu, Xuan; Xing, Chungen; He, Nongyue; Li, Kai; Feng, Yan; Zhang, Jia

2018-03-01

In the past decades, significant progresses have been achieved in genetic engineering of nucleases. Among the genetically engineered nucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases, and CRIPSPR/Cas9 system form a new field of gene editing. The gene editing efficiency or targeting effect and the off-target effect are the two major determinant factors in evaluating the usefulness of a new enzyme. Engineering strategies in improving these gene editing enzymes, particularly in minimizing their off-target effects, are the focus of this paper. Examples of using these genetically engineered enzymes in genome modification are discussed in order to better understand the requirement of engineering efforts in obtaining more powerful and useful gene editing enzymes. In addition, the identification of naturally existed anti-Cas proteins has been employed in minimizing off-target effects. Considering the future application in human gene therapy, optimization of these well recognized gene editing enzymes and exploration of more novel enzymes are both required. Before people find an ideal gene editing system having virtually no off-target effect, technologies used to screen and identify off-target effects are of importance in clinical trials employing gene therapy.

[Clinical Tests Testing New Therapies for Stargardt Disease].

PubMed

Kousal, B; Ďuďáková, Ľ; Hlavatá, L; Lišková, P

2016-02-01

To provide information on currently ongoing clinical trials for Stargardt disease. We have searched the clinical trial register (www.clinicaltrials.gov) for the keyword "Stargardt" and list active ongoing studies. There are currently eight registered clinical trials enrolling patients with Stargardt disease; all in phase I or II aiming at four mechanisms of action: inhibition of the production of vitamin A toxic dimers, gene therapy restoring wild type transcription of the ABCA4 gene, neuroprotection preventing retinal cells from oxidative damage, and replacement of the damaged retinal pigment epithelium using stem cell therapy. The basic prerequisite for enrolment in the vast majority of clinical trials is confirmation of the clinical diagnosis by mutational analysis. The wide variety of therapies that are registered as clinical trials for Stargardt disease significantly raises the possibility that effective treatments will be available in the near future for this currently incurable condition and that molecular genetic testing should be increasingly considered. Stargardt disease, clinical trial, ABCA4, mutation.

Preclinical and clinical experience in vascular gene therapy: advantages over conservative/standard therapy.

PubMed

Nikol, S; Huehns, T Y

2001-04-01

No systemic pharmacological treatment has been shown to convincingly reduce the incidence of restenosis after angioplasty or increase the formation of collaterals in ischemic tissue in patients. The lack of success of many pharmaceutical agents in reducing restenosis rates or in inducing angiogenesis post-angioplasty and following stent implantation has encouraged the development of new technological treatment approaches. Gene therapy is a novel strategy with the potential to prevent some of the sequelae after arterial injury, particularly cell proliferation, and to induce growth of new vessels or remodeling of pre-existing vessel branches, which may help patients with critical ischemia. Gene therapy strategies have the advantage of minimizing systemic side effects and may have a long-term effect as the encoded protein is released. Most clinical trials investigating gene therapy for vascular disease have been uncontrolled phase I and IIa trials. Gene therapy into vessels with the genes for growth factors has been demonstrated to be feasible and efficient. Local drug delivery devices have been used in combination with gene therapy in several trials to maximize safety and efficiency. Data from experimental animal work indicates that gene therapy may modify intimal hyperplasia after arterial injury, but there are few clinical trials on restenosis in patients. Preliminary clinical results show only limited success in altering restenosis rates. In vitro and experimental in vivo investigations into gene therapy for angiogenesis demonstrate increased formation of collaterals and functional improvement of limb ischemia. There is some evidence of increased collateral formation and clinical improvement in patients with critical limb ischemia. Results of placebo-controlled and double-blind trials of gene therapy for vascular disease are awaited.

Human Gene Therapy: Genes without Frontiers?

ERIC Educational Resources Information Center

Simon, Eric J.

2002-01-01

Describes the latest advancements and setbacks in human gene therapy to provide reference material for biology teachers to use in their science classes. Focuses on basic concepts such as recombinant DNA technology, and provides examples of human gene therapy such as severe combined immunodeficiency syndrome, familial hypercholesterolemia, and…

Phage-Mediated Gene Therapy.

PubMed

Hosseinidoust, Zeinab

2017-01-01

Bacteriophages (bacterial viruses) have long been under investigation as vectors for gene therapy. Similar to other viral vectors, the phage coat proteins have evolved over millions of years to protect the viral genome from degradation post injection, offering protection for the valuable therapeutic sequence. However, what sets phage apart from other viral gene delivery vectors is their safety for human use and the relative ease by which foreign molecules can be expressed on the phage outer surface, enabling highly targeted gene delivery. The latter property also makes phage a popular choice for gene therapy target discovery through directed evolution. Although promising, phage-mediated gene therapy faces several outstanding challenges, the most notable being lower gene delivery efficiency compared to animal viruses, vector stability, and nondesirable immune stimulation. This review presents a critical review of promises and challenges of employing phage as gene delivery vehicles as well as an introduction to the concept of phage-based microbiome therapy as the new frontier and perhaps the most promising application of phage-based gene therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Gene therapy on the move

PubMed Central

Kaufmann, Kerstin B; Büning, Hildegard; Galy, Anne; Schambach, Axel; Grez, Manuel

2013-01-01

The first gene therapy clinical trials were initiated more than two decades ago. In the early days, gene therapy shared the fate of many experimental medicine approaches and was impeded by the occurrence of severe side effects in a few treated patients. The understanding of the molecular and cellular mechanisms leading to treatment- and/or vector-associated setbacks has resulted in the development of highly sophisticated gene transfer tools with improved safety and therapeutic efficacy. Employing these advanced tools, a series of Phase I/II trials were started in the past few years with excellent clinical results and no side effects reported so far. Moreover, highly efficient gene targeting strategies and site-directed gene editing technologies have been developed and applied clinically. With more than 1900 clinical trials to date, gene therapy has moved from a vision to clinical reality. This review focuses on the application of gene therapy for the correction of inherited diseases, the limitations and drawbacks encountered in some of the early clinical trials and the revival of gene therapy as a powerful treatment option for the correction of monogenic disorders. PMID:24106209

Synthesis of galactosyl compounds for targeted gene delivery.

PubMed

Ren, T; Zhang, G; Liu, D

2001-11-01

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.

STAT3/NF-κB-Regulated Lentiviral TK/GCV Suicide Gene Therapy for Cisplatin-Resistant Triple-Negative Breast Cancer

PubMed Central

Kuo, Wei-Ying; Hwu, Luen; Wu, Chun-Yi; Lee, Jhih-Shian; Chang, Chi-Wei; Liu, Ren-Shyan

2017-01-01

Triple-negative breast cancer (TNBC) represents approximately 20% of all breast cancers and appears resistance to conventional cytotoxic chemotherapy, demonstrating a particularly poor prognosis and a significantly worse clinical outcome than other types of cancer. Suicide gene therapy has been used for the in vivo treatment of various solid tumors in recent clinical trials. In tumor microenvironment, STAT3/NF-κB pathways are constitutively activated in stromal cells as well as in cancer stem cells (CSCs). In this study, we have cloned a novel STAT3/NF-κB-based reporter system to drive the expression of herpes simplex virus thymidine kinase (HSV-TK) against breast cancer. Lentiviral vector expressing HSV-TK under the regulation of STAT3/NF-κB fused response element was developed. In this setting, we exploited the constitutive STAT3/NF-κB activation in tumors to achieve higher transgene expression than that driven by a constitutively active CMV promotor in vivo. An orthotropic MDA-MB-231 triple negative breast cancer mouse model was used for evaluating the feasibility of STAT3-NF-κB-TK/GCV suicide gene therapy system. The basal promoter activity of Lenti-CMV-TK and Lenti-STAT3-NF-κB-TK in MDA-MB-231 cells was compared by 3H-FEAU uptake assay. The Lenti-CMV-TK showed ~5 fold higher 3H-FEAU uptake then Lenti -STAT3-NF-κB-TK. In clonogenic assay, cells expressing Lenti-CMV-TK were 2-fold more sensitive to GCV than Lenti-STAT3-NF-κB-TK transduced cells. In vitro effect of STAT3-NF-κB-induced transgene expression was determined by 10ng/mL TNF-α induction and confirmed by western blot analysis and DsRedm fluorescent microscopy. In vivo evaluation of therapeutic effect by bioluminescence and [18F]FHBG microPET imaging indicated that Lenti-STAT3-NF-κB-TK showed more tumor growth retardation than Lenti-CMV-TK when GCV (20 mg/kg) was administered. The invasiveness and expression of cancer stem cell markers were both decreased after STAT3/NF-κB-regulated HSV-TK/GCV therapy. Moreover, STAT3/NF-κB signaling targeting could further sensitize tumor cells to cisplatin. This study successfully established a theranositic approach to treat triple-negative breast cancer via STAT3-NF-κB responsive element-driven suicide gene therapy. This platform may also be an alternative strategy to handle with drug-resistant cancer cells. PMID:28255357

A snapshot of gene therapy in Latin America

PubMed Central

Linden, Rafael; Matte, Ursula

2014-01-01

Gene therapy attempts the insertion and expression of exogenous genetic material in cells for therapeutic purposes. Conceived in the 1960s, gene therapy reached its first clinical trial at the end of the 1980s and by December 2013 around 600 genuine open clinical trials of gene therapy were registered at NIH Clinical Trials Database. Here, we summarize the current efforts towards the development of gene therapy in Latin America. Our survey shows that the number of scientists involved in the development of gene therapy and DNA vaccines in Latin America is still very low. Higher levels of investment in this technology are necessary to boost the advancement of innovation and intellectual property in this field in a way that would ease both the social and financial burden of various medical conditions in Latin America. PMID:24764763

Internal and External Triggering Mechanism of "Smart" Nanoparticle-Based DDSs in Targeted Tumor Therapy.

PubMed

Qiana, Xian-Ling; Li, Jun; Wei, Ran; Lin, Hui; Xiong, Li-Xia

2018-05-09

Anticancer chemotherapeutics have a lot of problems via conventional drug delivery systems (DDSs), including non-specificity, burst release, severe side-effects, and damage to normal cells. Owing to its potential to circumventing these problems, nanotechnology has gained increasing attention in targeted tumor therapy. Chemotherapeutic drugs or genes encapsulated in nanoparticles could be used to target therapies to the tumor site in three ways: "passive", "active", and "smart" targeting. To summarize the mechanisms of various internal and external "smart" stimulating factors on the basis of findings from in vivo and in vitro studies. A thorough search of PubMed was conducted in order to identify the majority of trials, studies and novel articles related to the subject. Activated by internal triggering factors (pH, redox, enzyme, hypoxia, etc.) or external triggering factors (temperature, light of different wavelengths, ultrasound, magnetic fields, etc.), "smart" DDSs exhibit targeted delivery to the tumor site, and controlled release of chemotherapeutic drugs or genes. In this review article, we summarize and classify the internal and external triggering mechanism of "smart" nanoparticle-based DDSs in targeted tumor therapy, and the most recent research advances are illustrated for better understanding. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Blocking the PI3K pathway enhances the efficacy of ALK-targeted therapy in EML4-ALK-positive nonsmall-cell lung cancer.

PubMed

Yang, Lin; Li, Guangchao; Zhao, Likun; Pan, Fei; Qiang, Jiankun; Han, Siqi

2014-10-01

Targeted therapy based on ALK tyrosine kinase inhibitors (ALK-TKIs) has made significant achievements in individuals with EML4-ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene) fusion positive nonsmall-cell lung cancer (NSCLC). However, a high fraction of patients receive inferior clinical response to such treatment in the initial therapy, and the exact mechanisms underlying this process need to be further investigated. In this study, we revealed a persistently activated PI3K/AKT signaling that mediates the drug ineffectiveness. We found that genetic or pharmacological inhibition of ALK markedly abrogated phosphorylated STAT3 and ERK, but it failed to suppress AKT activity or induce apoptosis, in EML4-ALK-positive H2228 cells. Furthermore, targeted RNA interference of PI3K pathway components restored sensitivity to TAE684 treatment at least partially due to increased apoptosis. Combined TAE684 with PI3K inhibitor synergistically inhibited the proliferation of EML4-ALK-positive cells in vitro and significantly suppressed the growth of H2228 xenografts in vivo, suggesting the potential clinical application of such combinatorial therapy regimens in patients with EML4-ALK positive lung cancer.

“Stealth” Adenoviruses Blunt Cell-Mediated and Humoral Immune Responses against the Virus and Allow for Significant Gene Expression upon Readministration in the Lung

PubMed Central

Croyle, Maria A.; Chirmule, Narendra; Zhang, Yi; Wilson, James M.

2001-01-01

Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy. PMID:11312351

Adeno-associated virus transfer of a gene encoding SNAP-25 resistant to botulinum toxin A attenuates neuromuscular paralysis associated with botulism.

PubMed

Raghunath, Arvind; Perez-Branguli, Francesc; Smith, Leonard; Dolly, J Oliver

2008-04-02

Advances in viral gene therapy have opened new possibilities for treating a range of motor neuron diseases, but these have not yet been translated into clinically applicable therapies because of difficulties in delivery to susceptible/damaged neurons, ambiguities in the identity of gene(s) implicated, and a paucity of means to quantify any physiological improvement. Most of these hurdles can be overcome by using the neuromuscular paralysis induced by botulinum neurotoxin type A (BoNT/A) as a prototype disease. Furthermore, because human botulism, occasionally fatal, causes prolonged muscle disablement as a result of the intraneuronal persistence of the toxin's SNAP-25 (S25)-cleaving protease, development of a genetic approach could lead to a potential treatment for this debilitating disease. Adeno-associated viral delivery of a cleavage-resistant S25 gene (S25-R198T) to chromaffin cells in vitro yielded exocytotically active S25-R198T that diminished subsequent blockade by BoNT/A of evoked catecholamine release. Evaluation in vivo, by administering this virus into rat spinal cord before injecting BoNT/A, showed a decreased inhibition of acetylcholine release as reflected in elevated retention of neuromuscular transmission. A similar, although smaller, protection of synaptic transmission from the toxin was seen after peripherally injecting the therapeutic virus. Such therapy also curtailed nerve sprouting normally induced by BoNT/A. This first demonstration of the utility of a DNA-based therapy for botulism paves the way for further advances in its treatment and for application to genetic disorders of motor neurons.

Challenges and opportunities in bioanalytical support for gene therapy medicinal product development.

PubMed

Ma, Mark; Balasubramanian, Nanda; Dodge, Robert; Zhang, Yan

2017-09-01

Gene and nucleic acid therapies have demonstrated patient benefits to address unmet medical needs. Beside considerations regarding the biological nature of the gene therapy, the quality of bioanalytical methods plays an important role in ensuring the success of these novel therapies. Inconsistent approaches among bioanalytical labs during preclinical and clinical phases have been observed. There are many underlying reasons for this inconsistency. Various platforms and reagents used in quantitative methods, lacking of detailed regulatory guidance on method validation and uncertainty of immunogenicity strategy in supporting gene therapy may all be influential. This review summarizes recent practices and considerations in bioanalytical support of pharmacokinetics/pharmacodynamics and immunogenicity evaluations in gene therapy development with insight into method design, development and validations.

76 FR 9028 - Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-16

...] Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability AGENCY: Food and... the availability of a document entitled ``Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products'' dated January 2011. The guidance document provides manufacturers of cellular and gene...

77 FR 63840 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-17

...] Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting AGENCY: Food and Drug... meeting will be closed to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory... to hear updates of research programs in the Gene Transfer and Immunogenicity Branch, Office of...

Towards a Safer, More Randomized Lentiviral Vector Integration Profile Exploring Artificial LEDGF Chimeras

PubMed Central

Vranckx, Lenard S.; Demeulemeester, Jonas; Debyser, Zeger

2016-01-01

The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia development in a subset of treated patients. Despite improvements in vector design, their use is still not risk-free. Lentiviral vector (LV) integration is directed into active transcription units by LEDGF/p75, a host-cell protein co-opted by the viral integrase. We engineered LEDGF/p75-based hybrid tethers in an effort to elicit a more random integration pattern to increase biosafety, and potentially reduce proto-oncogene activation. We therefore truncated LEDGF/p75 by deleting the N-terminal chromatin-reading PWWP-domain, and replaced this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells efficiently rescued LV transduction and resulted in LV integrations that distributed more randomly throughout the host-cell genome. In addition, when considering safe harbor criteria, LV integration sites for these LEDGF-hybrids distributed more safely compared to LEDGF/p75-mediated integration in wild-type cells. This approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as patient-specific iPS cells. PMID:27788138

Viability of long-term gene therapy in the cochlea.

PubMed

Atkinson, Patrick J; Wise, Andrew K; Flynn, Brianna O; Nayagam, Bryony A; Richardson, Rachael T

2014-04-22

Gene therapy has been investigated as a way to introduce a variety of genes to treat neurological disorders. An important clinical consideration is its long-term effectiveness. This research aims to study the long-term expression and effectiveness of gene therapy in promoting spiral ganglion neuron survival after deafness. Adenoviral vectors modified to express brain derived neurotrophic factor or neurotrophin-3 were unilaterally injected into the guinea pig cochlea one week post ototoxic deafening. After six months, persistence of gene expression and significantly greater neuronal survival in neurotrophin-treated cochleae compared to the contralateral cochleae were observed. The long-term gene expression observed indicates that gene therapy is potentially viable; however the degeneration of the transduced cells as a result of the original ototoxic insult may limit clinical effectiveness. With further research aimed at transducing stable cochlear cells, gene therapy may be an efficacious way to introduce neurotrophins to promote neuronal survival after hearing loss.

Gene Therapy for Pancreatic Cancer: Specificity, Issues and Hopes

PubMed Central

Rouanet, Marie; Lebrin, Marine; Gross, Fabian; Bournet, Barbara; Cordelier, Pierre; Buscail, Louis

2017-01-01

A recent death projection has placed pancreatic ductal adenocarcinoma as the second cause of death by cancer in 2030. The prognosis for pancreatic cancer is very poor and there is a great need for new treatments that can change this poor outcome. Developments of therapeutic innovations in combination with conventional chemotherapy are needed urgently. Among innovative treatments the gene therapy offers a promising avenue. The present review gives an overview of the general strategy of gene therapy as well as the limitations and stakes of the different experimental in vivo models, expression vectors (synthetic and viral), molecular tools (interference RNA, genome editing) and therapeutic genes (tumor suppressor genes, antiangiogenic and pro-apoptotic genes, suicide genes). The latest developments in pancreatic carcinoma gene therapy are described including gene-based tumor cell sensitization to chemotherapy, vaccination and adoptive immunotherapy (chimeric antigen receptor T-cells strategy). Nowadays, there is a specific development of oncolytic virus therapies including oncolytic adenoviruses, herpes virus, parvovirus or reovirus. A summary of all published and on-going phase-1 trials is given. Most of them associate gene therapy and chemotherapy or radiochemotherapy. The first results are encouraging for most of the trials but remain to be confirmed in phase 2 trials. PMID:28594388

Overexpression of the growth arrest-specific homeobox gene Gax inhibits proliferation, migration, cell cycle progression, and apoptosis in serum-induced vascular smooth muscle cells.

PubMed

Zheng, H; Xue, S; Hu, Z L; Shan, J G; Yang, W G

2014-03-24

The Gax gene has been implicated in a variety of cell-developmental and biological processes, and aberrant Gax expression is linked to many diseases. In this study, to provide important insights for Gax-based gene therapy in vein graft restenosis and its anti-restenotic mechanism, we used rabbit vascular smooth muscle cells (VSMCs) to investigate the effects of Gax overexpression on proliferation, migration, cell cycle, and apoptosis in a serum-stimulated culture. Rabbit VSMC lines that stably overexpressed Gax were established by transfection with recombinant adenoviral vector Ad5-Gax. The effect of Gax overexpression on in vitro serum-induced VSMCs proliferation, migration, cell cycle, and apoptosis was assessed by MTT, wound healing, and flow cytometry assays, respectively. To investigate the effect of Gax overexpression on PCNA and MMP-2 in serum-induced VSMCs, immunocytochemistry, RT-PCR, and gelatin zymography were performed. The results clearly showed that Gax overexpression decreases PCNA expression in serum-induced VSMCs. Gax overexpression also significantly inhibited cell proliferation by blocking entry into the S-phase of the cell cycle, promoted cell apoptosis, and reduced cell migration activity by downregulating MMP-2 release and activity. These findings indicate that Gax would be an optimal target gene for gene therapy to treat vein graft restenosis.

Chimeric adeno-associated virus and bacteriophage: a potential targeted gene therapy vector for malignant glioma.

PubMed

Asavarut, Paladd; O'Neill, Kevin; Syed, Nelofer; Hajitou, Amin

2014-01-01

The incipient development of gene therapy for cancer has fuelled its progression from bench to bedside in mere decades. Of all malignancies that exist, gliomas are the largest class of brain tumors, and are renowned for their aggressiveness and resistance to therapy. In order for gene therapy to achieve clinical success, a multitude of barriers ranging from glioma tumor physiology to vector biology must be overcome. Many viral gene delivery systems have been subjected to clinical investigation; however, with highly limited success. In this review, the current progress and challenges of gene therapy for malignant glioma are discussed. Moreover, we highlight the hybrid adeno-associated virus and bacteriophage vector as a potential candidate for targeted gene delivery to brain tumors.

Investor Outlook: Significance of the Positive LCA2 Gene Therapy Phase III Results.

PubMed

Schimmer, Joshua; Breazzano, Steven

2015-12-01

Spark Therapeutics recently reported positive phase III results for SPK-RPE65 targeting the treatment of visual impairment caused by RPE65 gene mutations (often referred to as Leber congenital amaurosis type 2, or LCA2, but may include other retinal disorders), marking an important inflection point for the field of gene therapy. The results highlight the ability to successfully design and execute a randomized trial of a gene therapy and also reinforce the potentially predictive nature of early preclinical and clinical data. The results are expected to pave the way for the first approved gene therapy product in the United States and should sustain investor interest and confidence in gene therapy for many approaches, including retina targeting and beyond.

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period ofmore » pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.« less

Gene Editing: Regulatory and Translation to Clinic.

PubMed

Ando, Dale; Meyer, Kathleen

2017-10-01

The clinical application and regulatory strategy of genome editing for ex vivo cell therapy is derived from the intersection of two fields of study: viral vector gene therapy trials; and clinical trials with ex vivo purification and engraftment of CD34 +  hematopoietic stem cells, T cells, and tumor cell vaccines. This article covers the regulatory and translational preclinical activities needed for a genome editing clinical trial modifying hematopoietic stem cells and the genesis of this current strategy based on previous clinical trials using genome-edited T cells. The SB-728 zinc finger nuclease platform is discussed because this is the most clinically advanced genome editing technology. Copyright © 2017 Elsevier Inc. All rights reserved.

Gene therapy for hemophilia

PubMed Central

Rogers, Geoffrey L.; Herzog, Roland W.

2015-01-01

Hemophilia is an X-linked inherited bleeding disorder consisting of two classifications, hemophilia A and hemophilia B, depending on the underlying mutation. Although the disease is currently treatable with intravenous delivery of replacement recombinant clotting factor, this approach represents a significant cost both monetarily and in terms of quality of life. Gene therapy is an attractive alternative approach to the treatment of hemophilia that would ideally provide life-long correction of clotting activity with a single injection. In this review, we will discuss the multitude of approaches that have been explored for the treatment of both hemophilia A and B, including both in vivo and ex vivo approaches with viral and nonviral delivery vectors. PMID:25553466

Virus-mediated EpoR76E Therapy Slows Optic Nerve Axonopathy in Experimental Glaucoma.

PubMed

Bond, Wesley S; Hines-Beard, Jessica; GoldenMerry, YPaul L; Davis, Mara; Farooque, Alma; Sappington, Rebecca M; Calkins, David J; Rex, Tonia S

2016-02-01

Glaucoma, a common cause of blindness, is currently treated by intraocular pressure (IOP)-lowering interventions. However, this approach is insufficient to completely prevent vision loss. Here, we evaluate an IOP-independent gene therapy strategy using a modified erythropoietin, EPO-R76E, which has reduced erythropoietic function. We used two models of glaucoma, the murine microbead occlusion model and the DBA/2J mouse. Systemic recombinant adeno-associated virus-mediated gene delivery of EpoR76E (rAAV.EpoR76E) was performed concurrent with elevation of IOP. Axon structure and active anterograde transport were preserved in both models. Vision, as determined by the flash visual evoked potential, was preserved in the DBA/2J. These results show that systemic EpoR76E gene therapy protects retinal ganglion cells from glaucomatous degeneration in two different models. This suggests that EPO targets a component of the neurodegenerative pathway that is common to both models. The efficacy of rAAV.EpoR76E delivered at onset of IOP elevation supports clinical relevance of this treatment.

Isolated and combined effects of photobiomodulation therapy, topical nonsteroidal anti-inflammatory drugs, and physical activity in the treatment of osteoarthritis induced by papain

NASA Astrophysics Data System (ADS)

Tomazoni, Shaiane Silva; Leal-Junior, Ernesto Cesar Pinto; Frigo, Lúcio; Pallotta, Rodney Capp; Teixeira, Simone; de Almeida, Patricia; Bjordal, Jan Magnus; Lopes-Martins, Rodrigo Álvaro Brandão

2016-10-01

Osteoarthritis (OA) is a chronic inflammatory disease and is characterized as a degenerative process. This study aimed to evaluate and compare the effects of a topical nonsteroidal anti-inflammatory drug (NSAID), physical activity, and photobiomodulation therapy (PBMT) applied alone and/or in combination between them in an experimental model of knee OA. OA was induced by injection of papain in the knees of rats. After 21 days, the animals started to be treated with the above treatment. Histological analysis shows that the experimental model of OA induction causes morphological changes consistent with the disease, and among treatments, the PBMT is the most effective for reducing these changes. Moreover, the results demonstrate that PBMT and NSAID reduce the total number of cells in the inflammatory infiltrate (p<0.05) and PBMT was the most effective for reducing the activity of myeloperoxidase (p<0.05). Finally, we observed that both NSAID and PBMT were effective for reducing the gene expression of MMP-3 (p<0.05), but in relation to the gene expression of MMP-13, PBMT was the most effective treatment (p<0.05). The results of this study indicate that PBMT is the most effective therapy in stopping disease progression, and improving inflammatory conditions observed in OA.

Prolonged lymphocytosis during ibrutinib therapy is associated with distinct molecular characteristics and does not indicate a suboptimal response to therapy

PubMed Central

Smucker, Kelly; Smith, Lisa L.; Lozanski, Arletta; Zhong, Yiming; Ruppert, Amy S.; Lucas, David; Williams, Katie; Zhao, Weiqiang; Rassenti, Laura; Ghia, Emanuela; Kipps, Thomas J.; Mantel, Rose; Jones, Jeffrey; Flynn, Joseph; Maddocks, Kami; O’Brien, Susan; Furman, Richard R.; James, Danelle F.; Clow, Fong; Lozanski, Gerard; Johnson, Amy J.; Byrd, John C.

2014-01-01

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has outstanding activity in patients with chronic lymphocytic leukemia. Most patients experience lymphocytosis, representing lymphocyte egress from nodal compartments. This resolves within 8 months in the majority of patients, but a subgroup has lymphocytosis lasting >12 months. Here we report a detailed characterization of patients with persistent lymphocytosis during ibrutinib therapy. Signaling evaluation showed that while BTK is inhibited, downstream mediators of B-cell receptor (BCR) signaling are activated in persistent lymphocytes. These cells cannot be stimulated through the BCR and do not show evidence of target gene activation. Flow cytometry for κ and λ expression, IGHV sequencing, Zap-70 methylation, and targeted gene sequencing in these patients are identical at baseline and later time points, suggesting that persistent lymphocytes do not represent clonal evolution. In vitro treatment with targeted kinase inhibitors shows that they are not addicted to a single survival pathway. Finally, progression-free survival is not inferior for patients with prolonged lymphocytosis vs those with traditional responses. Thus, prolonged lymphocytosis is common following ibrutinib treatment, likely represents the persistence of a quiescent clone, and does not predict a subgroup of patients likely to relapse early. PMID:24415539

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

PubMed

Polstein, Lauren R; Perez-Pinera, Pablo; Kocak, D Dewran; Vockley, Christopher M; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

2015-08-01

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. © 2015 Polstein et al.; Published by Cold Spring Harbor Laboratory Press.

Evaluation of genome-wide expression profiles of blood and sputum neutrophils in cystic fibrosis patients before and after antibiotic therapy.

PubMed

Conese, Massimo; Castellani, Stefano; Lepore, Silvia; Palumbo, Orazio; Manca, Antonio; Santostasi, Teresa; Polizzi, Angela Maria; Copetti, Massimiliano; Di Gioia, Sante; Casavola, Valeria; Guerra, Lorenzo; Diana, Anna; Montemurro, Pasqualina; Mariggiò, Maria Addolorata; Gallo, Crescenzio; Maffione, Angela Bruna; Carella, Massimo

2014-01-01

In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to "healthy" condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis.

Evaluation of Genome-Wide Expression Profiles of Blood and Sputum Neutrophils in Cystic Fibrosis Patients Before and After Antibiotic Therapy

PubMed Central

Conese, Massimo; Castellani, Stefano; Lepore, Silvia; Palumbo, Orazio; Manca, Antonio; Santostasi, Teresa; Polizzi, Angela Maria; Copetti, Massimiliano; Di Gioia, Sante; Casavola, Valeria; Guerra, Lorenzo; Diana, Anna; Montemurro, Pasqualina; Mariggiò, Maria Addolorata; Gallo, Crescenzio; Maffione, Angela Bruna; Carella, Massimo

2014-01-01

In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to “healthy” condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis. PMID:25084273

Epigenomic study identifies a novel mesenchyme homeobox2-GLI1 transcription axis involved in cancer drug resistance, overall survival and therapy prognosis in lung cancer patients

PubMed Central

Armas-López, Leonel; Piña-Sánchez, Patricia; Arrieta, Oscar; de Alba, Enrique Guzman; Ortiz-Quintero, Blanca; Santillán-Doherty, Patricio; Christiani, David C.; Zúñiga, Joaquín; Ávila-Moreno, Federico

2017-01-01

Several homeobox-related gene (HOX) transcription factors such as mesenchyme HOX-2 (MEOX2) have previously been associated with cancer drug resistance, malignant progression and/or clinical prognostic responses in lung cancer patients; however, the mechanisms involved in these responses have yet to be elucidated. Here, an epigenomic strategy was implemented to identify novel MEOX2 gene promoter transcription targets and propose a new molecular mechanism underlying lung cancer drug resistance and poor clinical prognosis. Chromatin immunoprecipitation (ChIP) assays derived from non-small cell lung carcinomas (NSCLC) hybridized on gene promoter tiling arrays and bioinformatics analyses were performed, and quantitative, functional and clinical validation were also carried out. We statistically identified a common profile consisting of 78 gene promoter targets, including Hedgehog-GLI1 gene promoter sequences (FDR≤0.1 and FDR≤0.2). The GLI-1 gene promoter region from −2,192 to −109 was occupied by MEOX2, accompanied by transcriptionally active RNA Pol II and was epigenetically linked to the active histones H3K27Ac and H3K4me3; these associations were quantitatively validated. Moreover, siRNA genetic silencing assays identified a MEOX2-GLI1 axis involved in cellular cytotoxic resistance to cisplatinum in a dose-dependent manner, as well as cellular migration and proliferation. Finally, Kaplan-Maier survival analyses identified significant MEOX2-dependent GLI-1 protein expression associated with clinical progression and poorer overall survival using an independent cohort of NSCLC patients undergoing platinum-based oncological therapy with both epidermal growth factor receptor (EGFR)-non-mutated and EGFR-mutated status. In conclusion, this is the first study to investigate epigenome-wide MEOX2-transcription factor occupation identifying a novel overexpressed MEOX2-GLI1 axis and its clinical association with platinum-based cancer drug resistance and EGFR-tyrosine kinase inhibitor (TKI)-based therapy responses in NSCLC patients. PMID:28978016

Human gene therapy and slippery slope arguments.

PubMed Central

McGleenan, T

1995-01-01

Any suggestion of altering the genetic makeup of human beings through gene therapy is quite likely to provoke a response involving some reference to a 'slippery slope'. In this article the author examines the topography of two different types of slippery slope argument, the logical slippery slope and the rhetorical slippery slope argument. The logical form of the argument suggests that if we permit somatic cell gene therapy then we are committed to accepting germ line gene therapy in the future because there is no logically sustainable distinction between them. The rhetorical form posits that allowing somatic cell therapy now will be taking the first step on a slippery slope which will ultimately lead to the type of genocide perpetrated by the Nazis. The author tests the validity of these lines of argument against the facts of human gene therapy and concludes that because of their dependence on probabilities that cannot be empirically proven they should be largely disregarded in the much more important debate on moral line-drawing in gene therapy. PMID:8778459

Human gene therapy and slippery slope arguments.

PubMed

McGleenan, T

1995-12-01

Any suggestion of altering the genetic makeup of human beings through gene therapy is quite likely to provoke a response involving some reference to a 'slippery slope'. In this article the author examines the topography of two different types of slippery slope argument, the logical slippery slope and the rhetorical slippery slope argument. The logical form of the argument suggests that if we permit somatic cell gene therapy then we are committed to accepting germ line gene therapy in the future because there is no logically sustainable distinction between them. The rhetorical form posits that allowing somatic cell therapy now will be taking the first step on a slippery slope which will ultimately lead to the type of genocide perpetrated by the Nazis. The author tests the validity of these lines of argument against the facts of human gene therapy and concludes that because of their dependence on probabilities that cannot be empirically proven they should be largely disregarded in the much more important debate on moral line-drawing in gene therapy.

Gene therapy strategies for urological dysfunction.

PubMed

Chancellor, M B; Yoshimura, N; Pruchnic, R; Huard, J

2001-07-01

Novel molecular techniques such as conventional and ex vivo gene therapy, and tissue engineering have only recently been introduced to the field of urology. The lower urinary tract is ideally suited for minimally invasive therapy, and also ex vivo approaches would limit the risk of systemic side effects. Muscle-derived stem cells have been used successfully to treat stress incontinence, and rats with diabetic bladder dysfunction benefited from nerve growth factor (NGF)-based gene therapy. Nitric oxide synthase and capase-7 might provide suitable gene therapy targets for erectile dysfunction and benign prostatic hyperplasia, respectively.

78 FR 69690 - Draft Guidance for Industry: Considerations for the Design of Early-Phase Clinical Trials of...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-20

... and Gene Therapy Products; Extension of Comment Period AGENCY: Food and Drug Administration, HHS...: Considerations for the Design of Early-Phase Clinical Trials of Cellular and Gene Therapy Products'' that... sponsors of Investigational New Drug Applications for cellular therapy (CT) and gene therapy (GT) products...

Regulatory considerations in application of encapsulated cell therapies.

PubMed

van Zanten, J; de Vos, Paul

2010-01-01

The encapsulation of tissue in semi-permeable membranes is a technology with high potential and in due time several new therapies based on this technology will be tested in clinical trials. Recent, new legislation requires that these investigational medicinal products used in clinical trials Phase I must be produced according to Good Manufacturing Practice (GMP). Consequently, the activities of GMP are expanding to the field of research and researchers might need to change developed protocols in order to meet GMP legislation. This chapters gives an overview of the overall guidelines covering GMP and more specific guidelines dealing with cell based therapies and gene therapy.

The "SWOT" of BRAF inhibition in melanoma: RAF inhibitors, MEK inhibitors or both?

PubMed

Nissan, Moriah H; Solit, David B

2011-12-01

Activating mutations in the BRAF gene are among the most prevalent kinase mutations in human cancer. BRAF mutations are most frequent in patients with melanoma where they occur in approximately 50% of patients with advanced disease. Remarkable clinical activity has recently been reported with highly selective RAF inhibitors in melanoma patients whose tumors harbor V600E BRAF mutations. The response rates of RAF inhibitors in patients with BRAF-mutant melanomas far exceed the activity level of any prior therapy studied in this disease. The results suggest that we have entered an era of personalized therapy for patients with metastatic melanoma in which treatment selection will be guided by BRAF mutational status. This review will discuss the strengths, weaknesses, opportunities and threats ("SWOT") of developing RAF and MEK selective inhibitors as anti-cancer therapies, recent insights into the mechanisms of intrinsic and acquired resistance to these agents, and current efforts to develop mechanism-based combination therapies.

The hopes and fears of in utero gene therapy for genetic disease--a review.

PubMed

Coutelle, C; Themis, M; Waddington, S; Gregory, L; Nivsarkar, M; Buckley, S; Cook, T; Rodeck, C; Peebles, D; David, A

2003-10-01

Somatic gene delivery in utero is a novel approach to gene therapy for genetic disease. It is based on the concept that application of gene therapy vectors to the fetus in utero may prevent the development of early disease related tissue damage, may allow targeting of otherwise inaccessible organs, tissues and still expanding stem cell populations and may also provide postnatal tolerance against the therapeutic transgenic protein. This review outlines the hypothesis and scientific background of in utero gene therapy and addresses some of the frequently expressed concerns raised by this still experimental, potentially preventive gene therapy approach. We describe and discuss the choice of vectors, of animal models and routes of administration to the fetus. We address potential risk factors of prenatal gene therapy such as vector toxicity, inadvertent germ line modification, developmental aberration and oncogenesis as well as specific risks of this procedure for the fetus and mother and discuss their ethical implications.

Investor Outlook: Rising from the Ashes; GSK's European Approval of Strimvelis for ADA-SCID.

PubMed

Schimmer, Joshua; Breazzano, Steven

2016-06-01

GlaxoSmithKline's (GSK) and partner San Raffaele Telethon Institute for Gene Therapy's recent positive European approval for Strimvelis for treatment of severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) represents the second EU-approved gene therapy and the first γ-retrovirus and first ex vivo gene therapy. In this article we discuss the significance and implications of this historic approval for the broader gene therapy field.

Systemic Gene Therapy for Tuberous Sclerosis

DTIC Science & Technology

2017-07-01

especially for children and LAM patients. Our group is focused on developing gene therapy for TSC which has the potential for single application and low-to...neurologic diseases in adults and children , and AAV9 can deliver genes not only to peripheral tissues, but also to the brain in mice and non-human...therapies, especially for children and LAM patients. Our group is focused on developing gene therapy for TSC which has the potential for single

Radiation increases the activity of oncolytic adenovirus cancer gene therapy vectors that overexpress the ADP (E3-11.6K) protein.

PubMed

Toth, Karoly; Tarakanova, Vera; Doronin, Konstantin; Ward, Peter; Kuppuswamy, Mohan; Locke, Jacob E; Dawson, Julie E; Kim, Han J; Wold, William S M

2003-03-01

We have described three potential adenovirus type 5 (Ad5)-based replication-competent cancer gene therapy vectors named KD1, KD3, and VRX-007. All three vectors overexpress an Ad5 protein named Adenovirus Death Protein (ADP, also named E3-11.6 K protein). ADP is required for efficient lysis of Ad5-infected cells and spread of virus from cell to cell, and thus its overexpression increases the oncolytic activity of the vectors. KD1 and KD3 contain mutations in the Ad5 E1A gene that knock out binding of the E1A proteins to cellular p300/CBP and pRB; these mutations allow KD1 and KD3 to grow well in cancer cells but not in normal cells. VRX-007 has wild-type E1A. Here we report that radiation increases the oncolytic activity of KD1, KD3, and VRX-007. This increased activity was observed in cultured cells, and it was not because of radiation-induced replication of the vectors. The combination of radiation plus KD3 suppressed the growth of A549 lung adenocarcinoma xenografts in nude mice more efficiently than radiation alone or KD3 alone. The combination of ADP-overexpressing vectors and radiation may have potential in treating cancer.

Inhibitory Monoclonal Antibodies against Mouse Proteases Raised in Gene-Deficient Mice Block Proteolytic Functions in vivo

PubMed Central

Lund, Ida K.; Rasch, Morten G.; Ingvarsen, Signe; Pass, Jesper; Madsen, Daniel H.; Engelholm, Lars H.; Behrendt, Niels; Høyer-Hansen, Gunilla

2012-01-01

Identification of targets for cancer therapy requires the understanding of the in vivo roles of proteins, which can be derived from studies using gene-targeted mice. An alternative strategy is the administration of inhibitory monoclonal antibodies (mAbs), causing acute disruption of the target protein function(s). This approach has the advantage of being a model for therapeutic targeting. mAbs for use in mouse models can be obtained through immunization of gene-deficient mice with the autologous protein. Such mAbs react with both species-specific epitopes and epitopes conserved between species. mAbs against proteins involved in extracellular proteolysis, including plasminogen activators urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA), their inhibitor PAI-1, the uPA receptor (uPAR), two matrix metalloproteinases (MMP9 and MMP14), as well as the collagen internalization receptor uPARAP, have been developed. The inhibitory mAbs against uPA and uPAR block plasminogen activation and thereby hepatic fibrinolysis in vivo. Wound healing, another plasmin-dependent process, is delayed by an inhibitory mAb against uPA in the adult mouse. Thromboembolism can be inhibited by anti-PAI-1 mAbs in vivo. In conclusion, function-blocking mAbs are well-suited for targeted therapy in mouse models of different diseases, including cancer. PMID:22754528

Genetic modification of hematopoietic stem cells: recent advances in the gene therapy of inherited diseases.

PubMed

Bueren, Juan A; Guenechea, Guillermo; Casado, José A; Lamana, María Luisa; Segovia, José C

2003-01-01

Hematopoietic stem cells constitute a rare population of precursor cells with remarkable properties for being used as targets in gene therapy protocols. The last years have been particularly productive both in the fields of gene therapy and stem cell biology. Results from ongoing clinical trials have shown the first unquestionable clinical benefits of immunodeficient patients transplanted with genetically modified autologous stem cells. On the other hand, severe side effects in a few patients treated with gene therapy have also been reported, indicating the usefulness of further improving the vectors currently used in gene therapy clinical trials. In the field of stem cell biology, evidence showing the plastic potential of adult hematopoietic stem cells and data indicating the multipotency of adult mesenchymal precursor cells have been presented. Also, the generation of embryonic stem cells by means of nuclear transfer techniques has appeared as a new methodology with direct implications in gene therapy.

Cardiac gene therapy: Recent advances and future directions.

PubMed

Mason, Daniel; Chen, Yu-Zhe; Krishnan, Harini Venkata; Sant, Shilpa

2015-10-10

Gene therapy has the potential to serve as an adaptable platform technology for treating various diseases. Cardiovascular disease is a major cause of mortality in the developed world and genetic modification is steadily becoming a more plausible method to repair and regenerate heart tissue. Recently, new gene targets to treat cardiovascular disease have been identified and developed into therapies that have shown promise in animal models. Some of these therapies have advanced to clinical testing. Despite these recent successes, several barriers must be overcome for gene therapy to become a widely used treatment of cardiovascular diseases. In this review, we evaluate specific genetic targets that can be exploited to treat cardiovascular diseases, list the important delivery barriers for the gene carriers, assess the most promising methods of delivering the genetic information, and discuss the current status of clinical trials involving gene therapies targeted to the heart. Copyright © 2015 Elsevier B.V. All rights reserved.

Transcription Factor Activities Enhance Markers of Drug Sensitivity in Cancer.

PubMed

Garcia-Alonso, Luz; Iorio, Francesco; Matchan, Angela; Fonseca, Nuno; Jaaks, Patricia; Peat, Gareth; Pignatelli, Miguel; Falcone, Fiammetta; Benes, Cyril H; Dunham, Ian; Bignell, Graham; McDade, Simon S; Garnett, Mathew J; Saez-Rodriguez, Julio

2018-02-01

Transcriptional dysregulation induced by aberrant transcription factors (TF) is a key feature of cancer, but its global influence on drug sensitivity has not been examined. Here, we infer the transcriptional activity of 127 TFs through analysis of RNA-seq gene expression data newly generated for 448 cancer cell lines, combined with publicly available datasets to survey a total of 1,056 cancer cell lines and 9,250 primary tumors. Predicted TF activities are supported by their agreement with independent shRNA essentiality profiles and homozygous gene deletions, and recapitulate mutant-specific mechanisms of transcriptional dysregulation in cancer. By analyzing cell line responses to 265 compounds, we uncovered numerous TFs whose activity interacts with anticancer drugs. Importantly, combining existing pharmacogenomic markers with TF activities often improves the stratification of cell lines in response to drug treatment. Our results, which can be queried freely at dorothea.opentargets.io, offer a broad foundation for discovering opportunities to refine personalized cancer therapies. Significance: Systematic analysis of transcriptional dysregulation in cancer cell lines and patient tumor specimens offers a publicly searchable foundation to discover new opportunities to refine personalized cancer therapies. Cancer Res; 78(3); 769-80. ©2017 AACR . ©2017 American Association for Cancer Research.

Targeting the Hippo Signaling Pathway for Tissue Regeneration and Cancer Therapy

PubMed Central

Juan, Wen Chun; Hong, Wanjin

2016-01-01

The Hippo signaling pathway is a highly-conserved developmental pathway that plays an essential role in organ size control, tumor suppression, tissue regeneration and stem cell self-renewal. The YES-associated protein (YAP) and the transcriptional co-activator with PDZ-binding motif (TAZ) are two important transcriptional co-activators that are negatively regulated by the Hippo signaling pathway. By binding to transcription factors, especially the TEA domain transcription factors (TEADs), YAP and TAZ induce the expression of growth-promoting genes, which can promote organ regeneration after injury. Therefore, controlled activation of YAP and TAZ can be useful for regenerative medicine. However, aberrant activation of YAP and TAZ due to deregulation of the Hippo pathway or overexpression of YAP/TAZ and TEADs can promote cancer development. Hence, pharmacological inhibition of YAP and TAZ may be a useful approach to treat tumors with high YAP and/or TAZ activity. In this review, we present the mechanisms regulating the Hippo pathway, the role of the Hippo pathway in tissue repair and cancer, as well as a detailed analysis of the different strategies to target the Hippo signaling pathway and the genes regulated by YAP and TAZ for regenerative medicine and cancer therapy. PMID:27589805

Targeting the Hippo Signaling Pathway for Tissue Regeneration and Cancer Therapy.

PubMed

Juan, Wen Chun; Hong, Wanjin

2016-08-30

The Hippo signaling pathway is a highly-conserved developmental pathway that plays an essential role in organ size control, tumor suppression, tissue regeneration and stem cell self-renewal. The YES-associated protein (YAP) and the transcriptional co-activator with PDZ-binding motif (TAZ) are two important transcriptional co-activators that are negatively regulated by the Hippo signaling pathway. By binding to transcription factors, especially the TEA domain transcription factors (TEADs), YAP and TAZ induce the expression of growth-promoting genes, which can promote organ regeneration after injury. Therefore, controlled activation of YAP and TAZ can be useful for regenerative medicine. However, aberrant activation of YAP and TAZ due to deregulation of the Hippo pathway or overexpression of YAP/TAZ and TEADs can promote cancer development. Hence, pharmacological inhibition of YAP and TAZ may be a useful approach to treat tumors with high YAP and/or TAZ activity. In this review, we present the mechanisms regulating the Hippo pathway, the role of the Hippo pathway in tissue repair and cancer, as well as a detailed analysis of the different strategies to target the Hippo signaling pathway and the genes regulated by YAP and TAZ for regenerative medicine and cancer therapy.

Nonviral vectors for cancer gene therapy: prospects for integrating vectors and combination therapies.

PubMed

Ohlfest, John R; Freese, Andrew B; Largaespada, David A

2005-12-01

Gene therapy has the potential to improve the clinical outcome of many cancers by transferring therapeutic genes into tumor cells or normal host tissue. Gene transfer into tumor cells or tumor-associated stroma is being employed to induce tumor cell death, stimulate anti-tumor immune response, inhibit angiogenesis, and control tumor cell growth. Viral vectors have been used to achieve this proof of principle in animal models and, in select cases, in human clinical trials. Nevertheless, there has been considerable interest in developing nonviral vectors for cancer gene therapy. Nonviral vectors are simpler, more amenable to large-scale manufacture, and potentially safer for clinical use. Nonviral vectors were once limited by low gene transfer efficiency and transient or steadily declining gene expression. However, recent improvements in plasmid-based vectors and delivery methods are showing promise in circumventing these obstacles. This article reviews the current status of nonviral cancer gene therapy, with an emphasis on combination strategies, long-term gene transfer using transposons and bacteriophage integrases, and future directions.

[Gene therapy for vision restoration in patients with Leber congenital amaurosis (LCA) due to RPE65 gene mutations: beginning the phase IV trial].

PubMed

Chacón-Camacho, Óscar Francisco; Zenteno, Juan Carlos

This is a significant time moment in the field of gene therapy in humans. Recently, results from a phase III clinical trial were published, demonstrating the first gene therapy success for a genetic disease. A clinical trial was carried out in patients suffering a hereditary blindness disease named Leber congenital amaurosis, caused by mutations in the RPE65 gene. Participating subjects received a subretinal injection of the normal RPE65 gene and one year after exhibited a significant improvement in visual acuity. It is expected that this gene therapy treatment will be approved by the FDA and commercialized in the USA in 2017.

Anti-Inflammatory Effects of Modified Adenoviral Vectors for Gene Therapy: A View through Animal Models Tested.

PubMed

Castañeda-Lopez, M E; Garza-Veloz, I; Lopez-Hernandez, Y; Barbosa-Cisneros, O Y; Martinez-Fierro, M L

2016-07-01

The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases.

High-Throughput Screening of a Luciferase Reporter of Gene Silencing on the Inactive X Chromosome.

PubMed

Keegan, Alissa; Plath, Kathrin; Damoiseaux, Robert

2018-01-01

Assays of luciferase gene activity are a sensitive and quantitative reporter system suited to high-throughput screening. We adapted a luciferase assay to a screening strategy for identifying factors that reactivate epigenetically silenced genes. This epigenetic luciferase reporter is subject to endogenous gene silencing mechanisms on the inactive X chromosome (Xi) in primary mouse cells and thus captures the multilayered nature of chromatin silencing in development. Here, we describe the optimization of an Xi-linked luciferase reactivation assay in 384-well format and adaptation of the assay for high-throughput siRNA and chemical screening. Xi-luciferase reactivation screening has applications in stem cell biology and cancer therapy. We have used the approach described here to identify chromatin-modifying proteins and to identify drug combinations that enhance the gene reactivation activity of the DNA demethylating drug 5-aza-2'-deoxycytidine.

[Gene Therapy for Inherited RETINAL AND OPTIC NERVE Disorders: Current Knowledge].

PubMed

Ďuďáková, Ľ; Kousal, B; Kolářová, H; Hlavatá, L; Lišková, P

The aim of this review is to provide a comprehensive summary of current gene therapy clinical trials for monogenic and optic nerve disorders.The number of genes for which gene-based therapies are being developed is growing. At the time of writing this review gene-based clinical trials have been registered for Leber congenital amaurosis 2 (LCA2), retinitis pigmentosa 38, Usher syndrome 1B, Stargardt disease, choroideremia, achromatopsia, Leber hereditary optic neuropathy (LHON) and X-linked retinoschisis. Apart from RPE65 gene therapy for LCA2 and MT-ND4 for LHON which has reached phase III, all other trials are in investigation phase I and II, i.e. testing the efficacy and safety.Because of the relatively easy accessibility of the retina and its ease of visualization which allows monitoring of efficacy, gene-based therapies for inherited retinal disorders represent a very promising treatment option. With the development of novel therapeutic approaches, the importance of establishing not only clinical but also molecular genetic diagnosis is obvious.Key words: gene therapy, monogenic retinal diseases, optic nerve atrophy, mitochondrial disease.

Gene therapy: light is finally in the tunnel.

PubMed

Cao, Huibi; Molday, Robert S; Hu, Jim

2011-12-01

After two decades of ups and downs, gene therapy has recently achieved a milestone in treating patients with Leber's congenital amaurosis (LCA). LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy. Mutations in several genes, including RPE65, cause the disease. Using adeno-associated virus as a vector, three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects. However, considering the whole field of gene therapy, there are still major obstacles to clinical applications for other diseases. These obstacles include innate and immune barriers to vector delivery, toxicity of vectors and the lack of sustained therapeutic gene expression. Therefore, new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy. In this article, we shall review the major advancements over the past two decades and, using lung gene therapy as an example, discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.

The DcpS inhibitor RG3039 improves motor function in SMA mice

PubMed Central

Van Meerbeke, James P.; Gibbs, Rebecca M.; Plasterer, Heather L.; Miao, Wenyan; Feng, Zhihua; Lin, Ming-Yi; Rucki, Agnieszka A.; Wee, Claribel D.; Xia, Bing; Sharma, Shefali; Jacques, Vincent; Li, Darrick K.; Pellizzoni, Livio; Rusche, James R.; Ko, Chien-Ping; Sumner, Charlotte J.

2013-01-01

Spinal muscular atrophy (SMA) is caused by mutations of the survival motor neuron 1 (SMN1) gene, retention of the survival motor neuron 2 (SMN2) gene and insufficient expression of full-length survival motor neuron (SMN) protein. Quinazolines increase SMN2 promoter activity and inhibit the ribonucleic acid scavenger enzyme DcpS. The quinazoline derivative RG3039 has advanced to early phase clinical trials. In preparation for efficacy studies in SMA patients, we investigated the effects of RG3039 in severe SMA mice. Here, we show that RG3039 distributed to central nervous system tissues where it robustly inhibited DcpS enzyme activity, but minimally activated SMN expression or the assembly of small nuclear ribonucleoproteins. Nonetheless, treated SMA mice showed a dose-dependent increase in survival, weight and motor function. This was associated with improved motor neuron somal and neuromuscular junction synaptic innervation and function and increased muscle size. RG3039 also enhanced survival of conditional SMA mice in which SMN had been genetically restored to motor neurons. As this systemically delivered drug may have therapeutic benefits that extend beyond motor neurons, it could act additively with SMN-restoring therapies delivered directly to the central nervous system such as antisense oligonucleotides or gene therapy. PMID:23727836

In vitro and in vivo gene therapy with CMV vector-mediated presumed dog beta-nerve growth factor in pyridoxine-induced neuropathy dogs.

PubMed

Chung, Jin Young; Choi, Jung Hoon; Shin, Il Seob; Choi, Eun Wha; Hwang, Cheol Yong; Lee, Sang Koo; Youn, Hwa Young

2008-12-01

Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog beta-nerve growth factor (pdbeta-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdbeta-NGF protein reacted with a human beta-NGF antibody and showed bioactivity in PC12 cells. The pdbeta-NGF was shown to have similar bioactivity to the dog beta-NGF. The recombinant pdbeta-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs' body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L4 were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L4 and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.

Human gene therapy: a brief overview of the genetic revolution.

PubMed

Misra, Sanjukta

2013-02-01

Advances in biotechnology have brought gene therapy to the forefront of medical research. The prelude to successful gene therapy i.e. the efficient transfer and expression of a variety of human gene into target cells has already been accomplished in several systems. Safe methods have been devised to do this, using several viral and no-viral vectors. Two main approaches emerged: in vivo modification and ex vivo modification. Retrovirus, adenovirus, adeno-associated virus are suitable for gene therapeutic approaches which are based on permanent expression of the therapeutic gene. Non-viral vectors are far less efficient than viral vectors, but they have advantages due to their low immunogenicity and their large capacity for therapeutic DNA. To improve the function of non-viral vectors, the addition of viral functions such as receptor mediated uptake and nuclear translocation of DNA may finally lead to the development of an artificial virus. Gene transfer protocols have been approved for human use in inherited diseases, cancers and acquired disorders. In 1990, the first successful clinical trial of gene therapy was initiated for adenosine deaminase deficiency. Since then, the number of clinical protocols initiated worldwide has increased exponentially. Although preliminary results of these trials are somewhat disappointing, but human gene therapy dreams of treating diseases by replacing or supplementing the product of defective or introducing novel therapeutic genes. So definitely human gene therapy is an effective addition to the arsenal of approaches to many human therapies in the 21st century.

Gene editing for skin diseases: designer nucleases as tools for gene therapy of skin fragility disorders.

PubMed

March, Oliver P; Reichelt, Julia; Koller, Ulrich

2018-04-01

What is the topic of this review? This review concerns current gene editing strategies for blistering skin diseases with respect to individual genetic constellations and distinct conditions. What advances does it highlight? Specificity and safety dominate the discussion of gene editing applications for gene therapy, where a number of tools are implemented. Recent developments in this rapidly progressing field pose further questions regarding which tool is best suited for each particular use. The current treatment of inherited blistering skin diseases, such as epidermolysis bullosa (EB), is largely restricted to wound care and pain management. More effective therapeutic strategies are urgently required, and targeting the genetic basis of these severe diseases is now within reach. Here, we describe current gene editing tools and their potential to correct gene function in monogenetic blistering skin diseases. We present the features of the most frequently used gene editing techniques, transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), determining their preferential application for specific genetic conditions, including the type of mutational inheritance, the targeting site within the gene or the possibility to target the mutation specifically. Both tools have traits beneficial in specific situations. Promising developments in the field engender gene editing as a potentially powerful therapeutic option for future clinical applications. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

A Look to Future Directions in Gene Therapy Research for Monogenic Diseases

PubMed Central

Porteus, Matthew H; Connelly, Jon P; Pruett, Shondra M

2006-01-01

The concept of gene therapy has long appealed to biomedical researchers and clinicians because it promised to treat certain diseases at their origins. In the last several years, there have been several trials in which patients have benefited from gene therapy protocols. This progress, however, has revealed important problems, including the problem of insertional oncogenesis. In this review, which focuses on monogenic diseases, we discuss the problem of insertional oncogenesis and identify areas for future research, such as developing more quantitative assays for risk and efficacy, and ways of minimizing the genotoxic effects of gene therapy protocols, which will be important if gene therapy is to fulfill its conceptual promise. PMID:17009872

Heterogeneous activation of the TGFβ pathway in glioblastomas identified by gene expression-based classification using TGFβ-responsive genes

PubMed Central

Xu, Xie L; Kapoun, Ann M

2009-01-01

Background TGFβ has emerged as an attractive target for the therapeutic intervention of glioblastomas. Aberrant TGFβ overproduction in glioblastoma and other high-grade gliomas has been reported, however, to date, none of these reports has systematically examined the components of TGFβ signaling to gain a comprehensive view of TGFβ activation in large cohorts of human glioma patients. Methods TGFβ activation in mammalian cells leads to a transcriptional program that typically affects 5–10% of the genes in the genome. To systematically examine the status of TGFβ activation in high-grade glial tumors, we compiled a gene set of transcriptional response to TGFβ stimulation from tissue culture and in vivo animal studies. These genes were used to examine the status of TGFβ activation in high-grade gliomas including a large cohort of glioblastomas. Unsupervised and supervised classification analysis was performed in two independent, publicly available glioma microarray datasets. Results Unsupervised and supervised classification using the TGFβ-responsive gene list in two independent glial tumor gene expression data sets revealed various levels of TGFβ activation in these tumors. Among glioblastomas, one of the most devastating human cancers, two subgroups were identified that showed distinct TGFβ activation patterns as measured from transcriptional responses. Approximately 62% of glioblastoma samples analyzed showed strong TGFβ activation, while the rest showed a weak TGFβ transcriptional response. Conclusion Our findings suggest heterogeneous TGFβ activation in glioblastomas, which may cause potential differences in responses to anti-TGFβ therapies in these two distinct subgroups of glioblastomas patients. PMID:19192267

Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader–Willi syndrome

PubMed Central

Kim, Yuna; Lee, Hyeong-Min; Xiong, Yan; Sciaky, Noah; Hulbert, Samuel W; Cao, Xinyu; Everitt, Jeffrey I; Jin, Jian; Roth, Bryan L; Jiang, Yong-hui

2017-01-01

Prader–Willi syndrome (PWS) is an imprinting disorder caused by a deficiency of paternally expressed gene(s) in the 15q11–q13 chromosomal region. The regulation of imprinted gene expression in this region is coordinated by an imprinting center (PWS-IC). In individuals with PWS, genes responsible for PWS on the maternal chromosome are present, but repressed epigenetically, which provides an opportunity for the use of epigenetic therapy to restore expression from the maternal copies of PWS-associated genes. Through a high-content screen (HCS) of >9,000 small molecules, we discovered that UNC0638 and UNC0642—two selective inhibitors of euchromatic histone lysine N-methyltransferase-2 (EHMT2, also known as G9a)—activated the maternal (m) copy of candidate genes underlying PWS, including the SnoRNA cluster SNORD116, in cells from humans with PWS and also from a mouse model of PWS carrying a paternal (p) deletion from small nuclear ribonucleoprotein N (Snrpn (S)) to ubiquitin protein ligase E3A (Ube3a (U)) (mouse model referred to hereafter as m+/pΔS−U). Both UNC0642 and UNC0638 caused a selective reduction of the dimethylation of histone H3 lysine 9 (H3K9me2) at PWS-IC, without changing DNA methylation, when analyzed by bisulfite genomic sequencing. This indicates that histone modification is essential for the imprinting of candidate genes underlying PWS. UNC0642 displayed therapeutic effects in the PWS mouse model by improving the survival and the growth of m+/pΔS−U newborn pups. This study provides the first proof of principle for an epigenetics-based therapy for PWS. PMID:28024084

A VEGF-dependent gene signature enriched in mesenchymal ovarian cancer predicts patient prognosis.

PubMed

Yin, Xia; Wang, Xiaojie; Shen, Boqiang; Jing, Ying; Li, Qing; Cai, Mei-Chun; Gu, Zhuowei; Yang, Qi; Zhang, Zhenfeng; Liu, Jin; Li, Hongxia; Di, Wen; Zhuang, Guanglei

2016-08-08

We have previously reported surrogate biomarkers of VEGF pathway activities with the potential to provide predictive information for anti-VEGF therapies. The aim of this study was to systematically evaluate a new VEGF-dependent gene signature (VDGs) in relation to molecular subtypes of ovarian cancer and patient prognosis. Using microarray profiling and cross-species analysis, we identified 140-gene mouse VDGs and corresponding 139-gene human VDGs, which displayed enrichment of vasculature and basement membrane genes. In patients who received bevacizumab therapy and showed partial response, the expressions of VDGs (summarized to yield VDGs scores) were markedly decreased in post-treatment biopsies compared with pre-treatment baselines. In contrast, VDGs scores were not significantly altered following bevacizumab treatment in patients with stable or progressive disease. Analysis of VDGs in ovarian cancer showed that VDGs as a prognostic signature was able to predict patient outcome. Correlation estimation of VDGs scores and molecular features revealed that VDGs was overrepresented in mesenchymal subtype and BRCA mutation carriers. These findings highlighted the prognostic role of VEGF-mediated angiogenesis in ovarian cancer, and proposed a VEGF-dependent gene signature as a molecular basis for developing novel diagnostic strategies to aid patient selection for VEGF-targeted agents.

Hypoxia as a target for tissue specific gene therapy.

PubMed

Rhim, Taiyoun; Lee, Dong Yun; Lee, Minhyung

2013-12-10

Hypoxia is a hallmark of various ischemic diseases such as ischemic heart disease, ischemic limb, ischemic stroke, and solid tumors. Gene therapies for these diseases have been developed with various therapeutic genes including growth factors, anti-apoptotic genes, and toxins. However, non-specific expression of these therapeutic genes may induce dangerous side effects in the normal tissues. To avoid the side effects, gene expression should be tightly regulated in an oxygen concentration dependent manner. The hypoxia inducible promoters and enhancers have been evaluated as a transcriptional regulation tool for hypoxia inducible gene therapy. The hypoxia inducible UTRs were also used in gene therapy for spinal cord injury as a translational regulation strategy. In addition to transcriptional and translational regulations, post-translational regulation strategies have been developed using the HIF-1α ODD domain. Hypoxia inducible transcriptional, translational, and post-translational regulations are useful for tissue specific gene therapy of ischemic diseases. In this review, hypoxia inducible gene expression systems are discussed and their applications are introduced. Copyright © 2013 Elsevier B.V. All rights reserved.

Genome-Editing Technologies in Adoptive T Cell Immunotherapy for Cancer.

PubMed

Singh, Nathan; Shi, Junwei; June, Carl H; Ruella, Marco

2017-12-01

In this review, we discuss the most recent developments in gene-editing technology and discuss their application to adoptive T cell immunotherapy. Engineered T cell therapies targeting cancer antigens have demonstrated significant efficacy in specific patient populations. Most impressively, CD19-directed chimeric antigen receptor T cells (CART19) have led to impressive responses in patients with B-cell leukemia and lymphoma. CTL019, or KYMRIAH™ (tisagenlecleucel), a CD19 CAR T cell product developed by Novartis and the University of Pennsylvania, was recently approved for clinical use by the Food and Drug Administration, representing a landmark in the application of adoptive T cell therapies. As CART19 enters routine clinical use, improving the efficacy of this exciting platform is the next step in broader application. Novel gene-editing technologies like CRISPR-Cas9 allow facile editing of specific genes within the genome, generating a powerful platform to further optimize the activity of engineered T cells.

Shock wave induced sonoporation and gene transfer

NASA Astrophysics Data System (ADS)

Miller, Douglas L.

2003-10-01

During shockwave (SW) treatment, cavitation activity can be applied for cell killing. A bonus is that some surviving cells appear to be briefly permeabilized, or sonoporated, allowing them to take up large molecules including DNA. In vitro research has indicated that as the number of SW increased, survival declined exponentially but the number of sonoporated cells increased to better than 50% of survivors for 1000 SW. In vivo tests have demonstrated SW-induced tumor ablation could indeed be accompanied by the transfection of marker plasmids into mouse B16 melanoma tumors in vivo. With intratumor injection of plasmid DNA and air bubbles, significant results were obtained for only 400 SW. In a trial of cancer therapy, the effects of 500 SW combined with interleukin-12 immuno-gene therapy was observed on the progression of two mouse tumors, B16 melanoma and RENCA renal carcinoma. The combination of SW and IL-12 plasmid injection provided a statistically significant inhibition of tumor growth relative to SW alone for both tumor models, demonstrating feasibility for this treatment method. In the future, the development of intravenous gene delivery and improved transfection, together with image-guided ultrasound treatment, should lead to the clinical application of ultrasound enhanced gene therapy. [Work supported by NIH Grant No. EB002782.

Remission of Diabetes by Insulin Gene Therapy Using a Hepatocyte-specific and Glucose-responsive Synthetic Promoter

PubMed Central

Han, Jaeseok; McLane, Brienne; Kim, Eung-Hwi; Yoon, Ji-Won; Jun, Hee-Sook

2011-01-01

Efficient production of insulin in response to changes in glucose levels has been a major issue for insulin gene therapy to treat diabetes. To express target genes in response to glucose specifically in hepatocytes, we generated a synthetic promoter library containing hepatocyte nuclear factor-1, CAAT/enhancer-binding protein (C/EBP) response element, and glucose-response element. Combinations of these three cis-elements in 3-, 6-, or 9-element configurations were screened for transcriptional activity and then glucose responsiveness in vitro. The most effective promoter (SP23137) was selected for further study. Intravenous administration of a recombinant adenovirus expressing furin-cleavable rat insulin under control of the SP23137 promoter into streptozotocin (STZ)-induced diabetic mice resulted in normoglycemia, which was maintained for >30 days. Glucose tolerance tests showed that treated mice produced insulin in response to glucose and cleared exogenous glucose from the blood in a manner similar to nondiabetic control mice, although the clearance was somewhat delayed. Insulin expression was seen specifically in the liver and not in other organs. These observations indicate the potential of this synthetic, artificial promoter to regulate glucose-responsive insulin production and remit hyperglycemia, thus providing a new method of liver-directed insulin gene therapy for type 1 diabetes. PMID:21119621

Remission of diabetes by insulin gene therapy using a hepatocyte-specific and glucose-responsive synthetic promoter.

PubMed

Han, Jaeseok; McLane, Brienne; Kim, Eung-Hwi; Yoon, Ji-Won; Jun, Hee-Sook

2011-03-01

Efficient production of insulin in response to changes in glucose levels has been a major issue for insulin gene therapy to treat diabetes. To express target genes in response to glucose specifically in hepatocytes, we generated a synthetic promoter library containing hepatocyte nuclear factor-1, CAAT/enhancer-binding protein (C/EBP) response element, and glucose-response element. Combinations of these three cis-elements in 3-, 6-, or 9-element configurations were screened for transcriptional activity and then glucose responsiveness in vitro. The most effective promoter (SP23137) was selected for further study. Intravenous administration of a recombinant adenovirus expressing furin-cleavable rat insulin under control of the SP23137 promoter into streptozotocin (STZ)-induced diabetic mice resulted in normoglycemia, which was maintained for >30 days. Glucose tolerance tests showed that treated mice produced insulin in response to glucose and cleared exogenous glucose from the blood in a manner similar to nondiabetic control mice, although the clearance was somewhat delayed. Insulin expression was seen specifically in the liver and not in other organs. These observations indicate the potential of this synthetic, artificial promoter to regulate glucose-responsive insulin production and remit hyperglycemia, thus providing a new method of liver-directed insulin gene therapy for type 1 diabetes.

Therapeutic Effect of Angiostatin Gene Transfer in a Murine Model of Endometriosis

PubMed Central

Dabrosin, Charlotta; Gyorffy, Steve; Margetts, Peter; Ross, Catherine; Gauldie, Jack

2002-01-01

Endometriosis, the growth of ectopic endometrial tissue, is a chronic recurrent disease affecting 10% of the female population causing dyspareunia, pelvic pain, dysmenorrhea, and infertility. Suppression of ovarian activity is the cornerstone of medical therapy with limited benefit and severe adverse effects. Angiogenesis plays a major role in the development of endometriosis suggesting that anti-angiogenic therapy would offer a new therapeutic approach. We report successful treatment of endometriosis in estrogen-supplemented ovariectomized mice by transient overexpression (6 to 10 days of duration) of the gene for a natural angiogenesis inhibitor angiostatin, delivered to the peritoneum by a replication-deficient adenovirus vector (AdAngiostatin). Established endometriosis was eradicated in 14 of 14 AdAngiostatin-treated animals, whereas 11 of 13 control animals showed full disease development. Administered to normal cycling mice for the same transient period, AdAngiostatin caused impaired ovarian function with suppressed corpus luteum development, decreased production of estradiol and progesterone, decreased ovarian and uterine weight, and increased body weight. AdAngiostatin treatment lowered the levels of sex steroids but did not induce total castration. Gene therapy with angiogenic inhibitors is a highly effective treatment for endometriosis, even in a host with preserved estrogen levels. However, local or targeted delivery of the gene must be considered to avoid prolonged systemic effects and impaired ovarian function. PMID:12213719

Activation of an IL-6:STAT3-dependent Transcriptome in Pediatric-onset Inflammatory Bowel Disease

PubMed Central

Carey, Rebecca; Jurickova, Ingrid; Ballard, Edgar; Bonkowski, Erin; Han, Xiaonan; Xu, Huan; Denson, Lee A.

2008-01-01

Background: While activation of the IL-6-dependent transcription factor signal transducer and activator of transcription 3 (STAT3) has been implicated in the pathogenesis of inflammatory bowel disease (IBD), a direct effect on mucosal gene expression and inflammation has not been shown. We hypothesized that a proinflammatory IL-6:STAT3-dependent biological network would be up regulated in pediatric-onset IBD patients, and would be associated with the severity of mucosal inflammation. Methods: Patients with pediatric-onset IBD were enrolled at diagnosis and during therapy. Serum cytokine analysis was performed using Bioplex. STAT3 phosphorylation (pSTAT3) in peripheral blood leukocytes (PBLs) was assessed by flow cytometry. Immunohistochemistry of colonic mucosa was used to localize pSTAT3 and STAT3 target genes. Microarray analysis was used to determine RNA expression profiles from colon biopsies. Results: Circulating IL-6 was upregulated in active IBD patients at diagnosis and during therapy. STAT3 activation was increased in PB granulocytes, IL-6-stimulated CD3+/CD4+ lymphocytes, and affected colon biopsies of IBD patients. The frequency of pSTAT3+PB granulocytes and colon epithelial and lamina propria cells was highly correlated with the degree of mucosal inflammation. Microarray and Ingenuity Systems bioinformatics analysis identified IL-6:STAT3-dependent biological networks upregulated in IBD patients which control leukocyte recruitment, HLA expression, angiogenesis, and tissue remodeling. Conclusions: A proinflammatory IL6:STAT3 biologic network is upregulated in active pediatric IBD patients at diagnosis and during therapy. Specific targeting of this network may be effective in reducing mucosal inflammation. PMID:18069684

Interferon-alpha, immune activation and immune dysfunction in treated HIV infection

PubMed Central

Cha, Lilian; Berry, Cassandra M; Nolan, David; Castley, Allison; Fernandez, Sonia; French, Martyn A

2014-01-01

Type I interferons (IFNs) exert anti-viral effects through the induction of numerous IFN-stimulated genes and an immunomodulatory effect on innate and adaptive immune responses. This is beneficial in controlling virus infections but prolonged IFN-α activity in persistent virus infections, such as HIV infection, may contribute to immune activation and have a detrimental effect on the function of monocytes and T and B lymphocytes. Activation of monocytes, associated with increased IFN-α activity, contributes to atherosclerotic vascular disease, brain disease and other ‘age-related diseases' in HIV patients treated with long-term antiretroviral therapy (ART). In HIV patients receiving ART, the anti-viral effects of IFN-α therapy have the potential to contribute to eradication of HIV infection while IFN-α inhibitor therapy is under investigation for the treatment of immune activation. The management of HIV patients receiving ART will be improved by understanding more about the opposing effects of IFN-α on HIV infection and disease and by developing methods to assess IFN-α activity in clinical practice. PMID:25505958

Plasticity of the human visual system after retinal gene therapy in patients with Leber’s congenital amaurosis

PubMed Central

Ashtari, Manzar; Zhang, Hui; Cook, Philip A.; Cyckowski, Laura L.; Shindler, Kenneth S.; Marshall, Kathleen A.; Aravand, Puya; Vossough, Arastoo; Gee, James C.; Maguire, Albert M.; Baker, Chris I.; Bennett, Jean

2015-01-01

Much of our knowledge of the mechanisms underlying plasticity in the visual cortex in response to visual impairment, vision restoration, and environmental interactions comes from animal studies. We evaluated human brain plasticity in a group of patients with Leber’s congenital amaurosis (LCA), who regained vision through gene therapy. Using non-invasive multimodal neuroimaging methods, we demonstrated that reversing blindness with gene therapy promoted long-term structural plasticity in the visual pathways emanating from the treated retina of LCA patients. The data revealed improvements and normalization along the visual fibers corresponding to the site of retinal injection of the gene therapy vector carrying the therapeutic gene in the treated eye compared to the visual pathway for the untreated eye of LCA patients. After gene therapy, the primary visual pathways (for example, geniculostriate fibers) in the treated retina were similar to those of sighted control subjects, whereas the primary visual pathways of the untreated retina continued to deteriorate. Our results suggest that visual experience, enhanced by gene therapy, may be responsible for the reorganization and maturation of synaptic connectivity in the visual pathways of the treated eye in LCA patients. The interactions between the eye and the brain enabled improved and sustained long-term visual function in patients with LCA after gene therapy. PMID:26180100

Association of killer cell immunoglobulin-like receptor polymorphisms with chronic hepatitis C and responses to therapy in Brazil

PubMed Central

de Vasconcelos, Janaina Mota; de Jesus Maués Pereira Móia, Lizomar; Amaral, Ivanete do Socorro Abraçado; Miranda, Esther Castello Branco Mello; CicaliseTakeshita, Louise Yukari; de Oliveira, Layanna Freitas; de Araújo Melo Mendes, Lilian; Sastre, Danuta; Tamegão-Lopes, Bruna Pedroso; de Aquino Pedroza, Larysse Santa Rosa; Batista dos Santos, Sidney Emanuel; Soares, Manoel do Carmo Pereira; de Araújo, Marialva Tereza Ferreira; Bandeira, Camila Lucas; de Sousa da Silva, Adriana Maria Paixão; de Medeiros, Zilene Lameira; Sena, Leonardo; Demachki, Samia; dos Santos, Eduardo José Melo

2013-01-01

Soroprevalence for Hepatitis C virus is reported as 2.12% in Northern Brazil, with about 50% of the patients exhibiting a sustained virological response (SVR). Aiming to associate polymorphisms in Killer Cell Immunoglobulin-like Receptors (KIR) with chronic hepatitis C and therapy responses we investigated 125 chronic patients and 345 controls. Additionally, 48 ancestry markers were genotyped to control for population stratification. The frequency of the KIR2DL2 and KIR2DL2+HLA-CAsp80 gene and ligand was higher in chronic infected patients than in controls (p < 0.0009, OR = 3.4; p = 0.001, OR = 3.45). In fact, KIR2DL3 is a weaker inhibitor of NK activity than KIR2DL2, which could explain the association of KIR2DL2 with chronic infection. Moreover, KIR2DS2 and KIR2DS2+HLA-CAsp80 (p < 0.0001, OR = 2.51; p = 0.0084, OR = 2.62) and KIR2DS3 (p < 0.0001; OR = 2.57) were associated with chronic infection, independently from KIR2DL2. No differences in ancestry composition were observed between control and patients, even with respect to therapy response groups. The allelic profile KIR2DL2/KIR2DS2/KIR2DS3 was associated with the chronic hepatitis C (p < 0.0001; OR = 3). Furthermore, the patients also showed a higher mean number of activating genes and a lower frequency of the homozygous AA profile, which is likely secondary to the association with non-AA and/or activating genes. In addition, the KIR2DS5 allele was associated with SVR (p = 0.0261; OR = 0.184).The ancestry analysis of samples ruled out any effects of population substructuring and did not evidence interethnic differences in therapy response, as suggested in previous studies. PMID:23569404

Progress toward Gene Therapy for Duchenne Muscular Dystrophy.

PubMed

Chamberlain, Joel R; Chamberlain, Jeffrey S

2017-05-03

Duchenne muscular dystrophy (DMD) has been a major target for gene therapy development for nearly 30 years. DMD is among the most common genetic diseases, and isolation of the defective gene (DMD, or dystrophin) was a landmark discovery, as it was the first time a human disease gene had been cloned without knowledge of the protein product. Despite tremendous obstacles, including the enormous size of the gene and the large volume of muscle tissue in the human body, efforts to devise a treatment based on gene replacement have advanced steadily through the combined efforts of dozens of labs and patient advocacy groups. Progress in the development of DMD gene therapy has been well documented in Molecular Therapy over the past 20 years and will be reviewed here to highlight prospects for success in the imminent human clinical trials planned by several groups. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Role of the NFκB-signaling pathway in cancer

PubMed Central

Zhou, Yujuan; Lin, Jingguan; Wang, Heran; Oyang, Linda; Tian, Yutong; Liu, Lu; Su, Min; Wang, Hui; Cao, Deliang; Liao, Qianjin

2018-01-01

Cancer is a group of cells that malignantly grow and proliferate uncontrollably. At present, treatment modes for cancer mainly comprise surgery, chemotherapy, radiotherapy, molecularly targeted therapy, gene therapy, and immunotherapy. However, the curative effects of these treatments have been limited thus far by specific characteristics of tumors. Abnormal activation of signaling pathways is involved in tumor pathogenesis and plays critical roles in growth, progression, and relapse of cancers. Targeted therapies against effectors in oncogenic signaling have improved the outcomes of cancer patients. NFκB is an important signaling pathway involved in pathogenesis and treatment of cancers. Excessive activation of the NFκB-signaling pathway has been documented in various tumor tissues, and studies on this signaling pathway for targeted cancer therapy have become a hot topic. In this review, we update current understanding of the NFκB-signaling pathway in cancer. PMID:29695914

Intratumoral gene therapy versus intravenous gene therapy for distant metastasis control with 2-diethylaminoethyl-dextran methyl methacrylate copolymer non-viral vector-p53.

PubMed

Baliaka, A; Zarogoulidis, P; Domvri, K; Hohenforst-Schmidt, W; Sakkas, A; Huang, H; Le Pivert, P; Koliakos, G; Koliakou, E; Kouzi-Koliakos, K; Tsakiridis, K; Chioti, A; Siotou, E; Cheva, A; Zarogoulidis, K; Sakkas, L

2014-02-01

Lung cancer still remains to be challenged by novel treatment modalities. Novel locally targeted routes of administration are a methodology to enhance treatment and reduce side effects. Intratumoral gene therapy is a method for local treatment and could be used either in early-stage lung cancer before surgery or at advanced stages as palliative care. Novel non-viral vectors are also in demand for efficient gene transfection to target local cancer tissue and at the same time protect the normal tissue. In the current study, C57BL/6 mice were divided into three groups: (a) control, (b) intravenous and (c) intatumoral gene therapy. The novel 2-Diethylaminoethyl-Dextran Methyl Methacrylate Copolymer Non-Viral Vector (Ryujyu Science Corporation) was conjugated with plasmid pSicop53 from the company Addgene for the first time. The aim of the study was to evaluate the safety and efficacy of targeted gene therapy in a Lewis lung cancer model. Indeed, although the pharmacokinetics of the different administration modalities differs, the intratumoral administration presented increased survival and decreased distant metastasis. Intratumoral gene therapy could be considered as an efficient local therapy for lung cancer.

Lentiviral vectors for gene therapy of heart disease.

PubMed

Higuchi, Koji; Medin, Jeffrey A

2007-01-01

Technological advances in genetic engineering developed over the past few years have been applied to the research and treatment of cardiovascular diseases. In many animal models, gene therapy has been shown to be an effective treatment schema. Some of these gene therapy treatments are now being applied in clinical trials. Also, as the science of gene therapy has progressed, alternative vector systems such as lentiviruses have been developed and implemented. Here we focus on the emerging role of lentiviral vectors in the treatment of cardiovascular disease.

Advancing Translational Research Through the NHLBI Gene Therapy Resource Program (GTRP)

PubMed Central

Benson, Janet; Cornetta, Kenneth; Diggins, Margaret; Johnston, Julie C.; Sepelak, Susan; Wang, Gensheng; Wilson, James M.; Wright, J. Fraser; Skarlatos, Sonia I.

2013-01-01

Abstract Translational research is a lengthy, complex, and necessary endeavor in order to bring basic science discoveries to clinical fruition. The NIH offers several programs to support translational research including an important resource established specifically for gene therapy researchers—the National Heart, Lung, and Blood Institute (NHLBI) Gene Therapy Resource Program (GTRP). This paper reviews the core components of the GTRP and describes how the GTRP provides researchers with resources that are critical to advancing investigational gene therapy products into clinical testing. PMID:23692378

Taking Stock of Retinal Gene Therapy: Looking Back and Moving Forward.

PubMed

Bennett, Jean

2017-05-03

Over the past 20 years, there has been tremendous progress in retinal gene therapy. The safety and efficacy results in one early-onset severe blinding disease may lead to the first gene therapy drug approval in the United States. Here, we review how far the field has come over the past two decades and speculate on the directions that the field will take in the future. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Germ-line gene therapy and the medical imperative.

PubMed

Munson, Ronald; Davis, Lawrence H

1992-06-01

Somatic cell gene therapy has yielded promising results. If germ cell gene therapy can be developed, the promise is even greater: hundreds of genetic diseases might be virtually eliminated. But some claim the procedure is morally unacceptable. We thoroughly and sympathetically examine several possible reasons for this claim but find them inadequate. There is no moral reason, then, not to develop and employ germ-line gene therapy. Taking the offensive, we argue next that medicine has a prima facie moral obligation to do so.

Silicon nanostructures for cancer diagnosis and therapy.

PubMed

Peng, Fei; Cao, Zhaohui; Ji, Xiaoyuan; Chu, Binbin; Su, Yuanyuan; He, Yao

2015-01-01

The emergence of nanotechnology suggests new and exciting opportunities for early diagnosis and therapy of cancer. During the recent years, silicon-based nanomaterials featuring unique properties have received great attention, showing high promise for myriad biological and biomedical applications. In this review, we will particularly summarize latest representative achievements on the development of silicon nanostructures as a powerful platform for cancer early diagnosis and therapy. First, we introduce the silicon nanomaterial-based biosensors for detecting cancer markers (e.g., proteins, tumor-suppressor genes and telomerase activity, among others) with high sensitivity and selectivity under molecular level. Then, we summarize in vitro and in vivo applications of silicon nanostructures as efficient nanoagents for cancer therapy. Finally, we discuss the future perspective of silicon nanostructures for cancer diagnosis and therapy.

Delivery of Na/I symporter gene into skeletal muscle using nanobubbles and ultrasound: visualization of gene expression by PET.

PubMed

Watanabe, Yukiko; Horie, Sachiko; Funaki, Yoshihito; Kikuchi, Youhei; Yamazaki, Hiromichi; Ishii, Keizo; Mori, Shiro; Vassaux, Georges; Kodama, Tetsuya

2010-06-01

The development of nonviral gene delivery systems is essential in gene therapy, and the use of a minimally invasive imaging methodology can provide important clinical endpoints. In the current study, we present a new methodology for gene therapy-a delivery system using nanobubbles and ultrasound as a nonviral gene delivery method. We assessed whether the gene transfer allowed by this methodology was detectable by PET and bioluminescence imaging. Two kinds of reported vectors (luciferase and human Na/I symporter [hNIS]) were transfected or cotransfected into the skeletal muscles of normal mice (BALB/c) using the ultrasound-nanobubbles method. The kinetics of luciferase gene expression were analyzed in vivo using bioluminescence imaging. At the peak of gene transfer, PET of hNIS expression was performed using our recently developed PET scanner, after (124)I injection. The imaging data were confirmed using reverse-transcriptase polymerase chain reaction amplification, biodistribution, and a blocking study. The imaging potential of the 2 methodologies was evaluated in 2 mouse models of human pathology (McH/lpr-RA1 mice showing vascular disease and C57BL/10-mdx Jic mice showing muscular dystrophy). Peak luciferase gene activity was observed in the skeletal muscle 4 d after transfection. On day 2 after hNIS and luciferase cotransfection, the expression of these genes was confirmed by reverse-transcriptase polymerase chain reaction on a muscle biopsy. PET of the hNIS gene, biodistribution, the blocking study, and autoradiography were performed on day 4 after transfection, and it was indicated that hNIS expression was restricted to the site of plasmid administration (skeletal muscle). Similar localized PET and (124)I accumulation were successfully obtained in the disease-model mice. The hNIS gene was delivered into the skeletal muscle of healthy and disease-model mice by the ultrasound-nanobubbles method, and gene expression was successfully visualized with PET. The combination of ultrasound-nanobubble gene transfer and PET may be applied to gene therapy clinical protocols.

Nanoparticles for Retinal Gene Therapy

PubMed Central

Conley, Shannon M.; Naash, Muna I.

2010-01-01

Ocular gene therapy is becoming a well-established field. Viral gene therapies for the treatment of Leber’s congentinal amaurosis (LCA) are in clinical trials, and many other gene therapy approaches are being rapidly developed for application to diverse ophthalmic pathologies. Of late, development of non-viral gene therapies has been an area of intense focus and one technology, polymer-compacted DNA nanoparticles, is especially promising. However, development of pharmaceutically and clinically viable therapeutics depends not only on having an effective and safe vector but also on a practical treatment strategy. Inherited retinal pathologies are caused by mutations in over 220 genes, some of which contain over 200 individual disease-causing mutations, which are individually very rare. This review will focus on both the progress and future of nanoparticles and also on what will be required to make them relevant ocular pharmaceutics. PMID:20452457

[Ethical guidelines on genetic testing and gene therapy].

PubMed

Fukushima, Yoshimitsu

2005-03-01

According to the recent and rapid advances in molecular genetics research, genetic testing and gene therapy have a potential of giving unexpected influence to the human beings. To prevent and to solve various ethical, legal and social implementations (ELSI) of genetic testing and gene therapy, several guidelines have been established. In Japan, all researchers and all clinicians have to know and keep the following three guidelines on genetic testing and a guideline on gene therapy: 1) "Guidelines for Researches on Human Genome and Gene (2001)" by the three Ministries (Education, Health and Economy), 2) "Guidelines for Genetic Testing (2001)" by the Genetic--medicine--related 10 societies, 3) "Ethical Principles on Entrusted Genetic Testing (2001)" by the Japan Registered Clinical Laboratories Association, and 4) "Guidelines for Clinical Research on Gene Therapy (2002)" by the two Ministries (Health and Education).

Long-term safety and efficacy of AAV gene therapy in the canine model of glycogen storage disease type Ia.

PubMed

Lee, Young Mok; Conlon, Thomas J; Specht, Andrew; Coleman, Kirsten E; Brown, Laurie M; Estrella, Ana M; Dambska, Monika; Dahlberg, Kathryn R; Weinstein, David A

2018-05-25

Viral mediated gene therapy has progressed after overcoming early failures, and gene therapy has now been approved for several conditions in Europe and the USA. Glycogen storage disease (GSD) type Ia, caused by a deficiency of glucose-6-phosphatase-α, has been viewed as an outstanding candidate for gene therapy. This follow-up report describes the long-term outcome for the naturally occurring GSD-Ia dogs treated with rAAV-GPE-hG6PC-mediated gene therapy. A total of seven dogs were treated with rAAV-GPE-hG6PC-mediated gene therapy. The first four dogs were treated at birth, and three dogs were treated between 2 and 6 months of age to assess the efficacy and safety in animals with mature livers. Blood and urine samples, radiographic studies, histological evaluation, and biodistribution were assessed. Gene therapy improved survival in the GSD-Ia dogs. With treatment, the biochemical studies normalized for the duration of the study (up to 7 years). None of the rAAV-GPE-hG6PC-treated dogs had focal hepatic lesions or renal abnormalities. Dogs treated at birth required a second dose of rAAV after 2-4 months; gene therapy after hepatic maturation resulted in improved efficacy after a single dose. rAAV-GPE-hG6PC treatment in GSD-Ia dogs was found to be safe and efficacious. GSD-Ia is an attractive target for human gene therapy since it is a monogenic disorder with limited tissue involvement. Blood glucose and lactate monitoring can be used to assess effectiveness and as a biomarker of success. GSD-Ia can also serve as a model for other hepatic monogenic disorders.

Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe's Disease after Global Gene Therapy.

PubMed

Marshall, Michael S; Issa, Yazan; Jakubauskas, Benas; Stoskute, Monika; Elackattu, Vince; Marshall, Jeffrey N; Bogue, Wil; Nguyen, Duc; Hauck, Zane; Rue, Emily; Karumuthil-Melethil, Subha; Zaric, Violeta; Bosland, Maarten; van Breemen, Richard B; Givogri, Maria I; Gray, Steven J; Crocker, Stephen J; Bongarzone, Ernesto R

2018-03-07

We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving nearly complete correction of GALC's metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Gene therapy in animal models of autosomal dominant retinitis pigmentosa

PubMed Central

Rossmiller, Brian; Mao, Haoyu

2012-01-01

Gene therapy for dominantly inherited genetic disease is more difficult than gene-based therapy for recessive disorders, which can be treated with gene supplementation. Treatment of dominant disease may require gene supplementation partnered with suppression of the expression of the mutant gene either at the DNA level, by gene repair, or at the RNA level by RNA interference or transcriptional repression. In this review, we examine some of the gene delivery approaches used to treat animal models of autosomal dominant retinitis pigmentosa, focusing on those models associated with mutations in the gene for rhodopsin. We conclude that combinatorial approaches have the greatest promise for success. PMID:23077406

Azadirachtin interacts with retinoic acid receptors and inhibits retinoic acid-mediated biological responses.

PubMed

Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B; Sureshkumar, Chitta; Manna, Sunil K

2011-02-11

Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency.

PubMed

Sauer, Aisha V; Mrak, Emanuela; Hernandez, Raisa Jofra; Zacchi, Elena; Cavani, Francesco; Casiraghi, Miriam; Grunebaum, Eyal; Roifman, Chaim M; Cervi, Maria C; Ambrosi, Alessandro; Carlucci, Filippo; Roncarolo, Maria Grazia; Villa, Anna; Rubinacci, Alessandro; Aiuti, Alessandro

2009-10-08

Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling.

Gene Therapy for Hemophilia and Duchenne Muscular Dystrophy in China.

PubMed

Liu, Xionghao; Liu, Mujun; Wu, Lingqian; Liang, Desheng

2018-02-01

Gene therapy is a new technology that provides potential for curing monogenic diseases caused by mutations in a single gene. Hemophilia and Duchenne muscular dystrophy (DMD) are ideal target diseases of gene therapy. Important advances have been made in clinical trials, including studies of adeno-associated virus vectors in hemophilia and antisense in DMD. However, issues regarding the high doses of viral vectors required and limited delivery efficiency of antisense oligonucleotides have not yet been fully addressed. As an alternative strategy to classic gene addition, genome editing based on programmable nucleases has also shown promise to correct mutations in situ. This review describes the recent progress made by Chinese researchers in gene therapy for hemophilia and DMD.

The Status of RPE65 Gene Therapy Trials: Safety and Efficacy

PubMed Central

Pierce, Eric A.; Bennett, Jean

2015-01-01

Several groups have reported the results of clinical trials of gene augmentation therapy for Leber congenital amaurosis (LCA) because of mutations in the RPE65 gene. These studies have used subretinal injection of adeno-associated virus (AAV) vectors to deliver the human RPE65 cDNA to the retinal pigment epithelial (RPE) cells of the treated eyes. In all of the studies reported to date, this approach has been shown to be both safe and effective. The successful clinical trials of gene augmentation therapy for retinal degeneration caused by mutations in the RPE65 gene sets the stage for broad application of gene therapy to treat retinal degenerative disorders. PMID:25635059

Investor Outlook: Gene Therapy Picking up Steam; At a Crossroads.

PubMed

Schimmer, Joshua; Breazzano, Steven

2016-09-01

The gene therapy field continues to pick up steam with recent successes in a number of different therapeutic indications that highlight the potential for the platform. As the field continues to make progress, a growing data set of long-term safety and efficacy data will continue to define gene therapy's role, determining ultimately how widely it may be used beyond rare, serious diseases with high unmet needs. New technologies often take unanticipated twists and turns as patient exposure accumulates, and gene therapy may be no exception. That said, with many diseases that have no other treatment options beyond gene therapy and that present considerable morbidity and mortality, the field appears poised to withstand some minor and even major bumps in the road should they emerge.

Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics

PubMed Central

Cideciyan, Artur V.; Aleman, Tomas S.; Boye, Sanford L.; Schwartz, Sharon B.; Kaushal, Shalesh; Roman, Alejandro J.; Pang, Ji-jing; Sumaroka, Alexander; Windsor, Elizabeth A. M.; Wilson, James M.; Flotte, Terence R.; Fishman, Gerald A.; Heon, Elise; Stone, Edwin M.; Byrne, Barry J.; Jacobson, Samuel G.; Hauswirth, William W.

2008-01-01

The RPE65 gene encodes the isomerase of the retinoid cycle, the enzymatic pathway that underlies mammalian vision. Mutations in RPE65 disrupt the retinoid cycle and cause a congenital human blindness known as Leber congenital amaurosis (LCA). We used adeno-associated virus-2-based RPE65 gene replacement therapy to treat three young adults with RPE65-LCA and measured their vision before and up to 90 days after the intervention. All three patients showed a statistically significant increase in visual sensitivity at 30 days after treatment localized to retinal areas that had received the vector. There were no changes in the effect between 30 and 90 days. Both cone- and rod-photoreceptor-based vision could be demonstrated in treated areas. For cones, there were increases of up to 1.7 log units (i.e., 50 fold); and for rods, there were gains of up to 4.8 log units (i.e., 63,000 fold). To assess what fraction of full vision potential was restored by gene therapy, we related the degree of light sensitivity to the level of remaining photoreceptors within the treatment area. We found that the intervention could overcome nearly all of the loss of light sensitivity resulting from the biochemical blockade. However, this reconstituted retinoid cycle was not completely normal. Resensitization kinetics of the newly treated rods were remarkably slow and required 8 h or more for the attainment of full sensitivity, compared with <1 h in normal eyes. Cone-sensitivity recovery time was rapid. These results demonstrate dramatic, albeit imperfect, recovery of rod- and cone-photoreceptor-based vision after RPE65 gene therapy. PMID:18809924

Transducing Airway Basal Cells with a Helper-Dependent Adenoviral Vector for Lung Gene Therapy.

PubMed

Cao, Huibi; Ouyang, Hong; Grasemann, Hartmut; Bartlett, Claire; Du, Kai; Duan, Rongqi; Shi, Fushan; Estrada, Marvin; Seigel, Kyle E; Coates, Allan L; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Moraes, Theo J; Hu, Jim

2018-06-01

A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells, of which basal cells are the major type in human airways. In this study, helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery, and direct bronchoscopic instillation, respectively. Vector transduction was assessed by immunostaining of lung tissue sections, which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition, efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore, we successfully delivered the human CFTR gene to airway basal cells from CF patients, and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases.

Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice.

PubMed

Iyer, Meera; Berenji, Manijeh; Templeton, Nancy S; Gambhir, Sanjiv S

2002-10-01

Gene therapy involves the safe and effective delivery of one or more genes of interest to target cells in vivo. The advantages of using nonviral delivery systems include ease of preparation, low toxicity, and weak immunogenicity. Nonviral delivery methods, when combined with a noninvasive, clinically applicable imaging assay, will greatly aid in the optimization of gene therapy approaches for cancer. We demonstrate cationic lipid-mediated noninvasive monitoring of reporter gene expression of firefly (Photinus pyralis) luciferase (fl) and a mutant herpes simplex virus type I thymidine kinase (HSV1-sr39tk, tk) in living mice using a cooled charge coupled device (CCD) camera and positron emission tomography (PET), respectively. We observe a high level of fl and tk reporter gene expression predominantly in the lungs after a single injection of the extruded DOTAP:cholesterol DNA liposome complexes by way of the tail vein, seen to be time- and dose-dependent. We observe a good correlation between the in vivo bioluminescent signal and the ex vivo firefly luciferase enzyme (FL) activity in different organs. We further demonstrate the feasibility of noninvasively imaging both optical and PET reporter gene expression in the same animal using the CCD camera and microPET, respectively.

Growth Suppression and Therapy Sensitization of Breast Cancer

DTIC Science & Technology

2000-07-01

determined by performed on two independent occasions. PCR amplification of a given housekeeping gene have been shown to correspond to determinations of...h incubation in the presence or absence of 1 mM cisplatin expressed housekeeping gene, dihydrofolate reductase (DHFR). (Platinol, aqueous solution at... G3PDH :j G3PDH Figure 9. A549 cells were treated with 3 different antisense oligonucleotides complementary to JNKI mRNA (including the active antisense

Orexin gene therapy restores the timing and maintenance of wakefulness in narcoleptic mice.

PubMed

Kantor, Sandor; Mochizuki, Takatoshi; Lops, Stefan N; Ko, Brian; Clain, Elizabeth; Clark, Erika; Yamamoto, Mihoko; Scammell, Thomas E

2013-08-01

Narcolepsy is caused by selective loss of the orexin/hypocretin-producing neurons of the hypothalamus. For patients with narcolepsy, chronic sleepiness is often the most disabling symptom, but current therapies rarely normalize alertness and do not address the underlying orexin deficiency. We hypothesized that the sleepiness of narcolepsy would substantially improve if orexin signaling were restored in specific brain regions at appropriate times of day. We used gene therapy to restore orexin signaling in a mouse model of narcolepsy. In these Atx mice, expression of a toxic protein (ataxin-3) selectively kills the orexin neurons. To induce ectopic expression of the orexin neuropeptides, we microinjected an adeno-associated viral vector coding for prepro-orexin plus a red fluorescence protein (AAV-orexin) into the mediobasal hypothalamus of Atx and wild-type mice. Control mice received an AAV coding only for red fluorescence protein. Two weeks later, we recorded sleep/wake behavior, locomotor activity, and body temperature and examined the patterns of orexin expression. Atx mice rescued with AAV-orexin produced long bouts of wakefulness and had a normal diurnal pattern of arousal, with the longest bouts of wake and the highest amounts of locomotor activity in the first hours of the night. In addition, AAV-orexin improved the timing of rapid eye movement sleep and the consolidation of nonrapid eye movement sleep in Atx mice. These substantial improvements in sleepiness and other symptoms of narcolepsy demonstrate the effectiveness of orexin gene therapy in a mouse model of narcolepsy. Additional work is needed to optimize this approach, but in time, AAV-orexin could become a useful therapeutic option for patients with narcolepsy.

Gene therapy for cardiovascular disease mediated by ultrasound and microbubbles

PubMed Central

2013-01-01

Gene therapy provides an efficient approach for treatment of cardiovascular disease. To realize the therapeutic effect, both efficient delivery to the target cells and sustained expression of transgenes are required. Ultrasound targeted microbubble destruction (UTMD) technique has become a potential strategy for target-specific gene and drug delivery. When gene-loaded microbubble is injected, the ultrasound-mediated microbubble destruction may spew the transported gene to the targeted cells or organ. Meanwhile, high amplitude oscillations of microbubbles increase the permeability of capillary and cell membrane, facilitating uptake of the released gene into tissue and cell. Therefore, efficiency of gene therapy can be significantly improved. To date, UTMD has been successfully investigated in many diseases, and it has achieved outstanding progress in the last two decades. Herein, we discuss the current status of gene therapy of cardiovascular diseases, and reviewed the progress of the delivery of genes to cardiovascular system by UTMD. PMID:23594865

The Pathway From Genes to Gene Therapy in Glaucoma: A Review of Possibilities for Using Genes as Glaucoma Drugs

PubMed Central

Borrás, Teresa

2018-01-01

Treatment of diseases with gene therapy is advancing rapidly. The use of gene therapy has expanded from the original concept of replacing the mutated gene causing the disease to the use of genes to control nonphysiological levels of expression or to modify pathways known to affect the disease. Genes offer numerous advantages over conventional drugs. They have longer duration of action and are more specific. Genes can be delivered to the target site by naked DNA, cells, nonviral, and viral vectors. The enormous progress of the past decade in molecular biology and delivery systems has provided ways for targeting genes to the intended cell/tissue and safe, long-term vectors. The eye is an ideal organ for gene therapy. It is easily accessible and it is an immune-privileged site. Currently, there are clinical trials for diseases affecting practically every tissue of the eye, including those to restore vision in patients with Leber congenital amaurosis. However, the number of eye trials compared with those for systemic diseases is quite low (1.8%). Nevertheless, judging by the vast amount of ongoing preclinical studies, it is expected that such number will increase considerably in the near future. One area of great need for eye gene therapy is glaucoma, where a long-term gene drug would eliminate daily applications and compliance issues. Here, we review the current state of gene therapy for glaucoma and the possibilities for treating the trabecular meshwork to lower intraocular pressure and the retinal ganglion cells to protect them from neurodegeneration. PMID:28161916

The important role of von Willebrand factor in platelet-derived FVIII gene therapy for murine hemophilia A in the presence of inhibitory antibodies.

PubMed

Shi, Q; Schroeder, J A; Kuether, E L; Montgomery, R R

2015-07-01

Our previous studies have demonstrated that targeting FVIII expression to platelets results in FVIII storage together with von Willebrand factor (VWF) in platelet α-granules and that platelet-derived FVIII (2bF8) corrects the murine hemophilia A phenotype even in the presence of high-titer anti-FVIII inhibitory antibodies (inhibitors). To explore how VWF has an impact on platelet gene therapy for hemophilia A with inhibitors. 2bF8 transgenic mice in the FVIII(-/-) background (2bF8(tg+/-) F8(-/-) ) with varying VWF phenotypes were used in this study. Animals were analyzed by VWF ELISA, FVIII activity assay, Bethesda assay and tail clip survival test. Only 18% of 2bF8(tg+/-) F8(-/-) VWF(-/-) animals, in which VWF was deficient, survived the tail clip challenge with inhibitor titers of 3-8000 BU mL(-1) . In contrast, 82% of 2bF8(tg+/-) F8(-/-) VWF(+/+) mice, which had normal VWF levels, survived tail clipping with inhibitor titers of 10-50,000 BU mL(-1) . All 2bF8(tg+/-) F8(-/-) VWF(-/-) mice without inhibitors survived tail clipping and no VWF(-/-) F8(-/-) mice survived this challenge. Because VWF is synthesized by endothelial cells and megakaryocytes and is distributed in both plasma and platelets in peripheral blood, we further investigated the effect of each compartment of VWF on platelet-FVIII gene therapy for hemophilia A with inhibitors. In the presence of inhibitors, 42% of animals survived tail clipping in the group with plasma-VWF and 50% survived in the platelet-VWF group. VWF is essential for platelet gene therapy for hemophilia A with inhibitors. Both platelet-VWF and plasma-VWF are required for optimal platelet-derived FVIII gene therapy for hemophilia A in the presence of inhibitors. © 2015 International Society on Thrombosis and Haemostasis.

Synergistic and Antagonistic Interplay between Myostatin Gene Expression and Physical Activity Levels on Gene Expression Patterns in Triceps Brachii Muscles of C57/BL6 Mice

PubMed Central

Caetano-Anollés, Kelsey; Mishra, Sanjibita; Rodriguez-Zas, Sandra L.

2015-01-01

Levels of myostatin expression and physical activity have both been associated with transcriptome dysregulation and skeletal muscle hypertrophy. The transcriptome of triceps brachii muscles from male C57/BL6 mice corresponding to two genotypes (wild-type and myostatin-reduced) under two conditions (high and low physical activity) was characterized using RNA-Seq. Synergistic and antagonistic interaction and ortholog modes of action of myostatin genotype and activity level on genes and gene pathways in this skeletal muscle were uncovered; 1,836, 238, and 399 genes exhibited significant (FDR-adjusted P-value < 0.005) activity-by-genotype interaction, genotype and activity effects, respectively. The most common differentially expressed profiles were (i) inactive myostatin-reduced relative to active and inactive wild-type, (ii) inactive myostatin-reduced and active wild-type, and (iii) inactive myostatin-reduced and inactive wild-type. Several remarkable genes and gene pathways were identified. The expression profile of nascent polypeptide-associated complex alpha subunit (Naca) supports a synergistic interaction between activity level and myostatin genotype, while Gremlin 2 (Grem2) displayed an antagonistic interaction. Comparison between activity levels revealed expression changes in genes encoding for structural proteins important for muscle function (including troponin, tropomyosin and myoglobin) and for fatty acid metabolism (some linked to diabetes and obesity, DNA-repair, stem cell renewal, and various forms of cancer). Conversely, comparison between genotype groups revealed changes in genes associated with G1-to-S-phase transition of the cell cycle of myoblasts and the expression of Grem2 proteins that modulate the cleavage of the myostatin propeptide. A number of myostatin-feedback regulated gene products that are primarily regulatory were uncovered, including microRNA impacting central functions and Piezo proteins that make cationic current-controlling mechanosensitive ion channels. These important findings extend hypotheses of myostatin and physical activity master regulation of genes and gene pathways, impacting medical practices and therapies associated with muscle atrophy in humans and companion animal species and genome-enabled selection practices applied to food-production animal species. PMID:25710176

Patient-derived Hormone-naive Prostate Cancer Xenograft Models Reveal Growth Factor Receptor Bound Protein 10 as an Androgen Receptor-repressed Gene Driving the Development of Castration-resistant Prostate Cancer.

PubMed

Hao, Jun; Ci, Xinpei; Xue, Hui; Wu, Rebecca; Dong, Xin; Choi, Stephen Yiu Chuen; He, Haiqing; Wang, Yu; Zhang, Fang; Qu, Sifeng; Zhang, Fan; Haegert, Anne M; Gout, Peter W; Zoubeidi, Amina; Collins, Colin; Gleave, Martin E; Lin, Dong; Wang, Yuzhuo

2018-06-01

Although androgen deprivation therapy is initially effective in controlling growth of hormone-naive prostate cancers (HNPCs) in patients, currently incurable castration-resistant prostate cancer (CRPC) inevitably develops. To identify CRPC driver genes that may provide new targets to enhance CRPC therapy. Patient-derived xenografts (PDXs) of HNPCs that develop CRPC following host castration were examined for changes in expression of genes at various time points after castration using transcriptome profiling analysis; particular attention was given to pre-CRPC changes in expression indicative of genes acting as potential CRPC drivers. The functionality of a potential CRPC driver was validated via its knockdown in cultured prostate cancer cells; its clinical relevance was established using data from prostate cancer patient databases. Eighty genes were found to be significantly upregulated at the CRPC stage, while seven of them also showed elevated expression prior to CRPC development. Among the latter, growth factor receptor bound protein 10 (GRB10) was the most significantly and consistently upregulated gene. Moreover, elevated GRB10 expression in clinical prostate cancer samples correlated with more aggressive tumor types and poorer patient treatment outcome. GRB10 knockdown markedly reduced prostate cancer cell proliferation and activity of AKT, a well-established CRPC mediator. A positive correlation between AKT activity and GRB10 expression was also found in clinical cohorts. GRB10 acts as a driver of CRPC and sensitizes androgen receptor pathway inhibitors, and hence GRB10 targeting provides a novel therapeutic strategy for the disease. Development of castration-resistant prostate cancer (CRPC) is a major problem in the management of the disease. Using state-of-the-art patient-derived hormone-naive prostate cancer xenograft models, we found and validated the growth factor receptor bound protein 10 gene as a driver of CRPC, indicating that it may be used as a new molecular target to enhance current CRPC therapy. Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.

HER2 activating mutations are targets for colorectal cancer treatment

PubMed Central

Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron

2015-01-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863

Heat shock proteins and heat shock factor 1 in carcinogenesis and tumor development: an update

PubMed Central

2013-01-01

Heat shock proteins (HSP) are a subset of the molecular chaperones, best known for their rapid and abundant induction by stress. HSP genes are activated at the transcriptional level by heat shock transcription factor 1 (HSF1). During the progression of many types of cancer, this heat shock transcriptional regulon becomes co-opted by mechanisms that are currently unclear, although evidently triggered in the emerging tumor cell. Concerted activation of HSF1 and the accumulation of HSPs then participates in many of the traits that permit the malignant phenotype. Thus cancers of many histologies exhibit activated HSF1 and increased HSP levels that may help to deter tumor suppression and evade therapy in the clinic. We review here the extensive work that has been carried out and is still in progress aimed at: (1) understanding the oncogenic mechanisms by which HSP genes are switched on, (2) determining the roles of HSF1 / HSP in malignant transformation and, (3) discovering approaches to therapy based on disrupting the influence of the HSF1 controlled transcriptome in cancer. PMID:22885793

A cluster of noncoding RNAs activates the ESR1 locus during breast cancer adaptation.

PubMed

Tomita, Saori; Abdalla, Mohamed Osama Ali; Fujiwara, Saori; Matsumori, Haruka; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Iwase, Hirotaka; Saitoh, Noriko; Nakao, Mitsuyoshi

2015-04-29

Estrogen receptor-α (ER)-positive breast cancer cells undergo hormone-independent proliferation after deprivation of oestrogen, leading to endocrine therapy resistance. Up-regulation of the ER gene (ESR1) is critical for this process, but the underlying mechanisms remain unclear. Here we show that the combination of transcriptome and fluorescence in situ hybridization analyses revealed that oestrogen deprivation induced a cluster of noncoding RNAs that defined a large chromatin domain containing the ESR1 locus. We termed these RNAs as Eleanors (ESR1 locus enhancing and activating noncoding RNAs). Eleanors were present in ER-positive breast cancer tissues and localized at the transcriptionally active ESR1 locus to form RNA foci. Depletion of one Eleanor, upstream (u)-Eleanor, impaired cell growth and transcription of intragenic Eleanors and ESR1 mRNA, indicating that Eleanors cis-activate the ESR1 gene. Eleanor-mediated gene activation represents a new type of locus control mechanism and plays an essential role in the adaptation of breast cancer cells.

A cluster of noncoding RNAs activates the ESR1 locus during breast cancer adaptation

PubMed Central

Tomita, Saori; Abdalla, Mohamed Osama Ali; Fujiwara, Saori; Matsumori, Haruka; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Iwase, Hirotaka; Saitoh, Noriko; Nakao, Mitsuyoshi

2015-01-01

Estrogen receptor-α (ER)-positive breast cancer cells undergo hormone-independent proliferation after deprivation of oestrogen, leading to endocrine therapy resistance. Up-regulation of the ER gene (ESR1) is critical for this process, but the underlying mechanisms remain unclear. Here we show that the combination of transcriptome and fluorescence in situ hybridization analyses revealed that oestrogen deprivation induced a cluster of noncoding RNAs that defined a large chromatin domain containing the ESR1 locus. We termed these RNAs as Eleanors (ESR1 locus enhancing and activating noncoding RNAs). Eleanors were present in ER-positive breast cancer tissues and localized at the transcriptionally active ESR1 locus to form RNA foci. Depletion of one Eleanor, upstream (u)-Eleanor, impaired cell growth and transcription of intragenic Eleanors and ESR1 mRNA, indicating that Eleanors cis-activate the ESR1 gene. Eleanor-mediated gene activation represents a new type of locus control mechanism and plays an essential role in the adaptation of breast cancer cells. PMID:25923108

Clinical applications of retinal gene therapy.

PubMed

Lipinski, Daniel M; Thake, Miriam; MacLaren, Robert E

2013-01-01

Many currently incurable forms of blindness affecting the retina have a genetic etiology and several others, such as those resulting from retinal vascular disturbances, respond to repeated, potentially indefinite administration of molecular based treatments. The recent clinical advances in retinal gene therapy have shown that viral vectors can deliver genes safely to the retina and the promising initial results from a number of clinical trials suggest that certain diseases may potentially be treatable. Gene therapy provides a means of expressing proteins within directly transduced cells with far greater efficacy than might be achieved by traditional systemic pharmacological approaches. Recent developments have demonstrated how vector gene expression may be regulated and further improvements to vector design have limited side effects and improved safety profiles. These recent steps have been most significant in bringing gene therapy into the mainstream of ophthalmology. Nevertheless translating retinal gene therapy from animal research into clinical trials is still a lengthy process, including complexities in human retinal diseases that have been difficult to model in the laboratory. The focus of this review is to summarize the genetic background of the most common retinal diseases, highlight current concepts of gene delivery technology, and relate those technologies to pre-clinical and clinical gene therapy studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Negotiating Gene Therapy: Controversies

ERIC Educational Resources Information Center

Sadler, Troy; Zeidler, Dana L.

2004-01-01

According to research, students often perceive the ethical implications of issues such as genetic engineering, but sometimes they are not equipped to handle multiple perspectives and articulate well-reasoned positions. A modified jigsaw activity, appropriate for secondary and introductory college biology classes, that introduces students to human…

Genome editing systems in novel therapies.

PubMed

Jang, Yoon-Young; Cai, Liuhong; Ye, Zhaohui

2016-01-01

Genome editing is the process in which DNA sequences at precise genomic locations are modified. In the past three decades, genome editing by homologous recombination has been successfully performed in mouse for generating genetic models. The low efficiency of this process in human cells, however, had prevented its clinical application until the recent advancements in designer endonuclease technologies. The significantly improved genome editing efficiencies aided by ZFN, TALEN, and CRISPR systems provide unprecedented opportunities not only for biomedical research, but also for developing novel therapies. Applications based on these genome editing tools to disrupt deleterious genes, correct genetic mutations, deliver functional transgenes more effectively or even modify the epigenetic landscape are being actively investigated for gene and cell therapy purposes. Encouraging results have been obtained in limited clinical trials in the past two years. While most of the applications are still in proof-of-principle or preclinical development stages, it is anticipated that the coming years will see increasing clinical success in novel therapies based on the modern genome editing technologies. It should be noted that critical issues still remain before the technologies can be translated into more reliable therapies. These key issues include off-target evaluation, establishing appropriate preclinical models and improving the currently low efficiency of homology-based precise gene replacement. In this review we discuss the preclinical and clinical studies aiming at translating the genome editing technologies as well as the issues that are important for more successful translation.

Gene and cell therapy for children — New medicines, new challenges?☆

PubMed Central

Buckland, Karen F.; Bobby Gaspar, H.

2014-01-01

The range of possible gene and cell therapy applications is expanding at an extremely rapid rate and advanced therapy medicinal products (ATMPs) are currently the hottest topic in novel medicines, particularly for inherited diseases. Paediatric patients stand to gain enormously from these novel therapies as it now seems plausible to develop a gene or cell therapy for a vast number of inherited diseases. There are a wide variety of potential gene and cell therapies in various stages of development. Patients who received first gene therapy treatments for primary immune deficiencies (PIDs) are reaching 10 and 15 years post-treatment, with robust and sustained immune recovery. Cell therapy clinical trials are underway for a variety of tissues including corneal, retinal and muscle repair and islet cell transplantation. Various cell therapy approaches are also being trialled to enhance the safety of bone marrow transplants, which should improve survival rates in childhood cancers and PIDs. Progress in genetic engineering of lymphocyte populations to target and kill cancerous cells is also described. If successful these ATMPs may enhance or replace the existing chemo-ablative therapy for several paediatric cancers. Emerging applications of gene therapy now include skin and neurological disorders such as epidermolysis bullosa, epilepsy and leukodystrophy. Gene therapy trials for haemophilia, muscular dystrophy and a range of metabolic disorders are underway. There is a vast array of potential advanced therapy medicinal products (ATMPs), and these are likely to be more cost effective than existing medicines. However, the first clinical trials have not been without setbacks and some of the key adverse events are discussed. Furthermore, the arrival of this novel class of therapies brings many new challenges for the healthcare industry. We present a summary of the key non-clinical factors required for successful delivery of these potential treatments. Technological advances are needed in vector design, raw material manufacture, cell culture and transduction methodology, and particularly in making all these technologies readily scalable. PMID:24583376

PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity.

PubMed

Stocco, Gabriele; Yang, Wenjian; Crews, Kristine R; Thierfelder, William E; Decorti, Giuliana; Londero, Margherita; Franca, Raffaella; Rabusin, Marco; Valsecchi, Maria Grazia; Pei, Deqing; Cheng, Cheng; Paugh, Steven W; Ramsey, Laura B; Diouf, Barthelemy; McCorkle, Joseph Robert; Jones, Terreia S; Pui, Ching-Hon; Relling, Mary V; Evans, William E

2012-11-01

Treatment-related toxicity can be life-threatening and is the primary cause of interruption or discontinuation of chemotherapy for acute lymphoblastic leukemia (ALL), leading to an increased risk of relapse. Mercaptopurine is an essential component of continuation therapy in all ALL treatment protocols worldwide. Genetic polymorphisms in thiopurine S-methyltransferase (TPMT) are known to have a marked effect on mercaptopurine metabolism and toxicity; however, some patients with wild-type TPMT develop toxicity during mercaptopurine treatment for reasons that are not well understood. To identify additional genetic determinants of mercaptopurine toxicity, a genome-wide analysis was performed in a panel of human HapMap cell lines to identify trans-acting genes whose expression and/or single-nucleotide polymorphisms (SNPs) are related to TPMT activity, then validated in patients with ALL. The highest ranking gene with both mRNA expression and SNPs associated with TPMT activity in HapMap cell lines was protein kinase C and casein kinase substrate in neurons 2 (PACSIN2). The association of a PACSIN2 SNP (rs2413739) with TPMT activity was confirmed in patients and knock-down of PACSIN2 mRNA in human leukemia cells (NALM6) resulted in significantly lower TPMT activity. Moreover, this PACSIN2 SNP was significantly associated with the incidence of severe gastrointestinal (GI) toxicity during consolidation therapy containing mercaptopurine, and remained significant in a multivariate analysis including TPMT and SLCO1B1 as covariates, consistent with its influence on TPMT activity. The association with GI toxicity was also validated in a separate cohort of pediatric patients with ALL. These data indicate that polymorphism in PACSIN2 significantly modulates TPMT activity and influences the risk of GI toxicity associated with mercaptopurine therapy.

PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

PubMed Central

Stocco, Gabriele; Yang, Wenjian; Crews, Kristine R.; Thierfelder, William E.; Decorti, Giuliana; Londero, Margherita; Franca, Raffaella; Rabusin, Marco; Valsecchi, Maria Grazia; Pei, Deqing; Cheng, Cheng; Paugh, Steven W.; Ramsey, Laura B.; Diouf, Barthelemy; McCorkle, Joseph Robert; Jones, Terreia S.; Pui, Ching-Hon; Relling, Mary V.; Evans, William E.

2012-01-01

Treatment-related toxicity can be life-threatening and is the primary cause of interruption or discontinuation of chemotherapy for acute lymphoblastic leukemia (ALL), leading to an increased risk of relapse. Mercaptopurine is an essential component of continuation therapy in all ALL treatment protocols worldwide. Genetic polymorphisms in thiopurine S-methyltransferase (TPMT) are known to have a marked effect on mercaptopurine metabolism and toxicity; however, some patients with wild-type TPMT develop toxicity during mercaptopurine treatment for reasons that are not well understood. To identify additional genetic determinants of mercaptopurine toxicity, a genome-wide analysis was performed in a panel of human HapMap cell lines to identify trans-acting genes whose expression and/or single-nucleotide polymorphisms (SNPs) are related to TPMT activity, then validated in patients with ALL. The highest ranking gene with both mRNA expression and SNPs associated with TPMT activity in HapMap cell lines was protein kinase C and casein kinase substrate in neurons 2 (PACSIN2). The association of a PACSIN2 SNP (rs2413739) with TPMT activity was confirmed in patients and knock-down of PACSIN2 mRNA in human leukemia cells (NALM6) resulted in significantly lower TPMT activity. Moreover, this PACSIN2 SNP was significantly associated with the incidence of severe gastrointestinal (GI) toxicity during consolidation therapy containing mercaptopurine, and remained significant in a multivariate analysis including TPMT and SLCO1B1 as covariates, consistent with its influence on TPMT activity. The association with GI toxicity was also validated in a separate cohort of pediatric patients with ALL. These data indicate that polymorphism in PACSIN2 significantly modulates TPMT activity and influences the risk of GI toxicity associated with mercaptopurine therapy. PMID:22846425

Chitosan nanoparticle-based delivery of fused NKG2D–IL-21 gene suppresses colon cancer growth in mice

PubMed Central

Tan, Lunmei; Han, Sen; Ding, Shizhen; Xiao, Weiming; Ding, Yanbing; Qian, Li; Wang, Chenming; Gong, Weijuan

2017-01-01

Nanoparticles can be loaded with exogenous DNA for the potential expression of cytokines with immune-stimulatory function. NKG2D identifies major histocompatibility complex class I chain-related protein in human and retinoic acid early induced transcript-1 in mouse, which acts as tumor-associated antigens. Biologic agents based on interleukin 21 (IL-21) have displayed antitumor activities through lymphocyte activation. The NKG2D–IL-21 fusion protein theoretically identifies tumor cells through NKG2D moiety and activates T cells through IL-21 moiety. In this study, double-gene fragments that encode the extracellular domains of NKG2D and IL-21 genes were connected and then inserted into the pcDNA3.1(−) plasmid. PcDNA3.1–dsNKG2D–IL-21 plasmid nanoparticles based on chitosan were generated. Tumor cells pretransfected with dsNKG2D–IL-21 gene nanoparticles can activate natural killer (NK) and CD8+ T cells in vitro. Serum IL-21 levels were enhanced in mice intramuscularly injected with the gene nanoparticles. DsNKG2D–IL-21 gene nanoparticles accumulated in tumor tissues after being intravenously injected for ~4–24 h. Treatment of dsNKG2D–IL-21 gene nanoparticles also retarded tumor growth and elongated the life span of tumor-bearing mice by activating NK and T cells in vivo. Thus, the dsNKG2D–IL-21 gene nanoparticles exerted efficient antitumor activities and would be potentially used for tumor therapy. PMID:28450784

Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko

Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicidemore » gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV-TK under the control of the ARF promoter shows lower cytotoxicity than that of the E2F1 promoter, in normal growing fibroblasts but has equivalent cytotoxicity in cancer cell lines. These results suggest that the ARF promoter, which is specifically activated by deregulated E2F activity, is an excellent candidate to drive therapeutic cytotoxic gene expression, specifically in cancer cells.« less

Progesterone receptor isoforms, agonists and antagonists differentially reprogram estrogen signaling

PubMed Central

Singhal, Hari; Greene, Marianne E.; Zarnke, Allison L.; Laine, Muriel; Al Abosy, Rose; Chang, Ya-Fang; Dembo, Anna G.; Schoenfelt, Kelly; Vadhi, Raga; Qiu, Xintao; Rao, Prakash; Santhamma, Bindu; Nair, Hareesh B.; Nickisch, Klaus J.; Long, Henry W.; Becker, Lev; Brown, Myles; Greene, Geoffrey L.

2018-01-01

Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Additionally, the two PR isoforms PRA and PRB, bind distinct but overlapping genomic sites and interact with different sets of co-regulators to differentially modulate estrogen signaling to be either pro- or anti-tumorigenic. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Differential gene expression was observed in PRA and PRB-rich patient tumors and PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. The better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher in vivo anti-tumor activity of therapies that combine tamoxifen with PR antagonists and modulators. This study suggests that distinguishing common effects observed due to concomitant interaction of another receptor with its ligand (agonist or antagonist), from unique isoform and ligand-specific effects will inform the development of biomarkers for patient selection and translation of PR-targeted therapies to the clinic. PMID:29435103

New Type of BACE1 siRNA Delivery to Cells

PubMed Central

Jabłkowski, Maciej; Szemraj, Maciej; Oszajca, Katarzyna; Janiszewska, Grażyna; Bartkowiak, Jacek; Szemraj, Janusz

2014-01-01

Background Small interfering RNA (siRNA) gene therapy is a new molecular approach in the search for an efficient therapy for Alzheimer disease (AD), based on the principle of RNA interference. Reducing BACE activity can have great therapeutic potential for the treatment of AD. In this study, receptor-mediated delivery was used to deliver opioid peptide-conjugated BACE 1 to INR-32 human neuroblastoma cells. Material/Methods An INR-32 human neuroblastoma cell line was stably transfected to express the APP cDNA coding fragment containing the predicted sites for cleavage by α, β, or γ-secretase. This was then treated with BACE 1 siRNA to silence BACE gene expression. BACE gene transcription and translation was determined using BACE-1 siRNA cross-linked with opioid peptide, together with RT-PCR, Western blot analysis, and ELISA. Results Receptor-mediated delivery was used to introduce BACE1 siRNA to the APP – INR 32 human neuroblastoma cells. Decreased BACE mRNA and protein expression were observed after the cells were transfected with BACE1 siRNA. Conclusions Delivery of BACE1 siRNA appears to specifically reduce the cleavage of APP by inhibiting BACE1 activity. PMID:25491230

Epigenetic regulation by selected dietary phytochemicals in cancer chemoprevention.

PubMed

Shukla, Samriddhi; Meeran, Syed M; Katiyar, Santosh K

2014-12-01

The growing interest in cancer epigenetics is largely due to the reversible nature of epigenetic changes which tend to alter during the course of carcinogenesis. Major epigenetic changes including DNA methylation, chromatin modifications and miRNA regulation play important roles in tumorigenic process. There are several epigenetically active synthetic molecules such as DNA methyltransferase (DNMTs) and histone deacetylases (HDACs) inhibitors, which are either approved or, are under clinical trials for the treatment of various cancers. However, most of the synthetic inhibitors have shown adverse side effects, narrow in their specificity and also expensive. Hence, bioactive phytochemicals, which are widely available with lesser toxic effects, have been tested for their role in epigenetic modulatory activities in gene regulation for cancer prevention and therapy. Encouragingly, many bioactive phytochemicals potentially altered the expression of key tumor suppressor genes, tumor promoter genes and oncogenes through modulation of DNA methylation and chromatin modification in cancer. These bioactive phytochemicals either alone or in combination with other phytochemicals showed promising results against various cancers. Here, we summarize and discuss the role of some commonly investigated phytochemicals and their epigenetic targets that are of particular interest in cancer prevention and cancer therapy. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Epigenetic regulation by selected dietary phytochemicals in cancer chemoprevention

PubMed Central

Shukla, Samriddhi; Meeran, Syed M.; Katiyar, Santosh K.

2014-01-01

The growing interest in cancer epigenetics is largely due to the reversible nature of epigenetic changes which tend to alter during the course of carcinogenesis. Major epigenetic changes including DNA methylation, chromatin modifications and miRNA regulation play important roles in tumorigenic process. There are several epigenetically active synthetic molecules such as DNA methyltransferase (DNMTs) and histone deacetylases (HDACs) inhibitors, which are either approved or, are under clinical trials for the treatment of various cancers. However, most of the synthetic inhibitors have shown adverse side effects, narrow in their specificity and also expensive. Hence, bioactive phytochemicals, which are widely available with lesser toxic effects, have been tested for their role in epigenetic modulatory activities in gene regulation for cancer prevention and therapy. Encouragingly, many bioactive phytochemicals potentially altered the expression of key tumor suppressor genes, tumor promoter genes and oncogenes through modulation of DNA methylation and chromatin modification in cancer. These bioactive phytochemicals either alone or in combination with other phytochemicals showed promising results against various cancers. Here, we summarize and discuss the role of some commonly investigated phytochemicals and their epigenetic targets that are of particular interest in cancer prevention and cancer therapy. PMID:25236912

[Gene therapy in Germany: from past to present].

PubMed

Kim, Young; Schmidt-Wolf, Ingo G H

2015-04-01

In 1994, the first clinical gene therapy trial was performed in Germany. Since then more than 2000 clinical gene therapy trials have been performed worldwide. After 20 years, a short résumé is drawn here. © Georg Thieme Verlag KG Stuttgart · New York.

The path to successful commercialization of cell and gene therapies: empowering patient advocates.

PubMed

Bauer, Gerhard; Abou-El-Enein, Mohamed; Kent, Alastair; Poole, Brian; Forte, Miguel

2017-02-01

Often, novel gene and cell therapies provide hope for many people living with incurable diseases. To facilitate and accelerate a successful regulatory approval and commercialization path for effective, safe and affordable cell and gene therapies, the involvement of patient advocacy groups (PAGs) should be considered early in the development process. This report provides a thorough overview of the various roles PAGs play in the clinical translation of cell and gene therapies and how they can bring about positive changes in the regulatory process, infrastructure improvements and market stability. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Insulin gene therapy for type 1 diabetes mellitus.

PubMed

Handorf, Andrew M; Sollinger, Hans W; Alam, Tausif

2015-04-01

Type 1 diabetes mellitus is an autoimmune disease resulting from the destruction of pancreatic β cells. Current treatments for patients with type 1 diabetes mellitus include daily insulin injections or whole pancreas transplant, each of which are associated with profound drawbacks. Insulin gene therapy, which has shown great efficacy in correcting hyperglycemia in animal models, holds great promise as an alternative strategy to treat type 1 diabetes mellitus in humans. Insulin gene therapy refers to the targeted expression of insulin in non-β cells, with hepatocytes emerging as the primary therapeutic target. In this review, we present an overview of the current state of insulin gene therapy to treat type 1 diabetes mellitus, including the need for an alternative therapy, important features dictating the success of the therapy, and current obstacles preventing the translation of this treatment option to a clinical setting. In so doing, we hope to shed light on insulin gene therapy as a viable option to treat type 1 diabetes mellitus.

Polycation-based gene therapy: current knowledge and new perspectives.

PubMed

Tiera, Marcio J; Shi, Qin; Winnik, Françoise M; Fernandes, Julio C

2011-08-01

At present, gene transfection insufficient efficiency is a major drawback of non-viral gene therapy. The 2 main types of delivery systems deployed in gene therapy are based on viral or non-viral gene carriers. Several non-viral modalities can transfer foreign genetic material into the human body. To do so, polycation-based gene delivery methods must achieve sufficient efficiency in the transportation of therapeutic genes across various extracellular and intracellular barriers. These barriers include interactions with blood components, vascular endothelial cells and uptake by the reticuloendothelial system. Furthermore, the degradation of therapeutic DNA by serum nucleases is a potential obstacle for functional delivery to target cells. Cationic polymers constitute one of the most promising approaches to the use of viral vectors for gene therapy. A better understanding of the mechanisms by which DNA can escape from endosomes and traffic to enter the nucleus has triggered new strategies of synthesis and has revitalized research into new polycation-based systems. The objective of this review is to address the state of the art in gene therapy with synthetic and natural polycations and the latest advances to improve gene transfer efficiency in cells.

The European hospital exemption clause-new option for gene therapy?

PubMed

Buchholz, Christian J; Sanzenbacher, Ralf; Schüle, Silke

2012-01-01

Gene-therapy medicinal products are currently applied to patients enrolled in authorized clinical trials to demonstrate safety and efficacy. Given a positive outcome, marketing authorization can subsequently be achieved via the centralized procedure coordinated by the European Medicines Agency. With Regulation (EC) No. 1394/2007 in force, advanced therapy medicinal products, including gene- and cell-therapy products, can be excepted from the obligation of obtaining a marketing authorization via the centralized procedure under specific conditions (so-called "hospital exemption"). This hospital exemption allows the application of gene-therapy medicinal products prepared on a non-routine basis for an individual patient and used under the exclusive professional responsibility of a medical practitioner. Here, we explain the requirements to be fulfilled in order to fall under this exemption, the implementation of this regulation into the German national legislation, and its impact on gene-therapy product development in the future.

Recent advances in the use of ZFN-mediated gene editing for human gene therapy.

PubMed

Chandrasegaran, Srinivasan

2017-01-01

Targeted genome editing with programmable nucleases has revolutionized biomedical research. The ability to make site-specific modifications to the human genome, has invoked a paradigm shift in gene therapy. Using gene editing technologies, the sequence in the human genome can now be precisely engineered to achieve a therapeutic effect. Zinc finger nucleases (ZFNs) were the first programmable nucleases designed to target and cleave custom sites. This article summarizes the advances in the use of ZFN-mediated gene editing for human gene therapy and discusses the challenges associated with translating this gene editing technology into clinical use.

Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice.

PubMed

Peng, Baowei; Ye, Peiqing; Blazar, Bruce R; Freeman, Gordon J; Rawlings, David J; Ochs, Hans D; Miao, Carol H

2008-09-01

Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid-treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS(+)CD4(+) T cells and activation of CD25(+)Foxp3(+) Tregs in the peripheral blood, spleen, and lymph nodes. CD4(+) T cells from anti-ICOS-treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-beta. Moreover, CD4(+)CD25(+) Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS-treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Phix 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

Therapeutic gene targeting approaches for the treatment of dyslipidemias and atherosclerosis.

PubMed

Mäkinen, Petri I; Ylä-Herttuala, Seppo

2013-04-01

Despite improved therapies, cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Therefore, new therapeutic approaches are still needed. In the gene therapy field, RNA interference (RNAi) and regulation of microRNAs (miRNAs) have gained a lot of attention in addition to traditional overexpression based strategies. Here, recent findings in therapeutic gene silencing and modulation of small RNA expression related to atherogenesis and dyslipidemia are summarized. Novel gene therapy approaches for the treatment of hyperlipidemia have been addressed. Antisense oligonucleotide and RNAi-based therapies against apolipoprotein B100 and proprotein convertase subtilisin/kexin type 9 have shown already efficacy in preclinical and clinical trials. In addition, several miRNAs dysregulated in atherosclerotic lesions and regulating cholesterol homeostasis have been found, which may represent novel targets for future therapies. New therapies for lowering lipid levels are now being tested in clinical trials, and both antisense oligonucleotide and RNAi-based therapies have shown promising results in lowering cholesterol levels. However, the modulation of inflammatory component in atherosclerosis by gene therapy and targeting of the effects to plaques are still difficult challenges.

Expression of tolerance associated gene-1, a mitochondrial protein inhibiting T cell activation, can be used to predict response to immune modulating therapies.

PubMed

Keeren, Kathrin; Friedrich, Markus; Gebuhr, Inga; Philipp, Sandra; Sabat, Robert; Sterry, Wolfram; Brandt, Christine; Meisel, Christian; Grütz, Gerald; Volk, Hans-Dieter; Sawitzki, Birgit

2009-09-15

Immune modulating therapies gain increasing importance in treatment of patients with autoimmune diseases such as psoriasis. None of the currently applied biologics achieves significant clinical improvement in all treated patients. Because the therapy with biologics is cost intensive and sometimes associated with side effects, noninvasive diagnostic tools for early prediction of responders are of major interest. We studied the effects of Alefacept (LFA3Ig), an approved drug for treatment of psoriasis, on leukocytes in vitro and in vivo to identify gene markers predictive for treatment response and to further investigate its molecular mechanisms of action. In an open-label study, 20 psoriasis patients were treated weekly with 15 mg Alefacept over 12 wk. We demonstrate that transcription of the tolerance-associated gene (TOAG-1) is significantly up-regulated whereas receptor for hyaluronic acid mediated migration (RHAMM) transcription is down-regulated in PBMCs of responding patients before clinical improvement. TOAG-1 is exclusively localized within mitochondria. Overexpression of TOAG-1 in murine T cells leads to increased susceptibility to apoptosis. Addition of Alefacept to stimulated human T cells in vitro resulted in reduced frequencies of activated CD137(+) cells, increased TOAG-1 but reduced RHAMM expression. This was accompanied by reduced proliferation and enhanced apoptosis. Inhibition of proliferation was dependent on enhanced PDL1 expression of APCs. Thus, peripheral changes of TOAG-1 and RHAMM expression can be used to predict clinical response to Alefacept treatment in psoriasis patients. In the presence of APCs Alefacept can inhibit T cell activation and survival by increasing expression of TOAG-1 on T cells and PDL1 on APCs.

Plasticity of the human visual system after retinal gene therapy in patients with Leber's congenital amaurosis.

PubMed

Ashtari, Manzar; Zhang, Hui; Cook, Philip A; Cyckowski, Laura L; Shindler, Kenneth S; Marshall, Kathleen A; Aravand, Puya; Vossough, Arastoo; Gee, James C; Maguire, Albert M; Baker, Chris I; Bennett, Jean

2015-07-15

Much of our knowledge of the mechanisms underlying plasticity in the visual cortex in response to visual impairment, vision restoration, and environmental interactions comes from animal studies. We evaluated human brain plasticity in a group of patients with Leber's congenital amaurosis (LCA), who regained vision through gene therapy. Using non-invasive multimodal neuroimaging methods, we demonstrated that reversing blindness with gene therapy promoted long-term structural plasticity in the visual pathways emanating from the treated retina of LCA patients. The data revealed improvements and normalization along the visual fibers corresponding to the site of retinal injection of the gene therapy vector carrying the therapeutic gene in the treated eye compared to the visual pathway for the untreated eye of LCA patients. After gene therapy, the primary visual pathways (for example, geniculostriate fibers) in the treated retina were similar to those of sighted control subjects, whereas the primary visual pathways of the untreated retina continued to deteriorate. Our results suggest that visual experience, enhanced by gene therapy, may be responsible for the reorganization and maturation of synaptic connectivity in the visual pathways of the treated eye in LCA patients. The interactions between the eye and the brain enabled improved and sustained long-term visual function in patients with LCA after gene therapy. Copyright © 2015, American Association for the Advancement of Science.

Special Issue: Gene Therapy with Emphasis on RNA Interference

PubMed Central

Lundstrom, Kenneth

2015-01-01

Gene therapy was originally thought to cover replacement of malfunctioning genes in treatment of various diseases. Today, the field has been expanded to application of viral and non-viral vectors for delivery of recombinant proteins for the compensation of missing or insufficient proteins, anti-cancer genes and proteins for destruction of tumor cells, immunostimulatory genes and proteins for stimulation of the host defense system against viral agents and tumors. Recently, the importance of RNA interference and its application in gene therapy has become an attractive alternative for drug development. PMID:26447255

Gene transfer as a strategy to achieve permanent cardioprotection I: rAAV-mediated gene therapy with inducible nitric oxide synthase limits infarct size 1 year later without adverse functional consequences.

PubMed

Li, Qianhong; Guo, Yiru; Wu, Wen-Jian; Ou, Qinghui; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Chen, Ning; Dawn, Buddhadeb; Luo, Li; O'Brien, Erin; Bolli, Roberto

2011-11-01

The ultimate goal of prophylactic gene therapy is to confer permanent protection against ischemia. Although gene therapy with inducible nitric oxide synthase (iNOS) is known to protect against myocardial infarction at 3 days and up to 2 months, the long-term effects on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the iNOS gene (rAAV/iNOS), which enables long-lasting transgene expression. The ability of rAAV/iNOS to direct the expression of functional iNOS protein was confirmed in COS-7 cells before in vivo gene transfer. Mice received injections in the anterior LV wall of rAAV/LacZ or rAAV/iNOS; 1 year later, they underwent a 30-min coronary occlusion (O) and 4 h of reperfusion (R). iNOS gene transfer resulted in elevated iNOS protein expression (+3-fold vs. the LacZ group, n = 6; P < 0.05) and iNOS activity (+4.4-fold vs. the LacZ group, n = 6; P < 0.05) 1 year later. Infarct size (% of risk region) was dramatically reduced at 1 year after iNOS gene transfer (13.5 ± 2.2%, n = 12, vs. 41.7 ± 2.9%, n = 10, in the LacZ group; P < 0.05). The infarct-sparing effect of iNOS gene therapy at 1 year was as powerful as that observed 24 h after ischemic preconditioning (six 4-min O/4-min R cycles) (19.3 ± 2.3%, n = 11; P < 0.05). Importantly, compared with the LacZ group (n = 11), iNOS gene transfer (n = 10) had no effect on LV dimensions or function for up to 1 year (at 1 year: FS 34.5 ± 2.0 vs. 34.6 ± 2.6%, EF 57.0 ± 2.0 vs. 59.7 ± 2.9%, LVEDD 4.3 ± 0.1 vs. 4.2 ± 0.2 mm, LVESD 2.8 ± 0.1 vs. 2.9 ± 0.2 mm) (echocardiography). These data demonstrate, for the first time, that rAAV-mediated iNOS gene transfer affords long-term, probably permanent (1 year), cardioprotection without adverse functional consequences, providing a strong rationale for further preclinical testing of prophylactic gene therapy.

Gene Therapy for Bone Defects in Oral and Maxillofacial Surgery: A Systematic Review and Meta-Analysis of Animal Studies.

PubMed

Fliefel, Riham; Kühnisch, Jan; Ehrenfeld, Michael; Otto, Sven

2017-02-15

Craniofacial bone defects are challenging problems for maxillofacial surgeons over the years. With the development of cell and molecular biology, gene therapy is a breaking new technology with the aim of regenerating tissues by acting as a delivery system for therapeutic genes in the craniofacial region rather than treating genetic disorders. A systematic review was conducted summarizing the articles reporting gene therapy in maxillofacial surgery to answer the question: Was gene therapy successfully applied to regenerate bone in the maxillofacial region? Electronic searching of online databases was performed in addition to hand searching of the references of included articles. No language or time restrictions were enforced. Meta-analysis was done to assess significant bone formation after delivery of gene material in the surgically induced maxillofacial defects. The search identified 2081 articles, of which 57 were included with 1726 animals. Bone morphogenetic proteins were commonly used proteins for gene therapy. Viral vectors were the universally used vectors. Sprague-Dawley rats were the frequently used animal model in experimental studies. The quality of the articles ranged from excellent to average. Meta-analysis results performed on 21 articles showed that defects favored bone formation by gene therapy. Funnel plot showed symmetry with the absence of publication bias. Gene therapy is on the top list of innovative strategies that developed in the last 10 years with the hope of developing a simple chair-side protocol in the near future, combining improvement of gene delivery as well as knowledge of the molecular basis of oral and maxillofacial structures.

FUNCTIONAL NANOPARTICLES FOR MOLECULAR IMAGING GUIDED GENE DELIVERY

PubMed Central

Liu, Gang; Swierczewska, Magdalena; Lee, Seulki; Chen, Xiaoyuan

2010-01-01

Gene therapy has great potential to bring tremendous changes in treatment of various diseases and disorders. However, one of the impediments to successful gene therapy is the inefficient delivery of genes to target tissues and the inability to monitor delivery of genes and therapeutic responses at the targeted site. The emergence of molecular imaging strategies has been pivotal in optimizing gene therapy; since it can allow us to evaluate the effectiveness of gene delivery noninvasively and spatiotemporally. Due to the unique physiochemical properties of nanomaterials, numerous functional nanoparticles show promise in accomplishing gene delivery with the necessary feature of visualizing the delivery. In this review, recent developments of nanoparticles for molecular imaging guided gene delivery are summarized. PMID:22473061

77 FR 73472 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-10

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2012-N-0001] Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting AGENCY: Food and Drug... closed to the public. Name of Committee: Cellular, Tissue and Gene Therapies Advisory Committee. General...

76 FR 64951 - Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-19

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2011-N-0002] Cellular, Tissue and Gene Therapies Advisory Committee; Notice of Meeting AGENCY: Food and Drug...: Cellular, Tissue and Gene Therapies Advisory Committee. General Function of the Committee: To provide...

Multilocus Genotypes of Relevance for Drug Metabolizing Enzymes and Therapy with Thiopurines in Patients with Acute Lymphoblastic Leukemia

PubMed Central

Stocco, Gabriele; Franca, Raffaella; Verzegnassi, Federico; Londero, Margherita; Rabusin, Marco; Decorti, Giuliana

2013-01-01

Multilocus genotypes have been shown to be of relevance for using pharmacogenomic principles to individualize drug therapy. As it relates to thiopurine therapy, genetic polymorphisms of TPMT are strongly associated with the pharmacokinetics and clinical effects of thiopurines (mercaptopurine and azathioprine), influencing their toxicity and efficacy. We have recently demonstrated that TPMT and ITPA genotypes constitute a multilocus genotype of pharmacogenetic relevance for children with acute lymphoblastic leukemia (ALL) receiving thiopurine therapy. The use of high-throughput genomic analysis allows identification of additional candidate genetic factors associated with pharmacogenetic phenotypes, such as TPMT enzymatic activity: PACSIN2 polymorphisms have been identified by a genome-wide analysis, combining evaluation of polymorphisms and gene expression, as a significant determinant of TPMT activity in the HapMap CEU cell lines and the effects of PACSIN2 on TPMT activity and mercaptopurine induced adverse effects were confirmed in children with ALL. Combination of genetic factors of relevance for thiopurine metabolizing enzyme activity, based on the growing understanding of their association with drug metabolism and efficacy, is particularly promising for patients with pediatric ALL. The knowledge basis and clinical applications for multilocus genotypes of importance for therapy with mercaptopurine in pediatric ALL is discussed in the present review. PMID:23335936

Gene therapy for immune disorders: good news tempered by bad news.

PubMed

Puck, Jennifer M; Malech, Harry L

2006-04-01

After a dozen years of human gene therapy trials characterized by minimal gene correction and disappointing clinical impact, the field of gene therapy received some good news in 2000. Infants with X-linked severe combined immunodeficiency who received retroviral gene addition to cells from their bone marrow developed impressive immune reconstitution. During the following 2 years, additional patients were treated and the news was even better-babies receiving gene therapy had sustained T-cell production and in several cases developed better cell function than most patients treated with standard bone marrow transplants. Unfortunately, bad news followed. Three of the patients experienced leukemic T-cell expansions, found to be associated with retroviral insertions into genomic DNA. Where does the field stand today?

Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID.

PubMed

Sauer, Aisha V; Brigida, Immacolata; Carriglio, Nicola; Hernandez, Raisa Jofra; Scaramuzza, Samantha; Clavenna, Daniela; Sanvito, Francesca; Poliani, Pietro L; Gagliani, Nicola; Carlucci, Filippo; Tabucchi, Antonella; Roncarolo, Maria Grazia; Traggiai, Elisabetta; Villa, Anna; Aiuti, Alessandro

2012-02-09

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.

Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

PubMed Central

Liu, Geyi; Aronovich, Elena L.; Cui, Zongbin; Whitley, Chester B.; Hackett, Perry B.

2007-01-01

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase–transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals. PMID:15133768

Hippocampal gene expression in a rat model of depression after electroacupuncture at the Baihui and Yintang acupoints

PubMed Central

Duan, Dongmei; Yang, Xiuyan; Ya, Tu; Chen, Liping

2014-01-01

Preliminary basic research and clinical findings have demonstrated that electroacupuncture therapy exhibits positive effects in ameliorating depression. However, most studies of the underlying mechanism are at the single gene level; there are few reports regarding the mechanism at the whole-genome level. Using a rat genomic gene-chip, we profiled hippocampal gene expression changes in rats after electroacupuncture therapy. Electroacupuncture therapy alleviated depression-related manifestations in the model rats. Using gene-chip analysis, we demonstrated that electroacupuncture at Baihui (DU20) and Yintang (EX-HN3) regulates the expression of 21 genes. Real-time PCR showed that the genes Vgf, Igf2, Tmp32, Loc500373, Hif1a, Folr1, Nmb, and Rtn were upregulated or downregulated in depression and that their expression tended to normalize after electroacupuncture therapy. These results indicate that electroacupuncture at Baihui and Yintang modulates depression by regulating the expression of particular genes. PMID:25206746

Design and testing of regulatory cassettes for optimal activity in skeletal and cardiac muscles.

PubMed

Himeda, Charis L; Chen, Xiaolan; Hauschka, Stephen D

2011-01-01

Gene therapy for muscular dystrophies requires efficient gene delivery to the striated musculature and specific, high-level expression of the therapeutic gene in a physiologically diverse array of muscles. This can be achieved by the use of recombinant adeno-associated virus vectors in conjunction with muscle-specific regulatory cassettes. We have constructed several generations of regulatory cassettes based on the enhancer and promoter of the muscle creatine kinase gene, some of which include heterologous enhancers and individual elements from other muscle genes. Since the relative importance of many control elements varies among different anatomical muscles, we are aiming to tailor these cassettes for high-level expression in cardiac muscle, and in fast and slow skeletal muscles. With the achievement of efficient intravascular gene delivery to isolated limbs, selected muscle groups, and heart in large animal models, the design of cassettes optimized for activity in different muscle types is now a practical goal. In this protocol, we outline the key steps involved in the design of regulatory cassettes for optimal activity in skeletal and cardiac muscle, and testing in mature muscle fiber cultures. The basic principles described here can also be applied to engineering tissue-specific regulatory cassettes for other cell types.

Linking transgene expression of engineered mesenchymal stem cells and angiopoietin-1-induced differentiation to target cancer angiogenesis.

PubMed

Conrad, Claudius; Hüsemann, Yves; Niess, Hanno; von Luettichau, Irene; Huss, Ralf; Bauer, Christian; Jauch, Karl-Walter; Klein, Christoph A; Bruns, Christiane; Nelson, Peter J

2011-03-01

To specifically target tumor angiogenesis by linking transgene expression of engineered mesenchymal stem cells to angiopoietin-1-induced differentiation. Mesenchymal stem cells (MSCs) have been used to deliver therapeutic genes into solid tumors. These strategies rely on their homing mechanisms only to deliver the therapeutic agent. We engineered murine MSC to express reporter genes or therapeutic genes under the selective control of the Tie2 promoter/enhancer. This approach uses the differentiative potential of MSCs induced by the tumor microenvironment to drive therapeutic gene expression only in the context of angiogenesis. When injected into the peripheral circulation of mice with either, orthotopic pancreatic or spontaneous breast cancer, the engineered MSCs were actively recruited to growing tumor vasculature and induced the selective expression of either reporter red florescent protein or suicide genes [herpes simplex virus-thymidine kinase (TK) gene] when the adoptively transferred MSC developed endothelial-like characteristics. The TK gene product in combination with the prodrug ganciclovir (GCV) produces a potent toxin, which affects replicative cells. The homing of engineered MSC with selective induction of TK in concert with GCV resulted in a toxic tumor-specific environment. The efficacy of this approach was demonstrated by significant reduction in primary tumor growth and prolongation of life in both tumor models. This "Trojan Horse" combined stem cell/gene therapy represents a novel treatment strategy for tailored therapy of solid tumors.

Hematopoietic Stem Cell Gene Therapy for Storage Disease: Current and New Indications.

PubMed

Biffi, Alessandra

2017-05-03

Lysosomal storage disorders (LSDs) are a broad class of monogenic diseases with an overall incidence of 1:7,000 newborns, due to the defective activity of one or more lysosomal hydrolases or related proteins resulting in storage of un-degraded substrates in the lysosomes. The over 40 different known LSDs share a life-threatening nature, but they are present with extremely variable clinical manifestations, determined by the characteristics and tissue distribution of the material accumulating due to the lysosomal dysfunction. The majority of LSDs lack a curative treatment. This is particularly true for LSDs severely affecting the CNS. Based on current preclinical and clinical evidences, among other treatment modalities, hematopoietic stem cell gene therapy could potentially result in robust therapeutic benefit for LSD patients, with particular indication for those characterized by severe brain damage. Optimization of current approaches and technology, as well as implementation of clinical trials for novel indications, and prolonged and more extensive follow-up of the already treated patients will allow translating this promise into new medicinal products. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Vascular Repair by Circumferential Cell Therapy Using Magnetic Nanoparticles and Tailored Magnets.

PubMed

Vosen, Sarah; Rieck, Sarah; Heidsieck, Alexandra; Mykhaylyk, Olga; Zimmermann, Katrin; Bloch, Wilhelm; Eberbeck, Dietmar; Plank, Christian; Gleich, Bernhard; Pfeifer, Alexander; Fleischmann, Bernd K; Wenzel, Daniela

2016-01-26

Cardiovascular disease is often caused by endothelial cell (EC) dysfunction and atherosclerotic plaque formation at predilection sites. Also surgical procedures of plaque removal cause irreversible damage to the EC layer, inducing impairment of vascular function and restenosis. In the current study we have examined a potentially curative approach by radially symmetric re-endothelialization of vessels after their mechanical denudation. For this purpose a combination of nanotechnology with gene and cell therapy was applied to site-specifically re-endothelialize and restore vascular function. We have used complexes of lentiviral vectors and magnetic nanoparticles (MNPs) to overexpress the vasoprotective gene endothelial nitric oxide synthase (eNOS) in ECs. The MNP-loaded and eNOS-overexpressing cells were magnetic, and by magnetic fields they could be positioned at the vascular wall in a radially symmetric fashion even under flow conditions. We demonstrate that the treated vessels displayed enhanced eNOS expression and activity. Moreover, isometric force measurements revealed that EC replacement with eNOS-overexpressing cells restored endothelial function after vascular injury in eNOS(-/-) mice ex and in vivo. Thus, the combination of MNP-based gene and cell therapy with custom-made magnetic fields enables circumferential re-endothelialization of vessels and improvement of vascular function.

Synthetic activation of caspases: Artificial death switches

PubMed Central

MacCorkle, Rebecca A.; Freeman, Kevin W.; Spencer, David M.

1998-01-01

The development of safe vectors for gene therapy requires fail-safe mechanisms to terminate therapy or remove genetically altered cells. The ideal “suicide switch” would be nonimmunogenic and nontoxic when uninduced and able to trigger cell death independent of tissue type or cell cycle stage. By using chemically induced dimerization, we have developed powerful death switches based on the cysteine proteases, caspase-1 ICE (interleukin-1β converting enzyme) and caspase-3 YAMA. In both cases, aggregation of the target protein is achieved by a nontoxic lipid-permeable dimeric FK506 analog that binds to the attached FK506-binding proteins, FKBPs. We find that intracellular cross-linking of caspase-1 or caspase-3 is sufficient to trigger rapid apoptosis in a Bcl-xL-independent manner, suggesting that these conditional proapoptotic molecules can bypass intracellular checkpoint genes, such as Bcl-xL, that limit apoptosis. Because these chimeric molecules are derived from autologous proteins, they should be nonimmunogenic and thus ideal for long-lived gene therapy vectors. These properties should also make chemically induced apoptosis useful for developmental studies, for treating hyperproliferative disorders, and for developing animal models to a wide variety of diseases. PMID:9520421

Enhanced Incretin Effects of Exendin-4 Expressing Chimeric Plasmid Based On Two-Step Transcription Amplification System with Dendritic Bioreducible Polymer for the Treatment of Type 2 Diabetes

PubMed Central

Kim, Pyung-Hwan; Lee, Minhyung; Nam, Kihoon; Kim, Sung Wan

2014-01-01

Glucagon-like peptide 1 (GLP-1) agonist, exenxdin-4, is currently being advanced as a promising diabetes remedy via a variety of incretin actions similar with GLP-1. In this study, we investigated an effective anti-diabetic therapy via exendin-4 expressing chimeric plasmid based on two-step transcription amplification (TSTA) system with dendrimer-type bioreducible polymer for more improved incretin-based gene therapy. Arginine-grafted poly (cystaminebisacrylamide-diaminohexane) (ABP)-conjugated poly (amido amine) (PAMAM) dendrimer (PAM-ABP) was used as gene carrier. PAM-ABP/chimeric DNA polyplex was markedly elevated exendin-4 expression in ectopic cells as well as increased insulin production through an enhanced activation of protein kinase K (PKA) induced by up-regulation of exendin-4-stimulated cyclic adenosine monophosphate (cAMP) in pancreatic β-cell. Consistent with these results, intravenous administration of PAM-ABP/chimeric DNA polyplex improved glucoregulotory effects, as well as increased insulin secretion by high expression of exendin-4 in blood in type 2 diabetic mice with no any toxicity. Our exendin-4 system can be attributed to provide a potential diabetes therapeutic agent for improved incretin gene therapy. PMID:24839613

Recent progress and considerations for AAV gene therapies targeting the central nervous system.

PubMed

Lykken, Erik Allen; Shyng, Charles; Edwards, Reginald James; Rozenberg, Alejandra; Gray, Steven James

2018-05-18

Neurodevelopmental disorders, as a class of diseases, have been particularly difficult to treat even when the underlying cause(s), such as genetic alterations, are understood. What treatments do exist are generally not curative and instead seek to improve quality of life for affected individuals. The advent of gene therapy via gene replacement offers the potential for transformative therapies to slow or even stop disease progression for current patients and perhaps minimize or prevent the appearance of symptoms in future patients. This review focuses on adeno-associated virus (AAV) gene therapies for diseases of the central nervous system. An overview of advances in AAV vector design for therapy is provided, along with a description of current strategies to develop AAV vectors with tailored tropism. Next, progress towards treatment of neurodegenerative diseases is presented at both the pre-clinical and clinical stages, focusing on a few select diseases to highlight broad categories of therapeutic parameters. Special considerations for more challenging cases are then discussed in addition to the immunological aspects of gene therapy. With the promising clinical trial results that have been observed for the latest AAV gene therapies and continued pre-clinical successes, the question is no longer whether a therapy can be developed for certain neurodevelopmental disorders, but rather, how quickly.

Combined Gene Therapy Using AdsVEGFR2 and AdsTie2 With Chemotherapy Reduces the Growth of Human Ovarian Cancer and Formation of Ascites in Mice.

PubMed

Tuppurainen, Laura; Sallinen, Hanna; Karvonen, Anni; Valkonen, Elina; Laakso, Hanne; Liimatainen, Timo; Hytönen, Elisa; Hämäläinen, Kirsi; Kosma, Veli-Matti; Anttila, Maarit; Ylä-Herttuala, Seppo

2017-06-01

Ovarian cancer is highly dependent on tumor microvessels and angiogenesis regulated by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) and angiopoietins (Ang) and their Tie receptors. We studied the efficacy of adenoviral (Ad) gene therapy with soluble VEGFR2 and Tie2 combined with paclitaxel and carboplatin for the treatment of ovarian cancer. An intraperitoneal human ovarian cancer xenograft model in nude mice (n = 44) was used in this study. Gene therapy was given intravenously when the presence of sizable tumors was confirmed in magnetic resonance imaging. The study groups were as follows: AdCMV as a control (group I), AdCMV with chemotherapy (group II), AdsVEGFR2 and AdsTie2 (group III), and AdsVEGFR2 and AdsTie2 with chemotherapy (group IV). Antitumor effectiveness was assessed by overall tumor growth, ascites, immunohistochemistry, microvessel density, and sequential magnetic resonance imaging analyses. AdsVEGFR2 and AdsTie2 gene therapy (group III) significantly reduced tumor weights as compared with group II (P = 0.007). Accumulation of ascites was significantly reduced when the mice were treated with AdsVEGFR2 and AdsTie2 gene therapy or with combined gene therapy and chemotherapy as compared with controls (P = 0.029 and P = 0.010, respectively). Vascular endothelial growth factor and Ang2 levels in ascites fluid were elevated after the gene therapy. Combined inhibition of VEGF/VEGFR2 and Ang/Tie2 pathways provided efficient therapy for ovarian cancer in mice. In addition, antiangiogenic gene therapy has potential as a treatment for the accumulation of ascites.

Computational Models of HIV-1 Resistance to Gene Therapy Elucidate Therapy Design Principles

PubMed Central

Aviran, Sharon; Shah, Priya S.; Schaffer, David V.; Arkin, Adam P.

2010-01-01

Gene therapy is an emerging alternative to conventional anti-HIV-1 drugs, and can potentially control the virus while alleviating major limitations of current approaches. Yet, HIV-1's ability to rapidly acquire mutations and escape therapy presents a critical challenge to any novel treatment paradigm. Viral escape is thus a key consideration in the design of any gene-based technique. We develop a computational model of HIV's evolutionary dynamics in vivo in the presence of a genetic therapy to explore the impact of therapy parameters and strategies on the development of resistance. Our model is generic and captures the properties of a broad class of gene-based agents that inhibit early stages of the viral life cycle. We highlight the differences in viral resistance dynamics between gene and standard antiretroviral therapies, and identify key factors that impact long-term viral suppression. In particular, we underscore the importance of mutationally-induced viral fitness losses in cells that are not genetically modified, as these can severely constrain the replication of resistant virus. We also propose and investigate a novel treatment strategy that leverages upon gene therapy's unique capacity to deliver different genes to distinct cell populations, and we find that such a strategy can dramatically improve efficacy when used judiciously within a certain parametric regime. Finally, we revisit a previously-suggested idea of improving clinical outcomes by boosting the proliferation of the genetically-modified cells, but we find that such an approach has mixed effects on resistance dynamics. Our results provide insights into the short- and long-term effects of gene therapy and the role of its key properties in the evolution of resistance, which can serve as guidelines for the choice and optimization of effective therapeutic agents. PMID:20711350

Gene therapy for metachromatic leukodystrophy.

PubMed

Rosenberg, Jonathan B; Kaminsky, Stephen M; Aubourg, Patrick; Crystal, Ronald G; Sondhi, Dolan

2016-11-01

Leukodystrophies (LDs) are rare, often devastating genetic disorders with neurologic symptoms. There are currently no disease-specific therapeutic approaches for these diseases. In this review we use metachromatic leukodystrophy as an example to outline in the brief the therapeutic approaches to MLD that have been tested in animal models and in clinical trials, such as enzyme-replacement therapy, bone marrow/umbilical cord blood transplants, ex vivo transplantation of genetically modified hematopoietic stem cells, and gene therapy. These studies suggest that to be successful the ideal therapy for MLD must provide persistent and high level expression of the deficient gene, arylsulfatase A in the CNS. Gene therapy using adeno-associated viruses is therefore the ideal choice for clinical development as it provides the best balance of potential for efficacy with reduced safety risk. Here we have summarized the published preclinical data from our group and from others that support the use of a gene therapy with AAVrh.10 serotype for clinical development as a treatment for MLD, and as an example of the potential of gene therapy for LDs especially for Krabbe disease, which is the focus of this special issue. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Curcumin Modulates the Radiosensitivity of Colorectal Cancer Cells by Suppressing Constitutive and Inducible NF-{kappa}B Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sandur, Santosh K.; Deorukhkar, Amit; Pandey, Manoj K.

2009-10-01

Purpose: Radiation therapy is an integral part of the preoperative treatment of rectal cancers. However, only a minority of patients achieve a complete pathologic response to therapy because of resistance of these tumors to radiation therapy. This resistance may be mediated by constitutively active pro-survival signaling pathways or by inducible/acquired mechanisms in response to radiation therapy. Simultaneous inhibition of these pathways can sensitize these tumors to radiation therapy. Methods and Materials: Human colorectal cancer cells were exposed to clinically relevant doses of gamma rays, and the mechanism of their radioresistance was investigated. We characterized the transcription factor nuclear factor-{kappa}B (NF-{kappa}B)more » activation as a mechanism of inducible radioresistance in colorectal cancer and used curcumin, the active ingredient in the yellow spice turmeric, to overcome this resistance. Results: Curcumin inhibited the proliferation and the post-irradiation clonogenic survival of multiple colorectal cancer cell lines. Radiation stimulated NF-{kappa}B activity in a dose- and time-dependent manner, whereas curcumin suppressed this radiation-induced NF-{kappa}B activation via inhibition of radiation-induced phosphorylation and degradation of inhibitor of {kappa}B alpha, inhibition of inhibitor of {kappa}B kinase activity, and inhibition of Akt phosphorylation. Curcumin also suppressed NF-{kappa}B-regulated gene products (Bcl-2, Bcl-x{sub L}, inhibitor of apoptosis protein-2, cyclooxygenase-2, and cyclin D1). Conclusions: Our results suggest that transient inducible NF-{kappa}B activation provides a prosurvival response to radiation that may account for development of radioresistance. Curcumin blocks this signaling pathway and potentiates the antitumor effects of radiation therapy.« less

Nitric Oxide Donor-Based Cancer Therapy: Advances and Prospects.

PubMed

Huang, Zhangjian; Fu, Junjie; Zhang, Yihua

2017-09-28

The increasing understanding of the role of nitric oxide (NO) in cancer biology has generated significant progress in the use of NO donor-based therapy to fight cancer. These advances strongly suggest the potential adoption of NO donor-based therapy in clinical practice, and this has been supported by several clinical studies in the past decade. In this review, we first highlight several types of important NO donors, including recently developed NO donors bearing a dinitroazetidine skeleton, represented by RRx-001, with potential utility in cancer therapy. Special emphasis is then given to the combination of NO donor(s) with other therapies to achieve synergy and to the hybridization of NO donor(s) with an anticancer drug/agent/fragment to enhance the activity or specificity or to reduce toxicity. In addition, we briefly describe inducible NO synthase gene therapy and nanotechnology, which have recently entered the field of NO donor therapy.

Modalities and future prospects of gene therapy in heart transplantation.

PubMed

Vassalli, Giuseppe; Roehrich, Marc-Estienne; Vogt, Pierre; Pedrazzini, Giovanni B; Siclari, Francesco; Moccetti, Tiziano; von Segesser, Ludwig K

2009-06-01

Heart transplantation is the treatment of choice for many patients with end-stage heart failure. Its success, however, is limited by organ shortage, side effects of immunosuppressive drugs, and chronic rejection. Gene therapy is conceptually appealing for applications in transplantation, as the donor organ is genetically manipulated ex vivo before transplantation. Localised expression of immunomodulatory genes aims to create a state of immune privilege within the graft, which could eliminate the need for systemic immunosuppression. In this review, recent advances in the development of gene therapy in heart transplantation are discussed. Studies in animal models have demonstrated that genetic modification of the donor heart with immunomodulatory genes attenuates ischaemia-reperfusion injury and rejection. Alternatively, bone marrow-derived cells genetically engineered with donor-type major histocompatibility complex (MHC) class I or II promote donor-specific hyporesponsiveness. Genetic engineering of naïve T cells or dendritic cells may induce regulatory T cells and regulatory dendritic cells. Despite encouraging results in animal models, however, clinical gene therapy trials in heart transplantation have not yet been started. The best vector and gene to be delivered remain to be identified. Pre-clinical studies in non-human primates are needed. Nonetheless, the potential of gene therapy as an adjunct therapy in transplantation is essentially intact.

A Guide to Approaching Regulatory Considerations for Lentiviral-Mediated Gene Therapies.

PubMed

White, Michael; Whittaker, Roger; Gándara, Carolina; Stoll, Elizabeth A

2017-08-01

Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is nonpathogenic, and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy. The focus is on European regulations and how they are administered in the United Kingdom, although many of the principles will be similar for other regions, including the United States. The report justifies the rationale for using third-generation lentiviral vectors to achieve gene delivery for in vivo and ex vivo applications; briefly summarizes the extant regulatory guidance for gene therapies, categorized as advanced therapeutic medicinal products (ATMPs); provides guidance on specific regulatory issues regarding gene therapies; presents an overview of the key stakeholders to be approached when pursuing clinical trials authorization for an ATMP; and includes a brief catalogue of the documentation required to submit an application for regulatory approval of a new gene therapy.

Gene therapy for the eye focus on mutation-independent approaches.

PubMed

Dalkara, Deniz; Duebel, Jens; Sahel, José-Alain

2015-02-01

This review will discuss retinal gene therapy strategies with a focus on mutation-independent approaches to treat a large number of patients without knowledge of the mutant gene. These approaches rely on the secretion of neurotrophic factors to slow down retinal degeneration and the use of optogenetics to restore vision in late-stage disease. Success in clinical application of adeno-associated virus (AAV)-mediated gene therapy for Leber's congenital amaurosis established the feasibility of retinal gene therapy. More clinical trials are currently on their way for recessive diseases with known mutations. However, the genetic and mechanistic diversity of the retinal diseases presents an enormous obstacle for the development of gene therapies tailored to each patient-specific mutation. To extend gene therapy's promise to a large number of patients, evidence suggests retina-specific trophic factors, such as rod-derived cone viability factor, can be used to slow down loss of cone cells responsible for our high acuity vision. In parallel, it has been shown that microbial opsins are able to restore light sensitivity when expressed in blind retinas. Recent findings imply that using the viral technology that has been demonstrated as well tolerated in patients, there are opportunities to develop widely applicable gene therapeutic interventions in clinical ophthalmology.

A Guide to Approaching Regulatory Considerations for Lentiviral-Mediated Gene Therapies

PubMed Central

White, Michael; Whittaker, Roger; Gándara, Carolina; Stoll, Elizabeth A.

2017-01-01

Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is nonpathogenic, and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy. The focus is on European regulations and how they are administered in the United Kingdom, although many of the principles will be similar for other regions, including the United States. The report justifies the rationale for using third-generation lentiviral vectors to achieve gene delivery for in vivo and ex vivo applications; briefly summarizes the extant regulatory guidance for gene therapies, categorized as advanced therapeutic medicinal products (ATMPs); provides guidance on specific regulatory issues regarding gene therapies; presents an overview of the key stakeholders to be approached when pursuing clinical trials authorization for an ATMP; and includes a brief catalogue of the documentation required to submit an application for regulatory approval of a new gene therapy. PMID:28817344

Cellular and molecular mechanisms of HIV-1 integration targeting.

PubMed

Engelman, Alan N; Singh, Parmit K

2018-07-01

Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral integrase protein interacts with transcriptional co-activator lens epithelium-derived growth factor p75 to principally position integration within gene bodies. The integrase additionally senses target DNA distortion and nucleotide sequence to help fine-tune the specific phosphodiester bonds that are cleaved at integration sites. Research into virus-host interactions that underlie HIV-1 integration targeting has aided the development of a novel class of integrase inhibitors and may help to improve the safety of viral-based gene therapy vectors.

Gene therapy for cancer: regulatory considerations for approval.

PubMed

Husain, S R; Han, J; Au, P; Shannon, K; Puri, R K

2015-12-01

The rapidly changing field of gene therapy promises a number of innovative treatments for cancer patients. Advances in genetic modification of cancer and immune cells and the use of oncolytic viruses and bacteria have led to numerous clinical trials for cancer therapy, with several progressing to late-stage product development. At the time of this writing, no gene therapy product has been approved by the United States Food and Drug Administration (FDA). Some of the key scientific and regulatory issues include understanding of gene transfer vector biology, safety of vectors in vitro and in animal models, optimum gene transfer, long-term persistence or integration in the host, shedding of a virus and ability to maintain transgene expression in vivo for a desired period of time. Because of the biological complexity of these products, the FDA encourages a flexible, data-driven approach for preclinical safety testing programs. The clinical trial design should be based on the unique features of gene therapy products, and should ensure the safety of enrolled subjects. This article focuses on regulatory considerations for gene therapy product development and also discusses guidance documents that have been published by the FDA.

Gene therapy for cancer: regulatory considerations for approval

PubMed Central

Husain, S R; Han, J; Au, P; Shannon, K; Puri, R K

2015-01-01

The rapidly changing field of gene therapy promises a number of innovative treatments for cancer patients. Advances in genetic modification of cancer and immune cells and the use of oncolytic viruses and bacteria have led to numerous clinical trials for cancer therapy, with several progressing to late-stage product development. At the time of this writing, no gene therapy product has been approved by the United States Food and Drug Administration (FDA). Some of the key scientific and regulatory issues include understanding of gene transfer vector biology, safety of vectors in vitro and in animal models, optimum gene transfer, long-term persistence or integration in the host, shedding of a virus and ability to maintain transgene expression in vivo for a desired period of time. Because of the biological complexity of these products, the FDA encourages a flexible, data-driven approach for preclinical safety testing programs. The clinical trial design should be based on the unique features of gene therapy products, and should ensure the safety of enrolled subjects. This article focuses on regulatory considerations for gene therapy product development and also discusses guidance documents that have been published by the FDA. PMID:26584531

Molecular Targeting of Prostate Cancer During Androgen Ablation: Inhibition of CHES1/FOXN3

DTIC Science & Technology

2010-05-10

target of rapamycin ( mTOR ) and hypoxia-inducible factor-1α (HIF-1α) target genes (12). Additionally, transcriptional activation of Bcl-2 by NF-κB...in resistance to hormone therapy (19). Elevated expression of genes encoding PI3K/Akt/ mTOR pathway components has also been implicated in androgen... mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat Med, 10

SENESCENCE (AGEING) @ 2011

PubMed Central

Nigam, Anjana

2011-01-01

Ageing, also called as senescence, is one of the most complex, intrinsic, biological processes of growing older and resulting into reduced functional ability of the organism. Telomerase, environment, low calorie diets, free radicals, etc., are all believed to affect this ageing process. A number of genetic components of ageing have been identified using model organisms. Genes, mainly the sirtuins, regulate the ageing speed by indirection and controlling organism resistance to damages by exogenous and endogenous stresses. In higher organisms, ageing is likely to be regulated, in part, through the insulin/insulin-like growth factor 1 pathway. Besides this, the induction of apoptosis in stem and progenitor cells, increased p53 activity, and autophagy is also thought to trigger premature organismal ageing. Ageing has also been shown to upregulate expression of inflammatory mediators in mouse adipose tissue. The understanding of pathophysiology of ageing over the past few years has posed tremendous challenges for the development of anti-ageing medicine for targeted therapy. Future research areas must include targeted role of systemic inflammatory markers such as C-reactive protein and interleukin 6 and other biochemical and genetic studies including gene signaling pathways, gene microarray analysis, gene modulation, gene therapy, and development of animal/human models for potential therapeutic measures and evaluations. PMID:22345758

Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome.

PubMed

Kuo, Caroline Y; Long, Joseph D; Campo-Fernandez, Beatriz; de Oliveira, Satiro; Cooper, Aaron R; Romero, Zulema; Hoban, Megan D; Joglekar, Alok V; Lill, Georgia R; Kaufman, Michael L; Fitz-Gibbon, Sorel; Wang, Xiaoyan; Hollis, Roger P; Kohn, Donald B

2018-05-29

X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Gene Therapy by Targeted Adenovirus-mediated Knockdown of Pulmonary Endothelial Tph1 Attenuates Hypoxia-induced Pulmonary Hypertension

PubMed Central

Morecroft, Ian; White, Katie; Caruso, Paola; Nilsen, Margaret; Loughlin, Lynn; Alba, Raul; Reynolds, Paul N; Danilov, Sergei M; Baker, Andrew H; MacLean, Margaret R

2012-01-01

Serotonin is produced by pulmonary arterial endothelial cells (PAEC) via tryptophan hydroxylase-1 (Tph1). Pathologically, serotonin acts on underlying pulmonary arterial cells, contributing to vascular remodeling associated with pulmonary arterial hypertension (PAH). The effects of hypoxia on PAEC-Tph1 activity are unknown. We investigated the potential of a gene therapy approach to PAH using selective inhibition of PAEC-Tph1 in vivo in a hypoxic model of PAH. We exposed cultured bovine pulmonary arterial smooth muscle cells (bPASMCs) to conditioned media from human PAECs (hPAECs) before and after hypoxic exposure. Serotonin levels were increased in hypoxic PAEC media. Conditioned media evoked bPASMC proliferation, which was greater with hypoxic PAEC media, via a serotonin-dependent mechanism. In vivo, adenoviral vectors targeted to PAECs (utilizing bispecific antibody to angiotensin-converting enzyme (ACE) as the selective targeting system) were used to deliver small hairpin Tph1 RNA sequences in rats. Hypoxic rats developed PAH and increased lung Tph1. PAEC-Tph1 expression and development of PAH were attenuated by our PAEC-Tph1 gene knockdown strategy. These results demonstrate that hypoxia induces Tph1 activity and selective knockdown of PAEC-Tph1 attenuates hypoxia-induced PAH in rats. Further investigation of pulmonary endothelial-specific Tph1 inhibition via gene interventions is warranted. PMID:22525513

Safety of gene therapy: new insights to a puzzling case.

PubMed

Rothe, Michael; Schambach, Axel; Biasco, Luca

2014-01-01

Over the last few years, the transfer of therapeutic genes via gammaretro- or lentiviral vector systems has proven its virtue as an alternative treatment for a series of genetic disorders. The number of approved phase I/II clinical trials, especially for rare diseases, is steadily increasing, but the overall hurdles to become a broadly acceptable therapy remain numerous. The efforts by clinicians and basic scientists have tremendously improved the knowledge available about feasibility and biosafety of gene therapy. Nonetheless, despite the generation of a plethora of clinical and preclinical safety data, we still lack sufficiently powerful assays to predictively assess the exact levels of toxicity that might be observed in any given clinical gene therapy. Insertional mutagenesis is one of the major concerns when using integrating vectors for permanent cell modification, and the occurrence of adverse events related to genotoxicity, in early gene therapy trials, has refrained the field of gene therapy from emerging further. In this review, we provided a comprehensive overview on the basic principles and potential co-factors concurring in the generation of adverse events reported in gene therapy clinical trials using integrating vectors. Additionally, we summarized the available systems to assess genotoxicity at the preclinical level and we shed light on the issues affecting the predictive value of these assays when translating their results into the clinical arena. In the last section of the review we briefly touched on the future trends and how they could increase the safety of gene therapy employing integrating vector technology to take it to the next level.

Utilizing social media to study information-seeking and ethical issues in gene therapy.

PubMed

Robillard, Julie M; Whiteley, Louise; Johnson, Thomas Wade; Lim, Jonathan; Wasserman, Wyeth W; Illes, Judy

2013-03-04

The field of gene therapy is rapidly evolving, and while hopes of treating disorders of the central nervous system and ethical concerns have been articulated within the academic community, little is known about views and opinions of different stakeholder groups. To address this gap, we utilized social media to investigate the kind of information public users are seeking about gene therapy and the hopes, concerns, and attitudes they express. We conducted a content analysis of questions containing the keywords "gene therapy" from the Q&A site "Yahoo! Answers" for the 5-year period between 2006 and 2010. From the pool of questions retrieved (N=903), we identified those containing at least one theme related to ethics, environment, economics, law, or society (n=173) and then characterized the content of relevant answers (n=399) through emergent coding. The results show that users seek a wide range of information regarding gene therapy, with requests for scientific information and ethical issues at the forefront of enquiry. The question sample reveals high expectations for gene therapy that range from cures for genetic and nongenetic diseases to pre- and postnatal enhancement of physiological attributes. Ethics questions are commonly expressed as fears about the impact of gene therapy on self and society. The answer sample echoes these concerns but further suggests that the acceptability of gene therapy varies depending on the specific application. Overall, the findings highlight the powerful role of social media as a rich resource for research into attitudes toward biomedicine and as a platform for knowledge exchange and public engagement for topics relating to health and disease.

Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain

PubMed Central

Lee, Jeffrey C; Vivanco, Igor; Beroukhim, Rameen; Huang, Julie H. Y; Feng, Whei L; DeBiasi, Ralph M; Yoshimoto, Koji; King, Jennifer C; Nghiemphu, Phioanh; Yuza, Yuki; Xu, Qing; Greulich, Heidi; Thomas, Roman K; Paez, J. Guillermo; Peck, Timothy C; Linhart, David J; Glatt, Karen A; Getz, Gad; Onofrio, Robert; Ziaugra, Liuda; Levine, Ross L; Gabriel, Stacey; Kawaguchi, Tomohiro; O'Neill, Keith; Khan, Haumith; Liau, Linda M; Nelson, Stanley F; Rao, P. Nagesh; Mischel, Paul; Pieper, Russell O; Cloughesy, Tim; Leahy, Daniel J; Sellers, William R; Sawyers, Charles L; Meyerson, Matthew; Mellinghoff, Ingo K

2006-01-01

Background Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma. PMID:17177598

Lentiviral-mediated gene therapy results in sustained expression of β-glucuronidase for up to 12 months in the gus(mps/mps) and up to 18 months in the gus(tm(L175F)Sly) mouse models of mucopolysaccharidosis type VII.

PubMed

Derrick-Roberts, Ainslie L K; Pyragius, Carmen E; Kaidonis, Xenia M; Jackson, Matilda R; Anson, Donald S; Byers, Sharon

2014-09-01

A number of mucopolysaccharidosis type VII (MPS VII) mouse models with different levels of residual enzyme activity have been created replicating the range of clinical phenotypes observed in human MPS VII patients. In this study, a lentivirus encoding murine β-glucuronidase was administered intravenously at birth to both the severe (Gus(mps/mps) strain) and attenuated (Gus(tm(L175F)Sly) strain) mouse models of MPS VII. Circulating enzyme levels were normalized in the Gus(mps/mps) mice and were 3.5-fold higher than normal in the Gus(tm(L175F)Sly) mouse 12 and 18 months after administration. Tissue β-glucuronidase activity increased over untreated levels in all tissues evaluated in both strains at 12 months, and the elevated level was maintained in Gus(tm(L175F)Sly) tissues at 18 months. These elevated enzyme levels reduced glycosaminoglycan storage in the liver, spleen, kidney, and heart in both models. Bone mineral volume decreased toward normal in both models after 12 months of therapy and after 18 months in the Gus(tm(L175F)Sly) mouse. Open-field exploration was improved in 18-month-old treated Gus(tm(L175F)Sly) mice, while spatial learning improved in both 12- and 18-month-old treated Gus(tm(L175F)Sly) mice. Overall, neonatal administration of lentiviral gene therapy resulted in sustained enzyme expression for up to 18 months in murine models of MPS VII. Significant improvements in biochemistry and enzymology as well as functional improvement of bone and behavior deficits in the Gus(tm(L175F)Sly) model were observed. Therapy significantly increased the lifespan of Gus(mps/mps) mice, with 12 months being the longest reported lentiviral treatment for this strain. It is important to assess the long-term outcome on enzyme levels and effect on pathology for lentiviral gene therapy to be a potential therapy for MPS patients.

Is cystic fibrosis genetic medicine's canary?

PubMed

Lindee, Susan; Mueller, Rebecca

2011-01-01

In 1989 the gene that causes cystic fibrosis (CF) was identified in a search accompanied by intense anticipation that the gene, once discovered, would lead rapidly to gene therapy. Many hoped that the disease would effectively disappear. Those affected were going to inhale vectors packed with functioning genes, which would go immediately to work in the lungs. It was a bewitching image, repeatedly invoked in both scientific and popular texts. Gene therapy clinical trials were carried out with a range of strategies and occasionally success seemed close, but by 1996 the idea that gene therapy for CF would quickly provide a cure was being abandoned by the communities engaged with treatment and research. While conventional wisdom holds that the death of Jesse Gelsinger in an unrelated gene therapy trial in 1999 produced new skepticism about gene therapy, the CF story suggests a different trajectory, and some different lessons. This article considers the rise and fall of gene therapy for CF and suggests that CF may provide a particularly compelling case study of a failed genomic technology, perhaps even of a medical "canary." The story of CF might be a kind of warning to us that genetic medicine may create as many problems as it solves, and that moving forward constructively with these techniques and practices requires many kinds of right information, not just about biology, but also about values, priorities, market forces, uncertainty, and consumer choice.

Immunostimulatory AdCD40L gene therapy combined with low-dose cyclophosphamide in metastatic melanoma patients

PubMed Central

Loskog, Angelica; Maleka, Aglaia; Mangsbo, Sara; Svensson, Emma; Lundberg, Christina; Nilsson, Anders; Krause, Johan; Agnarsdóttir, Margrét; Sundin, Anders; Ahlström, Håkan; Tötterman, Thomas H; Ullenhag, Gustav

2016-01-01

Background: Current approaches for treating metastatic malignant melanoma (MM) are not effective enough and are associated with serious adverse events. Due to its immunogenicity, melanoma is an attractive target for immunostimulating therapy. In this phase I/IIa study, local AdCD40L immunostimulatory gene therapy was evaluated in patients with MM. Methods: AdCD40L is an adenovirus carrying the gene for CD40 ligand. Patients that failed standard treatments were enrolled. Six patients received four weekly intratumoral AdCD40L injections. Next, nine patients received low-dose cyclophosphamide conditioning before the first and fourth AdCD40L injection. The blood samples were collected at multiple time points for chemistry, haematology and immunology evaluations. Radiology was performed at enrolment and repeated twice after the treatment. Results: AdCD40L was safe with mild transient reactions. No objective responses were recorded by MRI, however, local and distant responses were seen on FDG-PET. The overall survival at 6 months was significantly better when cyclophosphamide was added to AdCD40L. The patients with the best survival developed the highest levels of activated T cells and experienced a pronounced decrease of intratumoral IL8. Conclusions: AdCD40L therapy for MM was well tolerated. Local and distant responses along with better survival in the low-dose cyclophosphamide group are encouraging. PMID:27031851

Increased sensitivity to apoptosis induced by methotrexate is mediated by Jun N-terminal kinase

PubMed Central

Spurlock, Charles F.; Aune, Zachary T.; Tossberg, John T.; Collins, Patrick L.; Aune, Jessica P.; Huston, Joseph W.; Crooke, Philip S.; Olsen, Nancy J.; Aune, Thomas M.

2011-01-01

Objective Low-dose methotrexate [MTX] is an effective therapy for rheumatoid arthritis yet its mechanism of action is incompletely understood. Here, we explored induction of apoptosis by MTX. Methods We employed flow cytometry to assess changes in levels of intracellular proteins, reactive oxygen species [ROS], and apoptosis.Quantitative polymerase chain reaction was usedtoassess changes in transcript levels of select target genes in response to MTX. Results MTX does not directly induce apoptosis but rather ‘primes’ cells for markedly increased sensitivity to apoptosis via either mitochondrial or death receptor pathways by a Jun N-terminal kinase [JNK]-dependent mechanism. Increased sensitivity to apoptosis is mediated, at least in part, by MTX-dependent production of reactive oxygen species, JNK activation and JNK-dependent induction of genes whose protein products promote apoptosis. Supplementation with tetrahydrobiopterin blocks these methotrexate-induced effects. Subjects with rheumatoid arthritis on low-dose MTX therapy express elevated levels of the JNK-target gene, JUN. Conclusions Our results support a model whereby methotrexate inhibits reduction of dihydrobiopterin to tetrahydrobiopterin resulting in increased production of ROS, increased JNK activity and increased sensitivity to apoptosis. The finding of increased JUN levels in subjects with RA taking low-dose MTX supports the notion that this pathway is activated by MTX, in vivo, and may contribute to efficacy of MTX in inflammatory disease. PMID:21618198

Monogenic diseases associated with intestinal inflammation: implications for the understanding of inflammatory bowel disease.

PubMed

Uhlig, Holm H

2013-12-01

Inflammatory bowel disease (IBD), encompassing Crohn's disease and ulcerative colitis, has multifactorial aetiology with complex interactions between genetic and environmental factors. Over 150 genetic loci are associated with IBD. The genetic contribution of the majority of those loci towards explained heritability is low. Recent studies have reported an increasing spectrum of human monogenic diseases that can present with IBD-like intestinal inflammation. A substantial proportion of patients with those genetic defects present with very early onset of intestinal inflammation. The 40 monogenic defects with IBD-like pathology selected in this review can be grouped into defects in intestinal epithelial barrier and stress response, immunodeficiencies affecting granulocyte and phagocyte activity, hyper- and autoinflammatory disorders as well as defects with disturbed T and B lymphocyte selection and activation. In addition, there are defects in immune regulation affecting regulatory T cell activity and interleukin (IL)-10 signalling. Related to the variable penetrance of the IBD-like phenotype, there is a likely role for modifier genes and gene-environment interactions. Treatment options in this heterogeneous group of disorders range from anti-inflammatory and immunosuppressive therapy to blockade of tumour necrosis factor α and IL-1β, surgery, haematopoietic stem cell transplantation or gene therapy. Understanding of prototypic monogenic 'orphan' diseases cannot only provide treatment options for the affected patients but also inform on immunological mechanisms and complement the functional understanding of the pathogenesis of IBD.

Virus-directed enzyme prodrug therapy and the assessment of the cytotoxic impact of some benzimidazole derivatives.

PubMed

Szewczuk, Michał; Boguszewska, Karolina; Żebrowska, Marta; Balcerczak, Ewa; Stasiak, Marta; Świątkowska, Maria; Błaszczak-Świątkiewicz, Katarzyna

2017-07-01

Virus-directed enzyme prodrug therapy is one of the major strategy of increasing cytotoxicity of bioreductive agents. This research intended to examine new selected benzimidazole derivatives as a substrate for nitroreductase, the enzyme involved in nitroreduction which is responsible to the production of cytotoxic metabolites. In this way, the selectivity and strength of cytotoxicity can be raised. The effect of benzimidazoles on virus transfected cells and non-virus transfected cells A549 cell line was established by Annexin V + propidium iodide test, western blot, and polymerase chain reaction analysis of specific pro- and anti-apoptotic proteins in the corresponding gene expression and additionally nitroreductase gene expression. Our results proved the pro-apoptotic properties of all tested compounds in normoxia and hypoxia, especially according to virused A549 cells where the time of exposition was reduced from 48 to 4 h. In this shorten period of time, the strongest activity was shown by N-oxide compounds with nitro-groups. The apoptosis was confirmed by generation of BAX gene and protein and reduction of BCL2 gene and protein.

Prospects for Gene Therapy in the Fragile X Syndrome

ERIC Educational Resources Information Center

Rattazzi, Mario C.; LaFauci, Giuseppe; Brown, W. Ted

2004-01-01

Gene therapy is unarguably the definitive way to treat, and possibly cure, genetic diseases. A straightforward concept in theory, in practice it has proven difficult to realize, even when directed to easily accessed somatic cell systems. Gene therapy for diseases in which the central nervous system (CNS) is the target organ presents even greater…

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Chen; Jie, Leng; Yongqi, Wang

Nanoparticles are becoming promising carriers for gene delivery because of their high capacity in gene loading and low cell cytotoxicity. In this study, a chitosan-based nanoparticle encapsulated within a recombinant pcDNA3.1-dsNKG2D-IL-15 plasmid was generated. The fused dsNKG2D-IL-15 gene fragment consisted of double extracellular domains of NKG2D with IL-15 gene at downstream. The average diameter of the gene nanoparticles ranged from 200 nm to 400 nm, with mean zeta potential value of 53.8 ± 6.56 mV. The nanoparticles which were loaded with the dsNKG2D-IL-15 gene were uptaken by tumor cells with low cytotoxicity. Tumor cells pre-transfected by gene nanopartilces stimulated NK and T cells in vitro. Intramuscularmore » injection of gene nanoparticles suppressed tumor growth and prolonged survival of tumor-bearing mice through activation of NK and CD8{sup +} T cells. Thus, chitosan-based nanoparticle delivery of dsNKG2D-IL-15 gene vaccine can be potentially used for tumor therapy. - Highlights: • Generation of a nanoparticle for delivery of dsNKG2D-IL-15 gene. • Characterization of the gene nanoparticle. • Antitumor activity mediated by the gene nanoparticle.« less

Gene Therapy for Metachromatic Leukodystrophy

PubMed Central

Rosenberg, Jonathan B.; Kaminsky, Stephen M.; Aubourg, Patrick; Crystal, Ronald G.; Sondhi, Dolan

2016-01-01

Summary Leukodystrophies are rare white matter genetic disorders of the central nervous system (CNS) with progressive neurologic deterioration. One approach to the treatment of leukodystrophies is by gene therapy. Using metachromatic leukodystrophy (MLD), a leukodystrophy resulting from deficiency of a lysosomal catabolic enzyme arylsulfatase A (ARSA) as the example, this review is focused on the current status of preclinical and clinical development of gene therapy as a viable treatment option for leukodystrophies. In MLD, mutations in the ARSA gene result in excess buildup of sulfatides, which triggers apoptosis of glia and neurons. The disease is characterized by severe cerebral demyelination and atrophy, with progressive loss of oligodendrocytes, neurons and Schwann cells. The optimal therapy for MLD would provide persistent and high level expression of ARSA in the CNS. Gene therapy using adeno-associated virus (AAV) is an ideal choice for clinical development as it provides the best balance of potential for efficacy with a reduced safety risk profile. In this review, we have summarized preclinical data that support the use of a gene therapy with the AAVrh.10 serotype for clinical development as a treatment for MLD. PMID:27638601

17th Annual Meeting of the German Society for Gene Therapy.

PubMed

Büning, Hildegard; Baum, Christopher; Ehrhardt, Anja; Nettelbeck, Dirk M; Ogris, Manfred

2011-01-01

The 17th Annual Meeting of the German Society for Gene Therapy was held at the Chemistry and Pharmacy Campus of the University of Munich in conjunction with and supported by the British Society for Gene Therapy, the Viral Vectors Study Group of the German Society for Virology, the Research Priority Program SPP1230, the Nanosystems Initiative Munich and the Helmholtz Center Munich. The German Research Foundation provided financial support for the invited international speakers. In addition to 25 invited lectures, 21 oral presentations were selected out of more than 100 submitted abstracts. State-of-the-art advances in the field of gene therapy were presented, a field that has considerably evolved within recent years. More than 200 researchers from Germany and other European countries, as well as the USA, Canada and Japan attended the meeting. Prior to the official meeting, a public day was organized, in which the interested public could participate in talks and discussions concerning gene therapy issues. Furthermore, at the 'kids workshop' young scientists aged 8-10 years were discovering cellular and genetic mechanisms and the principles of gene therapy.

Targeting the p53 signaling pathway in cancer therapy - The promises, challenges, and perils

PubMed Central

Stegh, Alexander H.

2012-01-01

Introduction Research over the past three decades has identified p53 as a multifunctional transcription factor, which regulates the expression of >2,500 target genes. p53 impacts myriad, highly diverse cellular processes, including the maintenance of genomic stability and fidelity, metabolism, longevity, and represents one of the most important and extensively studied tumor suppressors. Activated by various stresses, foremost genotoxic damage, hypoxia, heat shock and oncogenic assault, p53 blocks cancer progression by provoking transient or permanent growth arrest, by enabling DNA repair or by advancing cellular death programs. This potent and versatile anti-cancer activity profile, together with genomic and mutational analyses documenting inactivation of p53 in more than 50% of human cancers, motivated drug development efforts to (re-) activate p53 in established tumors. Areas covered In this review the complexities of p53 signaling in cancer are summarized. Current strategies and challenges to restore p53’s tumor suppressive function in established tumors, i.e. adenoviral gene transfer and small molecules to activate p53, to inactivate p53 inhibitors and to restore wild type function of p53 mutant proteins are discussed. Expert opinion It is indubitable that p53 represents an attractive target for the development of anti-cancer therapies. Whether p53 is ‘druggable’, however, remains an area of active research and discussion, as p53 has pro-survival functions and chronic p53 activation accelerates aging, which may compromise the long-term homeostasis of an organism. Thus, the complex biology and dual functions of p53 in cancer prevention and age-related cellular responses pose significant challenges on the development of p53-targeting cancer therapies. PMID:22239435

Multivariate inference of pathway activity in host immunity and response to therapeutics

PubMed Central

Goel, Gautam; Conway, Kara L.; Jaeger, Martin; Netea, Mihai G.; Xavier, Ramnik J.

2014-01-01

Developing a quantitative view of how biological pathways are regulated in response to environmental factors is central for understanding of disease phenotypes. We present a computational framework, named Multivariate Inference of Pathway Activity (MIPA), which quantifies degree of activity induced in a biological pathway by computing five distinct measures from transcriptomic profiles of its member genes. Statistical significance of inferred activity is examined using multiple independent self-contained tests followed by a competitive analysis. The method incorporates a new algorithm to identify a subset of genes that may regulate the extent of activity induced in a pathway. We present an in-depth evaluation of specificity, robustness, and reproducibility of our method. We benchmarked MIPA's false positive rate at less than 1%. Using transcriptomic profiles representing distinct physiological and disease states, we illustrate applicability of our method in (i) identifying gene–gene interactions in autophagy-dependent response to Salmonella infection, (ii) uncovering gene–environment interactions in host response to bacterial and viral pathogens and (iii) identifying driver genes and processes that contribute to wound healing and response to anti-TNFα therapy. We provide relevant experimental validation that corroborates the accuracy and advantage of our method. PMID:25147207

Microneedles As a Delivery System for Gene Therapy

PubMed Central

Chen, Wei; Li, Hui; Shi, De; Liu, Zhenguo; Yuan, Weien

2016-01-01

Gene delivery systems can be divided to two major types: vector-based (either viral vector or non-viral vector) and physical delivery technologies. Many physical carriers, such as electroporation, gene gun, ultrasound start to be proved to have the potential to enable gene therapy. A relatively new physical delivery technology for gene delivery consists of microneedles (MNs), which has been studied in many fields and for many molecule types and indications. Microneedles can penetrate the stratum corneum, which is the main barrier for drug delivery through the skin with ease of administration and without significant pain. Many different kinds of MNs, such as metal MNs, coated MNs, dissolving MNs have turned out to be promising in gene delivery. In this review, we discussed the potential as well as the challenges of utilizing MNs to deliver nucleic acids for gene therapy. We also proposed that a combination of MNs and other gene delivery approaches may lead to a better delivery system for gene therapy. PMID:27303298

Gene Therapy for Cardiovascular Disease

PubMed Central

2003-01-01

The last decade has seen substantial advances in the development of gene therapy strategies and vector technology for the treatment of a diverse number of diseases, with a view to translating the successes observed in animal models into the clinic. Perhaps the overwhelming drive for the increase in vascular gene transfer studies is the current lack of successful long-term pharmacological treatments for complex cardiovascular diseases. The increase in cardiovascular disease to epidemic proportions has also led many to conclude that drug therapy may have reached a plateau in its efficacy and that gene therapy may represent a realistic solution to a long-term problem. Here, we discuss gene delivery approaches and target diseases. PMID:12721517

Deacetylases and NF-κB in Redox Regulation of Cigarette Smoke induced Lung Inflammation: Implications in Pathogenesis of COPD

PubMed Central

Rajendrasozhan, Saravanan; Yang, Se-Ran; Edirisinghe, Indika; Yao, Hongwei; Adenuga, David; Rahman, Irfan

2009-01-01

Oxidative stress has been implicated in the pathogenesis of several inflammatory lung disorders including chronic obstructive pulmonary disease (COPD) due to its effect on pro-inflammatory gene transcription. Cigarette smoke-mediated oxidative stress activates NF-κB-dependent transcription of pro-inflammatory mediators either through activation of inhibitor κB-α kinase (IKK) and/or the enhanced recruitment and activation of transcriptional co-activators. Enhanced NF-κB-co-activator complex formation results in targeted increase in chromatin modifications, such as histone acetylation leading to inflammatory gene transcription. NF-κB-dependent gene expression, at least in part, is regulated by changes in deacetylases such as histone deacetylases (HDACs) and sirtuins. Cigarette smoke and oxidants also alter the levels/activity of HDAC by post-translational modifications and in doing so further induces gene expression of pro-inflammatory mediators. In addition, cigarette smoke/oxidants can reduce glucocorticoid sensitivity by attenuating HDAC2 activity and expression, which may account for the glucocorticoid insensitivity in patients with COPD. Understanding the mechanisms of NF-κB regulation, and the balance between histone acetylation and deacetylation may lead to the development of novel therapies based on the pharmacological manipulation of IKK and deacetylases in lung inflammation and injury. PMID:18220485

Gene therapy comes of age.

PubMed

Dunbar, Cynthia E; High, Katherine A; Joung, J Keith; Kohn, Donald B; Ozawa, Keiya; Sadelain, Michel

2018-01-12

After almost 30 years of promise tempered by setbacks, gene therapies are rapidly becoming a critical component of the therapeutic armamentarium for a variety of inherited and acquired human diseases. Gene therapies for inherited immune disorders, hemophilia, eye and neurodegenerative disorders, and lymphoid cancers recently progressed to approved drug status in the United States and Europe, or are anticipated to receive approval in the near future. In this Review, we discuss milestones in the development of gene therapies, focusing on direct in vivo administration of viral vectors and adoptive transfer of genetically engineered T cells or hematopoietic stem cells. We also discuss emerging genome editing technologies that should further advance the scope and efficacy of gene therapy approaches. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Gene doping: gene delivery for olympic victory

PubMed Central

Gould, David

2013-01-01

With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. PMID:23082866

Gene therapy in dentistry: tool of genetic engineering. Revisited.

PubMed

Gupta, Khushboo; Singh, Saurabh; Garg, Kavita Nitish

2015-03-01

Advances in biotechnology have brought gene therapy to the forefront of medical research. The concept of transferring genes to tissues for clinical applications has been discussed nearly half a century, but the ability to manipulate genetic material via recombinant DNA technology has brought this goal to reality. The feasibility of gene transfer was first demonstrated using tumour viruses. This led to development of viral and nonviral methods for the genetic modification of somatic cells. Applications of gene therapy to dental and oral problems illustrate the potential impact of this technology on dentistry. Preclinical trial results regarding the same have been very promising. In this review we will discuss methods, vectors involved, clinical implication in dentistry and scientific issues associated with gene therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Potential large animal models for gene therapy of human genetic diseases of immune and blood cell systems.

PubMed

Bauer, Thomas R; Adler, Rima L; Hickstein, Dennis D

2009-01-01

Genetic mutations involving the cellular components of the hematopoietic system--red blood cells, white blood cells, and platelets--manifest clinically as anemia, infection, and bleeding. Although gene targeting has recapitulated many of these diseases in mice, these murine homologues are limited as translational models by their small size and brief life span as well as the fact that mutations induced by gene targeting do not always faithfully reflect the clinical manifestations of such mutations in humans. Many of these limitations can be overcome by identifying large animals with genetic diseases of the hematopoietic system corresponding to their human disease counterparts. In this article, we describe human diseases of the cellular components of the hematopoietic system that have counterparts in large animal species, in most cases carrying mutations in the same gene (CD18 in leukocyte adhesion deficiency) or genes in interacting proteins (DNA cross-link repair 1C protein and protein kinase, DNA-activated catalytic polypeptide in radiation-sensitive severe combined immunodeficiency). Furthermore, we describe the potential of these animal models to serve as disease-specific preclinical models for testing the efficacy and safety of clinical interventions such as hematopoietic stem cell transplantation or gene therapy before their use in humans with the corresponding disease.

Integration of high-risk human papillomavirus into cellular cancer-related genes in head and neck cancer cell lines.

PubMed

Walline, Heather M; Goudsmit, Christine M; McHugh, Jonathan B; Tang, Alice L; Owen, John H; Teh, Bin T; McKean, Erin; Glover, Thomas W; Graham, Martin P; Prince, Mark E; Chepeha, Douglas B; Chinn, Steven B; Ferris, Robert L; Gollin, Susanne M; Hoffmann, Thomas K; Bier, Henning; Brakenhoff, Ruud; Bradford, Carol R; Carey, Thomas E

2017-05-01

Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines. E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017. © 2017 Wiley Periodicals, Inc.

Synergistically combined gene delivery for enhanced VEGF secretion and anti-apoptosis

PubMed Central

Won, Young-Wook; Lee, Minhyung; Kim, Hyun Ah; Nam, Kihoon; Bull, David A.; Kim, Sung Wan

2013-01-01

With current pharmacological treatments, preventing the remodeling of the left ventricle and the progression to heart failure is a difficult task. Gene therapy is considered to provide a direct treatment to the long-term complications of ischemic heart diseases. Although current gene therapies that use single molecular targets seem potentially possible, they have not achieved a success in the treatment of ischemic diseases. With an efficient polymeric gene carrier, PAM-ABP, we designed a synergistically combined gene delivery strategy to enhance vascular endothelial growth factor (VEGF) secretion and prolong anti-apoptotic effects. A hypoxia-inducible plasmid expressing both hypoxia-inducible heme oxygenase-1 (HO-1) and the Src homology domain-2 containing tyrosine phosphatase-1 microRNA (miSHP 1) and a hypoxia-responsive VEGF plasmid were combined in this study. The positive feedback circuit between HO-1 and VEGF, and the negative regulatory role of SHP-1 in angiogenesis enhance VEGF secretion synergistically. The synergy in VEGF secretion as a consequence of the gene combination and the prolonged HO-1 activity was confirmed in hypoxic cardiomyocytes and cardiomyocyte apoptosis under hypoxia, and was decreased synergistically. These results suggest that the synergistic combination of VEGF, HO-1, and miSHP-1 may be promising for the clinical treatment of ischemic diseases. PMID:24007285

Subthalamic hGAD65 Gene Therapy and Striatum TH Gene Transfer in a Parkinson’s Disease Rat Model

PubMed Central

Zheng, Deyu; Jiang, Xiaohua; Zhao, Junpeng; Duan, Deyi; Zhao, Huanying; Xu, Qunyuan

2013-01-01

The aim of the present study is to detect a combination method to utilize gene therapy for the treatment of Parkinson’s disease (PD). Here, a PD rat model is used for the in vivo gene therapy of a recombinant adeno-associated virus (AAV2) containing a human glutamic acid decarboxylase 65 (rAAV2-hGAD65) gene delivered to the subthalamic nucleus (STN). This is combined with the ex vivo gene delivery of tyrosine hydroxylase (TH) by fibroblasts injected into the striatum. After the treatment, the rotation behavior was improved with the greatest efficacy in the combination group. The results of immunohistochemistry showed that hGAD65 gene delivery by AAV2 successfully led to phenotypic changes of neurons in STN. And the levels of glutamic acid and GABA in the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr) were obviously lower than the control groups. However, hGAD65 gene transfer did not effectively protect surviving dopaminergic neurons in the SNc and VTA. This study suggests that subthalamic hGAD65 gene therapy and combined with TH gene therapy can alleviate symptoms of the PD model rats, independent of the protection the DA neurons from death. PMID:23738148

Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant.

PubMed

Folcher, Marc; Oesterle, Sabine; Zwicky, Katharina; Thekkottil, Thushara; Heymoz, Julie; Hohmann, Muriel; Christen, Matthias; Daoud El-Baba, Marie; Buchmann, Peter; Fussenegger, Martin

2014-11-11

Synthetic devices for traceless remote control of gene expression may provide new treatment opportunities in future gene- and cell-based therapies. Here we report the design of a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly programme the transgene expression in human cells. An electroencephalography (EEG)-based brain-computer interface (BCI) processing mental state-specific brain waves programs an inductively linked wireless-powered optogenetic implant containing designer cells engineered for near-infrared (NIR) light-adjustable expression of the human glycoprotein SEAP (secreted alkaline phosphatase). The synthetic optogenetic signalling pathway interfacing the BCI with target gene expression consists of an engineered NIR light-activated bacterial diguanylate cyclase (DGCL) producing the orthogonal second messenger cyclic diguanosine monophosphate (c-di-GMP), which triggers the stimulator of interferon genes (STING)-dependent induction of synthetic interferon-β promoters. Humans generating different mental states (biofeedback control, concentration, meditation) can differentially control SEAP production of the designer cells in culture and of subcutaneous wireless-powered optogenetic implants in mice.

Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene.

PubMed

De Rocco, Davide; Pompili, Barbara; Castellani, Stefano; Morini, Elena; Cavinato, Luca; Cimino, Giuseppe; Mariggiò, Maria A; Guarnieri, Simone; Conese, Massimo; Del Porto, Paola; Ascenzioni, Fiorentina

2018-04-17

Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR , supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease.

Telomerase gene therapy rescues telomere length, bone marrow aplasia, and survival in mice with aplastic anemia.

PubMed

Bär, Christian; Povedano, Juan Manuel; Serrano, Rosa; Benitez-Buelga, Carlos; Popkes, Miriam; Formentini, Ivan; Bobadilla, Maria; Bosch, Fatima; Blasco, Maria A

2016-04-07

Aplastic anemia is a fatal bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia. The disease can be hereditary or acquired and develops at any stage of life. A subgroup of the inherited form is caused by replicative impairment of hematopoietic stem and progenitor cells due to very short telomeres as a result of mutations in telomerase and other telomere components. Abnormal telomere shortening is also described in cases of acquired aplastic anemia, most likely secondary to increased turnover of bone marrow stem and progenitor cells. Here, we test the therapeutic efficacy of telomerase activation by using adeno-associated virus (AAV)9 gene therapy vectors carrying the telomerase Tert gene in 2 independent mouse models of aplastic anemia due to short telomeres (Trf1- and Tert-deficient mice). We find that a high dose of AAV9-Tert targets the bone marrow compartment, including hematopoietic stem cells. AAV9-Tert treatment after telomere attrition in bone marrow cells rescues aplastic anemia and mouse survival compared with mice treated with the empty vector. Improved survival is associated with a significant increase in telomere length in peripheral blood and bone marrow cells, as well as improved blood counts. These findings indicate that telomerase gene therapy represents a novel therapeutic strategy to treat aplastic anemia provoked or associated with short telomeres. © 2016 by The American Society of Hematology.

Blood-Derived Smooth Muscle Cells as a Target for Gene Delivery

PubMed Central

Yang, Zhe; Shao, Hongwei; Tan, Yaohong; Eton, Darwin; Yu, Hong

2008-01-01

Objective To examine the feasibility of using blood-derived smooth muscle cells (BD-SMCs) as a target for to deliver therapeutic proteins. Materials and Methods Mononuclear cells (MNC) were isolated from peripheral blood. The outgrowth colonies from MNC culture were differentiated into BD-SMCs in media containing platelet-derived growth factor BB. Phenotypic characterization of BD-SMCs was assessed by immunocytochemistry. Cell proliferation, gene transfer efficiency with a retroviral vector, apoptosis, and the biological activity of the transduced gene product from the BD-SMCs were evaluated in vitro and in vivo in comparison with vascular derived SMC (VSMCs). Results BD-SMCs stained positive for SMC markers. No significant difference was observed between BD-SMCs and VSMCs in cell proliferation, migration, adhesiveness, and gene transfer efficiency. After BD-SMCs were transduced with a retroviral vector carrying the secreted alkaline phosphatase gene (SEAP), 174 ± 50 μg biologically active SEAP was produced per 106 cells over 24 hrs. After injecting 5×106 cells expressing SEAP intravenously into rabbits, SEAP concentration increased significantly in the circulation from 0.14 ± 0.04 μg/ml to 2.34 ± 0.16 μg/ml 3 days after cell injection (P<0.01, n=3). Circulating levels of SEAP decreased to 1.76 μg /ml one week later and remained at this level up to 8 weeks, then declined to pre-cell injection level at 12 weeks. VSMC in vivo gene expression data were equivalent. Conclusion BD-SMCs have similar characteristics to mature VSMCs, and can be used as a novel target for gene transfer to deliver a therapeutic protein. Clinical relevance Cell-based therapy strategies offer the potential to correct a wide spectrum of inherited and acquired human diseases. Translation to a clinical trial will require a detailed pre-clinical study to understand the characteristics of the isolated cells. BD-SMC are practical and effective targets for ex vivo genetic engineering. They are obtained with ease by phlebotomy, eliminating the need for surgical tissue explantation. This study tested the suitability of BD-SMC in vivo as a target for gene therapy. The outcome of the study has direct application in progenitor cell-based therapy. PMID:18241767

Electroporation in veterinary oncology.

PubMed

Impellizeri, J; Aurisicchio, L; Forde, P; Soden, D M

2016-11-01

Cancer treatments in veterinary medicine continue to evolve beyond the established standard therapies of surgery, chemotherapy and radiation therapy. New technologies in cancer therapy include a targeted mechanism to open the cell membrane based on electroporation, driving therapeutic agents, such as chemotherapy (electro-chemotherapy), for local control of cancer, or delivery of gene-based products (electro-gene therapy), directly into the cancer cell to achieve systemic control. This review examines electrochemotherapy and electro-gene therapy in veterinary medicine and considers future directions and applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

The single IGF-1 partial deficiency is responsible for mitochondrial dysfunction and is restored by IGF-1 replacement therapy.

PubMed

Olleros Santos-Ruiz, M; Sádaba, M C; Martín-Estal, I; Muñoz, U; Sebal Neira, C; Castilla-Cortázar, I

2017-08-01

We previously described in cirrhosis and aging, both conditions of IGF-1 deficiency, a clear hepatic mitochondrial dysfunction with increased oxidative damage. In both conditions, the hepatic mitochondrial function was improved with low doses of IGF-1. The aim of this work was to explore if the only mere IGF-1 partial deficiency, without any exogenous insult, is responsible for hepatic mitochondrial dysfunction. Heterozygous (igf1 +/- ) mice were divided into two groups: untreated and treated mice with low doses of IGF-1. WT group was used as controls. Parameters of hepatic mitochondrial function were determined by flow cytometry, antioxidant enzyme activities were determined by spectrophotometry, and electron chain transport enzyme levels were determined by immunohistochemistry and immunofluorescence analyses. Liver expression of genes coding for proteins involved in mitochondrial protection and apoptosis was studied by microarray analysis and RT-qPCR. Hz mice showed a significant reduction in hepatic mitochondrial membrane potential (MMP) and ATPase activity, and an increase in intramitochondrial free radical production and proton leak rates, compared to controls. These parameters were normalized by IGF-1 replacement therapy. No significant differences were found between groups in oxygen consumption and antioxidant enzyme activities, except for catalase, whose activity was increased in both Hz groups. Relevant genes coding for proteins involved in mitochondrial protection and survival were altered in Hz group and were reverted to normal in Hz+IGF-1 group. The mere IGF-1 partial deficiency is per se associated with hepatic mitochondrial dysfunction sensitive to IGF-1 replacement therapy. Results in this work prove that IGF-1 is involved in hepatic mitochondrial protection, because it is able to reduce free radical production, oxidative damage and apoptosis. All these IGF-1 actions are mediated by the modulation of the expression of genes encoding citoprotective and antiapoptotic proteins. Copyright © 2017. Published by Elsevier Ltd.