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Sample records for all-in-one lentiviral vector

  1. Tightly regulated 'all-in-one' lentiviral vectors for protection of human hematopoietic cells from anticancer chemotherapy.

    PubMed

    Lachmann, N; Brennig, S; Hillje, R; Schermeier, H; Phaltane, R; Dahlmann, J; Gruh, I; Heinz, N; Schiedlmeier, B; Baum, C; Moritz, T

    2015-11-01

    Successful application of gene therapy strategies may require stringently regulated transgene expression. Along this line, we describe a doxycycline (Dox)-inducible 'all-in-one' lentiviral vector design using the pTET-T11 (TII) minimal-promoter and a reverse transactivator protein (rtTA2S-M2) driven by the phosphoglycerate kinase promoter allowing for tight regulation of transgene expression (Lv.TII vectors). Vector design was evaluated in human hematopoietic cells in the context of cytidine deaminase (hCDD)-based myeloprotective gene therapy. Upon Dox administration, a rapid (16-24 h) and dose-dependent (>0.04 μg ml(-1) Dox) onset of transgene expression was detected in Lv.TII.CDD gene-modified K562 cells as well as in primary human CD34(+) hematopoietic cells. Importantly, in both cell models low background transgene expression was observed in the absence of Dox. Functionality of Dox-inducible hCDD expression was demonstrated by >10-fold increase in cytosine arabinoside (1-β-d-arabinofuranosylcytosine, Ara-C) resistance of Lv.TII.CDD-transduced K562 cells. In addition, Lv.TII.CDD-transduced CD34(+)-derived myeloid cells were protected from up to 300 nm Ara-C (control affected from 50 nm onwards). These data clearly demonstrate the suitability of our self-inactivating lentiviral vector to induce robust, tightly regulated transgene expression in human hematopoietic cells with minimal background activity and highlight the potential of our construct in myeloprotective gene therapy strategies.

  2. Lentiviral vectors.

    PubMed

    Giry-Laterrière, Marc; Verhoeyen, Els; Salmon, Patrick

    2011-01-01

    Lentiviral vectors have evolved over the last decade as powerful, reliable, and safe tools for stable gene transfer in a wide variety of mammalian cells. Contrary to other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells. In particular, lentivectors (LVs) derived from HIV-1 have gradually evolved to display many desirable features aimed at increasing both their safety and their versatility. This is why lentiviral vectors are becoming the most useful and promising tools for genetic engineering, to generate cells that can be used for research, diagnosis, and therapy. This chapter describes protocols and guidelines, for production and titration of LVs, which can be implemented in a research laboratory setting, with an emphasis on standardization in order to improve transposability of results between laboratories. We also discuss latest designs in LV technology.

  3. Production of lentiviral vectors

    PubMed Central

    Merten, Otto-Wilhelm; Hebben, Matthias; Bovolenta, Chiara

    2016-01-01

    Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented. PMID:27110581

  4. Lentiviral vectors in cancer immunotherapy.

    PubMed

    Oldham, Robyn Aa; Berinstein, Elliot M; Medin, Jeffrey A

    2015-01-01

    Basic science advances in cancer immunotherapy have resulted in various treatments that have recently shown success in the clinic. Many of these therapies require the insertion of genes into cells to directly kill them or to redirect the host's cells to induce potent immune responses. Other analogous therapies work by modifying effector cells for improved targeting and enhanced killing of tumor cells. Initial studies done using γ-retroviruses were promising, but safety concerns centered on the potential for insertional mutagenesis have highlighted the desire to develop other options for gene delivery. Lentiviral vectors (LVs) have been identified as potentially more effective and safer alternative delivery vehicles. LVs are now in use in clinical trials for many different types of inherited and acquired disorders, including cancer. This review will discuss current knowledge of LVs and the applications of this viral vector-based delivery vehicle to cancer immunotherapy.

  5. Advances in lentiviral vectors: a patent review.

    PubMed

    Picanco-Castro, Virginia; de Sousa Russo-Carbolante, Elisa Maria; Tadeu Covas, Dimas

    2012-08-01

    Lentiviral vectors are at the forefront of gene delivery systems for research and clinical applications. These vectors have the ability to efficiently transduce nondividing and dividing cells, to insert large genetic segment in the host chromatin, and to sustain stable long-term transgene expression. Most of lentiviral vectors systems in use are derived from HIV-1. Numerous modifications in the basic HIV structure have been made to ensure safety and to promote efficiency to vectors. Lentiviral vectors can be pseudotyped with distinct viral envelopes that influence vector tropism and transduction efficiency. Moreover, these vectors can be used to reprogram cells and generate induced pluripotent stem cells. This review aims to show the patents that resulted in improved safety and efficacy of lentiviral vector with important implications for clinical trials.

  6. Targeting lentiviral vectors for cancer immunotherapy

    PubMed Central

    Arce, Frederick; Breckpot, Karine; Collins, Mary; Escors, David

    2012-01-01

    Delivery of tumour-associated antigens (TAA) in a way that induces effective, specific immunity is a challenge in anti-cancer vaccine design. Circumventing tumour-induced tolerogenic mechanisms in vivo is also critical for effective immunotherapy. Effective immune responses are induced by professional antigen presenting cells, in particular dendritic cells (DC). This requires presentation of the antigen to both CD4+ and CD8+ T cells in the context of strong co-stimulatory signals. Lentiviral vectors have been tested as vehicles, for both ex vivo and in vivo delivery of TAA and/or activation signals to DC, and have been demonstrated to induce potent T cell mediated immune responses that can control tumour growth. This review will focus on the use of lentiviral vectors for in vivo gene delivery to DC, introducing strategies to target DC, either targeting cell entry or gene expression to improve safety of the lentiviral vaccine or targeting dendritic cell activation pathways to enhance performance of the lentiviral vaccine. In conclusion, this review highlights the potential of lentiviral vectors as a generally applicable ‘off-the-shelf’ anti-cancer immunotherapeutic. PMID:22983382

  7. Lentiviral Vector Gene Transfer to Porcine Airways

    PubMed Central

    Sinn, Patrick L; Cooney, Ashley L; Oakland, Mayumi; Dylla, Douglas E; Wallen, Tanner J; Pezzulo, Alejandro A; Chang, Eugene H; McCray, Paul B

    2012-01-01

    In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF). PMID:23187455

  8. The Inside Out of Lentiviral Vectors

    PubMed Central

    Durand, Stéphanie; Cimarelli, Andrea

    2011-01-01

    Lentiviruses induce a wide variety of pathologies in different animal species. A common feature of the replicative cycle of these viruses is their ability to target non-dividing cells, a property that constitutes an extremely attractive asset in gene therapy. In this review, we shall describe the main basic aspects of the virology of lentiviruses that were exploited to obtain efficient gene transfer vectors. In addition, we shall discuss some of the hurdles that oppose the efficient genetic modification mediated by lentiviral vectors and the strategies that are being developed to circumvent them. PMID:22049307

  9. Production and titration of lentiviral vectors.

    PubMed

    Salmon, Patrick; Trono, Didier

    2006-11-01

    Lentiviral vectors have emerged over the last decade as powerful, reliable and safe tools for stable gene transfer in a wide variety of mammalian cells. Unlike other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells, including neurons. This is why LVs are becoming the most useful and promising tools in the field of neuroscience, not only for research, but also for future gene and cell therapy approaches. Lentivectors (LVs) derived from HIV-1 have gradually evolved to display many desirable features aimed at increasing both their safety and their versatility. These latest designs are reviewed in this unit. This unit also describes protocols for production and titration of LVs that can be implemented in a research laboratory setting, with an emphasis on standardization to improve transposability of results between laboratories.

  10. Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases

    PubMed Central

    Hu, Biliang; Tai, April; Wang, Pin

    2011-01-01

    Summary The increasing level of understanding of the lentivirus biology has been instrumental in shaping the design strategy of creating therapeutic lentiviral delivery vectors. As a result, lentiviral vectors have become one of the most powerful gene transfer vehicles. They are widely used for therapeutic purposes as well as in studies of basic biology, due to their unique characteristics. Lentiviral vectors have been successfully employed to mediate durable and efficient antigen expression and presentation in dendritic cells both in vitro and in vivo, leading to the activation of cellular immunity and humoral responses. This capability makes the lentiviral vector an ideal choice for immunizations that target a wide range of cancers and infectious diseases. Further advances into optimizing the vector system and understanding the relationship between the immune system and diseases pathogenesis will only augment the potential benefits and utility of lentiviral vaccines for human health. PMID:21198664

  11. Lentiviral Vectors and Cystic Fibrosis Gene Therapy

    PubMed Central

    Castellani, Stefano; Conese, Massimo

    2010-01-01

    Cystic fibrosis (CF) is a chronic autosomic recessive syndrome, caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, a chloride channel expressed on the apical side of the airway epithelial cells. The lack of CFTR activity brings a dysregulated exchange of ions and water through the airway epithelium, one of the main aspects of CF lung disease pathophysiology. Lentiviral (LV) vectors, of the Retroviridae family, show interesting properties for CF gene therapy, since they integrate into the host genome and allow long-lasting gene expression. Proof-of-principle that LV vectors can transduce the airway epithelium and correct the basic electrophysiological defect in CF mice has been given. Initial data also demonstrate that LV vectors can be repeatedly administered to the lung and do not give rise to a gross inflammatory process, although they can elicit a T cell-mediated response to the transgene. Future studies will clarify the efficacy and safety profile of LV vectors in new complex animal models with CF, such as ferrets and pigs. PMID:21994643

  12. Titration of feline immunodeficiency virus-based lentiviral vector preparations.

    PubMed

    Saenz, Dyana T; Barraza, Román; Loewen, Nils; Teo, Wulin; Poeschla, Eric M

    2012-01-01

    Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes methods for measuring and calculating vector titers in transducing units (TU)/mL. Alternate methods are provided for green fluorescent protein (GFP) vectors and for β-galactosidase vectors.

  13. [Transfection of HL-60 cells by Venus lentiviral vector].

    PubMed

    Li, Zheng; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2013-06-01

    In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

  14. Lentiviral vector production, titration, and transduction of primary neurons.

    PubMed

    Ding, Baojin; Kilpatrick, Daniel L

    2013-01-01

    Lentiviral vectors have become very useful tools for transgene delivery. Based on their ability to transduce both dividing and nondividing cells and to produce long-term transgene expression, lentiviruses have found numerous applications in the biomedical sciences, including developmental neuroscience. This protocol describes how to prepare lentiviral vectors by calcium phosphate transfection and to concentrate viral particles by ultracentrifugation. Functional vector titers can then be determined by methods such as fluorescence-activated cell sorting or immunostaining. Effective titers in the range of 10(8)-10(9) infectious units/ml can be routinely obtained using these protocols. Finally, we describe the infection of primary neuronal cultures with lentiviral vectors resulting in 85-90 % cell transduction using appropriate multiplicities of infection.

  15. Genetic engineering of human embryonic stem cells with lentiviral vectors.

    PubMed

    Xiong, Chen; Tang, Dong-Qi; Xie, Chang-Qing; Zhang, Li; Xu, Ke-Feng; Thompson, Winston E; Chou, Wayne; Gibbons, Gary H; Chang, Lung-Ji; Yang, Li-Jun; Chen, Yuqing E

    2005-08-01

    Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

  16. Factors influencing the titer and infectivity of lentiviral vectors.

    PubMed

    Logan, Aaron C; Nightingale, Sarah J; Haas, Dennis L; Cho, Gerald J; Pepper, Karen A; Kohn, Donald B

    2004-10-01

    Lentiviral vectors have undergone several generations of design improvement to enhance their biosafety and expression characteristics, and have been approved for use in human clinical studies. Most preclinical studies with these vectors have employed easily assayed marker genes for the purpose of determining vector titers and transduction efficiencies. Naturally, the adaptation of these vector systems to clinical use will increasingly involve the transfer of genes whose products may not be easily measured, meaning that the determination of vector titer will be more complicated. One method for determining vector titer that can be universally employed on all human immunodeficiency virus type 1-based lentiviral vector supernatants involves the measurement of Gag (p24) protein concentration in vector supernatants by immunoassay. We have studied the effects that manipulation of several variables involved in vector design and production by transient transfection have on vector titer and infectivity. We have determined that manipulation of the amount of transfer vector, packaging, and envelope plasmids used to transfect the packaging cells does not alter vector infectivity, but does influence vector titer. We also found that modifications to the transfer vector construct, such as replacing the internal promoter or transgene, do not generally alter vector infectivity, whereas inclusion of the central polypurine tract in the transfer vector increases vector infectivity on HEK293 cells and human umbilical cord blood CD34+ hematopoietic progenitor cells (HPCs). The infectivities of vector supernatants can also be increased by harvesting at early time points after the initiation of vector production, collection in serum-free medium, and concentration by ultracentrifugation. For the transduction of CD34+ HPCs, we found that the simplest method of increasing vector infectivity is to pseudotype vector particles with the RD114 envelope instead of vesicular stomatitis virus G

  17. Biosafety challenges for use of lentiviral vectors in gene therapy.

    PubMed

    Rothe, Michael; Modlich, Ute; Schambach, Axel

    2013-12-01

    Lentiviral vectors are promising tools for the genetic modification of cells in biomedical research and gene therapy. Their use in recent clinical trials for the treatment of adrenoleukodystrophy, β-thalassemia, Wiskott-Aldrich- Syndrome and metachromatic leukodystrophy underlined their efficacy for therapies especially in case of hereditary diseases. In comparison to gammaretroviral LTR-driven vectors, which were employed in the first clinical trials, lentiviral vectors present with some favorable features like the ability to transduce also non-dividing cells and a potentially safer insertion profile. However, genetic modification with viral vectors in general and stable integration of the therapeutic gene into the host cell genome bear concerns with respect to different levels of personal or environmental safety. Among them, insertional mutagenesis by enhancer mediated dysregulation of neighboring genes or aberrant splicing is still the biggest concern. However, also risks like immunogenicity of vector particles, the phenotoxicity of the transgene and potential vertical or horizontal transmission by replication competent retroviruses need to be taken into account. This review will give an overview on biosafety aspects that are relevant to the use of lentiviral vectors for genetic modification and gene therapy. Furthermore, assay systems aiming at evaluating biosafety in preclinical settings and recent promising clinical trials including efforts of monitoring of patients after gene therapy will be discussed.

  18. Using Pulmozyme DNase treatment in lentiviral vector production.

    PubMed

    Shaw, Aaron; Bischof, Daniela; Jasti, Aparna; Ernstberger, Aaron; Hawkins, Troy; Cornetta, Kenneth

    2012-02-01

    In the production of lentiviral vector for clinical studies the purity of the final product is of vital importance. To remove plasmid and producer cell line DNA, investigators have incubated the vector product with Benzonase, a bacterially derived DNase. As an alternative we investigated the use of Pulmozyme, a U.S. Food and Drug Administration-approved human DNase for the treatment of cystic fibrosis, by comparing the efficiency of DNA removal from lentiviral vector preparations. A green fluorescent protein-expressing lentiviral vector was prepared by transient calcium phosphate transfection of HEK 293T cells and DNA removal was compared when treating vector after harvest or immediately after transfection. The effectiveness of DNase treatment was measured by quantitative PCR using primers for vesicular stomatitis virus glycoprotein G viral envelope plasmid. When treating the final product, 1-hr incubations (37°C) with Pulmozyme at 20 U/ml reduced plasmid DNA to undetectable levels. Longer incubations (up to 4 hr) did not improve DNA removal at lower concentrations and the effectiveness was equivalent to or better than Benzonase at 50 U/ml. Attempting to use Pulmozyme immediately after transfection, but before final medium change, as a means to decrease Pulmozyme concentration in the final product provided a 2-log reduction in DNA but was inferior to treatment at the end of production. Pulmozyme, at concentrations up to 100 U/ml, had no measurable effect on infectious titer of the final vector product. The use of Pulmozyme is likely to increase the cost of DNase treatment when preparing vector product and should be considered when generating clinical-grade vector products. PMID:22428981

  19. The feasibility of incorporating Vpx into lentiviral gene therapy vectors

    PubMed Central

    McAllery, Samantha A; Ahlenstiel, Chantelle L; Suzuki, Kazuo; Symonds, Geoff P; Kelleher, Anthony D; Turville, Stuart G

    2016-01-01

    While current antiretroviral therapy has significantly improved, challenges still remain in life-long targeting of HIV-1 reservoirs. Lentiviral gene therapy has the potential to deliver protective genes into the HIV-1 reservoir. However, inefficient reverse transcription (RT) occurs in HIV-1 reservoirs during lentiviral gene delivery. The viral protein Vpx is capable of increasing lentiviral RT by antagonizing the restriction factor SAMHD1. Incorporating Vpx into lentiviral vectors could substantially increase gene delivery into the HIV-1 reservoir. The feasibility of this Vpx approach was tested in resting cell models utilizing macrophages and dendritic cells. Our results showed Vpx exposure led to increased permissiveness of cells over a period that exceeded 2 weeks. Consequently, significant lower potency of HIV-1 antiretrovirals inhibiting RT and integration was observed. When Vpx was incorporated with anti-HIV-1 genes inhibiting either pre-RT or post-RT stages of the viral life-cycle, transduction levels significantly increased. However, a stronger antiviral effect was only observed with constructs that inhibit pre-RT stages of the viral life cycle. In conclusion this study demonstrates a way to overcome the major delivery obstacle of gene delivery into HIV-1 reservoir cell types. Importantly, incorporating Vpx with pre-RT anti-HIV-1 genes, demonstrated the greatest protection against HIV-1 infection. PMID:27790625

  20. Integration-deficient Lentiviral Vectors: A Slow Coming of Age

    PubMed Central

    Wanisch, Klaus; Yáñez-Muñoz, Rafael J

    2009-01-01

    Lentiviral vectors are very efficient at transducing dividing and quiescent cells, which makes them highly useful tools for genetic analysis and gene therapy. Traditionally this efficiency was considered dependent on provirus integration in the host cell genome; however, recent results have challenged this view. So called integration-deficient lentiviral vectors (IDLVs) can be produced through the use of integrase mutations that specifically prevent proviral integration, resulting in the generation of increased levels of circular vector episomes in transduced cells. These lentiviral episomes lack replication signals and are gradually lost by dilution in dividing cells, but are stable in quiescent cells. Compared to integrating lentivectors, IDLVs have a greatly reduced risk of causing insertional mutagenesis and a lower risk of generating replication-competent recombinants (RCRs). IDLVs can mediate transient gene expression in proliferating cells, stable expression in nondividing cells in vitro and in vivo, specific immune responses, RNA interference, homologous recombination (gene repair, knock-in, and knock-out), site-specific recombination, and transposition. IDLVs can be converted into replicating episomes, suggesting that if a clinically applicable system can be developed they would also become highly appropriate for stable transduction of proliferating tissues in therapeutic applications. PMID:19491821

  1. Visualization of targeted transduction by engineered lentiviral vectors.

    PubMed

    Joo, K-I; Wang, P

    2008-10-01

    We have reported a method to target lentiviral vectors to specific cell types. This method requires the incorporation of two distinct molecules on the viral vector surface: one is an antibody that renders the targeting specificity for the engineered vector, and the other is a fusogenic protein that allows the engineered vector to enter the target cell. However, the molecular mechanism that controls the targeted infection needs to be defined. In this report, we tracked the individual lentiviral particles by labeling the virus with the GFP-Vpr fusion protein. We were able to visualize the surface-displayed proteins on a single virion as well as antibody-directed targeting to a desired cell type. We also demonstrated the dynamics of virus fusion with endosomes and monitored endosome-associated transport of viruses in target cells. Our results suggest that the fusion between the engineered lentivirus and endosomes takes place at the early endosome level, and that the release of the viral core into the cytosol at the completion of the virus-endosome fusion is correlated with the endosome maturation process. This imaging study sheds some light on the infection mechanism of the engineered lentivirus and can be beneficial to the design of more efficient gene delivery vectors.

  2. Lentiviral vectors for cancer immunotherapy and clinical applications.

    PubMed

    Liechtenstein, Therese; Perez-Janices, Noemi; Escors, David

    2013-09-01

    The success of immunotherapy against infectious diseases has shown us the powerful potential that such a treatment offers, and substantial work has been done to apply this strategy in the fight against cancer. Cancer is however a fiercer opponent than pathogen-caused diseases due to natural tolerance towards tumour associated antigens and tumour-induced immunosuppression. Recent gene therapy clinical trials with viral vectors have shown clinical efficacy in the correction of genetic diseases, HIV and cancer. The first successful gene therapy clinical trials were carried out with onco(γ-)retroviral vectors but oncogenesis by insertional mutagenesis appeared as a serious complication. Lentiviral vectors have emerged as a potentially safer strategy, and recently the first clinical trial of patients with advanced leukemia using lentiviral vectors has proven successful. Additionally, therapeutic lentivectors have shown clinical efficacy for the treatment of HIV, X-linked adrenoleukodystrophy, and β-thalassaemia. This review aims at describing lentivectors and how they can be utilized to boost anti-tumour immune responses by manipulating the effector immune cells. PMID:24078865

  3. New methods to titrate EIAV-based lentiviral vectors.

    PubMed

    Martin-Rendon, Enca; White, Linda J; Olsen, Anna; Mitrophanous, Kyriacos A; Mazarakis, Nicholas D

    2002-05-01

    Ideally, gene transfer vectors used in clinical protocols should only express the gene of interest. So far most vectors have contained marker genes to aid their titration. We have used quantitative real-time PCR to titrate equine infectious anemia virus (EIAV) vectors for gene therapy applications. Viral RNA was isolated from vector preparations and analyzed in a one-step RT-PCR reaction in which reverse transcription and amplification were combined in one tube. The PCR assay of vector stocks was quantitative and linear over four orders of magnitude. In tandem, the integration efficiency of these vectors has also been determined by real-time PCR, measuring the number of vector genomes in the target cells. We have found that these methods permit reliable and sensitive titration of lentiviral vectors independent from the expression of a transgene. They also allow us to determine the integration efficiency of different vector genomes. This technology has proved very useful, especially in the absence of marker genes and where vectors express multiple genes.

  4. Lentiviral vector-mediated transduction of goat undifferentiated spermatogonia.

    PubMed

    Abbasi, Hassan; Hosseini, Sayyed Morteza; Hajian, Mahdi; Nasiri, Zahra; Bahadorani, Mehrnoosh; Tahmoorespur, Mojtaba; Nasiri, Mohammad Reza; Nasr-Esfahani, Mohammad Hossein

    2015-12-01

    Recent studies show that spermatogonial stem cells (SSCs) are able to colonize and form mature spermatozoa following transplantation into germ cell depleted testes of recipient males. Therefore, efficient ways for enrichment and gene transfer into SSCs provides a powerful tool for production of transgenic animals. In order to adapt the technique to goats, three issues were addressed: (i) enrichment of the undifferentiated spermatogonia including SSCs using magnetic activated cell sorting (MACS), (ii) lentiviral vector-mediated transduction of an enhanced green fluorescent protein (EGFP) transgene into enriched cells, and (iii) transplantation of transduced undifferentiated spermatogonia into the germ cell depleted testes of immune-suppressed mice to assess for migration and colony formation ability. Enriched cells were transduced by lentiviral vectors and subsequently analyzed for expression of THY1, PLZF, VASA, UCHL1 and BCL6B genes. Cells were also analyzed for GFP and PLZF by flow cytometry. Enriched transduced cells were transplanted into germ cell depleted mice testis. Quantitative analysis of transcripts revealed that MACS-enrichment significantly increased the expression of SSC-characteristic genes THY1, PLZF, VASA, UCHL1 and BCL6B compared to non-enriched population (P≤0.05). EGFP transduction did not affect the expression levels of SSC-characteristic genes. Flow cytometry revealed that 72% of transduced-enriched cells were positive for EGFP. Finally, transduced-enriched goat SSCs could colonize within the cells into the seminiferous tubules of germ cell depleted recipient mice at higher frequency than non-enriched cells. The results indicated that enrichment of goat undifferentiated spermatogonia by magnetic-activated cell sorting for THY1 antibody combined with lentiviral vector-mediated transduction has the potential to be used for production of transgenic goats.

  5. Development of Lentiviral Vectors for Targeted Integration and Protein Delivery.

    PubMed

    Schenkwein, Diana; Ylä-Herttuala, Seppo

    2016-01-01

    The method in this chapter describes the design of human immunodeficiency virus type 1 (HIV-1) integrase (IN)-fusion proteins which we have developed to transport different proteins into the nuclei of lentiviral vector (LV)-transduced cells. The IN-fusion protein cDNA is incorporated into the LV packaging plasmid, which leads to its incorporation into vector particles as part of a large Gag-Pol polyprotein. This specific feature of protein packaging enables also the incorporation of cytotoxic and proapoptotic proteins, such as frequently cutting endonucleases and P53. The vectors can hence be used for various protein transduction needs. An outline of the necessary methods is also given to study the functionality of a chosen IN-fusion protein in a cell culture assay. PMID:27317182

  6. Lentiviral Vector-Mediated RNA Silencing in the Central Nervous System

    PubMed Central

    Foster, Edmund; Moon, Lawrence D.F.

    2014-01-01

    Abstract RNA silencing is an established method for investigating gene function and has attracted particular interest because of the potential for generating RNA-based therapeutics. Using lentiviral vectors as an efficient delivery system that offers stable, long-term expression in postmitotic cells further enhances the applicability of an RNA-based gene therapy for the CNS. In this review we provide an overview of both lentiviral vectors and RNA silencing along with design considerations for generating lentiviral vectors capable of RNA silencing. We go on to describe the current preclinical data regarding lentiviral vector-mediated RNA silencing for CNS disorders and discuss the concerns of side effects associated with lentiviral vectors and small interfering RNAs and how these might be mitigated. PMID:24090197

  7. Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity.

    PubMed

    Gay, Virginie; Moreau, Karen; Hong, Saw-See; Ronfort, Corinne

    2012-02-01

    A human immunodeficiency virus type (HIV-1)-based lentiviral vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein and encoding the GFP reporter gene was used to evaluate different methods of lentiviral vector titration. GFP expression, viral DNA quantification and the efficiency of vector DNA integration were assayed after infection of conventional HIV-1-permissive cell lines and human primary adult fibroblasts with the vector. We found that vector titers based on GFP expression determined by flow cytometry may vary by more than 50-fold depending on the cell type and the promoter-cell combination used. Interestingly, we observed that the viral integration process in primary HDFa cells was significantly more efficient compared to that in SupT1 or 293T cells. We propose that determination of the amount of integrated viral DNA by quantitative PCR be used in combination with the reporter gene expression assay.

  8. Real-time quantitative PCR for the design of lentiviral vector analytical assays.

    PubMed

    Delenda, C; Gaillard, C

    2005-10-01

    From the recent and emerging concerns for approving lentiviral vector-mediated gene transfer in human clinical applications, several analytical methods have been applied in preclinical models to address the lentiviral vector load in batches, cells or tissues. This review points out the oldest generation methods (blots, RT activity, standard PCR) as well as a full description of the newest real-time quantitative PCR (qPCR) applications. Combinations of primer and probe sequences, which have worked in the lentiviral amplification context, have been included in the effort to dress an exhaustive list. Also, great variations have been observed from interlaboratory results, we have tempted to compare between them the different analytical methods that have been used to consider (i) the titration of lentiviral vector batches, (ii) the absence of the susceptible emerging replicative lentiviruses or (iii) the lentiviral vector biodistribution in the organism.

  9. A Large U3 Deletion Causes Increased In Vivo Expression from a Nonintegrating Lentiviral Vector

    PubMed Central

    Bayer, Matthew; Kantor, Boris; Cockrell, Adam; Ma, Hong; Zeithaml, Brian; Li, Xiangping; McCown, Thomas; Kafri, Tal

    2008-01-01

    The feasibility of employing nonintegrating lentiviral vectors has been demonstrated by recent studies showing the ability of nonintegrating lentiviral vectors to maintain transgene expression in vitro and in vivo. Furthermore, HIV-1 vectors packaged with a mutated integrase were able to correct retinal disease in a mouse model. Interestingly, these results differ from earlier studies in which first-generation nonintegrating lentiviral vectors yielded insignificant levels of transduction. However, to date a rigorous characterization of transgene expression from the currently used self-inactivating (SIN) nonintegrating lentiviral vectors has not been published. Here we characterize transgene expression from SIN nonintegrating lentiviral vectors. Overall, we found that nonintegrating vectors express transgenes at a significantly lower level than their integrating counterparts. Expression from nonintegrating vectors was improved upon introducing a longer deletion in the vector’s U3 region. A unique shuttle-vector assay indicated that the relative abundance of the different episomal forms was not altered by the longer U3 deletion. Interestingly, the longer U3 deletion did not enhance expression in the corpus callosum of the rat brain, suggesting that the extent of silencing of episomal transcription is influenced by tissue-specific factors. Finally, and for the first time, episomal expression in the mouse liver was potent and sustained. PMID:18797449

  10. Optimization of the transductional efficiency of lentiviral vectors: effect of sera and polycations.

    PubMed

    Denning, Warren; Das, Suvendu; Guo, Siqi; Xu, Jun; Kappes, John C; Hel, Zdenek

    2013-03-01

    Lentiviral vectors are widely used as effective gene-delivery vehicles. Optimization of the conditions for efficient lentiviral transduction is of a high importance for a variety of research applications. Presence of positively charged polycations reduces the electrostatic repulsion forces between a negatively charged cell and an approaching enveloped lentiviral particle resulting in an increase in the transduction efficiency. Although a variety of polycations are commonly used to enhance the transduction with retroviruses, the relative effect of various types of polycations on the efficiency of transduction and on the potential bias in the determination of titer of lentiviral vectors is not fully understood. Here, we present data suggesting that DEAE-dextran provides superior results in enhancing lentiviral transduction of most tested cell lines and primary cell cultures. Specific type and source of serum affects the efficiency of transduction of target cell populations. Non-specific binding of enhanced green fluorescent protein (EGFP)-containing membrane aggregates in the presence of DEAE-dextran does not significantly affect the determination of the titer of EGFP-expressing lentiviral vectors. In conclusion, various polycations and types of sera should be tested when optimizing lentiviral transduction of target cell populations.

  11. Packaging of HCV-RNA into lentiviral vector

    SciTech Connect

    Caval, Vincent; Piver, Eric; Ivanyi-Nagy, Roland; Darlix, Jean-Luc; Pages, Jean-Christophe

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Description of HCV-RNA Core-D1 interactions. Black-Right-Pointing-Pointer In vivo evaluation of the packaging of HCV genome. Black-Right-Pointing-Pointer Determination of the role of the three basic sub-domains of D1. Black-Right-Pointing-Pointer Heterologous system involving HIV-1 vector particles to mobilise HCV genome. Black-Right-Pointing-Pointer Full length mobilisation of HCV genome and HCV-receptor-independent entry. -- Abstract: The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. Molecular interactions of the domain 1 (D1) of HCV Core protein and HCV RNA have been described in vitro. Since compaction of genetic information within HCV genome has hampered conventional mutational approach to study packaging in vivo, we developed a novel heterologous system to evaluate the interactions between HCV RNA and Core D1. For this, we took advantage of the recruitment of Vpr fusion-proteins into HIV-1 particles. By fusing HCV Core D1 to Vpr we were able to package and transfer a HCV subgenomic replicon into a HIV-1 based lentiviral vector. We next examined how deletion mutants of basic sub-domains of Core D1 influenced HCV RNA recruitment. The results emphasized the crucial role of the first and third basic regions of D1 in packaging. Interestingly, the system described here allowed us to mobilise full-length JFH1 genome in CD81 defective cells, which are normally refractory to HCV infection. This finding paves the way to an evaluation of the replication capability of HCV in various cell types.

  12. A comparison of foamy and lentiviral vector genotoxicity in SCID-repopulating cells shows foamy vectors are less prone to clonal dominance

    PubMed Central

    Everson, Elizabeth M; Olzsko, Miles E; Leap, David J; Hocum, Jonah D; Trobridge, Grant D

    2016-01-01

    Hematopoietic stem cell (HSC) gene therapy using retroviral vectors has immense potential, but vector-mediated genotoxicity limits use in the clinic. Lentiviral vectors are less genotoxic than gammaretroviral vectors and have become the vector of choice in clinical trials. Foamy retroviral vectors have a promising integration profile and are less prone to read-through transcription than gammaretroviral or lentiviral vectors. Here, we directly compared the safety and efficacy of foamy vectors to lentiviral vectors in human CD34+ repopulating cells in immunodeficient mice. To increase their genotoxic potential, foamy and lentiviral vectors with identical transgene cassettes with a known genotoxic spleen focus forming virus promoter were used. Both vectors resulted in efficient marking in vivo and a total of 825 foamy and 460 lentiviral vector unique integration sites were recovered in repopulating cells 19 weeks after transplantation. Foamy vector proviruses were observed less often near RefSeq gene and proto-oncogene transcription start sites than lentiviral vectors. The foamy vector group were also more polyclonal with fewer dominant clones (two out of six mice) than the lentiviral vector group (eight out of eight mice), and only lentiviral vectors had integrants near known proto-oncogenes in dominant clones. Our data further support the relative safety of foamy vectors for HSC gene therapy. PMID:27579335

  13. A comparison of foamy and lentiviral vector genotoxicity in SCID-repopulating cells shows foamy vectors are less prone to clonal dominance.

    PubMed

    Everson, Elizabeth M; Olzsko, Miles E; Leap, David J; Hocum, Jonah D; Trobridge, Grant D

    2016-01-01

    Hematopoietic stem cell (HSC) gene therapy using retroviral vectors has immense potential, but vector-mediated genotoxicity limits use in the clinic. Lentiviral vectors are less genotoxic than gammaretroviral vectors and have become the vector of choice in clinical trials. Foamy retroviral vectors have a promising integration profile and are less prone to read-through transcription than gammaretroviral or lentiviral vectors. Here, we directly compared the safety and efficacy of foamy vectors to lentiviral vectors in human CD34(+) repopulating cells in immunodeficient mice. To increase their genotoxic potential, foamy and lentiviral vectors with identical transgene cassettes with a known genotoxic spleen focus forming virus promoter were used. Both vectors resulted in efficient marking in vivo and a total of 825 foamy and 460 lentiviral vector unique integration sites were recovered in repopulating cells 19 weeks after transplantation. Foamy vector proviruses were observed less often near RefSeq gene and proto-oncogene transcription start sites than lentiviral vectors. The foamy vector group were also more polyclonal with fewer dominant clones (two out of six mice) than the lentiviral vector group (eight out of eight mice), and only lentiviral vectors had integrants near known proto-oncogenes in dominant clones. Our data further support the relative safety of foamy vectors for HSC gene therapy. PMID:27579335

  14. Large-scale production of lentiviral vector in a closed system hollow fiber bioreactor

    PubMed Central

    Sheu, Jonathan; Beltzer, Jim; Fury, Brian; Wilczek, Katarzyna; Tobin, Steve; Falconer, Danny; Nolta, Jan; Bauer, Gerhard

    2015-01-01

    Lentiviral vectors are widely used in the field of gene therapy as an effective method for permanent gene delivery. While current methods of producing small scale vector batches for research purposes depend largely on culture flasks, the emergence and popularity of lentiviral vectors in translational, preclinical and clinical research has demanded their production on a much larger scale, a task that can be difficult to manage with the numbers of producer cell culture flasks required for large volumes of vector. To generate a large scale, partially closed system method for the manufacturing of clinical grade lentiviral vector suitable for the generation of induced pluripotent stem cells (iPSCs), we developed a method employing a hollow fiber bioreactor traditionally used for cell expansion. We have demonstrated the growth, transfection, and vector-producing capability of 293T producer cells in this system. Vector particle RNA titers after subsequent vector concentration yielded values comparable to lentiviral iPSC induction vector batches produced using traditional culture methods in 225 cm2 flasks (T225s) and in 10-layer cell factories (CF10s), while yielding a volume nearly 145 times larger than the yield from a T225 flask and nearly three times larger than the yield from a CF10. Employing a closed system hollow fiber bioreactor for vector production offers the possibility of manufacturing large quantities of gene therapy vector while minimizing reagent usage, equipment footprint, and open system manipulation. PMID:26151065

  15. Optimization of lentiviral vector production using polyethylenimine-mediated transfection.

    PubMed

    Tang, Yong; Garson, Kenneth; Li, Li; Vanderhyden, Barbara C

    2015-01-01

    The aim of the present study was to optimize the polyethylenimine (PEI)-mediated transfection method in order to simplify the efficient production of lentiviral vectors (LvVs), and to compare the CaPO4- and PEI-mediated transfection methods for producing LvVs. Different titration methods of LvV stocks, as well as different culture media, culture durations, cell densities and DNA quantities were compared to obtain an optimized procedure for the production of LvVs. Optimization of the production method for LvVs was achieved using PEI-mediated transient transfections. Serum-free Opti-MEM(®) was used to directly produce LvVs that could be harvested 48 h after transfection. Furthermore, a cell density of 15×10(6) cells/10-cm plate and a DNA concentration of 1X were selected for the optimum production of LvVs. The optimized LvV titration method was simple and direct; it involved LvVs carrying fluorescent reporters, which proved to be faster than the standard methods but equally as sensitive. In conclusion, a scalable process for production of LvVs by PEI-mediated transfection was established and optimized. The optimized PEI-mediated transfection method was easy to use, as well as providing greater reliability with a higher degree of reproducibility and consistency. Despite using less DNA, the PEI-mediated transfection method resulted in viral titers that were the same as those achieved using the CaPO4-mediated method.

  16. A large U3 deletion causes increased in vivo expression from a nonintegrating lentiviral vector.

    PubMed

    Bayer, Matthew; Kantor, Boris; Cockrell, Adam; Ma, Hong; Zeithaml, Brian; Li, Xiangping; McCown, Thomas; Kafri, Tal

    2008-12-01

    The feasibility of using nonintegrating lentiviral vectors has been demonstrated by recent studies showing their ability to maintain transgene expression both in vitro and in vivo. Furthermore, human immunodeficiency virus-1 (HIV-1) vectors packaged with a mutated integrase were able to correct retinal disease in a mouse model. Interestingly, these results differ from earlier studies in which first-generation nonintegrating lentiviral vectors yielded insignificant levels of transduction. However, to date, a rigorous characterization of transgene expression from the currently used self-inactivating (SIN) nonintegrating lentiviral vectors has not been published. In this study, we characterize transgene expression from SIN nonintegrating lentiviral vectors. Overall, we found that nonintegrating vectors express transgenes at a significantly lower level than their integrating counterparts. Expression from nonintegrating vectors was improved upon introducing a longer deletion in the vector's U3 region. A unique shuttle-vector assay indicated that the relative abundance of the different episomal forms was not altered by the longer U3 deletion. Interestingly, the longer U3 deletion did not enhance expression in the corpus callosum of the rat brain, suggesting that the extent of silencing of episomal transcription is influenced by tissue-specific factors. Finally, and for the first time, episomal expression in the mouse liver was potent and sustained.

  17. Altering Entry Site Preference of Lentiviral Vectors into Neuronal Cells by Pseudotyping with Envelope Glycoproteins.

    PubMed

    Kobayashi, Kenta; Kato, Shigeki; Inoue, Ken-Ichi; Takada, Masahiko; Kobayashi, Kazuto

    2016-01-01

    A lentiviral vector system provides a powerful strategy for gene therapy trials against a variety of neurological and neurodegenerative disorders. Pseudotyping of lentiviral vectors with different envelope glycoproteins not only confers the neurotropism to the vectors, but also alters the preference of sites of vector entry into neuronal cells. One major group of lentiviral vectors is a pseudotype with vesicular stomatitis virus glycoprotein (VSV-G) that enters preferentially cell body areas (somata/dendrites) of neurons and transduces them. Another group contains lentiviral vectors pseudotyped with fusion envelope glycoproteins composed of different sets of rabies virus glycoprotein and VSV-G segments that enter predominantly axon terminals of neurons and are transported through axons retrogradely to their cell bodies, resulting in enhanced retrograde gene transfer. This retrograde gene transfer takes a considerable advantage of delivering the transgene into neuronal cell bodies situated in regions distant from the injection site of the vectors. The rational use of these two vector groups characterized by different entry mechanisms will further extend the strategy for gene therapy of neurological and neurodegenerative disorders.

  18. Construction of stable packaging cell lines for clinical lentiviral vector production

    PubMed Central

    Sanber, Khaled S.; Knight, Sean B.; Stephen, Sam L.; Bailey, Ranbir; Escors, David; Minshull, Jeremy; Santilli, Giorgia; Thrasher, Adrian J.; Collins, Mary K.; Takeuchi, Yasuhiro

    2015-01-01

    Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 106 transducing units/ml can be harvested from the final producer clones, which can be increased to 108 TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors. PMID:25762005

  19. Polyploidization without mitosis improves in vivo liver transduction with lentiviral vectors.

    PubMed

    Pichard, Virginie; Couton, Dominique; Desdouets, Chantal; Ferry, Nicolas

    2013-02-01

    Lentiviral vectors are efficient gene delivery vehicles for therapeutic and research applications. In contrast to oncoretroviral vectors, they are able to infect most nonproliferating cells. In the liver, induction of cell proliferation dramatically improved hepatocyte transduction using all types of retroviral vectors. However, the precise relationship between hepatocyte division and transduction efficiency has not been determined yet. Here we compared gene transfer efficiency in the liver after in vivo injection of recombinant lentiviral or Moloney murine leukemia viral (MoMuLV) vectors in hepatectomized rats treated or not with retrorsine, an alkaloid that blocks hepatocyte division and induces megalocytosis. Partial hepatectomy alone resulted in a similar increase in hepatocyte transduction using either vector. In retrorsine-treated and partially hepatectomized rats, transduction with MoMuLV vectors dropped dramatically. In contrast, we observed that retrorsine treatment combined with partial hepatectomy increased lentiviral transduction to higher levels than hepatectomy alone. Analysis of nuclear ploidy in single cells showed that a high level of transduction was associated with polyploidization. In conclusion, endoreplication could be exploited to improve the efficiency of liver-directed lentiviral gene therapy.

  20. Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors

    PubMed Central

    Trobridge, Grant D.; Beard, Brian C.; Gooch, Christina; Wohlfahrt, Martin; Olsen, Philip; Fletcher, James; Malik, Punam

    2008-01-01

    Lentiviral vectors are attractive for hematopoietic stem cell (HSC) gene therapy because they do not require mitosis for nuclear entry, they efficiently transduce hematopoietic repopulating cells, and self-inactivating (SIN) designs can be produced at high titer. Experiments to evaluate HIV-derived lentiviral vectors in nonhuman primates prior to clinical trials have been hampered by low transduction frequencies due in part to host restriction by TRIM5α. We have established conditions for efficient transduction of pigtailed macaque (Macaca nemestrina) long-term repopulating cells using VSV-G–pseudotyped HIV-based lentiviral vectors. Stable, long-term, high-level gene marking was observed in 3 macaques using relatively low MOIs (5-10) in a 48-hour ex vivo transduction protocol. All animals studied had rapid neutrophil engraftment with a median of 10.3 days to a count greater than 0.5 × 109/L (500/μL). Expression was detected in all lineages, with long-term marking levels in granulocytes at approximately 20% to 30%, and in lymphocytes at approximately 12% to 23%. All animals had polyclonal engraftment as determined by analysis of vector integration sites. These data suggest that lentiviral vectors should be highly effective for HSC gene therapy, particularly for diseases in which maintaining the engraftment potential of stem cells using short-term ex vivo transduction protocols is critical. PMID:18388180

  1. Transfer and Expression of Small Interfering RNAs in Mammalian Cells Using Lentiviral Vectors.

    PubMed

    Lebedev, T D; Spirin, P V; Prassolov, V S

    2013-04-01

    RNA interference is a convenient tool for modulating gene expression. The widespread application of RNA interference is made difficult because of the imperfections of the methods used for efficient target cell delivery of whatever genes are under study. One of the most convenient and efficient gene transfer and expression systems is based on the use of lentiviral vectors, which direct the synthesis of small hairpin RNAs (shRNAs), the precursors of siRNAs. The application of these systems enables one to achieve sustainable and long-term shRNA expression in cells. This review considers the adaptation of the processing of artificial shRNA to the mechanisms used by cellular microRNAs and simultaneous expression of several shRNAs as potential approaches for producing lentiviral vectors that direct shRNA synthesis. Approaches to using RNA interference for the treatment of cancer, as well as hereditary and viral diseases, are under active development today. The improvement made to the methods for constructing lentiviral vectors and the investigation into the mechanisms of processing of small interfering RNA allow one to now consider lentiviral vectors that direct shRNA synthesis as one of the most promising tools for delivering small interfering RNAs.

  2. Transfer and Expression of Small Interfering RNAs in Mammalian Cells Using Lentiviral Vectors

    PubMed Central

    Lebedev, T. D.; Spirin, P. V.; Prassolov, V. S.

    2013-01-01

    RNA interference is a convenient tool for modulating gene expression. The widespread application of RNA interference is made difficult because of the imperfections of the methods used for efficient target cell delivery of whatever genes are under study. One of the most convenient and efficient gene transfer and expression systems is based on the use of lentiviral vectors, which direct the synthesis of small hairpin RNAs (shRNAs), the precursors of siRNAs. The application of these systems enables one to achieve sustainable and long-term shRNA expression in cells. This review considers the adaptation of the processing of artificial shRNA to the mechanisms used by cellular microRNAs and simultaneous expression of several shRNAs as potential approaches for producing lentiviral vectors that direct shRNA synthesis. Approaches to using RNA interference for the treatment of cancer, as well as hereditary and viral diseases, are under active development today. The improvement made to the methods for constructing lentiviral vectors and the investigation into the mechanisms of processing of small interfering RNA allow one to now consider lentiviral vectors that direct shRNA synthesis as one of the most promising tools for delivering small interfering RNAs. PMID:23819033

  3. Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells.

    PubMed

    Yang, Guanghua; Kramer, M Gabriela; Fernandez-Ruiz, Veronica; Kawa, Milosz P; Huang, Xin; Liu, Zhongmin; Prieto, Jesus; Qian, Cheng

    2015-11-27

    Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties.

  4. Lentiviral vectors express chondroitinase ABC in cortical projections and promote sprouting of injured corticospinal axons.

    PubMed

    Zhao, Rong-Rong; Muir, Elizabeth M; Alves, João Nuno; Rickman, Hannah; Allan, Anna Y; Kwok, Jessica C; Roet, Kasper C D; Verhaagen, Joost; Schneider, Bernard L; Bensadoun, Jean-Charles; Ahmed, Sherif G; Yáñez-Muñoz, Rafael J; Keynes, Roger J; Fawcett, James W; Rogers, John H

    2011-09-30

    Several diseases and injuries of the central nervous system could potentially be treated by delivery of an enzyme, which might most effectively be achieved by gene therapy. In particular, the bacterial enzyme chondroitinase ABC is beneficial in animal models of spinal cord injury. We have adapted the chondroitinase gene so that it can direct secretion of active chondroitinase from mammalian cells, and inserted it into lentiviral vectors. When injected into adult rat brain, these vectors lead to extensive secretion of chondroitinase, both locally and from long-distance axon projections, with activity persisting for more than 4 weeks. In animals which received a simultaneous lesion of the corticospinal tract, the vector reduced axonal die-back and promoted sprouting and short-range regeneration of corticospinal axons. The same beneficial effects on damaged corticospinal axons were observed in animals which received the chondroitinase lentiviral vector directly into the vicinity of a spinal cord lesion.

  5. Mobilization and Mechanism of Transcription of Integrated Self-Inactivating Lentiviral Vectors

    PubMed Central

    Hanawa, Hideki; Persons, Derek A.; Nienhuis, Arthur W.

    2005-01-01

    Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3′ long terminal repeat (LTR) is copied over into the 5′ LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector. PMID:15956585

  6. Efficient transduction of liver and muscle after in utero injection of lentiviral vectors with different pseudotypes.

    PubMed

    MacKenzie, Tippi C; Kobinger, Gary P; Kootstra, Neeltje A; Radu, Antoneta; Sena-Esteves, Miguel; Bouchard, Sarah; Wilson, James M; Verma, Inder M; Flake, Alan W

    2002-09-01

    In this study we investigate the efficacy of lentiviral vectors of different pseudotypes for gene transfer to tissues of the preimmune fetus. BALB/c fetuses at 14-15 days' gestation received lentiviral vectors carrying the transgene lacZ under the control of the human cytomegalovirus (CMV) promoter by intramuscular (i.m.) or intrahepatic (i.h.) injection. We pseudotyped the lentiviral vectors with vesicular stomatitis virus (VSV-G), with Mokola virus, or with Ebola virus envelope glycoproteins. We harvested the pups at time points between 5 days and 9 months following injection and performed a detailed histologic assessment. The efficiency and distribution of transduction after in utero administration was highly dependent upon the route of administration and the pseudotype of vector used. Biodistribution studies showed widespread distribution of vector sequences in multiple tissues, albeit at very low levels, and transduced cells were found in significant numbers only in liver, heart, and muscle. Overall, VSV-G was the most efficient in transducing hepatocytes, whereas Mokola and Ebola were more efficient in transducing myocytes. Transduction of cardiomyocytes was observed after both i.m. and i.h. injection of all three vectors. Our findings of long-term transduction of skeletal myocytes and cardiomyocytes after in utero administration suggest a novel strategy for the treatment of congenital muscular dystrophies. PMID:12231171

  7. Insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors.

    PubMed

    Modlich, Ute; Navarro, Susana; Zychlinski, Daniela; Maetzig, Tobias; Knoess, Sabine; Brugman, Martijn H; Schambach, Axel; Charrier, Sabine; Galy, Anne; Thrasher, Adrian J; Bueren, Juan; Baum, Christopher

    2009-11-01

    Gene transfer vectors may cause clonal imbalance and even malignant cell transformation by insertional upregulation of proto-oncogenes. Lentiviral vectors (LV) with their preferred integration in transcribed genes are considered less genotoxic than gammaretroviral vectors (GV) with their preference for integration next to transcriptional start sites and regulatory gene regions. Using a sensitive cell culture assay and a series of self-inactivating (SIN) vectors, we found that the lentiviral insertion pattern was approximately threefold less likely than the gammaretroviral to trigger transformation of primary hematopoietic cells. However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites (CIS) in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents a CIS for both GV and LV, despite major differences in their integration mechanisms. Altering the vectors' enhancer-promoter elements had a greater effect on safety than the retroviral insertion pattern. Clinical grade LV expressing the Wiskott-Aldrich syndrome (WAS) protein under control of its own promoter had no transforming potential. Mechanistic studies support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells, opening rational strategies for risk prevention.

  8. Measurement of lentiviral vector titre and copy number by cross-species duplex quantitative PCR.

    PubMed

    Christodoulou, I; Patsali, P; Stephanou, C; Antoniou, M; Kleanthous, M; Lederer, C W

    2016-01-01

    Lentiviruses are the vectors of choice for many preclinical studies and clinical applications of gene therapy. Accurate measurement of biological vector titre before treatment is a prerequisite for vector dosing, and the calculation of vector integration sites per cell after treatment is as critical to the characterisation of modified cell products as it is to long-term follow-up and the assessment of risk and therapeutic efficiency in patients. These analyses are typically based on quantitative real-time PCR (qPCR), but as yet compromise accuracy and comparability between laboratories and experimental systems, the former by using separate simplex reactions for the detection of endogene and lentiviral sequences and the latter by designing different PCR assays for analyses in human cells and animal disease models. In this study, we validate in human and murine cells a qPCR system for the single-tube assessment of lentiviral vector copy numbers that is suitable for analyses in at least 33 different mammalian species, including human and other primates, mouse, pig, cat and domestic ruminants. The established assay combines the accuracy of single-tube quantitation by duplex qPCR with the convenience of one-off assay optimisation for cross-species analyses and with the direct comparability of lentiviral transduction efficiencies in different species. PMID:26202078

  9. Elements of lentiviral vector design toward gene therapy for treating mucopolysaccharidosis I.

    PubMed

    Ou, Li; Przybilla, Michael J; Koniar, Brenda L; Whitley, Chester B

    2016-09-01

    Mucopolysaccharidosis type I (MPS I) is a lysosomal disease caused by α-l-iduronidase (IDUA) deficiency and accumulation of glycosaminoglycans (GAG). Lentiviral vector encoding correct IDUA cDNA could be used for treating MPS I. To optimize the lentiviral vector design, 9 constructs were designed by combinations of various promoters, enhancers, and codon optimization. After in vitro transfection into 293FT cells, 5 constructs achieved the highest IDUA activities (5613 to 7358 nmol/h/mg protein). These 5 candidate vectors were then tested by injection (1 × 10(7) TU/g) into neonatal MPS I mice. After 30 days, one vector, CCEoIDW, achieved the highest IDUA levels: 2.6% of wildtype levels in the brain, 9.9% in the heart, 200% in the liver and 257% in the spleen. CCEoIDW achieved the most significant GAG reduction: down 49% in the brain, 98% in the heart, 100% in the liver and 95% in the spleen. Further, CCEoIDW had the lowest transgene frequency, especially in the gonads (0.03 ± 0.01 copies/100 cells), reducing the risk of insertional mutagenesis and germ-line transmission. Therefore, CCEoIDW is selected as the optimal lentiviral vector for treating MPS I disease and will be applied in large animal preclinical studies. Further, taken both in vitro and in vivo comparisons together, codon optimization, use of EF-1α promoter and woodchuck hepatitis virus posttranscriptional response element (WPRE) could enhance transgene expression. These results provided a better understanding of factors contributing efficient transgene expression in lentiviral gene therapies. PMID:27556013

  10. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells.

    PubMed

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A F V

    2013-03-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.

  11. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells

    PubMed Central

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M.; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A. F. V.

    2013-01-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 ‘safe harbor’ locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform. PMID:23275534

  12. Transgenic chimera quail production by microinjecting lentiviral vector into the blood vessel of the early embryo.

    PubMed

    Sun, Peng; Zhang, Zifu; Wu, Guojin; Yan, Li; Yuan, Fang; Zhang, Wenxin; Gao, Junshuang; Jin, Wenjing; Li, Zandong

    2012-04-01

    In the past, several strategies have been used to generate transgenic birds. The most successful method has proven to be injection of lentiviral vector into the subgerminal cavity of the newly laid egg. In this study, we directly injected lentiviral vector into the blood vessel of HH13-15 quail embryos to produce transgenic chimeras. In the manipulated, hatched birds, the green fluorescent protein (GFP) gene driven by a cytomegalovirus (CMV) promoter was extensively expressed. All tissues analyzed were GFP-positive, and gonad cells from some of the manipulated embryos expressed GFP. The semen genome of 21.4% of mature male birds was determined to be GFP-positive by PCR, indicating these male birds were transgenic chimeras.

  13. A rapid and efficient branched DNA hybridization assay to titer lentiviral vectors.

    PubMed

    Nair, Ayyappan; Xie, Jinger; Joshi, Sarasijam; Harden, Paul; Davies, Joan; Hermiston, Terry

    2008-11-01

    A robust assay to titer lentiviral vectors is imperative to qualifying their use in drug discovery, target validation and clinical applications. In this study, a novel branched DNA based hybridization assay was developed to titer lentiviral vectors by quantifying viral RNA genome copy numbers from viral lysates without having to purify viral RNA, and this approach was compared with other non-functional (p24 protein ELISA and viral RT-qPCR) and a functional method (reporter gene expression) used commonly. The RT-qPCR method requires purification of viral RNA and the accuracy of titration therefore depends on the efficiency of purification; this requirement is ameliorated in the hybridization assay as RNA is measured directly in viral lysates. The present study indicates that the hybridization based titration assay performed on viral lysates was more accurate and has additional advantages of being rapid, robust and not dependent on transduction efficiency in different cell types.

  14. Lentiviral vector transduction of spermatozoa as a tool for the study of early development.

    PubMed

    Chandrashekran, Anil; Isa, Ihsan; Dudhia, Jayesh; Thrasher, Adrian J; Dibb, Nicholas; Casimir, Colin; Readhead, Carol; Winston, Robert

    2014-01-01

    Spermatozoa and lentiviruses are two of nature's most efficient gene delivery vehicles. Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders. Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies. Lentiviral vectors encoding Green Fluorescent Protein (GFP) were shown to be efficiently processed and expressed in sperm. When these transduced sperm were used in in vitro fertilisation studies, GFP expression was observed in arising blastocysts. This simple technique of directly transducing spermatozoa has potential to be a powerful tool for the study of early and pre-implantation development and could be used as a technique in transgenic development and vertical viral transmission studies. PMID:24918038

  15. Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy

    PubMed Central

    Negro-Demontel, María Luciana; Saccardo, Paolo; Giacomini, Cecilia; Yáñez-Muñoz, Rafael Joaquín; Ferrer-Miralles, Neus; Vazquez, Esther; Villaverde, Antonio; Peluffo, Hugo

    2014-01-01

    Traumatic brain injury (TBI) remains as one of the leading causes of mortality and morbidity worldwide and there are no effective treatments currently available. Gene therapy applications have emerged as important alternatives for the treatment of diverse nervous system injuries. New strategies are evolving with the notion that each particular pathological condition may require a specific vector. Moreover, the lack of detailed comparative studies between different vectors under similar conditions hampers the selection of an ideal vector for a given pathological condition. The potential use of lentiviral vectors versus several modular protein-based nanovectors was compared using a controlled cortical impact model of TBI under the same gene therapy conditions. We show that variables such as protein/DNA ratio, incubation volume, and presence of serum or chloroquine in the transfection medium impact on both nanovector formation and transfection efficiency in vitro. While lentiviral vectors showed GFP protein 1 day after TBI and increased expression at 14 days, nanovectors showed stable and lower GFP transgene expression from 1 to 14 days. No toxicity after TBI by any of the vectors was observed as determined by resulting levels of IL-1β or using neurological sticky tape test. In fact, both vector types induced functional improvement per se. PMID:26015985

  16. Lentiviral Vectors for the Engineering of Implantable Cells Secreting Recombinant Antibodies.

    PubMed

    Lathuilière, Aurélien; Schneider, Bernard L

    2016-01-01

    The implantation of genetically modified cells is considered for the chronic delivery of therapeutic recombinant proteins in vivo. In the context of gene therapy, the genetic engineering of cells faces two main challenges. First, it is critical to generate expandable cell sources, which can maintain stable high productivity of the recombinant protein of interest over time, both in culture and after transplantation. In addition, gene transfer techniques need to be developed to engineer cells synthetizing complex polypeptides, such as recombinant monoclonal antibodies, to broaden the range of potential therapeutic applications. Here, we provide a workflow for the use of lentiviral vectors as a flexible tool to generate antibody-producing cells. In particular, lentiviral vectors can be used to genetically engineer the cell types compatible with encapsulation devices protecting the implanted cells from the host immune system. Detailed methods are provided for the design and production of lentiviral vectors, optimization of cell transduction, as well as for the quantification and quality control of the produced recombinant antibody. PMID:27317179

  17. Priming of hepatocytes enhances in vivo liver transduction with lentiviral vectors in adult mice.

    PubMed

    Pichard, Virginie; Boni, Sébastien; Baron, William; Nguyen, Tuan Huy; Ferry, Nicolas

    2012-02-01

    Lentiviral vectors are promising tools for liver disease gene therapy, because they can achieve protracted expression of transgenes in hepatocytes. However, the question as to whether cell division is required for optimal hepatocyte transduction has still not been completely answered. Liver gene-transfer efficiency after in vivo administration of recombinant lentiviral vectors carrying a green fluorescent protein reporter gene under the control of a liver-specific promoter in mice that were either hepatectomized or treated with cholic acid or phenobarbital was compared. Phenobarbital is known as a weak inducer of hepatocyte proliferation, whereas cholic acid has no direct effect on the cell cycle. This study shows that cholic acid is able to prime hepatocytes without mitosis induction. Both phenobarbital and cholic acid significantly increased hepatocyte transduction six- to ninefold, although cholic acid did not modify the mitotic index or cell-cycle entry. However, the effect of either compound was weaker than that observed after partial hepatectomy. In no cases was there a correlation between the expression of cell-cycle marker and transduction efficiency. We conclude that priming of hepatocytes should be considered a clinically applicable strategy to enhance in vivo liver gene therapy with lentiviral vectors.

  18. Cytotoxicity associated with artemis overexpression after lentiviral vector-mediated gene transfer.

    PubMed

    Multhaup, Megan; Karlen, Andrea D; Swanson, Debra L; Wilber, Andrew; Somia, Nikunj V; Cowan, Morton J; McIvor, R Scott

    2010-07-01

    Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1alpha promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T(-)B(-) phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A. PMID:20163250

  19. Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates

    PubMed Central

    Murphy, Michele E; Vin, Chintan D; Slough, Megan M; Gombotz, Wayne R; Kelley-Clarke, Brenna

    2016-01-01

    Using lentiviral vector products in clinical applications requires an accurate method for measuring transduction titer. For vectors lacking a marker gene, quantitative polymerase chain reaction is used to evaluate the number of vector DNA copies in transduced target cells, from which a transduction titer is calculated. Immune Design previously described an integration-deficient lentiviral vector pseudotyped with a modified Sindbis virus envelope for use in cancer immunotherapy (VP02, of the ZVex platform). Standard protocols for titering integration-competent lentiviral vectors employ commercial spin columns to purify vector DNA from transduced cells, but such columns are not optimized for isolation of extrachromosomal (nonintegrated) DNA. Here, we describe a 96-well transduction titer assay in which DNA extraction is performed in situ in the transduction plate, yielding quantitative recovery of extrachromosomal DNA. Vector titers measured by this method were higher than when commercial spin columns were used for DNA isolation. Evaluation of the method’s specificity, linear range, and precision demonstrate that it is suitable for use as a lot release assay to support clinical trials with VP02. Finally, the method is compatible with titering both integrating and nonintegrating lentiviral vectors, suggesting that it may be used to evaluate the transduction titer for any lentiviral vector. PMID:26942209

  20. Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

    PubMed Central

    Mao, Yingying; Yan, Renhe; Li, Andrew; Zhang, Yanling; Li, Jinlong; Du, Hongyan; Chen, Baihong; Wei, Wenjin; Zhang, Yi; Sumners, Colin; Zheng, Haifa; Li, Hongwei

    2015-01-01

    Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors. Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution. Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction

  1. Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

    SciTech Connect

    Zhou Qi; Schneider, Irene C.; Gallet, Manuela; Kneissl, Sabrina; Buchholz, Christian J.

    2011-05-10

    The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential.

  2. Initial Characterization of Integrase-Defective Lentiviral Vectors for Pancreatic Cancer Gene Therapy.

    PubMed

    Hanoun, Naima; Gayral, Marion; Pointreau, Adeline; Buscail, Louis; Cordelier, Pierre

    2016-02-01

    The vast majority (85%) of pancreatic ductal adenocarcinomas (PDACs) are discovered at too of a late stage to allow curative surgery. In addition, PDAC is highly resistant to conventional methods of chemotherapy and radiotherapy, which only offer a marginal clinical benefit. Consequently, the prognosis of this cancer is devastating, with a 5-year survival rate of less than 5%. In this dismal context, we recently demonstrated that PDAC gene therapy using nonviral vectors is safe and feasible, with early signs of efficacy in selected patients. Our next step is to transfer to the clinic HIV-1-based lentiviral vectors (LVs) that outshine other therapeutic vectors to treat experimental models of PDAC. However, a primary safety issue presented by LVs that may delay their use in patients is the risk of oncogenesis after vector integration in the host's cell DNA. Thus, we developed a novel anticancerous approach based on integrase-defective lentiviral vectors (IDLVs) and demonstrated that IDLVs can be successfully engineered to transiently deliver therapeutic genes to inhibit pancreatic cancer cells proliferation. This work stems for the use of therapeutic IDLVs for the management of PDAC, in forthcoming early phase gene therapy clinical trial for this disease with no cure. PMID:26731312

  3. A Lentiviral Vector Expressing Desired Gene Only in Transduced Cells: An Approach for Suicide Gene Therapy.

    PubMed

    Mohammadi, Zahra; Shariati, Laleh; Khanahmad, Hossein; Kolahdouz, Mahsa; Kianpoor, Fariborz; Ghanbari, Jahan Afrooz; Hejazi, Zahra; Salehi, Mansoor; Nikpour, Parvaneh; Tabatabaiefar, Mohammad Amin

    2015-09-01

    Suicide gene therapy is a therapeutic strategy, in which cell suicide inducing transgenes are introduced into target cells. Inserting a toxin-encoding gene into a lentiviral vector leads to decreased efficiency of virus production due to lethal effect of toxin on packaging cells. In this study, we designed and constructed a transfer vector to express the toxin in transduced cells but not in packaging cells. Plasmid pLenti-F/GFP was constructed by cutting out R 5'LTR-R 3'LTR fragment with the AflII restriction endonuclease from a plasmid pLenti4-GW/H1/TO-laminshRNA, followed by ligating R 5'LTR-R 3'LTR fragment, constructed by three PCR stages. The promoter and GFP CDS were inserted in opposite strand. For lentiviral production, the HEK293T cell line was co-transfected with the PMD2G, psPAX2, and pLenti-F/GFP plasmids (envelope, packaging, and transfer plasmids).Viral vector titers were assayed. The HEK293T cell line was transduced with this virus. PCR was performed to confirm the presence of the promoter fragment between the R and U5 in 3'LTR. The lentivirus titers were approximately 2 × 10(5). The GFP expression was seen in 51 % of the HEK293T cells transduced with lentivirus. The PCR product size was 1440 bp confirming the promoter fragment position between the R and U5 in 3'LTR. The strategy enables us to use a broad spectrum of toxin genes in gene therapy and helps avoid the death of the packaging cells with lentiviral vectors carrying a toxin-encoding gene, thereby increasing the efficiency of viral production in packaging cells.

  4. Generation of a lentiviral vector producer cell clone for human Wiskott-Aldrich syndrome gene therapy.

    PubMed

    Wielgosz, Matthew M; Kim, Yoon-Sang; Carney, Gael G; Zhan, Jun; Reddivari, Muralidhar; Coop, Terry; Heath, Richard J; Brown, Scott A; Nienhuis, Arthur W

    2015-01-01

    We have developed a producer cell line that generates lentiviral vector particles of high titer. The vector encodes the Wiskott-Aldrich syndrome (WAS) protein. An insulator element has been added to the long terminal repeats of the integrated vector to limit proto-oncogene activation. The vector provides high-level, stable expression of WAS protein in transduced murine and human hematopoietic cells. We have also developed a monoclonal antibody specific for intracellular WAS protein. This antibody has been used to monitor expression in blood and bone marrow cells after transfer into lineage negative bone marrow cells from WAS mice and in a WAS negative human B-cell line. Persistent expression of the transgene has been observed in transduced murine cells 12-20 weeks following transplantation. The producer cell line and the specific monoclonal antibody will facilitate the development of a clinical protocol for gene transfer into WAS protein deficient stem cells. PMID:26052531

  5. Generation of a lentiviral vector producer cell clone for human Wiskott-Aldrich syndrome gene therapy.

    PubMed

    Wielgosz, Matthew M; Kim, Yoon-Sang; Carney, Gael G; Zhan, Jun; Reddivari, Muralidhar; Coop, Terry; Heath, Richard J; Brown, Scott A; Nienhuis, Arthur W

    2015-01-01

    We have developed a producer cell line that generates lentiviral vector particles of high titer. The vector encodes the Wiskott-Aldrich syndrome (WAS) protein. An insulator element has been added to the long terminal repeats of the integrated vector to limit proto-oncogene activation. The vector provides high-level, stable expression of WAS protein in transduced murine and human hematopoietic cells. We have also developed a monoclonal antibody specific for intracellular WAS protein. This antibody has been used to monitor expression in blood and bone marrow cells after transfer into lineage negative bone marrow cells from WAS mice and in a WAS negative human B-cell line. Persistent expression of the transgene has been observed in transduced murine cells 12-20 weeks following transplantation. The producer cell line and the specific monoclonal antibody will facilitate the development of a clinical protocol for gene transfer into WAS protein deficient stem cells.

  6. Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector.

    PubMed

    Burke, Bryan P; Levin, Bernard R; Zhang, Jane; Sahakyan, Anna; Boyer, Joshua; Carroll, Maria V; Colón, Joanna Camba; Keech, Naomi; Rezek, Valerie; Bristol, Gregory; Eggers, Erica; Cortado, Ruth; Boyd, Maureen P; Impey, Helen; Shimizu, Saki; Lowe, Emily L; Ringpis, Gene-Errol E; Kim, Sohn G; Vatakis, Dimitrios N; Breton, Louis R; Bartlett, Jeffrey S; Chen, Irvin S Y; Kitchen, Scott G; An, Dong Sung; Symonds, Geoff P

    2015-01-01

    We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC) either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection. PMID:25872029

  7. Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations

    PubMed Central

    Cesana, Daniela; Sgualdino, Jacopo; Rudilosso, Laura; Merella, Stefania; Naldini, Luigi; Montini, Eugenio

    2012-01-01

    Gamma-retroviral/lentiviral vectors (γRV/LV) with self-inactivating (SIN) long terminal repeats (LTRs) and internal moderate cellular promoters pose a reduced risk of insertional mutagenesis when compared with vectors with active LTRs. Yet, in a recent LV-based clinical trial for β-thalassemia, vector integration within the HMGA2 gene induced the formation of an aberrantly spliced mRNA form that appeared to cause clonal dominance. Using a method that we developed, cDNA linear amplification-mediated PCR, in combination with high-throughput sequencing, we conducted a whole transcriptome analysis of chimeric LV-cellular fusion transcripts in transduced human lymphoblastoid cells and primary hematopoietic stem/progenitor cells. We observed a surprising abundance of read-through transcription originating outside and inside the provirus and identified the vector sequences contributing to the aberrant splicing process. We found that SIN LV has a sharply reduced propensity to engage in aberrant splicing compared with that of vectors carrying active LTRs. Moreover, by recoding the identified vector splice sites, we reduced residual read-through transcription and demonstrated an effective strategy for improving vectors. Characterization of the mechanisms and genetic features underlying vector-induced aberrant splicing will enable the generation of safer vectors, with low impact on the cellular transcriptome. PMID:22523064

  8. Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors

    PubMed Central

    Askou, Anne Louise; Aagaard, Lars; Kostic, Corinne; Arsenijevic, Yvan; Hollensen, Anne Kruse; Bek, Toke; Jensen, Thomas Gryesten; Mikkelsen, Jacob Giehm; Corydon, Thomas Juhl

    2015-01-01

    Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration. PMID:26052532

  9. Genetic engineering of cell lines using lentiviral vectors to achieve antibody secretion following encapsulated implantation.

    PubMed

    Lathuilière, Aurélien; Bohrmann, Bernd; Kopetzki, Erhard; Schweitzer, Christoph; Jacobsen, Helmut; Moniatte, Marc; Aebischer, Patrick; Schneider, Bernard L

    2014-01-01

    The controlled delivery of antibodies by immunoisolated bioimplants containing genetically engineered cells is an attractive and safe approach for chronic treatments. To reach therapeutic antibody levels there is a need to generate renewable cell lines, which can long-term survive in macroencapsulation devices while maintaining high antibody specific productivity. Here we have developed a dual lentiviral vector strategy for the genetic engineering of cell lines compatible with macroencapsulation, using separate vectors encoding IgG light and heavy chains. We show that IgG expression level can be maximized as a function of vector dose and transgene ratio. This approach allows for the generation of stable populations of IgG-expressing C2C12 mouse myoblasts, and for the subsequent isolation of clones stably secreting high IgG levels. Moreover, we demonstrate that cell transduction using this lentiviral system leads to the production of a functional glycosylated antibody by myogenic cells. Subsequent implantation of antibody-secreting cells in a high-capacity macroencapsulation device enables continuous delivery of recombinant antibodies in the mouse subcutaneous tissue, leading to substantial levels of therapeutic IgG detectable in the plasma.

  10. Highly Efficient Large-Scale Lentiviral Vector Concentration by Tandem Tangential Flow Filtration

    PubMed Central

    Cooper, Aaron R.; Patel, Sanjeet; Senadheera, Shantha; Plath, Kathrin; Kohn, Donald B.; Hollis, Roger P.

    2014-01-01

    Large-scale lentiviral vector (LV) concentration can be inefficient and time consuming, often involving multiple rounds of filtration and centrifugation. This report describes a simpler method using two tangential flow filtration (TFF) steps to concentrate liter-scale volumes of LV supernatant, achieving in excess of 2000-fold concentration in less than 3 hours with very high recovery (>97%). Large volumes of LV supernatant can be produced easily through the use of multi-layer flasks, each having 1720 cm2 surface area and producing ~560 mL of supernatant per flask. Combining the use of such flasks and TFF greatly simplifies large-scale production of LV. As a demonstration, the method is used to produce a very high titer LV (>1010 TU/mL) and transduce primary human CD34+ hematopoietic stem/progenitor cells at high final vector concentrations with no overt toxicity. A complex LV (STEMCCA) for induced pluripotent stem cell generation is also concentrated from low initial titer and used to transduce and reprogram primary human fibroblasts with no overt toxicity. Additionally, a generalized and simple multiplexed real- time PCR assay is described for lentiviral vector titer and copy number determination. PMID:21784103

  11. Restoring ciliary function to differentiated Primary Ciliary Dyskinesia cells with a lentiviral vector

    PubMed Central

    Ostrowski, Lawrence E; Yin, Weining; Patel, Manij; Sechelski, John; Rogers, Troy; Burns, Kimberlie; Grubb, Barbara R; Olsen, John C

    2014-01-01

    Primary ciliary dyskinesia is a genetically heterogeneous autosomal recessive disease in which mutations disrupt ciliary function, leading to impaired mucociliary clearance and life-long lung disease. Mouse tracheal cells with a targeted deletion in the axonemal dynein intermediated chain gene Dnaic1 differentiate normally in culture but lack ciliary activity. Gene transfer to undifferentiated cultures of mouse Dnaic1−/− cells with a lentiviral vector pseudotyped with avian influenza hemagglutinin restored Dnaic1 expression and ciliary activity. Importantly, apical treatment of well-differentiated cultures of mouse Dnaic1−/− with lentiviral vector also restored ciliary activity, demonstrating successful gene transfer from the apical surface. Treatment of Dnaic1flox/flox mice expressing an estrogen responsive Cre recombinase with different doses of tamoxifen indicated that restoration of ~20% of ciliary activity may be sufficient to prevent the development of rhinosinusitis. However, while administration of a β-galactosidase expressing vector to control mice demonstrated efficient gene transfer to the nasal epithelium, treatment of Dnaic1−/− mice resulted in a low level of gene transfer, demonstrating that the severe rhinitis present in these animals impedes gene transfer. The results demonstrate that gene replacement therapy may be a viable treatment option for primary ciliary dyskinesia, but further improvements in the efficiency of gene transfer are necessary. PMID:24451115

  12. Transduction of Murine Hematopoietic Stem Cells with Tetracycline-regulated Lentiviral Vectors.

    PubMed

    Stahlhut, Maike; Schambach, Axel; Kustikova, Olga S

    2016-01-01

    Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells (HSCs). This approach combines the stable transgene insertion into a host genome with the opportunity for time- and dose-controlled reversible transgene expression in HSCs. Here, we describe the step-by-step protocol for transduction of murine stem-cell enriched populations of bone marrow cells, such as lineage negative cells (Lin(-)), with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP) under the control of the tetracycline-regulated promoter. This chapter explains how to establish in vitro and in vivo systems to study transgene dose-dependent mechanisms affecting cell fate decisions of genetically modified hematopoietic cells. PMID:27317173

  13. Lentiviral vectors in gene therapy: their current status and future potential.

    PubMed

    Escors, David; Breckpot, Karine

    2010-04-01

    The concept of gene therapy originated in the mid twentieth century and was perceived as a revolutionary technology with the promise to cure almost any disease of which the molecular basis was understood. Since then, several gene vectors have been developed and the feasibility of gene therapy has been shown in many animal models of human disease. However, clinical efficacy could not be demonstrated until the beginning of the new century in a small-scale clinical trial curing an otherwise fatal immunodeficiency disorder in children. This first success, achieved after retroviral therapy, was later overshadowed by the occurrence of vector-related leukemia in a significant number of the treated children, demonstrating that the future success of gene therapy depends on our understanding of vector biology. This has led to the development of later-generation vectors with improved efficiency, specificity, and safety. Amongst these are HIV-1 lentivirus-based vectors (lentivectors), which are being increasingly used in basic and applied research. Human gene therapy clinical trials are currently underway using lentivectors in a wide range of human diseases. The intention of this review is to describe the main scientific steps leading to the engineering of HIV-1 lentiviral vectors and place them in the context of current human gene therapy.

  14. Lentiviral vectors in gene therapy: their current status and future potential

    PubMed Central

    Escors, David; Breckpot, Karine

    2010-01-01

    Summary The concept of gene therapy originated in the mid 20th century and was perceived as a revolutionary technology with the promise to cure almost any disease of which the molecular basis was understood. Since then, several gene vectors have been developed, and the feasibility of gene therapy shown in many animal models of human disease. However, clinical efficacy could not be demonstrated until the beginning of the new century in a small-scale clinical trial curing an otherwise fatal immunodeficiency disorder in children. This first success – achieved after retroviral therapy - was later on overshadowed by the occurrence of vector-related leukaemia in a significant number of the treated children, demonstrating that the future success of gene therapy depends on our understanding of vector biology. This has led to the development of later generation vectors with improved efficiency, specificity and safety. Amongst these are HIV-1 lentivirus-based vectors (lentivectors), which are being increasingly used in basic and applied research. Human gene therapy clinical trials are currently under way using lentivectors in a wide range of human diseases. The intention of this review is to describe the main scientific steps leading to the engineering of HIV-1 lentiviral vectors, and place them in the context of current human gene therapy. PMID:20143172

  15. Lentiviral vector-mediated genetic modification of cell substrates for the manufacture of proteins and other biologics.

    PubMed

    Baranyi, Lajos; Roy, Andre; Embree, Heather D; Dropulic, Boro

    2010-01-01

    Transduction with Lentiviral vectors has been shown to be the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Lentiviral vectors have been widely used in research and have recently shown success in clinical trials for human gene therapy. In this paper, we describe the use of lentiviral vectors to generate genetically modified cell substrates for the manufacture of proteins and other complex biologics. The use of lentiviral vectors for the generation of genetically modified cell substrates for the production of biologic material has several advantages over other systems: (1) highly productive mammalian cell lines can be rapidly generated without selection or gene amplification; (2) the high number of vector copies are distributed throughout the open chromatin of the genome, resulting in cell lines that are extremely stable for high levels of gene expression and, consequently, protein production; and (3) high levels of protein glycosylation are maintained despite very high levels of protein production. These advantages offer the potential to significantly improve the quality, time-to-market, and manufacturing cost of biologics for human use.

  16. Visualization of DC-SIGN-mediated entry pathway of engineered lentiviral vectors in target cells.

    PubMed

    Liu, Yarong; Tai, April; Joo, Kye-Il; Wang, Pin

    2013-01-01

    Dendritic cells (DCs) are potent antigen-presenting cells and therefore have enormous potential as vaccine targets. We have previously developed an engineered lentiviral vector (LV) that is pseudotyped with a mutated Sindbis virus glycoprotein (SVGmu), which is capable of targeting DCs through Dendritic Cell-specific ICAM3-grabbing Nonintegrin (DC-SIGN), a receptor that is predominantly expressed by DCs. In this study, we aimed to elucidate the internalization and trafficking mechanisms of this viral vector system through direct visualization of GFP-Vpr-tagged viral particles in target DCs, which was further corroborated by drug inhibition and dominant-negative mutants of cellular proteins that regulate the endocytic traffic. We demonstrated that our engineered LVs enter the cell via receptor-mediated clathrin- and dynamin-dependent endocytosis. Microtubule networks were also involved in a productive infection. Viral vector fusion was low-pH-dependent and occurred in the early endosomal stage of the intracellular transport. Autophagy was also examined for its effect on transduction efficiency, and we observed that enhanced autophage activity reduced vector infectivity, while suppressed autophagy boosted transduction efficiency. This study shed some light on the internalization and trafficking mechanisms of DC-directed LVs and offers some strategies to further improve the efficiency of LV-mediated gene therapy.

  17. Stable and Efficient Gene Transfer into the Retina Using an HIV-Based Lentiviral Vector

    NASA Astrophysics Data System (ADS)

    Miyoshi, Hiroyuki; Takahashi, Masayo; Gage, Fred H.; Verma, Inder M.

    1997-09-01

    The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.

  18. Surface engineering of lentiviral vectors for gene transfer into gene therapy target cells.

    PubMed

    Lévy, Camille; Verhoeyen, Els; Cosset, François-Loïc

    2015-10-01

    Since they allow gene integration into their host genome, lentiviral vectors (LVs) have strong therapeutic potentials, as emphasized by recent clinical trials. The surface-display of the pantropic vesicular stomatitis virus G glycoprotein (VSV-G) on LVs resulted in powerful tools for fundamental and clinical research. However, improved LVs are required either to genetically modify cell types not permissive to classical VSV-G-LVs or to restrict entry to specific cell types. Incorporation of heterologous viral glycoproteins (gps) on LVs often require modification of their cytoplasmic tails and ligands can be inserted into their ectodomain to target LVs to specific receptors. Recently, measles virus (MV) gps have been identified as strong candidates for LV-retargeting to multiple cell types, with the potential to evolve toward clinical applications.

  19. Immune modulation by genetic modification of dendritic cells with lentiviral vectors.

    PubMed

    Liechtenstein, Therese; Perez-Janices, Noemi; Bricogne, Christopher; Lanna, Alessio; Dufait, Inès; Goyvaerts, Cleo; Laranga, Roberta; Padella, Antonella; Arce, Frederick; Baratchian, Mehdi; Ramirez, Natalia; Lopez, Natalia; Kochan, Grazyna; Blanco-Luquin, Idoia; Guerrero-Setas, David; Breckpot, Karine; Escors, David

    2013-09-01

    Our work over the past eight years has focused on the use of HIV-1 lentiviral vectors (lentivectors) for the genetic modification of dendritic cells (DCs) to control their functions in immune modulation. DCs are key professional antigen presenting cells which regulate the activity of most effector immune cells, including T, B and NK cells. Their genetic modification provides the means for the development of targeted therapies towards cancer and autoimmune disease. We have been modulating with lentivectors the activity of intracellular signalling pathways and co-stimulation during antigen presentation to T cells, to fine-tune the type and strength of the immune response. In the course of our research, we have found unexpected results such as the surprising immunosuppressive role of anti-viral signalling pathways, and the close link between negative co-stimulation in the immunological synapse and T cell receptor trafficking. Here we review our major findings and put them into context with other published work.

  20. Role of the mammalian target of rapamycin pathway and rapamycin in lentiviral vector gene transduction of hematopoietic stem cells

    PubMed Central

    Wang, Cathy X.; Torbett, Bruce E.

    2015-01-01

    Purpose of review A major goal in repopulating hematopoietic stem cell (HSC) gene therapies is achieving high-efficacy gene transfer, while maintaining robust HSC engraftment and differentiation in vivo. Recent studies have documented that rapamycin treatment of HSC during lentiviral vector transduction enhances gene transfer to human and mouse HSCs and maintains engraftment capacity. In this review, we place into context the role of mammalian target of rapamycin (mTOR) pathways in HSC quiescence and function, endocytic regulation, and lentiviral gene delivery. Recent findings Lentiviral vector transduction of human and mouse HSCs is considerably enhanced by rapamycin treatment. Furthermore, rapamycin preserves long-term engraftment of human and mouse HSCs. Investigations of cellular mechanisms that contribute to increased transduction in HSCs uncovered a role for mTOR inhibition-dependent activation of endocytosis. Summary Rapamycin enhances lentiviral vector transduction of HSCs through regulation of endocytic activity via mTOR inhibition. An important attribute of rapamycin treatment during transduction is the preservation of HSC function, allowing reconstitution of long-term hematopoiesis in vivo in murine models. PMID:26049750

  1. Lentiviral vector-mediated over-expression of Sox9 protected chondrocytes from IL-1β induced degeneration and apoptosis

    PubMed Central

    Lu, Huading; Zeng, Chun; Chen, Mingwei; Lian, Liyi; Dai, Yuhu; Zhao, Huiqing

    2015-01-01

    To explore whether the over-expression of Sry-related HMG box (Sox9) in degenerative chondrocytes is able to improve cell regeneration and protects cells from inflammation induced apoptosis, we generated a Sox9 over-expressing vector delivery system in which the Sox9 gene was inserted into a lentiviral vector. After infecting mouse chondrocytes with the Sox9-encoding vector, we observed a high level of gene transduction efficiency and achieved a high level of Sox9 expression in the infected chondrocytes. To explore whether over-expression of Sox9 is able to induce cell regeneration and improve cell survival, we induced Sox9 over-expression by lentiviral vector infection 48 hours before IL-1β treatment. The cells were infected with the reporter gene GFP-encoded lentiviral vector as a negative control or left uninfected. 48-hours after IL-1β treatment, the chrondrocytes treated with IL-1β alone, underwent a degenerative process, with elevated expression of MMP-3, MMP-13, ADAMTS-5 and ALP, but the cell specific anabolic proteins collagen II and aggrecan were significantly suppressed. The cells infected with the GFP reporter vector had no increased regeneration after IL-1β treatment. The results indicated that Sox9 is an important chondrocyte transcription factor, promoting chondrocyte regeneration and cell survival, which were mediated through affecting multiple cell differentiation as well as anti-apoptotic signaling pathways. PMID:26617711

  2. 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

  3. A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs

    PubMed Central

    Souque, Philippe; Frenkiel, Marie-Pascale; Paulous, Sylvie; Garcìa-Nicolàs, Obdulio; Summerfield, Artur; Charneau, Pierre; Desprès, Philippe

    2015-01-01

    Background Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a “one health” strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. Methodology/Principal Findings A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. Conclusions/Significance Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great

  4. Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

    PubMed

    Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

    2007-07-30

    Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

  5. Lentiviral vectors with CMV or MHCII promoters administered in vivo: immune reactivity versus persistence of expression.

    PubMed

    Kimura, Takahiro; Koya, Richard C; Anselmi, Laura; Sternini, Catia; Wang, He-Jing; Comin-Anduix, Begonya; Prins, Robert M; Faure-Kumar, Emmanuelle; Rozengurt, Nora; Cui, Yan; Kasahara, Noriyuki; Stripecke, Renata

    2007-07-01

    Lentiviral vectors (LVs) are potential tools for genetic vaccination. To improve the safety of LV vaccines, we evaluated the selectivity, bio-distribution, persistence of expression, and immune potency of vesicular stomatitis virus G (VSV-G)-pseudotyped vectors transcriptionally targeted to antigen presenting cells (APCs) through a major histocompatibility complex class II (MHCII) promoter. Control vectors contained the ubiquitous cytomegalovirus (CMV) promoter. Bio-distribution studies after intravenous injections of LVs expressing green fluorescent protein (GFP) or luciferase were conducted by a combination of flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (RT-Q-PCR) and whole-body bioluminescence analyses. GFP-expressing vectors showed selective expression in MHCII(+) cells of spleen and LV-CMV-GFP administration produced noticeable spleen inflammation, whereas LV-MHCII-GFP did not. Long-term optical imaging analyses of C57BL/6 mice injected with LV-CMV-LUC showed diminishing luciferase expression in the liver and spleen over time. Vaccination/boost with LV-CMV expressing the melanoma antigen tyrosinase-related protein 2 (TRP2) yielded dose-dependent antigen-specific CD8(+) T-cell reactivity and high protection against B16 melanoma challenge. Unexpectedly, administration of LVs containing the MHCII promoter resulted in persistence of luciferase expression and viral integration in MHCII(+) splenocytes and virtually no CD8(+) T-cell responses against TRP2. These studies reveal that APC transduction by LVs could lead to immune reactivity (LV-CMV) or persistence of transgene expression (LV-MHCII), providing a relevant paradigm for vaccination and gene replacement approaches. PMID:17505480

  6. Intercellular delivery of a herpes simplex virus VP22 fusion protein from cells infected with lentiviral vectors

    PubMed Central

    Lai, Zhennan; Han, Ina; Zirzow, Gregory; Brady, Roscoe O.; Reiser, Jakob

    2000-01-01

    Effective gene therapy depends on the efficient transfer of therapeutic genes and their protein products to target cells. Lentiviral vectors appear promising for virus-mediated gene delivery and long-term expression in nondividing cells. The herpes simplex virus type 1 tegument protein VP22 has recently been shown to mediate intercellular transport of proteins, raising the possibility that it may be helpful in a setting where the global delivery of therapeutic proteins is desired. To investigate the effectiveness of lentiviral vectors to deliver genes encoding proteins fused to VP22, and to test whether the system is sufficiently potent to allow protein delivery from transduced cells in vitro and in vivo, fusion constructs of VP22 and the enhanced green fluorescent protein (EGFP) were prepared and delivered into target cells by using HIV-1-based lentiviral vectors. To follow the spread of VP22-EGFP to other cells, transduced COS-7 cells were coplated with a number of different cell types, including brain choroid plexus cells, human endothelial cells, H9 cells, and HeLa cells. We found that VP22-EGFP fusion proteins were transported from transduced cells to recipient cells and that such fusion proteins accumulated in the nucleus and in the cytoplasm of such cells. To determine the ability to deliver fusion proteins in vivo, we injected transduced H9 cells as well as the viral vector directly into the brain of mice. We present evidence that VP22-EGFP fusion proteins were transported effectively from lentivirus transduced cells in vivo. We also show that the VP22-EGFP fusion protein encoded by the lentivirus is transported between cells. Our data indicate that such fusion proteins are present in the nucleus and in the cytoplasm of neighboring cells. Therefore, lentiviral vectors may provide a potent biological system for delivering genes encoding therapeutic proteins fused to VP22. PMID:11027330

  7. Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors: assessment of immunophenotype and differentiation capacity in vitro.

    PubMed

    van Vollenstee, Fiona A; Jackson, Carlo; Hoffmann, Danie; Potgieter, Marnie; Durandt, Chrisna; Pepper, Michael S

    2016-10-01

    Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.

  8. Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter.

    PubMed

    Cooper, Aaron R; Lill, Georgia R; Gschweng, Eric H; Kohn, Donald B

    2015-01-01

    Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors.

  9. Lentiviral Delivery of Proteins for Genome Engineering.

    PubMed

    Cai, Yujia; Mikkelsen, Jacob Giehm

    2016-01-01

    Viruses have evolved to traverse cellular barriers and travel to the nucleus by mechanisms that involve active transport through the cytoplasm and viral quirks to resist cellular restriction factors and innate immune responses. Virus-derived vector systems exploit the capacity of viruses to ferry genetic information into cells, and now - more than three decades after the discovery of HIV - lentiviral vectors based on HIV-1 have become instrumental in biomedical research and gene therapies that require genomic insertion of transgenes. By now, the efficacy of lentiviral gene delivery to stem cells, cells of the immune system including T cells, hepatic cells, and many other therapeutically relevant cell types is well established. Along with nucleic acids, HIV-1 virions carry the enzymatic tools that are essential for early steps of infection. Such capacity to package enzymes, even proteins of nonviral origin, has unveiled new ways of exploiting cellular intrusion of HIV-1. Based on early findings demonstrating the packaging of heterologous proteins into virus particles as part of the Gag and GagPol polypeptides, we have established lentiviral protein transduction for delivery of DNA transposases and designer nucleases. This strategy for delivering genome-engineering proteins facilitates high enzymatic activity within a short time frame and may potentially improve the safety of genome editing. Exploiting the full potential of lentiviral vectors, incorporation of foreign protein can be combined with the delivery of DNA transposons or a donor sequence for homology-directed repair in so-called 'all-in-one' lentiviral vectors. Here, we briefly describe intracellular restrictions that may affect lentiviral gene and protein delivery and review the current status of lentiviral particles as carriers of tool kits for genome engineering. PMID:27228988

  10. Low titer lentiviral transgenesis in rodents with simian immundeficiency virus vector.

    PubMed

    Bender, Balázs; Hoffmann, Orsolya Ivett; Negre, Didier; Kvell, Krisztián; Bősze, Zsuzsanna; Hiripi, László

    2013-09-01

    Efficient production of transgenic animals using low-titer lentiviral constructs remains challenging. Here we demonstrate that microinjection of simian immundeficiency virus-derived lentiviral constructs can produce transgenic mice and rats with high efficiency even when using low-titer virus preparations.

  11. Lentiviral Vector-Mediated Correction of a Mouse Model of Leukocyte Adhesion Deficiency Type I

    PubMed Central

    Leon-Rico, Diego; Aldea, Montserrat; Sanchez-Baltasar, Raquel; Mesa-Nuñez, Cristina; Record, Julien; Burns, Siobhan O.; Santilli, Giorgia; Thrasher, Adrian J.; Bueren, Juan A.; Almarza, Elena

    2016-01-01

    Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene and is characterized by recurrent and life-threatening bacterial infections. These mutations lead to defective or absent expression of β2 integrins on the leukocyte surface, compromising adhesion and extravasation at sites of infection. Three different lentiviral vectors (LVs) conferring ubiquitous or preferential expression of CD18 in myeloid cells were constructed and tested in human and mouse LAD-I cells. All three hCD18-LVs restored CD18 and CD11a membrane expression in LAD-I patient-derived lymphoblastoid cells. Corrected cells recovered the ability to aggregate and bind to sICAM-1 after stimulation. All vectors induced stable hCD18 expression in hematopoietic cells from mice with a hypomorphic Itgb2 mutation (CD18HYP), both in vitro and in vivo after transplantation of corrected cells into primary and secondary CD18HYP recipients. hCD18+ hematopoietic cells from transplanted CD18HYP mice also showed restoration of mCD11a surface co-expression. The analysis of in vivo neutrophil migration in CD18HYP mice subjected to two different inflammation models demonstrated that the LV-mediated gene therapy completely restored neutrophil extravasation in response to inflammatory stimuli. Finally, these vectors were able to correct the phenotype of human myeloid cells derived from CD34+ progenitors defective in ITGB2 expression. These results support for the first time the use of hCD18-LVs for the treatment of LAD-I patients in clinical trials. PMID:27056660

  12. Targeted, homology-driven gene insertion in stem cells by ZFN-loaded ‘all-in-one’ lentiviral vectors

    PubMed Central

    Cai, Yujia; Laustsen, Anders; Zhou, Yan; Sun, Chenglong; Anderson, Mads Valdemar; Li, Shengting; Uldbjerg, Niels; Luo, Yonglun; Jakobsen, Martin R; Mikkelsen, Jacob Giehm

    2016-01-01

    Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et al., 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZFN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded ‘all-in-one’ IDLVs for site-directed gene insertion in stem cell-based gene therapies. DOI: http://dx.doi.org/10.7554/eLife.12213.001 PMID:27278774

  13. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

    PubMed

    Kabadi, Ami M; Ousterout, David G; Hilton, Isaac B; Gersbach, Charles A

    2014-10-29

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

  14. Use of a Closed Culture System to Improve the Safety of Lentiviral Vector Production.

    PubMed

    Wu, Tao; Bour, Gaëtan; Durand, Sarah; Lindner, Véronique; Gossé, Francine; Zona, Laetitia; Certoux, Jean-Marie; Diana, Michele; Baumert, Thomas F; Marescaux, Jacques; Mutter, Didier; Pessaux, Patrick; Robinet, Eric

    2015-12-01

    We evaluated the possibility of introducing a combination of six oncogenes into primary porcine hepatocytes (PPH) using a lentiviral vector (LV)-mediated gene transfer in order to develop a porcine hepatocellular carcinoma model based on autologous transplantation of ex vivo-transformed hepatocytes. The six oncogenes were introduced into three plasmids, hence enabling the production of LVs encoding a luciferase reporter gene and hTERT+p53(DD), cyclinD1+CDK4(R24C), and c-myc(T58A)+HRas(G21V) genes, respectively. In order to improve the protection of the laboratory personnel manipulating such LVs, we used a compact cell culture cassette (CliniCell(®) device) as a closed cell culture system. We demonstrated that the CliniCell device allows to produce LVs, through plasmid transfection of 293T cells, and, after transfer to a second cassette, to transduce PPH with a similar efficacy as conventional open cell culture systems such as flasks or Petri dishes. Additionally, it is possible to cryopreserve at -80°C the transduced cells, directly in the CliniCell device used for the transduction. In conclusion, the use of a closed culture system for the safe handling of oncogene-encoding LVs lays the foundation for the development of porcine tumor models based on the autologous transplantation of ex vivo-transformed primary cells. PMID:26467420

  15. Design of a regulated lentiviral vector for hematopoietic stem cell gene therapy of globoid cell leukodystrophy

    PubMed Central

    Ungari, Silvia; Montepeloso, Annita; Morena, Francesco; Cocchiarella, Fabienne; Recchia, Alessandra; Martino, Sabata; Gentner, Bernhard; Naldini, Luigi; Biffi, Alessandra

    2015-01-01

    Globoid cell leukodystrophy (GLD) is a demyelinating lysosomal storage disease due to the deficiency of the galactocerebrosidase (GALC) enzyme. The favorable outcome of hematopoietic stem and progenitor cell (HSPC)-based approaches in GLD and other similar diseases suggests HSPC gene therapy as a promising therapeutic option for patients. The path to clinical development of this strategy was hampered by a selective toxicity of the overexpressed GALC in the HSPC compartment. Here, we presented the optimization of a lentiviral vector (LV) in which miR-126 regulation was coupled to codon optimization of the human GALC cDNA to obtain a selective and enhanced enzymatic activity only upon transduced HSPCs differentiation. The safety of human GALC overexpression driven by this LV was extensively demonstrated in vitro and in vivo on human HSPCs from healthy donors. No perturbation in the content of proapoptotic sphingolipids, gene expression profile, and capability of engraftment and mutlilineage differentiation in chimeric mice was observed. The therapeutic potential of this LV was then assessed in a severe GLD murine model that benefited from transplantation of corrected HSPCs with longer survival and ameliorated phenotype as compared to untreated siblings. This construct has thus been selected as a candidate for clinical translation. PMID:26509184

  16. Design of a regulated lentiviral vector for hematopoietic stem cell gene therapy of globoid cell leukodystrophy.

    PubMed

    Ungari, Silvia; Montepeloso, Annita; Morena, Francesco; Cocchiarella, Fabienne; Recchia, Alessandra; Martino, Sabata; Gentner, Bernhard; Naldini, Luigi; Biffi, Alessandra

    2015-01-01

    Globoid cell leukodystrophy (GLD) is a demyelinating lysosomal storage disease due to the deficiency of the galactocerebrosidase (GALC) enzyme. The favorable outcome of hematopoietic stem and progenitor cell (HSPC)-based approaches in GLD and other similar diseases suggests HSPC gene therapy as a promising therapeutic option for patients. The path to clinical development of this strategy was hampered by a selective toxicity of the overexpressed GALC in the HSPC compartment. Here, we presented the optimization of a lentiviral vector (LV) in which miR-126 regulation was coupled to codon optimization of the human GALC cDNA to obtain a selective and enhanced enzymatic activity only upon transduced HSPCs differentiation. The safety of human GALC overexpression driven by this LV was extensively demonstrated in vitro and in vivo on human HSPCs from healthy donors. No perturbation in the content of proapoptotic sphingolipids, gene expression profile, and capability of engraftment and mutlilineage differentiation in chimeric mice was observed. The therapeutic potential of this LV was then assessed in a severe GLD murine model that benefited from transplantation of corrected HSPCs with longer survival and ameliorated phenotype as compared to untreated siblings. This construct has thus been selected as a candidate for clinical translation.

  17. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

    PubMed

    Kabadi, Ami M; Ousterout, David G; Hilton, Isaac B; Gersbach, Charles A

    2014-10-29

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types. PMID:25122746

  18. Reduction of liver macrophage transduction by pseudotyping lentiviral vectors with a fusion envelope from Autographa californica GP64 and Sendai virus F2 domain

    PubMed Central

    Markusic, David M; van Til, Niek P; Hiralall, Johan K; Elferink, Ronald PJ Oude; Seppen, Jurgen

    2009-01-01

    Background Lentiviral vectors are well suited for gene therapy because they can mediate long-term expression in both dividing and nondividing cells. However, lentiviral vectors seem less suitable for liver gene therapy because systemically administered lentiviral vectors are preferentially sequestered by liver macrophages. This results in a reduction of available virus and might also increase the immune response to the vector and vector products. Reduction of macrophage sequestration is therefore essential for efficient lentiviral liver gene therapy. Results Fusions were made of Autographa californica GP64 and the hepatocyte specific Sendai Virus envelope proteins. Lentiviral vectors were produced with either wild type GP64, Sendai-GP64, or both wild type GP64 and Sendai-GP64 and tested in vitro and in vivo for hepatocyte and macrophage gene transfer. Sendai-GP64 pseudotyped vectors showed specific gene transfer to HepG2 hepatoma cells, with no detectable transduction of HeLa cervical carcinoma cells, and a decreased affinity for RAW mouse macrophages. Co-expression of wild type GP64 and Sendai-GP64 resulted in improved viral titers while retaining increased affinity for HepG2 cells. In vivo, the Sendai-GP64 vectors also showed decreased transduction of murine liver macrophages. Conclusion We demonstrate reduced macrophage transduction in vitro and in vivo with GP64/Sendai chimeric envelope proteins. PMID:19811629

  19. Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer.

    PubMed

    Mostoslavsky, Gustavo; Fabian, Attila J; Rooney, Sean; Alt, Frederick W; Mulligan, Richard C

    2006-10-31

    Artemis gene mutations are responsible for the development of a severe combined immunodeficiency [radiation-sensitive (RS) SCID] characterized by a severe B and T cell deficiency and a normal natural killer cell population. To establish the feasibility of a gene therapy approach to the treatment of RS-SCID, we generated a series of lentiviral vectors expressing human Artemis from different promoters and used them to transduce highly purified hematopoietic stem cells (HSCs) from Artemis knockout mice. HSCs transduced by the different viruses were transplanted into either lethally irradiated Rag-1-deficient animals or Artemis knockout mice treated with a nonmyeloablative dose of Busulfan. In both models, transplantation of HSCs transduced by a vector that used a murine phosphoglycerate kinase (PGK) promoter led to a complete functional correction of the immunodeficiency. Corrected animals displayed rescue of mature B cells with normal levels of serum immunoglobulins, together with complete rescue of the T cell compartment as evidenced by the presence of mature T lymphocytes in peripheral blood as well as normal values of thymocytes in thymus. Those B and T cells were capable of activation, as shown both by in vitro stimulation responses and in vivo after immune challenge. Overall, the results indicate that a gene therapy approach for RS-SCID involving the transplantation of genetically modified HSCs is indeed feasible. Furthermore, our studies suggest the possibility that nonmyeloablative conditioning regimens might be effectively used to promote engraftment of genetically modified cells in the case of diseases where standard irradiation-based myeloablative bone marrow transplantation protocols may prove problematic. PMID:17062750

  20. Optimization of lentiviral vector transduction into peripheral blood mononuclear cells in combination with the fibronectin fragment CH-296 stimulation.

    PubMed

    Chono, Hideto; Goto, Yumi; Yamakawa, Satoko; Tanaka, Shinya; Tosaka, Yasuhiro; Nukaya, Ikuei; Mineno, Junichi

    2011-03-01

    Large scale T-cell expansion and efficient gene transduction are required for adoptive T-cell gene therapy. Based on our previous observations, human peripheral blood mononuclear cells (PBMCs) can be expanded efficiently while conserving a naïve phenotype by stimulating with both recombinant human fibronectin fragment (CH-296) and anti-CD3 monoclonal antibodies. In this article, we explored the possibility of using this co-stimulation method to generate engineered T cells using lentiviral vector. Human PBMCs were stimulated with anti-CD3 together with immobilized CH-296 or anti-CD28 antibody as well as anti-CD3/anti-CD28 conjugated beads and transduced with lentiviral vector simultaneously. Co-stimulation with CH-296 gave superior transduction efficiency than with anti-CD28. Next, PBMCs were stimulated and transduced with anti-CD3/CH-296 or with anti-CD3/CD28 beads. T-cell expansion, gene transfer efficiencies and immunophenotypes were analysed. Stimulation with anti-CD3/CH-296 resulted in more than 10-times higher cell expansion and higher gene transfer efficiency with conservation of the naïve phenotype compared with anti-CD3/CD28 stimulation method. Thus, lentiviral transduction with anti-CD3/CH-296 co-stimulation is an efficient way to generate large numbers of genetically modified T cells and may be suitable for many gene therapy protocols that use adoptive T-cell transfer therapy.

  1. Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation.

    PubMed

    Dhamne, Hemant; Chande, Ajit G; Mukhopadhyaya, Robin

    2014-01-01

    Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance.

  2. Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation.

    PubMed

    Dhamne, Hemant; Chande, Ajit G; Mukhopadhyaya, Robin

    2014-01-01

    Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance. PMID:24325878

  3. Factors that influence VSV-G pseudotyping and transduction efficiency of lentiviral vectors-in vitro and in vivo implications.

    PubMed

    Farley, Daniel C; Iqball, Sharifah; Smith, Joanne C; Miskin, James E; Kingsman, Susan M; Mitrophanous, Kyriacos A

    2007-05-01

    Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.

  4. Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A.

    PubMed

    Wang, Xuefeng; Shin, Simon C; Chiang, Andy F J; Khan, Iram; Pan, Dao; Rawlings, David J; Miao, Carol H

    2015-04-01

    Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency. PMID:25655313

  5. Intraosseous Delivery of Lentiviral Vectors Targeting Factor VIII Expression in Platelets Corrects Murine Hemophilia A

    PubMed Central

    Wang, Xuefeng; Shin, Simon C; Chiang, Andy F J; Khan, Iram; Pan, Dao; Rawlings, David J; Miao, Carol H

    2015-01-01

    Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage-Sca1+c-Kit+ hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency. PMID:25655313

  6. Efficient Downregulation of Multiple mRNA Targets with a Single shRNA-Expressing Lentiviral Vector

    PubMed Central

    Chumakov, Stepan P.; Kravchenko, Julia E.; Prassolov, Vladimir S.; Frolova, Elena I.; Chumakov, Peter M.

    2010-01-01

    Gene silencing based on RNA interference is widely used in fundamental research and in practical applications. However, a commonly incomplete functional suppression represents a serious drawback of this technology. We describe a series of lentiviral vectors each containing a single or multiple shRNA expression cassette(s) driven by a RNA polymerase III specific promoter and localized within the 3′-LTR of the lentiviral DNA backbone. The vectors also contain an antibiotic-resistance gene that allows positive selection of recipient cells. The combined expression of three different shRNAs specific to a single mRNA was shown to improve dramatically the level of mRNA inhibition, while the use of three different RNA polymerase III specific promoters avoids the loss of shRNA expression cassettes through the homologous recombination. The vector system was used for successful simultaneous suppression of three related SESN1, SESN2 and SESN3 genes, which suggests its particular value for testing phenotypes of functionally redundant genes. PMID:20064551

  7. Efficient downregulation of multiple mRNA targets with a single shRNA-expressing lentiviral vector.

    PubMed

    Chumakov, Stepan P; Kravchenko, Julia E; Prassolov, Vladimir S; Frolova, Elena I; Chumakov, Peter M

    2010-05-01

    Gene silencing based on RNA interference is widely used in fundamental research and in practical applications. However, a commonly incomplete functional suppression represents a serious drawback of this technology. We describe a series of lentiviral vectors each containing a single or multiple shRNA-expression cassette(s) driven by a RNA-polymerase III specific promoter and localized within the 3'-LTR of the lentiviral DNA backbone. The vectors also contain an antibiotic-resistance gene that allows positive selection of recipient cells. The combined expression of three different shRNAs specific to a single mRNA was shown to improve dramatically the level of mRNA inhibition, while the use of three different RNA-polymerase III specific promoters avoids the loss of shRNA-expression cassettes through the homologous recombination. The vector system was used for successful simultaneous suppression of three related SESN1, SESN2 and SESN3 genes, which suggests its particular value for testing phenotypes of functionally redundant genes.

  8. Construction of a lentiviral vector encoding heme oxygenase 1 and its introduction into mouse adipose tissue-derived stem cells.

    PubMed

    Zhu, C H; Lei, W; Chen, Z R

    2015-09-09

    Many studies exist concerning the use of stem cells as delivery vehicles in gene therapy, expressing genes such as vascular endothelial growth factor 165 and hepatocyte growth factor. However, few reports regarding adipose tissue-derived stem cells (ADSCs) and the heme oxygenase 1 (HO-1) gene have been published. Therefore, we established a lentiviral vector encoding HO-1 and used this to infect ADSCs with the aim of producing therapeutic seed cells. In this study, ADSCs were isolated from mouse adipose tissue (AT), cultured, and identified according to the expression of antigens on their cell surface and their capacity for multilineage differentiation. A lentiviral vector encoding HO-1 was constructed, ADSCs were infected with this, and HO-1 protein expression was examined by western blotting. Our results show that ADSCs can be isolated from mouse AT, while DNA sequencing demonstrated that HO-1 was successfully transferred to the vector fused with GFP. Following 293T cell transfection, lentivirus titers were approximately 3 x 10(8) TU/mL. Fluorescence microscopy confirmed the expression of the HO-1 construct in lentivirus-infected ADSCs and the overexpression of HO-1 protein in these cells was verified by western blot. The production of ADSCs overexpressing HO-1 described in this study may aid in the development of a novel method for the treatment of asthma.

  9. Characterization and comparative performance of lentiviral vector preparations concentrated by either one-step ultrafiltration or ultracentrifugation.

    PubMed

    Papanikolaou, Eleni; Kontostathi, Georgia; Drakopoulou, Ekati; Georgomanoli, Maria; Stamateris, Evangelos; Vougas, Kostas; Vlahou, Antonia; Maloy, Andrew; Ware, Mark; Anagnou, Nicholas P

    2013-07-01

    Gene therapy utilizing lentiviral vectors (LVs) constitutes a real therapeutic alternative for many inherited monogenic diseases. Therefore, the generation of functional vectors using fast, non-laborious and cost-effective strategies is imperative. Among the available concentration methods for VSV-G pseudotyped lentiviruses to achieve high therapeutic titers, ultracentrifugation represents the most common approach. However, the procedure requires special handling and access to special instrumentation, it is time-consuming, and most importantly, it is cost-ineffective due to the high maintenance expenses and consumables of the ultracentrifuge apparatus. Here we describe an improved protocol in which vector stocks are prepared by transient transfection using standard cell culture media and are then concentrated by ultrafiltration, resulting in functional vector titers of up to 6×10(9) transducing units per millilitre (TU/ml) without the involvement of any purification step. Although ultrafiltration per se for concentrating viruses is not a new procedure, our work displays one major novelty; we characterized the nature and the constituents of the viral batches produced by ultrafiltration using peptide mass fingerprint analysis. We also determined the viral functional titer by employing flow cytometry and evaluated the actual viral particle size and concentration in real time by using laser-based nanoparticle tracking analysis based on Brownian motion. Vectors generated by this production method are contained in intact virions and when tested to transduce in vitro either murine total bone marrow or human CD34(+) hematopoietic stem cells, resulted in equal transduction efficiency and reduced toxicity, compared to lentiviral vectors produced using standard ultracentrifugation-based methods. The data from this study can eventually lead to the improvement of protocols and technical modifications for the clinical trials for gene therapy.

  10. Development of an Equine-Tropic Replication-Competent Lentivirus Assay for Equine Infectious Anemia Virus-Based Lentiviral Vectors

    PubMed Central

    Bannister, Richard; Leroux-Carlucci, Marie A.; Evans, Nerys E.; Miskin, James E.; Mitrophanous, Kyriacos A.

    2012-01-01

    Abstract The release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (RCL). Vectors derived from the nonprimate lentivirus equine infectious anemia virus (EIAV) have been directly administered to patients in several clinical trials, with no toxicity observed to date. Because EIAV does not replicate in human cells, and because putative RCLs derived from vector components within human vector production cells would most likely be human cell-tropic, we previously developed an RCL assay using amphotropic murine leukemia virus (MLV) as a surrogate positive control and human cells as RCL amplification/indicator cells. Here we report an additional RCL assay that tests for the presence of theoretical “equine-tropic” RCLs. This approach provides further assurance of safety by detecting putative RCLs with an equine cell-specific tropism that might not be efficiently amplified by the human cell-based RCL assay. We tested the ability of accessory gene-deficient EIAV mutant viruses to replicate in a highly permissive equine cell line to direct our choice of a suitable EIAV-derived positive control. In addition, we report for the first time the mathematical rationale for use of the Poisson distribution to calculate minimal infectious dose of positive control virus and for use in monitoring assay positive/spike control failures in accumulating data sets. No RCLs have been detected in Good Manufacturing Practice (GMP)-compliant RCL assays to date, further demonstrating that RCL formation is highly unlikely in contemporary minimal lentiviral vector systems. PMID:23121195

  11. High-titer lentiviral vectors stimulate fetal calf serum-specific human CD4 T-cell responses: implications in human gene therapy.

    PubMed

    Bao, L; Guo, H; Huang, X; Tammana, S; Wong, M; McIvor, R S; Zhou, X

    2009-06-01

    Human immunodeficiency virus-1-derived lentiviral vectors have been increasingly used for gene delivery in both pre-clinical and clinical models. Numerous studies have shown that dendritic cells (DC) transduced with concentrated lentiviral vectors can induce primary T-cell responses to viral and tumor antigens. In this study, we attempted to generate influenza hemagglutinin-specific CD4 T cells using lentiviral vectors containing the signal sequence and human lysosome-associated membrane protein to target hemagglutinin to the major histocompatibility complex class II processing pathway. Autologous dendritic cells were generated in serum-free medium and transduced with concentrated, high-titer lentiviruses to stimulate autologous T cells. Unexpectedly, we failed to generate influenza hemagglutinin-specific CD4 T cells rather than T cells specific for fetal calf serum (FCS). By limiting dilution, we established several FCS-specific CD4 T-cell clones restricted by human leukocyte antigen-DR1 and human leukocyte antigen-DR4. Lentiviruses produced in human serum-adapted 293 cells or in serum-free medium were unable to sensitize dendritic cells for recognition by FCS-specific CD4 T-cell clones. Our results indicate that residual FCS in concentrated lentiviral pellets is, in part, responsible for its immunogenicity. These FCS-specific CD4 T cells may be useful in testing clinical grade lentiviral vectors for the presence of contaminating FCS.

  12. Generation and usage of aequorin lentiviral vectors for Ca(2+) measurement in sub-cellular compartments of hard-to-transfect cells.

    PubMed

    Lim, Dmitry; Bertoli, Alessandro; Sorgato, M Catia; Moccia, Francesco

    2016-05-01

    Targeted aequorin-based Ca(2+) probes represent an unprecedented tool for the reliable measurement of Ca(2+) concentration and dynamics in different sub-cellular compartments. The main advantages of aequorin are its proteinaceous nature, which allows attachment of a signal peptide for targeting aequorin to virtually any sub-cellular compartment; its low Ca(2+)-binding capacity; the wide range of Ca(2+) concentrations that can be measured, ranging from sub-micromolar to millimolar; its robust performance in aggressive environments, e.g., the strong acidic pH of the lysosomal lumen. Lentiviral vectors represent a popular tool to transduce post-mitotic or hard-to-transfect cells both in vitro and in vivo. Furthermore, it has great potential for gene therapy. Last generation lentiviral vectors represent a perfect compromise for combining large insert size, ease of production and handling, and high degree of biosafety. Here, we describe strategies for cloning aequorin probes - targeted to the cytosol, sub-plasma membrane cytosolic domains, the mitochondrial matrix, and the endoplasmic reticulum lumen - into lentiviral vectors. We describe methods for the production of lentiviral particles, and provide examples of measuring Ca(2+) dynamics by such aequorin-encoding lentiviral vectors in sub-cellular compartments of hard-to-transfect cells, including immortalized striatal neurons, primary cerebellar granule neurons and endothelial progenitor cells, which provide suitable in vitro models for the study of different human diseases. PMID:26992273

  13. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.

    PubMed

    Merten, Otto-Wilhelm; Charrier, Sabine; Laroudie, Nicolas; Fauchille, Sylvain; Dugué, Céline; Jenny, Christine; Audit, Muriel; Zanta-Boussif, Maria-Antonietta; Chautard, Hélène; Radrizzani, Marina; Vallanti, Giuliana; Naldini, Luigi; Noguiez-Hellin, Patricia; Galy, Anne

    2011-03-01

    From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.

  14. Integrase-Deficient Lentiviral Vectors Mediate Efficient Gene Transfer to Human Vascular Smooth Muscle Cells with Minimal Genotoxic Risk

    PubMed Central

    Chick, Helen E.; Nowrouzi, Ali; Fronza, Raffaele; McDonald, Robert A.; Kane, Nicole M.; Alba, Raul; Delles, Christian; Sessa, William C.; Schmidt, Manfred; Thrasher, Adrian J.

    2012-01-01

    Abstract We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer. PMID:22931362

  15. Dual promoter lentiviral vector generates transgenic mice expressing E2-CSFV glycoprotein in their milk, but impairs early identification of transgenic embryos.

    PubMed

    Ramos, Oliberto Sánchez; Carratalá, Yanet Prieto; Puerta, Silvia Gómez; Pereira, Natalie C Parra; Amarán, Lester Suárez; Chaves, Silvana P Jimenez; Alonso, Jorge R Toledo

    2011-04-15

    Lentiviral vectors containing the green fluorescent protein gene have been successfully used to select transgenic embryos before transfer to a surrogate mother. However, there are apparently no reports regarding early detection of transgenic embryos using a lentiviral vector carrying an additional transcription unit for tissue-specific expression of a valuable protein. In this study, two HIV-based lentiviral vectors were constructed. The first one contained the green fluorescent protein (GFP) coding sequence driven by the early SV40 promoter (Lv-G), whereas the other contained an additional transcription unit for the expression of E2 glycoprotein from classical swine fever virus, driven by a 1.5 kb αS1casein promoter from water buffalo (Lv-αS1cE2hisG). Microinjection of single-cell mouse embryos with Lv-G lentiviral vector rendered embryos which were GFP-positive, beginning at the four-cell stage. Of 33 mice born, 28 (81%) carried the transgene DNA and 15 (55.5%) were GFP-positive. Microinjection of Lv-αS1cE2hisG lentiviral vector yielded 28 mice born; although 24 (85%) carried the transgene DNA, none were GFP-positive, suggesting that the tissue-specific expression cassette interfered with expression of the ubiquitous trancriptional unit. In Lv-αS1cE2hisG transgenic mice, E2his was expressed in milk as a homodimer (at concentrations ≤ 0.422 mg/mL). This was apparently the first report of expression of a recombinant protein in the milk of transgenic animals generated by lentiviral transgenesis.

  16. Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors

    PubMed Central

    Hu, Peirong; Li, Yedda; Sands, Mark S; McCown, Thomas; Kafri, Tal

    2015-01-01

    The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors. Here, we describe the development of the first tetracycline-inducible stable IDLV packaging cell line comprising the D64E integrase mutant and the VSV-G envelope protein. A conditional self-inactivating (cSIN) vector and a novel polypurine tract (PPT)-deleted vector were incorporated into the newly developed stable packaging cell line by transduction and stable transfection, respectively. High-titer (~107 infectious units (IU)/ml) cSIN vectors were routinely generated. Furthermore, screening of single-cell clones stably transfected with PPT-deleted vector DNA resulted in the identification of highly efficient producer cell lines generating IDLV titers higher than 108 IU/mL, which upon concentration increased to 1010 IU/ml. IDLVs generated by stable producer lines efficiently transduce CNS tissues of rodents. Overall, the availability of high-titer IDLV lentivirus packaging cell line described here will significantly facilitate IDLV-based basic science research, as well as preclinical and clinical applications. PMID:26229972

  17. Innocuous full-length botulinum neurotoxin targets and promotes the expression of lentiviral vectors in central and autonomic neurons.

    PubMed

    O'Leary, V B; Ovsepian, S V; Raghunath, A; Huo, Q; Lawrence, G W; Smith, L; Dolly, J O

    2011-07-01

    Fragments of botulinum neurotoxin (BoNT) have been explored as potential targeting moieties and carriers of biomolecules into neurons, although with lower binding and translocation efficiency compared with intact proteins. This study exploits a detoxified recombinant form of full-length BoNT/B (BoTIM/B) fused with core streptavidin (CS-BoTIM/B) for lentiviral targeting to central and autonomic neurons. CS-BoTIM/B underwent an activity-dependent entry into cultured spinal cord neurons. Coupling CS-BoTIM/B to biotinylated lentivirus-encoding green fluorescent protein (GFP) endowed considerable neuron selectivity to the vector as evident from the preferential expression of the reporter in neurons co-cultured with skeletal muscle cells. CS-BoTIM/B-guided lentiviral transduction with the expression of a SNARE protein, SNAP-25 (S25), rendered non-susceptible to proteolysis by three BoNT serotypes, yielded a sizable decrease in cleaved S25 upon exposure of spinal cord neurons to these toxins. This was accompanied by synaptic transmission being spared from blockade by BoNT/A or BoNT/E, reflecting adequate translation and functional competence of recombinant multi-toxin-resistant S25. The augmented neurotropism conveyed on the lentivirus by CS-BoTIM/B was also demonstrated in vivo through enhanced expression of a reporter in intramural ganglionic neurons in the rat trachea, after injection of the targeted GFP-encoding lentivirus. Thus, a novel and realistic prospect for gene therapy of peripheral neuropathies is offered in this study through lentiviral targeting to neurons by CS-BoTIM/B.

  18. Comparative Genomic Integration Profiling of Sleeping Beauty Transposons Mobilized With High Efficacy From Integrase-defective Lentiviral Vectors in Primary Human Cells

    PubMed Central

    Moldt, Brian; Miskey, Csaba; Staunstrup, Nicklas Heine; Gogol-Döring, Andreas; Bak, Rasmus O; Sharma, Nynne; Mátés, Lajos; Izsvák, Zsuzsanna; Chen, Wei; Ivics, Zoltán; Mikkelsen, Jacob Giehm

    2011-01-01

    It has been previously shown that integrase-defective HIV-1-based gene vectors can serve, with moderate efficiency, as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) transposase. Here, we describe the enhanced gene transfer properties of a HIV-1/SB hybrid vector that allows efficient DNA transposition, facilitated by the hyperactive SB100X transposase, from vector DNA intermediates in primary human cells. Potent transposase-dependent integration of genetic cargo carried by the hybrid HIV-1/SB vector (up to 160-fold above background) is reported in human cell lines as well as in primary human fibroblasts and keratinocytes. The efficiency of transgene integration in context of the newly developed hybrid vector is comparable with that of conventional lentiviral vectors (LVs). Integration profiles of integrating HIV-1-derived vectors and SB transposons mobilized from LVs are investigated by deep sequencing of a large number of integration sites. A significant bias of lentiviral integrations in genes is reported, confirming that biological properties of the viral integration machinery facilitate preferred insertion into actively transcribed genomic regions. In sharp contrast, lentiviral insertions catalyzed by the SB100X transposase are far more random with respect to genes. Based on these properties, HIV-1/SB vectors may become valuable tools for genetic engineering and therapeutic gene transfer. PMID:21468003

  19. Virological and preclinical characterization of a dendritic cell targeting, integration-deficient lentiviral vector for cancer immunotherapy.

    PubMed

    Odegard, Jared M; Kelley-Clarke, Brenna; Tareen, Semih U; Campbell, David J; Flynn, Patrick A; Nicolai, Christopher J; Slough, Megan M; Vin, Chintan D; McGowan, Patrick J; Nelson, Lisa T; Ter Meulen, Jan; Dubensky, Thomas W; Robbins, Scott H

    2015-01-01

    Dendritic cells (DCs) are essential antigen-presenting cells for the initiation of cytotoxic T-cell responses and therefore attractive targets for cancer immunotherapy. We have developed an integration-deficient lentiviral vector termed ID-VP02 that is designed to deliver antigen-encoding nucleic acids selectively to human DCs in vivo. ID-VP02 utilizes a genetically and glycobiologically engineered Sindbis virus glycoprotein to target human DCs through the C-type lectin DC-SIGN (CD209) and also binds to the homologue murine receptor SIGNR1. Specificity of ID-VP02 for antigen-presenting cells in the mouse was confirmed through biodistribution studies showing that following subcutaneous administration, transgene expression was only detectable at the injection site and the draining lymph node. A single immunization with ID-VP02 induced a high level of antigen-specific, polyfunctional effector and memory CD8 T-cell responses that fully protected against vaccinia virus challenge. Upon homologous readministration, ID-VP02 induced a level of high-quality secondary effector and memory cells characterized by stable polyfunctionality and expression of IL-7Rα. Importantly, a single injection of ID-VP02 also induced robust cytotoxic responses against an endogenous rejection antigen of CT26 colon carcinoma cells and conferred both prophylactic and therapeutic antitumor efficacy. ID-VP02 is the first lentiviral vector which combines integration deficiency with DC targeting and is currently being investigated in a phase I trial in cancer patients. PMID:25658613

  20. Co-transduction of lentiviral and adenoviral vectors for co-delivery of growth factor and shRNA genes in mesenchymal stem cells-based chondrogenic system.

    PubMed

    Zhang, Feng; Yao, Yongchang; Su, Kai; Fang, Yu; Citra, Fudiman; Wang, Dong-An

    2015-09-01

    Gene delivery takes advantage of cellular mechanisms to express gene products and is an efficient way to deliver them into cells, influencing cellular behaviours and expression patterns. Among the delivery methods, viral vectors are applied due to their high efficiency. Two typical viral vectors for gene delivery include lentiviral vector for integrative transduction and adenoviral vector for transient episomal transduction, respectively. The selection and formulation of proper viral vectors applied to cells can modulate gene expression profiles and further impact the downstream pathways. In this study, recombinant lentiviral and adenoviral vectors were co-transduced in a synovial mesenchymal stem cells (SMSCs)-based articular chondrogenic system by which two transgenes were co-delivered - the gene for transforming growth factor (TGF)β3, to facilitate SMSC chondrogenesis, and the gene for small hairpin RNA (shRNA), targeting the mRNA of type I collagen (Col I) α1 chain to silence Col I expression and minimize fibrocartilage formation. Delivery of either gene could be achieved with either lentiviral or adenoviral vectors. Therefore, co-delivery of the two transgenes via the two types of vectors was performed to determine which combination was optimal for three-dimensional (3D) articular chondrogenesis to construct articular hyaline cartilage tissue. Suppression of Col I and expression of cartilage markers, including type II collagen, aggrecan and cartilage oligomeric matrix protein (COMP), were assessed at both the transcriptome and protein phenotypic levels. It was concluded that the combination of lentiviral-mediated TGFβ3 release and adenoviral-mediated shRNA expression (LV-T + Ad-sh) generally demonstrated optimal efficacy in engineered articular cartilage with SMSCs.

  1. Transduction of Photoreceptors With Equine Infectious Anemia Virus Lentiviral Vectors: Safety and Biodistribution of StarGen for Stargardt Disease

    PubMed Central

    Binley, Katie; Widdowson, Peter; Loader, Julie; Kelleher, Michelle; Iqball, Sharifah; Ferrige, Georgina; de Belin, Jackie; Carlucci, Marie; Angell-Manning, Diana; Hurst, Felicity; Ellis, Scott; Miskin, James; Fernandes, Alcides; Wong, Paul; Allikmets, Rando; Bergstrom, Christopher; Aaberg, Thomas; Yan, Jiong; Kong, Jian; Gouras, Peter; Prefontaine, Annick; Vezina, Mark; Bussieres, Martin; Naylor, Stuart; Mitrophanous, Kyriacos A.

    2013-01-01

    Purpose. StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed. Methods. Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery. Results. Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment. Conclusions. In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration. PMID:23620430

  2. MAC-T cells as a tool to evaluate lentiviral vector construction targeting recombinant protein expression in milk.

    PubMed

    Monzani, Paulo S; Guemra, Samuel; Adona, Paulo R; Ohashi, Otavio M; Meirelles, Flávio V; Wheeler, Matthew B

    2015-01-01

    Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk. PMID:25380466

  3. Transduction of Human CD34+ Repopulating Cells with a Self-Inactivating Lentiviral Vector for SCID-X1 Produced at Clinical Scale by a Stable Cell Line

    PubMed Central

    Lockey, Timothy; Mehta, Perdeep K.; Kim, Yoon-Sang; Eldridge, Paul W.; Gray, John T.; Sorrentino, Brian P.

    2012-01-01

    Abstract Self-inactivating (SIN)-lentiviral vectors have safety and efficacy features that are well suited for transduction of hematopoietic stem cells (HSCs), but generation of vector at clinical scale has been challenging. Approximately 280 liters of an X-Linked Severe Combined Immunodeficiency Disorder (SCID-X1) SIN-lentiviral vector in two productions from a stable cell line were concentrated to final titers of 4.5 and 7.2×108 tu/ml. These two clinical preparations and three additional development-scale preparations were evaluated in human CD34+ hematopoietic cells in vitro using colony forming cell (CFU-C) assay and in vivo using the NOD/Lt-scid/IL2Rγnull (NSG) mouse xenotransplant model. A 40-hour transduction protocol using a single vector exposure conferred a mean NSG repopulating cell transduction of 0.23 vector genomes/human genome with a mean myeloid vector copy number of 3.2 vector genomes/human genome. No adverse effects on engraftment were observed from vector treatment. Direct comparison between our SIN-lentiviral vector using a 40-hour protocol and an MFGγc γ-retroviral vector using a five-day protocol demonstrated equivalent NSG repopulating cell transduction efficiency. Clonality survey by linear amplification-mediated polymerase chain reaction (LAM-PCR) with Illumina sequencing revealed common clones in sorted myeloid and lymphoid populations from engrafted mice demonstrating multipotent cell transduction. These vector preparations will be used in two clinical trials for SCID-X1. PMID:23075105

  4. Exclusive Transduction of Human CD4+ T Cells upon Systemic Delivery of CD4-Targeted Lentiviral Vectors.

    PubMed

    Zhou, Qi; Uhlig, Katharina M; Muth, Anke; Kimpel, Janine; Lévy, Camille; Münch, Robert C; Seifried, Janna; Pfeiffer, Anett; Trkola, Alexandra; Coulibaly, Cheick; von Laer, Dorothee; Wels, Winfried S; Hartwig, Udo F; Verhoeyen, Els; Buchholz, Christian J

    2015-09-01

    Playing a central role in both innate and adaptive immunity, CD4(+) T cells are a key target for genetic modifications in basic research and immunotherapy. In this article, we describe novel lentiviral vectors (CD4-LV) that have been rendered selective for human or simian CD4(+) cells by surface engineering. When applied to PBMCs, CD4-LV transduced CD4(+) but not CD4(-) cells. Notably, also unstimulated T cells were stably genetically modified. Upon systemic or intrasplenic administration into mice reconstituted with human PBMCs or hematopoietic stem cells, reporter gene expression was predominantly detected in lymphoid organs. Evaluation of GFP expression in organ-derived cells and blood by flow cytometry demonstrated exclusive gene transfer into CD4(+) human lymphocytes. In bone marrow and spleen, memory T cells were preferentially hit. Toward therapeutic applications, we also show that CD4-LV can be used for HIV gene therapy, as well as for tumor therapy, by delivering chimeric Ag receptors. The potential for in vivo delivery of the FOXP3 gene was also demonstrated, making CD4-LV a powerful tool for inducible regulatory T cell generation. In summary, our work demonstrates the exclusive gene transfer into a T cell subset upon systemic vector administration opening an avenue toward novel strategies in immunotherapy.

  5. Vaccination with lentiviral vector expressing the nfa1 gene confers a protective immune response to mice infected with Naegleria fowleri.

    PubMed

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Yang, Hee-Jong; Chwae, Yong-Joon; Kim, Kyongmin; Park, Sun; Shin, Ho-Joon

    2013-07-01

    Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection. PMID:23677321

  6. Optimization of lentiviral vectors generation for biomedical and clinical research purposes: contemporary trends in technology development and applications.

    PubMed

    Kumar, Pankaj; Woon-Khiong, Chan

    2011-04-01

    Classical non-viral methods of gene transfer, such as chemical transfection, have met with limited success of instillation of genetic material into non-proliferating cells in vitro. Among the different kinds of viral vectors, Lentiviral vectors (LVs) have emerged as robust and versatile tool for ex vivo and in vivo gene delivery into multiple cell types including non-dividing cells such as neurons. The capacity of LVs to maintain stable, long-term transgene expression and the substantial flexibility in the design of the expression cassettes account for their increasing use in various pre-clinical and clinical applications. Additionally, LVs have been hugely successful in reprogramming induced pluripotent stem cells (iPSCs). Recent development using LVs in conjunction with a Cre-Lox based reversible system has opened up many new possibilities towards therapeutic application of iPSC technology in various clinical settings. Moreover, improvements in term of biosafety and efficacy, achieved either by modifying the vector design or by involving integration-deficient LVs (IDLVs), have important implications for adoption of LV as the vector of choice for clinical trials. Several human gene therapy clinical trials evaluating the use of LVs for treatment: of human diseases such as Parkinson's disease, β-thalassemia, X-linked adrenoleukodystrophy (ALD), and AIDS are currently ongoing. This review will describe the state of the art achieved by LV technology, its impact on biomedical research, and implications to human clinical trials as therapeutic gene delivery vehicle for a wide range of infectious and genetic diseases.

  7. Vaccination with lentiviral vector expressing the nfa1 gene confers a protective immune response to mice infected with Naegleria fowleri.

    PubMed

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Yang, Hee-Jong; Chwae, Yong-Joon; Kim, Kyongmin; Park, Sun; Shin, Ho-Joon

    2013-07-01

    Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection.

  8. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

    PubMed

    Hart, Bryan E; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D; Lukose, Regy; Souther, Sommer J R; Rayasam, Swati D G; Saelens, Joseph W; Chen, Ching-Ju; Seay, Sarah A; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E; Ng, Tony W; Tobin, David M; Porcelli, Steven A; Larsen, Michelle H; Schmitz, Joern E; Haynes, Barton F; Jacobs, William R; Lee, Sunhee; Frothingham, Richard

    2015-07-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. PMID:25924766

  9. Lentiviral vectors carrying enhancer elements of Hb9 promoter drive selective transgene expression in mouse spinal cord motor neurons.

    PubMed

    Peviani, Marco; Kurosaki, Mami; Terao, Mineko; Lidonnici, Dario; Gensano, Francesco; Battaglia, Elisa; Tortarolo, Massimo; Piva, Roberto; Bendotti, Caterina

    2012-03-30

    Recombinant lentiviral vectors (rLVs) have emerged as versatile tools for gene delivery applications due to a number of favorable features, such as the possibility to maintain long-term transgene expression, the flexibility in the design of the expression cassettes and recent improvements in their biosafety profile. Since rLVs are able to infect multiple cell types including post-mitotic cells such as neurons and skeletal muscle cells, several studies have been exploring their application for the study and cure of neurodegenerative diseases. In particular, the introduction of rLVs carrying cell-type specific promoters could restrict the transgene expression either to neuronal or glial cells, thus helping to better dissect in vivo the role played by these cell populations in several neurodegenerative processes. In this study we developed rLVs carrying motor neuron specific regulatory sequences derived from the promoter of homeobox gene Hb9, and demonstrated that these constructs can represent a suitable platform for selective gene-targeting of murine spinal cord motor neurons, in vivo. This tool could be instrumental in the dissection of the molecular mechanisms involved in the selective degeneration of motor neurons occurring in Motor Neuron Diseases.

  10. In utero lung gene transfer using adeno-associated viral and lentiviral vectors in mice.

    PubMed

    Joyeux, Luc; Danzer, Enrico; Limberis, Maria P; Zoltick, Philip W; Radu, Antoneta; Flake, Alan W; Davey, Marcus G

    2014-06-01

    Virus-mediated gene transfer to the fetal lung epithelium holds considerable promise for the therapeutic management of prenatally diagnosed, potentially life-threatening inherited lung diseases. In this study we hypothesized that efficient and life-long lung transduction can be achieved by in utero gene therapy, using viral vectors. To facilitate diffuse entry into the lung, viral vector was injected into the amniotic sac of C57BL/6 mice on embryonic day 16 (term, ∼ 20 days) in a volume of 10 μl. Vectors investigated included those based on adeno-associated virus (AAV) (serotypes 5, 6.2, 9, rh.64R1) and vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1-based lentivirus (LV). All vectors expressed green fluorescent protein (GFP) under the transcriptional control of various promoters including chicken β-actin (CB) or cytomegalovirus (CMV) for AAV and CMV or MND (myeloproliferative sarcoma virus enhancer, negative control region deleted) for LV. Pulmonary GFP gene expression was detected by fluorescence stereoscopic microscopy and immunohistochemistry for up to 9 months after birth. At equivalent vector doses (mean, 12 × 10(10) genome copies per fetus) three AAV vectors resulted in long-term (up to 9 months) pulmonary epithelium transduction. AAV2/6.2 transduced predominantly cells of the conducting airway epithelium, although transduction decreased 2 months after vector delivery. AAV2/9-transduced cells of the alveolar epithelium with a type 1 pneumocyte phenotype for up to 6 months. Although minimal levels of GFP expression were observed with AAV2/5 up to 9 months, the transduced cells immunostained positive for F480 and were retrievable by bronchoalveolar lavage, confirming an alveolar macrophage phenotype. No GFP expression was observed in lung epithelial cells after AAV2/rh.64R1 and VSV-G-LV vector-mediated gene transfer. We conclude that these experiments demonstrate that prenatal lung gene transfer with AAV vectors engineered to target

  11. The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

    PubMed Central

    Papanikolaou, Eleni; Georgomanoli, Maria; Stamateris, Evangelos; Panetsos, Fottes; Karagiorga, Markisia; Tsaftaridis, Panagiotis; Graphakos, Stelios

    2012-01-01

    Abstract To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated self-inactivating (SIN) lentiviral vector, termed GGHI, carrying the Aγ-globin gene with the −117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of Aγ-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34+ hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with β-thalassemia. Our results show that GGHI increased the production of γ-globin by 32.9% as measured by high-performance liquid chromatography (p=0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of γ-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro. PMID:21875313

  12. Transgenic expression of human glial cell line-derived neurotrophic factor from integration-deficient lentiviral vectors is neuroprotective in a rodent model of Parkinson's disease.

    PubMed

    Lu-Nguyen, Ngoc B; Broadstock, Martin; Schliesser, Maximilian G; Bartholomae, Cynthia C; von Kalle, Christof; Schmidt, Manfred; Yáñez-Muñoz, Rafael J

    2014-07-01

    Standard integration-proficient lentiviral vectors (IPLVs) are effective at much lower doses than other vector systems and have shown promise for gene therapy of Parkinson's disease (PD). Their main drawback is the risk of insertional mutagenesis. The novel biosafety-enhanced integration-deficient lentiviral vectors (IDLVs) may offer a significant enhancement in biosafety, but have not been previously tested in a model of a major disease. We have assessed biosafety and transduction efficiency of IDLVs in a rat model of PD, using IPLVs as a reference. Genomic insertion of lentivectors injected into the lesioned striatum was studied by linear amplification-mediated polymerase chain reaction (PCR), followed by deep sequencing and insertion site analysis, demonstrating lack of significant IDLV integration. Reporter gene expression studies showed efficient, long-lived, and transcriptionally targeted expression from IDLVs injected ahead of lesioning in the rat striatum, although at somewhat lower expression levels than from IPLVs. Transgenic human glial cell line-derived neurotrophic factor (hGDNF) expression from IDLVs was used for a long-term investigation of lentivector-mediated, transcriptionally targeted neuroprotection in this PD rat model. Vectors were injected before striatal lesioning, and the results showed improvements in nigral dopaminergic neuron survival and behavioral tests regardless of lentiviral integration proficiency, although they confirmed lower expression levels of hGDNF from IDLVs. These data demonstrate the effectiveness of IDLVs in a model of a major disease and indicate that these vectors could provide long-term PD treatment at low dose, combining efficacy and biosafety for targeted central nervous system applications.

  13. Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects.

    PubMed

    Hu, Peirong; Li, Yedda; Nikolaishvili-Feinberg, Nana; Scesa, Giuseppe; Bi, Yanmin; Pan, Dao; Moore, Dominic; Bongarzone, Ernesto R; Sands, Mark S; Miller, Ryan; Kafri, Tal

    2016-11-01

    Currently, presymtomatic hematopoietic stem and progenitor cell transplantation (HSPCT) is the only therapeutic modality that alleviates Krabbe's disease (KD)-induced central nervous system damage. However, all HSPCT-treated patients exhibit severe deterioration in peripheral nervous system function characterized by major motor and expressive language pathologies. We hypothesize that a combination of several mechanisms contribute to this phenomenon, including 1) nonoptimal conditioning protocols with consequent inefficient engraftment and biodistribution of donor-derived cells and 2) insufficient uptake of donor cell-secreted galactocerebrosidease (GALC) secondary to a naturally low expression level of the cation-independent mannose 6-phosphate-receptor (CI-MPR). We have characterized the effects of a busulfan (Bu) based conditioning regimen on the efficacy of HSPCT in prolonging twi mouse average life span. There was no correlation between the efficiency of bone marrow engraftment of donor cells and twi mouse average life span. HSPCT prolonged the average life span of twi mice, which directly correlated with the aggressiveness of the Bu-mediated conditioning protocols. HSPC transduced with lentiviral vectors carrying the GALC cDNA under control of cell-specific promoters were efficiently engrafted in twi mouse bone marrow. To facilitate HSPCT-mediated correction of GALC deficiency in target cells expressing low levels of CI-MPR, a novel GALC fusion protein including the ApoE1 receptor was developed. Efficient cellular uptake of the novel fusion protein was mediated by a mannose-6-phosphate-independent mechanism. The novel findings described here elucidate some of the cellular mechanisms that impede the cure of KD patients by HSPCT and concomitantly open new directions to enhance the therapeutic efficacy of HSPCT protocols for KD. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc. PMID:27638600

  14. Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects.

    PubMed

    Hu, Peirong; Li, Yedda; Nikolaishvili-Feinberg, Nana; Scesa, Giuseppe; Bi, Yanmin; Pan, Dao; Moore, Dominic; Bongarzone, Ernesto R; Sands, Mark S; Miller, Ryan; Kafri, Tal

    2016-11-01

    Currently, presymtomatic hematopoietic stem and progenitor cell transplantation (HSPCT) is the only therapeutic modality that alleviates Krabbe's disease (KD)-induced central nervous system damage. However, all HSPCT-treated patients exhibit severe deterioration in peripheral nervous system function characterized by major motor and expressive language pathologies. We hypothesize that a combination of several mechanisms contribute to this phenomenon, including 1) nonoptimal conditioning protocols with consequent inefficient engraftment and biodistribution of donor-derived cells and 2) insufficient uptake of donor cell-secreted galactocerebrosidease (GALC) secondary to a naturally low expression level of the cation-independent mannose 6-phosphate-receptor (CI-MPR). We have characterized the effects of a busulfan (Bu) based conditioning regimen on the efficacy of HSPCT in prolonging twi mouse average life span. There was no correlation between the efficiency of bone marrow engraftment of donor cells and twi mouse average life span. HSPCT prolonged the average life span of twi mice, which directly correlated with the aggressiveness of the Bu-mediated conditioning protocols. HSPC transduced with lentiviral vectors carrying the GALC cDNA under control of cell-specific promoters were efficiently engrafted in twi mouse bone marrow. To facilitate HSPCT-mediated correction of GALC deficiency in target cells expressing low levels of CI-MPR, a novel GALC fusion protein including the ApoE1 receptor was developed. Efficient cellular uptake of the novel fusion protein was mediated by a mannose-6-phosphate-independent mechanism. The novel findings described here elucidate some of the cellular mechanisms that impede the cure of KD patients by HSPCT and concomitantly open new directions to enhance the therapeutic efficacy of HSPCT protocols for KD. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

  15. Efficient Expression of Igf-1 from Lentiviral Vectors Protects In Vitro but Does Not Mediate Behavioral Recovery of a Parkinsonian Lesion in Rats.

    PubMed

    Lu-Nguyen, Ngoc B; Broadstock, Martin; Yáñez-Muñoz, Rafael J

    2015-11-01

    Gene therapy approaches delivering neurotrophic factors have offered promising results in both preclinical and clinical trials of Parkinson's disease (PD). However, failure of glial cell line-derived neurotrophic factor in phase 2 clinical trials has sparked a search for other trophic factors that may retain efficacy in the clinic. Direct protein injections of one such factor, insulin-like growth factor (IGF)-1, in a rodent model of PD has demonstrated impressive protection of dopaminergic neurons against 6-hydroxydopamine (6-OHDA) toxicity. However, protein infusion is associated with surgical risks, pump failure, and significant costs. We therefore used lentiviral vectors to deliver Igf-1, with a particular focus on the novel integration-deficient lentiviral vectors (IDLVs). A neuron-specific promoter, from the human synapsin 1 gene, excellent for gene expression from IDLVs, was additionally used to enhance Igf-1 expression. An investigation of neurotrophic effects on primary rat neuronal cultures demonstrated that neurons transduced with IDLV-Igf-1 vectors had complete protection on withdrawal of exogenous trophic support. Striatal transduction of such vectors into 6-OHDA-lesioned rats, however, provided neither protection of dopaminergic substantia nigra neurons nor improvement of animal behavior.

  16. Transfer of three transcription factors via a lentiviral vector ameliorates spatial learning and memory impairment in a mouse model of Alzheimer's disease.

    PubMed

    Chen, Pin; Yan, Qing; Wang, Songtao; Wang, Cunzu; Zhao, Peng

    2016-08-01

    Alzheimer's disease (AD) is an irreversible and progressive neurodegenerative disorder with observable memory impairment. The present study was performed to evaluate the beneficial effects of lentiviral vector-mediated overexpression of a combination of three transcription regulators, ABN (Ascl1, Brn2 and Ngn2), on learning and memory loss in a mouse model of AD. The AD model was established by injecting Aβ1-42 bilaterally into the mouse hippocampus. Lentiviral ABN was delivered bilaterally into the hippocampus of mice. Animals injected with LV-ABN showed significantly improved spatial learning and memory in the water maze test. Additionally, antibody array analysis indicated that intrahippocampal LV-ABN delivery significantly altered the expression levels of some proteins that were identified as inflammatory factors or neuroprotective and growth factors. In conclusion, our data suggest that LV-ABN delivery can ameliorate spatial learning and memory impairment in an AD mouse model, and the beneficial effect of ABN gene treatment could be linked to inhibition of the neuroinflammatory response and enhancement of neuroprotection and neurogenesis. Thus, these findings indicate that lentiviral ABN gene delivery has potential therapeutic applications for AD.

  17. Production of functional coagulation factor VIII from iPSCs using a lentiviral vector.

    PubMed

    Kashiwakura, Y; Ohmori, T; Mimuro, J; Madoiwa, S; Inoue, M; Hasegawa, M; Ozawa, K; Sakata, Y

    2014-01-01

    The use of induced pluripotent stem cells (iPSCs) as an autologous cell source has shed new light on cell replacement therapy with respect to the treatment of numerous hereditary disorders. We focused on the use of iPSCs for cell-based therapy of haemophilia. We generated iPSCs from mesenchymal stem cells that had been isolated from C57BL/6 mice. The mouse iPSCs were generated through the induction of four transcription factor genes Oct3/4, Klf-4, Sox-2 and c-Myc. The derived iPSCs released functional coagulation factor VIII (FVIII) following transduction with a simian immunodeficiency virus vector. The subcutaneous transplantation of iPSCs expressing FVIII into nude mice resulted in teratoma formation, and significantly increased plasma levels of FVIII. The plasma concentration of FVIII was at levels appropriate for human therapy at 2-4 weeks post transplantation. Our data suggest that iPSCs could be an attractive and prospective autologous cell source for the production of coagulation factor, and that engineered iPSCs expressing coagulation factor might provide a cell-based therapeutic strategy appropriate for haemophilia.

  18. Gene therapy for canine leukocyte adhesion deficiency with lentiviral vectors using the murine stem cell virus and human phosphoglycerate kinase promoters.

    PubMed

    Hunter, Michael J; Zhao, Huifen; Tuschong, Laura M; Bauer, Thomas R; Burkholder, Tanya H; Persons, Derek A; Hickstein, Dennis D

    2011-06-01

    Children with leukocyte adhesion deficiency type 1 (LAD-1) and dogs with canine LAD (CLAD) develop life-threatening bacterial infections due to mutations in the leukocyte integrin CD18. Here, we compared the human phosphoglycerate kinase (hPGK) promoter to the murine stem cell virus (MSCV) promoter/enhancer in a self-inactivating HIV-1-derived lentiviral vector to treat animals with CLAD. Four CLAD dogs were infused with CD34(+) cells transduced with the hPGK vector, and two CLAD dogs received MSCV vector-transduced CD34(+) cells. Infusions were preceded by a nonmyeloablative dose of 200 cGy total body irradiation. Comparable numbers of transduced cells were infused in each group of animals. Only one of four CLAD animals treated with the hPGK-cCD18 vector had reversal of CLAD, whereas both MSCV-cCD18 vector-treated dogs had reversal of the phenotype. Correction of CLAD depends both upon the percentage of CD18(+) myeloid cells and the level of expression of CD18 on individual myeloid cells. In this regard, the hPGK promoter directed low levels of expression of CD18 on neutrophils compared to the MSCV promoter, likely contributing to the suboptimal clinical outcome with the hPGK vector. PMID:21275758

  19. Design and in vitro characterization of a single regulatory module for efficient control of gene expression in both plasmid DNA and a self-inactivating lentiviral vector.

    PubMed Central

    Ogueta, S. B.; Yao, F.; Marasco, W. A.

    2001-01-01

    BACKGROUND: Regulation of transgene expression in target cells represents a critical and challenging aspect of gene therapy. Recently, a two-plasmid tetracycline-inducible system was developed in which the tetracycline repressor (tetR) alone, rather than the tetR-VP16 fusion derivative, was shown to function as a potent trans-modulator of a second plasmid that contains two tandem repeats of the tetracycline operator (tetO) inserted between the TATA box and the transcription start site of the hCMV major immediate-early promoter. A technological advance in this area would be the development of a single autoregulatory cassette that incorporates both of these components into nonviral and viral gene transfer vectors. For the latter, an inducible lentiviral vector that is capable of temporal and quantitative control of gene expression in either dividing or nondividing cells is highly desirable. MATERIALS AND METHODS: A one-piece inducible (1Pi) autoregulatory cassette was constructed to provide IRES-mediated translation of the tetR as well as tight control over the tetO unit preventing transcription initiation of the first cistron in the absence of the tetracycline. To increase efficiency of tetR-mediated repression, a nuclear localization signal was incorporated at the 3' end of the tetR gene. Regulation of gene expression at the transcriptional and protein level was analyzed in transient transfection experiments using plasmid DNA. Construction of a self-inactivating lentiviral vector containing this 1Pi cassette allowed the study of its long-term effectiveness in primary human cells. RESULTS: The 1Pi autoregulatory cassette when incorporated into plasmid DNA allows efficient control of the secretable hEGF as well as eGFP expression in a variety of cell types. Transient transfection studies demonstrated that the time course of repression is different for the 1Pi and two-plasmid system (2Pi). In the 2Pi system, greater repression is seen with the first 24-48 hr; however

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

    SciTech Connect

    Strauss, Bryan E.; Vieira de Carvalho, Anna Carolina; Bajgelman, Marcio C.

    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 this 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.

  1. A Modular Lentiviral and Retroviral Construction System to Rapidly Generate Vectors for Gene Expression and Gene Knockdown In Vitro and In Vivo

    PubMed Central

    Geiling, Benjamin; Vandal, Guillaume; Posner, Ada R.; de Bruyns, Angeline; Dutchak, Kendall L.; Garnett, Samantha; Dankort, David

    2013-01-01

    The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems. PMID:24146852

  2. Long-Term Fate Mapping Using Conditional Lentiviral Vectors Reveals a Continuous Contribution of Radial Glia-Like Cells to Adult Hippocampal Neurogenesis in Mice

    PubMed Central

    Aelvoet, Sarah-Ann; Pascual-Brazo, Jesus; Libbrecht, Sarah; Reumers, Veerle; Gijsbers, Rik; Van den Haute, Chris; Baekelandt, Veerle

    2015-01-01

    Newborn neurons are generated throughout life in two neurogenic regions, the subventricular zone and the hippocampal dentate gyrus. Stimulation of adult neurogenesis is considered as an attractive endogenous repair mechanism to treat different neurological disorders. Although tremendous progress has been made in our understanding of adult hippocampal neurogenesis, important questions remain unanswered, regarding the identity and the behavior of neural stem cells in the dentate gyrus. We previously showed that conditional Cre-Flex lentiviral vectors can be used to label neural stem cells in the subventricular zone and to track the migration of their progeny with non-invasive bioluminescence imaging. Here, we applied these Cre-Flex lentiviral vectors to study neurogenesis in the dentate gyrus with bioluminescence imaging and histological techniques. Stereotactic injection of the Cre-Flex vectors into the dentate gyrus of transgenic Nestin-Cre mice resulted in specific labeling of the nestin-positive neural stem cells. The labeled cell population could be detected with bioluminescence imaging until 9 months post injection, but no significant increase in the number of labeled cells over time was observed with this imaging technique. Nevertheless, the specific labeling of the nestin-positive neural stem cells, combined with histological analysis at different time points, allowed detailed analysis of their neurogenic potential. This long-term fate mapping revealed that a stable pool of labeled nestin-positive neural stem cells continuously contributes to the generation of newborn neurons in the mouse brain until 9 months post injection. In conclusion, the Cre-Flex technology is a valuable tool to address remaining questions regarding neural stem cell identity and behavior in the dentate gyrus. PMID:26600383

  3. A lentiviral vector-based therapeutic vaccine encoding Ag85B-Rv3425 potently increases resistance to acute tuberculosis infection in mice.

    PubMed

    Yang, Enzhuo; Wang, Feifei; Xu, Ying; Wang, Honghai; Hu, Yong; Shen, Hongbo; Chen, Zheng W

    2015-08-01

    Few treatment options for multidrug-resistant tuberculosis (TB) and extensively drug-resistant TB call attention to the development of novel therapeutic approaches for TB. Therapeutic vaccines are promising candidates because they can induce antigen-specific cellular immune responses, which play an important role in the elimination of Mycobacterium tuberculosis (MTB). In this study, a novel lentiviral vector therapeutic vaccine for delivering MTB-specific fusion protein Ag85B-Rv3425 was constructed. Results showed that one single-injection of this recombinant lentivirus vaccine could trigger antigen-specific Th1-type immune responses in mice. More importantly, mice with acute infection benefited a lot from a single-dose administration of this vaccine by markedly reduced MTB burdens in lungs and spleens as well as attenuated lesions in lungs compared with untreated mice. These results displayed good prospects of this novel vaccine for the immunotherapy of TB.

  4. Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis.

    PubMed

    Weber, K; Mock, U; Petrowitz, B; Bartsch, U; Fehse, B

    2010-04-01

    Vector-encoded fluorescent proteins (FPs) facilitate unambiguous identification or sorting of gene-modified cells by fluorescence-activated cell sorting (FACS). Exploiting this feature, we have recently developed lentiviral gene ontology (LeGO) vectors (www.LentiGO-Vectors.de) for multi-gene analysis in different target cells. In this study, we extend the LeGO principle by introducing 10 different drug-selectable FPs created by fusing one of the five selection marker (protecting against blasticidin, hygromycin, neomycin, puromycin and zeocin) and one of the five FP genes (Cerulean, eGFP, Venus, dTomato and mCherry). All tested fusion proteins allowed both fluorescence-mediated detection and drug-mediated selection of LeGO-transduced cells. Newly generated codon-optimized hygromycin- and neomycin-resistance genes showed improved expression as compared with their ancestors. New LeGO constructs were produced at titers >10(6) per ml (for non-concentrated supernatants). We show efficient combinatorial marking and selection of various cells, including mesenchymal stem cells, simultaneously transduced with different LeGO constructs. Inclusion of the cytomegalovirus early enhancer/chicken beta-actin promoter into LeGO vectors facilitated robust transgene expression in and selection of neural stem cells and their differentiated progeny. We suppose that the new drug-selectable markers combining advantages of FACS and drug selection are well suited for numerous applications and vector systems. Their inclusion into LeGO vectors opens new possibilities for (stem) cell tracking and functional multi-gene analysis.

  5. Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

    PubMed

    Spanevello, Francesca; Calistri, Arianna; Del Vecchio, Claudia; Mantelli, Barbara; Frasson, Chiara; Basso, Giuseppe; Palù, Giorgio; Cavazzana, Marina; Parolin, Cristina

    2016-04-19

    Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4(+) T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.

  6. Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector

    PubMed Central

    Shimizu, Saki; Yadav, Swati Seth; An, Dong Sung

    2016-01-01

    RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC. PMID:26472455

  7. RD2-MolPack-Chim3, a Packaging Cell Line for Stable Production of Lentiviral Vectors for Anti-HIV Gene Therapy

    PubMed Central

    Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo

    2013-01-01

    Abstract Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology. PMID:23767932

  8. RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.

    PubMed

    Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

    2013-08-01

    Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

  9. Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector.

    PubMed

    Shimizu, Saki; Yadav, Swati Seth; An, Dong Sung

    2016-01-01

    RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC. PMID:26472455

  10. Optimized lentiviral vectors for HIV gene therapy: multiplexed expression of small RNAs and inclusion of MGMT(P140K) drug resistance gene.

    PubMed

    Chung, Janet; Scherer, Lisa J; Gu, Angel; Gardner, Agnes M; Torres-Coronado, Monica; Epps, Elizabeth W; Digiusto, David L; Rossi, John J

    2014-05-01

    Gene therapy with hematopoietic stem and progenitor cells is a promising approach to engineering immunity to human immunodeficiency virus (HIV) that may lead to a functional cure for acquired immunodeficiency syndrome (AIDS). In support of this approach, we created lentiviral vectors with an engineered polycistronic platform derived from the endogenous MCM7 gene to express a diverse set of small antiviral RNAs and a drug resistance MGMT(P140K) marker. Multiple strategies for simultaneous expression of up to five RNA transgenes were tested. The placement and orientation of each transgene and its promoter were important determinants for optimal gene expression. Antiviral RNA expression from the MCM7 platform with a U1 promoter was sufficient to provide protection from R5-tropic HIV in macrophages and resulted in reduced hematopoietic toxicity compared with constructs expressing RNA from independent RNA polymerase III promoters. The addition of an HIV entry inhibitor and nucleolar TAR RNA decoy did not enhance antiviral potency over constructs that targeted only viral RNA transcripts. We also demonstrated selective enrichment of gene-modified cells in vivo using a humanized mouse model. The use of these less toxic, potent anti-HIV vectors expressing a drug selection marker is likely to enhance the in vivo efficacy of our stem cell gene therapy approach in treating HIV/AIDS.

  11. Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

    PubMed Central

    Bandaranayake, Ashok D.; Correnti, Colin; Ryu, Byoung Y.; Brault, Michelle; Strong, Roland K.; Rawlings, David J.

    2011-01-01

    A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20–100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications. PMID:21911364

  12. Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors.

    PubMed

    Bandaranayake, Ashok D; Correnti, Colin; Ryu, Byoung Y; Brault, Michelle; Strong, Roland K; Rawlings, David J

    2011-11-01

    A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications. PMID:21911364

  13. Construction and verification of the targeted uPA-shRNA lentiviral vector and evaluation of the transfection and silencing rate

    PubMed Central

    WANG, WEI-SHAN; GUO, FENG-JING; LI, CHANG-JUN; ZHANG, ZHEN-DONG; SHI, CHEN-HUI

    2014-01-01

    Urokinase-type plasminogen activator (uPA) receptors, which are released by the synovial tissue, are responsible for the activation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). RNA interference (RNAi) technology has emerged as a potent tool to generate cellular knockdown phenotypes of a desired gene. The aims of the present study were to investigate the effect of siRNA specific to the uPA gene on chondrocytes and to investigate the possible mechanisms of OA. Firstly, four types of small hairpin RNA (shRNA) sequence (P1, P2, P3 and P4) were obtained from the targeted uPA gene of the New Zealand rabbit, based on siRNA theory. The sequences were designed, constructed and subjected to restriction enzyme digestion, transformation, polymerase chain reaction (PCR) identification, positive clone sequencing and lentivirus packaging. Secondly, primary culturing cartilage cells from the New Zealand rabbit were transfected with P1, P2, P3 or P4 to observe the transfection rate under a fluorescence microscope. The mRNA expression levels of uPA were analyzed in cartilage cells using quantitative PCR, while protein expression levels were analyzed in the cartilage cells using western blot technology. Four types of uPA-shRNA lentiviral vectors were constructed successfully, which were all able to be transfected into the primary culturing cartilage cells. The transfection rate was as high as 85% when the multiplicity of infection was 100, which demonstrated that P1, P2, P3 and P4 were all capable of inhibiting the mRNA and protein expression of uPA in cartilage cells. In addition, among the four sequences, the P2 sequence exhibited the highest silencing rate of 70%. Statistical significance (P<0.05) was observed when analyzing the silencing rate of P2 compared to the other three groups. The most efficient targeted uPA-shRNA sequence was identified following screening. The results strongly verified

  14. Lentiviral vectors encoding human immunodeficiency virus type 1 (HIV-1)-specific T-cell receptor genes efficiently convert peripheral blood CD8 T lymphocytes into cytotoxic T lymphocytes with potent in vitro and in vivo HIV-1-specific inhibitory activity.

    PubMed

    Joseph, Aviva; Zheng, Jian Hua; Follenzi, Antonia; Dilorenzo, Teresa; Sango, Kaori; Hyman, Jaime; Chen, Ken; Piechocka-Trocha, Alicja; Brander, Christian; Hooijberg, Erik; Vignali, Dario A; Walker, Bruce D; Goldstein, Harris

    2008-03-01

    The human immunodeficiency virus type 1 (HIV-1)-specific CD8 cytotoxic T-lymphocyte (CTL) response plays a critical role in controlling HIV-1 replication. Augmenting this response should enhance control of HIV-1 replication and stabilize or improve the clinical course of the disease. Although cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection in immunocompromised patients can be treated by adoptive transfer of ex vivo-expanded CMV- or EBV-specific CTLs, adoptive transfer of ex vivo-expanded, autologous HIV-1-specific CTLs had minimal effects on HIV-1 replication, likely a consequence of the inherently compromised qualitative function of HIV-1-specific CTLs derived from HIV-1-infected individuals. We hypothesized that this limitation could be circumvented by using as an alternative source of HIV-1-specific CTLs, autologous peripheral CD8(+) T lymphocytes whose antigen specificity is redirected by transduction with lentiviral vectors encoding HIV-1-specific T-cell receptor (TCR) alpha and beta chains, an approach used successfully in cancer therapy. To efficiently convert peripheral CD8 lymphocytes into HIV-1-specific CTLs that potently suppress in vivo HIV-1 replication, we constructed lentiviral vectors encoding the HIV-1-specific TCR alpha and TCR beta chains cloned from a CTL clone specific for an HIV Gag epitope, SL9, as a single transcript linked with a self-cleaving peptide. We demonstrated that transduction with this lentiviral vector efficiently converted primary human CD8 lymphocytes into HIV-1-specific CTLs with potent in vitro and in vivo HIV-1-specific activity. Using lentiviral vectors encoding an HIV-1-specific TCR to transform peripheral CD8 lymphocytes into HIV-1-specific CTLs with defined specificities represents a new immunotherapeutic approach to augment the HIV-1-specific immunity of infected patients.

  15. Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach

    PubMed Central

    Spanevello, Francesca; Calistri, Arianna; Del Vecchio, Claudia; Mantelli, Barbara; Frasson, Chiara; Basso, Giuseppe; Palù, Giorgio; Cavazzana, Marina; Parolin, Cristina

    2016-01-01

    Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4+ T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application. PMID:27093170

  16. In Vitro Generation of IL-35-expressing Human Wharton's Jelly-derived Mesenchymal Stem Cells Using Lentiviral Vector.

    PubMed

    Amari, Afshin; Ebtekar, Massoumeh; Moazzeni, Seyed Mohammad; Soleimani, Masoud; Mohammadi Amirabad, Leila; Tahoori, Mohammad Taher; Massumi, Mohammad

    2015-08-01

    Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease. Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit. IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127 low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells. Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.

  17. Targeted Inhibitory Effect of Lenti-SM22alpha-p27-EGFP Recombinant Lentiviral Vectors on Proliferation of Vascular Smooth Muscle Cells without Compromising Re-Endothelialization in a Rat Carotid Artery Balloon Injury Model

    PubMed Central

    Zhang, Shuangshuang; Xie, Minjie; Tian, Daishi; Luo, Xiang; Wang, Daowen; Ning, Qin; Lü, Jiagao; Wang, Wei

    2015-01-01

    Aims In-stent restenosis remains a serious problem after the implantation of drug-eluting stents, which is attributable to neointima formation and re-endothelialization. Here, we tried to find a new method which aims at selectively inhibiting proliferation of vascular smooth muscle cells (VSMC) proliferation without inhibition of re-endothelialization. Methods and Results We used the smooth muscle-specific SM22alpha promoter in a recombinant lentiviral vector to drive overexpression of cell-cycle inhibitor, p27, in VSMCs. p27 effectively inhibited VSMC proliferation mediated by cell cycle arrest at the G0/G1 checkpoint. The SM22alpha-p27 lentiviral vector inhibited VSMC proliferation more effectively than paclitaxel. Rats infected with Lenti-SM22alpha-p27 had a significantly lower intima/media (I/M) ratio and also showed inhibition of restenosis on day 28 after balloon injury. Moreover, the repair of injured endothelium, and re-endothelialization of the carotid artery wall, was not affected by the smooth muscle cell-specific expression of p27. Conclusion A recombinant lentiviral vector carrying the SM22alpha promoter was used to effectively infect and selectively overexpress p27 protein in VSMCs, leading to inhibition of intimal hyperplasia without compromising endothelial repair. PMID:25760326

  18. A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice.

    PubMed

    Frecha, Cecilia; Costa, Caroline; Nègre, Didier; Amirache, Fouzia; Trono, Didier; Rio, Paula; Bueren, Juan; Cosset, François-Loïc; Verhoeyen, Els

    2012-02-01

    In vivo lentiviral vector (LV)-mediated gene delivery would represent a great step forward in the field of gene therapy. Therefore, we have engineered a novel LV displaying SCF and a mutant cat endogenous retroviral glycoprotein, RDTR. These RDTR/SCF-LVs outperformed RDTR-LVs for transduction of human CD34(+) cells (hCD34(+)). For in vivo gene therapy, these novel RDTR/SCF-displaying LVs can distinguish between the target hCD34(+) cells of interest and nontarget cells. Indeed, they selectively targeted transduction to 30%-40% of the hCD34(+) cells in cord blood mononuclear cells and in the unfractionated BM of healthy and Fanconi anemia donors, resulting in the correction of CD34(+) cells in the patients. Moreover, RDTR/SCF-LVs targeted transduction to CD34(+) cells with 95-fold selectivity compared with T cells in total cord blood. Remarkably, in vivo injection of the RDTR/SCF-LVs into the BM cavity of humanized mice resulted in the highly selective transduction of candidate hCD34(+)Lin(-) HSCs. In conclusion, this new LV will facilitate HSC-based gene therapy by directly targeting these primitive cells in BM aspirates or total cord blood. Most importantly, in the future, RDTR/SCF-LVs might completely obviate ex vivo handling and simplify gene therapy for many hematopoietic defects because of their applicability to direct in vivo inoculation.

  19. LV305, a dendritic cell-targeting integration-deficient ZVexTM-based lentiviral vector encoding NY-ESO-1, induces potent anti-tumor immune response

    PubMed Central

    Albershardt, Tina Chang; Campbell, David James; Parsons, Andrea Jean; Slough, Megan Merrill; ter Meulen, Jan; Berglund, Peter

    2016-01-01

    We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1. PMID:27626061

  20. LV305, a dendritic cell-targeting integration-deficient ZVex(TM)-based lentiviral vector encoding NY-ESO-1, induces potent anti-tumor immune response.

    PubMed

    Albershardt, Tina Chang; Campbell, David James; Parsons, Andrea Jean; Slough, Megan Merrill; Ter Meulen, Jan; Berglund, Peter

    2016-01-01

    We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1. PMID:27626061

  1. A new system for parallel drug screening against multiple-resistant HIV mutants based on lentiviral self-inactivating (SIN) vectors and multi-colour analyses

    PubMed Central

    2013-01-01

    Background Despite progress in the development of combined antiretroviral therapies (cART), HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed. Methods We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN) LeGO vector technology. Results We demonstrated the successful use of this approach for screening compounds against up to four HIV gag-pol variants (wild-type and three mutants) simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment. Conclusions The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs. PMID:23286882

  2. Pretransplant Mobilization with Granulocyte Colony-Stimulating Factor Improves B-Cell Reconstitution by Lentiviral Vector Gene Therapy in SCID-X1 Mice

    PubMed Central

    Huston, Marshall W.; Riegman, Adriaan R.A.; Yadak, Rana; van Helsdingen, Yvette; de Boer, Helen; van Til, Niek P.

    2014-01-01

    Abstract Hematopoietic stem cell (HSC) gene therapy is a demonstrated effective treatment for X-linked severe combined immunodeficiency (SCID-X1), but B-cell reconstitution and function has been deficient in many of the gene therapy treated patients. Cytoreductive preconditioning is known to improve HSC engraftment, but in general it is not considered for SCID-X1 since the poor health of most of these patients at diagnosis and the risk of toxicity preclude the conditioning used in standard bone marrow stem cell transplantation. We hypothesized that mobilization of HSC by granulocyte colony-stimulating factor (G-CSF) should create temporary space in bone marrow niches to improve engraftment and thereby B-cell reconstitution. In the present pilot study supplementing our earlier preclinical evaluation (Huston et al., 2011), Il2rg−/− mice pretreated with G-CSF were transplanted with wild-type lineage negative (Lin−) cells or Il2rg−/− Lin− cells transduced with therapeutic IL2RG lentiviral vectors. Mice were monitored for reconstitution of lymphocyte populations, level of donor cell chimerism, and antibody responses as compared to 2 Gy total body irradiation (TBI), previously found effective in promoting B-cell reconstitution. The results demonstrate that G-CSF promotes B-cell reconstitution similar to low-dose TBI and provides proof of principle for an alternative approach to improve efficacy of gene therapy in SCID patients without adverse effects associated with cytoreductive conditioning. PMID:25222508

  3. LV305, a dendritic cell-targeting integration-deficient ZVex(TM)-based lentiviral vector encoding NY-ESO-1, induces potent anti-tumor immune response.

    PubMed

    Albershardt, Tina Chang; Campbell, David James; Parsons, Andrea Jean; Slough, Megan Merrill; Ter Meulen, Jan; Berglund, Peter

    2016-01-01

    We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1.

  4. An all-in-one nanopore battery array.

    PubMed

    Liu, Chanyuan; Gillette, Eleanor I; Chen, Xinyi; Pearse, Alexander J; Kozen, Alexander C; Schroeder, Marshall A; Gregorczyk, Keith E; Lee, Sang Bok; Rubloff, Gary W

    2014-12-01

    A single nanopore structure that embeds all components of an electrochemical storage device could bring about the ultimate miniaturization in energy storage. Self-alignment of electrodes within each nanopore may enable closer and more controlled spacing between electrodes than in state-of-art batteries. Such an 'all-in-one' nanopore battery array would also present an alternative to interdigitated electrode structures that employ complex three-dimensional geometries with greater spatial heterogeneity. Here, we report a battery composed of an array of nanobatteries connected in parallel, each composed of an anode, a cathode and a liquid electrolyte confined within the nanopores of anodic aluminium oxide, as an all-in-one nanosize device. Each nanoelectrode includes an outer Ru nanotube current collector and an inner nanotube of V₂O₅ storage material, forming a symmetric full nanopore storage cell with anode and cathode separated by an electrolyte region. The V₂O₅ is prelithiated at one end to serve as the anode, with pristine V₂O₅ at the other end serving as the cathode, forming a battery that is asymmetrically cycled between 0.2 V and 1.8 V. The capacity retention of this full cell (relative to 1 C values) is 95% at 5 C and 46% at 150 C, with a 1,000-cycle life. From a fundamental point of view, our all-in-one nanopore battery array unveils an electrochemical regime in which ion insertion and surface charge mechanisms for energy storage become indistinguishable, and offers a testbed for studying ion transport limits in dense nanostructured electrode arrays.

  5. Rapid generation of stable cell lines expressing high levels of erythropoietin, factor VIII, and an antihuman CD20 antibody using lentiviral vectors.

    PubMed

    Baranyi, Lajos; Doering, Christopher B; Denning, Gabriella; Gautney, Richard E; Harris, Kyle T; Spencer, H Trent; Roy, Andre; Zayed, Hatem; Dropulic, Boro

    2013-08-01

    Lentiviral vectors (LVs) are widely recognized as the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Using erythropoietin (EPO), recombinant factor VIII (fVIII), and an anti-CD20 antibody as model proteins, we demonstrate advantages of LV-based gene delivery to achieve high production levels by transduced cells. Highly productive cell clones were able to incorporate up to 100 vector copies per cellular genome, without selection or gene amplification, and were isolated without extensive screening of a large number of clones. The LV transgenes were shown to be distributed throughout the genome, as visualized by fluorescent in situ hybridization. High-expressing clones producing 100-200 pg/cell/day of EPO were isolated and characterized. EPO production was demonstrated for at least 5½ months of continuous culture without selection, during which all the clones displayed high levels of glycosylation despite production levels at 10-20 g/liter. To demonstrate the utility of LV technology for multiple classes of proteins, cell lines producing fVIII and an anti-CD20 antibody were also developed. Cell clones demonstrating high levels of fVIII (100 clot units/ml and anti-CD20 antibody as high as 40-100 pg/cell/day) were isolated and characterized. LV-transduced cells and plasmid-transfected cells were compared for protein production per transgene copy. LV-transduced cells produced significantly higher levels of protein per copy of transgene than plasmid-transfected cells did. This study demonstrates the utility of LV technology for rapid generation of highly productive and stable cell lines over conventional plasmid transfection methods, significantly decreasing the time, cost, and risk of the manufacture of proteins and other complex biological molecules.

  6. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition.

    PubMed

    Qiu, Huiling; Zhong, Jiasheng; Luo, Lan; Liu, Nian; Kang, Kang; Qu, Junle; Peng, Wenda; Gou, Deming

    2015-01-01

    DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo. PMID:26624995

  7. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition

    PubMed Central

    Luo, Lan; Liu, Nian; Kang, Kang; Qu, Junle; Peng, Wenda; Gou, Deming

    2015-01-01

    DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo. PMID:26624995

  8. Striatal modulation of BDNF expression using microRNA124a-expressing lentiviral vectors impairs ethanol-induced conditioned-place preference and voluntary alcohol consumption.

    PubMed

    Bahi, Amine; Dreyer, Jean-Luc

    2013-07-01

    Alcohol abuse is a major health, economic and social concern in modern societies, but the exact molecular mechanisms underlying ethanol addiction remain elusive. Recent findings show that small non-coding microRNA (miRNA) signaling contributes to complex behavioral disorders including drug addiction. However, the role of miRNAs in ethanol-induced conditioned-place preference (CPP) and voluntary alcohol consumption has not yet been directly addressed. Here, we assessed the expression profile of miR124a in the dorsal striatum of rats upon ethanol intake. The results show that miR124a was downregulated in the dorso-lateral striatum (DLS) following alcohol drinking. Then, we identified brain-derived neurotrophic factor (BDNF) as a direct target of miR124a. In fact, BDNF mRNA was upregulated following ethanol drinking. We used lentiviral vector (LV) gene transfer technology to further address the role of miR124a and its direct target BDNF in ethanol-induced CPP and alcohol consumption. Results reveal that stereotaxic injection of LV-miR124a in the DLS enhances ethanol-induced CPP as well as voluntary alcohol consumption in a two-bottle choice drinking paradigm. Moreover, miR124a-silencer (LV-siR124a) as well as LV-BDNF infusion in the DLS attenuates ethanol-induced CPP as well as voluntary alcohol consumption. Importantly, LV-miR124a, LV-siR124a and LV-BDNF have no effect on saccharin and quinine intake. Our findings indicate that striatal miR124a and BDNF signaling have crucial roles in alcohol consumption and ethanol conditioned reward.

  9. Comparison of Lentiviral Packaging Mixes and Producer Cell Lines for RNAi Applications.

    PubMed

    Albrecht, Christian; Hosiner, Stefanie; Tichy, Brigitte; Aldrian, Silke; Hajdu, Stefan; Nürnberger, Sylvia

    2015-06-01

    Lentiviral transduction is a highly efficient DNA delivery method for RNA interference applications. However, obtaining high lentiviral titers of shRNA and miRNA encoding vectors is challenging, since shRNA and miRNA cassettes have been shown to reduce lentiviral titers. In this study, we compare four commercially available packaging mixes and two producer cell lines in order to optimize lentiviral production for gene silencing experiments. Lentiviral vectors encoding a miRNA sequence and emerald green fluorescence protein were co-transfected with ViraPower™, Lenti-X™ HTX, MISSION(®) Lentiviral or Trans-Lentiviral™ packaging mix in HEK-293T or 293FT cells. After transducing HeLa cells with virus-containing supernatant, lentiviral titers were determined by flow cytomerty. In both cell lines, the highest lentiviral titer was obtained with MISSION(®) Lentiviral packaging mix, followed by ViraPower™, Lenti-X™ HTX, and Trans-Lentiviral™. On average, HEK-293T cells produced 6.2-fold higher lentiviral titers than 293FT cells (p < 0.001). With the combination of MISSION(®) Lentiviral packaging mix and HEK-293T cells, an up to 48.5-fold higher lentiviral titer was reached compared to other packaging mixes and producer cell lines. The optimized selection of packaging mix and cell line described in this work should facilitate the production of high-titer lentiviruses for gene silencing experiments. PMID:25616840

  10. Conditional RNAi Using the Lentiviral GLTR System.

    PubMed

    Pfeiffenberger, Elisabeth; Sigl, Reinhard; Geley, Stephan

    2016-01-01

    RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success depends on the effective expression of target gene-specific RNAi-inducing small double-stranded interfering RNA molecules (siRNAs). Here, were describe the use of a recently developed lentiviral RNAi system that allows the rapid generation of stable cell lines with inducible RNAi based on conditional expression of double-stranded short hairpin RNA (shRNA). These lentiviral vectors can be generated rapidly using the GATEWAY recombination cloning technology. Conditional cell lines can be established by using either a two-vector system in which the regulator is encoded by a separate vector or by a one-vector system. The available different lentiviral vectors for conditional shRNA expression cassette delivery co-express additional genes that allow (1) the use of fluorescent proteins for color-coded combinatorial RNAi or monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one-vector system (pGLTR-X). PMID:27317178

  11. Inhibition of in vivo HIV infection in humanized mice by gene therapy of human hematopoietic stem cells with a lentiviral vector encoding a broadly neutralizing anti-HIV antibody.

    PubMed

    Joseph, Aviva; Zheng, Jian Hua; Chen, Ken; Dutta, Monica; Chen, Cindy; Stiegler, Gabriela; Kunert, Renate; Follenzi, Antonia; Goldstein, Harris

    2010-07-01

    Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/gamma(c)(null) mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/gamma(c)(null) mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1(JR-CSF), mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/gamma(c)(null) mice inoculated with equivalent high-titer HIV-1(JR-CSF). These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.

  12. Increased Locomotor Activity and Non-Selective Attention and Impaired Learning Ability in SD Rats after Lentiviral Vector-Mediated RNA Interference of Homer 1a in the Brain

    PubMed Central

    Hong, Qin; Yang, Lei; Zhang, Min; Pan, Xiao-Qin; Guo, Mei; Fei, Li; Tong, Mei-Ling; Chen, Rong-Hua; Guo, Xi-Rong; Chi, Xia

    2013-01-01

    Our previous studies found that Homer 1a, a scaffolding protein localized at the post-synaptic density (PSD) of glutamatergic excitatory synapses, is significantly down-regulated in the brain of spontaneous hypertensive rats (SHR), an animal model of attention deficit hyperactivity disorder (ADHD). Furthermore, a first-line treatment drug for ADHD, methylphenidate, can up-regulate the expression of Homer 1a. To investigate the possible role of Homer 1a in the etiology and pathogenesis of ADHD, a lentiviral vector containing miRNA specific for Homer 1a was constructed in this study. Intracerebroventricular injection of this vector into the brain of Sprague Dawley (SD) rats significantly decreased Homer 1a mRNA and protein expression levels. Compared to their negative controls, these rats displayed a range of abnormal behaviors, including increased locomotor activity and non-selective attention and impaired learning ability. Our results indicated that Homer 1a down-regulation results in deficits in control over behavioral output and learning similar to ADHD. PMID:23289010

  13. All-in-one construct for genome engineering using Cre-lox technology

    PubMed Central

    Mariscal, Ana M.; González-González, Luis; Querol, Enrique; Piñol, Jaume

    2016-01-01

    Mycoplasma genitalium is an appealing model of a minimal cell and synthetic biology study, and it was one of the first organisms whose genome was fully sequenced and chemically synthesized. Despite its usefulness as a model organism, many genetic tools well established for other microorganisms are not currently available in mycoplasmas. We have developed several vectors to adapt the Cre-lox technology for genome engineering in M. genitalium, providing an all-in-one construct that could be also useful to obtain unmarked genetic modifications in many other slow growing microorganisms. This construct contains a modified promoter sequence based in TetR system that exhibits an enhanced control on Cre recombinase expression, virtually abolishing the presence of this recombinase in the absence of inducer. This allows to introduce the Cre recombinase gene and the desired genetic modification in a single transformation step. In addition, this inducible promoter may be a very promising tool for a wide range of molecular applications. PMID:27084897

  14. The effect of lentiviral vector-mediated RNA interference targeting hypoxia-inducible factor 1α on the uptake of fluorodeoxyglucose ((18)f) in the human pancreatic cancer cell line, patu8988.

    PubMed

    Fan, Guanglei; Bo, Jingli; Wan, Renming; Peng, Mingya; Luan, Yufen; Deng, Minbin; Xu, Longbao

    2015-05-01

    Hypoxia can stimulate (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in cultured tumor cells. This study has investigated the effect of lentiviral vector-mediated RNA interference (RNAi) targeting hypoxia-inducible factor 1α (HIF-1α) on the changes in HIF-1 and glucose transporter 1 (Glut-1) expression, the cell growth, and the uptake of (18)F-FDG in the human pancreatic cancer cell line, Patu8988. Lentiviral RNAi vector targeting the HIF-1α gene (LV-HIF-1αRNAi) was constructed and used to treat cells at various concentrations (25-200 nM). The expression changes of HIF-1α and Glut-1 in hypoxic Patu8988 cells after RNAi treatment were determined using real time reverse transcription-polymerase chain reaction (real-time PCR). The inhibition rate of cell proliferation 48 hours after the addition of 10 μL of different concentrations of LV-HIF-1αRNAi (25-200 nM) was assayed using the MTT method. Meanwhile, the cell uptake of (18)F-FDG was also assessed. After RNAi transfection, the relative expression levels of HIF-1α mRNA and Glut-1 under hypoxia were reduced and the relative expression levels of HIF-1α protein also decreased. Compared with the control group, the inhibition rates of cell proliferation under different viral dosages were 5.98%, 15.65%, 26.42%, and 40.81%, respectively, positively correlated with the viral doses (r=0.558, p<0.05). Under hypoxia, Glut-1 mRNA expression in Patu8988 cells treated with 200 nM of LV-HIF-1αRNAi for 24, 48, and 72 hours, respectively, was positively correlated with the inhibition rate of cell proliferation (r=0.618, p<0.05) as well as the inhibition rate of (18)F-FDG uptake (r=0.664, p<0.05), while the latter two displayed a positive correlation with each other too (r=0.582, p<0.05). Under hypoxia, RNAi targeting HIF-1α significantly inhibited the expression of Glut-1 mRNA in Patu8988 pancreatic cancer cells and their uptake of (18)F-FDG. These results suggest that LV-HIF-1αRNAi may form a new treatment for

  15. Lentiviral transgenesis--a versatile tool for basic research and gene therapy.

    PubMed

    Pfeifer, Alexander

    2006-08-01

    Transgenic animals are of outstanding relevance for medical sciences, because they can be used to model human diseases and to develop gene therapy strategies. A recent development is lentiviral transgenesis: The generation of transgenic animals by lentiviral transduction of oocytes or early embryos. Lentiviral transgenesis is an efficient method to express transgenes in mice and rats as well as in biomedically relevant livestock. Thus, the applications of this technology range from the generation of disease models to gene pharming for human proteins. An important extension of viral transgenesis is the combination of lentiviral gene transfer with RNA interference. Thereby, expression of specific genes can be silenced and loss-of-function models can be generated. Finally, lentiviral transgenic animals can be used to directly evaluate gene therapy strategies that are based on lentiviral vectors prior to their use in humans.

  16. Lentiviral transgenesis--a versatile tool for basic research and gene therapy.

    PubMed

    Pfeifer, Alexander

    2006-08-01

    Transgenic animals are of outstanding relevance for medical sciences, because they can be used to model human diseases and to develop gene therapy strategies. A recent development is lentiviral transgenesis: The generation of transgenic animals by lentiviral transduction of oocytes or early embryos. Lentiviral transgenesis is an efficient method to express transgenes in mice and rats as well as in biomedically relevant livestock. Thus, the applications of this technology range from the generation of disease models to gene pharming for human proteins. An important extension of viral transgenesis is the combination of lentiviral gene transfer with RNA interference. Thereby, expression of specific genes can be silenced and loss-of-function models can be generated. Finally, lentiviral transgenic animals can be used to directly evaluate gene therapy strategies that are based on lentiviral vectors prior to their use in humans. PMID:16918338

  17. Intravenous delivery of HIV-based lentiviral vectors preferentially transduces F4/80+ and Ly-6C+ cells in spleen, important target cells in autoimmune arthritis.

    PubMed

    van den Brand, Ben T; Vermeij, Eline A; Waterborg, Claire E J; Arntz, Onno J; Kracht, Michael; Bennink, Miranda B; van den Berg, Wim B; van de Loo, Fons A J

    2013-01-01

    Antigen presenting cells (APCs) play an important role in arthritis and APC specific gene therapeutic targeting will enable intracellular modulation of cell activity. Viral mediated overexpression is a potent approach to achieve adequate transgene expression levels and lentivirus (LV) is useful for sustained expression in target cells. Therefore, we studied the feasibility of lentiviral mediated targeting of APCs in experimental arthritis. Third generation VSV-G pseudotyped self-inactivating (SIN)-LV were injected intravenously and spleen cells were analyzed with flow cytometry for green fluorescent protein (GFP) transgene expression and cell surface markers. Collagen-induced arthritis (CIA) was induced by immunization with bovine collagen type II in complete Freund's adjuvant. Effect on inflammation was monitored macroscopically and T-cell subsets in spleen were analyzed by flow cytometry. Synovium from arthritic knee joints were analyzed for proinflammatory cytokine expression. Lentiviruses injected via the tail vein preferentially infected the spleen and transduction peaks at day 10. A dose escalating study showed that 8% of all spleen cells were targeted and further analysis showed that predominantly Ly6C+ and F4/80+ cells in spleen were targeted by the LV. To study the feasibility of blocking TAK1-dependent pathways by this approach, a catalytically inactive mutant of TAK1 (TAK1-K63W) was overexpressed during CIA. LV-TAK1-K63W significantly reduced incidence and arthritis severity macroscopically. Further histological analysis showed a significant decrease in bone erosion in LV-TAK1-K63W treated animals. Moreover, systemic Th17 levels were decreased by LV-TAK1-K63W treatment in addition to diminished IL-6 and KC production in inflamed synovium. In conclusion, systemically delivered LV efficiently targets monocytes and macrophages in spleen that are involved in autoimmune arthritis. Moreover, this study confirms efficacy of TAK1 targeting in arthritis. This

  18. Driving DNA transposition by lentiviral protein transduction

    PubMed Central

    Cai, Yujia; Mikkelsen, Jacob Giehm

    2014-01-01

    Gene vectors derived from DNA transposable elements have become powerful molecular tools in biomedical research and are slowly moving into the clinic as carriers of therapeutic genes. Conventional uses of DNA transposon-based gene vehicles rely on the intracellular production of the transposase protein from transfected nucleic acids. The transposase mediates mobilization of the DNA transposon, which is typically provided in the context of plasmid DNA. In recent work, we established lentiviral protein transduction from Gag precursors as a new strategy for direct delivery of the transposase protein. Inspired by the natural properties of infecting viruses to carry their own enzymes, we loaded lentivirus-derived particles not only with vector genomes carrying the DNA transposon vector but also with hundreds of transposase subunits. Such particles were found to drive efficient transposition of the piggyBac transposable element in a range of different cell types, including primary cells, and offer a new transposase delivery approach that guarantees short-term activity and limits potential cytotoxicity. DNA transposon vectors, originally developed and launched as a non-viral alternative to viral integrating vectors, have truly become viral. Here, we briefly review our findings and speculate on the perspectives and potential advantages of transposase delivery by lentiviral protein transduction. PMID:25057443

  19. Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase

    PubMed Central

    Moldt, Brian; Staunstrup, Nicklas H; Jakobsen, Maria; Yáñez-Muñoz, Rafael J; Mikkelsen, Jacob G

    2008-01-01

    Background Circular forms of viral genomic DNA are generated during infection of cells with retroviruses like HIV-1. Such circles are unable to replicate and are eventually lost as a result of cell division, lending support to the prevalent notion that episomal retroviral DNA forms are dead-end products of reverse transcription. Results We demonstrate that circular DNA generated during transduction with HIV-1-based lentiviral vectors can be utilized as substrate for gene insertion directed by nonviral recombinases co-expressed in the target cells. By packaging of lentiviral genomic RNA in integrase-defective lentiviral vectors, harboring an inactive form of the viral integrase, the normal pathway for viral integration is blocked and circular vector DNA accumulates in transduced cells as a result. We find that the amount of DNA circles is increased 4-fold in cells transduced with integration-defective vectors relative to cells treated with integrase-proficient vectors. By transduction of target cells harboring engineered recognition sites for the yeast Flp recombinase with integration-defective lentiviral vectors containing an ATG-deficient hygromycin B selection gene we demonstrate precise integration of lentiviral vector-derived DNA circles in a drug-selective approach. Moreover, it is demonstrated that trans-acting Flp recombinase can be delivered by Flp-encoding transfected plasmid DNA or, alternatively, by co-transduced integrase-defective lentiviral vectors carrying a Flp expression cassette. Conclusion Our data provide proof-of-principle that nonviral recombinases, like Flp, produced by plasmid DNA or non-integrating lentiviral vectors can gain access to circular viral recombination substrates and facilitate site-directed genomic insertion of such episomal DNA forms. Replacement of the normal viral integration machinery with nonviral mediators of integration represents a new platform for creation of lentiviral vectors with an altered integration profile. PMID

  20. In vitro incorporation of a cell-binding protein to a lentiviral vector using an engineered split intein enables targeted delivery of genetic cargo.

    PubMed

    Chamoun-Emanuelli, Ana M; Wright, Gus; Roger, Smith; Münch, Robert C; Buchholz, Christian J; Chen, Zhilei

    2015-12-01

    Gene therapy represents a promising therapeutic paradigm for addressing many disorders, but the absence of a vector that can be robustly and reproducibly functionalized with cell-homing functionality to mediate the delivery of genetic cargo specifically to target cells following systemic administration has stood as a major impediment. In this study, a high-affinity protein-protein pair comprising a splicing-deficient naturally split intein was used as molecular Velcro to append a HER2/neu-binding protein (DARPin) onto the surface of a binding-deficient, fusion-competent lentivirus. HER2/neu-specific lentiviruses created using this in vitro pseudotyping approach were able to deliver their genetic reporter cargo specifically to cells that express the target receptor at high levels in a co-culture. We envision that the described technology could provide a powerful, broadly applicable platform for the incorporation of cell-targeting functionality onto viral vectors.

  1. Gene delivery to rat and human Schwann cells and nerve segments: a comparison of AAV 1-9 and lentiviral vectors.

    PubMed

    Hoyng, S A; De Winter, F; Gnavi, S; van Egmond, L; Attwell, C L; Tannemaat, M R; Verhaagen, J; Malessy, M J A

    2015-10-01

    Schwann cells (SCs) in an injured peripheral nerve form pathways for regenerating axons. Although these cells initially support regeneration, SCs lose their pro-regenerative properties following a prolonged period of denervation. Gene transfer to SC can enhance their therapeutic potential. In this article, we compared adeno-associated viral (AAV) vectors based on serotypes 1-9 for their capability to transduce cultured primary rat and human SCs and nerve segments. AAV1 is the best serotype to transduce rat SCs, whereas AAV2 and AAV6 performed equally well in human SCs. Transduction of monolayers of cultured rat and human SCs did not accurately predict the transduction efficiency in nerve segments. Rat nerve segments could be genetically modified equally well by a set of four AAV vectors (AAV1, AAV5, AAV7, AAV9), whereas AAV2 was superior in human nerve segments. The current experiments were undertaken as a first step towards future clinical implementation of ex vivo AAV-based gene therapy in surgical nerve repair. The transduction of rat and human SCs and nerve segments by entirely different AAV serotypes, as documented here, highlights one of the challenges of translating gene therapy from experimental animals to human patients.

  2. RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope.

    PubMed

    Marin, Virna; Stornaiuolo, Anna; Piovan, Claudia; Corna, Stefano; Bossi, Sergio; Pema, Monika; Giuliani, Erica; Scavullo, Cinzia; Zucchelli, Eleonora; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

    2016-01-01

    To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications. PMID:27222840

  3. RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope.

    PubMed

    Marin, Virna; Stornaiuolo, Anna; Piovan, Claudia; Corna, Stefano; Bossi, Sergio; Pema, Monika; Giuliani, Erica; Scavullo, Cinzia; Zucchelli, Eleonora; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

    2016-01-01

    To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.

  4. RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope

    PubMed Central

    Marin, Virna; Stornaiuolo, Anna; Piovan, Claudia; Corna, Stefano; Bossi, Sergio; Pema, Monika; Giuliani, Erica; Scavullo, Cinzia; Zucchelli, Eleonora; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

    2016-01-01

    To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications. PMID:27222840

  5. The Development of an All-in-One Virtual Campus from Ground Zero.

    ERIC Educational Resources Information Center

    Farmer, Shanan W.; Sobieszcyk, Frank; Farmer, Rachelle

    This paper describes the authors' experiences in developing an all-in-one virtual university. The Defense Acquisition University (DAU) is a consortium of Department of Defense education and training institutions and organizations that provides mandatory and assignment-specific courses for military and civilian personnel serving in 11 acquisition…

  6. Lentiviral delivery of short hairpin RNAs

    PubMed Central

    Manjunath, N; Haoquan, Wu; Sandesh, Subramanya; Premlata, Shankar

    2009-01-01

    In less than a decade after discovery, RNA interference-mediated gene silencing is already being tested as potential therapy in clinical trials for a number of diseases. Lentiviral vectors provide a means to express short hairpin RNA (shRNA) to induce stable and long-term gene silencing in both dividing and non-dividing cells and thus, are being intensively investigated for this purpose. However, induction of long-term shRNA expression can also cause toxicities by inducing off target effects and interference with the endogenous micro RNA (miRNA) pathway that regulates cellular gene expression. Recently, several advances have been made in the shRNA vector design to mimic cellular miRNA processing and to express multiplex siRNAs in a tightly regulated and reversible manner to overcome toxicities. In this review we describe some of these advances, focusing on the progress made in the development of lentiviral shRNA delivery strategies to combat viral infections. PMID:19341774

  7. Antibacterial Effect of All-in-one Self-etch Adhesives on Enterococcus faecalis

    PubMed Central

    Ebrahimi Chaharom, Mohammad Esmaeel; Ajami, Amir Ahmad; Abed Kahnamouei, Mehdi; Jafari Navimipour, Elmira; Tehranchi, Pardis; Zand, Vahid; Sadeghi, Mohammad Reza; Sohrabi, Aydin

    2014-01-01

    Background and aims. The aim of this study was to evaluate the antibacterial activity of one-step self-etch adhesives on Enterococcus faecalis on days 1, 7 and 14 with the use of modified direct contact test. Materials and methods. The modified directcontact test was used to evaluate the antibacterial effect of Adper Easy One, Bond Force, Clearfil S3 Bond, Futurabond M, G-Bond, iBond and OptiBond All-in-one adhesives on Enterococcus faecalis after aging the samples in phosphate-buffered saline for one, seven and fourteen days. Data were analyzed using one-way ANOVA and post hoc Tukey tests. Aging effect of each adhesive was evaluated by paired-sample test. In this study, P<0.05 was considered significant. Results. All the tested adhesives exhibited antibacterial activity after one day and had significant differences with the positive control group (P<0.05). After one week, OptiBond All-in-one, iBond and Futurabond M exhibited significant differences in bacterial growth from other groups (P<0.05). There were no significant differences between the groups in two weeks (P>0.05). Conclusion. iBond exhibited the highest antibacterial effect on E. faecalis after one week. Futurabond and OptiBond All-in-one exhibited antibacterial effects against E. faecalis for one week. PMID:25587384

  8. All-in-one detector of circulating mRNA based on a smartphone

    NASA Astrophysics Data System (ADS)

    Cmiel, Vratislav; Gumulec, Jaromir; Svoboda, Ondrej; Raudenska, Martina; Hudcova, Kristyna; Sekora, Jiri; Balogh, Jaroslav; Masarik, Michal; Provaznik, Ivo

    2016-03-01

    Metallothionein is significantly elevated in various tumors, notably in prostate cancer on both mRNA and protein level. We demonstrated a strong predictive potential of free circulating metallothionein 2A isoform mRNA for patients with this cancer. Circulating mRNA detection relies on expensive equipment and requires high level of expertise. In this work we developed compact "all-in-one" laboratory system which replace microvolume spectrophotometer, thermocycler and realtime PCR machines. We managed to design and construct a microprocessor controlled heating/cooling chamber that ensures required temperature gradient. The chamber includes implemented optical system to enable fluorescence excitation and fluorescence analysis using a smart-phone.

  9. Surface modification via strain-promoted click reaction facilitates targeted lentiviral transduction.

    PubMed

    Chu, Yanjie; Oum, Yoon Hyeun; Carrico, Isaac S

    2016-01-01

    As a result of their ability to integrate into the genome of both dividing and non-dividing cells, lentiviruses have emerged as a promising vector for gene delivery. Targeted gene transduction of specific cells and tissues by lentiviral vectors has been a major goal, which has proven difficult to achieve. We report a novel targeting protocol that relies on the chemoselective attachment of cancer specific ligands to unnatural glycans on lentiviral surfaces. This strategy exhibits minimal perturbation on virus physiology and demonstrates remarkable flexibility. It allows for targeting but can be more broadly useful with applications such as vector purification and immunomodulation. PMID:26499046

  10. Surface modification via strain-promoted click reaction facilitates targeted lentiviral transduction.

    PubMed

    Chu, Yanjie; Oum, Yoon Hyeun; Carrico, Isaac S

    2016-01-01

    As a result of their ability to integrate into the genome of both dividing and non-dividing cells, lentiviruses have emerged as a promising vector for gene delivery. Targeted gene transduction of specific cells and tissues by lentiviral vectors has been a major goal, which has proven difficult to achieve. We report a novel targeting protocol that relies on the chemoselective attachment of cancer specific ligands to unnatural glycans on lentiviral surfaces. This strategy exhibits minimal perturbation on virus physiology and demonstrates remarkable flexibility. It allows for targeting but can be more broadly useful with applications such as vector purification and immunomodulation.

  11. Developing and Implementing All-in-One Standard Paediatric Parenteral Nutrition

    PubMed Central

    Meyer, Rosan; Timmermann, Meike; Schulzke, Sven; Kiss, Caroline; Sidler, Marc A.; Furlano, Raoul I.

    2013-01-01

    Parenteral nutrition (PN) is a feeding mode suitable for children that do not achieve requirements via the enteral route. For this intervention to be successful, healthcare professionals require: knowledge on nutrient requirements; access to an aseptic compounding facility; and a system that ensures adequate and safe delivery of PN. Previously, it was thought that individualised PN was the “gold standard” for delivering nutrients to children; however, studies have highlighted concerns regarding inadequate delivery of nutrients, prescribing and compounding errors. We, therefore, set out to develop and implement all-in-one (AIO) paediatric PN solutions. Through a systematic approach, four AIO PN solutions were developed: birth–two months of age (Ped 1); two months–10 kg (Ped 2); 11–15 kg (Ped 3); and 16–30 kg (Ped 4). We implemented them with the help of a teaching pack, over a one month time period, and reviewed usage at six months. At that time, five children initially received standard PN without electrolyte changes; but after a few days, electrolytes needed amendments, and three required individualised PN. A change to AIO PN is feasible and safe; however, some may require electrolyte changes, and there will always be those that will require individualised PN. PMID:23739142

  12. Development of an all-in-one gamma camera/CCD system for safeguard verification

    NASA Astrophysics Data System (ADS)

    Kim, Hyun-Il; An, Su Jung; Chung, Yong Hyun; Kwak, Sung-Woo

    2014-12-01

    For the purpose of monitoring and verifying efforts at safeguarding radioactive materials in various fields, a new all-in-one gamma camera/charged coupled device (CCD) system was developed. This combined system consists of a gamma camera, which gathers energy and position information on gamma-ray sources, and a CCD camera, which identifies the specific location in a monitored area. Therefore, 2-D image information and quantitative information regarding gamma-ray sources can be obtained using fused images. A gamma camera consists of a diverging collimator, a 22 × 22 array CsI(Na) pixelated scintillation crystal with a pixel size of 2 × 2 × 6 mm3 and Hamamatsu H8500 position-sensitive photomultiplier tube (PSPMT). The Basler scA640-70gc CCD camera, which delivers 70 frames per second at video graphics array (VGA) resolution, was employed. Performance testing was performed using a Co-57 point source 30 cm from the detector. The measured spatial resolution and sensitivity were 4.77 mm full width at half maximum (FWHM) and 7.78 cps/MBq, respectively. The energy resolution was 18% at 122 keV. These results demonstrate that the combined system has considerable potential for radiation monitoring.

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

    PubMed Central

    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. PMID:27006970

  14. Advanced Modular "All in One" Battery System with Intelligent Autonomous Cell Balancing Management

    NASA Astrophysics Data System (ADS)

    Petitdidier, X.; Pasquier, E.; Defer, M.; Koch, M.; Knorr, W.

    2008-09-01

    A new generation of energy storage systems based on Li-ion technology emerged at the end of the last century.To perform the first tests in safe conditions, Saft designed a simple electronic.Today, all Li-ion batteries for autonomous applications such as drones, launchers, missiles, torpedoes and "human" applications such as cellular, laptop, hybrid vehicle and nearly sub-marines need a Battery Management System.The minimum in terms of functions is the overcharge and over-discharge protections.For a battery made of 2 cells connected in series or more, a balancing system is added to maintain the available energy during all the life of the battery. For stringent/demanding applications, the state of charge and state of health are calculated by one or more computers.It is now time to take benefit of the past 10 years of Saft's experience in the domain to re-evaluate the constraints of Li-ion batteries and provide customers with improved products by optimizing the battery management.Benefits of electronic for satellite applications:• Full control over battery.• Confidence whatever the possible change of conditions in environment.• The battery system can resist long exposure to gradient conditions with mitigated and stabilized impact on performances.• The balancing function allow to use all the energy of all the cells: optimize of installed energy (compact design, mass saving). It started out with the basic fact that electrochemists are not intended to be space rated electronic experts and vice versa, even if Saft has a good heritage in the electronic battery management system. Consequently, considering heritage and expertise in their respective core businesses, Saft and ASP teamed up.It became necessary to provide an "all in one" modular energy storage system with intelligent autonomous cell balancing management.

  15. Thermally Cured Dual Functional Viologen-Based All-in-One Electrochromic Devices with Panchromatic Modulation.

    PubMed

    Kao, Sheng-Yuan; Lu, Hsin-Che; Kung, Chung-Wei; Chen, Hsin-Wei; Chang, Ting-Hsiang; Ho, Kuo-Chuan

    2016-02-17

    Vinyl benzyl viologen (VBV) was synthesized and utilized to obtain all-in-one thermally cured electrochromic devices (ECDs). The vinyl moiety of VBV monomer could react with methyl methacrylate (MMA) to yield bulky VBV/poly(methyl methacrylate) (PMMA) chains and even cross-linked network without the assistance of additional cross-linker. Both the bulky VBV/PMMA chains and the resulting polymer network can hinder the aggregation of the viologens and reduce the possibility of dimerization, rendering enhanced cycling stability. Large transmittance changes (ΔT) over 60% at both 570 and 615 nm were achieved when the VBV-based ECD was switched from 0 V to a low potential bias of 0.5 V. Ultimately, the dual functional of VBV molecules, serving simultaneously as a promising electrochromic material and a cross-linker, is fully utilized in the proposed electrochromic system, making its fabrication process much easier. Negligible decays in ΔT at both wavelengths were observed for the cured ECD after being subjected to 1000 repetitive cycles, while 17.1% and 22.0% decays were noticed at 570 and 615 nm, respectively, for the noncured ECD. In addition, the low voltage-driven feature of the VBV-based ECD enables it to be incorporated with phenyl viologen (PV), further expanding the absorption range of the ECD. Panchromatic characteristic of the proposed PV/VBV-based ECD was demonstrated while exhibiting ΔT over 60% at both wavelengths. Only 5.3% and 6.9% decays, corresponding at 570 and 615 nm, respectively, were observed in the PV/VBV-based ECD after 10 000 continuous cycles at bleaching/coloring voltages of 0/0.5 V with an interval of 10 s for both bleaching and coloring processes. PMID:26807824

  16. One-year water sorption and solubility of "all-in-one" adhesives.

    PubMed

    Walter, Ricardo; Feiring, Andrew E; Boushell, Lee W; Braswell, Krista; Bartholomew, Whitley; Chung, Yunro; Phillips, Ceib; Pereira, Patricia N R; Swift, Edward J

    2013-01-01

    The aim of this study was to evaluate the water sorption and solubility of different adhesives. Adper Easy Bond, Adper Single Bond Plus, Bond Force, Clearfil SE Bond (bonding resin only), and Xeno IV were the materials evaluated. Ten disks of each adhesive were made in Teflon molds and evaporation of any volatile components was allowed. The disks were weighed daily in an analytical balance until a constant mass was obtained (m1). Disks were then immersed in water for 12 months when their wet weight was recorded (m2). The disks were again weighed daily until a constant mass was obtained and the final weight recorded (m3). Water sorption and solubility (percentages) were calculated using the recorded mass values. Kruskal-Wallis tests were used to compare the average water sorption and solubility among the different adhesives. Mann-Whitney tests with a Bonferroni correction were used to determine the pairwise differences between adhesives in water sorption and solubility. The level of significance was set at 0.05. Water sorption and solubility were significantly different among the groups (p<0.05). Pairwise comparisons showed no significant differences (p>0.05) between Adper Single Bond Plus and Bond Force, or between Clearfil SE Bond and Xeno IV in either water sorption or solubility. Xeno IV did not differ from Adper Easy Bond in water sorption (p>0.05). Water sorption and solubility of all-in-one adhesives increased with time, and the rates of increase were composition-dependent. The results suggest that monomers other than HEMA contribute to water sorption and solubility of adhesive systems from different categories. PMID:24173253

  17. Lentiviral gene transfer into human and murine hematopoietic stem cells: size matters.

    PubMed

    Canté-Barrett, Kirsten; Mendes, Rui D; Smits, Willem K; van Helsdingen-van Wijk, Yvette M; Pieters, Rob; Meijerink, Jules P P

    2016-01-01

    Contemporary biomedical research increasingly depends on techniques to induce or to inhibit expression of genes in hematopoietic stem cells (HSCs) or other primary cells to assess their roles on cellular processes including differentiation, apoptosis and migration. Surprisingly little information is available to optimize lentiviral transduction of HSCs. We have therefore carefully optimized transduction of murine and human HSCs by optimizing vector design, serum-free virus production and virus quantitation. We conclude that the viral RNA length, even in relatively small vectors, is an important factor affecting the lentiviral gene transfer on the level of both the virus production and the cellular transduction efficiency. Efficient transfer of large gene sequences into difficult-to-transduce primary cells will benefit from reducing the lentiviral construct size. PMID:27306375

  18. Quantitative analysis of lentiviral transgene expression in mice over seven generations.

    PubMed

    Wang, Yong; Song, Yong-tao; Liu, Qin; Liu, Cang'e; Wang, Lu-lu; Liu, Yu; Zhou, Xiao-yang; Wu, Jun; Wei, Hong

    2010-10-01

    Lentiviral transgenesis is now recognized as an extremely efficient and cost-effective method to produce transgenic animals. Transgenes delivered by lentiviral vectors exhibited inheritable expression in many species including those which are refractory to genetic modification such as non-human primates. However, epigenetic modification was frequently observed in lentiviral integrants, and transgene expression found to be inversely correlated with methylation density. Recent data showed that about one-third lentiviral integrants exhibited hypermethylation and low expression, but did not demonstrate whether those integrants with high expression could remain constant expression and hypomethylated during long term germline transmission. In this study, using lentiviral eGFP transgenic mice as the experimental animals, lentiviral eGFP expression levels and its integrant numbers in genome were quantitatively analyzed by fluorescent quantitative polymerase-chain reaction (FQ-PCR), using the house-keeping gene ribosomal protein S18 (Rps18) and the single copy gene fatty acid binding protein of the intestine (Fabpi) as the internal controls respectively. The methylation densities of the integrants were quantitatively analyzed by bisulfite sequencing. We found that the lentiviral integrants with high expression exhibited a relative constant expression level per integrant over at least seven generations. Besides, the individuals containing these integrants exhibited eGFP expression levels which were positively and almost linearly correlated with the integrant numbers in their genomes, suggesting that no remarkable position effect on transgene expression of the integrants analyzed was observed. In addition, over seven generations the methylation density of these integrants did not increase, but rather decreased remarkably, indicating that these high expressing integrants were not subjected to de novo methylation during at least seven generations of germline transmission. Taken

  19. Polybrene: Observations on cochlear hair cell necrosis and minimal lentiviral transduction of cochlear hair cells.

    PubMed

    Han, Miaomiao; Yu, Dongzhen; Song, Qiang; Wang, Jiping; Dong, Pin; He, Jingchun

    2015-07-23

    Polybrene is widely used to enhance viral transduction; however, little is known about the utility thereof, in enhancing lentiviral transduction of cochlear cells. In the present study, we examined the cytotoxic effects of polybrene, and the further effects thereof, on lentiviral transduction of cochlear cells, especially sensory hair cells. Cochlear basilar membranes of newborn rats were cultured and treated with 0.1-10 μg/mL polybrene for 24h to explore the potential development of ototoxicity. PI staining and TUNEL detection were used to evaluate necrosis or apoptosis of hair cell. Various doses of lentivirus-GFP were added to cochlear organotypic cultures with safe concentrations of polybrene, incubated for 24h, and cultured (in the absence of the virus and polybrene) for a further 48 h. Transduction efficiencies were evaluated. The results showed that polybrene at 0.1 μg/mL was safe to cochlear cells, and 0.5-10 μg/mL concentration induced hair cell necrosis in a dose-dependent manner. However, supporting cells were not damaged. Lentiviral vectors transduced into cochlear cells and 0.1 μg/mL polybrene enhanced transduction efficiency. However, hair cells were hardly transduced with lentiviral vectors either alone or in the presence of 0.1 μg/mL polybrene. The use of polybrene to aid lentiviral transduction of cochlear hair cells requires further attention.

  20. Polybrene: Observations on cochlear hair cell necrosis and minimal lentiviral transduction of cochlear hair cells.

    PubMed

    Han, Miaomiao; Yu, Dongzhen; Song, Qiang; Wang, Jiping; Dong, Pin; He, Jingchun

    2015-07-23

    Polybrene is widely used to enhance viral transduction; however, little is known about the utility thereof, in enhancing lentiviral transduction of cochlear cells. In the present study, we examined the cytotoxic effects of polybrene, and the further effects thereof, on lentiviral transduction of cochlear cells, especially sensory hair cells. Cochlear basilar membranes of newborn rats were cultured and treated with 0.1-10 μg/mL polybrene for 24h to explore the potential development of ototoxicity. PI staining and TUNEL detection were used to evaluate necrosis or apoptosis of hair cell. Various doses of lentivirus-GFP were added to cochlear organotypic cultures with safe concentrations of polybrene, incubated for 24h, and cultured (in the absence of the virus and polybrene) for a further 48 h. Transduction efficiencies were evaluated. The results showed that polybrene at 0.1 μg/mL was safe to cochlear cells, and 0.5-10 μg/mL concentration induced hair cell necrosis in a dose-dependent manner. However, supporting cells were not damaged. Lentiviral vectors transduced into cochlear cells and 0.1 μg/mL polybrene enhanced transduction efficiency. However, hair cells were hardly transduced with lentiviral vectors either alone or in the presence of 0.1 μg/mL polybrene. The use of polybrene to aid lentiviral transduction of cochlear hair cells requires further attention. PMID:26071903

  1. Development of a doxycycline-inducible lentiviral plasmid with an instant regulatory feature.

    PubMed

    Yang, Tian; Burrows, Christopher; Park, Jeong Hyeon

    2014-03-01

    Lentiviruses provide highly efficient gene delivery vehicles in both dividing and non-dividing cells. Inducible gene expression systems often employ a specific cell line that constitutively expresses a regulatory protein for transgene expression. As one of such inducible expression systems the Tet-On system uses a cell line expressing reverse tetracycline-responsive transcriptional activator (rtTA). The rtTA protein binds to the tetracycline-responsive element (TRE) in the promoter and activates transcription of a transgene in a doxycycline-dependent manner. To establish a universal and instant regulatory system without generating Tet-On cell lines, the cDNAs of rtTA and a testing target gene (PPM1B) were cloned in the bi-directional TRE-containing promoters. Here, we examined whether a basal leaky expression of rtTA allows instantly inducible expression of both rtTA itself and the target gene, PPM1B in a single plasmid using the two mini-CMV promoters. Transient transfection of the lentiviral plasmids into human embryonic kidney HEK293T cells showed a significant induction of PPM1B expression in response to doxycycline, suggesting that these lentiviral plasmids can be used as an instantly inducible mammalian expression vector. However, the expression of rtTA by lentiviral transduction shows a minimal expression without a consistent response to doxycycline, suggesting that the utility of these lentiviral vectors is limited. A potential solution to overcome lentiviral transgene inactivation is proposed. PMID:24727543

  2. Cell cycle requirements for transduction by foamy virus vectors compared to those of oncovirus and lentivirus vectors.

    PubMed

    Trobridge, Grant; Russell, David W

    2004-03-01

    Retroviral vectors based on foamy viruses (FV) are efficient gene delivery vehicles for therapeutic and research applications. While previous studies have shown that FV vectors transduce quiescent cell cultures more efficiently than oncoviral vectors, their specific cell cycle requirements have not been determined. Here we compare the transduction frequencies of FV vectors with those of onco- and lentiviral vectors in nondividing and dividing normal human fibroblasts by several methods. FV vectors transduced serum-deprived fibroblast cultures more efficiently than oncoretroviral vectors and at rates comparable to those of lentiviral vectors. However, in these cultures FV vectors only transduced a subpopulation of proliferating cells, as determined by bromodeoxyuridine staining for DNA synthesis. In contrast to lentiviral vectors, FV vectors were unable to transduce human fibroblasts arrested by aphidicolin (G(1)/S phase) or gamma-irradiation (G(2) phase), and a partial cell cycle that included mitosis but not DNA synthesis was required. We could not determine if mitosis facilitated nuclear entry of FV vectors, since cell-free vector preparations contained long terminal repeat circles, precluding their use as nuclear markers. In contrast to oncoviral vectors, both FV and lentiviral vectors efficiently transduced G(0) fibroblasts that were later stimulated to divide. In the case of FV vectors, this was due to the persistence of a stable transduction intermediate in quiescent cells. Our findings support the use of FV vectors as a safe and effective alternative to lentiviral vectors for ex vivo transduction of stem cells that are quiescent during culture but divide following transplantation.

  3. Recent Advances in Lentiviral Vaccines for HIV-1 Infection.

    PubMed

    Norton, Thomas D; Miller, Elizabeth A

    2016-01-01

    The development of an effective HIV vaccine to prevent and/or cure HIV remains a global health priority. Given their central role in the initiation of adaptive immune responses, dendritic cell (DC)-based vaccines are being increasingly explored as immunotherapeutic strategies to enhance HIV-specific T cells in infected individuals and, thus, promote immune responses that may help facilitate a functional cure. HIV-1-based lentiviral (LV) vectors have inherent advantages as DC vaccine vectors due to their ability to transduce non-dividing cells and integrate into the target cell genomic DNA, allowing for expression of encoded antigens over the lifespan of the cell. Moreover, LV vectors may express additional immunostimulatory and immunoregulatory proteins that enhance DC function and direct antigen-specific T cells responses. Recent basic and clinical research efforts have broadened our understanding of LV vectors as DC-based vaccines. In this review, we provide an overview of the pre-clinical and clinical LV vector vaccine studies for treating HIV to date. We also discuss advances in LV vector designs that have enhanced DC transduction efficiency, target cell specificity, and immunogenicity, and address potential safety concerns regarding LV vector-based vaccines. PMID:27446074

  4. Recent Advances in Lentiviral Vaccines for HIV-1 Infection

    PubMed Central

    Norton, Thomas D.; Miller, Elizabeth A.

    2016-01-01

    The development of an effective HIV vaccine to prevent and/or cure HIV remains a global health priority. Given their central role in the initiation of adaptive immune responses, dendritic cell (DC)-based vaccines are being increasingly explored as immunotherapeutic strategies to enhance HIV-specific T cells in infected individuals and, thus, promote immune responses that may help facilitate a functional cure. HIV-1-based lentiviral (LV) vectors have inherent advantages as DC vaccine vectors due to their ability to transduce non-dividing cells and integrate into the target cell genomic DNA, allowing for expression of encoded antigens over the lifespan of the cell. Moreover, LV vectors may express additional immunostimulatory and immunoregulatory proteins that enhance DC function and direct antigen-specific T cells responses. Recent basic and clinical research efforts have broadened our understanding of LV vectors as DC-based vaccines. In this review, we provide an overview of the pre-clinical and clinical LV vector vaccine studies for treating HIV to date. We also discuss advances in LV vector designs that have enhanced DC transduction efficiency, target cell specificity, and immunogenicity, and address potential safety concerns regarding LV vector-based vaccines. PMID:27446074

  5. Lentiviral-mediated phenotypic correction of cystic fibrosis pigs

    PubMed Central

    Cooney, Ashley L.; Abou Alaiwa, Mahmoud H.; Shah, Viral S.; Bouzek, Drake C.; Stroik, Mallory R.; Powers, Linda S.; Gansemer, Nick D.; Meyerholz, David K.; Welsh, Michael J.; Stoltz, David A.; Sinn, Patrick L.; McCray, Paul B.

    2016-01-01

    Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in CF transmembrane conductance regulator (CFTR), resulting in defective anion transport. Regardless of the disease-causing mutation, gene therapy is a strategy to restore anion transport to airway epithelia. Indeed, viral vector–delivered CFTR can complement the anion channel defect. In this proof-of-principle study, functional in vivo CFTR channel activity was restored in the airways of CF pigs using a feline immunodeficiency virus–based (FIV-based) lentiviral vector pseudotyped with the GP64 envelope. Three newborn CF pigs received aerosolized FIV-CFTR to the nose and lung. Two weeks after viral vector delivery, epithelial tissues were analyzed for functional correction. In freshly excised tracheal and bronchus tissues and cultured ethmoid sinus cells, we observed a significant increase in transepithelial cAMP-stimulated current, evidence of functional CFTR. In addition, we observed increases in tracheal airway surface liquid pH and bacterial killing in CFTR vector–treated animals. Together, these data provide the first evidence to our knowledge that lentiviral delivery of CFTR can partially correct the anion channel defect in a large-animal CF model and validate a translational strategy to treat or prevent CF lung disease.

  6. Lentiviral-mediated phenotypic correction of cystic fibrosis pigs

    PubMed Central

    Cooney, Ashley L.; Abou Alaiwa, Mahmoud H.; Shah, Viral S.; Bouzek, Drake C.; Stroik, Mallory R.; Powers, Linda S.; Gansemer, Nick D.; Meyerholz, David K.; Welsh, Michael J.; Stoltz, David A.; Sinn, Patrick L.; McCray, Paul B.

    2016-01-01

    Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in CF transmembrane conductance regulator (CFTR), resulting in defective anion transport. Regardless of the disease-causing mutation, gene therapy is a strategy to restore anion transport to airway epithelia. Indeed, viral vector–delivered CFTR can complement the anion channel defect. In this proof-of-principle study, functional in vivo CFTR channel activity was restored in the airways of CF pigs using a feline immunodeficiency virus–based (FIV-based) lentiviral vector pseudotyped with the GP64 envelope. Three newborn CF pigs received aerosolized FIV-CFTR to the nose and lung. Two weeks after viral vector delivery, epithelial tissues were analyzed for functional correction. In freshly excised tracheal and bronchus tissues and cultured ethmoid sinus cells, we observed a significant increase in transepithelial cAMP-stimulated current, evidence of functional CFTR. In addition, we observed increases in tracheal airway surface liquid pH and bacterial killing in CFTR vector–treated animals. Together, these data provide the first evidence to our knowledge that lentiviral delivery of CFTR can partially correct the anion channel defect in a large-animal CF model and validate a translational strategy to treat or prevent CF lung disease. PMID:27656681

  7. A novel one lobe technique of thulium laser enucleation of the prostate: 'All-in-One' technique

    PubMed Central

    Kim, Yeon Joo; Lee, Yoon Hyung; Kwon, Joon Beom; Cho, Sung Ryong

    2015-01-01

    Purpose The thulium laser is the most recently introduced technology for the surgical treatment of benign prostatic hyperplasia (BPH). Until recently, most thulium laser enucleation of the prostate (ThuLEP) was performed by use of the three-lobe technique. We introduce a novel one-lobe enucleation technique for ThuLEP called the "All-in-One" technique. We report our initial experiences here. Materials and Methods From June 2013 to May 2014, a total of 47 patients underwent the All-in-One technique of ThuLEP for symptomatic BPH performed by a single surgeon. All patients were assessed with the International Prostate Symptom Score (IPSS), transrectal ultrasonography, serum prostate-specific antigen (PSA), maximal urine flow rate (Qmax), and postvoid residual urine volume (PVR) before and 1 month after surgery. We reassessed IPSS, Qmax, and PVR 3 months after surgery. To assess the efficacy of the All-in-One technique, we checked the PSA reduction ratio, transitional zone volume reduction ratio, and enucleation failure rate. Results The mean operative time was 82.1±33.3 minutes. The mean enucleation time and morcellation time were 52.7±21.7 minutes and 8.2±7.0 minutes, respectively. The mean resected tissue weight and decrease in hemoglobin were 36.9±24.6 g and 0.4±0.8 g/dL, respectively. All perioperative parameters showed significant improvement (p<0.05). No major complications were observed. The PSA reduction ratio, transitional zone volume reduction ratio, and enucleation failure rate were 0.81, 0.92, and 4.3%, respectively. Conclusions The All-in-One technique of ThuLEP showed efficacy and effectiveness comparable to that of other techniques. We expect that this new technique could reduce the operation time and the bleeding and improve the effectiveness of enucleation. PMID:26568795

  8. Biosafety in Ex Vivo Gene Therapy and Conditional Ablation of Lentivirally Transduced Hepatocytes in Nonhuman Primates

    PubMed Central

    Menzel, Olivier; Birraux, Jacques; Wildhaber, Barbara E; Jond, Caty; Lasne, Françoise; Habre, Walid; Trono, Didier; Nguyen, Tuan H; Chardot, Christophe

    2009-01-01

    Ex vivo gene therapy is an interesting alternative to orthotopic liver transplantation (OLT) for treating metabolic liver diseases. In this study, we investigated its efficacy and biosafety in nonhuman primates. Hepatocytes isolated from liver lobectomy were transduced in suspension with a bicistronic liver-specific lentiviral vector and immediately autotransplanted (SLIT) into three cynomolgus monkeys. The vector encoded cynomolgus erythropoietin (EPO) and the conditional suicide gene herpes simplex virus-thymidine kinase (HSV-TK). Survival of transduced hepatocytes and vector dissemination were evaluated by detecting transgene expression and vector DNA. SLIT was safely performed within a day in all three subjects. Serum EPO and hematocrit rapidly increased post-SLIT and their values returned to baseline within about 1 month. Isoforms of EPO detected in monkeys' sera differed from the physiological renal EPO. In liver biopsies at months 8 and 15, we detected EPO protein, vector mRNA and DNA, demonstrating long-term survival and functionality of transplanted lentivirally transduced hepatocytes. Valganciclovir administration resulted in complete ablation of the transduced hepatocytes. We demonstrated the feasibility and biosafety of SLIT, and the long term (>1 year) functionality of lentivirally transduced hepatocytes in nonhuman primates. The HSV-TK/valganciclovir suicide strategy can increase the biosafety of liver gene therapy protocols by safely and completely ablating transduced hepatocytes on demand. PMID:19568222

  9. Effect of Thermocycling, Degree of Conversion, and Cavity Configuration on the Bonding Effectiveness of All-in-One Adhesives.

    PubMed

    El-Damanhoury, H M; Gaintantzopoulou, M

    2015-01-01

    The aim of this study was to compare five all-in-one bonding agents with respect to microleakage, microtensile bond strength (μTBS), degree of conversion (DC) and the impact of cavity configuration. The materials tested were Adper Easy Bond, Clearfil S3 Bond, iBond, Optibond All-in-One, Xeno IV, and Adper Single Bond Plus as a control. The DC of each adhesive was measured on the surfaces of dentin discs (n=5) by attenuated total reflectance Fourier transform infrared spectroscopy. One hundred and forty-four extracted human molars were randomly divided and assigned to one of the five tested adhesives and the control group. The μTBS to dentin was measured on flat occlusal dentin with and without thermocycling and to the gingival floor dentin of class II cavities (n=8). All specimens were restored with Filtek Z250 resin composite. Class II samples were immersed in a 5% methylene blue dye solution for 24 hours, and microleakage was examined under a stereomicroscope. Micromorphological analysis of demineralized/deproteinized specimens was done using scanning electron microscopy. The DC and microleakage data were statistically analyzed by one-way analysis of variance (ANOVA) and μTBS data by two-way ANOVA followed by a Bonferroni multiple comparison post hoc test (α=0.05) and Weibull-distribution survival analysis. The relation between different variables and μTBS and microleakage was tested by the Pearson correlation coefficient and regression statistics. A moderate direct relation between DC and μTBS durability was found for all the adhesives tested. Significant wide variations exist among the results obtained for single-bottle adhesives tested regarding their μTBS and microleakage. Some of the all-in-one materials tested have shown significantly inferior results under a high C-factor or after aging. The use of these materials should be carefully considered. PMID:25748210

  10. Comparison of two all-in-one adhesives bonded to non-carious cervical lesions--results at 3 years.

    PubMed

    Burrow, Michael F; Tyas, Martin J

    2012-08-01

    The aim of this study was to evaluate the clinical performance of S(3) Bond (Kuraray Corp., Japan) and G-Bond (GC Corp., Japan) all-in-one bonding agents, over 3 years in non-carious cervical lesions (NCCLs). Ethics Committee approval was obtained, and 60 restorations were placed in 11 patients aged 45-84 years (mean 60.5 years), using either Clearfil ST resin composite (Kuraray) and S(3) Bond or Gradia resin composite (GC) and G-Bond alternately, without phosphoric acid etch on the uncut enamel margins. Patients were recalled at 6 months, 1 year, 2 years and 3 years, and photographs were taken for assessment of colour match and marginal discoloration. One patient was not available at 3 years, resulting in 54 restorations being available for evaluation. One restoration of S(3)/Clearfil ST was lost at 2 years, giving retention rates of 97% for S(3) and 100% for G-Bond. At 3 years, six restorations for S(3)/Clearfil ST showed slight marginal discoloration and one restoration pronounced marginal staining. For G-Bond/Gradia at 3 years, 11 restorations exhibited slight marginal staining and one restoration pronounced marginal staining. Most restorations were bonded to sclerotic dentin. Statistical analysis of marginal staining showed no significant difference between the two restoration groups. The degree of marginal staining was almost identical for both materials and tended to be in larger restorations. Both S(3) and G-Bond all-in-one bonding systems appear to be good adhesives for the restoration of NCCL for the length of the current study. Restoration of NCCLs with the newer all-in-one adhesives appears to be a viable alternative technique to more complicated adhesive materials. PMID:21789590

  11. A novel all-in-one magnetic pump and power harvester design for bio-medical applications

    NASA Astrophysics Data System (ADS)

    Kim, Sung Hoon; Shin, Jaewon; Hashi, Shuichiro; Ishiyama, Kazushi

    2011-03-01

    This paper presents a magnetic centrifugal pump with a magnetic power harvester (all-in-one system) for medical applications. The proposed pump is driven by an external rotating magnetic field. To produce pressure and electrical power, an all-in-one device consisting of a pump and a power harvester was designed. It consists of a multi-stage impeller, a disc type NdFeB permanent magnet, and a fixed wound coil on the pump case. The rotation of the rotor creates a continuous flow of liquid through the pump, with a pressure head, and an electrical power is generated in the wound coil because of the rotating magnetic field. The maximum flow rate and pressure are 5000 ml min-1 and 16 kPa, respectively, at 100 Hz. These results meet the requirements of an artificial heart assistance blood pump. Under these operating conditions, the harvested voltage can reach a maximum of 8.2 Vp-p. With this configuration and control method, wireless and battery-free operation is possible, which is required in the medical field. Moreover, the power harvester can monitor the pump conditions without additional electrical power and can provide electrical power to other implanted electrical devices. The performances of the pump and power harvester were verified in a laboratory experiment. Overall, the proposed system acts as a pump and a power harvester that is fully wireless and battery-free.

  12. Effect of EDTA Conditioning and Carbodiimide Pretreatment on the Bonding Performance of All-in-One Self-Etch Adhesives

    PubMed Central

    Singh, Shipra; Nagpal, Rajni; Tyagi, Shashi Prabha; Manuja, Naveen

    2015-01-01

    Objective. This study evaluated the effect of ethylenediaminetetraacetic acid (EDTA) conditioning and carbodiimide (EDC) pretreatment on the shear bond strength of two all-in-one self-etch adhesives to dentin. Methods. Flat coronal dentin surfaces were prepared on one hundred and sixty extracted human molars. Teeth were randomly divided into eight groups according to two different self-etch adhesives used [G-Bond and OptiBond-All-In-One] and four different surface pretreatments: (a) adhesive applied following manufacturer's instructions; (b) dentin conditioning with 24% EDTA gel prior to application of adhesive; (c) EDC pretreatment followed by application of adhesive; (d) application of EDC on EDTA conditioned dentin surface followed by application of adhesive. Composite restorations were placed in all the samples. Ten samples from each group were subjected to immediate and delayed (6-month storage in artificial saliva) shear bond strength evaluation. Data collected was subjected to statistical analysis using three-way ANOVA and post hoc Tukey's test at a significance level of p < 0.05.  Results and Conclusion. EDTA preconditioning as well as EDC pretreatment alone had no significant effect on the immediate and delayed bond strengths of either of the adhesives. However, EDC pretreatment on EDTA conditioned dentin surface resulted in preservation of resin-dentin bond strength of both adhesives with no significant fall over six months. PMID:26557850

  13. Performance Evaluation of a Multichannel All-In-One Phantom Dosimeter for Dose Measurement of Diagnostic X-ray Beam

    PubMed Central

    Jeon, Hyesu; Yoo, Wook Jae; Shin, Sang Hun; Kwon, Guwon; Kim, Mingeon; Kim, Hye Jin; Song, Young Beom; Jang, Kyoung Won; Youn, Won Sik; Lee, Bongsoo

    2015-01-01

    We developed a multichannel all-in-one phantom dosimeter system composed of nine sensing probes, a chest phantom, an image intensifier, and a complementary metal-oxide semiconductor (CMOS) image sensor to measure the dose distribution of an X-ray beam used in radiation diagnosis. Nine sensing probes of the phantom dosimeter were fabricated identically by connecting a plastic scintillating fiber (PSF) to a plastic optical fiber (POF). To measure the planar dose distribution on a chest phantom according to exposure parameters used in clinical practice, we divided the top of the chest phantom into nine equal parts virtually and then installed the nine sensing probes at each center of the nine equal parts on the top of the chest phantom as measuring points. Each scintillation signal generated in the nine sensing probes was transmitted through the POFs and then intensified by the image intensifier because the scintillation signal normally has a very low light intensity. Real-time scintillation images (RSIs) containing the intensified scintillation signals were taken by the CMOS image sensor with a single lens optical system and displayed through a software program. Under variation of the exposure parameters, we measured RSIs containing dose information using the multichannel all-in-one phantom dosimeter and compared the results with the absorbed doses obtained by using a semiconductor dosimeter (SCD). From the experimental results of this study, the light intensities of nine regions of interest (ROI) in the RSI measured by the phantom dosimeter were similar to the dose distribution obtained using the SCD. In conclusion, we demonstrated that the planar dose distribution including the entrance surface dose (ESD) can be easily measured by using the proposed phantom dosimeter system. PMID:26569252

  14. All-in-One Beaker Method for Large-Scale Production of Metal Oxide Hollow Nanospheres Using Nanoscale Kirkendall Diffusion.

    PubMed

    Cho, Jung Sang; Kang, Yun Chan

    2016-02-17

    A simple and easily scalable process for the formation of metal oxide hollow nanospheres using nanoscale Kirkendall diffusion called the "all-in-one beaker method" is introduced. The Fe2O3, SnO2, NiO, and Co3O4 hollow nanospheres are successfully prepared by the all-in-one beaker method. The detailed formation mechanism of aggregate-free hematite hollow nanospheres is studied. Dimethylformamide solution containing Fe acetate, polyacrylonitrile (PAN), and polystyrene (PS) transforms into aggregate-free Fe2O3 hollow nanospheres. The porous structure formed by the combustion of PS provides a good pathway for the reducing gas. The carbon matrix formed from PAN acts as a barrier, which can prevent the aggregation of metallic Fe nanopowders by surrounding each particle. The Fe-C bulk material formed as an intermediate product transforms into aggregate-free Fe2O3 hollow nanospheres by the nanoscale Kirkendall diffusion process. The mean size and shell thickness of the hollow Fe2O3 nanospheres measured from the TEM images are 52 and 9 nm, respectively. The discharge capacities of the Fe2O3 nanopowders with hollow and dense structures and the bulk material for the 200th cycle at a current density of 0.5 A g(-1) are 1012, 498, and 637 mA h g(-1), respectively, and their capacity retentions calculated compared to those in the second cycles are 92, 45, and 59%, respectively. Additionally, Fe2O3 hollow nanospheres cycled at 1 A g(-1) after 1000 cycles showed a high discharge capacity of 871 mA h g(-1) (capacity retention was 80% from the second cycle). The Fe2O3, SnO2, NiO, and Co3O4 hollow nanospheres show excellent cycling performances for lithium-ion storage because they have a high contact area with the liquid electrolyte and space for accommodating a huge volume change during cycling.

  15. Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

    PubMed

    Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G; Corydon, Thomas J; Mikkelsen, Jacob Giehm; Aagaard, Lars

    2015-08-01

    Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo. PMID:26204415

  16. DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors

    PubMed Central

    Cai, Yujia; Bak, Rasmus O.; Krogh, Louise Bechmann; Staunstrup, Nicklas H.; Moldt, Brian; Corydon, Thomas J.; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

    2014-01-01

    DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies. PMID:24270790

  17. B-cell reconstitution after lentiviral vector–mediated gene therapy in patients with Wiskott-Aldrich syndrome

    PubMed Central

    Castiello, Maria Carmina; Scaramuzza, Samantha; Pala, Francesca; Ferrua, Francesca; Uva, Paolo; Brigida, Immacolata; Sereni, Lucia; van der Burg, Mirjam; Ottaviano, Giorgio; Albert, Michael H.; Grazia Roncarolo, Maria; Naldini, Luigi; Aiuti, Alessandro; Villa, Anna; Bosticardo, Marita

    2015-01-01

    Background Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene–corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach. Objective Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration. Methods We evaluated B-cell counts, B-cell subset distribution, B cell–activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein. Results After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19+CD21−CD35− and CD21low B cells and a reduction in B cell–activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients. Conclusions We provide evidence that lentiviral vector-mediated GT induces transgene expression in the B-cell compartment, resulting in ameliorated B-cell development and functionality and contributing to immunologic

  18. Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency.

    PubMed

    De Ravin, Suk See; Wu, Xiaolin; Moir, Susan; Anaya-O'Brien, Sandra; Kwatemaa, Nana; Littel, Patricia; Theobald, Narda; Choi, Uimook; Su, Ling; Marquesen, Martha; Hilligoss, Dianne; Lee, Janet; Buckner, Clarissa M; Zarember, Kol A; O'Connor, Geraldine; McVicar, Daniel; Kuhns, Douglas; Throm, Robert E; Zhou, Sheng; Notarangelo, Luigi D; Hanson, I Celine; Cowan, Mort J; Kang, Elizabeth; Hadigan, Coleen; Meagher, Michael; Gray, John T; Sorrentino, Brian P; Malech, Harry L

    2016-04-20

    X-linked severe combined immunodeficiency (SCID-X1) is a profound deficiency of T, B, and natural killer (NK) cell immunity caused by mutations inIL2RGencoding the common chain (γc) of several interleukin receptors. Gamma-retroviral (γRV) gene therapy of SCID-X1 infants without conditioning restores T cell immunity without B or NK cell correction, but similar treatment fails in older SCID-X1 children. We used a lentiviral gene therapy approach to treat five SCID-X1 patients with persistent immune dysfunction despite haploidentical hematopoietic stem cell (HSC) transplant in infancy. Follow-up data from two older patients demonstrate that lentiviral vector γc transduced autologous HSC gene therapy after nonmyeloablative busulfan conditioning achieves selective expansion of gene-marked T, NK, and B cells, which is associated with sustained restoration of humoral responses to immunization and clinical improvement at 2 to 3 years after treatment. Similar gene marking levels have been achieved in three younger patients, albeit with only 6 to 9 months of follow-up. Lentiviral gene therapy with reduced-intensity conditioning appears safe and can restore humoral immune function to posthaploidentical transplant older patients with SCID-X1. PMID:27099176

  19. Comparison of Lentiviral and Sleeping Beauty Mediated αβ T Cell Receptor Gene Transfer

    PubMed Central

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm’s tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies. PMID:23840834

  20. Lentiviral-based approach for the validation of cancer therapeutic targets in vivo.

    PubMed

    Ambrogio, Chiara; Stern, Patrick; Scuoppo, Claudio; Kranz, Harald; Barbacid, Mariano; Santamaría, David

    2014-10-01

    Despite the pressing need for novel cancer treatments, our improved understanding of tumor biology is not being successfully translated into better therapies. Here we present a lentiviral vector that enables in vivo validation of cancer therapeutic targets when combined with existing cancer animal models that faithfully reproduce the natural history of human disease. Unlike the conventional genetic approaches with targeted alleles, the outlined experimental strategy could be used to assess the preclinical efficacy of a growing number of putative therapeutic hits in a rapid and cost-effective manner. PMID:25312087

  1. All-in-One Chip

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Under an SBIR contract, EMC Technology, Inc., was funded to develop a family of Temperature Compensating Attenuators. These devices are used to compensate for changes in amplifier gain with temperatures. Special thermistor materials were developed that not only satisfied NASA requirements, but also proved useful on several new commercial products. New Thermopad devices with greater compensation and lower loss were developed and used by companies such as Hughes Space and Communications, Motorola, Lucent, Ericsson, and General Instrument. A Power Sensing Termination (Smartload) was also developed for use in communication systems that require accurate, reliable, low cost power detection for level control and alarm circuits.

  2. Optimized Lentiviral Transduction Protocols by Use of a Poloxamer Enhancer, Spinoculation, and scFv-Antibody Fusions to VSV-G.

    PubMed

    Anastasov, Nataša; Höfig, Ines; Mall, Sabine; Krackhardt, Angela M; Thirion, Christian

    2016-01-01

    Lentiviral vectors (LV) are widely used to successfully transduce cells for research and clinical applications. This optimized LV infection protocol includes a nontoxic poloxamer-based adjuvant combined with antibody-retargeted lentiviral particles. The novel poloxamer P338 demonstrates superior characteristics for enhancing lentiviral transduction over the best-in-class polybrene-assisted transduction. Poloxamer P338 exhibited dual benefits of low toxicity and high efficiency of lentiviral gene delivery into a range of different primary cell cultures. One of the major advantages of P338 is its availability in pharma grade and applicability as cell culture medium additive in clinical protocols. Lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) can be produced to high titers and mediate high transduction efficiencies in vitro. For clinical applications the need for optimized transduction protocols, especially for transduction of primary T and stem cells, is high. The successful use of retronectin, the second lentivirus enhancer available as GMP material, requires the application of specific coating protocols not applicable in all processes, and results in the need of a relatively high multiplicity of infection (MOI) to achieve effective transduction efficiencies for hematopoietic cells (e.g., CD34+ hematopoietic stem cells). Cell specificity of lentiviral vectors was successfully increased by displaying different ratios of scFv-fused VSV-G glycoproteins on the viral envelope. The system has been validated with human CD30+ lymphoma cells, resulting in preferential gene delivery to CD30+ cells, which was increased fourfold in mixed cell cultures, by presenting scFv antibody fragments binding to respective surface markers. A combination of spinoculation and poloxamer-based chemical adjuvant increases the transduction of primary T-cells by greater than twofold. The combination of poloxamer-based and scFv-retargeted LVs increased

  3. Lentiviral gene therapy using cellular promoters cures type 1 Gaucher disease in mice.

    PubMed

    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

    2015-05-01

    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.

  4. Development of a Multipurpose GATEWAY-Based Lentiviral Tetracycline-Regulated Conditional RNAi System (GLTR)

    PubMed Central

    Shivalingaiah, Giridhar; Kofler, Reinhard; Geley, Stephan

    2014-01-01

    RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success, however, depends on the effective expression of RNAi-inducing small double-stranded interfering RNA molecules (siRNAs) in target cells. In many cell types, RNAi can be achieved by transfection of chemically synthesised siRNAs, which results in transient knockdown of protein expression. Expression of double-stranded short hairpin RNA (shRNA) provides another means to induce RNAi in cells that are hard to transfect. To facilitate the generation of stable, conditional RNAi cell lines, we have developed novel one- and two-component vector GATEWAY-compatible lentiviral tetracycline-regulated RNAi (GLTR) systems. The combination of a modified RNA-polymerase-III-dependent H1 RNA promoter (designated ‘THT’) for conditional shRNA expression with different lentiviral delivery vectors allows (1) the use of fluorescent proteins for colour-coded combinatorial RNAi or for monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one vector system (pGLTR-X). All three systems were found to be suitable for the analysis of essential genes, such as CDC27, a component of the mitotic ubiquitin ligase APC/C, in cell lines and primary human cells. PMID:24841113

  5. Lentiviral gene therapy using cellular promoters cures type 1 Gaucher disease in mice.

    PubMed

    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

    2015-05-01

    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

  6. Bioinspired Synthesis of All-in-One Organic-Inorganic Hybrid Nanoflowers Combined with a Handheld pH Meter for On-Site Detection of Food Pathogen.

    PubMed

    Ye, Ranfeng; Zhu, Chengzhou; Song, Yang; Lu, Qian; Ge, Xiaoxiao; Yang, Xu; Zhu, Mei-Jun; Du, Dan; Li, He; Lin, Yuehe

    2016-06-01

    With a mild elaborately bioinspired one-pot process, Con A-GOx-CaHPO4 nanoflowers are prepared. Employing the as-prepared all-in-one hybrid nanoflowers as signal tags, a simple but potentially powerful amplification biosensing technology for the detection of food pathogen with excellent simplicity, portability, sensitivity, and adaptability is achieved.

  7. Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients

    PubMed Central

    Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Luz Lozano, M; Lamana, Maria Luisa; Sevilla, Julian; Olive, Teresa; Diaz-Heredia, Cristina; Badell, Isabel; Estella, Jesus; Madero, Luis; Guenechea, Guillermo; Casado, José; Segovia, Jose C; Bueren, Juan A

    2009-01-01

    Previous clinical trials based on the genetic correction of purified CD34+ cells with γ-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34+ cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34+ cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34− mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs). PMID:19277017

  8. A minimally invasive, lentiviral based method for the rapid and sustained genetic manipulation of renal tubules.

    PubMed

    Espana-Agusti, Judit; Tuveson, David A; Adams, David J; Matakidou, Athena

    2015-01-01

    The accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation. Direct, somatic, genetic engineering approaches allow for accelerated and flexible genetic manipulation and represent an attractive alternative to GEMMs. In this study we investigated the feasibility, safety and efficiency of a minimally invasive, lentiviral based approach for the sustained in-vivo modification of renal tubular epithelial cells. Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma. We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection. Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences. This study demonstrates that ultrasound-guided injection of lentiviral vectors provides a safe and efficient method for the genetic manipulation of renal tubules, representing a quick and versatile alternative to GEMMs for the functional characterisation of disease-related genes. PMID:26046460

  9. Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy.

    PubMed

    Meneghini, Vasco; Lattanzi, Annalisa; Tiradani, Luigi; Bravo, Gabriele; Morena, Francesco; Sanvito, Francesca; Calabria, Andrea; Bringas, John; Fisher-Perkins, Jeanne M; Dufour, Jason P; Baker, Kate C; Doglioni, Claudio; Montini, Eugenio; Bunnell, Bruce A; Bankiewicz, Krystof; Martino, Sabata; Naldini, Luigi; Gritti, Angela

    2016-05-02

    Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non-human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe-affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

  10. Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy.

    PubMed

    Meneghini, Vasco; Lattanzi, Annalisa; Tiradani, Luigi; Bravo, Gabriele; Morena, Francesco; Sanvito, Francesca; Calabria, Andrea; Bringas, John; Fisher-Perkins, Jeanne M; Dufour, Jason P; Baker, Kate C; Doglioni, Claudio; Montini, Eugenio; Bunnell, Bruce A; Bankiewicz, Krystof; Martino, Sabata; Naldini, Luigi; Gritti, Angela

    2016-01-01

    Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non-human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe-affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD. PMID

  11. Efficient biotechnological approach for lentiviral transduction of induced pluripotent stem cells.

    PubMed

    Zare, Mehrak; Soleimani, Masoud; Mohammadian, Mozhdeh; Akbarzadeh, Abolfazl; Havasi, Parvaneh; Zarghami, Nosratollah

    2016-01-01

    Induced pluripotent stem (iPS) cells are generated from differentiated adult somatic cells by reprogramming them. Unlimited self-renewal, and the potential to differentiate into any cell type, make iPS cells very promising candidates for basic and clinical research. Furthermore, iPS cells can be genetically manipulated for use as therapeutic tools. DNA can be introduced into iPS cells, using lentiviral vectors, which represent a helpful choice for efficient transduction and stable integration of transgenes. In this study, we compare two methods of lentiviral transduction of iPS cells, namely, the suspension method and the hanging drop method. In contrast to the conventional suspension method, in the hanging drop method, embryoid body (EB) formation and transduction occur concurrently. The iPS cells were cultured to form EBs, and then transduced with lentiviruses, using the conventional suspension method and the hanging drop method, to express miR-128 and green fluorescent protein (GFP). The number of transduced cells were assessed by fluorescent microscopy and flow cytometry. MTT assay and real-time PCR were performed to determine the cell viability and transgene expression, respectively. Morphologically, GFP+ cells were more detectable in the hanging drop method, and this finding was quantified by flow cytometric analysis. According to the results of the MTT assay, cell viability was considerably higher in the hanging drop method, and real-time PCR represented a higher relative expression of miR-128 in the iPS cells introduced with lentiviruses in drops. Altogether, it seems that lentiviral transduction of challenging iPS cells using the hanging drop method offers a suitable and sufficient strategy in their gene transfer, with less toxicity than the conventional suspension method.

  12. A job for quantum dots: use of a smartphone and 3D-printed accessory for all-in-one excitation and imaging of photoluminescence.

    PubMed

    Petryayeva, Eleonora; Algar, W Russ

    2016-04-01

    Point-of-care (POC) diagnostic technologies are needed to improve global health and smartphones are a prospective platform for these technologies. While many fluorescence or photoluminescence-based smartphone assays have been reported in the literature, common shortcomings are the requirement of an excitation light source external to the smartphone and complicated integration of that excitation source with the smartphone. Here, we show that the photographic flash associated with the smartphone camera can be utilized to enable all-in-one excitation and imaging of photoluminescence (PL), thus eliminating the need for an excitation light source external to the smartphone. A simple and low-cost 3D-printed accessory was designed to create a dark environment and direct excitation light from the smartphone flash onto a sample. Multiple colors and compositions of semiconductor quantum dot (QD) were evaluated as photoluminescent materials for all-in-one smartphone excitation and imaging of PL, and these were compared with fluorescein and R-phycoerythrin (R-PE), which are widely utilized molecular and protein materials for fluorescence-based bioanalysis. The QDs were found to exhibit much better brightness and have the best potential for two-color detection. A model protein binding assay with a sub-microgram per milliliter detection limit and a Förster resonance energy transfer (FRET) assay for proteolytic activity were demonstrated, including imaging with serum as a sample matrix. In addition, FRET within tandem conjugates of a QD donor and fluorescent dye acceptor enabled smartphone detection of dye fluorescence that was otherwise unobservable without the QD to enhance its brightness. The ideal properties of photoluminescent materials for all-in-one smartphone excitation and imaging are discussed in the context of several different materials, where QDs appear to be the best overall material for this application.

  13. All-in-one assembly based on 3D-intertangled and cross-jointed architectures of Si/Cu 1D-nanowires for lithium ion batteries

    PubMed Central

    Hwang, Chihyun; Kim, Tae-Hee; Cho, Yoon-Gyo; Kim, Jieun; Song, Hyun-Kon

    2015-01-01

    All-in-one assemblies of separator, electrode and current collector (SECA) for lithium ion batteries are presented by using 1D nanowires of Si and Cu (nwSi and nwCu). Even without binders, integrity of SECA is secured via structural joints based on ductility of Cu as well as entanglement of nwSi and nwCu. By controlling the ratio of the nanowires, the number of contact points and voids accommodating volume expansion of Si active material are tunable. Zero volume expansion and high energy density are simultaneously achievable by the architecture. PMID:25720334

  14. A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

    PubMed

    Gonçalves, Cristine; Gross, Fabian; Guégan, Philippe; Cheradame, Hervé; Midou, Patrick

    2014-11-01

    Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications. PMID:25215936

  15. Ultrastructural observation of the acid-base resistant zone of all-in-one adhesives using three different acid-base challenges.

    PubMed

    Tsujimoto, Miho; Nikaido, Toru; Inoue, Go; Sadr, Alireza; Tagami, Junji

    2010-11-01

    The aim of this study was to analyze the ultrastructure of the dentin-adhesive interface using two all-in-one adhesive systems (Clearfil Tri-S Bond, TB; Tokuyama Bond Force, BF) after different acid-base challenges. Three solutions were used as acidic solutions for the acid-base challenges: a demineralizing solution (DS), a phosphoric acid solution (PA), and a hydrochloric acid solution (HCl). After the acid-base challenges, the bonded interfaces were examined by scanning electron microscopy. Thickness of the acid-base resistant zone (ABRZ) created in PA and HCl was thinner than in DS for both adhesive systems. For BF adhesive, an eroded area was observed beneath the ABRZ after immersion in PA and HCl, but not in DS. Conversely for TB adhesive, the eroded area was observed only after immersion in PA. In conclusion, although the ABRZ was observed for both all-in-one adhesive systems, its morphological features were influenced by the ingredients of both the adhesive material and acidic solution.

  16. Thin resin coating by dual-application of all-in-one adhesives improves dentin bond strength of resin cements for indirect restorations.

    PubMed

    Takahashi, Rena; Nikaido, Toru; Ariyoshi, Meu; Kitayama, Shuzo; Sadr, Alireza; Foxton, Richard M; Tagami, Junji

    2010-10-01

    This study was evaluated the tensile bond strength (TBS) of resin cements to bovine dentin resin-coated with all-in-one adhesive systems. Each of the dual-polymerizing resin cements; Link Max, Clearfil Esthetic Cement, Bistite II and Chemiace II were used to bond indirect resin disks to bovine dentin, as control, or coated by single-application or by dual-application of an adhesive system from the same manufacturer; G-Bond, Clearfil Tri-S Bond, Tokuyama Bond Force and Hybrid-Coat (n=10). After 24-hour water storage, TBSs were measured. The fracture pattern and the adhesive interface were observed using an SEM. Dual-application of the adhesive yielded significantly higher TBSs compared to control and single-application groups for all materials (p<0.001). From the limited information of this study, it was concluded that dual-application of all-in-one adhesive systems created a thin coating on dentin, and significantly improved the bond strengths of resin cements.

  17. Thin resin coating by dual-application of all-in-one adhesives improves dentin bond strength of resin cements for indirect restorations.

    PubMed

    Takahashi, Rena; Nikaido, Toru; Ariyoshi, Meu; Kitayama, Shuzo; Sadr, Alireza; Foxton, Richard M; Tagami, Junji

    2010-10-01

    This study was evaluated the tensile bond strength (TBS) of resin cements to bovine dentin resin-coated with all-in-one adhesive systems. Each of the dual-polymerizing resin cements; Link Max, Clearfil Esthetic Cement, Bistite II and Chemiace II were used to bond indirect resin disks to bovine dentin, as control, or coated by single-application or by dual-application of an adhesive system from the same manufacturer; G-Bond, Clearfil Tri-S Bond, Tokuyama Bond Force and Hybrid-Coat (n=10). After 24-hour water storage, TBSs were measured. The fracture pattern and the adhesive interface were observed using an SEM. Dual-application of the adhesive yielded significantly higher TBSs compared to control and single-application groups for all materials (p<0.001). From the limited information of this study, it was concluded that dual-application of all-in-one adhesive systems created a thin coating on dentin, and significantly improved the bond strengths of resin cements. PMID:20823621

  18. Decitabine suspends human CD34+ cell differentiation and proliferation during lentiviral transduction.

    PubMed

    Uchida, Naoya; Hsieh, Matthew M; Platner, Charlotte; Saunthararajah, Yogen; Tisdale, John F

    2014-01-01

    Efficient ex vivo transduction of hematopoietic stem cells (HSCs) is encumbered by differentiation which reduces engraftment. We hypothesized that inhibiting DNA methyltransferase with decitabine would block differentiation of transduced CD34+ cells under cytokine stimulation and thus improve transduction efficiency for engrafting HSCs. Human CD34+ cells in cytokine-containing media were treated with or without decitabine for 24 or 48 hours, and then these cells were transduced with a GFP-expressing lentiviral vector. Utilizing decitabine pre-treatment for 48 hours, we observed an equivalent percentage of successfully transduced cells (GFP-positivity) and a higher percentage of cells that retained CD34 positivity, compared to no decitabine exposure. Cell proliferation was inhibited after decitabine exposure. Similar results were observed among CD34+ cells from six different donors. Repopulating activity was evaluated by transplantation into NOD/SCID/IL2Rγnull mice and demonstrated an equivalent percentage of GFP-positivity in human cells from decitabine-treated samples and a trend for higher human cell engraftment (measured 20-24 weeks after transplantation), compared to no decitabine exposure. In conclusion, ex vivo decitabine exposure inhibits both differentiation and proliferation in transduced human CD34+ cells and modestly increases the engraftment ability in xenograft mice, while the transduction efficiency is equivalent in decitabine exposure, suggesting improvement of lentiviral transduction for HSCs.

  19. RNAi technology and lentiviral delivery as a powerful tool to suppress Tpr-Met-mediated tumorigenesis.

    PubMed

    Taulli, Riccardo; Accornero, Paolo; Follenzi, Antonia; Mangano, Tony; Morotti, Alessandro; Scuoppo, Claudio; Forni, Paolo E; Bersani, Francesca; Crepaldi, Tiziana; Chiarle, Roberto; Naldini, Luigi; Ponzetto, Carola

    2005-05-01

    Tpr-Met, the oncogenic counterpart of the Met receptor, has been detected in gastric cancers, as well as in precursor lesions and in the adjacent normal gastric mucosa. This has prompted the suggestion that Tpr-Met may predispose to the development of gastric tumors. Given the sequence specificity of RNA interference, oncogenes activated by point mutation or rearrangements can be targeted while spearing the product of the wild-type allele. In this work, we report specific suppression of Tpr-Met expression and inhibition of Tpr-Met-mediated transformation and tumorigenesis by means of a short interfering RNA (siRNA) directed toward the Tpr-Met junction (anti-TM2). When delivered by a lentiviral vector, anti-TM2 siRNA was effective also in mouse embryonal fibroblasts or epithelial cells expressing high levels of Tpr-Met. Our results suggest that lentiviral-mediated delivery of anti-TM2 siRNA may be developed into a powerful tool to treat Tpr-Met-positive cancers.

  20. Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning.

    PubMed

    Monzani, P S; Sangalli, J R; De Bem, T H C; Bressan, F F; Fantinato-Neto, P; Pimentel, J R V; Birgel-Junior, E H; Fontes, A M; Covas, D T; Meirelles, F V

    2013-02-28

    Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine β-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.

  1. Lung endothelial HO-1 targeting in vivo using lentiviral miRNA regulates apoptosis and autophagy during oxidant injury

    PubMed Central

    Zhang, Yi; Jiang, Ge; Sauler, Maor; Lee, Patty J.

    2013-01-01

    The lung endothelium is a major target for inflammatory and oxidative stress. Heme oxygenase-1 (HO-1) induction is a crucial defense mechanism during oxidant challenges, such as hyperoxia. The role of lung endothelial HO-1during hyperoxia in vivo is not well defined. We engineered lentiviral vectors with microRNA (miRNA) sequences controlled by vascular endothelium cadherin (VE-cad) to study the specific role of lung endothelial HO-1. Wild-type (WT) murine lung endothelial cells (MLECs) or WT mice were treated with lentivirus and exposed to hyperoxia (95% oxygen). We detected HO-1 knockdown (∼55%) specifically in the lung endothelium. MLECs and lungs showed approximately a 2-fold increase in apoptosis and ROS generation after HO-1 silencing. We also demonstrate for the first time that silencing endothelial HO-1 has the same effect on lung injury and survival as silencing HO-1 in multiple lung cell types and that HO-1 regulates caspase 3 activation and autophagy in endothelium during hyperoxia. These studies demonstrate the utility of endothelial-targeted gene silencing in vivo using lentiviral miRNA constructs to assess gene function and that endothelial HO-1 is an important determinant of survival during hyperoxia.—Zhang, Y., Jiang, G., Sauler, M., Lee, P. J. Lung endothelial HO-1 targeting in vivo using lentiviral miRNA regulates apoptosis and autophagy during oxidant injury. PMID:23771928

  2. Effect of sodium ascorbate on the bond strength of all-in-one adhesive systems to NaOCl-treated dentin

    PubMed Central

    Ebrahimi-Chaharom, Mohammad-Esmaeel; Kimyai, Soodabeh; Mohammadi, Narmin; Oskoee, Parnian-Alizadeh; Daneshpuy, Mehdi

    2015-01-01

    Background Ascorbic acid and its salts are low-toxicity products, which are routinely used in food industries as antioxidants. The aim of the present study was to evaluate the effect of 10% sodium ascorbate on the bond strength of two all-in-one adhesive systems to NaOCl-treated dentin. Material and Methods After exposing the dentin on the facial surface of 90 sound human premolars and mounting in an acrylic resin mold, the exposed dentin surfaces were polished with 600-grit SiC paper under running water. Then the samples were randomly divided into 6 groups of 15. Groups 1 and 4 were the controls, in which no surface preparation was carried out. In groups 2 and 5 the dentin surfaces were treated with 5.25% NaOCl alone for 10 minutes and in groups 3 and 6 with 5.25% NaOCl for 10 minutes followed by 10% sodium ascorbate for 10 minutes. Then composite resin cylinders, measuring 2 mm in diameter and 2 mm in height, were bonded on the dentin surfaces in groups 1, 2 and 3 with Clearfil S3 Bond and in groups 4, 5 and 6 with Adper Easy One adhesive systems according to manufacturers’ instructions. The samples were stored in distilled water for 24 hours at 37°C and then thermocycled. Finally, the samples underwent shear bond strength test in a universal testing machine at a strain rate of 1 mm/min. Data were analyzed with two-way ANOVA and post hoc Tukey tests at α=0.05. Results The differences between groups 1 and 2 (P=0.01), 1 and 5 (P=0.003). 1 and 6 (P=0.03) and 4 and 5 (P=0.03) were statistically significant. Two-by-two comparisons did not reveal any significant difference between other groups (P>0.05). Conclusions Use of 10% sodium ascorbate for 10 minutes restored the decreased bond strength of the adhesive systems to that of the control groups. Key words:Sodium ascorbate, adhesive systems, all-in-one, bond strength, sodium hypochlorite. PMID:26644835

  3. Effect of functional monomers in all-in-one adhesive systems on formation of enamel/dentin acid-base resistant zone.

    PubMed

    Nikaido, Toru; Ichikawa, Chiaki; Li, Na; Takagaki, Tomohiro; Sadr, Alireza; Yoshida, Yasuhiro; Suzuki, Kazuomi; Tagami, Junji

    2011-01-01

    This study aimed at evaluating the effect of functional monomers in all-in-one adhesive systems on formation of acid-base resistant zone (ABRZ) in enamel and dentin. Experimental adhesive systems containing one of three functional monomers; MDP, 3D-SR and 4-META were applied to enamel or dentin surface and light-cured. A universal resin composite was then placed. The specimens were subjected to a demineralizing solution (pH 4.5) and 5% NaClO for acid-base challenge and then observed by SEM. The ABRZ was clearly observed in both enamel and dentin interfaces. However, enamel ABRZ was thinner than dentin ABRZ in all adhesives. Morphology of the ABRZ was different between enamel and dentin, and also among the adhesives. Funnel-shaped erosion was observed only in the enamel specimen with the 4-META adhesive. The formation of enamel/dentin ABRZ was confirmed in all adhesives, but the morphology was influenced by the functional monomers.

  4. Bioinspired, Highly Stretchable, and Conductive Dry Adhesives Based on 1D-2D Hybrid Carbon Nanocomposites for All-in-One ECG Electrodes.

    PubMed

    Kim, Taehoon; Park, Junyong; Sohn, Jongmoo; Cho, Donghwi; Jeon, Seokwoo

    2016-04-26

    Here we propose a concept of conductive dry adhesives (CDA) combining a gecko-inspired hierarchical structure and an elastomeric carbon nanocomposite. To complement the poor electrical percolation of 1D carbon nanotube (CNT) networks in an elastomeric matrix at a low filler content (∼1 wt %), a higher dimensional carbon material (i.e., carbon black, nanographite, and graphene nanopowder) is added into the mixture as an aid filler. The co-doped graphene and CNT in the composite show the lowest volume resistance (∼100 ohm·cm) at an optimized filler ratio (1:9, total filler content: 1 wt %) through a synergetic effect in electrical percolation. With an optimized conductive elastomer, gecko-inspired high-aspect-ratio (>3) microstructures over a large area (∼4 in.(2)) are successfully replicated from intaglio-patterned molds without collapse. The resultant CDA pad shows a high normal adhesion force (∼1.3 N/cm(2)) even on rough human skin and an excellent cycling property for repeatable use over 30 times without degradation of adhesion force, which cannot be achieved by commercial wet adhesives. The body-attachable CDA can be used as a metal-free, all-in-one component for measuring biosignals under daily activity conditions (i.e., underwater, movements) because of its superior conformality and water-repellent characteristic. PMID:26986477

  5. Helicenes as All-in-One Organic Materials for Application in OLEDs: Synthesis and Diverse Applications of Carbo- and Aza[5]helical Diamines.

    PubMed

    Jhulki, Samik; Mishra, Abhaya Kumar; Chow, Tahsin J; Moorthy, Jarugu Narasimha

    2016-06-27

    A set of eight helical diamines were designed and synthesized to demonstrate their relevance as all-in-one materials for multifarious applications in organic light-emitting diodes (OLEDs), that is, as hole-transporting materials (HTMs), EMs, bifunctional hole transporting + emissive materials, and host materials. Azahelical diamines function very well as HTMs. Indeed, with high Tg values (127-214 °C), they are superior alternatives to popular N,N'-di(1-naphthyl)-N,N'-diphenyl-(1,1'-biphenyl)-4,4'-diamine (NPB). All the helical diamines exhibit emissive properties when employed in nondoped as well as doped devices, the performance characteristics being superior in the latter. One of the carbohelical diamines (CHTPA) serves the dual function of hole transport as well as emission in simple double-layer devices; the efficiencies observed were better by quite some margin than those of other emissive helicenes reported. The twisting endows helical diamines with significantly high triplet energies such that they also function as host materials for red and green phosphors, that is, [Ir(btp)2 acac] (btp=2-(2'-benzothienyl)pyridine; acac=acetylacetonate) and [Ir(ppy)3 ] (ppy=2-phenylpyridine), respectively. The results of device fabrications demonstrate how helicity/ helical scaffold may be diligently exploited to create molecular systems for maneuvering diverse applications in OLEDs. PMID:27244223

  6. Helicenes as All-in-One Organic Materials for Application in OLEDs: Synthesis and Diverse Applications of Carbo- and Aza[5]helical Diamines.

    PubMed

    Jhulki, Samik; Mishra, Abhaya Kumar; Chow, Tahsin J; Moorthy, Jarugu Narasimha

    2016-06-27

    A set of eight helical diamines were designed and synthesized to demonstrate their relevance as all-in-one materials for multifarious applications in organic light-emitting diodes (OLEDs), that is, as hole-transporting materials (HTMs), EMs, bifunctional hole transporting + emissive materials, and host materials. Azahelical diamines function very well as HTMs. Indeed, with high Tg values (127-214 °C), they are superior alternatives to popular N,N'-di(1-naphthyl)-N,N'-diphenyl-(1,1'-biphenyl)-4,4'-diamine (NPB). All the helical diamines exhibit emissive properties when employed in nondoped as well as doped devices, the performance characteristics being superior in the latter. One of the carbohelical diamines (CHTPA) serves the dual function of hole transport as well as emission in simple double-layer devices; the efficiencies observed were better by quite some margin than those of other emissive helicenes reported. The twisting endows helical diamines with significantly high triplet energies such that they also function as host materials for red and green phosphors, that is, [Ir(btp)2 acac] (btp=2-(2'-benzothienyl)pyridine; acac=acetylacetonate) and [Ir(ppy)3 ] (ppy=2-phenylpyridine), respectively. The results of device fabrications demonstrate how helicity/ helical scaffold may be diligently exploited to create molecular systems for maneuvering diverse applications in OLEDs.

  7. Lentiviral Hematopoietic Stem Cell Gene Therapy in Inherited Metabolic Disorders

    PubMed Central

    2014-01-01

    Abstract After more than 20 years of development, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and storage disorders. This brief review summarizes the development and applications, focusing on the lysosomal enzyme deficiencies, especially Pompe disease. PMID:25184354

  8. First all-in-one diagnostic tool for DNA intelligence: genome-wide inference of biogeographic ancestry, appearance, relatedness, and sex with the Identitas v1 Forensic Chip.

    PubMed

    Keating, Brendan; Bansal, Aruna T; Walsh, Susan; Millman, Jonathan; Newman, Jonathan; Kidd, Kenneth; Budowle, Bruce; Eisenberg, Arthur; Donfack, Joseph; Gasparini, Paolo; Budimlija, Zoran; Henders, Anjali K; Chandrupatla, Hareesh; Duffy, David L; Gordon, Scott D; Hysi, Pirro; Liu, Fan; Medland, Sarah E; Rubin, Laurence; Martin, Nicholas G; Spector, Timothy D; Kayser, Manfred

    2013-05-01

    When a forensic DNA sample cannot be associated directly with a previously genotyped reference sample by standard short tandem repeat profiling, the investigation required for identifying perpetrators, victims, or missing persons can be both costly and time consuming. Here, we describe the outcome of a collaborative study using the Identitas Version 1 (v1) Forensic Chip, the first commercially available all-in-one tool dedicated to the concept of developing intelligence leads based on DNA. The chip allows parallel interrogation of 201,173 genome-wide autosomal, X-chromosomal, Y-chromosomal, and mitochondrial single nucleotide polymorphisms for inference of biogeographic ancestry, appearance, relatedness, and sex. The first assessment of the chip's performance was carried out on 3,196 blinded DNA samples of varying quantities and qualities, covering a wide range of biogeographic origin and eye/hair coloration as well as variation in relatedness and sex. Overall, 95 % of the samples (N = 3,034) passed quality checks with an overall genotype call rate >90 % on variable numbers of available recorded trait information. Predictions of sex, direct match, and first to third degree relatedness were highly accurate. Chip-based predictions of biparental continental ancestry were on average ~94 % correct (further support provided by separately inferred patrilineal and matrilineal ancestry). Predictions of eye color were 85 % correct for brown and 70 % correct for blue eyes, and predictions of hair color were 72 % for brown, 63 % for blond, 58 % for black, and 48 % for red hair. From the 5 % of samples (N = 162) with <90 % call rate, 56 % yielded correct continental ancestry predictions while 7 % yielded sufficient genotypes to allow hair and eye color prediction. Our results demonstrate that the Identitas v1 Forensic Chip holds great promise for a wide range of applications including criminal investigations, missing person investigations, and for national security

  9. Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

    SciTech Connect

    Cheng, Lei; Liu, Yi; Zhao, Hua; Zhang, Wen; Guo, Ying-Jun; Nie, Lin

    2013-10-18

    Highlights: •CDNF was successfully transfected by a lentiviral vector into the distal sciatic nerve. •CDNF improved S-100, NF200 expression and nerve regeneration after sciatic injury. •CDNF improved the remyelination and thickness of the regenerated sciatic nerve. •CDNF improved gastrocnemius muscle weight and sciatic functional recovery. -- Abstract: Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a

  10. Role of Transgene Regulation in Ex Vivo Lentiviral Correction of Artemis Deficiency

    PubMed Central

    Multhaup, Megan M.; Podetz-Pedersen, Kelly M.; Karlen, Andrea D.; Olson, Erik R.; Gunther, Roland; Somia, Nikunj V.; Blazar, Bruce R.; Cowan, Morton J.

    2015-01-01

    Abstract Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1α-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the APro-Artemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID. PMID:25738323

  11. Vectofusin-1, a new viral entry enhancer, strongly promotes lentiviral transduction of human hematopoietic stem cells.

    PubMed

    Fenard, David; Ingrao, Dina; Seye, Ababacar; Buisset, Julien; Genries, Sandrine; Martin, Samia; Kichler, Antoine; Galy, Anne

    2013-05-07

    Gene transfer into hCD34(+) hematopoietic stem/progenitor cells (HSCs) using human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) has several promising therapeutic applications. Yet, efficiency, safety, and cost of LV gene therapy could be ameliorated by enhancing target cell transduction levels and reducing the amount of LV used on the cells. Several transduction enhancers already exist such as fibronectin fragments and cationic compounds, but all present limitations. In this study, we describe a new transduction enhancer called Vectofusin-1, which is a short cationic peptide, active on several LV pseudotypes. Vectofusin-1 is used as a soluble additive to safely increase the frequency of transduced HSCs and to augment the level of transduction to one or two copies of vector per cell in a vector dose-dependent manner. Vectofusin-1 acts at the entry step by promoting the adhesion and the fusion between viral and cellular membranes. Vectofusin-1 is therefore a promising additive that could significantly ameliorate hCD34(+) cell-based gene therapy.Molecular Therapy-Nucleic Acids (2013) 2, e90; doi:10.1038/mtna.2013.17; published online 7 May 2013.

  12. Role of transgene regulation in ex vivo lentiviral correction of artemis deficiency.

    PubMed

    Multhaup, Megan M; Podetz-Pedersen, Kelly M; Karlen, Andrea D; Olson, Erik R; Gunther, Roland; Somia, Nikunj V; Blazar, Bruce R; Cowan, Morton J; McIvor, R Scott

    2015-04-01

    Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1α-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the APro-Artemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID. PMID:25738323

  13. Lentiviral Delivery of RNAi for In Vivo Lineage-Specific Modulation of Gene Expression in Mouse Lung Macrophages

    PubMed Central

    Wilson, Andrew A; Kwok, Letty W; Porter, Emily L; Payne, Julie G; McElroy, Gregory S; Ohle, Sarah J; Greenhill, Sara R; Blahna, Matthew T; Yamamoto, Kazuko; Jean, Jyh C; Mizgerd, Joseph P; Kotton, Darrell N

    2013-01-01

    Although RNA interference (RNAi) has become a ubiquitous laboratory tool since its discovery 12 years ago, in vivo delivery to selected cell types remains a major technical challenge. Here, we report the use of lentiviral vectors for long-term in vivo delivery of RNAi selectively to resident alveolar macrophages (AMs), key immune effector cells in the lung. We demonstrate the therapeutic potential of this approach by RNAi-based downregulation of p65 (RelA), a component of the pro-inflammatory transcriptional regulator, nuclear factor κB (NF-κB) and a key participant in lung disease pathogenesis. In vivo RNAi delivery results in decreased induction of NF-κB and downstream neutrophilic chemokines in transduced AMs as well as attenuated lung neutrophilia following stimulation with lipopolysaccharide (LPS). Through concurrent delivery of a novel lentiviral reporter vector (lenti-NF-κB-luc-GFP) we track in vivo expression of NF-κB target genes in real time, a critical step towards extending RNAi-based therapy to longstanding lung diseases. Application of this system reveals that resident AMs persist in the airspaces of mice following the resolution of LPS-induced inflammation, thus allowing these localized cells to be used as effective vehicles for prolonged RNAi delivery in disease settings. PMID:23403494

  14. Endogenous Lentiviral Elements in the Weasel Family (Mustelidae)

    PubMed Central

    Han, Guan-Zhu; Worobey, Michael

    2012-01-01

    Endogenous retroviruses provide molecular fossils for studying the ancient evolutionary history of retroviruses. Here, we report our independent discovery and analysis of endogenous lentiviral insertions (Mustelidae endogenous lentivirus [MELV]) within the genomes of weasel family (Mustelidae). Genome-scale screening identified MELV elements in the domestic ferret (Mustela putorius furo) genome (MELVmpf). MELVmpf exhibits a typical lentiviral genomic organization. Phylogenetic analyses position MELVmpf basal to either primate lentiviruses or feline immunodeficiency virus. Moreover, we verified the presence of MELV insertions in the genomes of several species of the Lutrinae and Mustelinae subfamilies but not the Martinae subfamily, suggesting that the invasion of MELV into the Mustelidae genomes likely took place between 8.8 and 11.8 Ma. The discovery of MELV in weasel genomes extends the host range of lentiviruses to the Caniformia (order Carnivora) and provides important insights into the prehistoric diversity of lentiviruses. PMID:22522310

  15. Endogenous lentiviral elements in the weasel family (Mustelidae).

    PubMed

    Han, Guan-Zhu; Worobey, Michael

    2012-10-01

    Endogenous retroviruses provide molecular fossils for studying the ancient evolutionary history of retroviruses. Here, we report our independent discovery and analysis of endogenous lentiviral insertions (Mustelidae endogenous lentivirus [MELV]) within the genomes of weasel family (Mustelidae). Genome-scale screening identified MELV elements in the domestic ferret (Mustela putorius furo) genome (MELVmpf). MELVmpf exhibits a typical lentiviral genomic organization. Phylogenetic analyses position MELVmpf basal to either primate lentiviruses or feline immunodeficiency virus. Moreover, we verified the presence of MELV insertions in the genomes of several species of the Lutrinae and Mustelinae subfamilies but not the Martinae subfamily, suggesting that the invasion of MELV into the Mustelidae genomes likely took place between 8.8 and 11.8 Ma. The discovery of MELV in weasel genomes extends the host range of lentiviruses to the Caniformia (order Carnivora) and provides important insights into the prehistoric diversity of lentiviruses.

  16. Improved lentiviral transduction of ALS motoneurons in vivo via dual targeting.

    PubMed

    O'Leary, Valerie B; Ovsepian, Saak V; Bodeker, Macdara; Dolly, J Oliver

    2013-11-01

    Treatment of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, is hampered by its complex etiology and lack of efficient means for targeted transfer of therapeutics into motoneurons. The objective of this research was engineering of a versatile motoneuron targeting adapter--a full-length atoxic tetanus toxin fused to core-streptavidin (CS-TeTIM)--for retro-axonal transduction of viral vectors; validation of the targeting efficiency of CS-TeTIM in vivo, by expression of green fluorescence protein (GFP) reporter in motoneurons of presymptomatic and symptomatic ALS-like SOD1(G93A) mice, and comparison with age-matched controls; and appraisal of lentiviral transduction with CS-TeTIM relative to (1) a HC binding fragment of tetanus toxin CS-TeTx(HC), (2) rabies glycoprotein (RG), and (3) a CS-TeTIM-RG dual targeting approach. CS-TeTIM and CS-TeTx(HC) were engineered using recombinant technology and site-directed mutagenesis. Biotinylated vectors, pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) or RG, were linked to these adaptors and injected intraperitoneally (ip) into presymptomatic (12 weeks old), symptomatic SOD1(G93A) (22 weeks old) or wild type control mice, followed by monitoring of GFP expression in the spinal cord and supraspinal motor structures with quantitative PCR and immuno-histochemistry. Transcripts were detected in the spinal cord and supraspinal motor structures of all mice 2 weeks after receiving a single ip injection, although in symptomatic SOD1(G93A) animals reporter RNA levels were lower compared to presymptomatic and wild-type controls irrespective of the targeting approach. GFP transduction with CS-TeTIM proved more efficient than CS-TeTx(HC) across all groups while CS-TeTIM-RG dual-targeted vectors yielded the highest transcript numbers. Importantly, in both wild-type and presymptomatic SOD1(G93A) mice strong colabeling of choline-acetyltransferase (ChAT) and GFP was visualized in neurons of the

  17. Improved lentiviral transduction of ALS motoneurons in vivo via dual targeting.

    PubMed

    O'Leary, Valerie B; Ovsepian, Saak V; Bodeker, Macdara; Dolly, J Oliver

    2013-11-01

    Treatment of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, is hampered by its complex etiology and lack of efficient means for targeted transfer of therapeutics into motoneurons. The objective of this research was engineering of a versatile motoneuron targeting adapter--a full-length atoxic tetanus toxin fused to core-streptavidin (CS-TeTIM)--for retro-axonal transduction of viral vectors; validation of the targeting efficiency of CS-TeTIM in vivo, by expression of green fluorescence protein (GFP) reporter in motoneurons of presymptomatic and symptomatic ALS-like SOD1(G93A) mice, and comparison with age-matched controls; and appraisal of lentiviral transduction with CS-TeTIM relative to (1) a HC binding fragment of tetanus toxin CS-TeTx(HC), (2) rabies glycoprotein (RG), and (3) a CS-TeTIM-RG dual targeting approach. CS-TeTIM and CS-TeTx(HC) were engineered using recombinant technology and site-directed mutagenesis. Biotinylated vectors, pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) or RG, were linked to these adaptors and injected intraperitoneally (ip) into presymptomatic (12 weeks old), symptomatic SOD1(G93A) (22 weeks old) or wild type control mice, followed by monitoring of GFP expression in the spinal cord and supraspinal motor structures with quantitative PCR and immuno-histochemistry. Transcripts were detected in the spinal cord and supraspinal motor structures of all mice 2 weeks after receiving a single ip injection, although in symptomatic SOD1(G93A) animals reporter RNA levels were lower compared to presymptomatic and wild-type controls irrespective of the targeting approach. GFP transduction with CS-TeTIM proved more efficient than CS-TeTx(HC) across all groups while CS-TeTIM-RG dual-targeted vectors yielded the highest transcript numbers. Importantly, in both wild-type and presymptomatic SOD1(G93A) mice strong colabeling of choline-acetyltransferase (ChAT) and GFP was visualized in neurons of the

  18. Combination of grafted Schwann cells and lentiviral-mediated prevention of glial scar formation improve recovery of spinal cord injured rats.

    PubMed

    Do-Thi, Anh; Perrin, Florence E; Desclaux, Mathieu; Saillour, Paulette; Amar, Lahouari; Privat, Alain; Mallet, Jacques

    2016-10-01

    The present study was intended to combine three therapeutic approaches in a well-defined rat model of spinal cord injury, a lateral hemisection at thoracic level. A guidance channel was implanted at the lesion site. This channel was seeded with native Schwann cells or Schwann cells that had been previously transduced with a lentiviral vector carrying the GDNF gene. Thereafter, these experiences were reproduced in animals injected with lentiviral vectors carrying a shRNA for GFAP (Lv-shGFAP), which has recently been shown to block glial scar formation. Functional evaluations showed that Lv-shGFAP induced a significant improvement in recovery in animals grafted with Schwann cells. Histological studies demonstrated the outgrowth of axons in the guidance channel containing Schwann cells transduced or not with GDNF. This axonal growth was enhanced in rats receiving Lv-shGFAP vector. Also, a significant increase of serotonergic innervation of the injured hemicord, distal to the lesion, was found only in animals treated with Lv-shGFAP vectors. Importantly, this study confirms that glial scar formation is a major impediment for axonal sprouting after spinal cord injury, and emphasizes the importance of serotonergic innervation for locomotor function. Moreover we show a significant additive effect of a combinatorial approach to axonal regeneration in the injured spinal cord.

  19. Lentiviral-mediated silencing of glial fibrillary acidic protein and vimentin promotes anatomical plasticity and functional recovery after spinal cord injury.

    PubMed

    Desclaux, Mathieu; Perrin, Florence E; Do-Thi, Anh; Prieto-Cappellini, Monica; Gimenez Y Ribotta, Minerva; Mallet, Jacques; Privat, Alain

    2015-01-01

    In spinal cord injury (SCI), absence of functional recovery and lack of spontaneous axonal regeneration are attributed, among other factors, to the formation of a glial scar that forms both physical and chemical barriers. The glial scar is composed mainly of reactive astrocytes that overexpress two intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin (VIM). To promote regeneration and sprouting of spared axons after spinal cord trauma and with the objective of translation to clinics, we designed an original in vivo gene transfer strategy to reduce glial scar formation after SCI, based on the RNA interference (RNAi)-mediated inhibition of GFAP and VIM. We first show that direct injection of lentiviral vectors expressing short hairpin RNA (shRNA) against GFAP and VIM in a mouse model of SCI allows efficient and specific targeting of astrocytes. We then demonstrate that the lentiviral-mediated and stable expression of shGFAP and shVIM leads to a strong reduction of astrogliosis, improves functional motor recovery, and promotes axonal regrowth and sprouting of spared axons. This study thus examplifies how the nonneuronal environment might be a major target within the lesioned central nervous system to promote axonal regeneration (and sprouting) and validates the use of lentiviral-mediated RNAi in SCI.

  20. Retroviral vectors elevate coexpressed protein levels in trans through cap-dependent translation

    PubMed Central

    Gou, Yongqiang; Byun, Hyewon; Zook, Adam E.; B. Singh, Gurvani; Nash, Andrea K.; Lozano, Mary M.; Dudley, Jaquelin P.

    2015-01-01

    Retroviruses cause immunodeficiency and cancer but also are used as vectors for the expression of heterologous genes. Nevertheless, optimal translation of introduced genes often is not achieved. Here we show that transfection into mammalian cells of lentiviral or gammaretroviral vectors, including those with specific shRNAs, increased expression of a cotransfected gene relative to standard plasmid vectors. Levels of most endogenous cellular proteins were unchanged. Transfer of lentiviral vector sequences into a standard plasmid conferred the ability to give increased expression of cotransfected genes (superinduction). Superinduction by the retroviral vector was not dependent on the cell type or species, the type of reporter gene, or the method of transfection. No differences were detected in the IFN, unfolded protein, or stress responses in the presence of retroviral vectors. RT-PCRs revealed that RNA levels of cotransfected genes were unchanged during superinduction, yet Western blotting, pulse labeling, and the use of bicistronic vectors showed increased cap-dependent translation of cointroduced genes. Expression of the mammalian target of rapamycin (mTOR) kinase target 4E-BP1, but not the mTOR inhibitor Torin 1, preferentially inhibited superinduction relative to basal protein expression. Furthermore, transcription of lentiviral vector sequences from a doxycycline-inducible promoter eliminated superinduction, consistent with a DNA-triggered event. Thus, retroviral DNA increased translation of cointroduced genes in trans by an mTOR-independent signaling mechanism. Our experiments have broad applications for the design of retroviral vectors for transfections, DNA vaccines, and gene therapy. PMID:25737543

  1. All-in-One Movie Book.

    ERIC Educational Resources Information Center

    Petzold, Paul

    The amateur movie camera differs from a still camera on several important points. The author explores these differences and discusses the various ways they may be used to advantage. He describes in detail the workings of basic equipment--cameras, exposure meters, lenses, films, and lights--and demonstrates the proper use of each. Techniques such…

  2. Screening of Potential HIV-1 Inhibitors/Replication Blockers Using Secure Lentiviral in Vitro System.

    PubMed

    Prokofjeva, M M; Spirin, P V; Yanvarev, D V; Ivanov, A V; Novikov, M S; Stepanov, O A; Gottikh, M B; Kochetkov, S N; Fehse, B; Stocking, C; Prassolov, V S

    2011-10-01

    The development and usage of safe cell systems for testing agents which possess anti-HIV activity is a very important factor in the design of new drugs. We have described in detail a system we designed that is based on lentiviral vectors (Prokofjeva et. al.,Antiviral Therapy,in print) for swift and completely safe screening of potential HIV-1 replication inhibitors. The system enables one to test the efficiency of the inhibitory activity of compounds whose action is directed towards either wild-type HIV-1 reverse transcriptase or integrase, or mutant enzymes corresponding to the drug-resistant virus form. Testing results of a number of already known drugs, which correlate well with published data as well as data on newly synthesized compounds, were obtained. Application of this system substantially broadens the possibilities of preclinical anti-HIV drugs testing.

  3. Screening of Potential HIV-1 Inhibitors/Replication Blockers Using Secure Lentiviral in Vitro System

    PubMed Central

    Prokofjeva, M.M.; Spirin, P.V.; Yanvarev, D.V.; Ivanov, A.V.; Novikov, M.S.; Stepanov, O.A.; Gottikh, M.B.; Kochetkov, S.N.; Fehse, B.; Stocking, C.; Prassolov, V.S.

    2011-01-01

    The development and usage of safe cell systems for testing agents which possess anti-HIV activity is a very important factor in the design of new drugs. We have described in detail a system we designed that is based on lentiviral vectors (Prokofjeva et. al.,Antiviral Therapy,in print) for swift and completely safe screening of potential HIV-1 replication inhibitors. The system enables one to test the efficiency of the inhibitory activity of compounds whose action is directed towards either wild-type HIV-1 reverse transcriptase or integrase, or mutant enzymes corresponding to the drug-resistant virus form. Testing results of a number of already known drugs, which correlate well with published data as well as data on newly synthesized compounds, were obtained. Application of this system substantially broadens the possibilities of preclinical anti-HIV drugs testing. PMID:22649704

  4. Introducing Vectors.

    ERIC Educational Resources Information Center

    Roche, John

    1997-01-01

    Suggests an approach to teaching vectors that promotes active learning through challenging questions addressed to the class, as opposed to subtle explanations. Promotes introducing vector graphics with concrete examples, beginning with an explanation of the displacement vector. Also discusses artificial vectors, vector algebra, and unit vectors.…

  5. pLR: a lentiviral backbone series to stable transduction of bicistronic genes and exchange of promoters.

    PubMed

    Vargas, José Eduardo; Salton, Gabrielle; Sodré de Castro Laino, Andressa; Pires, Tiago Dalberto; Bonamino, Martin; Lenz, Guido; Delgado-Cañedo, Andrés

    2012-11-01

    Gene transfer based on lentiviral vectors allow the integration of exogenous genes into the genome of a target cell, turning these vectors into one of the most used methods for stable transgene expression in mammalian cells, in vitro and in vivo. Currently, there are no lentivectors that allow the cloning of different genes to be regulated by different promoters. Also, there are none that permit the analysis of the expression through an IRES (internal ribosome entry site)-- reporter gene system. In this work, we have generated a series of lentivectors containing: (1) a malleable structure to allow the cloning of different target genes in a multicloning site (mcs); (2) unique site to exchange promoters, and (3) IRES followed by one of two reporter genes: eGFP or DsRed. The series of the produced vectors were named pLR (for lentivirus and RSV promoter) and were fairly efficient with a strong fluorescence of the reporter genes in direct transfection and viral transduction experiments. This being said, the pLR series have been found to be powerful biotechnological tools for stable gene transfer and expression. PMID:22728068

  6. The Nef-Infectivity Enigma: Mechanisms of Enhanced Lentiviral Infection

    PubMed Central

    Vermeire, Jolien; Vanbillemont, Griet; Witkowski, Wojciech; Verhasselt, Bruno

    2011-01-01

    The Nef protein is an essential factor for lentiviral pathogenesis in humans and other simians. Despite a multitude of functions attributed to this protein, the exact role of Nef in disease progression remains unclear. One of its most intriguing functions is the ability of Nef to enhance the infectivity of viral particles. In this review we will discuss current insights in the mechanism of this well-known, yet poorly understood Nef effect. We will elaborate on effects of Nef, on both virion biogenesis and the early stage of the cellular infection, that might be involved in infectivity enhancement. In addition, we provide an overview of different HIV-1 Nef domains important for optimal infectivity and briefly discuss some possible sources of the frequent discrepancies in the field. Hereby we aim to contribute to a better understanding of this highly conserved and therapeutically attractive Nef function. PMID:22103831

  7. Development of hybrid viral vectors for gene therapy.

    PubMed

    Huang, Shuohao; Kamihira, Masamichi

    2013-01-01

    Adenoviral, retroviral/lentiviral, adeno-associated viral, and herpesviral vectors are the major viral vectors used in gene therapy. Compared with non-viral methods, viruses are highly-evolved, natural delivery agents for genetic materials. Despite their remarkable transduction efficiency, both clinical trials and laboratory experiments have suggested that viral vectors have inherent shortcomings for gene therapy, including limited loading capacity, immunogenicity, genotoxicity, and failure to support long-term adequate transgenic expression. One of the key issues in viral gene therapy is the state of the delivered genetic material in transduced cells. To address genotoxicity and improve the therapeutic transgene expression profile, construction of hybrid vectors have recently been developed. By adding new abilities or replacing certain undesirable elements, novel hybrid viral vectors are expected to outperform their conventional counterparts with improved safety and enhanced therapeutic efficacy. This review provides a comprehensive summary of current achievements in hybrid viral vector development and their impact on the field of gene therapy.

  8. Local long-term expression of lentivirally delivered IL-10 in the lung attenuates obliteration of intrapulmonary allograft airways.

    PubMed

    Hirayama, Shin; Sato, Masaaki; Liu, Mingyao; Loisel-Meyer, Severine; Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Zehong, Guan; Medin, Jeffrey A; Keshavjee, Shaf

    2011-11-01

    Obliterative bronchiolitis (OB) is a form of chronic rejection after lung transplantation. Lentiviral vectors (LVs) facilitate long-term gene transduction in many tissues and organs. We hypothesized that lentiviral gene transfer of interleukin (IL)-10, a potent immune-modulating cytokine, to the lung could modulate the alloimmune responses in the lung after transplantation. C57BL6 mice received LVs encoding luciferase, enhanced green fluorescent protein (eGFP), or human IL-10 (huIL-10) through airways and underwent repeated bioluminescent imaging, immunofluorescence imaging, or ELISA of lung tissues, respectively. Luciferase activities peaked at day 7 and were stable after day 28 to over 15 months. eGFP staining demonstrated LV-mediated gene transduction mainly in alveolar macrophages. LV-huIL-10 delivery resulted in stable long-term expression of huIL-10 in the lung tissue (average 3.66 pg/mg at 1 year). Intrapulmonary allograft tracheal transplantation (BALBc→C57BL6) was used as a model of OB. LV-huIL-10 or LV-eGFP were delivered 7 days before transplantation and compared with no LV-transfection group. Allograft airways at day 28 were almost completely obliterated in all the groups. However, at day 42, allograft airways treated with LV-huIL-10 showed a spectrum of attenuation in airway fibrosis ranging from complete obliteration through bubble-like partial opening to complete patency with epithelial coverage in association with a significantly reduced obliteration ratio compared with the other groups (p<0.05). In conclusion, lentivirus-mediated gene transduction is useful in achieving long-term transgene expression in the lung. Long-term IL-10 expression has the potential to attenuate allograft airway obliteration. LV-mediated gene therapy could be a useful strategy to prevent or treat OB after lung transplantation. PMID:21568692

  9. Lentiviral and adeno-associated vector-based therapy for motor neuron disease through RNAi.

    PubMed

    Towne, Chris; Aebischer, Patrick

    2009-01-01

    RNAi holds promise for neurodegenerative disorders caused by gain-of-function mutations. We and others have demonstrated proof-of-principle for viral-mediated RNAi in a mouse model of motor neuron disease. Lentivirus and adeno-associated virus have been used to knockdown levels of mutated superoxide dismutase 1 (SOD1) in the G93A SOD1 mouse model of familial amyotrophic lateral sclerosis (fALS) to result in beneficial therapeutic outcomes. This chapter describes the design, production, and titration of lentivirus and adeno-associated virus capable of mediating SOD1 knockdown in vivo. The delivery of the virus to the spinal cord directly, through intraspinal injection, or indirectly, through intramuscular injection, is also described, as well as the methods pertaining to the analysis of spinal cord transduction, SOD1 silencing, and determination of motor neuron protection.

  10. [Construction and identification of a lentiviral vector for RNA interference of human GLUT3 gene].

    PubMed

    Zheng, Chuanyi; Chen, Zhenggang; Bai, Enqi; Li, Zhengzheng; Yang, Kun

    2016-05-01

    目的:筛选出人GLUT3基因有效的RNA干扰(RNA interference,RNAi)序列,并构建出慢病毒RNAi载体。方法:根据GLUT3基因mRNA序列设计合成siRNA片段4个,分别定向克隆至pLV-shRNA载体上,并将构建的质粒转染HeLa细胞,运用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测GLUT3mRNA的表达量验证其干扰效果。筛选出其中有效的质粒与病毒包装质粒共转染293T细胞进行包装,收获并浓缩重组慢病毒颗粒,测定病毒颗粒滴度后,将病毒感染U251胶质瘤细胞以测定感染慢病毒干扰载体后胶质瘤细胞内GLUT3的表达情况。结果:重组RNAi质粒pLV-shRNA-GLUT3-1,pLV-shRNA-GLUT3-2,pLV-shRNA-GLUT3-3,pLV-shRNA- GLUT3-4经测序证实质粒载体构建成功;4个干扰质粒在HeLa细胞中均可以明显抑制GLUT3-mRNA的表达。pLV-shRNA-GLUT3可以在293T细胞中成功包装。收集293T细胞分泌的病毒上清浓缩后测定病毒颗粒LV-GLUT3滴度为1.5×109 TU/mL。与感染阴性对照慢病毒颗粒(0.3641±0.044)相比,胶质瘤U251细胞感染慢病毒颗粒LV- GLUT3后,GLUT3蛋白相对表达明显降低(0.108±0.016,t=16.267,P<0.001)。结论:成功构建了人GLUT3基因有效的慢病毒RNAi载体,为进一步研究GLUT3的生物学功能奠定了基础。.

  11. Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.

    PubMed

    Brunger, Jonathan M; Huynh, Nguyen P T; Guenther, Caitlin M; Perez-Pinera, Pablo; Moutos, Franklin T; Sanchez-Adams, Johannah; Gersbach, Charles A; Guilak, Farshid

    2014-03-01

    The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering. PMID:24550481

  12. Generation of Islet-like Cell Aggregates from Human Adipose Tissue-derived Stem Cells by Lentiviral Overexpression of PDX-1

    PubMed Central

    Bahrebar, M.; Soleimani, M.; Karimi, M. H.; Vahdati, A.; Yaghobi, R.

    2015-01-01

    Background: Pancreatic duodenal homeobox1 (PDX-1) is a transcription factor that is important in regulating pancreas development and maintaining β-cell function. β-cell replacement is an effective approach for the treatment of type 1 diabetes. Human adipose-mesenchymal stem cells (hAMSCs) are the ideal population cells for differentiating into insulin-producing cells. Objective: To determine if islet-like cell aggregates production could be generated from hAMSCs by lentiviral overexpression of PDX-1. Methods: After isolation of hAMSCs, characteristics of these cells were identified by flow-cytometic analysis and multilineage differentiation studies. PDX-1 gene delivered into hAMSCs through lentiviral vector for differentiating hAMSCs into insulin-producing cells (IPCs) at the utilized protocol for 14 days. Characteristics of IPCs were evaluated by immunocytofluorescence, dithizone staining, and quantitative reverse transcription PCR. In response to high glucose medium, insulin release was detected by chemiluminescence enzyme immunoassay. Results: The islet-like cell aggregates appeared about 10 days after introduction of PDX-1 into hAMSCs. PDX-1 induced its own expression (auto-induction), a number of islet-related genes such as Ngn3, Nkx2-2, and insulin. The insulin-positive cells were detected in the PDX-1 transduced cells. In response to glucose challenge test, secretion of insulin hormone in the medium with high glucose concentration significantly increased in the PDX-1-transduced cells related to medium with low glucose concentration. Conclusion: Introduction of lentiviral PDX-1 significantly induces hAMSCs to differentiate into islet-like cell aggregates, which may provide a source of adipose stem cells-derived insulin-producing cells for cell replacement therapy in type 1 diabetes. PMID:26082830

  13. Preventing and exploiting the oncogenic potential of integrating gene vectors.

    PubMed

    Modlich, Ute; Baum, Christopher

    2009-04-01

    Gene therapy requires efficient gene delivery to cure or prevent disease by modifying the genome of somatic cells. However, gene vectors, which insert themselves into the host genome in order to achieve persistent protein expression, can trigger oncogenesis by upregulating cellular protooncogenes. This adverse event, known as insertional mutagenesis, has become a major hurdle in the field. Vectors developed on the basis of lentiviruses are considered to be less genotoxic than the hitherto used gamma-retroviral vectors. For their report in this issue of the JCI, Montini et al. utilized a tumor-prone mouse model to identify the genetic determinants of insertional mutagenesis (see the related article beginning on page 964). They report that the lentiviral integration pattern and additional improvements in vector design reduce the genotoxic risk. These findings will inform future vector design with the goal of limiting genotoxicity for gene therapy or increasing genotoxicity for protooncogene discovery.

  14. Lentivirus-expressed siRNA vectors against Alzheimer disease.

    PubMed

    Peng, Kevin A; Masliah, Eliezer

    2010-01-01

    Amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease, and the accumulation of APP products ultimately leads to the familiar histopathological and clinical manifestations associated with this most common form of dementia. A protein that has been shown to promote APP accumulation is beta-secretase (beta-site APP cleaving enzyme 1, or BACE1), which is increased in the cerebrospinal fluid in those affected with Alzheimer disease. Through in vivo studies using APP transgenic mice, we demonstrated that decreasing the expression of BACE1 via lentiviral vector delivery of BACE1 siRNA has the potential for significantly reducing the cleavage of APP, accumulation of these products, and consequent neurodegeneration. As such, lentiviral-expressed siRNA against BACE1 is a therapeutic possibility in the treatment of Alzheimer disease. We detail the use of lentivirus-expressed siRNA as a method to ameliorate Alzheimer disease neuropathology in APP transgenic mice.

  15. Lentiviral-Mediated Overexpression of the 18 kDa Translocator Protein (TSPO) in the Hippocampal Dentate Gyrus Ameliorates LPS-Induced Cognitive Impairment in Mice

    PubMed Central

    Wang, Wei; Zhang, Liming; Zhang, Xiaoying; Xue, Rui; Li, Lei; Zhao, Weixing; Fu, Qiang; Mi, Weidong; Li, Yunfeng

    2016-01-01

    The 18 kDa translocator protein (TSPO) is involved in the immune/inflammatory response. However, the exact role that TSPO plays in neuroinflammation-induced cognitive impairment is still elusive. The purpose of our present study was to investigate the effects of lentiviral-mediated hippocampal overexpression of the TSPO in a mouse model of LPS-induced cognitive impairment. We established a mouse cognitive impairment model using systematic daily administration of lipopolysaccharide (LPS) (0.5 mg/kg). Microinjection of the dentate gyrus of the mouse with lentiviral vectors, which contained a cDNA targeting TSPO (Lv-TSPO), resulted in a significant increase in TSPO expression and allopregnanolone production. Mice treated with LPS showed cognitive deficits in the novel object recognition test and the Morris water maze test that could be ameliorated by TSPO overexpression. In addition, TSPO overexpression reversed LPS-induced microglial activation and accumulation of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Moreover, TSPO overexpression attenuated the LPS-induced impairment of hippocampal neurogenesis. Our results suggest that local overexpression of TSPO in the hippocampal dentate gyrus alleviated LPS-induced cognitive deficits, and its effects might be mediated by the attenuation of inflammatory cytokines, inhibition of microglial activation, and promotion of neurogenesis. PMID:27803668

  16. In vitro generation of glucose-responsive insulin producing cells using lentiviral based pdx-1 gene transduction of mouse (C57BL/6) mesenchymal stem cells.

    PubMed

    Rahmati, Saman; Alijani, Najva; Kadivar, Mehdi

    2013-08-01

    The objective of this study was to evaluate the potential of this type of recombinant lentivirus to generate glucose-responsive insulin producing cells in vitro. All steps of cloning were confirmed using restriction digests. After the transduction, mesenchymal stem cells gradually began to change their morphology and showed differentiation into islet like structures. RT-PCR results confirmed the expression of insulin1, insulin2 and pdx-1 in differentiated cells. Dithizone staining of mouse MSCs showed the concentration of glucose in islet like structures. ELISA analysis validated the insulin secretion of islet like structures which in the high-glucose medium (25mmol/l) was 7.44 fold higher than that secreted in the low-glucose medium (5mmol/l). Our results demonstrated that mouse mesenchymal stem cells can be differentiated into effective glucose-responsive insulin producing cells through our new recombinant lentiviral transduction of pdx-1 gene in vitro. This new lentiviral vector could be suggested as an effective candidate for using in gene therapy of type-1 diabetes.

  17. Transduction of the choroid plexus and ependyma in neonatal mouse brain by vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus type 5 vectors.

    PubMed

    Watson, Deborah J; Passini, Marco A; Wolfe, John H

    2005-01-01

    Evaluation of gene transfer into the developing mouse brain has shown that when adeno-associated virus serotype 1 (AAV1) or AAV2 vectors are injected into the cerebral lateral ventricles at birth, widespread parenchymal transduction occurs. Lentiviral vectors have not been tested by this route. In this study, we found that injection of lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) resulted in targeted transduction of the ependymal cells lining the ventricular system and the choroid plexus along the entire rostrocaudal axis of the brain, whereas a Mokola pseudotype transduced only a few cells after injection into the neonatal ventricle. In contrast, when lentiviral vectors pseudotyped with either VSV-G or Mokola glycoprotein are injected into the adult mouse brain, they transduce similar patterns of cells. An Ebola-Zaire-pseudotyped vector did not transduce any neonatal CNS cells, as was also the case for adult parenchymal injections. Long-term gene expression (12 months) occurred with a constitutively active mammalian promoter and a self-inactivating long terminal repeat (LTR), whereas the cytomegalovirus promoter in a vector with an intact LTR was expressed only in short-term experiments. We found that an AAV5 vector also targeted the ependymal and choroid plexus cells throughout the ventricular system. This vector exhibited limited penetration from the ventricle to other structures, which was significantly different from the previously reported patterns of transduction after intraventricular injection of AAV1 and AAV2 vectors. PMID:15703488

  18. Foamy virus vectors for gene transfer

    PubMed Central

    Trobridge, Grant D.

    2009-01-01

    Foamy virus (FV) vectors are efficient gene delivery vehicles that have shown great promise for gene therapy in preclinical animal models. FVs or spumaretroviruses are not endemic in humans, but are prevalent in nonhuman primates and in other mammals. They have evolved means for efficient horizontal transmission in their host species without pathology. FV vectors have several unique properties that make them well-suited for therapeutic gene transfer including a desirable safety profile, a broad tropism, a large transgene capacity, and the ability to persist in quiescent cells. They mediate efficient and stable gene transfer to hematopoietic stem cells (HSCs) in mouse models, and in the canine large animal model. Analysis of FV vector integration sites in vitro and in hematopoietic repopulating cells shows they have a unique integration profile, and suggests they may be safer than gammaretroviruses or lentiviral vectors. Here properties of FVs relevant to the safety and efficacy of FV vectors are discussed. The development of FV vector systems is described, and studies evaluating their potential in vitro, and in small and large animal models is reviewed. PMID:19743892

  19. Lentiviral Transduction of Neurons in Adult Brain: Evaluation of Inflammatory Response and Cognitive Effects in Mice.

    PubMed

    Kunitsyna, T A; Ivashkina, O I; Roshchina, M A; Toropova, K A; Anokhin, K V

    2016-06-01

    We evaluated the effect of hippocampal injection of lentiviral particles p156-CMV-EGFP on behavior, learning, and microglial Iba1(+) cells activation in mice. Testing in the open field and elevated plus-maze revealed higher anxiety levels in lentiviral-injected mice in comparison with animals injected with vehicle. At the same time, lentivirus injection did not change learning and memory of mice in the hippocampal-dependent fear conditioning task. Microglia density in lentivirus-injected mice was significantly higher than in vehicle-injected mice. Thus, hippocampal injection of lentiviral particles with minimum content of transgenes produced evident inflammation process, changed anxiety level of experimental animals, but had no effect on hippocampal-dependent learning and memory. PMID:27383167

  20. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.

    PubMed

    Koike-Yusa, Hiroko; Li, Yilong; Tan, E-Pien; Velasco-Herrera, Martin Del Castillo; Yusa, Kosuke

    2014-03-01

    Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.

  1. Lentiviral-mediated RNA interference targeting stathmin1 gene in human gastric cancer cells inhibits proliferation in vitro and tumor growth in vivo

    PubMed Central

    2013-01-01

    Background Gastric cancer is highly aggressive disease. Despite advances in diagnosis and therapy, the prognosis is still poor. Various genetic and molecular alterations are found in gastric cancer that underlies the malignant transformation of gastric mucosa during the multistep process of gastric cancer pathogenesis. The detailed mechanism of the gastric cancer development remains uncertain. In present study we investigated the potential role of stathmin1 gene in gastric cancer tumorigenesis and examined the usefulness of RNA interference (RNAi) targeting stathmin1 as a form of gastric cancer treatment. Methods A lentiviral vector encoding a short hairpin RNA (shRNA) targeted against stathmin1 was constructed and transfected into the packaging cells HEK 293 T and the viral supernatant was collected to transfect MKN-45 cells. The transwell chemotaxis assay and the CCK-8 assay were used to measure migration and proliferation of tumor cells, respectively. Quantitative real-time PCR and western blotting were used to detect the expression levels of stathmin1. Results Lentivirus mediated RNAi effectively reduced stathmin1 expression in gastric cells. Significant decreases in stathmin1 mRNA and protein expression were detected in gastric cells carrying lentiviral stathmin-shRNA vector and also significantly inhibited the proliferation, migration in gastric cancer cells and tumorigenicity in Xenograft Animal Models. Conclusions Our findings suggest that stathmin1 overexpression is common in gastric cancer and may play a role in its pathogenesis. Lentivirus mediated RNAi effectively reduced stathmin1 expression in gastric cells. In summary, shRNA targeting of stathmin1 can effectively inhibits human gastric cancer cell growth in vivo and may be a potential therapeutic strategy for gastric cancer. PMID:24040910

  2. Fe3O4@Graphene Oxide@Ag Particles for Surface Magnet Solid-Phase Extraction Surface-Enhanced Raman Scattering (SMSPE-SERS): From Sample Pretreatment to Detection All-in-One.

    PubMed

    Liu, Zhigang; Wang, Yi; Deng, Rong; Yang, Liyuan; Yu, Shihua; Xu, Shuping; Xu, Weiqing

    2016-06-01

    A multifunctional magnetic graphene surface-enhanced Raman scattering (SERS) substrate was fabricated successfully by the layer-by-layer assembly of silver and graphene oxide (GO) nanoparticles (NPs) on the magnetic ferroferric oxide particles (Fe3O4@GO@Ag). This ternary particle possesses magnetic properties, SERS activity, and adsorption ability simultaneously. Owing to the multifunction of this Fe3O4@GO@Ag ternary complex, we put forward a new method called a surface magnetic solid-phase extraction (SMSPE) technique, for the SERS detections of pesticide residues on the fruit peels. SMSPE integrates many sample pretreatment procedures, such as surface extraction, separation sample, and detection, all-in-one. So this method shows great superiority in simplicity, rapidity, and high efficiency above other standard methods. The whole detection process can be finished within 20 min including the sample pretreatment and SERS detection. Owing to the high density of Ag NPs, the detection sensitivity is high enough that the lowest detectable concentrations are 0.48 and 40 ng/cm(2) for thiram and thiabendazole, which are much lower than the maximal residue limits in fruit prescribed by the U.S. Environmental Protection Agency. This multifunctional ternary particle and its corresponding analytical method have been proven to be applicable for practical samples and also valuable for other surface analysis. PMID:27191584

  3. All-in-one bioprobe devised with hierarchical-ordered magnetic NiCo2O4 superstructure for ultrasensitive dual-readout immunosensor for logic diagnosis of tumor marker.

    PubMed

    Dai, Hong; Gong, Lingshan; Zhang, Shupei; Xu, Guifang; Li, Yilin; Hong, Zhensheng; Lin, Yanyu

    2016-03-15

    A new enzyme-free all-in-one bioprobe, consisted of hematin decorated magnetic NiCo2O4 superstructure (ATS-MNS-Hb), was designed for ultrasensitive photoelectrochemical and electrochemical dual-readout immunosensing of carcinoembryonic antigen (CEA) on carbon nanohorns (CNH) support. Herein, the MNS, possessed hierarchical-ordered structure, good porosity and magnetism, acted as nanocarrier to absorb abundant Hb molecular after functionalization, providing a convenient collection means by magnetic control as well as enhanced dual-readout sensing performances. CNH superstructures were employed as support to immobilize abounding captured antibodies, and then as-designed dual mode bioprobe, covalent binding with secondary antibody of CEA, was introduced for ultrasensitive detection of CEA by sandwich immunosensing. Photoelectrochemical response originated from plentiful hematin molecular, a excellent photosensitizer with good visible light harvesting efficiency, absorbed by functionalized porous MNS. The resultant concentration dependant linear calibration range was from 10 fg/mL to 1 ng/mL with ultralow detection limit of 10 fg/mL. For electrochemical process, catalase-like property of MNS was validated, moreover, MNS-Hb hybrid exhibited much higher mimic enzyme catalytic activity and evidently amplified electrocatalytic signal, performing a wide dynamic linear range from 1 ng/mL to 40 ng/mL with low detection limit of 1 ng/mL. Additionally, due to the improved accuracy of dual signals detection, the exact diagnoses of serum samples were gotten by operating resulting dual signals with AND logic system. This work demonstrated the promising application of MNS in developing ultrasensitive, cost-effective and environment friendly dual-readout immunosensor and accurate diagnoses strategy for tumor markers. PMID:26528807

  4. Solid lipid-PEI hybrid nanocarrier: an integrated approach to provide extended, targeted, and safer siRNA therapy of prostate cancer in an all-in-one manner.

    PubMed

    Xue, Hui-Yi; Wong, Ho-Lun

    2011-09-27

    Small-interfering RNA (siRNA) has a high application potential for therapeutic silencing of pathologic or drug-resistance genes. However, although recent research has led to several nonviral nucleic acid delivery systems with encouraging transfection performance, there remains a substantial gap between these systems and an ideal siRNA carrier that can be safely and effectively used for the more complex delivery tasks such as cancer management. We hypothesized that by integrating the high transfection performance of linear polyethylenimine (PEI) with the controlled release properties of solid lipid components, and complementing the resulting lipid-PEI hybrid nanocarrier (LPN) with receptor-targeting capability, multiple limitations of the conventional siRNA carriers would be simultaneously overcome. Data comparing this new hybrid system with the conventional siRNA-PEI polyplexes showed 15 to 21% less loss of siRNA, higher selectivity for prostate cancer cells over noncancerous prostate cells, and significant reduction in both acute and delayed carrier toxicity especially to the noncancerous RWPE1 cells (e.g., 71.2% of LPN-treated cells preserved proliferative capacity versus ≤30.2% in other groups). We further demonstrated sustained intracellular siRNA release from LPNs, which was shown translatable into extended in vitro and in vivo RNA-interference effects for a minimum of one week. Our findings generally support the use of LPN technology to achieve a longer-acting, less toxic, more efficient, and cancer-specific form of siRNA therapy in an "all-in-one" manner. This brings the nonviral siRNA delivery approach one important step closer to its clinical application.

  5. Lentiviral-mediated multiple gene transfer to chondrocytes promotes chondrocyte differentiation and bone formation in rabbit bone marrow-derived mesenchymal stem cells.

    PubMed

    Liu, Ping; Sun, Liang; Chen, Hui; Sun, Shui; Zhou, Dongsheng; Pang, Bo; Wang, Jian

    2015-11-01

    The aim of the present study was to provide a theoretical and experimental foundation on the differentiation of stem cells through the induction of multiple genes. The lentiviral vector carrying TGF-β1 and IL-10 genes was transfected to bone marrow-derived mesenchymal stem cells (BMSCs) which differentiated into chondrogenesis. Healthy New Zealand white rabbits, 2-3 months of age were used in the present study. A 6-8 ml of bone marrow was isolated from the iliac and tibial shaft of each rabbit. The BMSCs suspension was aspired following centrifugation of the bone marrow by percoll separating medium. The BMSCs were primarily cultured and subcultured in vitro, then divided into four groups according to the difference of lentivirus vectors: group A, receiving transforming growth factor β1 (TGF‑β1); group B, receiving TGF-β1 and Interleukin-10 (IL-10); group C, empty vector transfection; and group D, receiving no cell growth factor. Fluorescence expression was detected 12 h after transfecting the lentiviral vector carrying the TGF-β1 and IL-10 gene to BMSCs. The transfection efficiency was approximately 70% with a MOI=100 after 96 h. Expression of SOX-9 aggrecan and Type Ⅱ collagen in groups A-E on day 7 and 14 was detected by RT-PCR and western blot analysis. The expression level of three genes expressed in groups A and C were higher compared to the expression in groups B, D and E. The expression level of the three genes expressed in group B was higher compared to the expression in group D. The expression level of three genes expressed in group A and C showed no statistical difference. Cytokines therefore play an important role in cell proliferation and chondrogenic differentiation. TGF-β1 has a synergistic effect in the differentiation. In addition, IL-10 may have a protective role in the restoration of cartilaginous tissue. PMID:26328747

  6. Cloning vector

    DOEpatents

    Guilfoyle, R.A.; Smith, L.M.

    1994-12-27

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

  7. Cloning vector

    DOEpatents

    Guilfoyle, Richard A.; Smith, Lloyd M.

    1994-01-01

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

  8. Vector quantization

    NASA Technical Reports Server (NTRS)

    Gray, Robert M.

    1989-01-01

    During the past ten years Vector Quantization (VQ) has developed from a theoretical possibility promised by Shannon's source coding theorems into a powerful and competitive technique for speech and image coding and compression at medium to low bit rates. In this survey, the basic ideas behind the design of vector quantizers are sketched and some comments made on the state-of-the-art and current research efforts.

  9. Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.

    PubMed

    Crispo, M; Vilariño, M; dos Santos-Neto, P C; Núñez-Olivera, R; Cuadro, F; Barrera, N; Mulet, A P; Nguyen, T H; Anegón, I; Menchaca, A

    2015-02-01

    Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4%; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3%, 46/117) and IVF embryos (39.6%, 44/111). Pregnancy rate was detected in 50.0% (6/12) of recipient ewes with TG embryos, in 46.7% (7/15) with IVF embryos, and in 65.0% (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9%) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs.

  10. Generating Transgenic Mice by Lentiviral Transduction of Spermatozoa Followed by In Vitro Fertilization and Embryo Transfer.

    PubMed

    Chandrashekran, Anil; Casimir, Colin; Dibb, Nick; Readhead, Carol; Winston, Robert

    2016-01-01

    Most transgenic technologies rely on the oocyte as a substrate for genetic modification. Transgenics animals are usually generated by the injection of the gene constructs (including lentiviruses encoding gene constructs or modified embryonic stem cells) into the pronucleus of a fertilized egg followed by the transfer of the injected embryos into the uterus of a foster mother. Male germ cells also have potential as templates for transgenic development. We have previously shown that mature sperm can be utilized as template for lentiviral transduction and as such used to generate transgenic mice efficiently with germ line capabilities. We provide here a detailed protocol that is relatively simple, to establish transgenic mice using lentivirally transduced spermatozoa. This protocol employs a well-established lentiviral gene delivery system (usual for somatic cells) delivering a variety of transgenes to be directly used with sperm, and the subsequent use of these modified sperm in in vitro fertilization studies and embryo transfer into foster female mice, for the establishment of transgenic mice. PMID:27317176

  11. Cellular cofactors of lentiviral integrase: from target validation to drug discovery.

    PubMed

    Taltynov, Oliver; Desimmie, Belete A; Demeulemeester, Jonas; Christ, Frauke; Debyser, Zeger

    2012-01-01

    To accomplish their life cycle, lentiviruses make use of host proteins, the so-called cellular cofactors. Interactions between host cell and viral proteins during early stages of lentiviral infection provide attractive new antiviral targets. The insertion of lentiviral cDNA in a host cell chromosome is a step of no return in the replication cycle, after which the host cell becomes a permanent carrier of the viral genome and a producer of lentiviral progeny. Integration is carried out by integrase (IN), an enzyme playing also an important role during nuclear import. Plenty of cellular cofactors of HIV-1 IN have been proposed. To date, the lens epithelium-derived growth factor (LEDGF/p75) is the best studied cofactor of HIV-1 IN. Moreover, small molecules that block the LEDGF/p75-IN interaction have recently been developed for the treatment of HIV infection. The nuclear import factor transportin-SR2 (TRN-SR2) has been proposed as another interactor of HIV IN-mediating nuclear import of the virus. Using both proteins as examples, we will describe approaches to be taken to identify and validate novel cofactors as new antiviral targets. Finally, we will highlight recent advances in the design and the development of small-molecule inhibitors binding to the LEDGF/p75-binding pocket in IN (LEDGINs).

  12. Hematopoietic Stem cell transplantation and lentiviral vector‐based gene therapy for Krabbe's disease: Present convictions and future prospects

    PubMed Central

    Hu, Peirong; Li, Yedda; Nikolaishvili‐Feinberg, Nana; Scesa, Giuseppe; Bi, Yanmin; Pan, Dao; Moore, Dominic; Bongarzone, Ernesto R.; Sands, Mark S.; Miller, Ryan

    2016-01-01

    Currently, presymtomatic hematopoietic stem and progenitor cell transplantation (HSPCT) is the only therapeutic modality that alleviates Krabbe's disease (KD)‐induced central nervous system damage. However, all HSPCT‐treated patients exhibit severe deterioration in peripheral nervous system function characterized by major motor and expressive language pathologies. We hypothesize that a combination of several mechanisms contribute to this phenomenon, including 1) nonoptimal conditioning protocols with consequent inefficient engraftment and biodistribution of donor‐derived cells and 2) insufficient uptake of donor cell‐secreted galactocerebrosidease (GALC) secondary to a naturally low expression level of the cation‐independent mannose 6‐phosphate‐receptor (CI‐MPR). We have characterized the effects of a busulfan (Bu) based conditioning regimen on the efficacy of HSPCT in prolonging twi mouse average life span. There was no correlation between the efficiency of bone marrow engraftment of donor cells and twi mouse average life span. HSPCT prolonged the average life span of twi mice, which directly correlated with the aggressiveness of the Bu‐mediated conditioning protocols. HSPC transduced with lentiviral vectors carrying the GALC cDNA under control of cell‐specific promoters were efficiently engrafted in twi mouse bone marrow. To facilitate HSPCT‐mediated correction of GALC deficiency in target cells expressing low levels of CI‐MPR, a novel GALC fusion protein including the ApoE1 receptor was developed. Efficient cellular uptake of the novel fusion protein was mediated by a mannose‐6‐phosphate‐independent mechanism. The novel findings described here elucidate some of the cellular mechanisms that impede the cure of KD patients by HSPCT and concomitantly open new directions to enhance the therapeutic efficacy of HSPCT protocols for KD. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc. PMID:27638600

  13. Therapeutic benefit of lentiviral-mediated neonatal intracerebral gene therapy in a mouse model of globoid cell leukodystrophy.

    PubMed

    Lattanzi, Annalisa; Salvagno, Camilla; Maderna, Claudio; Benedicenti, Fabrizio; Morena, Francesco; Kulik, Willem; Naldini, Luigi; Montini, Eugenio; Martino, Sabata; Gritti, Angela

    2014-06-15

    Globoid cell leukodystrophy (GLD) is an inherited lysosomal storage disease caused by β-galactocerebrosidase (GALC) deficiency. Gene therapy (GT) should provide rapid, extensive and lifetime GALC supply in central nervous system (CNS) tissues to prevent or halt irreversible neurologic progression. Here we used a lentiviral vector (LV) to transfer a functional GALC gene in the brain of Twitcher mice, a severe GLD model. A single injection of LV.GALC in the external capsule of Twitcher neonates resulted in robust transduction of neural cells with minimal and transient activation of inflammatory and immune response. Importantly, we documented a proficient transduction of proliferating and post-mitotic oligodendroglia, a relevant target cell type in GLD. GALC activity (30-50% of physiological levels) was restored in the whole CNS of treated mice as early as 8 days post-injection. The early and stable enzymatic supply ensured partial clearance of storage and reduction of psychosine levels, translating in amelioration of histopathology and enhanced lifespan. At 6 months post-injection in non-affected mice, LV genome persisted exclusively in the injected region, where transduced cells overexpressed GALC. Integration site analysis in transduced brain tissues showed no aberrant clonal expansion and preferential targeting of neural-specific genes. This study establishes neonatal LV-mediated intracerebral GT as a rapid, effective and safe therapeutic intervention to correct CNS pathology in GLD and provides a strong rationale for its application in this and similar leukodystrophies, alone or in combination with therapies targeting the somatic pathology, with the final aim of providing an effective and timely treatment of these global disorders.

  14. Vector platforms for gene therapy of inherited retinopathies

    PubMed Central

    Trapani, Ivana; Puppo, Agostina; Auricchio, Alberto

    2014-01-01

    Inherited retinopathies (IR) are common untreatable blinding conditions. Most of them are inherited as monogenic disorders, due to mutations in genes expressed in retinal photoreceptors (PR) and in retinal pigment epithelium (RPE). The retina’s compatibility with gene transfer has made transduction of different retinal cell layers in small and large animal models via viral and non-viral vectors possible. The ongoing identification of novel viruses as well as modifications of existing ones based either on rational design or directed evolution have generated vector variants with improved transduction properties. Dozens of promising proofs of concept have been obtained in IR animal models with both viral and non-viral vectors, and some of them have been relayed to clinical trials. To date, recombinant vectors based on the adeno-associated virus (AAV) represent the most promising tool for retinal gene therapy, given their ability to efficiently deliver therapeutic genes to both PR and RPE and their excellent safety and efficacy profiles in humans. However, AAVs’ limited cargo capacity has prevented application of the viral vector to treatments requiring transfer of genes with a coding sequence larger than 5 kb. Vectors with larger capacity, i.e. nanoparticles, adenoviral and lentiviral vectors are being exploited for gene transfer to the retina in animal models and, more recently, in humans. This review focuses on the available platforms for retinal gene therapy to fight inherited blindness, highlights their main strengths and examines the efforts to overcome some of their limitations. PMID:25124745

  15. VISPA: a computational pipeline for the identification and analysis of genomic vector integration sites.

    PubMed

    Calabria, Andrea; Leo, Simone; Benedicenti, Fabrizio; Cesana, Daniela; Spinozzi, Giulio; Orsini, Massimilano; Merella, Stefania; Stupka, Elia; Zanetti, Gianluigi; Montini, Eugenio

    2014-01-01

    The analysis of the genomic distribution of viral vector genomic integration sites is a key step in hematopoietic stem cell-based gene therapy applications, allowing to assess both the safety and the efficacy of the treatment and to study the basic aspects of hematopoiesis and stem cell biology. Identifying vector integration sites requires ad-hoc bioinformatics tools with stringent requirements in terms of computational efficiency, flexibility, and usability. We developed VISPA (Vector Integration Site Parallel Analysis), a pipeline for automated integration site identification and annotation based on a distributed environment with a simple Galaxy web interface. VISPA was successfully used for the bioinformatics analysis of the follow-up of two lentiviral vector-based hematopoietic stem-cell gene therapy clinical trials. Our pipeline provides a reliable and efficient tool to assess the safety and efficacy of integrating vectors in clinical settings. PMID:25342980

  16. Construction of p66Shc gene interfering lentivirus vectors and its effects on alveolar epithelial cells apoptosis induced by hyperoxia

    PubMed Central

    Zhang, Chan; Dong, Wen-Bin; Zhao, Shuai; Li, Qing-Ping; Kang, Lan; Lei, Xiao-Ping; Guo, Lin; Zhai, Xue-Song

    2016-01-01

    Background The aim of this study is to observe the inhibitive effects of p66Shc gene interfering lentivirus vectors on the expression of p66Shc, and to explore its effects on alveolar epithelial cells apoptosis induced by hyperoxia. Methods The gene sequences were cloned into the pLenR-GPH-shRNA lentiviral vector, which was selected by Genebank searches. The pLenR-GPH-shRNA and lentiviral vector packaging plasmid mix were cotransfected into 293T cells to package lentiviral particles. Culture virus supernatant was harvested, and then the virus titer was determined by serial dilution assay. A549 cells were transduced with the constructed lentiviral vectors, and real-time polymerase chain reaction (RT-PCR) and Western blot were used to evaluate p66Shc expression. This study is divided into a control group, a hyperoxia group, an A549-p66ShcshRNA hyperoxia group, and a negative lentivirus group. Cell apoptosis was detected by flow cytometry after 24 hours; the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-9 were detected by immunohistochemistry assay. The production of reactive oxygen species and cellular mitochondria membrane potential (ΔΨm) were determined by fluorescence microscopy. Results We successfully established the p66Shc gene interfering lentivirus vectors, A549-p66ShcshRNA. The A549-p66ShcshRNA was transfected into alveolar epithelial cells, and the inhibitive effects on the expression of p66Shc were observed. Both RT-PCR and Western blot demonstrated downregulation of p66Shc expression in A549 cells. In the A549-p66ShcshRNA hyperoxia group, we found dampened oxidative stress. A549-p66ShcshRNA can cause p66Shc gene silencing, reduce mitochondrial reactive oxygen species generation, reduce membrane potential decrease, reduce the apoptosis of A549 cells, and reduce alveolar epithelial cell injury, while the lentiviral empty vector group had no such changes. Conclusion p66Shc gene interfering lentivirus vector can affect the

  17. Therapeutic effect of recombinant lentiviral vector containing succinate dehydrogenase iron-sulfur protein on the treatment of experimental autoimmunity myocarditis.

    PubMed

    Han, Lina; Wang, Chunxi; Guo, Shuli; Liu, Siyu; Yang, Liming

    2016-08-01

    Cardiac autoimmune reaction takes part in myocarditis, dilated cardiomyopathy and heart failure. Existing literature has confirmed that the occurrence of cardiomyopathy belongs to mitochondrial diseases and is related to the oxidative respiratory chain subunit. The special structure of iron-sulfur protein (ISP) is responsible for the oxidative stress in oxidative phosphorylation, which is also a target that is easily attacked by various damage factors. Using gene therapy technology to restore succinate dehydrogenase iron-sulfur protein (SDISP) function- and thus resume myocardial mitochondria function and myocardial function is hypothesized to alleviate the experimental autoimmunity myocarditis (EAM).

  18. CD19 and CD20 Targeted Vectors Induce Minimal Activation of Resting B Lymphocytes

    PubMed Central

    Schwenkert, Michael; Cosset, François-Loic; Verhoeyen, Els; Buchholz, Christian J.

    2013-01-01

    B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral

  19. Lentiviral transgenesis in mice via a simple method of viral concentration.

    PubMed

    Cheng, Pei-Hsun; Chang, Yu-Fan; Mao, Su-Han; Lin, Hsiu-Lien; Chen, Chuan-Mu; Yang, Shang-Hsun

    2016-10-01

    Transgenic animals are important in vivo models for biological research. However, low transgenic rates are commonly reported in the literature. Lentiviral transgenesis is a promising method that has greater efficiency with regard to generating transgenic animals, although the transgenic rate of this approach is highly dependent on different transgenes and concentrated lentiviruses. In this study, we modified a method to concentrate lentiviruses using a table centrifuge, commonly available in most laboratories, and carried out analysis of the transgenic efficiency in mice. Based on 26 individual constructs and 627 live pups, we found that the overall transgenic rate was more than 30%, which is higher than obtained with pronuclear microinjection. In addition, we did not find any significant differences in transgenic efficiency when the size of inserts was less than 5000 bp. These results not only show that our modified method can successfully generate transgenic mice but also suggest that this approach could be generally applied to different constructs when the size of inserts is less than 5000 bp. It is anticipated that the results of this study can help encourage the wider laboratory use of lentiviral transgenesis in mice. PMID:27264740

  20. Expression Profiles of Vpx/Vpr Proteins Are Co-related with the Primate Lentiviral Lineage

    PubMed Central

    Sakai, Yosuke; Miyake, Ariko; Doi, Naoya; Sasada, Hikari; Miyazaki, Yasuyuki; Adachi, Akio; Nomaguchi, Masako

    2016-01-01

    Viruses of human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency virus (SIV) lineages carry a unique accessory protein called Vpx. Vpx is essential or critical for viral replication in natural target cells such as macrophages and T lymphocytes. We have previously shown that a poly-proline motif (PPM) located at the C-terminal region of Vpx is required for its efficient expression in two strains of HIV-2 and SIVmac, and that the Vpx expression levels of the two clones are significantly different. Notably, the PPM sequence is conserved and confined to Vpx and Vpr proteins derived from certain lineages of HIV-2/SIVs. In this study, Vpx/Vpr proteins from diverse primate lentiviral lineages were experimentally and phylogenetically analyzed to obtain the general expression picture in cells. While both the level and PPM-dependency of Vpx/Vpr expression in transfected cells varied among viral strains, each viral group, based on Vpx/Vpr amino acid sequences, was found to exhibit a characteristic expression profile. Moreover, phylogenetic tree analyses on Gag and Vpx/Vpr proteins gave essentially the same results. Taken together, our study described here suggests that each primate lentiviral lineage may have developed a unique expression pattern of Vpx/Vpr proteins for adaptation to its hostile cellular and species environments in the process of viral evolution. PMID:27536295

  1. Tumor Therapy Mediated by Lentiviral Expression of shBcl-2 and S-TRAIL1

    PubMed Central

    Kock, Norman; Kasmieh, Randa; Weissleder, Ralph; Shah, Khalid

    2007-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells and, in combination with other agents, could enhance tumor therapy. We explored the combined therapeutic effects of a secretable form of (S) TRAIL-induced apoptosis and the downregulation of Bcl-2 in human gliomas. We constructed a lentiviral delivery system: 1) for the expression of short hairpin (sh) RNA to downregulate Bcl-2 and for the expression of S-TRAIL to induce apoptosis in glioma cells; and 2) to follow delivery in vitro and the fate of tumors in real time in vivo. We demonstrate that lentiviral-mediated simultaneous downregulation of Bcl-2 and S-TRAIL-induced apoptosis leads to an increased expression of activated caspase-3 and caspase-7, thus resulting in accelerated S-TRAIL-mediated apoptosis in glioma cells in vitro. Using a highly malignant human glioma model expressing EGFRvIII and firefly luciferase, we show that the combined effect of Bcl-2 downregulation and S-TRAIL-induced apoptosis results in complete eradication of gliomas compared to S-TRAIL monotherapy. These results show that simultaneous triggering of TRAIL-mediated death receptor pathway and downregulation of Bcl-2 by shRNA leads to enhanced eradication of gliomas and serves as a template in developing and monitoring combination therapies for the treatment of drug-resistant cancers. PMID:17534449

  2. Characterization and biodistribution of human mesenchymal stem cells transduced with lentiviral-mediated BMP2.

    PubMed

    Choi, Kyoung Suk; Ahn, Soon Young; Kim, Tek Seung; Kim, Jiseon; Kim, Byoung-Guk; Han, Kyung Ho; Ban, Sang Ja; Kim, Hyung Soo; Choi, Youngju; Lim, Chul-Joo

    2011-04-01

    Recently, the genetic modification of mesenchymal stem cells (MSCs) has led to increased differentiation potential. For the therapeutic application of genetically modified MSCs, it is crucial to evaluate their characteristics and safety. In this study, we investigated the effects of bone morphogenetic protein 2 (BMP2) gene transfer on the characteristics and biodistribution of human MSCs. Lentiviral-mediated BMP2 transduction to MSCs enhanced osteocyte differentiation and decreased adipocyte differentiation. Although there is no significant difference in cell proliferation capacity, MSCs transduced BMP2 proliferate somewhat higher than nontransduced or GFP transduced MSCs. No significant changes were observed in surface antigen expression in genetically modified MSCs. In vivo transplantation of lentiviral-mediated BMP2 gene transferred MSCs to nude mice did not result in tumor formation. To evaluate the biodistribution of genetically modified cells, MSCs carrying BMP2 were injected into the tail vein of femur fractured mice. The introduced MSCs were detected in the spleen, testis and fractured femur 28 days post-implantation. These findings suggest that diverse safety tests for genetically modified MSCs should be considered, particularly when a lentivirus mediated gene transfer method is used.

  3. Evolution Loses and Wins, All in One Day

    ERIC Educational Resources Information Center

    Cavanagh, Sean

    2005-01-01

    The forces seeking to subject the theory of evolution to greater criticism tasted both victory and defeat. Kansas officials approved an overhaul of their state science standards to do just that, while voters in a rural Pennsylvania district ousted advocates of "intelligent design" from the school board. Those two high-profile battles were just the…

  4. Carnivorous plants: trapping, digesting and absorbing all in one.

    PubMed

    Brownlee, Colin

    2013-09-01

    The Venus flytrap digests and absorbs its prey, but how does it coordinate digestion and absorption to maximise the efficiency of this highly evolved mechanism? A new study that combines direct recordings from cells within the trap along with molecular characterization of nutrient transport reveals a complex and coordinated suite of mechanisms that underlie this elegant process.

  5. Methyltriphenylphosphonium Methylcarbonate, an All-In-One Wittig Vinylation Reagent.

    PubMed

    Cattelan, Lisa; Noè, Marco; Selva, Maurizio; Demitri, Nicola; Perosa, Alvise

    2015-12-01

    The methyltriphenylphosphonium methylcarbonate salt [Ph3 PCH3 ][CH3 OCO2 ], obtained directly by quaternarization of triphenylphosphine with dimethylcarbonate, is a latent ylide that promotes Wittig vinylation of aldehydes and ketones. Alkenes are obtained simply by mixing [Ph3 PCH3 ][CH3 OCO2 ] and the carbonyl and heating in a solvent (no base, no halides, and no inorganic byproducts). Deuterium exchange experiments and the particularly short anion-cation distance measured by XRD in [Ph3 PCH3 ][CH3 OCO2 ] allowed to explain the nature and reactivity of this species. Green chemistry metrics (atom economy, mass index, environmental factor) indicate that this vinylation procedure is more efficient than comparable ones. Deuterated [Ph3 PCD3 ][CH3 OCO2 ] promoted the synthesis of deuterated olefins. PMID:26383135

  6. Retroviral vector silencing during iPS cell induction: an epigenetic beacon that signals distinct pluripotent states.

    PubMed

    Hotta, Akitsu; Ellis, James

    2008-11-01

    Retroviral vectors are transcriptionally silent in pluripotent stem cells. This feature has been potently applied in studies that reprogram somatic cells into induced pluripotent stem (iPS) cells. By delivering the four Yamanaka factors in retroviral vectors, high expression is obtained in fibroblasts to induce the pluripotent state. Partial reprogramming generates Class I iPS cells that express the viral transgenes and endogenous pluripotency genes. Full-reprogramming in Class II iPS cells silences the vectors as the endogenous genes maintain the pluripotent state. Thus, retroviral vector silencing serves as a beacon marking the fully reprogrammed pluripotent state. Here we review known silencer elements, and the histone modifying and DNA methylation pathways, that silence retroviral and lentiviral vectors in pluripotent stem cells. Both retroviral and lentiviral vectors are influenced by position effects and often exhibit variegated expression. The best vector designs facilitate full-reprogramming and subsequent retroviral silencing, which is required for directed-differentiation. Current retroviral reprogramming methods can be immediately applied to create patient-specific iPS cell models of human disease, however, future clinical applications will require novel chemical or other reprogramming methods that reduce or eliminate the integrated vector copy number load. Nevertheless, retroviral vectors will continue to play an important role in genetically correcting patient iPS cell models. We anticipate that novel pluripotent-specific reporter vectors will select for isolation of high quality human iPS cell lines, and select against undifferentiated pluripotent cells during regenerative medicine to prevent teratoma formation after transplantation.

  7. A quasi-lentiviral green fluorescent protein reporter exhibits nuclear export features of late human immunodeficiency virus type 1 transcripts

    SciTech Connect

    Graf, Marcus; Ludwig, Christine; Kehlenbeck, Sylvia; Jungert, Kerstin; Wagner, Ralf . E-mail: ralf.wagner@klinik.uni-regensburg.de

    2006-09-01

    We have previously shown that Rev-dependent expression of HIV-1 Gag from CMV immediate early promoter critically depends on the AU-rich codon bias of the gag gene. Here, we demonstrate that adaptation of the green fluorescent protein (GFP) reporter gene to HIV codon bias is sufficient to turn this hivGFP RNA into a quasi-lentiviral message following the rules of late lentiviral gene expression. Accordingly, GFP expression was significantly decreased in transfected cells strictly correlating with reduced RNA levels. In the presence of the HIV 5' major splice donor, the hivGFP RNAs were stabilized in the nucleus and efficiently exported to the cytoplasm following fusion of the 3' Rev-responsive element (RRE) and coexpression of HIV-1 Rev. This Rev-dependent translocation was specifically inhibited by leptomycin B suggesting export via the CRM1-dependent pathway used by late lentiviral transcripts. In conclusion, this quasi-lentiviral reporter system may provide a new platform for developing sensitive Rev screening assays.

  8. Leukemia induction after a single retroviral vector insertion in Evi1 or Prdm16.

    PubMed

    Modlich, U; Schambach, A; Brugman, M H; Wicke, D C; Knoess, S; Li, Z; Maetzig, T; Rudolph, C; Schlegelberger, B; Baum, C

    2008-08-01

    Insertional activation of cellular proto-oncogenes by replication-defective retroviral vectors can trigger clonal dominance and leukemogenesis in animal models and clinical trials. Here, we addressed the leukemogenic potential of vectors expressing interleukin-2 receptor common gamma-chain (IL2RG), the coding sequence required for correction of X-linked severe combined immunodeficiency. Similar to conventional gamma-retroviral vectors, self-inactivating (SIN) vectors with strong internal enhancers also triggered profound clonal imbalance, yet with a characteristic insertion preference for a window located downstream of the transcriptional start site. Controls including lentivirally transduced cells revealed that ectopic IL2RG expression was not sufficient to trigger leukemia. After serial bone marrow transplantation involving 106 C57Bl6/J mice monitored for up to 18 months, we observed leukemic progression of six distinct clones harboring gamma-retroviral long terminal repeat (LTR) or SIN vector insertions in Evi1 or Prdm16, two functionally related genes. Three leukemic clones had single vector integrations, and identical clones manifested with a remarkably similar latency and phenotype in independent recipients. We conclude that upregulation of Evi1 or Prdm16 was sufficient to initiate a leukemogenic cascade with consistent intrinsic dynamics. Our study also shows that insertional mutagenesis is required for leukemia induction by IL2RG vectors, a risk to be addressed by improved vector design.

  9. Cleavage of Hemagglutinin-Bearing Lentiviral Pseudotypes and Their Use in the Study of Influenza Virus Persistence

    PubMed Central

    Sawoo, Olivier; Dublineau, Amélie; Batéjat, Christophe; Zhou, Paul; Manuguerra, Jean-Claude; Leclercq, India

    2014-01-01

    Influenza A viruses (IAVs) are a major cause of infectious respiratory human diseases and their transmission is dependent upon the environment. However, the role of environmental factors on IAV survival outside the host still raises many questions. In this study, we used lentiviral pseudotypes to study the influence of the hemagglutinin protein in IAV survival. High-titered and cleaved influenza-based lentiviral pseudoparticles, through the use of a combination of two proteases (HAT and TMPRSS2) were produced. Pseudoparticles bearing hemagglutinin proteins derived from different H1N1, H3N2 and H5N1 IAV strains were subjected to various environmental parameters over time and tested for viability through single-cycle infectivity assays. We showed that pseudotypes with different HAs have different persistence profiles in water as previously shown with IAVs. Our results also showed that pseudotypes derived from H1N1 pandemic virus survived longer than those derived from seasonal H1N1 virus from 1999, at high temperature and salinity, as previously shown with their viral counterparts. Similarly, increasing temperature and salinity had a negative effect on the survival of the H3N2 and H5N1 pseudotypes. These results showed that pseudotypes with the same lentiviral core, but which differ in their surface glycoproteins, survived differently outside the host, suggesting a role for the HA in virus stability. PMID:25166303

  10. Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

    PubMed

    Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

    2015-01-01

    Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002-0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer. PMID:26290434

  11. Rotations with Rodrigues' Vector

    ERIC Educational Resources Information Center

    Pina, E.

    2011-01-01

    The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears…

  12. Adenoviral vector DNA for accurate genome editing with engineered nucleases.

    PubMed

    Holkers, Maarten; Maggio, Ignazio; Henriques, Sara F D; Janssen, Josephine M; Cathomen, Toni; Gonçalves, Manuel A F V

    2014-10-01

    Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.

  13. Prime-boost bacillus Calmette-Guérin vaccination with lentivirus-vectored and DNA-based vaccines expressing antigens Ag85B and Rv3425 improves protective efficacy against Mycobacterium tuberculosis in mice.

    PubMed

    Xu, Ying; Yang, Enzhuo; Wang, Jianguang; Li, Rui; Li, Guanghua; Liu, Guoyuan; Song, Na; Huang, Qi; Kong, Cong; Wang, Honghai

    2014-10-01

    To prevent the global spread of tuberculosis (TB), more effective vaccines and vaccination strategies are urgently needed. As a result of the success of bacillus Calmette-Guérin (BCG) in protecting children against miliary and meningeal TB, the majority of individuals will have been vaccinated with BCG; hence, boosting BCG-primed immunity will probably be a key component of future vaccine strategies. In this study, we compared the ability of DNA-, protein- and lentiviral vector-based vaccines that express the antigens Ag85B and Rv3425 to boost the effects of BCG in the context of immunity and protection against Mycobacterium tuberculosis in C57BL/6 mice. Our results demonstrated that prime-boost BCG vaccination with a lentiviral vector expressing the antigens Ag85B and Rv3425 significantly enhanced immune responses, including T helper type 1 and CD8(+) cytotoxic T lymphocyte responses, compared with DNA- and protein-based vaccines. However, lentivirus-vectored and DNA-based vaccines greatly improved the protective efficacy of BCG against M. tuberculosis, as indicated by a lack of weight loss and significantly reduced bacterial loads and histological damage in the lung. Our study suggests that the use of lentiviral or DNA vaccines containing the antigens Ag85B and Rv3425 to boost BCG is a good choice for the rational design of an efficient vaccination strategy against TB.

  14. Editing T cell specificity towards leukemia by zinc-finger nucleases and lentiviral gene transfer

    PubMed Central

    Lombardo, Angelo; Magnani, Zulma; Liu, Pei-Qi; Reik, Andreas; Chu, Victoria; Paschon, David E.; Zhang, Lei; Kuball, Jurgen; Camisa, Barbara; Bondanza, Attilio; Casorati, Giulia; Ponzoni, Maurilio; Ciceri, Fabio; Bordignon, Claudio; Greenberg, Philip D.; Holmes, Michael C.; Gregory, Philip D.; Naldini, Luigi; Bonini, Chiara

    2016-01-01

    The transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. We designed zinc-finger nucleases (ZFNs) promoting the disruption of endogenous TCR β and α chain genes. ZFN-treated lymphocytes lacked CD3/TCR surface expression and expanded with IL-7 and IL-15. Upon lentiviral transfer of a TCR for the WT1 tumor antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near-purity, and proved superior in specific antigen recognition to matched TCR-transferred cells. In contrast to TCR-transferred cells, TCR edited lymphocytes did not mediate off-target reactivity while maintaining anti-tumor activity in vivo, thus demonstrating that complete editing of T-cell specificity generate tumor-specific lymphocytes with improved biosafety profile. PMID:22466705

  15. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

    PubMed Central

    Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J

    2016-01-01

    Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223

  16. Optimizing Glioblastoma Temozolomide Chemotherapy Employing Lentiviral-based Anti-MGMT shRNA Technology

    PubMed Central

    Viel, Thomas; Monfared, Parisa; Schelhaas, Sonja; Fricke, Inga B; Kuhlmann, Michael T; Fraefel, Cornel; Jacobs, Andreas H

    2013-01-01

    Despite treatments combining surgery, radiation-, and chemotherapy, patients affected by glioblastoma (GBM) have a limited prognosis. Addition of temozolomide (TMZ) to radiation therapy is the standard therapy in clinical application, but effectiveness of TMZ is limited by the tumor's overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). The goal of this study was to use the highly specific and efficient RNA interference (RNAi) pathway to modulate MGMT expression to increase TMZ efficiency in chemotherapy resistant GBM. Using lentiviral-based anti-MGMT small hairpin RNA (shRNA) technology we observed a specific inhibition of the MGMT expression in GBM cell lines as well as in subcutaneous tumors. Tumor growth inhibition was observed following TMZ treatment of xenografts with low MGMT expression in contrast to xenografts with high MGMT expression. Bioluminescence imaging (BLI) measurements indicated that luciferase and shRNA-expressing lentiviruses were able to efficiently transduce the GBM xenografts in vivo. Treatment combining injection of a lentivirus expressing an anti-MGMT shRNA and TMZ induced a reduction of the size of the tumors, in contrast with treatment combining the lentivirus expressing the control shRNA and TMZ. Our data suggest that anti-MGMT shRNA therapy could be used in combination with TMZ chemotherapy in order to improve the treatment of resistant GBM. PMID:23319055

  17. Understanding Singular Vectors

    ERIC Educational Resources Information Center

    James, David; Botteron, Cynthia

    2013-01-01

    matrix yields a surprisingly simple, heuristical approximation to its singular vectors. There are correspondingly good approximations to the singular values. Such rules of thumb provide an intuitive interpretation of the singular vectors that helps explain why the SVD is so…

  18. Comparison of toxicogenomic profiles of two murine strains treated with HIV-1-based vectors for gene therapy

    SciTech Connect

    Zhao Yuan Keating, Kenneth; Thorpe, Robin

    2007-12-01

    Gene therapy vectors based on lentiviruses persist in the host and are ideally suited for long-term therapies of genetic disorders. However, recent incidences of T cell leukemia in X-SCID children receiving gene therapy reveal discrepancy among the preclinical and clinical studies. Divergent results on the potential oncogenic property of integrating vectors obtained from different animal models further raise concern about the relevance and sensitivity of available preclinical systems used to assess their toxicity. Evaluation of transcriptional responses to vector transduction in different in vivo models may provide us with early indications of the potential adverse effects of these vectors and comparative information on the sensitivity and suitability of these models. For this purpose, we used cDNA microarray to examine transcriptional changes in BALB/c and CD-1 (IRC) mice, the two common murine strains used in pharmacological and toxicological studies. Our results revealed a significant difference in the transcriptional responses to vector transduction between the two mouse strains. Modest gene changes, in terms of gene numbers and gene expression were observed in BALB/c mice, whereby expression of 15 oncogenes was up-regulated in CD-1 (ICR) mice. We confirmed the up-regulation of oncogenes, e.g., N-myc, A-Raf, Fli-1, Wnt-2b, and c-jun in CD-1 (IRC) mice using RT-PCR. This study provided an insight into the function of lentiviral transduction in different mouse strains. Distinctive toxicogenomic profiles of two mouse strains should be considered in the context of future development of sensitive models for toxicity evaluation of lentiviral vector products.

  19. Rhotrix Vector Spaces

    ERIC Educational Resources Information Center

    Aminu, Abdulhadi

    2010-01-01

    By rhotrix we understand an object that lies in some way between (n x n)-dimensional matrices and (2n - 1) x (2n - 1)-dimensional matrices. Representation of vectors in rhotrices is different from the representation of vectors in matrices. A number of vector spaces in matrices and their properties are known. On the other hand, little seems to be…

  20. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    NASA Astrophysics Data System (ADS)

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2014-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

  1. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    PubMed

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

  2. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    PubMed

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells. PMID:20164855

  3. MicroRNA-regulated viral vectors for gene therapy.

    PubMed

    Geisler, Anja; Fechner, Henry

    2016-05-20

    Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene. Besides traditional approaches, such as transcriptional and transductional targeting, microRNA-dependent post-transcriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. MicroRNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region (UTR) of the mRNA. To control exogenous transgene expression, tandem repeats of artificial microRNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene mRNA in cells expressing the corresponding microRNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying microRNA-regulation, highlights new developments in this field and gives an overview of applications of microRNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases. PMID:27226955

  4. MicroRNA-regulated viral vectors for gene therapy

    PubMed Central

    Geisler, Anja; Fechner, Henry

    2016-01-01

    Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene. Besides traditional approaches, such as transcriptional and transductional targeting, microRNA-dependent post-transcriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. MicroRNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3’ untranslated region (UTR) of the mRNA. To control exogenous transgene expression, tandem repeats of artificial microRNA target sites are usually incorporated into the 3’ UTR of the transgene expression cassette, leading to subsequent degradation of transgene mRNA in cells expressing the corresponding microRNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying microRNA-regulation, highlights new developments in this field and gives an overview of applications of microRNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases. PMID:27226955

  5. Production of Retrovirus-Based Vectors in Mildly Acidic pH Conditions.

    PubMed

    Holic, Nathalie; Fenard, David

    2016-01-01

    Gene transfer vectors based on retroviridae are increasingly becoming a tool of choice for biomedical research and for the development of biotherapies in rare diseases or cancers. To meet the challenges of preclinical and clinical production, different steps of the production process of self-inactivating γ-retroviral (RVs) and lentiviral vectors (LVs) have been improved (e.g., transfection, media optimization, cell culture conditions). However, the increasing need for mass production of such vectors is still a challenge and could hamper their availability for therapeutic use. Recently, we observed that the use of a neutral pH during vector production is not optimal. The use of mildly acidic pH conditions (pH 6) can increase by two- to threefold the production of RVs and LVs pseudotyped with the vesicular stomatitis virus G (VSV-G) or gibbon ape leukemia virus (GALV) glycoproteins. Here, we describe the production protocol in mildly acidic pH conditions of GALVTR- and VSV-G-pseudotyped LVs using the transient transfection of HEK293T cells and the production protocol of GALV-pseudotyped RVs produced from a murine producer cell line. These protocols should help to achieve higher titers of vectors, thereby facilitating experimental research and therapeutic applications. PMID:27317171

  6. Index Sets and Vectorization

    SciTech Connect

    Keasler, J A

    2012-03-27

    Vectorization is data parallelism (SIMD, SIMT, etc.) - extension of ISA enabling the same instruction to be performed on multiple data items simultaeously. Many/most CPUs support vectorization in some form. Vectorization is difficult to enable, but can yield large efficiency gains. Extra programmer effort is required because: (1) not all algorithms can be vectorized (regular algorithm structure and fine-grain parallelism must be used); (2) most CPUs have data alignment restrictions for load/store operations (obey or risk incorrect code); (3) special directives are often needed to enable vectorization; and (4) vector instructions are architecture-specific. Vectorization is the best way to optimize for power and performance due to reduced clock cycles. When data is organized properly, a vector load instruction (i.e. movaps) can replace 'normal' load instructions (i.e. movsd). Vector operations can potentially have a smaller footprint in the instruction cache when fewer instructions need to be executed. Hybrid index sets insulate users from architecture specific details. We have applied hybrid index sets to achieve optimal vectorization. We can extend this concept to handle other programming models.

  7. Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

    PubMed Central

    Friedrichs, Stephanie; Malan, Daniela; Voss, Yvonne; Sasse, Philipp

    2015-01-01

    Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening. PMID:26237021

  8. Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer

    PubMed Central

    Bierwolf, Jeanette; Volz, Tassilo; Lütgehetmann, Marc; Allweiss, Lena; Riecken, Kristoffer; Warlich, Michael; Fehse, Boris; Kalff, Joerg C.; Dandri, Maura

    2016-01-01

    Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo. PMID:27068494

  9. Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer.

    PubMed

    Bierwolf, Jeanette; Volz, Tassilo; Lütgehetmann, Marc; Allweiss, Lena; Riecken, Kristoffer; Warlich, Michael; Fehse, Boris; Kalff, Joerg C; Dandri, Maura; Pollok, Joerg-Matthias

    2016-05-01

    Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo. PMID:27068494

  10. Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells.

    PubMed

    Witkowski, Wojciech; Vermeire, Jolien; Landi, Alessia; Naessens, Evelien; Vanderstraeten, Hanne; Nauwynck, Hans; Favoreel, Herman; Verhasselt, Bruno

    2015-01-01

    The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting. PMID:26208151

  11. Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells

    PubMed Central

    Witkowski, Wojciech; Vermeire, Jolien; Landi, Alessia; Naessens, Evelien; Vanderstraeten, Hanne; Nauwynck, Hans; Favoreel, Herman; Verhasselt, Bruno

    2015-01-01

    The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting. PMID:26208151

  12. Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing

    PubMed Central

    Chiou, Shin-Heng; Winters, Ian P.; Wang, Jing; Naranjo, Santiago; Dudgeon, Crissy; Tamburini, Fiona B.; Brady, Jennifer J.; Yang, Dian; Grüner, Barbara M.; Chuang, Chen-Hua; Caswell, Deborah R.; Zeng, Hong; Chu, Pauline; Kim, Grace E.; Carpizo, Darren R.; Kim, Seung K.; Winslow, Monte M.

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer. PMID:26178787

  13. Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing.

    PubMed

    Chiou, Shin-Heng; Winters, Ian P; Wang, Jing; Naranjo, Santiago; Dudgeon, Crissy; Tamburini, Fiona B; Brady, Jennifer J; Yang, Dian; Grüner, Barbara M; Chuang, Chen-Hua; Caswell, Deborah R; Zeng, Hong; Chu, Pauline; Kim, Grace E; Carpizo, Darren R; Kim, Seung K; Winslow, Monte M

    2015-07-15

    Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer. PMID:26178787

  14. Retroviral vector production.

    PubMed

    Miller, A Dusty

    2014-01-01

    In this unit, the basic protocol generates stable cell lines that produce retroviral vectors that carry selectable markers. Also included are an alternate protocol that applies when the retroviral vector does not carry a selectable marker, and another alternate protocol for rapidly generating retroviral vector preparations by transient transfection. A support protocol describes construction of the retroviral vectors. The methods for generating virus from retroviral vector plasmids rely on the use of packaging cells that synthesize all of the retroviral proteins but do not produce replication-competent virus. Additional protocols detail plasmid transfection, virus titration, assay for replication-competent virus, and histochemical staining to detect transfer of a vector encoding alkaline phosphatase.

  15. Vectorization of a Treecode

    NASA Astrophysics Data System (ADS)

    Makino, Junichiro

    1990-03-01

    Vectorized algorithms for the force calculation and tree construction in the Barnes-Hut tree algorithm are described. The basic idea for the vectorization of the force calculation is to vectorize the tree traversal across particles, so that all particles in the system traverse the tree simultaneously. The tree construction algorithm also makes use of the fact that particles can be treated in parallel. Thus these algorithms take advantage of the internal parallelism in the N-body system and the tree algorithm most effectively. As a natural result, these algorithms can be used on a wide range of vector/parallel architectures, including current supercomputers and highly parallel architectures such as the Connection Machine. The vectorized code runs about five times faster than the non-vector code on a Cyber 205 for an N-body system with N = 8192.

  16. Support vector tracking.

    PubMed

    Avidan, Shai

    2004-08-01

    Support Vector Tracking (SVT) integrates the Support Vector Machine (SVM) classifier into an optic-flow-based tracker. Instead of minimizing an intensity difference function between successive frames, SVT maximizes the SVM classification score. To account for large motions between successive frames, we build pyramids from the support vectors and use a coarse-to-fine approach in the classification stage. We show results of using SVT for vehicle tracking in image sequences.

  17. Vectorized Monte Carlo

    SciTech Connect

    Brown, F.B.

    1981-01-01

    Examination of the global algorithms and local kernels of conventional general-purpose Monte Carlo codes shows that multigroup Monte Carlo methods have sufficient structure to permit efficient vectorization. A structured multigroup Monte Carlo algorithm for vector computers is developed in which many particle events are treated at once on a cell-by-cell basis. Vectorization of kernels for tracking and variance reduction is described, and a new method for discrete sampling is developed to facilitate the vectorization of collision analysis. To demonstrate the potential of the new method, a vectorized Monte Carlo code for multigroup radiation transport analysis was developed. This code incorporates many features of conventional general-purpose production codes, including general geometry, splitting and Russian roulette, survival biasing, variance estimation via batching, a number of cutoffs, and generalized tallies of collision, tracklength, and surface crossing estimators with response functions. Predictions of vectorized performance characteristics for the CYBER-205 were made using emulated coding and a dynamic model of vector instruction timing. Computation rates were examined for a variety of test problems to determine sensitivities to batch size and vector lengths. Significant speedups are predicted for even a few hundred particles per batch, and asymptotic speedups by about 40 over equivalent Amdahl 470V/8 scalar codes arepredicted for a few thousand particles per batch. The principal conclusion is that vectorization of a general-purpose multigroup Monte Carlo code is well worth the significant effort required for stylized coding and major algorithmic changes.

  18. Vector processing unit

    SciTech Connect

    Garcia, L.C.; Tjon-Pian-Gi, D.C.; Tucker, S.G.; Zajac, M.W.

    1988-12-13

    This patent describes a data processing system comprising: memory means for storing instruction words of operands; a central processing unit (CPU) connected to the memory means for fetching and decoding instructions and controlling execution of instructions, including transfer of operands to and from the memory means, the control of execution of instructions is effected by a CPU clock and microprogram control means connected to the CPU clock for generating periodic execution control signals in synchronism with the CPU clock; vector processing means tightly coupled to the CPU for effecting data processing on vector data; and interconnection means, connecting the CPU and the vector processing means, including operand transfer lines for transfer of vector data between the CPU and the vector processing means, control lines, status lines for signalling conditions of the vector processor means to the CPU, and a vector timing signal line connected to one of the execution control signals from the microprogram control means, whereby the vector processing means receives periodic execution control signals at the clock rate and is synchronized with the CPU clock on a clock pulse by clock pulse basis during execution of instructions.

  19. Vector generator scan converter

    DOEpatents

    Moore, J.M.; Leighton, J.F.

    1988-02-05

    High printing speeds for graphics data are achieved with a laser printer by transmitting compressed graphics data from a main processor over an I/O channel to a vector generator scan converter which reconstructs a full graphics image for input to the laser printer through a raster data input port. The vector generator scan converter includes a microprocessor with associated microcode memory containing a microcode instruction set, a working memory for storing compressed data, vector generator hardware for drawing a full graphic image from vector parameters calculated by the microprocessor, image buffer memory for storing the reconstructed graphics image and an output scanner for reading the graphics image data and inputting the data to the printer. The vector generator scan converter eliminates the bottleneck created by the I/O channel for transmitting graphics data from the main processor to the laser printer, and increases printer speed up to thirty fold. 7 figs.

  20. Vector generator scan converter

    DOEpatents

    Moore, James M.; Leighton, James F.

    1990-01-01

    High printing speeds for graphics data are achieved with a laser printer by transmitting compressed graphics data from a main processor over an I/O (input/output) channel to a vector generator scan converter which reconstructs a full graphics image for input to the laser printer through a raster data input port. The vector generator scan converter includes a microprocessor with associated microcode memory containing a microcode instruction set, a working memory for storing compressed data, vector generator hardward for drawing a full graphic image from vector parameters calculated by the microprocessor, image buffer memory for storing the reconstructed graphics image and an output scanner for reading the graphics image data and inputting the data to the printer. The vector generator scan converter eliminates the bottleneck created by the I/O channel for transmitting graphics data from the main processor to the laser printer, and increases printer speed up to thirty fold.

  1. Vector theories in cosmology

    SciTech Connect

    Esposito-Farese, Gilles; Pitrou, Cyril; Uzan, Jean-Philippe

    2010-03-15

    This article provides a general study of the Hamiltonian stability and the hyperbolicity of vector field models involving both a general function of the Faraday tensor and its dual, f(F{sup 2},FF-tilde), as well as a Proca potential for the vector field, V(A{sup 2}). In particular it is demonstrated that theories involving only f(F{sup 2}) do not satisfy the hyperbolicity conditions. It is then shown that in this class of models, the cosmological dynamics always dilutes the vector field. In the case of a nonminimal coupling to gravity, it is established that theories involving Rf(A{sup 2}) or Rf(F{sup 2}) are generically pathologic. To finish, we exhibit a model where the vector field is not diluted during the cosmological evolution, because of a nonminimal vector field-curvature coupling which maintains second-order field equations. The relevance of such models for cosmology is discussed.

  2. Vector generator scan converter

    SciTech Connect

    Moore, J.M.; Leighton, J.F.

    1990-04-17

    This patent describes high printing speeds for graphics data that are achieved with a laser printer by transmitting compressed graphics data from a main processor over an I/O (input/output) channel to a vector generator scan converter which reconstructs a full graphics image for input to the laser printer through a raster data input port. The vector generator scan converter includes a microprocessor with associated microcode memory containing a microcode instruction set, a working memory for storing compressed data, vector generator hardware for drawing a full graphic image from vector parameters calculated by the microprocessor, image buffer memory for storing the reconstructed graphics image and an output scanner for reading the graphics image data and inputting the data to the printer. The vector generator scan converter eliminates the bottleneck created by the I/O channel for transmitting graphics data from the main processor to the laser printer, and increases printer speed up to thirty fold.

  3. Prevention of Lethal Murine Hypophosphatasia by Neonatal Ex Vivo Gene Therapy Using Lentivirally Transduced Bone Marrow Cells.

    PubMed

    Iijima, Osamu; Miyake, Koichi; Watanabe, Atsushi; Miyake, Noriko; Igarashi, Tsutomu; Kanokoda, Chizu; Nakamura-Takahashi, Aki; Kinoshita, Hideaki; Noguchi, Taku; Abe, Shinichi; Narisawa, Sonoko; Millán, José Luis; Okada, Takashi; Shimada, Takashi

    2015-12-01

    Hypophosphatasia (HPP) is an inherited skeletal and dental disease caused by loss-of-function mutations in the gene that encodes tissue-nonspecific alkaline phosphatase (TNALP). The major symptoms of severe forms of the disease are bone defects, respiratory insufficiency, and epileptic seizures. In 2015, enzyme replacement therapy (ERT) using recombinant bone-targeted TNALP with deca-aspartate (D10) motif was approved to treat pediatric HPP patients in Japan, Canada, and Europe. However, the ERT requires repeated subcutaneous administration of the enzyme because of the short half-life in serum. In the present study, we evaluated the feasibility of neonatal ex vivo gene therapy in TNALP knockout (Akp2(-/-)) HPP mice using lentivirally transduced bone marrow cells (BMC) expressing bone-targeted TNALP in which a D10 sequence was linked to the C-terminus of soluble TNALP (TNALP-D10). The Akp2(-/-) mice usually die within 20 days because of growth failure, epileptic seizures, and hypomineralization. However, an intravenous transplantation of BMC expressing TNALP-D10 (ALP-BMC) into neonatal Akp2(-/-) mice prolonged survival of the mice with improved bone mineralization compared with untransduced BMC-transplanted Akp2(-/-) mice. The treated Akp2(-/-) mice were normal in appearance and experienced no seizures during the experimental period. The lentivirally transduced BMC were efficiently engrafted in the recipient mice and supplied TNALP-D10 continuously at a therapeutic level for at least 3 months. Moreover, TNALP-D10 overexpression did not affect multilineage reconstitution in the recipient mice. The plasma ALP activity was sustained at high levels in the treated mice, and tissue ALP activity was selectively detected on bone surfaces, not in the kidneys or other organs. No ectopic calcification was observed in the ALP-BMC-treated mice. These results indicate that lentivirally transduced BMC can serve as a reservoir for stem cell-based ERT to rescue the Akp2

  4. Tropism-modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy

    PubMed Central

    Sallach, Jessica; Di Pasquale, Giovanni; Larcher, Fernando; Niehoff, Nadine; Rübsam, Matthias; Huber, Anke; Chiorini, Jay; Almarza, David; Eming, Sabine A; Ulus, Hikmet; Nishimura, Stephen; Hacker, Ulrich T; Hallek, Michael; Niessen, Carien M; Büning, Hildegard

    2014-01-01

    Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvβ8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy. PMID:24468915

  5. Tailored HIV-1 vectors for genetic modification of primary human dendritic cells and monocytes.

    PubMed

    Durand, Stéphanie; Nguyen, Xuan-Nhi; Turpin, Jocelyn; Cordeil, Stephanie; Nazaret, Nicolas; Croze, Séverine; Mahieux, Renaud; Lachuer, Joël; Legras-Lachuer, Catherine; Cimarelli, Andrea

    2013-01-01

    Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.

  6. Down-regulation of CD81 tetraspanin in human cells producing retroviral-based particles: tailoring vector composition.

    PubMed

    Rodrigues, A F; Guerreiro, M R; Santiago, V M; Dalba, C; Klatzmann, D; Alves, P M; Carrondo, M J T; Coroadinha, A S

    2011-11-01

    Retroviral-derived biopharmaceuticals (RV) target numerous therapeutic applications, from gene therapy to virus-like particle (rVLP)-based vaccines. During particle formation, beside the pseudotyped envelope proteins, RV can incorporate proteins derived from the virus producer cells (VPC). This may be detrimental by reducing the amounts of the pseudotyped envelope and/or by incorporating protein capable of inducing immune responses when non-human VPC are used. Manipulating the repertoire of VPC proteins integrated onto the vector structure is an underexplored territory and should provide valuable insights on potential targets to improve vector pharmacokinetic and pharmacodynamic properties. In this work, human HEK 293 cells producing retrovirus-like particles (rVLPs) and infectious RV vectors were used to prove the concept of customizing RV composition by manipulating cellular protein content. The tetraspanin CD81 was chosen since it is significantly incorporated in the RV membrane, conferring to the vector significant immunogenicity when used in mice. RNA interference-mediated by shRNA lentiviral vector transduction was efficiently used to silence CD81 expression (up to 99%) and the rVLPs produced by knocked-down cells lack CD81. Silenced clones were analyzed for cell proliferation, morphological changes, susceptibility to oxidative stress conditions, and rVLP productivities. The results showed that the down-regulation of VPC proteins requires close monitoring for possible side effects on cellular production performance. Yet, they confirm that it is possible to change the composition of host-derived immunogens in RV by altering cellular protein content with no detriment for vector productivity and titers. This constitutes an important manipulation tool in vaccinology--by exploiting the potential adjuvant effect of VPC proteins or using them as fusion agents to other proteins of interest to be exposed on the vector membrane--and in gene therapy, by reducing the

  7. Line Integral of a Vector.

    ERIC Educational Resources Information Center

    Balabanian, Norman

    This programed booklet is designed for the engineering student who understands and can use vector and unit vector notation, components of a vector, parallel law of vector addition, and the dot product of two vectors. Content begins with work done by a force in moving a body a certain distance along some path. For each of the examples and problem…

  8. Species‐specific differences in the ability of feline lentiviral Vif to degrade feline APOBEC3 proteins

    PubMed Central

    Yoshikawa, Rokusuke; Nakano, Yusuke; Yamada, Eri; Izumi, Taisuke; Misawa, Naoko; Koyanagi, Yoshio

    2016-01-01

    ABSTRACT How host–virus co‐evolutionary relationships manifest is one of the most intriguing issues in virology. To address this topic, the mammal–lentivirus relationship can be considered as an interplay of cellular and viral proteins, particularly apolipoprotein B mRNA editing enzyme catalytic polypeptide‐like 3 (APOBEC3) and viral infectivity factor (Vif). APOBEC3s enzymatically restrict lentivirus replication, whereas Vif antagonizes the host anti‐viral action mediated by APOBEC3. In this study, the focus was on the interplay between feline APOBEC3 proteins and two feline immunodeficiency viruses in cats and pumas. To our knowledge, this study provides the first evidence of non‐primate lentiviral Vif being incapable of counteracting a natural host's anti‐viral activity mediated via APOBEC3 protein. PMID:26935128

  9. Polycistronic viral vectors.

    PubMed

    de Felipe, P

    2002-09-01

    Traditionally, vectors for gene transfer/therapy experiments were mono- or bicistronic. In the latter case, vectors express the gene of interest coupled with a marker gene. An increasing demand for more complex polycistronic vectors has arisen in recent years to obtain complex gene transfer/therapy effects. In particular, this demand is stimulated by the hope of a more powerful effect from combined gene therapy than from single gene therapy in a process whose parallels lie in the multi-drug combined therapies for cancer or AIDS. In the 1980's we had only splicing signals and internal promoters to construct such vectors: now a new set of biotechnological tools enables us to design new and more reliable bicistronic and polycistronic vectors. This article focuses on the description and comparison of the strategies for co-expression of two genes in bicistronic vectors, from the oldest to the more recently described: internal promoters, splicing, reinitiation, IRES, self-processing peptides (e.g. foot-and-mouth disease virus 2A), proteolytic cleavable sites (e.g. fusagen) and fusion of genes. I propose a classification of these strategies based upon either the use of multiple transcripts (with transcriptional mechanisms), or single transcripts (using translational/post-translational mechanisms). I also examine the different attempts to utilize these strategies in the construction of polycistronic vectors and the main problems encountered. Several potential uses of these polycistronic vectors, both in basic research and in therapy-focused applications, are discussed. The importance of the study of viral gene expression strategies and the need to transfer this knowledge to vector design is highlighted.

  10. Fractal vector optical fields.

    PubMed

    Pan, Yue; Gao, Xu-Zhen; Cai, Meng-Qiang; Zhang, Guan-Lin; Li, Yongnan; Tu, Chenghou; Wang, Hui-Tian

    2016-07-15

    We introduce the concept of a fractal, which provides an alternative approach for flexibly engineering the optical fields and their focal fields. We propose, design, and create a new family of optical fields-fractal vector optical fields, which build a bridge between the fractal and vector optical fields. The fractal vector optical fields have polarization states exhibiting fractal geometry, and may also involve the phase and/or amplitude simultaneously. The results reveal that the focal fields exhibit self-similarity, and the hierarchy of the fractal has the "weeding" role. The fractal can be used to engineer the focal field. PMID:27420485

  11. Bloch vector projection noise

    NASA Technical Reports Server (NTRS)

    Wang, Li-Jun; Bacon, A. M.; Zhao, H.-Z.; Thomas, J. E.

    1994-01-01

    In the optical measurement of the Bloch vector components describing a system of N two-level atoms, the quantum fluctuations in these components are coupled into the measuring optical field. This paper develops the quantum theory of optical measurement of Bloch vector projection noise. The preparation and probing of coherence in an effective two-level system consisting of the two ground states in an atomic three-level lambda-scheme are analyzed.

  12. Poynting-vector filter

    SciTech Connect

    Carrigan, Charles R.

    2011-08-02

    A determination is made of frequency components associated with a particular bearing or location resulting from sources emitting electromagnetic-wave energy for which a Poynting-Vector can be defined. The broadband frequency components associated with a specific direction or location of interest are isolated from other components in the power spectrum that are not associated with the direction or location of interest. The collection of pointing vectors can be used to characterize the source.

  13. A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors.

    PubMed

    Spirin, P V; Baskaran, D; Rubtsov, P M; Zenkova, M A; Vlassov, V V; Chernolovskaya, E L; Prassolov, V S

    2009-07-01

    RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it's widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ET O by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented.

  14. A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors

    PubMed Central

    Spirin, P.V.; Baskaran, D.; Rubtsov, P.M.; Zenkova, M.A.; Vlassov, V.V.; Chernolovskaya, E.L.

    2009-01-01

    RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it's widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ET O by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented. PMID:22649608

  15. A lentiviral vector-based genetic sensor system for comparative analysis of permeability and activity of vitamin D3 analogues in xenotransplanted human skin.

    PubMed

    Staunstrup, Nicklas Heine; Bak, Rasmus O; Cai, Yujia; Svensson, Lars; Petersen, Thomas K; Rosada, Cecilia; Stenderup, Karin; Bolund, Lars; Mikkelsen, Jacob Giehm

    2013-03-01

    Vitamin D3 analogues are widely used topical and oral remedies for various ailments such as psoriasis, osteoporosis and secondary hyperparathyroidism. In topical treatment, high skin permeability and cellular uptake are key criteria for beneficial effects due to the natural barrier properties of skin. In this study, we wish to establish an in vivo model that allows the comparison of permeability and activity of vitamin D3 analogues in human skin. We generate a bipartite, genetic sensor technology that combines efficient lentivirus-directed gene delivery to xenotransplanted human skin with vitamin D3-induced expression of a luciferase reporter gene and live imaging of animals by bioluminescence imaging. Based on the induction of a transcriptional activator consisting of the vitamin D receptor fused to the Gal4 DNA-binding domain, the vitamin D3-responsive sensor facilitates non-invasive and rapid assessment of permeability and functional properties of vitamin D3 analogues. By topical application of a panel of vitamin D3 analogues onto 'sensorized' human skin, the sensor produces a drug-induced readout with a magnitude and persistence that allow a direct comparative analysis of different analogues. This novel genetic tool has great potential as a non-invasive in vivo screening system for further development and refinement of vitamin D3 analogues.

  16. Syngeneic AAV pseudo-vectors potentiates full vector transduction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An excessive amount of empty capsids are generated during regular AAV vector production process. These pseudo-vectors often remain in final vectors used for animal studies or clinical trials. The potential effects of these pseudo-vectors on AAV transduction have been a major concern. In the current ...

  17. Vector and Axial Vector Pion Form Factors

    NASA Astrophysics Data System (ADS)

    Vitz, Michael; PEN Collaboration

    2015-04-01

    Radiative pion decay π+ -->e+ νγ (RPD) provides critical input to chiral perturbation theory (χPT). Aside from the uninteresting ``inner bremsstrahlung'' contribution from QED, the RPD rate contains ``structure dependent'' terms given by FV and FA, the vector and axial-vector pion form factors, respectively. The two appear in the decay rate in combinations FV -FA and FV +FA , i.e., in the so-called SD- and SD+ terms, respectively. The latter has been measured to high precision by the PIBETA collaboration. We report on the analysis of new data, measured by the PEN collaboration in runs between 2008 and 2010 at the Paul Scherrer Institute, Switzerland. We particularly focus on the possibility of improvement in the determination of the SD- term. Precise determinations of FV and FA test the validity of the CVC hypothesis, provide numerical input for the l9 +l10 terms in the χPT lagrangian, and constrain potential non-(V - A) terms, such as a possible tensor term FT. NSF grants PHY-0970013, 1307328, and others.

  18. Bunyavirus-vector interactions.

    PubMed

    Beaty, B J; Bishop, D H

    1988-06-01

    Recent advances in the genetics and molecular biology of bunyaviruses have been applied to understanding bunyavirus-vector interactions. Such approaches have revealed which virus gene and gene products are important in establishing infections in vectors and in transmission of viruses. However, much more information is required to understand the molecular mechanisms of persistent infections of vectors which are lifelong but apparently exert no untoward effect. In fact, it seems remarkable that LAC viral antigen can be detected in almost every cell in an ovarian follicle, yet no untoward effect on fecundity and no teratology is seen. Similarly the lifelong infection of the vector would seem to provide ample opportunity for bunyavirus evolution by genetic drift and, under the appropriate circumstances, by segment reassortment. The potential for bunyavirus evolution by segment reassortment in vectors certainly exists. For example the Group C viruses in a small forest in Brazil seem to constitute a gene pool, with the 6 viruses related alternately by HI/NT and CF reactions, which assay respectively M RNA and S RNA gene products (Casals and Whitman, 1960; Shope and Causey, 1962). Direct evidence for naturally occurring reassortant bunyaviruses has also been obtained. Oligonucleotide fingerprint analyses of field isolates of LAC virus and members of the Patois serogroup of bunyaviruses have demonstrated that reassortment does occur in nature (El Said et al., 1979; Klimas et al., 1981; Ushijima et al., 1981). Determination of the genotypic frequencies of viruses selected by the biological interactions of viruses and vectors after dual infection and segment reassortment is an important issue. Should a virus result that efficiently interacts with alternate vector species, the virus could be expressed in different circumstances with serious epidemiologic consequences. Dual infection of vectors with different viruses is not unlikely, because many bunyaviruses are sympatric in

  19. Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery

    PubMed Central

    Denk, Nora; Misra, Vikram; Sandmeyer, Lynne S.; Bauer, Bianca B.; Singh, Jaswant; Forsyth, George W.; Grahn, Bruce H.

    2015-01-01

    The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions. PMID:25673906

  20. Vector financial rogue waves

    NASA Astrophysics Data System (ADS)

    Yan, Zhenya

    2011-11-01

    The coupled nonlinear volatility and option pricing model presented recently by Ivancevic is investigated, which generates a leverage effect, i.e., stock volatility is (negatively) correlated to stock returns, and can be regarded as a coupled nonlinear wave alternative of the Black-Scholes option pricing model. In this Letter, we analytically propose vector financial rogue waves of the coupled nonlinear volatility and option pricing model without an embedded w-learning. Moreover, we exhibit their dynamical behaviors for chosen different parameters. The vector financial rogue wave (rogon) solutions may be used to describe the possible physical mechanisms for the rogue wave phenomena and to further excite the possibility of relative researches and potential applications of vector rogue waves in the financial markets and other related fields.

  1. Vectorized garbage collection

    SciTech Connect

    Appel, A.W.; Bendiksen, A.

    1988-01-01

    Garbage collection can be done in vector mode on supercomputers like the Cray-2 and the Cyber 205. Both copying collection and mark-and-sweep can be expressed as breadth-first searches in which the queue can be processed in parallel. The authors have designed a copying garbage collector whose inner loop works entirely in vector mode. The only significant limitation of the algorithm is that if the size of the records is not constant, the implementation becomes much more complicated. The authors give performance measurements of the algorithm as implemented for Lisp CONS cells on the Cyber 205. Vector-mode garbage collection performs up to 9 times faster than scalar-mode collection.

  2. Identification of a Human Respiratory Syncytial Virus Cell Entry Inhibitor by Using a Novel Lentiviral Pseudotype System

    PubMed Central

    Haid, Sibylle; Grethe, Christina; Bankwitz, Dorothea; Grunwald, Thomas

    2015-01-01

    ABSTRACT Lentiviral budding is governed by group-specific antigens (Gag proteins) and proceeds in the absence of cognate viral envelope proteins, which has been exploited to create pseudotypes incorporating envelope proteins from nonlentiviral families. Here, we report the generation of infectious lentiviral pseudoparticles incorporating human respiratory syncytial virus (hRSV) F protein alone (hRSV-Fpp) or carrying SH, G, and F proteins (hRSV-SH/G/Fpp). These particles recapitulate key infection steps of authentic hRSV particles, including utilization of glycosaminoglycans and low-pH-independent cell entry. Moreover, hRSV pseudoparticles (hRSVpp) can faithfully reproduce phenotypic resistance to a small-molecule fusion inhibitor in clinical development (BMS-433771) and a licensed therapeutic F protein-targeting antibody (palivizumab). Inoculation of several human cell lines from lung and liver revealed more than 30-fold differences in susceptibility to hRSVpp infection, suggesting differential expression of hRSV entry cofactors and/or restriction factors between these cell types. Moreover, we observed cell-type-dependent functional differences between hRSVpp carrying solely F protein or SH, G, and F proteins with regard to utilization of glycosaminoglycans. Using hRSVpp, we identified penta-O-galloyl-β-d-glucose (PGG) as a novel hRSV cell entry inhibitor. Moreover, we show that PGG also inhibits cell entry of hRSVpp carrying F proteins resistant to BMS-433771 or palivizumab. This work sheds new light on the mechanisms of hRSV cell entry, including possible strategies for antiviral intervention. Moreover, hRSVpp should prove valuable to dissect hRSV envelope protein functions, including the interaction with cell entry factors. IMPORTANCE Lentiviral pseudotypes are highly useful to specifically dissect the functions of viral and host factors in cell entry, which have been exploited for numerous viruses. Here, we successfully created hRSVpp and show that they

  3. Bunyavirus-Vector Interactions

    PubMed Central

    Horne, Kate McElroy; Vanlandingham, Dana L.

    2014-01-01

    The Bunyaviridae family is comprised of more than 350 viruses, of which many within the Hantavirus, Orthobunyavirus, Nairovirus, Tospovirus, and Phlebovirus genera are significant human or agricultural pathogens. The viruses within the Orthobunyavirus, Nairovirus, and Phlebovirus genera are transmitted by hematophagous arthropods, such as mosquitoes, midges, flies, and ticks, and their associated arthropods not only serve as vectors but also as virus reservoirs in many cases. This review presents an overview of several important emerging or re-emerging bunyaviruses and describes what is known about bunyavirus-vector interactions based on epidemiological, ultrastructural, and genetic studies of members of this virus family. PMID:25402172

  4. Support vector machines

    NASA Technical Reports Server (NTRS)

    Garay, Michael J.; Mazzoni, Dominic; Davies, Roger; Wagstaff, Kiri

    2004-01-01

    Support Vector Machines (SVMs) are a type of supervised learning algorith,, other examples of which are Artificial Neural Networks (ANNs), Decision Trees, and Naive Bayesian Classifiers. Supervised learning algorithms are used to classify objects labled by a 'supervisor' - typically a human 'expert.'.

  5. Vector potential methods

    NASA Technical Reports Server (NTRS)

    Hafez, M.

    1989-01-01

    Vector potential and related methods, for the simulation of both inviscid and viscous flows over aerodynamic configurations, are briefly reviewed. The advantages and disadvantages of several formulations are discussed and alternate strategies are recommended. Scalar potential, modified potential, alternate formulations of Euler equations, least-squares formulation, variational principles, iterative techniques and related methods, and viscous flow simulation are discussed.

  6. Killing vectors and anisotropy

    SciTech Connect

    Krisch, J. P.; Glass, E. N.

    2009-08-15

    We consider an action that can generate fluids with three unequal stresses for metrics with a spacelike Killing vector. The parameters in the action are directly related to the stress anisotropies. The field equations following from the action are applied to an anisotropic cosmological expansion and an extension of the Gott-Hiscock cosmic string.

  7. Gene transfer vector

    SciTech Connect

    Puhler, A.; Simon, R.

    1987-08-11

    A Tn-Mob vector is described comprising: (a) A replicon functional E. coli; and (b) A Tn-Mob element comprising a transposon containing (i) a functional selection marker, and (ii) a Mob-site and oriT located in a region of the transposon that is not essential to transposability.

  8. Redshifts and Killing vectors

    NASA Astrophysics Data System (ADS)

    Harvey, Alex; Schucking, Engelbert; Surowitz, Eugene J.

    2006-11-01

    Current approaches to physics stress the importance of conservation laws due to spacetime and internal symmetries. In special and general relativity the generators of these symmetries are known as Killing vectors. We use them for the rigorous determination of gravitational and cosmological redshifts.

  9. Vector-borne diseases.

    PubMed

    Gubler, D J

    2009-08-01

    Vector-borne diseases have been the scourge of man and animals since the beginning of time. Historically, these are the diseases that caused the great plagues such as the 'Black Death' in Europe in the 14th Century and the epidemics of yellow fever that plagued the development of the New World. Others, such as Nagana, contributed to the lack of development in Africa for many years. At the turn of the 20th Century, vector-borne diseases were among the most serious public and animal health problems in the world. For the most part, these diseases were controlled by the middle of the 20th Century through the application of knowledge about their natural history along with the judicious use of DDT (dichlorodiphenyltrichloroethane) and other residual insecticides to interrupt the transmission cycle between arthropod and vertebrate host. However, this success initiated a period of complacency in the 1960s and 1970s, which resulted in the redirection of resources away from prevention and control of vector-borne diseases. The 1970s was also a time in which there were major changes to public health policy. Global trends, combined with changes in animal husbandry, urbanisation, modern transportation and globalisation, have resulted in a global re-emergence of epidemic vector-borne diseases affecting both humans and animals over the past 30 years. PMID:20128467

  10. Vector-borne diseases.

    PubMed

    Gubler, D J

    2009-08-01

    Vector-borne diseases have been the scourge of man and animals since the beginning of time. Historically, these are the diseases that caused the great plagues such as the 'Black Death' in Europe in the 14th Century and the epidemics of yellow fever that plagued the development of the New World. Others, such as Nagana, contributed to the lack of development in Africa for many years. At the turn of the 20th Century, vector-borne diseases were among the most serious public and animal health problems in the world. For the most part, these diseases were controlled by the middle of the 20th Century through the application of knowledge about their natural history along with the judicious use of DDT (dichlorodiphenyltrichloroethane) and other residual insecticides to interrupt the transmission cycle between arthropod and vertebrate host. However, this success initiated a period of complacency in the 1960s and 1970s, which resulted in the redirection of resources away from prevention and control of vector-borne diseases. The 1970s was also a time in which there were major changes to public health policy. Global trends, combined with changes in animal husbandry, urbanisation, modern transportation and globalisation, have resulted in a global re-emergence of epidemic vector-borne diseases affecting both humans and animals over the past 30 years.

  11. Singular Vectors' Subtle Secrets

    ERIC Educational Resources Information Center

    James, David; Lachance, Michael; Remski, Joan

    2011-01-01

    Social scientists use adjacency tables to discover influence networks within and among groups. Building on work by Moler and Morrison, we use ordered pairs from the components of the first and second singular vectors of adjacency matrices as tools to distinguish these groups and to identify particularly strong or weak individuals.

  12. Variability in assays used for detection of lentiviral infection in bobcats (Lynx rufus), pumas (Puma concolor), and ocelots (Leopardus pardalis).

    PubMed

    Franklin, Samuel P; Troyer, Jennifer L; Terwee, Julie A; Lyren, Lisa M; Kays, Roland W; Riley, Seth P D; Boyce, Walter M; Crooks, Kevin R; Vandewoude, Sue

    2007-10-01

    Although lentiviruses similar to feline immunodeficiency virus (FIV) are known to infect numerous felid species, the relative utility of assays used for detecting lentiviral infection has not been compared for many of these hosts. We tested bobcats (Lynx rufus), pumas (Felis concolor), and ocelots (Leopardus pardalis) for exposure to lentivirus using five different assays: puma lentivirus (PLV), African lion lentivirus (LLV), and domestic cat FIV-based immunoblots, a commercially available enzyme-linked immunosorbent assay (ELISA) kit, and nested polymerase chain reaction (PCR). Puma lentivirus immunoblots identified more seropositive individuals than the other antibody-detection assays. The commercial ELISA provided a fair ability to recognize seropositive samples when compared with PLV immunoblot for screening bobcats and ocelots, but not pumas. Polymerase chain reaction identified fewer positive samples than PLV immunoblot for all three species. Immunoblot results were equivalent whether the sample tested was serum, plasma, or whole blood. The results from this study and previous investigations suggest that the PLV immunoblot has the greatest ability to detect reactive samples when screening wild felids of North America and is unlikely to produce false positive results. However, the commercial ELISA kit may provide an adequate alternative for screening of some species and is more easily adapted to field conditions.

  13. Variability in assays used for detection of lentiviral infection in bobcats (Lynx rufus), pumas (Puma concolor), and ocelots (Leopardus pardalis)

    USGS Publications Warehouse

    Franklin, S.P.; Troyer, J.L.; TerWee, J.A.; Lyren, L.M.; Kays, R.W.; Riley, S.P.D.; Boyce, W.M.; Crooks, K.R.; VandeWoude, S.

    2007-01-01

    Although lentiviruses similar to feline immunodeficiency virus (FIV) are known to infect numerous felid species, the relative utility of assays used for detecting lentiviral infection has not been compared for many of these hosts. We tested bobcats (Lynx rufus), pumas (Felis concolor), and ocelots (Leopardus pardalis) for exposure to lentivirus using five different assays: puma lentivirus (PLV), African lion lentivirus (LLV), and domestic cat FIV-based immunoblots, a commercially available enzyme-linked immunosorbent assay (ELISA) kit, and nested polymerase chain reaction (PCR). Puma lentivirus immunoblots identified more seropositive individuals than the other antibody-detection assays. The commercial ELISA provided a fair ability to recognize seropositive samples when compared with PLV immunoblot for screening bobcats and ocelots, but not pumas. Polymerase chain reaction identified fewer positive samples than PLV immunoblot for all three species. Immunoblot results were equivalent whether the sample tested was serum, plasma, or whole blood. The results from this study and previous investigations suggest that the PLV immunoblot has the greatest ability to detect reactive samples when screening wild felids of North America and is unlikely to produce false positive results. However, the commercial ELISA kit may provide ap adequate alternative for screening of some species and is more easily adapted to field conditions. ?? Wildlife Disease Association 2007.

  14. Lentiviral silencing of GSK-3β in adult dentate gyrus impairs contextual fear memory and synaptic plasticity

    PubMed Central

    Chew, Benjamin; Ryu, Jae Ryun; Ng, Teclise; Ma, Dongliang; Dasgupta, Ananya; Neo, Sin Hui; Zhao, Jing; Zhong, Zhong; Bichler, Zoë; Sajikumar, Sreedharan; Goh, Eyleen L. K.

    2015-01-01

    Attempts have been made to use glycogen synthase kinase-3 beta (GSK3β) inhibitors for prophylactic treatment of neurocognitive conditions. However the use of lithium, a non-specific inhibitor of GSK3β results in mild cognitive impairment in humans. The effects of global GSK3β inhibition or knockout on learning and memory in healthy adult mice are also inconclusive. Our study aims to better understand the role of GSK3β in learning and memory through a more regionally, targeted approach, specifically performing lentiviral-mediated knockdown of GSK3β within the dentate gyrus (DG). DG-GSK3β-silenced mice showed impaired contextual fear memory retrieval. However, cue fear memory, spatial memory, locomotor activity and anxiety levels were similar to control. These GSK3β-silenced mice also showed increased induction and maintenance of DG long-term potentiation (DG-LTP) compared to control animals. Thus, this region-specific, targeted knockdown of GSK3β in the DG provides better understanding on the role of GSK3β in learning and memory. PMID:26157370

  15. Measurement of Cancer Cell Growth Heterogeneity through Lentiviral Barcoding Identifies Clonal Dominance as a Characteristic of In Vivo Tumor Engraftment.

    PubMed

    Nolan-Stevaux, Olivier; Tedesco, Donato; Ragan, Seamus; Makhanov, Mikhail; Chenchik, Alex; Ruefli-Brasse, Astrid; Quon, Kim; Kassner, Paul D

    2013-01-01

    Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate in vitro and in vivo of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth in vitro was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term "clonal dominance". We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780(cis)) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic in vivo selection process we describe has important implications for the fields of in vivo shRNA screening and tumor initiating cells.

  16. [Vector control and malaria control].

    PubMed

    Carnevale, P; Mouchet, J

    1990-01-01

    Vector control is an integral part of malaria control. Limiting parasite transmission vector control must be considered as one of the main preventive measure. Indeed it prevents transmission of Plasmodium from man to vector and from vector to man. But vector control must be adapted to local situation to be efficient and feasible. Targets of vector control can be larval and/or adults stages. In both cases 3 main methods are currently available: physical (source reduction), chemical (insecticides) and biological tolls. Antilarval control is useful only in some particular circumstances (unstable malaria, island, oasis...) Antiadult control is mainly based upon house-spraying while pyrethroid treated bed nets is advocated regarding efficiency, simple technique and cheap price. Vector control measures could seem restricted but can be very efficient if political will is added to a right choice of adapted measures, a good training of involved personal and a large information of the population concerned with vector control.

  17. Elusive vector glueball

    SciTech Connect

    Suzuki, Mahiko

    2002-05-01

    If the vector glueball {Omicron} exists in the mass range that theory suggests, its resonant production cross section can be detected in e{sup +}e{sup -} annihilation only if the decay width is very narrow ({le} a few MeV). Otherwise {Omicron} will be observed only indirectly through its mixing with {psi}{prime}. We propose a few tests of the {Omicron}-{psi}{prime} mixing for future charm factories.

  18. Vector soliton fission.

    PubMed

    Lu, F; Lin, Q; Knox, W H; Agrawal, Govind P

    2004-10-29

    We investigate the vectorial nature of soliton fission in an isotropic nonlinear medium both theoretically and experimentally. As a specific example, we show that supercontinuum generation in a tapered fiber is extremely sensitive to the input state of polarization. Multiple vector solitons generated through soliton fission exhibit different states of elliptical polarization while emitting nonsolitonic radiation with complicated polarization features. Experiments performed with a tapered fiber agree with our theoretical description.

  19. Vector Magnetograph Design

    NASA Technical Reports Server (NTRS)

    Chipman, Russell A.

    1996-01-01

    This report covers work performed during the period of November 1994 through March 1996 on the design of a Space-borne Solar Vector Magnetograph. This work has been performed as part of a design team under the supervision of Dr. Mona Hagyard and Dr. Alan Gary of the Space Science Laboratory. Many tasks were performed and this report documents the results from some of those tasks, each contained in the corresponding appendix. Appendices are organized in chronological order.

  20. Vector representation of tourmaline compositions

    NASA Technical Reports Server (NTRS)

    Burt, Donald M.

    1989-01-01

    The vector method for representing mineral compositions of amphibole and mica groups is applied to the tourmaline group. Consideration is given to the methods for drawing the relevant vector diagrams, relating the exchange vectors to one another, and contouring the diagrams for constant values of Na, Ca, Li, Fe, Mg, Al, Si, and OH. The method is used to depict a wide range of possible tourmaline end-member compositions and solid solutions, starting from a single point. In addition to vector depictions of multicomponent natural tourmalines, vectors are presented for simpler systems such as (Na,Al)-tourmalines, alkali-free tourmalines, and elbaites.

  1. Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

    PubMed Central

    Börner, Kathleen; Niopek, Dominik; Cotugno, Gabriella; Kaldenbach, Michaela; Pankert, Teresa; Willemsen, Joschka; Zhang, Xian; Schürmann, Nina; Mockenhaupt, Stefan; Serva, Andrius; Hiet, Marie-Sophie; Wiedtke, Ellen; Castoldi, Mirco; Starkuviene, Vytaute; Erfle, Holger; Gilbert, Daniel F.; Bartenschlager, Ralf; Boutros, Michael; Binder, Marco; Streetz, Konrad; Kräusslich, Hans-Georg; Grimm, Dirk

    2013-01-01

    As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems. PMID:24049077

  2. Vector ecology of equine piroplasmosis.

    PubMed

    Scoles, Glen A; Ueti, Massaro W

    2015-01-01

    Equine piroplasmosis is a disease of Equidae, including horses, donkeys, mules, and zebras, caused by either of two protozoan parasites, Theileria equi or Babesia caballi. These parasites are biologically transmitted between hosts via tick vectors, and although they have inherent differences they are categorized together because they cause similar pathology and have similar morphologies, life cycles, and vector relationships. To complete their life cycle, these parasites must undergo a complex series of developmental events, including sexual-stage development in their tick vectors. Consequently, ticks are the definitive hosts as well as vectors for these parasites, and the vector relationship is restricted to a few competent tick species. Because the vector relationship is critical to the epidemiology of these parasites, we highlight current knowledge of the vector ecology of these tick-borne equine pathogens, emphasizing tick transmissibility and potential control strategies to prevent their spread.

  3. Lentiviral shRNA against KCa3.1 inhibits allergic response in allergic rhinitis and suppresses mast cell activity via PI3K/AKT signaling pathway

    PubMed Central

    Lin, Hai; Zheng, Chunquan; Li, Jing; Yang, Chen; Hu, Li

    2015-01-01

    Calcium-activated potassium ion channel-3.1 (KCa3.1) plays a pivotal role in the potassium-calcium exchange involved in atopy. This study aimed to explore the impact of lentiviral-mediated shRNA silencing KCa3.1 on allergic response in a murine allergic rhinitis (AR) model. The BALB/c mice were divided into four groups: untreated AR group, negative control AR group, lentiviral KCa3.1-shRNA treated AR group and normal control group. Concentrations of ovalbumin (OVA)-specific IgE, histamine and leukotrienes C4 (LTC4) in serum, and IL-4, IL-9 and IL-17 in nasal lavage fluid (NLF) were analyzed. Goblet cells and mast cells were counted. KCa3.1 positive cells were counted after immunolabelling by immunofluorescence method. KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction. Furthermore, P815 cell line was used to explore the role and mechanism of lentiviral KCa3.1-shRNA on mast cells. The results showed that LV-KCa3.1-shRNA intervention effectively attenuated allergic responses in LV-KCa3.1-shRNA treated mice. LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels. LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway. PMID:26272420

  4. A versatile modular vector system for rapid combinatorial mammalian genetics.

    PubMed

    Albers, Joachim; Danzer, Claudia; Rechsteiner, Markus; Lehmann, Holger; Brandt, Laura P; Hejhal, Tomas; Catalano, Antonella; Busenhart, Philipp; Gonçalves, Ana Filipa; Brandt, Simone; Bode, Peter K; Bode-Lesniewska, Beata; Wild, Peter J; Frew, Ian J

    2015-04-01

    Here, we describe the multiple lentiviral expression (MuLE) system that allows multiple genetic alterations to be introduced simultaneously into mammalian cells. We created a toolbox of MuLE vectors that constitute a flexible, modular system for the rapid engineering of complex polycistronic lentiviruses, allowing combinatorial gene overexpression, gene knockdown, Cre-mediated gene deletion, or CRISPR/Cas9-mediated (where CRISPR indicates clustered regularly interspaced short palindromic repeats) gene mutation, together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging. Examples of tumor engineering were used to illustrate the speed and versatility of performing combinatorial genetics using the MuLE system. By transducing cultured primary mouse cells with single MuLE lentiviruses, we engineered tumors containing up to 5 different genetic alterations, identified genetic dependencies of molecularly defined tumors, conducted genetic interaction screens, and induced the simultaneous CRISPR/Cas9-mediated knockout of 3 tumor-suppressor genes. Intramuscular injection of MuLE viruses expressing oncogenic H-RasG12V together with combinations of knockdowns of the tumor suppressors cyclin-dependent kinase inhibitor 2A (Cdkn2a), transformation-related protein 53 (Trp53), and phosphatase and tensin homolog (Pten) allowed the generation of 3 murine sarcoma models, demonstrating that genetically defined autochthonous tumors can be rapidly generated and quantitatively monitored via direct injection of polycistronic MuLE lentiviruses into mouse tissues. Together, our results demonstrate that the MuLE system provides genetic power for the systematic investigation of the molecular mechanisms that underlie human diseases.

  5. A versatile modular vector system for rapid combinatorial mammalian genetics

    PubMed Central

    Albers, Joachim; Danzer, Claudia; Rechsteiner, Markus; Lehmann, Holger; Brandt, Laura P.; Hejhal, Tomas; Catalano, Antonella; Busenhart, Philipp; Gonçalves, Ana Filipa; Brandt, Simone; Bode, Peter K.; Bode-Lesniewska, Beata; Wild, Peter J.; Frew, Ian J.

    2015-01-01

    Here, we describe the multiple lentiviral expression (MuLE) system that allows multiple genetic alterations to be introduced simultaneously into mammalian cells. We created a toolbox of MuLE vectors that constitute a flexible, modular system for the rapid engineering of complex polycistronic lentiviruses, allowing combinatorial gene overexpression, gene knockdown, Cre-mediated gene deletion, or CRISPR/Cas9-mediated (where CRISPR indicates clustered regularly interspaced short palindromic repeats) gene mutation, together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging. Examples of tumor engineering were used to illustrate the speed and versatility of performing combinatorial genetics using the MuLE system. By transducing cultured primary mouse cells with single MuLE lentiviruses, we engineered tumors containing up to 5 different genetic alterations, identified genetic dependencies of molecularly defined tumors, conducted genetic interaction screens, and induced the simultaneous CRISPR/Cas9-mediated knockout of 3 tumor-suppressor genes. Intramuscular injection of MuLE viruses expressing oncogenic H-RasG12V together with combinations of knockdowns of the tumor suppressors cyclin-dependent kinase inhibitor 2A (Cdkn2a), transformation-related protein 53 (Trp53), and phosphatase and tensin homolog (Pten) allowed the generation of 3 murine sarcoma models, demonstrating that genetically defined autochthonous tumors can be rapidly generated and quantitatively monitored via direct injection of polycistronic MuLE lentiviruses into mouse tissues. Together, our results demonstrate that the MuLE system provides genetic power for the systematic investigation of the molecular mechanisms that underlie human diseases. PMID:25751063

  6. A versatile modular vector system for rapid combinatorial mammalian genetics.

    PubMed

    Albers, Joachim; Danzer, Claudia; Rechsteiner, Markus; Lehmann, Holger; Brandt, Laura P; Hejhal, Tomas; Catalano, Antonella; Busenhart, Philipp; Gonçalves, Ana Filipa; Brandt, Simone; Bode, Peter K; Bode-Lesniewska, Beata; Wild, Peter J; Frew, Ian J

    2015-04-01

    Here, we describe the multiple lentiviral expression (MuLE) system that allows multiple genetic alterations to be introduced simultaneously into mammalian cells. We created a toolbox of MuLE vectors that constitute a flexible, modular system for the rapid engineering of complex polycistronic lentiviruses, allowing combinatorial gene overexpression, gene knockdown, Cre-mediated gene deletion, or CRISPR/Cas9-mediated (where CRISPR indicates clustered regularly interspaced short palindromic repeats) gene mutation, together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging. Examples of tumor engineering were used to illustrate the speed and versatility of performing combinatorial genetics using the MuLE system. By transducing cultured primary mouse cells with single MuLE lentiviruses, we engineered tumors containing up to 5 different genetic alterations, identified genetic dependencies of molecularly defined tumors, conducted genetic interaction screens, and induced the simultaneous CRISPR/Cas9-mediated knockout of 3 tumor-suppressor genes. Intramuscular injection of MuLE viruses expressing oncogenic H-RasG12V together with combinations of knockdowns of the tumor suppressors cyclin-dependent kinase inhibitor 2A (Cdkn2a), transformation-related protein 53 (Trp53), and phosphatase and tensin homolog (Pten) allowed the generation of 3 murine sarcoma models, demonstrating that genetically defined autochthonous tumors can be rapidly generated and quantitatively monitored via direct injection of polycistronic MuLE lentiviruses into mouse tissues. Together, our results demonstrate that the MuLE system provides genetic power for the systematic investigation of the molecular mechanisms that underlie human diseases. PMID:25751063

  7. Thrust vectoring systems

    NASA Technical Reports Server (NTRS)

    King, H. J.; Schnelker, D.; Ward, J. W.; Dulgeroff, C.; Vahrenkamp, R.

    1972-01-01

    The design, fabrication, and testing of thrust vectorable ion optical systems capable of controlling the thrust direction from both 5- and 30-cm diameter ion thrusters is described. Both systems are capable of greater than 10 deg thrust deflection in any azimuthal direction. The 5-cm system is electrostatic and hence has a short response time and minimal power consumption. It has recently been tested for more than 7500 hours on an operational thruster. The 30-cm system is mechanical, has a response time of the order of 1 min, and consumes less than 0.3% of the total system input power at full deflection angle.

  8. Vector potential photoelectron microscopy

    SciTech Connect

    Browning, R.

    2011-10-15

    A new class of electron microscope has been developed for the chemical microanalysis of a wide range of real world samples using photoelectron spectroscopy. Highly structured, three-dimensional samples, such as fiber mats and fracture surfaces can be imaged, as well as insulators and magnetic materials. The new microscope uses the vector potential field from a solenoid magnet as a spatial reference for imaging. A prototype instrument has demonstrated imaging of uncoated silk, magnetic steel wool, and micron-sized single strand tungsten wires.

  9. Deciphering the impact of parameters influencing transgene expression kinetics after repeated cell transduction with integration-deficient retroviral vectors.

    PubMed

    Schott, Juliane W; Jaeschke, Nico M; Hoffmann, Dirk; Maetzig, Tobias; Ballmaier, Matthias; Godinho, Tamaryin; Cathomen, Toni; Schambach, Axel

    2015-05-01

    Lentiviral and gammaretroviral vectors are state-of-the-art tools for transgene expression within target cells. The integration of these vectors can be deliberately suppressed to derive a transient gene expression system based on extrachromosomal circular episomes with intact coding regions. These episomes can be used to deliver DNA templates and to express RNA or protein. Importantly, transient gene transfer avoids the genotoxic side effects of integrating vectors. Restricting their applicability, episomes are rapidly lost upon dilution in dividing target cells. Addressing this limitation, we could establish comparably stable percentages of transgene-positive cells over prolonged time periods in proliferating cells by repeated transductions. Flow cytometry was applied for kinetic analyses to decipher the impact of individual parameters on the kinetics of fluoroprotein expression after episomal retransduction and to visualize sequential and simultaneous transfer of heterologous fluoroproteins. Expression windows could be exactly timed by the number of transduction steps. The kinetics of signal loss was affected by the cell proliferation rate. The transfer of genes encoding fluoroproteins with different half-lives revealed a major impact of protein stability on temporal signal distribution and accumulation, determining optimal retransduction intervals. In addition, sequential transductions proved broad applicability in different cell types and using different envelope pseudotypes without receptor overload. Stable percentages of cells coexpressing multiple transgenes could be generated upon repeated coadministration of different episomal vectors. Alternatively, defined patterns of transgene expression could be recapitulated by sequential transductions. Altogether, we established a methodology to control and adjust a temporally defined window of transgene expression using retroviral episomal vectors. Combined with the highly efficient cell entry of these vectors while

  10. A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

    PubMed

    Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

    2014-12-05

    Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

  11. A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets

    PubMed Central

    Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

    2014-01-01

    Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways. PMID:25475013

  12. Hyperbolic-symmetry vector fields.

    PubMed

    Gao, Xu-Zhen; Pan, Yue; Cai, Meng-Qiang; Li, Yongnan; Tu, Chenghou; Wang, Hui-Tian

    2015-12-14

    We present and construct a new kind of orthogonal coordinate system, hyperbolic coordinate system. We present and design a new kind of local linearly polarized vector fields, which is defined as the hyperbolic-symmetry vector fields because the points with the same polarization form a series of hyperbolae. We experimentally demonstrate the generation of such a kind of hyperbolic-symmetry vector optical fields. In particular, we also study the modified hyperbolic-symmetry vector optical fields with the twofold and fourfold symmetric states of polarization when introducing the mirror symmetry. The tight focusing behaviors of these vector fields are also investigated. In addition, we also fabricate micro-structures on the K9 glass surfaces by several tightly focused (modified) hyperbolic-symmetry vector fields patterns, which demonstrate that the simulated tightly focused fields are in good agreement with the fabricated micro-structures.

  13. Covariant Lyapunov vectors

    NASA Astrophysics Data System (ADS)

    Ginelli, Francesco; Chaté, Hugues; Livi, Roberto; Politi, Antonio

    2013-06-01

    Recent years have witnessed a growing interest in covariant Lyapunov vectors (CLVs) which span local intrinsic directions in the phase space of chaotic systems. Here, we review the basic results of ergodic theory, with a specific reference to the implications of Oseledets’ theorem for the properties of the CLVs. We then present a detailed description of a ‘dynamical’ algorithm to compute the CLVs and show that it generically converges exponentially in time. We also discuss its numerical performance and compare it with other algorithms presented in the literature. We finally illustrate how CLVs can be used to quantify deviations from hyperbolicity with reference to a dissipative system (a chain of Hénon maps) and a Hamiltonian model (a Fermi-Pasta-Ulam chain). This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Lyapunov analysis: from dynamical systems theory to applications’.

  14. Solar imaging vector magnetograph

    NASA Technical Reports Server (NTRS)

    Canfield, Richard C.

    1993-01-01

    This report describes an instrument which has been constructed at the University of Hawaii to make observations of the magnetic field in solar active regions. Detailed knowledge of active region magnetic structures is crucial to understanding many solar phenomena, because the magnetic field both defines the morphology of structures seen in the solar atmosphere and is the apparent energy source for solar flares. The new vector magnetograph was conceived in response to a perceived discrepancy between the capabilities of X ray imaging telescopes to be operating during the current solar maximum and those of existing magnetographs. There were no space-based magnetographs planned for this period; the existing ground-based instruments variously suffered from lack of sensitivity, poor time resolution, inadequate spatial resolution or unreliable sites. Yet the studies of flares and their relationship to the solar corona planned for the 1991-1994 maximum absolutely required high quality vector magnetic field measurements. By 'vector' measurements we mean that the observation attempts to deduce the complete strength and direction of the field at the measurement site, rather than just the line of sight component as obtained by a traditional longitudinal magnetograph. Knowledge of the vector field permits one to calculate photospheric electric currents, which might play a part in heating the corona, and to calculate energy stored in coronal magnetic fields as the result of such currents. Information about the strength and direction of magnetic fields in the solar atmosphere can be obtained in a number of ways, but quantitative data is best obtained by observing Zeeman-effect polarization in solar spectral lines. The technique requires measuring the complete state of polarization at one or more wavelengths within a magnetically sensitive line of the solar spectrum. This measurement must be done for each independent spatial point for which one wants magnetic field data. All the

  15. Enhanced Potency of a Broadly Neutralizing HIV-1 Antibody In Vitro Improves Protection against Lentiviral Infection In Vivo

    PubMed Central

    Rudicell, Rebecca S.; Kwon, Young Do; Ko, Sung-Youl; Pegu, Amarendra; Louder, Mark K.; Georgiev, Ivelin S.; Wu, Xueling; Zhu, Jiang; Boyington, Jeffrey C.; Chen, Xuejun; Shi, Wei; Yang, Zhi-yong; Doria-Rose, Nicole A.; McKee, Krisha; O'Dell, Sijy; Schmidt, Stephen D.; Chuang, Gwo-Yu; Druz, Aliaksandr; Soto, Cinque; Yang, Yongping; Zhang, Baoshan; Zhou, Tongqing; Todd, John-Paul; Lloyd, Krissey E.; Eudailey, Joshua; Roberts, Kyle E.; Donald, Bruce R.; Bailer, Robert T.; Ledgerwood, Julie; Mullikin, James C.; Shapiro, Lawrence; Koup, Richard A.; Graham, Barney S.; Nason, Martha C.; Connors, Mark; Haynes, Barton F.; Rao, Srinivas S.; Roederer, Mario; Kwong, Peter D.

    2014-01-01

    ABSTRACT Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans. IMPORTANCE In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope

  16. All-in-one readout electronics for the Belle-II Central Drift Chamber

    NASA Astrophysics Data System (ADS)

    Taniguchi, Nanae; Ikeno, Masahiro; Iwasaki, Yoshihito; Saito, Masatoshi; Shimazaki, Shoichi; Tanaka, Manobu; Uchida, Tomohisa; Uno, Shoji

    2013-12-01

    For the Belle-II experiment at KEK, a Central Drift Chamber (CDC) with readout electronics is required to be upgraded to cope with the design luminosity of 8×1035 cm-2 s-1. Not only the readout electronics will be completely replaced, but also the wire chamber itself. The new readout electronics system must handle higher trigger rates with less dead time. The front-end electronics are located close to detector and send digitized signal through optical fibers. The Amp-Shaper-Discriminator chips, FADC and FPGA are assembled on a single board. This paper will give an overview of the Belle-II CDC and present results of the performance of the readout board in a test beam.

  17. All in One Stop? The Accessibility of Work Support Programs at One-Stop Centers.

    ERIC Educational Resources Information Center

    Richer, Elise; Kubo, Hitomi; Frank, Abbey

    The accessibility of work support programs at one-stop centers was examined in a study during which 33 telephone directors or managers of one-stop centers in 22 states were interviewed by telephone. The interviews established the existence of extensive differences between one-stop centers from the standpoint of all aspects of their operation,…

  18. All-in-One and One-for-All: A Proposed Model of Lifelong Technology Education

    ERIC Educational Resources Information Center

    Lee, Lung-Sheng

    2008-01-01

    This paper proposes a lifelong model of technology education to promote the analysis and explanation of technology education. Including five stages, this model assumes that everyone in most countries/regions should have opportunities to learn about technology in every stage. Learning in the first three stages, awareness-literacy-specialization,…

  19. CEval: All-in-one software for data processing and statistical evaluations in affinity capillary electrophoresis.

    PubMed

    Dubský, Pavel; Ördögová, Magda; Malý, Michal; Riesová, Martina

    2016-05-01

    We introduce CEval software (downloadable for free at echmet.natur.cuni.cz) that was developed for quicker and easier electrophoregram evaluation and further data processing in (affinity) capillary electrophoresis. This software allows for automatic peak detection and evaluation of common peak parameters, such as its migration time, area, width etc. Additionally, the software includes a nonlinear regression engine that performs peak fitting with the Haarhoff-van der Linde (HVL) function, including automated initial guess of the HVL function parameters. HVL is a fundamental peak-shape function in electrophoresis, based on which the correct effective mobility of the analyte represented by the peak is evaluated. Effective mobilities of an analyte at various concentrations of a selector can be further stored and plotted in an affinity CE mode. Consequently, the mobility of the free analyte, μA, mobility of the analyte-selector complex, μAS, and the apparent complexation constant, K('), are first guessed automatically from the linearized data plots and subsequently estimated by the means of nonlinear regression. An option that allows two complexation dependencies to be fitted at once is especially convenient for enantioseparations. Statistical processing of these data is also included, which allowed us to: i) express the 95% confidence intervals for the μA, μAS and K(') least-squares estimates, ii) do hypothesis testing on the estimated parameters for the first time. We demonstrate the benefits of the CEval software by inspecting complexation of tryptophan methyl ester with two cyclodextrins, neutral heptakis(2,6-di-O-methyl)-β-CD and charged heptakis(6-O-sulfo)-β-CD.

  20. CancellationTools: All-in-one software for administration and analysis of cancellation tasks.

    PubMed

    Dalmaijer, Edwin S; Van der Stigchel, Stefan; Nijboer, Tanja C W; Cornelissen, Tim H W; Husain, Masud

    2015-12-01

    In a cancellation task, a participant is required to search for and cross out ("cancel") targets, which are usually embedded among distractor stimuli. The number of cancelled targets and their location can be used to diagnose the neglect syndrome after stroke. In addition, the organization of search provides a potentially useful way to measure executive control over multitarget search. Although many useful cancellation measures have been introduced, most fail to make their way into research studies and clinical practice due to the practical difficulty of acquiring such parameters from traditional pen-and-paper measures. Here we present new, open-source software that is freely available to all. It allows researchers and clinicians to flexibly administer computerized cancellation tasks using stimuli of their choice, and to directly analyze the data in a convenient manner. The automated analysis suite provides output that includes almost all of the currently existing measures, as well as several new ones introduced here. All tasks can be performed using either a computer mouse or a touchscreen as an input device, and an online version of the task runtime is available for tablet devices. A summary of the results is produced in a single A4-sized PDF document, including high quality data visualizations. For research purposes, batch analysis of large datasets is possible. In sum, CancellationTools allows users to employ a flexible, computerized cancellation task, which provides extensive benefits and ease of use.

  1. All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

    PubMed Central

    Werner, Melanie; Driftmann, Sabrina; Kleinehr, Kathrin; Kaiser, Gernot M.; Mathé, Zotlan; Treckmann, Juergen-Walter; Paul, Andreas; Skibbe, Kathrin; Timm, Joerg; Canbay, Ali; Gerken, Guido; Schlaak, Joerg F.; Broering, Ruth

    2015-01-01

    Background & Aims Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. Methods Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. Results Cell preparation yielded the following cell counts per gram of liver tissue: 2.0±0.4×107 hepatocytes, 1.8±0.5×106 Kupffer cells, 4.3±1.9×105 liver sinusoidal endothelial cells, and 3.2±0.5×105 stellate cells. Hepatocytes were identified by albumin (95.5±1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5±1.2%) and exhibited phagocytic activity, as determined with 1μm latex beads. Endothelial cells were CD146+ (97.8±1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1±1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. Conclusions Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease. PMID:26407160

  2. The All-in-One Human Resource Policy of Taiwan's Hand-in-Hand Program

    ERIC Educational Resources Information Center

    Ho, Hsuan-fu; Lin, Liang-Ching

    2012-01-01

    The Taiwan Government launched the Hand-in-Hand After-School Care Program in 2006 with several goals: In addition to providing disadvantaged students with supplemental learning opportunities, this highly ambitious government program hopes to provide increased employment opportunities for substitute teachers and low-income college students, and…

  3. New "all-in-one" device for mesh plug hernioplasty: the Trabucco repair.

    PubMed

    Gossetti, Francesco; Massa, Salvatore; Abbonante, Francesco; Calabria, Michele; Ceci, Francesca; Viarengo, Maria Antonietta; Manzi, Emy; D'Amore, Linda; Negro, Paolo

    2015-01-01

    Sebbene la tecnica Mesh Plug rappresenti una procedura di indubbia efficacia nel trattamento dell’ernia inguinale, tuttora permangono dubbi in relazione alla storia naturale del plug. In particolare, il plug può andare incontro a coartazione (shrinkage) con formazione di un “meshoma” responsabile di una sintomatologia dolorosa cronica. Più raramente, il plug può migrare e provocare erosione delle strutture anatomiche circostanti. Nel passato sono stati proposti alcuni devices per cercare di risolvere il problema della migrazione; nessuno di questi, tuttavia, ha raggiunto la popolarità dei plug conici o a forma di fiore. Il presente lavoro riporta i risultati di uno studio pilota condotto con l’impiego di un nuovo device tridimensionale, il NeT Plug & Patch. I risultati dopo 12 mesi di follow-up, hanno dimostrato una ridotta incidenza di dolore postoperatorio e cronico, senza rischio di migrazione. Inoltre è stata registrata una compliance soddisfacente sia dei pazienti che degli operatori. La protesi NeT Plug & Patch ha dimostrato di poter realizzare un’ernioplastica semplice ed efficace per la riparazione dell’ernia inguinale.

  4. GPU Accelerated Vector Median Filter

    NASA Technical Reports Server (NTRS)

    Aras, Rifat; Shen, Yuzhong

    2011-01-01

    Noise reduction is an important step for most image processing tasks. For three channel color images, a widely used technique is vector median filter in which color values of pixels are treated as 3-component vectors. Vector median filters are computationally expensive; for a window size of n x n, each of the n(sup 2) vectors has to be compared with other n(sup 2) - 1 vectors in distances. General purpose computation on graphics processing units (GPUs) is the paradigm of utilizing high-performance many-core GPU architectures for computation tasks that are normally handled by CPUs. In this work. NVIDIA's Compute Unified Device Architecture (CUDA) paradigm is used to accelerate vector median filtering. which has to the best of our knowledge never been done before. The performance of GPU accelerated vector median filter is compared to that of the CPU and MPI-based versions for different image and window sizes, Initial findings of the study showed 100x improvement of performance of vector median filter implementation on GPUs over CPU implementations and further speed-up is expected after more extensive optimizations of the GPU algorithm .

  5. Bubble vector in automatic merging

    NASA Technical Reports Server (NTRS)

    Pamidi, P. R.; Butler, T. G.

    1987-01-01

    It is shown that it is within the capability of the DMAP language to build a set of vectors that can grow incrementally to be applied automatically and economically within a DMAP loop that serves to append sub-matrices that are generated within a loop to a core matrix. The method of constructing such vectors is explained.

  6. Vectors on the Basketball Court

    ERIC Educational Resources Information Center

    Bergman, Daniel

    2010-01-01

    An Idea Bank published in the April/May 2009 issue of "The Science Teacher" describes an experiential physics lesson on vectors and vector addition (Brown 2009). Like its football predecessor, the basketball-based investigation presented in this Idea Bank addresses National Science Education Standards Content B, Physical Science, 9-12 (NRC 1996)…

  7. Rice Reoviruses in Insect Vectors.

    PubMed

    Wei, Taiyun; Li, Yi

    2016-08-01

    Rice reoviruses, transmitted by leafhopper or planthopper vectors in a persistent propagative manner, seriously threaten the stability of rice production in Asia. Understanding the mechanisms that enable viral transmission by insect vectors is a key to controlling these viral diseases. This review describes current understanding of replication cycles of rice reoviruses in vector cell lines, transmission barriers, and molecular determinants of vector competence and persistent infection. Despite recent breakthroughs, such as the discoveries of actin-based tubule motility exploited by viruses to overcome transmission barriers and mutually beneficial relationships between viruses and bacterial symbionts, there are still many gaps in our knowledge of transmission mechanisms. Advances in genome sequencing, reverse genetics systems, and molecular technologies will help to address these problems. Investigating the multiple interaction systems among the virus, insect vector, insect symbiont, and plant during natural infection in the field is a central topic for future research on rice reoviruses. PMID:27296147

  8. A neural support vector machine.

    PubMed

    Jändel, Magnus

    2010-06-01

    Support vector machines are state-of-the-art pattern recognition algorithms that are well founded in optimization and generalization theory but not obviously applicable to the brain. This paper presents Bio-SVM, a biologically feasible support vector machine. An unstable associative memory oscillates between support vectors and interacts with a feed-forward classification pathway. Kernel neurons blend support vectors and sensory input. Downstream temporal integration generates the classification. Instant learning of surprising events and off-line tuning of support vector weights trains the system. Emotion-based learning, forgetting trivia, sleep and brain oscillations are phenomena that agree with the Bio-SVM model. A mapping to the olfactory system is suggested.

  9. Vector Network Analysis

    1997-10-20

    Vector network analyzers are a convenient way to measure scattering parameters of a variety of microwave devices. However, these instruments, unlike oscilloscopes for example, require a relatively high degree of user knowledge and expertise. Due to the complexity of the instrument and of the calibration process, there are many ways in which an incorrect measurement may be produced. The Microwave Project, which is part of Sandia National Laboratories Primary Standards Laboratory, routinely uses check standardsmore » to verify that the network analyzer is operating properly. In the past, these measurements were recorded manually and, sometimes, interpretation of the results was problematic. To aid our measurement assurance process, a software program was developed to automatically measure a check standard and compare the new measurements with an historical database of measurements of the same device. The program acquires new measurement data from selected check standards, plots the new data against the mean and standard deviation of prior data for the same check standard, and updates the database files for the check standard. The program is entirely menu-driven requiring little additional work by the user.« less

  10. Chikungunya Virus–Vector Interactions

    PubMed Central

    Coffey, Lark L.; Failloux, Anna-Bella; Weaver, Scott C.

    2014-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever, a severe, debilitating disease that often produces chronic arthralgia. Since 2004, CHIKV has emerged in Africa, Indian Ocean islands, Asia, Europe, and the Americas, causing millions of human infections. Central to understanding CHIKV emergence is knowledge of the natural ecology of transmission and vector infection dynamics. This review presents current understanding of CHIKV infection dynamics in mosquito vectors and its relationship to human disease emergence. The following topics are reviewed: CHIKV infection and vector life history traits including transmission cycles, genetic origins, distribution, emergence and spread, dispersal, vector competence, vector immunity and microbial interactions, and co-infection by CHIKV and other arboviruses. The genetics of vector susceptibility and host range changes, population heterogeneity and selection for the fittest viral genomes, dual host cycling and its impact on CHIKV adaptation, viral bottlenecks and intrahost diversity, and adaptive constraints on CHIKV evolution are also discussed. The potential for CHIKV re-emergence and expansion into new areas and prospects for prevention via vector control are also briefly reviewed. PMID:25421891

  11. Successive refinement lattice vector quantization.

    PubMed

    Mukherjee, Debargha; Mitra, Sanjit K

    2002-01-01

    Lattice Vector quantization (LVQ) solves the complexity problem of LBG based vector quantizers, yielding very general codebooks. However, a single stage LVQ, when applied to high resolution quantization of a vector, may result in very large and unwieldy indices, making it unsuitable for applications requiring successive refinement. The goal of this work is to develop a unified framework for progressive uniform quantization of vectors without having to sacrifice the mean- squared-error advantage of lattice quantization. A successive refinement uniform vector quantization methodology is developed, where the codebooks in successive stages are all lattice codebooks, each in the shape of the Voronoi regions of the lattice at the previous stage. Such Voronoi shaped geometric lattice codebooks are named Voronoi lattice VQs (VLVQ). Measures of efficiency of successive refinement are developed based on the entropy of the indices transmitted by the VLVQs. Additionally, a constructive method for asymptotically optimal uniform quantization is developed using tree-structured subset VLVQs in conjunction with entropy coding. The methodology developed here essentially yields the optimal vector counterpart of scalar "bitplane-wise" refinement. Unfortunately it is not as trivial to implement as in the scalar case. Furthermore, the benefits of asymptotic optimality in tree-structured subset VLVQs remain elusive in practical nonasymptotic situations. Nevertheless, because scalar bitplane- wise refinement is extensively used in modern wavelet image coders, we have applied the VLVQ techniques to successively refine vectors of wavelet coefficients in the vector set-partitioning (VSPIHT) framework. The results are compared against SPIHT and the previous successive approximation wavelet vector quantization (SA-W-VQ) results of Sampson, da Silva and Ghanbari.

  12. Vector and Axial-Vector Structures of the Θ+

    NASA Astrophysics Data System (ADS)

    Kim, Hyun-Chul; Ledwig, Tim; Goeke, Klaus

    We present in this talk recent results of the vector and axial-vector transitions of the nucleon to the pentaquark baryon Θ+, based on the SU(3) chiral quark-soliton model. The results are summarized as follows: K*NΘ vector and tensor coupling constants turn out to be gK*NΘ ≃ 0.81 and fK*NΘ ≃ 0.84, respectively, and the KNΘ axial-vector coupling constant to be g*A ˜= 0.05. As a result, the total decay width for Θ+ → NK becomes very small: ΓΘ→NK ≃ 0.71 MeV, which is consistent with the DIANA result ΓΘ→NK = 0.36 ± 0.11 MeV.

  13. Colliders and brane vector phenomenology

    SciTech Connect

    Clark, T. E.; Love, S. T.; Xiong, C.; Nitta, Muneto; Veldhuis, T. ter

    2008-12-01

    Brane world oscillations manifest themselves as massive vector gauge fields. Their coupling to the standard model is deduced using the method of nonlinear realizations of the spontaneously broken higher dimensional space-time symmetries. Brane vectors are stable and weakly interacting and therefore escape particle detectors unnoticed. LEP and Tevatron data on the production of a single photon in conjunction with missing energy are used to delineate experimentally excluded regions of brane vector parameter space. The additional region of parameter space accessible to the LHC as well as a future lepton linear collider is also determined by means of this process.

  14. Initial conditions for vector inflation

    SciTech Connect

    Chiba, Takeshi

    2008-08-15

    Recently, a model of inflation using non-minimally coupled massive vector fields has been proposed. For a particular choice of non-minimal coupling parameter and for a flat Friedmann-Robertson-Walker model, the model is reduced to the model of chaotic inflation with massive scalar field. We study the effect of non-zero curvature of the universe on the onset of vector inflation. We find that in a curved universe the dynamics of vector inflation can be different from the dynamics of chaotic inflation, and the fraction of the initial conditions leading to inflationary solutions is reduced as compared with the chaotic inflation case.

  15. U1 snRNA as an effective vector for stable expression of antisense molecules and for the inhibition of the splicing reaction.

    PubMed

    Martone, Julie; De Angelis, Fernanda Gabriella; Bozzoni, Irene

    2012-01-01

    We report the use of the U1 snRNA as a vector for the stable expression of antisense molecules against the splice junctions of specific dystrophin exons. The single-stranded 5' terminus of U1 can be replaced by unrelated sequences as long as 50 nucleotides without affecting both the stability and the ability to assemble into snRNP particles. Effective exon skipping has been obtained for different dystrophin exons by antisense sequences against 5' and 3' splice sites alone or in combination with ESE sequences. The efficacy of these molecules has been studied both in in vitro systems and in animals. In both cases the chimeric molecules, delivered as part of lentiviral or AAV vectors (De Angelis et al. Proc Natl Acad Sci USA 99:9456-9461, 2002; Denti et al. Proc Natl Acad Sci USA 103: 3758-3763, 2006; Denti et al. Hum Gene Ther 17: 565-743, 2006; Denti et al. Hum Gene Ther 19: 601-608, 2008; Incitti et al. Mol Ther 18: 1675-1682, 2010), provided high skipping activity and efficient rescue of dystrophin synthesis. Moreover, the U1-antisense molecules, delivered to mice via systemic injection of recombinant AAV viruses, displayed body wide transduction, long-term expression, dystrophin rescue as well as morphological and functional benefit (Denti et al. Hum Gene Ther 19: 601-608, 2008). In this Chapter we report methods for producing U1-antisense expression cassettes in the backbone of lentiviral constructs and for testing their activity both in patients' derived myoblasts as well as in fibroblasts reprogrammed to muscle differentiation.

  16. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the βA(T87Q)-Globin Gene

    PubMed Central

    Negre, Olivier; Eggimann, Anne-Virginie; Beuzard, Yves; Ribeil, Jean-Antoine; Bourget, Philippe; Borwornpinyo, Suparerk; Hongeng, Suradej; Hacein-Bey, Salima; Cavazzana, Marina; Leboulch, Philippe; Payen, Emmanuel

    2016-01-01

    β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (βAT87Q-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. βAT87Q-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease. PMID:26886832

  17. Experiments With Magnetic Vector Potential

    ERIC Educational Resources Information Center

    Skinner, J. W.

    1975-01-01

    Describes the experimental apparatus and method for the study of magnetic vector potential (MVP). Includes a discussion of inherent errors in the calculations involved, precision of the results, and further applications of MVP. (GS)

  18. Electromagnetic structure of vector mesons

    NASA Astrophysics Data System (ADS)

    Adamuščín, C.; Dubnička, S.; Dubničková, A. Z.

    2014-11-01

    Electromagnetic structure of the complete nonet of vector mesons (ρ0, ρ+, ρ-, ω, ϕ, K*0, K*+, K¯*0, K*-) is investigated in the framework of the Unitary and Analytic model and insufficient experimental information on it is discussed.

  19. Polynomial interpretation of multipole vectors

    NASA Astrophysics Data System (ADS)

    Katz, Gabriel; Weeks, Jeff

    2004-09-01

    Copi, Huterer, Starkman, and Schwarz introduced multipole vectors in a tensor context and used them to demonstrate that the first-year Wilkinson microwave anisotropy probe (WMAP) quadrupole and octopole planes align at roughly the 99.9% confidence level. In the present article, the language of polynomials provides a new and independent derivation of the multipole vector concept. Bézout’s theorem supports an elementary proof that the multipole vectors exist and are unique (up to rescaling). The constructive nature of the proof leads to a fast, practical algorithm for computing multipole vectors. We illustrate the algorithm by finding exact solutions for some simple toy examples and numerical solutions for the first-year WMAP quadrupole and octopole. We then apply our algorithm to Monte Carlo skies to independently reconfirm the estimate that the WMAP quadrupole and octopole planes align at the 99.9% level.

  20. Brief history of vector Doppler

    NASA Astrophysics Data System (ADS)

    Dunmire, Barbrina; Beach, Kirk W.

    2001-05-01

    Since the development of the directional Doppler by McLeod in 1967, methods of acquiring, analyzing, and displaying blood velocity information have been under constant exploration. These efforts are motivated by a variety of interest and objectives including, to: a) simplify clinical examination, examiner training, and study interpretation, b) provide more hemodynamic information, and c) reduce examination variability and improve accuracy. The vector Doppler technique has been proposed as one potential avenue to achieve these objects. Vector Doppler systems are those that determine the true 2D or 3D blood flow velocity by combining multiple independent velocity component measurements. Most instruments can be divided into two broad categories: 1) cross-beam and 2) time-domain. This paper provides a brief synopsis of the progression of vector Doppler techniques, from its onset in 1970 to present, as well as possible avenues for future work. This is not intended to be a comprehensive review of all vector Doppler systems.

  1. Unsupervised learning of binary vectors

    NASA Astrophysics Data System (ADS)

    Copelli Lopes da Silva, Mauro

    In this thesis, unsupervised learning of binary vectors from data is studied using methods from Statistical Mechanics of disordered systems. In the model, data vectors are distributed according to a single symmetry-breaking direction. The aim of unsupervised learning is to provide a good approximation to this direction. The difference with respect to previous studies is the knowledge that this preferential direction has binary components. It is shown that sampling from the posterior distribution (Gibbs learning) leads, for general smooth distributions, to an exponentially fast approach to perfect learning in the asymptotic limit of large number of examples. If the distribution is non-smooth, then first order phase transitions to perfect learning are expected. In the limit of poor performance, a second order phase transition ("retarded learning") is predicted to occur if the data distribution is not biased. Using concepts from Bayesian inference, the center of mass of the Gibbs ensemble is shown to have maximal average (Bayes-optimal) performance. This upper bound for continuous vectors is extended to a discrete space, resulting in the clipped center of mass of the Gibbs ensemble having maximal average performance among the binary vectors. To calculate the performance of this best binary vector, the geometric properties of the center of mass of binary vectors are studied. The surprising result is found that the center of mass of infinite binary vectors which obey some simple constraints, is again a binary vector. When disorder is taken into account in the calculation, however, a vector with continuous components is obtained. The performance of the best binary vector is calculated and shown to always lie above that of Gibbs learning and below the Bayes-optimal performance. Making use of a variational approach under the replica symmetric ansatz, an optimal potential is constructed in the limits of zero temperature and mutual overlap 1. Minimization of this potential

  2. Redirecting T cells to Ewing's sarcoma family of tumors by a chimeric NKG2D receptor expressed by lentiviral transduction or mRNA transfection.

    PubMed

    Lehner, Manfred; Götz, Gabriel; Proff, Julia; Schaft, Niels; Dörrie, Jan; Full, Florian; Ensser, Armin; Muller, Yves A; Cerwenka, Adelheid; Abken, Hinrich; Parolini, Ornella; Ambros, Peter F; Kovar, Heinrich; Holter, Wolfgang

    2012-01-01

    We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8(pos) and also CD4(pos) cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection. PMID:22355347

  3. Effective Masses of Vector Polarons

    NASA Astrophysics Data System (ADS)

    Foell, Charles; Clougherty, Dennis

    2006-03-01

    We consider the vector polarons of a one-dimensional model of an electron in a doubly (or nearly) degenerate band that couples to two elastic distortions, as described previously by Clougherty and Foell [1]. A variational approach is used to analytically and numerically calculate effective masses of the three types of vector polarons. [1] D. P. Clougherty and C. A. Foell, Phys. Rev. B 70, 052301 (2004).

  4. Hybrid Lentivirus-phiC31-int-NLS Vector Allows Site-Specific Recombination in Murine and Human Cells but Induces DNA Damage

    PubMed Central

    Grandchamp, Nicolas; Altémir, Dorothée; Philippe, Stéphanie; Ursulet, Suzanna; Pilet, Héloïse; Serre, Marie-Claude; Lenain, Aude; Serguera, Che; Mallet, Jacques; Sarkis, Chamsy

    2014-01-01

    Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable. PMID:24956106

  5. Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

    PubMed

    Grandchamp, Nicolas; Altémir, Dorothée; Philippe, Stéphanie; Ursulet, Suzanna; Pilet, Héloïse; Serre, Marie-Claude; Lenain, Aude; Serguera, Che; Mallet, Jacques; Sarkis, Chamsy

    2014-01-01

    Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable. PMID:24956106

  6. Axisymmetric Coanda-assisted vectoring

    NASA Astrophysics Data System (ADS)

    Allen, Dustin; Smith, Barton L.

    2009-01-01

    An experimental demonstration of a jet vectoring technique used in our novel spray method called Coanda-assisted Spray Manipulation (CSM) is presented. CSM makes use of the Coanda effect on axisymmetric geometries through the interaction of two jets: a primary jet and a control jet. The primary jet has larger volume flow rate but generally a smaller momentum flux than the control jet. The primary jet flows through the center of a rounded collar. The control jet is parallel to the primary and is adjacent to the convex collar. The Reynolds number range for the primary jet at the exit plane was between 20,000 and 80,000. The flow was in the incompressible Mach number range (Mach < 0.3). The control jet attaches to the convex wall and vectors according to known Coanda effect principles, entraining and vectoring the primary jet, resulting in controllable r - θ directional spraying. Several annular control slots and collar radii were tested over a range of momentum flux ratios to determine the effects of these variables on the vectored jet angle and spreading. Two and Three-component Particle Image Velocimetry systems were used to determine the vectoring angle and the profile of the combined jet in each experiment. The experiments show that the control slot and expansion radius, along with the momentum ratios of the two jets predominantly affected the vectoring angle and profile of the combined jets.

  7. Vectoring of parallel synthetic jets

    NASA Astrophysics Data System (ADS)

    Berk, Tim; Ganapathisubramani, Bharathram; Gomit, Guillaume

    2015-11-01

    A pair of parallel synthetic jets can be vectored by applying a phase difference between the two driving signals. The resulting jet can be merged or bifurcated and either vectored towards the actuator leading in phase or the actuator lagging in phase. In the present study, the influence of phase difference and Strouhal number on the vectoring behaviour is examined experimentally. Phase-locked vorticity fields, measured using Particle Image Velocimetry (PIV), are used to track vortex pairs. The physical mechanisms that explain the diversity in vectoring behaviour are observed based on the vortex trajectories. For a fixed phase difference, the vectoring behaviour is shown to be primarily influenced by pinch-off time of vortex rings generated by the synthetic jets. Beyond a certain formation number, the pinch-off timescale becomes invariant. In this region, the vectoring behaviour is determined by the distance between subsequent vortex rings. We acknowledge the financial support from the European Research Council (ERC grant agreement no. 277472).

  8. Sustained expression from DNA vectors.

    PubMed

    Wong, Suet Ping; Argyros, Orestis; Harbottle, Richard P

    2015-01-01

    DNA vectors have the potential to become powerful medical tools for treatment of human disease. The human body has, however, developed a range of defensive strategies to detect and silence foreign or misplaced DNA, which is more typically encountered during infection or chromosomal damage. A clinically relevant human gene therapy vector must overcome or avoid these protections whilst delivering sustained levels of therapeutic gene product without compromising the vitality of the recipient host. Many non-viral DNA vectors trigger these defense mechanisms and are subsequently destroyed or rendered silent. Thus, without modification or considered design, the clinical utility of a typical DNA vector is fundamentally limited due to the transient nature of its transgene expression. The development of safe and persistently expressing DNA vectors is a crucial prerequisite for its successful clinical application and subsequently remains, therefore, one of the main strategic tasks of non-viral gene therapy research. In this chapter we will describe our current understanding of the mechanisms that can destroy or silence DNA vectors and discuss strategies, which have been utilized to improve their sustenance and the level and duration of their transgene expression.

  9. Are Bred Vectors The Same As Lyapunov Vectors?

    NASA Astrophysics Data System (ADS)

    Kalnay, E.; Corazza, M.; Cai, M.

    Regional loss of predictability is an indication of the instability of the underlying flow, where small errors in the initial conditions (or imperfections in the model) grow to large amplitudes in finite times. The stability properties of evolving flows have been studied using Lyapunov vectors (e.g., Alligood et al, 1996, Ott, 1993, Kalnay, 2002), singular vectors (e.g., Lorenz, 1965, Farrell, 1988, Molteni and Palmer, 1993), and, more recently, with bred vectors (e.g., Szunyogh et al, 1997, Cai et al, 2001). Bred vectors (BVs) are, by construction, closely related to Lyapunov vectors (LVs). In fact, after an infinitely long breeding time, and with the use of infinitesimal ampli- tudes, bred vectors are identical to leading Lyapunov vectors. In practical applications, however, bred vectors are different from Lyapunov vectors in two important ways: a) bred vectors are never globally orthogonalized and are intrinsically local in space and time, and b) they are finite-amplitude, finite-time vectors. These two differences are very significant in a dynamical system whose size is very large. For example, the at- mosphere is large enough to have "room" for several synoptic scale instabilities (e.g., storms) to develop independently in different regions (say, North America and Aus- tralia), and it is complex enough to have several different possible types of instabilities (such as barotropic, baroclinic, convective, and even Brownian motion). Bred vectors share some of their properties with leading LVs (Corazza et al, 2001a, 2001b, Toth and Kalnay, 1993, 1997, Cai et al, 2001). For example, 1) Bred vectors are independent of the norm used to define the size of the perturba- tion. Corazza et al. (2001) showed that bred vectors obtained using a potential enstro- phy norm were indistinguishable from bred vectors obtained using a streamfunction squared norm, in contrast with singular vectors. 2) Bred vectors are independent of the length of the rescaling period as long as the

  10. Black holes with vector hair

    NASA Astrophysics Data System (ADS)

    Fan, Zhong-Ying

    2016-09-01

    In this paper, we consider Einstein gravity coupled to a vector field, either minimally or non-minimally, together with a vector potential of the type V = 2{Λ}_0+1/2{m}^2{A}^2 + {γ}_4{A}^4 . For a simpler non-minimally coupled theory with Λ0 = m = γ4 = 0, we obtain both extremal and non-extremal black hole solutions that are asymptotic to Minkowski space-times. We study the global properties of the solutions and derive the first law of thermodynamics using Wald formalism. We find that the thermodynamical first law of the extremal black holes is modified by a one form associated with the vector field. In particular, due to the existence of the non-minimal coupling, the vector forms thermodynamic conjugates with the graviton mode and partly contributes to the one form modifying the first law. For a minimally coupled theory with Λ0 ≠ 0, we also obtain one class of asymptotically flat extremal black hole solutions in general dimensions. This is possible because the parameters ( m 2 , γ4) take certain values such that V = 0. In particular, we find that the vector also forms thermodynamic conjugates with the graviton mode and contributes to the corresponding first law, although the non-minimal coupling has been turned off. Thus all the extremal black hole solutions that we obtain provide highly non-trivial examples how the first law of thermodynamics can be modified by a either minimally or non-minimally coupled vector field. We also study Gauss-Bonnet gravity non-minimally coupled to a vector and obtain asymptotically flat black holes and Lifshitz black holes.

  11. VectorBase: a home for invertebrate vectors of human pathogens.

    PubMed

    Lawson, Daniel; Arensburger, Peter; Atkinson, Peter; Besansky, Nora J; Bruggner, Robert V; Butler, Ryan; Campbell, Kathryn S; Christophides, George K; Christley, Scott; Dialynas, Emmanuel; Emmert, David; Hammond, Martin; Hill, Catherine A; Kennedy, Ryan C; Lobo, Neil F; MacCallum, M Robert; Madey, Greg; Megy, Karine; Redmond, Seth; Russo, Susan; Severson, David W; Stinson, Eric O; Topalis, Pantelis; Zdobnov, Evgeny M; Birney, Ewan; Gelbart, William M; Kafatos, Fotis C; Louis, Christos; Collins, Frank H

    2007-01-01

    VectorBase (http://www.vectorbase.org/) is a web-accessible data repository for information about invertebrate vectors of human pathogens. VectorBase annotates and maintains vector genomes providing an integrated resource for the research community. Currently, VectorBase contains genome information for two organisms: Anopheles gambiae, a vector for the Plasmodium protozoan agent causing malaria, and Aedes aegypti, a vector for the flaviviral agents causing Yellow fever and Dengue fever.

  12. The biological control of disease vectors.

    PubMed

    Okamoto, Kenichi W; Amarasekare, Priyanga

    2012-09-21

    Vector-borne diseases are common in nature and can have a large impact on humans, livestock and crops. Biological control of vectors using natural enemies or competitors can reduce vector density and hence disease transmission. However, the indirect interactions inherent in host-vector disease systems make it difficult to use traditional pest control theory to guide biological control of disease vectors. This necessitates a conceptual framework that explicitly considers a range of indirect interactions between the host-vector disease system and the vector's biological control agent. Here we conduct a comparative analysis of the efficacy of different types of biological control agents in controlling vector-borne diseases. We report three key findings. First, highly efficient predators and parasitoids of the vector prove to be effective biological control agents, but highly virulent pathogens of the vector also require a high transmission rate to be effective. Second, biocontrol agents can successfully reduce long-term host disease incidence even though they may fail to reduce long-term vector densities. Third, inundating a host-vector disease system with a natural enemy of the vector has little or no effect on reducing disease incidence, but inundating the system with a competitor of the vector has a large effect on reducing disease incidence. The comparative framework yields predictions that are useful in developing biological control strategies for vector-borne diseases. We discuss how these predictions can inform ongoing biological control efforts for host-vector disease systems.

  13. Learning with LOGO: Logo and Vectors.

    ERIC Educational Resources Information Center

    Lough, Tom; Tipps, Steve

    1986-01-01

    This is the first of a two-part series on the general concept of vector space. Provides tool procedures to allow investigation of vector properties, vector addition and subtraction, and X and Y components. Lists several sources of additional vector ideas. (JM)

  14. Vector Encoding in Biochemical Networks

    NASA Astrophysics Data System (ADS)

    Potter, Garrett; Sun, Bo

    Encoding of environmental cues via biochemical signaling pathways is of vital importance in the transmission of information for cells in a network. The current literature assumes a single cell state is used to encode information, however, recent research suggests the optimal strategy utilizes a vector of cell states sampled at various time points. To elucidate the optimal sampling strategy for vector encoding, we take an information theoretic approach and determine the mutual information of the calcium signaling dynamics obtained from fibroblast cells perturbed with different concentrations of ATP. Specifically, we analyze the sampling strategies under the cases of fixed and non-fixed vector dimension as well as the efficiency of these strategies. Our results show that sampling with greater frequency is optimal in the case of non-fixed vector dimension but that, in general, a lower sampling frequency is best from both a fixed vector dimension and efficiency standpoint. Further, we find the use of a simple modified Ornstein-Uhlenbeck process as a model qualitatively captures many of our experimental results suggesting that sampling in biochemical networks is based on a few basic components.

  15. A generalized nonlocal vector calculus

    NASA Astrophysics Data System (ADS)

    Alali, Bacim; Liu, Kuo; Gunzburger, Max

    2015-10-01

    A nonlocal vector calculus was introduced in Du et al. (Math Model Meth Appl Sci 23:493-540, 2013) that has proved useful for the analysis of the peridynamics model of nonlocal mechanics and nonlocal diffusion models. A formulation is developed that provides a more general setting for the nonlocal vector calculus that is independent of particular nonlocal models. It is shown that general nonlocal calculus operators are integral operators with specific integral kernels. General nonlocal calculus properties are developed, including nonlocal integration by parts formula and Green's identities. The nonlocal vector calculus introduced in Du et al. (Math Model Meth Appl Sci 23:493-540, 2013) is shown to be recoverable from the general formulation as a special example. This special nonlocal vector calculus is used to reformulate the peridynamics equation of motion in terms of the nonlocal gradient operator and its adjoint. A new example of nonlocal vector calculus operators is introduced, which shows the potential use of the general formulation for general nonlocal models.

  16. Generalized Selection Weighted Vector Filters

    NASA Astrophysics Data System (ADS)

    Lukac, Rastislav; Plataniotis, Konstantinos N.; Smolka, Bogdan; Venetsanopoulos, Anastasios N.

    2004-12-01

    This paper introduces a class of nonlinear multichannel filters capable of removing impulsive noise in color images. The here-proposed generalized selection weighted vector filter class constitutes a powerful filtering framework for multichannel signal processing. Previously defined multichannel filters such as vector median filter, basic vector directional filter, directional-distance filter, weighted vector median filters, and weighted vector directional filters are treated from a global viewpoint using the proposed framework. Robust order-statistic concepts and increased degree of freedom in filter design make the proposed method attractive for a variety of applications. Introduced multichannel sigmoidal adaptation of the filter parameters and its modifications allow to accommodate the filter parameters to varying signal and noise statistics. Simulation studies reported in this paper indicate that the proposed filter class is computationally attractive, yields excellent performance, and is able to preserve fine details and color information while efficiently suppressing impulsive noise. This paper is an extended version of the paper by Lukac et al. presented at the 2003 IEEE-EURASIP Workshop on Nonlinear Signal and Image Processing (NSIP '03) in Grado, Italy.

  17. Vectors for cancer gene therapy.

    PubMed

    Zhang, J; Russell, S J

    1996-09-01

    Many viral and non-viral vector systems have now been developed for gene therapy applications. In this article, the pros and cons of these vector systems are discussed in relation to the different cancer gene therapy strategies. The protocols used in cancer gene therapy can be broadly divided into six categories including gene transfer to explanted cells for use as cell-based cancer vaccines; gene transfer to a small number of tumour cells in situ to achieve a vaccine effect; gene transfer to vascular endothelial cells (VECs) lining the blood vessels of the tumour to interfere with tumour angiogenesis; gene transfer to T lymphocytes to enhance their antitumour effector capability; gene transfer to haemopoietic stem cells (HSCs) to enhance their resistance to cytotoxic drugs and gene transfer to a large number of tumour cells in situ to achieve nonimmune tumour reduction with or without bystander effect. Each of the six strategies makes unique demands on the vector system and these are discussed with reference to currently available vectors. Aspects of vector biology that are in need of further development are discussed in some detail. The final section points to the potential use of replicating viruses as delivery vehicles for efficient in vivo gene transfer to disseminated cancers.

  18. Partial correction of sensitivity to oxidant stress in Friedreich ataxia patient fibroblasts by frataxin-encoding adeno-associated virus and lentivirus vectors.

    PubMed

    Fleming, Jane; Spinoulas, Afroditi; Zheng, Maolin; Cunningham, Sharon C; Ginn, Samantha L; McQuilty, Robert C; Rowe, Peter B; Alexander, Ian E

    2005-08-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of a number of debilitating inherited and acquired neurological conditions. The lack of effective treatments for many such conditions provides a strong rationale for exploring novel therapeutic approaches, including gene therapy. Friedreich ataxia (FRDA), a sensory neuropathy, is a progressive neurodegenerative disease associated with a loss of large sensory neurons from the dorsal root ganglia. Because a mouse model for this well-characterized disease has been generated, we elected to use FRDA as a model disease. In previous studies we achieved efficient and sustained delivery of a reporter gene to PNS sensory neurons, using recombinant adeno-associated viral (AAV) and lentiviral (LV) vectors. In the current study, AAV and LV vectors encoding the human frataxin cDNA were constructed and assessed for frataxin expression and function in primary FRDA patient fibroblast cell lines. FRDA fibroblasts have been shown to exhibit subtle biochemical changes, including increased mitochondrial iron and sensitivity to oxidant stress. Despite the inherent difficulty in working with primary cells, transduction of patient fibroblasts with either vector resulted in the expression of appropriately localized frataxin and partial reversal of phenotype.

  19. Gauge Theories of Vector Particles

    DOE R&D Accomplishments Database

    Glashow, S. L.; Gell-Mann, M.

    1961-04-24

    The possibility of generalizing the Yang-Mills trick is examined. Thus we seek theories of vector bosons invariant under continuous groups of coordinate-dependent linear transformations. All such theories may be expressed as superpositions of certain "simple" theories; we show that each "simple theory is associated with a simple Lie algebra. We may introduce mass terms for the vector bosons at the price of destroying the gauge-invariance for coordinate-dependent gauge functions. The theories corresponding to three particular simple Lie algebras - those which admit precisely two commuting quantum numbers - are examined in some detail as examples. One of them might play a role in the physics of the strong interactions if there is an underlying super-symmetry, transcending charge independence, that is badly broken. The intermediate vector boson theory of weak interactions is discussed also. The so-called "schizon" model cannot be made to conform to the requirements of partial gauge-invariance.

  20. Extrapolation methods for vector sequences

    NASA Technical Reports Server (NTRS)

    Smith, David A.; Ford, William F.; Sidi, Avram

    1987-01-01

    This paper derives, describes, and compares five extrapolation methods for accelerating convergence of vector sequences or transforming divergent vector sequences to convergent ones. These methods are the scalar epsilon algorithm (SEA), vector epsilon algorithm (VEA), topological epsilon algorithm (TEA), minimal polynomial extrapolation (MPE), and reduced rank extrapolation (RRE). MPE and RRE are first derived and proven to give the exact solution for the right 'essential degree' k. Then, Brezinski's (1975) generalization of the Shanks-Schmidt transform is presented; the generalized form leads from systems of equations to TEA. The necessary connections are then made with SEA and VEA. The algorithms are extended to the nonlinear case by cycling, the error analysis for MPE and VEA is sketched, and the theoretical support for quadratic convergence is discussed. Strategies for practical implementation of the methods are considered.

  1. Helper-Dependent Adenoviral Vectors

    PubMed Central

    Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

    2012-01-01

    Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology. PMID:24533227

  2. Requirements for airborne vector gravimetry

    NASA Technical Reports Server (NTRS)

    Schwarz, K. P.; Colombo, O.; Hein, G.; Knickmeyer, E. T.

    1992-01-01

    The objective of airborne vector gravimetry is the determination of the full gravity disturbance vector along the aircraft trajectory. The paper briefly outlines the concept of this method using a combination of inertial and GPS-satellite data. The accuracy requirements for users in geodesy and solid earth geophysics, oceanography and exploration geophysics are then specified. Using these requirements, accuracy specifications for the GPS subsystem and the INS subsystem are developed. The integration of the subsystems and the problems connected with it are briefly discussed and operational methods are indicated that might reduce some of the stringent accuracy requirements.

  3. Anisotropic inflation from vector impurity

    SciTech Connect

    Kanno, Sugumi; Kimura, Masashi; Soda, Jiro; Yokoyama, Shuichiro E-mail: mkimura@sci.osaka-cu.ac.jp E-mail: shu@a.phys.nagoya-u.ac.jp

    2008-08-15

    We study an inflationary scenario with a vector impurity. We show that the universe undergoes anisotropic inflationary expansion due to a preferred direction determined by the vector. Using the slow roll approximation, we find a formula for determining the anisotropy of the inflationary universe. We discuss possible observable predictions of this scenario. In particular, it is stressed that primordial gravitational waves can be induced from curvature perturbations. Hence, even in low scale inflation, a sizable amount of primordial gravitational waves may be produced during inflation.

  4. Complexity of vector spin glasses.

    PubMed

    Yeo, J; Moore, M A

    2004-08-13

    We study the annealed complexity of the m-vector spin glasses in the Sherrington-Kirkpatrick limit. The eigenvalue spectrum of the Hessian matrix of the Thouless-Anderson-Palmer free energy is found to consist of a continuous band of positive eigenvalues in addition to an isolated eigenvalue and (m-1) null eigenvalues due to rotational invariance. Rather surprisingly, the band does not extend to zero at any finite temperature. The isolated eigenvalue becomes zero in the thermodynamic limit, as in the Ising case (m=1), indicating that the same supersymmetry breaking recently found in Ising spin glasses occurs in vector spin glasses.

  5. Lentiviral Expression of Retinal Guanylate Cyclase-1 (RetGC1) Restores Vision in an Avian Model of Childhood Blindness

    PubMed Central

    Haire, Shannon E; Aleman, Tomas S; Cideciyan, Artur V; Sokal, Izabel; Palczewski, Krzysztof; Jacobson, Samuel G; Semple-Rowland, Susan L

    2006-01-01

    Background Leber congenital amaurosis (LCA) is a genetically heterogeneous group of retinal diseases that cause congenital blindness in infants and children. Mutations in the GUCY2D gene that encodes retinal guanylate cyclase–1 (retGC1) were the first to be linked to this disease group (LCA type 1 [LCA1]) and account for 10%–20% of LCA cases. These mutations disrupt synthesis of cGMP in photoreceptor cells, a key second messenger required for function of these cells. The GUCY1*B chicken, which carries a null mutation in the retGC1 gene, is blind at hatching and serves as an animal model for the study of LCA1 pathology and potential treatments in humans. Methods and Findings A lentivirus-based gene transfer vector carrying the GUCY2D gene was developed and injected into early-stage GUCY1*B embryos to determine if photoreceptor function and sight could be restored to these animals. Like human LCA1, the avian disease shows early-onset blindness, but there is a window of opportunity for intervention. In both diseases there is a period of photoreceptor cell dysfunction that precedes retinal degeneration. Of seven treated animals, six exhibited sight as evidenced by robust optokinetic and volitional visual behaviors. Electroretinographic responses, absent in untreated animals, were partially restored in treated animals. Morphological analyses indicated there was slowing of the retinal degeneration. Conclusions Blindness associated with loss of function of retGC1 in the GUCY1*B avian model of LCA1 can be reversed using viral vector-mediated gene transfer. Furthermore, this reversal can be achieved by restoring function to a relatively low percentage of retinal photoreceptors. These results represent a first step toward development of gene therapies for one of the more common forms of childhood blindness. PMID:16700630

  6. Primer vector theory and applications

    NASA Technical Reports Server (NTRS)

    Jezewski, D. J.

    1975-01-01

    A method developed to compute two-body, optimal, N-impulse trajectories was presented. The necessary conditions established define the gradient structure of the primer vector and its derivative for any set of boundary conditions and any number of impulses. Inequality constraints, a conjugate gradient iterator technique, and the use of a penalty function were also discussed.

  7. Interframe vector wavelet coding technique

    NASA Astrophysics Data System (ADS)

    Wus, John P.; Li, Weiping

    1997-01-01

    Wavelet coding is often used to divide an image into multi- resolution wavelet coefficients which are quantized and coded. By 'vectorizing' scalar wavelet coding and combining this with vector quantization (VQ), vector wavelet coding (VWC) can be implemented. Using a finite number of states, finite-state vector quantization (FSVQ) takes advantage of the similarity between frames by incorporating memory into the video coding system. Lattice VQ eliminates the potential mismatch that could occur using pre-trained VQ codebooks. It also eliminates the need for codebook storage in the VQ process, thereby creating a more robust coding system. Therefore, by using the VWC coding method in conjunction with the FSVQ system and lattice VQ, the formulation of a high quality very low bit rate coding systems is proposed. A coding system using a simple FSVQ system where the current state is determined by the previous channel symbol only is developed. To achieve a higher degree of compression, a tree-like FSVQ system is implemented. The groupings are done in this tree-like structure from the lower subbands to the higher subbands in order to exploit the nature of subband analysis in terms of the parent-child relationship. Class A and Class B video sequences from the MPEG-IV testing evaluations are used in the evaluation of this coding method.

  8. [Vector control, perspectives and realities].

    PubMed

    Carnevale, P

    1995-01-01

    In the WHO Global Strategy for Malaria Control, selective and sustainable vector control is one of the measures to be implemented to complement case management and for the control of epidemics. Vector control can be targeted against larvae and adults, but two elements must be recognized: -vector control measures must be selected according to the existing eco-epidemiological diversity, which has to be well understood before embarking upon any extensive action; -efficient tools are currently available, both for large scale and household use. House spraying is still the method of choice for epidemic control but must be carefully considered and used selectively in endemic countries for various well known reasons. The promotion of personal protection measures for malaria prevention is advocated because insecticide-impregnated mosquito nets and other materials have proved to be effective in different situations. Implementation, sustainability and large scale use of impregnated nets implies a strong community participation supported by well motivated community health workers, the availability of suitable materials (insecticide, mosquito nets), intersectorial collaboration at all levels, well trained health workers from central to the most peripheral level and appropriate educational messages (Knowledge, Attitude and Practices) adapted and elaborated after surveys. It has to be kept in mind that the evaluation of the impact of vector control activities will be made in epidemiological terms such as the reduction of malaria morbidity and mortality.

  9. Hydrogen as an energy vector

    NASA Technical Reports Server (NTRS)

    Powers, W. D.

    1975-01-01

    The feasibility of utilizing hydrogen as an energy vector is considered, with special attention given to means of hydrogen production. The state-of-the-art in thermochemical processes is reviewed, and criteria for the technical and economic feasibility of large-scale thermochemical water splitting processes are presented. The production of hydrogen from coal and from photolysis of water is discussed.

  10. Transcriptomics and disease vector control.

    PubMed

    Vontas, John; Ranson, Hilary; Alphey, Luke

    2010-01-01

    Next-generation sequencing can be used to compare transcriptomes under different conditions. A study in BMC Genomics applies this approach to investigating the effects of exposure to a range of xenobiotics on changes in gene expression in the larvae of Aedes aegypti, the mosquito vector of dengue fever.

  11. Vector ecology of equine piroplasmosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Equine piroplasmosis (EP) is a disease of equidae including horses, donkeys, mules and zebras caused by either of two protozoan parasites, Theileria equi or Babesia caballi. These parasites are biologically transmitted between hosts via tick-vectors and although they have inherent differences, they ...

  12. Portfolio Analysis for Vector Calculus

    ERIC Educational Resources Information Center

    Kaplan, Samuel R.

    2015-01-01

    Classic stock portfolio analysis provides an applied context for Lagrange multipliers that undergraduate students appreciate. Although modern methods of portfolio analysis are beyond the scope of vector calculus, classic methods reinforce the utility of this material. This paper discusses how to introduce classic stock portfolio analysis in a…

  13. Present status of vectorized Monte Carlo

    SciTech Connect

    Brown, F.B.

    1987-01-01

    Monte Carlo applications have traditionally been limited by the large amounts of computer time required to produce acceptably small statistical uncertainties, so the immediate benefit of vectorization is an increase in either the number of jobs completed or the number of particles processed per job, typically by one order of magnitude or more. This results directly in improved engineering design analyses, since Monte Carlo methods are used as standards for correcting more approximate methods. The relatively small number of vectorized programs is a consequence of the newness of vectorized Monte Carlo, the difficulties of nonportability, and the very large development effort required to rewrite or restructure Monte Carlo codes for vectorization. Based on the successful efforts to date, it may be concluded that Monte Carlo vectorization will spread to increasing numbers of codes and applications. The possibility of multitasking provides even further motivation for vectorizing Monte Carlo, since the step from vector to multitasked vector is relatively straightforward.

  14. Symbolic Vector Analysis in Plasma Physics

    SciTech Connect

    Qin, H.; Rewoldt, G.; Tang, W.M.

    1997-10-01

    Many problems in plasma physics involve substantial amounts of analytical vector calculation. The complexity usually originates from both the vector operations themselves and the choice of underlying coordinate system. A computer algebra package for symbolic vector analysis in general coordinate systems, GeneralVectorAnalysis (GVA), is developed using Mathematica. The modern viewpoint for 3D vector calculus, differential forms on 3-manifolds, is adopted to unify and systematize the vector calculus operations in general coordinate systems. This package will benefit physicists and applied mathematicians in their research where complicated vector analysis is required. It will not only save a huge amount of human brain-power and dramatically improve accuracy, but this package will also be an intelligent tool to assist researchers in finding the right approaches to their problems. Several applications of this symbolic vector analysis package to plasma physics are also given.

  15. Symbolic Vector Analysis in Plasma Physics

    SciTech Connect

    Qin, H.; Tang, W.M.; Rewoldt, G.

    1997-10-09

    Many problems in plasma physics involve substantial amounts of analytical vector calculation. The complexity usually originates from both the vector operations themselves and the choice of underlying coordinate system. A computer algebra package for symbolic vector analysis in general coordinate systems, General Vector Analysis (GVA), is developed using Mathematica. The modern viewpoint for 3D vector calculus, differential forms on 3-manifolds, is adopted to unify and systematize the vector calculus operations in general coordinate systems. This package will benefit physicists and applied mathematicians in their research where complicated vector analysis is required. It will not only save a huge amount of human brain-power and dramatically improve accuracy, but this package will also be an intelligent tool to assist researchers in finding the right approaches to their problems. Several applications of this symbolic vector analysis package to plasma physics are also given.

  16. Differential regulation of NF-κB-mediated proviral and antiviral host gene expression by primate lentiviral Nef and Vpu proteins.

    PubMed

    Sauter, Daniel; Hotter, Dominik; Van Driessche, Benoît; Stürzel, Christina M; Kluge, Silvia F; Wildum, Steffen; Yu, Hangxing; Baumann, Bernd; Wirth, Thomas; Plantier, Jean-Christophe; Leoz, Marie; Hahn, Beatrice H; Van Lint, Carine; Kirchhoff, Frank

    2015-02-01

    NF-κB is essential for effective transcription of primate lentiviral genomes and also activates antiviral host genes. Here, we show that the early protein Nef of most primate lentiviruses enhances NF-κB activation. In contrast, the late protein Vpu of HIV-1 and its simian precursors inhibits activation of NF-κB, even in the presence of Nef. Although this effect of Vpu did not correlate with its ability to interact with β-TrCP, it involved the stabilization of IκB and reduced nuclear translocation of p65. Interestingly, however, Vpu did not affect casein kinase II-mediated phosphorylation of p65. Lack of Vpu was associated with increased NF-κB activation and induction of interferon and interferon-stimulated genes (ISGs) in HIV-1-infected T cells. Thus, HIV-1 and its simian precursors employ Nef to boost NF-κB activation early during the viral life cycle to initiate proviral transcription, while Vpu is used to downmodulate NF-κB-dependent expression of ISGs at later stages.

  17. Rho kinase inhibitor Y-27632 prolongs the life span of adult human keratinocytes, enhances skin equivalent development, and facilitates lentiviral transduction.

    PubMed

    van den Bogaard, Ellen H; Rodijk-Olthuis, Diana; Jansen, Patrick A M; van Vlijmen-Willems, Ivonne M J J; van Erp, Piet E; Joosten, Irma; Zeeuwen, Patrick L J M; Schalkwijk, Joost

    2012-09-01

    The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes. PMID:22519508

  18. High-throughput screening using pseudotyped lentiviral particles: a strategy for the identification of HIV-1 inhibitors in a cell-based assay.

    PubMed

    Garcia, Jean-Michel; Gao, Anhui; He, Pei-Lan; Choi, Joyce; Tang, Wei; Bruzzone, Roberto; Schwartz, Olivier; Naya, Hugo; Nan, Fa-Jun; Li, Jia; Altmeyer, Ralf; Zuo, Jian-Ping

    2009-03-01

    Two decades after its discovery the human immunodeficiency virus (HIV) is still spreading worldwide and killing millions. There are 25 drugs formally approved for HIV currently on the market, but side effects as well as the emergence of HIV strains showing single or multiple resistances to current drug-therapy are causes for concern. Furthermore, these drugs target only 4 steps of the viral cycle, hence the urgent need for new drugs and also new targets. In order to tackle this problem, we have devised a cell-based assay using lentiviral particles to look for post-entry inhibitors of HIV-1. We report here the assay development, validation as well as confirmation of the hits using both wild-type and drug-resistant HIV-1 viruses. The screening was performed on an original library, rich in natural compounds and pure molecules from Traditional Chinese Medicine pharmacopoeia, which had never been screened for anti-HIV activity. The identified hits belong to four chemical sub-families that appear to be all non-nucleoside reverse transcriptase inhibitors (NNRTIs). Secondary tests with live viruses showed that there was good agreement with pseudotyped particles, confirming the validity of this approach for high-throughput drug screens. This assay will be a useful tool that can be easily adapted to screen for inhibitors of viral entry.

  19. Vector computer memory bank contention

    NASA Technical Reports Server (NTRS)

    Bailey, D. H.

    1985-01-01

    A number of vector supercomputers feature very large memories. Unfortunately the large capacity memory chips that are used in these computers are much slower than the fast central processing unit (CPU) circuitry. As a result, memory bank reservation times (in CPU ticks) are much longer than on previous generations of computers. A consequence of these long reservation times is that memory bank contention is sharply increased, resulting in significantly lowered performance rates. The phenomenon of memory bank contention in vector computers is analyzed using both a Markov chain model and a Monte Carlo simulation program. The results of this analysis indicate that future generations of supercomputers must either employ much faster memory chips or else feature very large numbers of independent memory banks.

  20. Vector computer memory bank contention

    NASA Technical Reports Server (NTRS)

    Bailey, David H.

    1987-01-01

    A number of vector supercomputers feature very large memories. Unfortunately the large capacity memory chips that are used in these computers are much slower than the fast central processing unit (CPU) circuitry. As a result, memory bank reservation times (in CPU ticks) are much longer than on previous generations of computers. A consequence of these long reservation times is that memory bank contention is sharply increased, resulting in significantly lowered performance rates. The phenomenon of memory bank contention in vector computers is analyzed using both a Markov chain model and a Monte Carlo simulation program. The results of this analysis indicate that future generations of supercomputers must either employ much faster memory chips or else feature very large numbers of independent memory banks.

  1. Medium Modification of Vector Mesons

    SciTech Connect

    Chaden Djalali, Michael Paolone, Dennis Weygand, Michael H. Wood, Rakhsha Nasseripour

    2011-03-01

    The theory of the strong interaction, Quantum Chromodynamics (QCD), has been remarkably successful in describing high-energy and short-distance-scale experiments involving quarks and gluons. However, applying QCD to low energy and large-distance scale experiments has been a major challenge. Various QCD-inspired models predict a partial restoration of chiral symmetry in nuclear matter with modifications of the properties of hadrons from their free-space values. Measurable changes such as a shift in mass and/or a change of width are predicted at normal nuclear density. Photoproduction of vector mesons off nuclei have been performed at different laboratories. The properties of the ρ, ω and φ mesons are investigated either directly by measuring their mass spectra or indirectly through transparency ratios. The latest results regarding medium modifications of the vector mesons in the nuclear medium will be discussed.

  2. Gene targeting with retroviral vectors

    SciTech Connect

    Ellis, J.; Bernstein, A. )

    1989-04-01

    The authors have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418/sup r/ cell per 3 x 10/sup 6/ infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.

  3. Benchmarking the IBM 3090 with Vector Facility

    SciTech Connect

    Brickner, R.G.; Wasserman, H.J.; Hayes, A.H.; Moore, J.W.

    1986-01-01

    The IBM 3090 with Vector Facility is an extremely interesting machine because it combines very good scaler performance with enhanced vector and multitasking performance. For many IBM installations with a large scientific workload, the 3090/vector/MTF combination may be an ideal means of increasing throughput at minimum cost. However, neither the vector nor multitasking capabilities are sufficiently developed to make the 3090 competitive with our current worker machines for our large-scale scientific codes.

  4. Ultrasonic Dynamic Vector Stress Sensor

    NASA Technical Reports Server (NTRS)

    Heyman, Joseph S.; Froggatt, Mark

    1992-01-01

    Stress inferred from measurements in specimens rather than in bonded gauges. Ultrasonic dynamic vector stress sensor (UDVSS) measures changes in dynamic directional stress occurring in material or structure at location touched by device when material or structure put under cyclic load. Includes phase-locked loop, synchronous amplifier, and contact probe. Useful among manufacturers of aerospace and automotive structures for stress testing and evaluation of designs.

  5. GAPS IN SUPPORT VECTOR OPTIMIZATION

    SciTech Connect

    STEINWART, INGO; HUSH, DON; SCOVEL, CLINT; LIST, NICOLAS

    2007-01-29

    We show that the stopping criteria used in many support vector machine (SVM) algorithms working on the dual can be interpreted as primal optimality bounds which in turn are known to be important for the statistical analysis of SVMs. To this end we revisit the duality theory underlying the derivation of the dual and show that in many interesting cases primal optimality bounds are the same as known dual optimality bounds.

  6. Quantum mechanics without state vectors

    NASA Astrophysics Data System (ADS)

    Weinberg, Steven

    2014-10-01

    Because the state vectors of isolated systems can be changed in entangled states by processes in other isolated systems, keeping only the density matrix fixed, it is proposed to give up the description of physical states in terms of ensembles of state vectors with various probabilities, relying only on density matrices. The density matrix is defined here by the formula giving the mean values of physical quantities, which implies the same properties as the usual definition in terms of state vectors and their probabilities. This change in the description of physical states opens up a large variety of new ways that the density matrix may transform under various symmetries, different from the unitary transformations of ordinary quantum mechanics. Such new transformation properties have been explored before, but so far only for the symmetry of time translations into the future, treated as a semigroup. Here, new transformation properties are studied for general symmetry transformations forming groups, not just semigroups. Arguments that such symmetries should act on the density matrix as in ordinary quantum mechanics are presented, but all of these arguments are found to be inconclusive.

  7. MISR Level 3 Cloud Motion Vector

    Atmospheric Science Data Center

    2013-07-10

    MISR Level 3 Cloud Motion Vector Level 3 Wednesday, November 7, 2012 ... A new version, F02_0002, of the MISR L3 CMV (Cloud Motion Vector) data product is now available. This new release provides finer ... coverage. These enhancements are the result of reorganizing motion vector information present in the recent Level 2 Cloud product as ...

  8. Visualizing vector field topology in fluid flows

    NASA Technical Reports Server (NTRS)

    Helman, James L.; Hesselink, Lambertus

    1991-01-01

    Methods of automating the analysis and display of vector field topology in general and flow topology in particular are discussed. Two-dimensional vector field topology is reviewed as the basis for the examination of topology in three-dimensional separated flows. The use of tangent surfaces and clipping in visualizing vector field topology in fluid flows is addressed.

  9. A drop theorem without vector topology

    NASA Astrophysics Data System (ADS)

    Wong, Chi-Wing

    2007-05-01

    Danes' drop theorem is extended to bornological vector spaces. An immediate application is to establish Ekeland-type variational principle and its equivalence, Caristi fixed point theorem, in bornological vector spaces. Meanwhile, since every locally convex space becomes a convex bornological vector space when equipped with the canonical von Neumann bornology, Qiu's generalization of Danes' work to locally convex spaces is recovered.

  10. Purification and Injection of Retroviral Vectors.

    PubMed

    Tashiro, Ayumu; Zhao, Chunmei; Suh, Hoonkyo; Gage, Fred H

    2015-10-01

    Retroviral vectors are powerful tools for genetic manipulation. This protocol discusses the production, purification, and use of replication-deficient retroviral vectors based on Moloney murine leukemia virus and lentivirus. It also describes the injection of a retroviral vector into the dentate gyrus of young adult mice to fluorescently label live murine brain tissue.

  11. Problems with the Method of Correlated Vectors

    ERIC Educational Resources Information Center

    Ashton, M.C.; Lee, K.

    2005-01-01

    The method of correlated vectors has been used widely to identify variables that are associated with general intelligence (g). Briefly, this method involves finding the correlation between the vector of intelligence subtests' g-loadings and the vector of those subtests' correlations with the variable in question. We describe two major problems…

  12. Utilizing a TLR5-Adjuvanted Cytomegalovirus as a Lentiviral Vaccine in the Nonhuman Primate Model for AIDS

    PubMed Central

    Deere, Jesse D.; Chang, W. L. William; Castillo, Luis D.; Schmidt, Kim A.; Kieu, Hung T.; Renzette, Nicholas; Kowalik, Timothy; Barthold, Stephen W.; Shacklett, Barbara L.; Barry, Peter A.; Sparger, Ellen E.

    2016-01-01

    Despite tremendous progress in our understanding of human immunodeficiency virus (HIV) natural history and advances in HIV treatment, there is neither an approved vaccine nor a cure for infection. Here, we describe the development and characterization of a novel replicating vaccine vector utilizing Cytomegalovirus (CMV) and a TLR5 adjuvant. After partial truncation of the central, immunodominant hypervariable domain, flagellin (fliC) from Salmonella was cloned downstream of a codon optimized gag gene from simian immunodeficiency virus (SIV) and transiently expressed in telomerized rhesus fibroblast (TeloRF) cells in culture. Lysates generated from these transfected cells induced the tumor necrosis factor alpha (TNF-α), in a mouse macrophage cell line, in a TLR5-dependent manner. The Gag/FliC expression construct was cloned into a bacterial artificial chromosome encoding the rhesus CMV (RhCMV) genome, and infectious RhCMV was generated following transfection of TeloRF cells. This virus stably expressed an SIV Gag/FliC fusion protein through four serial passages. Lysates generated from infected cells induced TNF-α in a TLR5-dependent manner. Western blot analysis of infected cell lysates verified expression of a Gag/FliC fusion protein using a SIV p27 capsid monoclonal antibody. Lastly, rhesus macaques inoculated with this novel RhCMV virus demonstrated increased inflammatory responses at the site of inoculation seven days post-infection when compared to the parental RhCMV. These results demonstrate that an artificially constructed replicating RhCMV expressing an SIV Gag/FliC fusion protein is capable of activating TLR5 in a macrophage cell line in vitro and induction of an altered inflammatory response in vivo. Ongoing animals studies are aimed at determining vaccine efficacy, including subsequent challenge with pathogenic SIV. PMID:27182601

  13. Gaussian statistics for palaeomagnetic vectors

    USGS Publications Warehouse

    Love, J.J.; Constable, C.G.

    2003-01-01

    With the aim of treating the statistics of palaeomagnetic directions and intensities jointly and consistently, we represent the mean and the variance of palaeomagnetic vectors, at a particular site and of a particular polarity, by a probability density function in a Cartesian three-space of orthogonal magnetic-field components consisting of a single (unimoda) non-zero mean, spherically-symmetrical (isotropic) Gaussian function. For palaeomagnetic data of mixed polarities, we consider a bimodal distribution consisting of a pair of such symmetrical Gaussian functions, with equal, but opposite, means and equal variances. For both the Gaussian and bi-Gaussian distributions, and in the spherical three-space of intensity, inclination, and declination, we obtain analytical expressions for the marginal density functions, the cumulative distributions, and the expected values and variances for each spherical coordinate (including the angle with respect to the axis of symmetry of the distributions). The mathematical expressions for the intensity and off-axis angle are closed-form and especially manageable, with the intensity distribution being Rayleigh-Rician. In the limit of small relative vectorial dispersion, the Gaussian (bi-Gaussian) directional distribution approaches a Fisher (Bingham) distribution and the intensity distribution approaches a normal distribution. In the opposite limit of large relative vectorial dispersion, the directional distributions approach a spherically-uniform distribution and the intensity distribution approaches a Maxwell distribution. We quantify biases in estimating the properties of the vector field resulting from the use of simple arithmetic averages, such as estimates of the intensity or the inclination of the mean vector, or the variances of these quantities. With the statistical framework developed here and using the maximum-likelihood method, which gives unbiased estimates in the limit of large data numbers, we demonstrate how to

  14. Comparative investigation of multiplane thrust vectoring nozzles

    NASA Technical Reports Server (NTRS)

    Capone, F.; Smereczniak, P.; Spetnagel, D.; Thayer, E.

    1992-01-01

    The inflight aerodynamic performance of multiplane vectoring nozzles is critical to development of advanced aircraft and flight control systems utilizing thrust vectoring. To investigate vectoring nozzle performance, subscale models of two second-generation thrust vectoring nozzle concepts currently under development for advanced fighters were integrated into an axisymmetric test pod. Installed drag and vectoring performance characteristics of both concepts were experimentally determined in wind tunnel testing. CFD analyses were conducted to understand the impact of internal flow turning on thrust vectoring characteristics. Both nozzles exhibited drag comparable with current nonvectoring axisymmetric nozzles. During vectored-thrust operations, forces produced by external flow effects amounted to about 25 percent of the total force measured.

  15. Nonviral Vectors for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Baoum, Abdulgader Ahmed

    2011-12-01

    The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,L-lactide- co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (˜200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium. On the other hand, a more simple method to synthesize 50-200 nm complexes capable of high transfection efficiency or high gene knockdown was

  16. Quantum Electrodynamics for Vector Mesons

    SciTech Connect

    Djukanovic, Dalibor; Schindler, Matthias R.; Scherer, Stefan; Gegelia, Jambul

    2005-07-01

    Quantum electrodynamics for {rho} mesons is considered. It is shown that, at the tree level, the value of the gyromagnetic ratio of the {rho}{sup +} is fixed to 2 in a self-consistent effective quantum field theory. Further, the mixing parameter of the photon and the neutral vector meson is equal to the ratio of electromagnetic and strong couplings, leading to the mass difference M{sub {rho}}{sub {sup 0}}-M{sub {rho}}{sub {sup {+-}}}{approx}1 MeV at tree order.

  17. Jet vectoring through nozzle asymmetry

    NASA Astrophysics Data System (ADS)

    Roh, Chris; Rosakis, Alexandros; Gharib, Morteza

    2015-11-01

    Previously, we explored the functionality of a tri-leaflet anal valve of a dragonfly larva. We saw that the dragonfly larva is capable of controlling the three leaflets independently to asymmetrically open the nozzle. Such control resulted in vectoring of the jet in various directions. To further understand the effect of asymmetric nozzle orifice, we tested jet flow through circular asymmetric nozzles. We report the relationship between nozzle asymmetry and redirecting of the jet at various Reynolds numbers. This material is based upon work supported by the National Science Foundation under Grant No. CBET-1511414; additional support by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1144469.

  18. Properties of Vector Preisach Models

    NASA Technical Reports Server (NTRS)

    Kahler, Gary R.; Patel, Umesh D.; Torre, Edward Della

    2004-01-01

    This paper discusses rotational anisotropy and rotational accommodation of magnetic particle tape. These effects have a performance impact during the reading and writing of the recording process. We introduce the reduced vector model as the basis for the computations. Rotational magnetization models must accurately compute the anisotropic characteristics of ellipsoidally magnetizable media. An ellipticity factor is derived for these media that computes the two-dimensional magnetization trajectory for all applied fields. An orientation correction must be applied to the computed rotational magnetization. For isotropic materials, an orientation correction has been developed and presented. For anisotropic materials, an orientation correction is introduced.

  19. Vector spaces, invariance, and camouflage

    NASA Astrophysics Data System (ADS)

    Arsenault, Henri H.; Garcia-Martinez, Pascuala

    2004-12-01

    We present a method based on an orthonormal vector space basis representation to detect camouflaged targets in natural environments. Because the method is intensity invariant we detect camouflage targets independently of the illumination conditions. The detection technique does not require knowing the exact camouflage pattern, but only the class of patterns (foliage, netting, woods...). We used nonlinear filtering based on the calculation of several correlations. Moreover, the nonlinearity of the filtering process allows a high discrimination against false targets. Several experiments confirm the target detectability where strong camouflage might delude even human viewers.

  20. Enhancement of the antigen-specific cytotoxic T lymphocyte-inducing ability in the PMDC11 leukemic plasmacytoid dendritic cell line via lentiviral vector-mediated transduction of the caTLR4 gene.

    PubMed

    Iwabuchi, Minami; Narita, Miwako; Uchiyama, Takayoshi; Iwaya, Shunpei; Oiwa, Eri; Nishizawa, Yoshinori; Hashimoto, Shigeo; Bonehill, Aude; Kasahara, Noriyuki; Takizawa, Jun; Takahashi, Masuhiro

    2015-08-01

    The aim of the present study was to enhance the efficiency of leukemia immunotherapy by increasing the antigen-specific cytotoxic T lymphocyte-inducing ability of leukemia cells. The leukemic plasmacytoid dendritic cell line PMDC05 containing the HLA-A02/24 antigen, which was previously established in our laboratory (Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Japan), was used in the present study. It exhibited higher expression levels of CD80 following transduction with lentiviruses encoding the CD80 gene. This CD80-expressing PMDC05 was named PMDC11. In order to establish a more potent antigen-presenting cell for cellular immunotherapy of tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) toll-like receptor 4 (TLR4) gene using the Tet-On system (caTLR4-PMDC11). CD8(+) T cells from healthy donors with HLA-A02 were co-cultured with mutant WT1 peptide-pulsed PMDC11, lipopolysaccharide (LPS)-stimulated PMDC11 or caTLR4-PMDC11 cells. Interleukin (IL)-2 (50 IU/ml) and IL-7 (10 ng/ml) were added on day three of culture. Priming with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 cells was conducted once per week and two thirds of the IL-2/IL-7 containing medium was replenished every 3-4 days. Immediately prior to the priming with these various PMDC11 cells, the cultured cells were analyzed for the secretion of interferon (IFN)-γ in addition to the percentage and number of CD8(+)/WT1 tetramer(+) T cells using flow cytometry. caTLR4-PMDC11 cells were observed to possess greater antigen-presenting abilities compared with those of PMDC11 or LPS-stimulated PMDC11 cells in a mixed leukocyte culture. CD8 T cells positive for the WT1 tetramer were generated following 3-4 weeks of culture and CD8(+)/WT1 tetramer+ T cells were markedly increased in caTLR4-PMDC11-primed CD8(+) T cell culture compared with PMDC11 or LPS-stimulated PMDC11-primed CD8(+) T cell culture. These CD8(+) T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to secrete IFN-γ and to be cytotoxic to WT1-expressing target cells. These data suggested that the antigen-specific cytotoxic T lymphocyte (CTL)-inducing ability of PMDC11 was potentiated via transduction of the caTLR4 gene. The present study also suggested that caTLR4-PMDC11 cells may be applied as potent antigen-presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infections.

  1. Enhancement of the antigen-specific cytotoxic T lymphocyte-inducing ability in the PMDC11 leukemic plasmacytoid dendritic cell line via lentiviral vector-mediated transduction of the caTLR4 gene

    PubMed Central

    IWABUCHI, MINAMI; NARITA, MIWAKO; UCHIYAMA, TAKAYOSHI; IWAYA, SHUNPEI; OIWA, ERI; NISHIZAWA, YOSHINORI; HASHIMOTO, SHIGEO; BONEHILL, AUDE; KASAHARA, NORIYUKI; TAKIZAWA, JUN; TAKAHASHI, MASUHIRO

    2015-01-01

    The aim of the present study was to enhance the efficiency of leukemia immunotherapy by increasing the antigen-specific cytotoxic T lymphocyte-inducing ability of leukemia cells. The leukemic plasmacytoid dendritic cell line PMDC05 containing the HLA-A02/24 antigen, which was previously established in our laboratory (Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Japan), was used in the present study. It exhibited higher expression levels of CD80 following transduction with lentiviruses encoding the CD80 gene. This CD80-expressing PMDC05 was named PMDC11. In order to establish a more potent antigen-presenting cell for cellular immunotherapy of tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) toll-like receptor 4 (TLR4) gene using the Tet-On system (caTLR4-PMDC11). CD8+ T cells from healthy donors with HLA-A02 were co-cultured with mutant WT1 peptide-pulsed PMDC11, lipopolysaccharide (LPS)-stimulated PMDC11 or caTLR4-PMDC11 cells. Interleukin (IL)-2 (50 IU/ml) and IL-7 (10 ng/ml) were added on day three of culture. Priming with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 cells was conducted once per week and two thirds of the IL-2/IL-7 containing medium was replenished every 3–4 days. Immediately prior to the priming with these various PMDC11 cells, the cultured cells were analyzed for the secretion of interferon (IFN)-γ in addition to the percentage and number of CD8+/WT1 tetramer+ T cells using flow cytometry. caTLR4-PMDC11 cells were observed to possess greater antigen-presenting abilities compared with those of PMDC11 or LPS-stimulated PMDC11 cells in a mixed leukocyte culture. CD8 T cells positive for the WT1 tetramer were generated following 3–4 weeks of culture and CD8+/WT1 tetramer+ T cells were markedly increased in caTLR4-PMDC11-primed CD8+ T cell culture compared with PMDC11 or LPS-stimulated PMDC11-primed CD8+ T cell culture. These CD8+ T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to secrete IFN-γ and to be cytotoxic to WT1-expressing target cells. These data suggested that the antigen-specific cytotoxic T lymphocyte (CTL)-inducing ability of PMDC11 was potentiated via transduction of the caTLR4 gene. The present study also suggested that caTLR4-PMDC11 cells may be applied as potent antigen-presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infections. PMID:25936433

  2. Volumetric Acoustic Vector Intensity Probe

    NASA Technical Reports Server (NTRS)

    Klos, Jacob

    2006-01-01

    A new measurement tool capable of imaging the acoustic intensity vector throughout a large volume is discussed. This tool consists of an array of fifty microphones that form a spherical surface of radius 0.2m. A simultaneous measurement of the pressure field across all the microphones provides time-domain near-field holograms. Near-field acoustical holography is used to convert the measured pressure into a volumetric vector intensity field as a function of frequency on a grid of points ranging from the center of the spherical surface to a radius of 0.4m. The volumetric intensity is displayed on three-dimensional plots that are used to locate noise sources outside the volume. There is no restriction on the type of noise source that can be studied. The sphere is mobile and can be moved from location to location to hunt for unidentified noise sources. An experiment inside a Boeing 757 aircraft in flight successfully tested the ability of the array to locate low-noise-excited sources on the fuselage. Reference transducers located on suspected noise source locations can also be used to increase the ability of this device to separate and identify multiple noise sources at a given frequency by using the theory of partial field decomposition. The frequency range of operation is 0 to 1400Hz. This device is ideal for the study of noise sources in commercial and military transportation vehicles in air, on land and underwater.

  3. Vector computer memory bank contention

    SciTech Connect

    Bailey, D.H.

    1987-03-01

    A number of recent vector supercomputer designs have featured main memories with very large capacities, and presumably even larger memories are planned for future generations. While the memory chips used in these computers can store much larger amounts of data than before, their operation speeds are rather slow when compared to the significantly faster CPU (central processing unit) circuitry in new supercomputer designs. A consequence of this speed disparity between CPU's and main memory is that memory access times and memory bank reservation times (as measured in CPU ticks) are sharply increased from previous generations. While it has been recognized that these longer memory operation times will reduce scalar performance, it has not been generally realized that vector performance could suffer as well, due to a sharp increase in memory bank contention. This paper examines this phenomenon using both a Markov chain mathematical model and a Monte Carlo simulation program. The potential for performance reduction is described and techniques for ameliorating this reduction are proposed.

  4. Genetics of Mosquito Vector Competence

    PubMed Central

    Beerntsen, Brenda T.; James, Anthony A.; Christensen, Bruce M.

    2000-01-01

    Mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. Efforts to control mosquito-borne diseases have been impeded, in part, by the development of drug-resistant parasites, insecticide-resistant mosquitoes, and environmental concerns over the application of insecticides. Therefore, there is a need to develop novel disease control strategies that can complement or replace existing control methods. One such strategy is to generate pathogen-resistant mosquitoes from those that are susceptible. To this end, efforts have focused on isolating and characterizing genes that influence mosquito vector competence. It has been known for over 70 years that there is a genetic basis for the susceptibility of mosquitoes to parasites, but until the advent of powerful molecular biological tools and protocols, it was difficult to assess the interactions of pathogens with their host tissues within the mosquito at a molecular level. Moreover, it has been only recently that the molecular mechanisms responsible for pathogen destruction, such as melanotic encapsulation and immune peptide production, have been investigated. The molecular characterization of genes that influence vector competence is becoming routine, and with the development of the Sindbis virus transducing system, potential antipathogen genes now can be introduced into the mosquito and their effect on parasite development can be assessed in vivo. With the recent successes in the field of mosquito germ line transformation, it seems likely that the generation of a pathogen-resistant mosquito population from a susceptible population soon will become a reality. PMID:10704476

  5. Attenuated Vesicular Stomatitis Viruses as Vaccine Vectors

    PubMed Central

    Roberts, Anjeanette; Buonocore, Linda; Price, Ryan; Forman, John; Rose, John K.

    1999-01-01

    We showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (VSV) expressing an influenza virus hemagglutinin (HA) protein provided complete protection from lethal challenge with influenza virus (A. Roberts, E. Kretzschmar, A. S. Perkins, J. Forman, R. Price, L. Buonocore, Y. Kawaoka, and J. K. Rose, J. Virol. 72:4704–4711, 1998). Because some pathogenesis was associated with the vector itself, in the present study we generated new VSV vectors expressing HA which are completely attenuated for pathogenesis in the mouse model. The first vector has a truncation of the cytoplasmic domain of the VSV G protein and expresses influenza virus HA (CT1-HA). This nonpathogenic vector provides complete protection from lethal influenza virus challenge after intranasal administration. A second vector with VSV G deleted and expressing HA (ΔG-HA) is also protective and nonpathogenic and has the advantage of not inducing neutralizing antibodies to the vector itself. PMID:10196265

  6. Gateway vectors for transformation of cereals.

    PubMed

    Karimi, Mansour; Inzé, Dirk; Van Lijsebettens, Mieke; Hilson, Pierre

    2013-01-01

    Until now, the availability of vectors for transgenic research in cereal crops has been rather limited. We present a novel collection of Agrobacterium tumefaciens binary T-DNA vectors compatible with Gateway recombinational cloning that facilitate the modular assembly of genes of interest together with new regulatory sequences, such as strong constitutive or endosperm-specific Brachypodium distachyon promoters. This resource aims at streamlining the creation of vectors and transgenes designed to explore gene functions in vital monocotyledonous crops.

  7. (New hosts and vectors for genome cloning)

    SciTech Connect

    Not Available

    1991-02-28

    The main goal of our project has been to develop new bacterial hosts and vectors for cloning human DNA in the bacterium E. coli. Because human DNA is both A+T-rich and highly repetitive, many human sequences are unstable as inserts in vectors that are propagated in bacteria. During the past eight months, we have (1) assessed what fraction of human DNA inserts in cosmid vectors is unstable, (2) developed new host strains that help stabilize unstable sequence features of human DNA, and (3) begun the development of a new generation of cloning vectors that should permit the more stable maintenance and more facile analysis of large human DNA inserts.

  8. Bacteriophage lambda-based expression vectors.

    PubMed

    Christensen, A C

    2001-03-01

    Bacteriophage lambda has been in use as a cloning vector for over 25 years, and has been used extensively as an expression vector. The efficiency of packaging and infection, and the simplicity of plaque screening are advantages of lambda as a cloning vector. A number of ingenious modifications help overcome the disadvantages associated with its mode of growth and its size. Some lambda vectors have been designed to be readily converted into plasmids or phagemids, and there are a variety of promoters and fusions that can be used to drive expression of foreign genes. Screening lambda libraries with antibodies or ligands is a powerful way of identifying novel genes. PMID:11434310

  9. Building mosaics of therapeutic plasmid gene vectors.

    PubMed

    Tolmachov, Oleg E

    2011-12-01

    Plasmids are circular or linear DNA molecules propagated extra-chromosomally in bacteria. Evolution shaped plasmids are inherently mosaic structures with individual functional units represented by distinct segments in the plasmid genome. The patchwork of plasmid genetic modules is a convenient template and a model for the generation of artificial plasmids used as vehicles for gene delivery into human cells. Plasmid gene vectors are an important tool in gene therapy and in basic biomedical research, where these vectors offer efficient transgene expression in many settings in vitro and in vivo. Plasmid vectors can be attached to nuclear directing ligands or transferred by electroporation as naked DNA to deliver the payload genes to the nuclei of the target cells. Transgene expression silencing by plasmid sequences of bacterial origin and immune stimulation by bacterial unmethylated CpG motifs can be avoided by the generation of plasmid-based minimized DNA vectors, such as minicircles. Systems of efficient site-specific integration into human chromosomes and stable episomal maintenance in human cells are being developed for further reduction of the chances for transgene silencing. The successful generation of plasmid vectors is governed by a number of vector design rules, some of which are common to all gene vectors, while others are specific to plasmid vectors. This review is focused both on the guiding principles and on the technical know-how of plasmid gene vector design. PMID:22023476

  10. Vector wind profile gust model

    NASA Technical Reports Server (NTRS)

    Adelfang, S. I.

    1981-01-01

    To enable development of a vector wind gust model suitable for orbital flight test operations and trade studies, hypotheses concerning the distributions of gust component variables were verified. Methods for verification of hypotheses that observed gust variables, including gust component magnitude, gust length, u range, and L range, are gamma distributed and presented. Observed gust modulus has been drawn from a bivariate gamma distribution that can be approximated with a Weibull distribution. Zonal and meridional gust components are bivariate gamma distributed. An analytical method for testing for bivariate gamma distributed variables is presented. Two distributions for gust modulus are described and the results of extensive hypothesis testing of one of the distributions are presented. The validity of the gamma distribution for representation of gust component variables is established.

  11. High power thrust vector actuation

    NASA Astrophysics Data System (ADS)

    Kittock, M. J.

    1993-06-01

    Modern missile programs are frequently favoring electro-mechanical (EM) thrust vector actuation (TVA) over hydraulic for a variety of reasons. However, actuation system performance requirements are not relaxed for EM systems. Thus the development of EM systems with greater power output is required. The configuration of EM actuator studied consists of a DC brushless motor driving a spur gear train, which drives a ballscrew that converts rotary motion to rectilinear motion. This design produces an actuator with high levels of performance in a compact mechanical package. Design for manufacturability and assembly (DFMA) was part of the design process, resulting in an actuator that can be assembled easily and will operate reliably. This paper will discuss the mechanical details of the resultant actuator and report test results on a prototype derivative.

  12. Vector wind profile gust model

    NASA Technical Reports Server (NTRS)

    Adelfang, S. I.

    1979-01-01

    Work towards establishing a vector wind profile gust model for the Space Transportation System flight operations and trade studies is reported. To date, all the statistical and computational techniques required were established and partially implemented. An analysis of wind profile gust at Cape Kennedy within the theoretical framework is presented. The variability of theoretical and observed gust magnitude with filter type, altitude, and season is described. Various examples are presented which illustrate agreement between theoretical and observed gust percentiles. The preliminary analysis of the gust data indicates a strong variability with altitude, season, and wavelength regime. An extension of the analyses to include conditional distributions of gust magnitude given gust length, distributions of gust modulus, and phase differences between gust components has begun.

  13. Slow deterministic vector rogue waves

    NASA Astrophysics Data System (ADS)

    Sergeyev, S. V.; Kolpakov, S. A.; Mou, Ch.; Jacobsen, G.; Popov, S.; Kalashnikov, V.

    2016-03-01

    For an erbium-doped fiber laser mode-locked by carbon nanotubes, we demonstrate experimentally and theoretically a new type of the vector rogue waves emerging as a result of the chaotic evolution of the trajectories between two orthogonal states of polarization on the Poincare sphere. In terms of fluctuation induced phenomena, by tuning polarization controller for the pump wave and in-cavity polarization controller, we are able to control the Kramers time, i.e. the residence time of the trajectory in vicinity of each orthogonal state of polarization, and so can cause the rare events satisfying rogue wave criteria and having the form of transitions from the state with the long residence time to the state with a short residence time.

  14. Introduction to Vector Field Visualization

    NASA Technical Reports Server (NTRS)

    Kao, David; Shen, Han-Wei

    2010-01-01

    Vector field visualization techniques are essential to help us understand the complex dynamics of flow fields. These can be found in a wide range of applications such as study of flows around an aircraft, the blood flow in our heart chambers, ocean circulation models, and severe weather predictions. The vector fields from these various applications can be visually depicted using a number of techniques such as particle traces and advecting textures. In this tutorial, we present several fundamental algorithms in flow visualization including particle integration, particle tracking in time-dependent flows, and seeding strategies. For flows near surfaces, a wide variety of synthetic texture-based algorithms have been developed to depict near-body flow features. The most common approach is based on the Line Integral Convolution (LIC) algorithm. There also exist extensions of LIC to support more flexible texture generations for 3D flow data. This tutorial reviews these algorithms. Tensor fields are found in several real-world applications and also require the aid of visualization to help users understand their data sets. Examples where one can find tensor fields include mechanics to see how material respond to external forces, civil engineering and geomechanics of roads and bridges, and the study of neural pathway via diffusion tensor imaging. This tutorial will provide an overview of the different tensor field visualization techniques, discuss basic tensor decompositions, and go into detail on glyph based methods, deformation based methods, and streamline based methods. Practical examples will be used when presenting the methods; and applications from some case studies will be used as part of the motivation.

  15. Vector Addition: Effect of the Context and Position of the Vectors

    NASA Astrophysics Data System (ADS)

    Barniol, Pablo; Zavala, Genaro

    2010-10-01

    In this article we investigate the effect of: 1) the context, and 2) the position of the vectors, on 2D vector addition tasks. We administered a test to 512 students completing introductory physics courses at a private Mexican university. In the first part, we analyze students' responses in three isomorphic problems: displacements, forces, and no physical context. Students were asked to draw two vectors and the vector sum. We analyzed students' procedures detecting the difficulties when drawing the vector addition and proved that the context matters, not only compared to the context-free case but also between the contexts. In the second part, we analyze students' responses with three different arrangements of the sum of two vectors: tail-to-tail, head-to-tail and separated vectors. We compared the frequencies of the errors in the three different positions to deduce students' conceptions in the addition of vectors.

  16. Promoter interference mediated by the U3 region in early-generation HIV-1-derived lentivirus vectors can influence detection of transgene expression in a cell-type and species-specific manner.

    PubMed

    Ginn, Samantha L; Fleming, Jane; Rowe, Peter B; Alexander, Ian E

    2003-08-10

    In a previous study using an early-generation VSV-G-pseudotyped lentivirus vector encoding enhanced green fluorescent protein (EGFP) under the transcriptional control of a human cytomegalovirus (CMV) immediate-early promoter, we examined transduction efficiency in dissociated dorsal root ganglia (DRG) cultures. In cultures of murine origin, transgene expression was observed solely in the sensory neurons with the stromal cell population failing to show evidence of transduction. In contrast, efficient and sustained transduction of both sensory neurons and the stromal cell population was observed in cultures of human origin. Given the widespread use of murine models in preclinical gene therapy studies, in the current study we investigated the basis of this apparent neuron specificity of lentivirus-mediated transduction in murine DRG cultures. The interspecies differences persisted at high multiplicities of infection, and irrespective of whether lentiviral vector stocks were packaged in the presence or absence of human immunodeficiency vir