Science.gov

Sample records for lentiviral vector-mediated transduction

  1. 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

  2. Prospects for Lentiviral Vector Mediated Prostaglandin F Synthase Gene Delivery in Monkey Eyes In vivo

    PubMed Central

    Lee, Eun Suk; Rasmussen, Carol A.; Filla, Mark S.; Slauson, Sarah R.; Kolb, Aaron W.; Peters, Donna M.; Kaufman, Paul L.; Gabelt, B’Ann True; Brandt, Curtis R.

    2014-01-01

    Currently, the most effective outflow drugs approved for clinical use are prostaglandin F2α analogues, but these require daily topical self-dosing and have various intraocular, ocular surface and extraocular side effects. Lentiviral vector-mediated delivery of the prostaglandin F synthase (PGFS) gene, resulting in long-term reduction of IOP, may eliminate off-target tissue effects and the need for daily topical PGF2α self-administration. Lentiviral vector-mediated delivery of the PGFS gene to the anterior segment has been achieved in cats and non-human primates. Although these results are encouraging, our studies have identified a number of challenges that need to be overcome for prostaglandin gene therapy to be translated into the clinic. Using examples from our work in non-human primates, where we were able to achieve a significant reduction in IOP (2 mm Hg) for 5 months after delivery of the cDNA for bovine PGF synthase, we identify and discuss these issues and consider several possible solutions. PMID:24559478

  3. 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

  4. 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.

  5. Cytotoxicity Associated with Artemis Overexpression After Lentiviral Vector-Mediated Gene Transfer

    PubMed Central

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

    2010-01-01

    Abstract 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-1α 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

  6. 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

  7. 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.

  8. 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

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

    PubMed

    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

    2015-09-01

    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. 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. 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. 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 CD21(low) B cells and a reduction in B cell-activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients. 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 improvement in patients with WAS. Copyright

  10. Correction of murine Rag1 deficiency by self-inactivating lentiviral vector-mediated gene transfer.

    PubMed

    Pike-Overzet, K; Rodijk, M; Ng, Y-Y; Baert, M R M; Lagresle-Peyrou, C; Schambach, A; Zhang, F; Hoeben, R C; Hacein-Bey-Abina, S; Lankester, A C; Bredius, R G M; Driessen, G J A; Thrasher, A J; Baum, C; Cavazzana-Calvo, M; van Dongen, J J M; Staal, F J T

    2011-09-01

    Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1-/- mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR Vβ gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B- as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1-/- mice using lentiviral SIN vectors.

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

    PubMed

    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.

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

    PubMed

    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

    2016-09-01

    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 (CD18(HYP)), both in vitro and in vivo after transplantation of corrected cells into primary and secondary CD18(HYP) recipients. hCD18(+) hematopoietic cells from transplanted CD18(HYP) mice also showed restoration of mCD11a surface co-expression. The analysis of in vivo neutrophil migration in CD18(HYP) 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.

  13. 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

  14. Preclinical demonstration of lentiviral vector-mediated correction of immunological and metabolic abnormalities in models of adenosine deaminase deficiency.

    PubMed

    Carbonaro, Denise A; Zhang, Lin; Jin, Xiangyang; Montiel-Equihua, Claudia; Geiger, Sabine; Carmo, Marlene; Cooper, Aaron; Fairbanks, Lynette; Kaufman, Michael L; Sebire, Neil J; Hollis, Roger P; Blundell, Michael P; Senadheera, Shantha; Fu, Pei-Yu; Sahaghian, Arineh; Chan, Rebecca Y; Wang, Xiaoyan; Cornetta, Kenneth; Thrasher, Adrian J; Kohn, Donald B; Gaspar, H Bobby

    2014-03-01

    Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA(-/-) mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA(-/-) mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34(+) cells transduced with 1-5 × 10(7) TU/ml had 1-3 vector copies/cell and expressed 1-2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis.

  15. Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cells.

    PubMed

    Wang, Yebo; Wang, Yingjia; Chang, Tammy; Huang, He; Yee, Jiing-Kuan

    2016-11-28

    Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs.

  16. Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cells

    PubMed Central

    Wang, Yebo; Wang, Yingjia; Chang, Tammy

    2017-01-01

    Abstract Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs. PMID:27899664

  17. Lysophosphatidylcholine as an adjuvant for lentiviral vector mediated gene transfer to airway epithelium: effect of acyl chain length

    PubMed Central

    2010-01-01

    Background Poor gene transfer efficiency has been a major problem in developing an effective gene therapy for cystic fibrosis (CF) airway disease. Lysophosphatidylcholine (LPC), a natural airway surfactant, can enhance viral gene transfer in animal models. We examined the electrophysiological and physical effect of airway pre-treatment with variants of LPC on lentiviral (LV) vector gene transfer efficiency in murine nasal airways in vivo. Methods Gene transfer was assessed after 1 week following nasal instillations of a VSV-G pseudotype LV vector pre-treated with a low and high dose of LPC variants. The electrophysiological effects of a range of LPC variants were assessed by nasal transepithelial potential difference measurements (TPD) to determine tight junction permeability. Any physical changes to the epithelium from administration of the LPC variants were noted by histological methods in airway tissue harvested after 1 hour. Results Gene transduction was significantly greater compared to control (PBS) for our standard LPC (palmitoyl/stearoyl mixture) treatment and for the majority of the other LPC variants with longer acyl chain lengths. The LPC variant heptadecanoyl also produced significantly greater LV gene transfer compared to our standard LPC mixture. LV gene transfer and the transepithelial depolarization produced by the 0.1% LPC variants at 1 hour were strongly correlated (r2 = 0.94), but at the 1% concentration the correlation was less strong (r2 = 0.59). LPC variants that displayed minor to moderate levels of disruption to the airway epithelium were clearly associated with higher LV gene transfer. Conclusions These findings show the LPC variants effect on airway barrier function and their correlation to the effectiveness of gene expression. The enhanced expression produced by a number of LPC variants should provide new options for preclinical development of efficient airway gene transfer techniques. PMID:20569421

  18. 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.

  19. Lentiviral vector-mediated RNA interference targeted against prohibitin inhibits apoptosis of the retinoic acid-resistant acute promyelocytic leukemia cell line NB4-R1.

    PubMed

    Liu, Yanfeng; He, Pengcheng; Zhang, Mei; Wu, Di

    2012-12-01

    To investigate the possibility of prohibitin (PHB) inhibition by lentiviral vector-mediated RNA interference (RNAi) and its influence on cell apoptosis in the retinoic acid-resistant acute promyelocytic leukemia cell line NB4-R1, a lentiviral vector encoding a short hairpin RNA (shRNA) targeted against PHB (pGCSIL-GFP-PHB) was constructed and transfected into the packaging cells 293T, and the viral supernatant was collected to transfect NB4-R1 cells. Quantitative real-time fluorescent PCR and western blotting were used to detect the expression levels of PHB. Flow cytometry and detection of enzymatic activity of caspase-3 by western blotting were employed to examine cell apoptosis. Our results provide evidence that the lentiviral vector pGCSIL-GFP-PHB was constructed successfully, and the PHB mRNA and the protein expression inhibitory rates were 90.3 and 95.8%, respectively. When compared to the control group, the activity of caspase-3 decreased significantly, which showed a 57.3% downregulation, and the apoptosis rate was reduced by 44.6% (P<0.05). In conclusion, downregulation of the PHB gene may inhibit apoptosis of NB4-R1 cells, and it is speculated that this was at least partly due to the downregulation of caspase-3, and PHB may be a novel target for gene therapy for retinoic acid-resistant acute promyelocytic leukemia.

  20. Targeting expression of the leukemogenic PML-RARα fusion protein by lentiviral vector-mediated small interfering RNA results in leukemic cell differentiation and apoptosis.

    PubMed

    Ward, Simone V; Sternsdorf, Thomas; Woods, Niels-Bjarne

    2011-12-01

    Acute promyelocytic leukemia (APL) results from a chromosomal translocation that gives rise to the leukemogenic fusion protein PML-RARα (promyelocytic leukemia-retinoic acid α receptor). Differentiation of leukemic cells and complete remission of APL are achieved by treatment of patients with pharmacological doses of all-trans retinoic acid (ATRA), making APL a model disease for differentiation therapy. However, because patients are resistant to further treatment with ATRA on relapse, it is necessary to develop alternative treatment strategies to specifically target APL. We therefore sought to develop a treatment strategy based on lentiviral vector-mediated delivery of small interfering RNA (siRNA) that specifically targets the breakpoint region of PML-RARα. Unlike treatment with ATRA, which resulted in differentiation of leukemic NB4 cells, delivery of siRNA targeting PML-RARα into NB4 cells resulted in both differentiation and apoptosis, consistent with the specific knockdown of PML-RARα. Intraperitoneal injection of NB4 cells transduced with lentiviral vectors delivering PML-RARα-specific siRNA but not control siRNA prevented development of disease in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Taken together, these results indicate that development of PML-RARα-specific siRNA may represent a promising treatment strategy for ATRA-resistant APL.

  1. Measles virus glycoprotein-pseudotyped lentiviral vector-mediated gene transfer into quiescent lymphocytes requires binding to both SLAM and CD46 entry receptors.

    PubMed

    Frecha, Cecilia; Lévy, Camille; Costa, Caroline; Nègre, Didier; Amirache, Fouzia; Buckland, Robin; Russell, Steven J; Cosset, François-Loïc; Verhoeyen, Els

    2011-06-01

    Gene transfer into quiescent T and B cells is of importance for gene therapy and immunotherapy approaches to correct hematopoietic disorders. Previously, we generated lentiviral vectors (LVs) pseudotyped with the Edmonston measles virus (MV) hemagglutinin and fusion glycoproteins (Hgps and Fgps) (H/F-LVs), which, for the first time, allowed efficient transduction of quiescent human B and T cells. These target cells express both MV entry receptors used by the vaccinal Edmonston strain, CD46 and signaling lymphocyte activation molecule (SLAM). Interestingly, LVs pseudotyped with an MV Hgp, blind for the CD46 binding site, were completely inefficient for resting-lymphocyte transduction. Similarly, SLAM-blind H mutants that recognize only CD46 as the entry receptor did not allow stable LV transduction of resting T cells. The CD46-tropic LVs accomplished vector-cell binding, fusion, entry, and reverse transcription at levels similar to those achieved by the H/F-LVs, but efficient proviral integration did not occur. Our results indicate that both CD46 and SLAM binding sites need to be present in cis in the Hgp to allow successful stable transduction of quiescent lymphocytes. Moreover, the entry mechanism utilized appears to be crucial: efficient transduction was observed only when CD46 and SLAM were correctly engaged and an entry mechanism that strongly resembles macropinocytosis was triggered. Taken together, our results suggest that although vector entry can occur through the CD46 receptor, SLAM binding and subsequent signaling are also required for efficient LV transduction of quiescent lymphocytes to occur.

  2. 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.

  3. Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol.

    PubMed

    Horn, Peter A; Keyser, Kirsten A; Peterson, Laura J; Neff, Tobias; Thomasson, Bobbie M; Thompson, Jesse; Kiem, Hans-Peter

    2004-05-15

    The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34(+) hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)- and granulocyte-colony stimulating factor (G-CSF)-primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.

  4. Real-time in vivo bioluminescence imaging of lentiviral vector-mediated gene transfer in mouse testis.

    PubMed

    Kim, T S; Choi, H S; Ryu, B Y; Gang, G T; Kim, S U; Koo, D B; Kim, J M; Han, J H; Park, C K; Her, S; Lee, D S

    2010-01-01

    Although much research has focused on transferring exogenous genes into living mouse testis to investigate specific gene functions in spermatogenic, Sertoli, and Leydig cells, relatively little is known regarding real-time gene expression in vivo. In this study, we constructed a bicistronic lentiviral vector (LV) encoding firefly luciferase and enhanced green fluorescence protein (EGFP); this was a highly efficient in vivo gene transfer tool. After microinjecting LV into the seminiferous tubules the ICR mouse testis, we detected luciferase and EGFP expression in vivo and ex vivo in the injected tubules using bioluminescence imaging (BLI) with the IVIS-200 system and fibered confocal fluorescence microscopy (CellViZio), respectively. In addition, with an in vivo BLI system, luciferase expression in the testis was detected for approximately 3 mo. Furthermore, EGFP expression in seminiferous tubules was confirmed in excised testes via three-dimensional fluorescent imaging with a confocal laser-scanning microscope. With immunostaining, EGFP expression was confirmed in several male germ cell types in the seminiferous tubules, as well as in Sertoli and Leydig cells. In conclusion, we demonstrated that real-time in vivo BLI analysis can be used to noninvasively (in vivo) monitor long-term luciferase expression in mouse testis, and we verified that EGFP expression is localized in seminiferous tubules after bicistronic LV-mediated gene transfer into mouse testes. Furthermore, we anticipate the future use of in vivo BLI technology for real-time study of specific genes involved in spermatogenesis.

  5. Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

    PubMed Central

    Kim, Yoon-Sang; Wielgosz, Matthew M; Hargrove, Phillip; Kepes, Steven; Gray, John; Persons, Derek A; Nienhuis, Arthur W

    2010-01-01

    Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34+ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34+ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34+ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45+ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector. PMID:20372106

  6. Transduction of human primitive repopulating hematopoietic cells with lentiviral vectors pseudotyped with various envelope proteins.

    PubMed

    Kim, Yoon-Sang; Wielgosz, Matthew M; Hargrove, Phillip; Kepes, Steven; Gray, John; Persons, Derek A; Nienhuis, Arthur W

    2010-07-01

    Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34(+) peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34(+) cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34(+) cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45(+) cells in total bone marrow were comparable to that of the control, mock-transduced group (37-45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the gamma-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the gamma-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.

  7. 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.

  8. Selective transduction of astrocytic and neuronal CNS subpopulations by lentiviral vectors pseudotyped with Chikungunya virus envelope.

    PubMed

    Eleftheriadou, Ioanna; Dieringer, Michael; Poh, Xuan Ying; Sanchez-Garrido, Julia; Gao, Yunan; Sgourou, Argyro; Simmons, Laura E; Mazarakis, Nicholas D

    2017-04-01

    Lentiviral vectors are gene delivery vehicles that integrate into the host genome of dividing and non-dividing mammalian cells facilitating long-term transgene expression. Lentiviral vector versatility is greatly increased by incorporating heterologous viral envelope proteins onto the vector particles instead of the native envelope, conferring on these pseudotyped vectors a modified tropism and host range specificity. We investigated the pseudotyping efficiency of HIV-1 based lentiviral vectors with alphaviral envelope proteins from the Chikungunya Virus (CHIKV-G) and Sindbis Virus (SINV-G). Following vector production optimisation, titres for the CHIKV-G pseudotype were comparable to the VSV-G pseudotype but those for the SINV-G pseudotype were significantly lower. High titre CHIKV-G pseudotyped vector efficiently transduced various human and mouse neural cell lines and normal human astrocytes (NHA) in vitro. Although transduction was broad, tropism for NHAs was observed. In vivo stereotaxic delivery in striatum, thalamus and hippocampus respectively in the adult rat brain revealed localised transduction restricted to striatal astrocytes and hippocampal dentate granule neurons. Transduction of different subtypes of granule neurons from precursor to post-mitotic stages of differentiation was evident in the sub-granular zone and dentate granule cell layer. No significant inflammatory response was observed, but comparable to that of VSV-G pseudotyped lentiviral vectors. Robust long-term expression followed for three months post-transduction along with absence of neuroinflammation, coupled to the selective and unique neuron/glial tropism indicates that these vectors could be useful for modelling and gene therapy studies in the CNS.

  9. Priming of Hepatocytes Enhances In Vivo Liver Transduction with Lentiviral Vectors in Adult Mice

    PubMed Central

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

    2012-01-01

    Abstract 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. PMID:22428976

  10. 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.

  11. Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers

    PubMed Central

    Castellani, Stefano; Orlando, Clara; Carbone, Annalucia; Di Gioia, Sante; Conese, Massimo

    2016-01-01

    Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions. PMID:27886077

  12. Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers.

    PubMed

    Castellani, Stefano; Orlando, Clara; Carbone, Annalucia; Di Gioia, Sante; Conese, Massimo

    2016-11-23

    Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.

  13. 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.

  14. Specific Retrograde Transduction of Spinal Motor Neurons Using Lentiviral Vectors Targeted to Presynaptic NMJ Receptors

    PubMed Central

    Eleftheriadou, I; Trabalza, A; Ellison, SM; Gharun, K; Mazarakis, ND

    2014-01-01

    To understand how receptors are involved in neuronal trafficking and to be able to utilize them for specific targeting via the peripheral route would be of great benefit. Here, we describe the generation of novel lentiviral vectors with tropism to motor neurons that were made by coexpressing onto the lentiviral surface a fusogenic glycoprotein (mutated sindbis G) and an antibody against a cell-surface receptor (Thy1.1, p75NTR, or coxsackievirus and adenovirus receptor) on the presynaptic terminal of the neuromuscular junction. These vectors exhibit binding specificity and efficient transduction of receptor positive cell lines and primary motor neurons in vitro. Targeting of each of these receptors conferred to these vectors the capability of being transported retrogradely from the axonal tip, leading to transduction of motor neurons in vitro in compartmented microfluidic cultures. In vivo delivery of coxsackievirus and adenovirus receptor-targeted vectors in leg muscles of mice resulted in predicted patterns of motor neuron labeling in lumbar spinal cord. This opens up the clinical potential of these vectors for minimally invasive administration of central nervous system-targeted therapeutics in motor neuron diseases. PMID:24670531

  15. Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases.

    PubMed

    Cai, Yujia; Bak, Rasmus O; Mikkelsen, Jacob Giehm

    2014-04-24

    Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001.

  16. Vectofusin-1, a New Viral Entry Enhancer, Strongly Promotes Lentiviral Transduction of Human Hematopoietic Stem Cells

    PubMed Central

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

    2013-01-01

    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. PMID:23653154

  17. Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.

    PubMed

    Huo, Wenying; Zhao, Guannan; Yin, Jinggang; Ouyang, Xuan; Wang, Yinan; Yang, Chuanhe; Wang, Baojing; Dong, Peixin; Wang, Zhixiang; Watari, Hidemichi; Chaum, Edward; Pfeffer, Lawrence M; Yue, Junming

    2017-01-01

    CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.

  18. Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells

    PubMed Central

    Huo, Wenying; Zhao, Guannan; Yin, Jinggang; Ouyang, Xuan; Wang, Yinan; Yang, Chuanhe; Wang, Baojing; Dong, Peixin; Wang, Zhixiang; Watari, Hidemichi; Chaum, Edward; Pfeffer, Lawrence M.; Yue, Junming

    2017-01-01

    CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells. PMID:28123598

  19. Efficient transduction of cytotoxic and anti-HIV-1 genes by a gene-regulatable lentiviral vector.

    PubMed

    Shinoda, Yasuhiko; Hieda, Kuniko; Koyanagi, Yoshio; Suzuki, Youichi

    2009-10-01

    Lentiviral vectors modified from human immunodeficiency virus type 1 (HIV-1) offer a promising approach for gene therapy, facilitating transduction of genes into non-dividing cells both in vitro and in vivo. When transducing cytotoxic or anti-HIV genes, however, the vector must avoid self-inhibition by the transgene that can lead to a disruption in production of infectious virions. In this study, we constructed two HIV-1-based lentiviral vectors harboring the mifepristone-inducible gene expression unit in either the forward or the reverse orientation with respect to the direction of viral genomic RNA. The ability of these vectors to transduce cytotoxic and anti-HIV genes was evaluated. When human CD14 was used as a transgene, infectious lentiviral vectors were produced by both forward and reverse vector systems. CD14 expression was efficiently induced in cells transduced by both lentiviral vectors following treatment with mifepristone. However, a higher level of basal transgene expression was observed in the forward vector system in the absence of mifepristone. In contrast, high titers of infectious lentiviral vector containing the cytotoxic vesicular stomatitis virus M gene were successfully generated using the reverse vector, but not the forward vector. In addition, when a VPS4Bdominant negative mutant against HIV-1 budding was cloned into the reverse vector, significant amounts of lentiviral vector were obtained. Subsequent transduction of cells with the VPS4B mutant resulted in approximately 50% inhibition of HIV-1 production only in the presence of mifepristone. Our study thus demonstrates that incorporation of a mifepristone-regulatable gene expression unit in the reverse orientation makes significant advances toward development of a lentiviral vector that allows transduction of harmful genes.

  20. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    PubMed

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

  1. A protocol for lentiviral transduction and downstream analysis of intestinal organoids.

    PubMed

    Van Lidth de Jeude, Jooske F; Vermeulen, Jacqueline L M; Montenegro-Miranda, Paula S; Van den Brink, Gijs R; Heijmans, Jarom

    2015-04-20

    Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

  2. A MicroRNA-regulated and GP64-pseudotyped Lentiviral Vector Mediates Stable Expression of FVIII in a Murine Model of Hemophilia A

    PubMed Central

    Matsui, Hideto; Hegadorn, Carol; Ozelo, Margareth; Burnett, Erin; Tuttle, Angie; Labelle, Andrea; McCray, Paul B; Naldini, Luigi; Brown, Brian; Hough, Christine; Lillicrap, David

    2011-01-01

    The objective to use gene therapy to provide sustained, therapeutic levels of factor VIII (FVIII) for hemophilia A is compromised by the emergence of inhibitory antibodies that prevent FVIII from performing its essential function as a cofactor for factor IX (FIX). FVIII appears to be more immunogenic than FIX and an immune response is associated more frequently with FVIII than FIX gene therapy strategies. We have evaluated a modified lentiviral delivery strategy that facilitates liver-restricted transgene expression and prevents off-target expression in hematopoietic cells by incorporating microRNA (miRNA) target sequences. In contrast to outcomes using this strategy to deliver FIX, this modified delivery strategy was in and of itself insufficient to prevent an anti-FVIII immune response in treated hemophilia A mice. However, pseudotyping the lentivirus with the GP64 envelope glycoprotein, in conjunction with a liver-restricted promoter and a miRNA-regulated FVIII transgene resulted in sustained, therapeutic levels of FVIII. These modifications to the lentiviral delivery system effectively restricted FVIII transgene expression to the liver. Plasma levels of FVIII could be increased to around 9% that of normal levels when macrophages were depleted prior to treating the hemophilia A mice with the modified lentiviral FVIII delivery system. PMID:21285959

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

    PubMed

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

    2013-11-04

    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

  4. Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector.

    PubMed

    Cmielewski, Patricia; Farrow, Nigel; Donnelley, Martin; McIntyre, Chantelle; Penny-Dimri, Jahan; Kuchel, Tim; Parsons, David

    2014-11-21

    The safety and efficiency of gene therapies for cystic fibrosis (CF) need to be assessed in pre-clinical models. Using the normal ferret, this study sought to determine whether ferret airway epithelia could be transduced with a lysophosphatidylcholine (LPC) pre-treatment followed by a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector, in preparation for future studies in CF ferrets. Six normal ferrets (7 -8 weeks old) were treated with a 150 μL LPC pre-treatment, followed one hour later by a 500 μL LV vector dose containing the LacZ transgene. LacZ gene expression in the conducting airways and lung was assessed by X-gal staining after 7 days. The presence of transduction in the lung, as well as off-target transduction in the liver, spleen and gonads, were assessed by qPCR. The levels of LV vector p24 protein bio-distribution in blood sera were assessed by ELISA at 0, 1, 3, 5 and 7 days. The dosing protocol was well tolerated. LacZ gene expression was observed en face in the trachea of all animals. Histology showed that ciliated and basal cells were transduced in the trachea, with rare LacZ transduced single cells noted in lung. p24 levels was not detectable in the sera of 5 of the 6 animals. The LacZ gene was not detected in the lung tissue and no off-target transduction was detected by qPCR. This study shows that ferret airway epithelia are transducible using our unique two-step pre-treatment and LV vector dosing protocol. We have identified a number of unusual anatomical factors that are likely to influence the level of transduction that can be achieved in ferret airways. The ability to transduce ferret airway epithelium is a promising step towards therapeutic LV-CFTR testing in a CF ferret model.

  5. Anti-Apoptotic Effects of Lentiviral Vector Transduction Promote Increased Rituximab Tolerance in Cancerous B-Cells

    PubMed Central

    Ranjbar, Benyamin; Krogh, Louise Bechmann; Laursen, Maria Bach; Primo, Maria Nascimento; Marques, Sara Correia; Dybkær, Karen; Mikkelsen, Jacob Giehm

    2016-01-01

    Diffuse large B-cell lymphoma (DLBCL) is characterized by great genetic and clinical heterogeneity which complicates prognostic prediction and influences treatment efficacy. The most common regimen, R-CHOP, consists of a combination of anthracycline- and immuno-based drugs including Rituximab. It remains elusive how and to which extent genetic variability impacts the response and potential tolerance to R-CHOP. Hence, an improved understanding of mechanisms leading to drug tolerance in B-cells is crucial, and modelling by genetic intervention directly in B-cells is fundamental in such investigations. Lentivirus-based gene vectors are widely used gene vehicles, which in B-cells are an attractive alternative to potentially toxic transfection-based methodologies. Here, we investigate the use of VSV-G-pseudotyped lentiviral vectors in B-cells for exploring the impact of microRNAs on tolerance to Rituximab. Notably, we find that robust lentiviral transduction of cancerous B-cell lines markedly and specifically enhances the resistance of transduced germinal center B-cells (GCBs) to Rituximab. Although Rituximab works partially through complement-mediated cell lysis, increased tolerance is not achieved through effects of lentiviral transduction on cell death mediated by complement. Rather, reduced levels of PARP1 and persistent high levels of CD43 in Rituximab-treated GCBs demonstrate anti-apoptotic effects of lentiviral transduction that may interfere with the outcome and interpretation of Rituximab tolerance studies. Our findings stress that caution should be exercised exploiting lentiviral vectors in studies of tolerance to therapeutics in DLBCL. Importantly, however, we demonstrate the feasibility of using the lentiviral gene delivery platform in studies addressing the impact of specific microRNAs on Rituximab responsiveness. PMID:27045839

  6. Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy.

    PubMed

    Sutlu, Tolga; Nyström, Sanna; Gilljam, Mari; Stellan, Birgitta; Applequist, Steven E; Alici, Evren

    2012-10-01

    Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approach for cancer treatment. Yet, optimization of highly efficient and clinically applicable gene transfer protocols for NK cells still presents a challenge. In this study, we aimed at identifying conditions under which optimum lentiviral gene transfer to NK cells can be achieved. Our results demonstrate that stimulation of NK cells with interleukin (IL)-2 and IL-21 supports efficient transduction using a VSV-G pseudotyped lentiviral vector. Moreover, we have identified that inhibition of innate immune receptor signaling greatly enhances transduction efficiency. We were able to boost the efficiency of lentiviral genetic modification on average 3.8-fold using BX795, an inhibitor of the TBK1/IKKɛ complex acting downstream of RIG-I, MDA-5, and TLR3. We have also observed that the use of BX795 enhances lentiviral transduction efficiency in a number of human and mouse cell lines, indicating a broadly applicable, practical, and safe approach that has the potential of being applicable to various gene therapy protocols.

  7. A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors.

    PubMed

    Jimenez-Moreno, Carmen Maria; Herrera-Gomez, Irene de Gracia; Lopez-Noriega, Livia; Lorenzo, Petra Isabel; Cobo-Vuilleumier, Nadia; Fuente-Martin, Esther; Mellado-Gil, Jose Manuel; Parnaud, Geraldine; Bosco, Domenico; Gauthier, Benoit Raymond; Martin-Montalvo, Alejandro

    2015-01-01

    Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional β-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes.

  8. Effects of vector backbone and pseudotype on lentiviral vector-mediated gene transfer: studies in infant ADA-deficient mice and rhesus monkeys.

    PubMed

    Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

    2014-10-01

    Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

  9. Lentiviral vector-mediated down-regulation of IL-17A receptor in hepatic stellate cells results in decreased secretion of IL-6

    PubMed Central

    Zhang, Sheng-Chu; Zheng, Yi-Hu; Yu, Pan-Pan; Min, Tan Hooi; Yu, Fu-Xiang; Ye, Chao; Xie, Yuan-Kang; Zhang, Qi-Yu

    2012-01-01

    AIM: To investigate the mechanism of interleukin (IL)-6 secretion through blocking the IL-17A/IL-17A receptor (IL-17RA) signaling pathway with a short hairpin RNA (shRNA) in hepatic stellate cells (HSCs) in vitro. METHODS: HSCs were derived from the livers of adult male Sprague-Dawley rats. IL-6 expression was evaluated using real-time quantitative polymerase chain reaction and enzyme linked immunosorbent assay. The phosphorylation activity of p38 mitogen activated protein kinases (MAPK) and extracellular regulated protein kinases (ERK) 1/2 upon induction by IL-17A and suppression by IL-17RA shRNA were examined using Western blotting. RESULTS: IL-6 expression induced by IL-17A was significantly increased compared to control in HSCs (P < 0.01 in a dose-dependent manner). Suppression of IL-17RA using lentiviral-mediated shRNA inhibited IL-6 expression induced by IL-17A compared to group with only IL-17A treatment (1.44 ± 0.17 vs 4.07 ± 0.43, P < 0.01). IL-17A induced rapid phosphorylation of p38 MAPK and ERK1/2 after 5 min exposure, and showed the strongest levels of phosphorylation of p38 MAPK and ERK1/2 at 15 min in IL-17A-treated HSCs. IL-6 mRNA expression induced by IL-17A (100 ng/mL) for 3 h exposure was inhibited by preincubation with specific inhibitors of p38 MAPK (SB-203580) and ERK1/2 (PD-98059) compared to groups without inhibitors preincubation (1.67 ± 0.24, 2.01 ± 0.10 vs 4.08 ± 0.59, P < 0.01). Moreover, Lentiviral-mediated IL-17RA shRNA 1 inhibited IL-17A-induced IL-6 mRNA expression compared to random shRNA in HSCs (1.44 ± 0.17 vs 3.98 ± 0.68, P < 0.01). Lentiviral-mediated IL-17RA shRNA 1 inhibited phosphorylation of p38 MAPK and ERK1/2 induced by 15 min IL-17A (100 ng/mL) exposure. CONCLUSION: Down-regulation of the IL-17RA receptor by shRNA decreased IL-6 expression induced by IL-17A via p38 MAPK and ERK1/2 phosphorylation in HSCs. Suppression of IL-17RA expression may be a strategy to reduce the inflammatory response induced by IL-17A in

  10. Induction of Human Blood Group A Antigen Expression on Mouse Cells, Using Lentiviral Gene Transduction

    PubMed Central

    Fan, Xiaohu; Lang, Haili; Zhou, Xianpei; Zhang, Li; Yin, Rong; Maciejko, Jessica; Giannitsos, Vasiliki; Motyka, Bruce; Medin, Jeffrey A.; Platt, Jeffrey L.

    2010-01-01

    Abstract The ABO histo-blood group system is the most important antigen system in transplantation medicine, yet no small animal model of the ABO system exists. To determine the feasibility of developing a murine model, we previously subcloned the human α-1,2-fucosyltransferase (H-transferase, EC 2.4.1.69) cDNA and the human α-1,3-N-acetylgalactosaminyltransferase (A-transferase, EC 2.4.1.40) cDNA into lentiviral vectors to study their ability to induce human histo-blood group A antigen expression on mouse cells. Herein we investigated the optimal conditions for human A and H antigen expression in murine cells. We determined that transduction of a bicistronic lentiviral vector (LvEF1-AH-trs) resulted in the expression of A antigen in a mouse endothelial cell line. We also studied the in vivo utility of this vector to induce human A antigen expression in mouse liver. After intrahepatic injection of LvEF1-AH-trs, A antigen expression was observed on hepatocytes as detected by immunohistochemistry and real-time RT-PCR. In human group A erythrocyte-sensitized mice, A antigen expression in the liver was associated with tissue damage, and deposition of antibody and complement. These results suggest that this gene transfer strategy can be used to simulate the human ABO blood group system in a murine model. This model will facilitate progress in the development of interventions for ABO-incompatible transplantation and transfusion scenarios, which are difficult to develop in clinical or large animal settings. PMID:20163247

  11. Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer

    PubMed Central

    Wilber, Andrew; Hargrove, Phillip W.; Kim, Yoon-Sang; Riberdy, Janice M.; Sankaran, Vijay G.; Papanikolaou, Eleni; Georgomanoli, Maria; Anagnou, Nicholas P.; Orkin, Stuart H.; Nienhuis, Arthur W.

    2011-01-01

    β-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α2β2;HbA). Increased levels of fetal hemoglobin (α2γ2;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34+ cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with β-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34+ cells demonstrated levels of HbF up to 21% per vector copy. For β-thalassemic CD34+ cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with β-globin deficiency. PMID:21156846

  12. Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease.

    PubMed

    Azzouz, Mimoun; Martin-Rendon, Enca; Barber, Robert D; Mitrophanous, Kyriacos A; Carter, Emma E; Rohll, Jonathan B; Kingsman, Susan M; Kingsman, Alan J; Mazarakis, Nicholas D

    2002-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra. This loss leads to complete dopamine depletion in the striatum and severe motor impairment. It has been demonstrated previously that a lentiviral vector system based on equine infectious anemia virus (EIAV) gives rise to highly efficient and sustained transduction of neurons in the rat brain. Therefore, a dopamine replacement strategy using EIAV has been investigated as a treatment in the 6-hydroxydopamine (6-OHDA) animal model of PD. A self-inactivating EIAV minimal lentiviral vector that expresses tyrosine hydroxylase (TH), aromatic amino acid dopa decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1) in a single transcription unit has been generated. In cultured striatal neurons transduced with this vector, TH, AADC, and CH1 proteins can all be detected. After stereotactic delivery into the dopamine-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each enzyme and effective production of catecholamines were detected, resulting in significant reduction of apomorphine-induced motor asymmetry compared with control animals (p < 0.003). Expression of each enzyme in the striatum was observed for up to 5 months after injection. These data indicate that the delivery of three catecholaminergic synthetic enzymes by a single lentiviral vector can achieve functional improvement and thus open the potential for the use of this vector for gene therapy of late-stage PD patients.

  13. A VSV-G Pseudotyped Last Generation Lentiviral Vector Mediates High Level and Persistent Gene Transfer in Models of Airway Epithelium In Vitro and In Vivo

    PubMed Central

    Copreni, Elena; Palmieri, Lucia; Castellani, Stefano; Conese, Massimo

    2010-01-01

    The aim of this work was to evaluate the efficiency and duration of gene expression mediated by a VSV-G pseudotyped last generation lentiviral (LV) vector. We studied LV efficiency in ex-vivo models of respiratory epithelial cells, obtained from bronchial biopsies and nasal polyps, by GFP epifluorescence and cytofluorimetry. In vivo efficiency and persistence of gene expression was investigated by GFP immunohistochemistry and luciferase activity in lung cryosections and homogenates, respectively, upon intranasal and intratracheal administration protocols in C57Bl/6 mice. Both primary bronchial and nasal epithelial cells were transduced up to 70–80% 72 hr after the LV infection. In vivo nasal luciferase expression was increased by lysophosphatidylcholine pre-treatment of the nose. Conversely, the bronchial epithelium was transduced in the absence of any pre-conditioning treatment and luciferase expression lasted for at least 6 months without any decline. We conclude that a last generation LV vector is a promising gene transfer agent in the target organ of genetic and acquired lung diseases, as in the case of cystic fibrosis. PMID:21994695

  14. CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells.

    PubMed

    Bellec, Jessica; Bacchetta, Marc; Losa, Davide; Anegon, Ignacio; Chanson, Marc; Nguyen, Tuan Huy

    2015-01-01

    Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation. Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells. shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion. We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

  15. Suppression of neovascularization of donor corneas by transduction with equine infectious anemia virus-based lentiviral vectors expressing endostatin and angiostatin.

    PubMed

    Parker, Maria; Bellec, Jessica; McFarland, Trevor; Scripps, Vicky; Appukuttan, Binoy; Hartzell, Matt; Yeager, Austen; Hady, Thomas; Mitrophanous, Kyriacos A; Stout, Tim; Ellis, Scott

    2014-05-01

    Corneal transplantation is the oldest and one of the most successful transplant procedures with a success rate in many studies in excess of 90%. The high success rate is mainly attributable to the relatively immune-privileged status of the eye and the fact that the cornea is largely avascular. However, the success rate in patients with failed grafts is much lower such that regrafting is frequently the top indication for corneal transplantation in many centers. Neovascularization is the most important risk factor for rejection, as it allows access of the immune system to the donor tissue, compromising immune privilege of the graft/eye. We have developed a process to modify donor corneal tissue to prevent rejection by a single exposure to a gene therapy vector before surgery (EncorStat(®)). The vector used is based on clinically relevant equine infectious anemia virus (EIAV)-derived lentiviral platform and contains genes for two potently angiostatic genes, endostatin and angiostatin. We show that incubation of rabbit, primate, and human corneal tissue with the EIAV vector mediates strong, stable expression in the corneal endothelium. We have optimized this process to maximize transduction and, once this is complete, maximize the removal of free vector before transplant. Rabbit corneas treated with two different antiangiogenic expression vectors (EIAV-EndoAngio and to a lesser extent EIAV-Endo:k5) significantly suppressed neovascularization in a rabbit model of corneal rejection. As a result, corneal opacity, edema, and inflammatory infiltrates were reduced in these corneas. This study demonstrates that angiogenesis is a suitable target to prevent corneal rejection, and provides the first proof-of-concept data for the development of EncorStat, an ex vivo gene therapy treatment to prevent corneal rejection.

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

    PubMed Central

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

    2014-01-01

    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

  17. 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

  18. Characterization of a third generation lentiviral vector pseudotyped with Nipah virus envelope proteins for endothelial cell transduction.

    PubMed

    Witting, S R; Vallanda, P; Gamble, A L

    2013-10-01

    Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, third generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared with vesicular stomatitis virus pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in third generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells.

  19. Clinical-scale lentiviral vector transduction of PBL for TCR gene therapy and potential for expression in less differentiated cells

    PubMed Central

    Yang, Shicheng; Rosenberg, Steven A.; Morgan, Richard A.

    2012-01-01

    Summary In human gene therapy applications, lentiviral vectors may have advantages over gamma-retroviral vectors because of their ability to transduce non-dividing cells, their resistance to gene silencing, and a lack of integration site preference. In this study, we utilized VSV-G pseudotype third generation lentiviral vectors harboring specific anti-tumor T-cell receptor (TCR) to establish clinical-scale lentiviral transduction of PBL. Spinoculation (1000 × g, 32°C for 2 h) in the presence of protamine sulfate represents the most efficient and economical approach to transduce a large number of PBLs compared to RetroNectin-based methods. Up to 20 million cells per well of a 6-well plate were efficiently transduced and underwent an average 50-fold expansion in two weeks. TCR transduced PBL mediated specific anti-tumor activities including IFN-γ release and cell lysis. Compared to gamma-retroviral vectors, the TCR transgene could be preferentially expressed on a less-differentiated cell population. PMID:18833004

  20. Modified HIV-1 based lentiviral vectors have an effect on viral transduction efficiency and gene expression in vitro and in vivo.

    PubMed

    Park, F; Kay, M A

    2001-09-01

    Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human alpha1-anti-trypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that incorporation of the central polypurine tract sequence (cppt) increased transduction efficiency in vitro while increasing the transduction of non-cell-cycling hepatocytes in vivo. C57Bl/6 scid mice that were administered lentiviral vectors devoid of the cppt (2 x 10(8) transducing units (T.U.)/mouse) had 81% of their lacZ-transduced hepatocytes colabeled with the cell cycle marker 5'-bromo-2'-deoxyuridine (BrdU). In contrast, inclusion of the cppt reduced the colabeling in mouse hepatocytes by 50%. Further modifications in the lentiviral vectors were performed to enhance viral titer and gene expression. We found that the inclusion of a matrix attachment region (MAR) from immunoglobulin-kappa (Igkappa) significantly increased the transduction efficiency, as measured by transgene protein expression and proviral DNA copy number, compared with vectors without Igkappa MAR. In vitro studies using human hepatoma cells demonstrated a significant increase (two- to fourfold) in human AAT and human FIX production when the Igkappa MAR was incorporated. In vivo transduction of partially hepatectomized C57Bl/6 mice given an optimized lentiviral vector containing the cppt and Igkappa MAR (2 x 10(8) T.U./mouse) resulted in sustained therapeutic levels of serum FIX (approximately 65 ng/ml). Our study demonstrates the importance of cis-acting elements to enhancing the transduction ability of lentiviral vectors and the expression of vector transgenes.

  1. 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.

  2. Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs☆

    PubMed Central

    Li, Duo; Schlaepfer, Erika; Audigé, Annette; Rochat, Mary-Aude; Ivic, Sandra; Knowlton, Caitlin N.; Kim, Baek; Keppler, Oliver T.; Speck, Roberto F.

    2016-01-01

    Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription. PMID:26207584

  3. Concurrent measures of fusion and transduction efficiency of primary CD34+ cells with human immunodeficiency virus 1-based lentiviral vectors reveal different effects of transduction enhancers.

    PubMed

    Ingrao, Dina; Majdoul, Saliha; Seye, Ababacar K; Galy, Anne; Fenard, David

    2014-02-01

    Lentiviral vectors (LVs) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking for precisely evaluating parameters that control the efficiency of transduction in relation to the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer-based human immunodeficiency virus-1 fusion assay to measure the entry of nonreplicative recombinant LVs in various cell types, including primary human hematopoietic stem progenitor cells (HSPCs), and to quantify the level of transduction of the same initially infected cells. The assay utilizes recombinant LVs containing β-lactamase (BLAM)-Vpr chimeric proteins (BLAM-LVs) and encoding a truncated form of the low-affinity nerve growth factor receptor (ΔNGFR). After infection of target cells with BLAM-LVs, the vector entry rapidly leads to BLAM-Vpr release into the cytoplasm, which is measured by cleavage of a fluorescent substrate using flow cytometry. Parallel cultures of the same infected cells show transduction efficiency resulting from ΔNGFR expression. This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing, for instance, the postentry restrictions of LVs known to occur in cells of hematopoietic origin, especially human HSPCs. Furthermore, this BLAM-LV assay allowed us to evaluate the effect of cytokine prestimulation of HSPCs on the entry step of LVs. The assay also shows that transduction enhancers such as Vectofusin-1 or Retronectin can partially relieve the postentry block, but their effects differ in how they promote LV entry. In conclusion, one such assay should be useful to study hematopoietic postentry restrictions directed against LVs and therefore should allow improvements in various LV-based gene therapy protocols.

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

    PubMed Central

    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

  5. Single-polarity recombinant adeno-associated virus 2 vector-mediated transgene expression in vitro and in vivo: mechanism of transduction.

    PubMed

    Zhong, Li; Zhou, Xiaohuai; Li, Yanjun; Qing, Keyun; Xiao, Xiao; Samulski, Richard Jude; Srivastava, Arun

    2008-02-01

    Recombinant adeno-associated virus 2 (AAV) vectors encapsidate single-stranded genomes of either polarity equally frequently in separate mature virions. Because viral genomes of either polarity are transcriptionally inactive, both the failure to undergo viral second-strand DNA synthesis and the failure to undergo DNA strand annealing have been proposed as possible reasons to account for the observed low efficiency of transgene expression. We compared the transduction efficiencies of conventional AAV vectors containing both [-] and [+] polarity genomes with those containing either the [-] or the [+] polarity genomes, in vitro as well as in vivo. We document that the transduction efficiency of single-polarity AAV vectors is significantly enhanced by (i) co-infection with adenovirus; (ii) small interfering RNA (siRNA)-mediated down-modulation of a cellular protein, FKBP52, tyrosine-phosphorylated forms of which inhibit AAV second-strand DNA synthesis; (iii) over-expression of a cellular protein tyrosine phosphatase, T cell protein tyrosine phosphatase (TC-PTP), which catalyzes tyrosine-dephosphorylation of FKBP52; and (iv) deliberate over-expression of TC-PTP, or the absence of FKBP52, respectively, in TC-PTP-transgenic mice and in FKBP52-knockout mice. These data confirm that viral second-strand DNA synthesis, rather than DNA strand annealing, is the rate-limiting step in efficient transduction by AAV vectors. This finding has implications in the use of these vectors in human gene therapy.

  6. Reversal of Diabetes Through Gene Therapy of Diabetic Rats by Hepatic Insulin Expression via Lentiviral Transduction

    PubMed Central

    Elsner, Matthias; Terbish, Taivankhuu; Jörns, Anne; Naujok, Ortwin; Wedekind, Dirk; Hedrich, Hans-Jürgen; Lenzen, Sigurd

    2012-01-01

    Due to shortage of donor tissue a cure for type 1 diabetes by pancreas organ or islet transplantation is an option only for very few patients. Gene therapy is an alternative approach to cure the disease. Insulin generation in non-endocrine cells through genetic engineering is a promising therapeutic concept to achieve insulin independence in patients with diabetes. In the present study furin-cleavable human insulin was expressed in the liver of autoimmune-diabetic IDDM rats (LEW.1AR1/Ztm-iddm) and streptozotocin-diabetic rats after portal vein injection of INS-lentivirus. Within 5–7 days after the virus injection of 7 × 109 INS-lentiviral particles the blood glucose concentrations were normalized in the treated animals. This glucose lowering effect remained stable for the 1 year observation period. Human C-peptide as a marker for hepatic release of human insulin was in the range of 50–100 pmol/ml serum. Immunofluorescence staining of liver tissue was positive for insulin showing no signs of transdifferentiation into pancreatic β-cells. This study shows that the diabetic state can be efficiently reversed by insulin release from non-endocrine cells through a somatic gene therapy approach. PMID:22354377

  7. Lentiviral transduction of CD34(+) cells induces genome-wide epigenetic modifications.

    PubMed

    Yamagata, Yoshiaki; Parietti, Véronique; Stockholm, Daniel; Corre, Guillaume; Poinsignon, Catherine; Touleimat, Nizar; Delafoy, Damien; Besse, Céline; Tost, Jörg; Galy, Anne; Paldi, András

    2012-01-01

    Epigenetic modifications may occur during in vitro manipulations of stem cells but these effects have remained unexplored in the context of cell and gene therapy protocols. In an experimental model of ex vivo gene modification for hematopoietic gene therapy, human CD34(+) cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34(+) cells to cytokines increased nuclear expression of epigenetic regulators SIRT1 or DNMT1 and caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34(+) cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols.

  8. In vitro rescue of FGA deletion by lentiviral transduction of an afibrinogenemic patient's hepatocytes.

    PubMed

    Stroka, D; Keogh, A; Vu, D; Fort, A; Stoffel, M H; Kühni-Boghenbor, K; Furer, C; Banz, V; Demarmels Biasiutti, F; Lämmle, B; Candinas, D; Neerman-Arbez, M

    2014-11-01

    Congenital afibrinogenemia is a rare inherited autosomal recessive disorder in which a mutation in one of three genes coding for the fibrinogen polypeptide chains Aα, Bβ and γ results in the absence of a functional coagulation protein. A patient with congenital afibrinogenemia, resulting from an FGA homozygous gene deletion, underwent an orthotopic liver transplant that resulted in complete restoration of normal hemostasis. The patient's explanted liver provided a unique opportunity to further investigate a potential novel treatment modality. To explore a targeted gene therapy approach for patients with congenital afibrinogenemia. At the time of transplant, the patient's FGA-deficient hepatocytes were isolated and transduced with lentiviral vectors encoding the human fibrinogen Aα-chain. FGA-transduced hepatocytes produced fully functional fibrinogen in vitro. Orthotopic liver transplantation is a possible rescue treatment for failure of on-demand fibrinogen replacement therapy. In addition, we provide evidence that hepatocytes homozygous for a large FGA deletion can be genetically modified to restore Aα-chain protein expression and secrete a functional fibrinogen hexamer. © 2014 International Society on Thrombosis and Haemostasis.

  9. Reversal of diabetes through gene therapy of diabetic rats by hepatic insulin expression via lentiviral transduction.

    PubMed

    Elsner, Matthias; Terbish, Taivankhuu; Jörns, Anne; Naujok, Ortwin; Wedekind, Dirk; Hedrich, Hans-Jürgen; Lenzen, Sigurd

    2012-05-01

    Due to shortage of donor tissue a cure for type 1 diabetes by pancreas organ or islet transplantation is an option only for very few patients. Gene therapy is an alternative approach to cure the disease. Insulin generation in non-endocrine cells through genetic engineering is a promising therapeutic concept to achieve insulin independence in patients with diabetes. In the present study furin-cleavable human insulin was expressed in the liver of autoimmune-diabetic IDDM rats (LEW.1AR1/Ztm-iddm) and streptozotocin-diabetic rats after portal vein injection of INS-lentivirus. Within 5-7 days after the virus injection of 7 × 10(9) INS-lentiviral particles the blood glucose concentrations were normalized in the treated animals. This glucose lowering effect remained stable for the 1 year observation period. Human C-peptide as a marker for hepatic release of human insulin was in the range of 50-100 pmol/ml serum. Immunofluorescence staining of liver tissue was positive for insulin showing no signs of transdifferentiation into pancreatic β-cells. This study shows that the diabetic state can be efficiently reversed by insulin release from non-endocrine cells through a somatic gene therapy approach.

  10. Impaired nuclear transport and uncoating limit recombinant adeno-associated virus 2 vector-mediated transduction of primary murine hematopoietic cells.

    PubMed

    Zhong, Li; Li, Weiming; Yang, Zuocheng; Qing, Keyun; Tan, Mengqun; Hansen, Jonathan; Li, Yanjun; Chen, Linyuan; Chan, Rebecca J; Bischof, Daniela; Maina, Njeri; Weigel-Kelley, Kirsten A; Zhao, Weihong; Larsen, Steven H; Yoder, Mervin C; Shou, Weinian; Srivastava, Arun

    2004-12-01

    Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

  11. Pre-immunization with an Intramuscular Injection of AAV9-Human Erythropoietin Vectors Reduces the Vector-Mediated Transduction following Re-Administration in Rat Brain

    PubMed Central

    Yang, Chun; Yang, Wei-Hua; Chen, Sha-Sha; Ma, Bao-Feng; Li, Bin; Lu, Tao; Qu, Ting-Yu; Klein, Ronald L.; Zhao, Li-Ru; Duan, Wei-Ming

    2013-01-01

    We have recently demonstrated that adeno-associated virus serotype 9 (AAV9)-mediated human erythropoietin (hEPO) gene delivery into the brain protects dopaminergic (DA) neurons in the substantia nigra in a rat model of Parkinson's disease. In the present study, we examined whether pre-exposure to AAV9-hEPO vectors with an intramuscular or intrastriatal injection would reduce AAV9-mediated hEPO transduction in rat brain. We first characterized transgene expression and immune responses against AAV9-hEPO vectors in rat striatum at 4 days, 3 weeks and 6 months, and with doses ranging from 1011 to 1013 viral genomes. To sensitize immune system, rats received an injection of AAV9-hEPO into either the muscle or the left striatum, and then sequentially an injection of AAV9-hEPO into the right striatum 3 weeks later. We observed that transgene expression exhibited in a time course and dose dependent manner, and inflammatory and immune responses displayed in a time course manner. Intramuscular, but not intrastriatal injections of AAV9-hEPO resulted in reduced levels of hEPO transduction and increased levels of the major histocompatibility complex (MHC) class I and class II antigen expression in the striatum following AAV9-hEPO re-administration. There were infiltration of the cluster of differentiation 4 (CD4)-and CD8-lymphacytes, and accumulation of activated microglial cells and astrocytes in the virally injected striatum. In addition, the sera from the rats with intramuscular injections of AAV9-hEPO contained greater levels of antibodies against both AAV9 capsid protein and hEPO protein than the other treatment groups. hEPO gene expression was negatively correlated with the levels of circulating antibodies against AAV9 capsid protein. Intramuscular and intrastriatal re-administration of AAV9-hEPO led to increased numbers of red blood cells in peripheral blood. Our results suggest that pre-immunization with an intramuscular injection can lead to the reduction of transgene

  12. 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

  13. 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

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

    2012-10-01

    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 7.2×10(8) 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/IL2Rγ(null) (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 MFGγ(c) 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.

  14. Comparison of HIV-1 and EIAV-based lentiviral vectors in corneal transduction.

    PubMed

    Beutelspacher, Sven Christoph; Ardjomand, Navid; Tan, Peng Hong; Patton, Gillian Sarah; Larkin, D Frank P; George, Andrew J T; McClure, Myra O

    2005-06-01

    In this study we compare the ability of self-inactivating Human Immunodeficiency Virus 1 (HIV-1) and Equine Infectious Anaemia Virus (EIAV)-based vectors to mediate gene transfer to rabbit and human corneas and to a murine corneal endothelial cell line. Both vectors were pseudotyped with vesicular stomatitis virus-G (VSV-G) envelope and contained marker transgenes under the control of an internal CMV promoter. For specificity of action, the heterologous promoter in the EIAV-vector was exchanged for an inducible E-Selectin promoter, previously shown to regulate gene-expression in a plasmid system. We show that EIAV is more efficient than HIV in transducing human and rabbit corneal endothelial cells. Rabbit corneal endothelial cells are transduced in higher quantity than human corneal endothelial cells. In the inducible system, however, we detected impairment between the vector and its internal E-Selectin promoter. Instead of controlled transgene expression or silencing of promoter activity, the U3-modified long-terminal-repeats (LTR) impaired the conditional activity of the E-Selectin promoter. Significant transgene expression was seen without stimulation of the inducible promoter. We show efficient transduction by lentiviruses of a corneal endothelial cell line and of full thickness corneas from different species, confirming that those vectors would be appropriate tools for gene therapy of selected corneal diseases. However, the modification within the U3-LTR did not adequately allow regulated transgene expression. These findings have important implications for vector design for diagnostic or therapeutic opportunities.

  15. HTLV type 1 Tax transduction in microglial cells and astrocytes by lentiviral vectors.

    PubMed

    Wrzesinski, S; Séguin, R; Liu, Y; Domville, S; Planelles, V; Massa, P; Barker, E; Antel, J; Feuer, G

    2000-11-01

    Infection with human T cell leukemia virus type 1 (HTLV-1) can result in the development of HAM/TSP, a nonfatal, chronic inflammatory disease involving neuronal degeneration and demyelination of the central nervous system. Elevated levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1 observed in the cerebrospinal fluid of HAM-TSP patients suggest that cytokine dysregulation within the CNS is involved in neuropathogenesis. HTLV-1 infection and enhanced expression of TNF-alpha by microglial cells, astrocytes, and macrophages has been hypothesized to lead to the destruction of myelin and oligodendrocytes in the CNS. Although the association of HTLV-2 infection and development of neurological disease is more tenuous, HTLV-2 has also been found to be associated with peripheral neuropathies. To investigate the roles of HTLV Tax(1) and Tax(2) in the induction of cytokine disregulation in these cell types, we are currently developing gene delivery vectors based on human immunodeficiency virus type-1 (HIV-1) capable of stably coexpressing the HTLV-1 or -2 tax and eGFP reporter genes in primary human cells. Transduction frequencies of up to 50%, as assessed by eGFP expression, can be achieved in human monocyte-derived macrophages and in explanted cultures of human microglia. Preliminary data suggest that Tax(1) expression is sufficient to up-regulate the proinflammatory cytokine profile in explanted human microglial cells. Future experiments will compare and evaluate the effect of tax(1) and tax(2) gene expression on the cellular proinflammatory cytokine expression profile, as well as demonstrate the effects of transducing human fetal astrocytes and PBMC-derived macrophages.

  16. Glycoprotein Ibalpha promoter drives megakaryocytic lineage-restricted expression after hematopoietic stem cell transduction using a self-inactivating lentiviral vector.

    PubMed

    Lavenu-Bombled, Cécile; Izac, Brigitte; Legrand, Faézeh; Cambot, Marie; Vigier, Agathe; Massé, Jean-Marc; Dubart-Kupperschmitt, Anne

    2007-06-01

    Megakaryocytic (MK) lineage is an attractive target for cell/gene therapy approaches, aiming at correcting platelet protein deficiencies. However, MK cells are short-lived cells, and their permanent modification requires modification of hematopoietic stem cells with an integrative vector such as a lentiviral vector. Glycoprotein (Gp) IIb promoter, the most studied among the MK regulatory sequences, is also active in stem cells. To strictly limit transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells with a lentiviral vector, we looked for a promoter activated later during MK differentiation. Human cord blood, bone marrow, and peripheral-blood mobilized CD34(+) cells were transduced with a human immunodeficiency virus-derived self-inactivating lentiviral vector encoding the green fluorescent protein (GFP) under the transcriptional control of GpIbalpha, GpIIb, or EF1alpha gene regulatory sequences. Both GpIbalpha and GpIIb promoters restricted GFP expression (analyzed by flow cytometry and immunoelectron microscopy) in MK cells among the maturing progeny of transduced cells. However, only the GpIbalpha promoter was strictly MK-specific, whereas GpIIb promoter was leaky in immature progenitor cells not yet engaged in MK cell lineage differentiation. We thus demonstrate the pertinence of using a 328-base-pair fragment of the human GpIbalpha gene regulatory sequence, in the context of a lentiviral vector, to tightly restrict transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells. Disclosure of potential conflicts of interest is found at the end of this article.

  17. 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.

  18. A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors: useful for making iPS cells and transduction of primary cells.

    PubMed

    Yang, Shaozhe; Shi, Haijun; Chu, Xinran; Zhou, Xiaoling; Sun, Pingnan

    2016-09-01

    To improve the efficiency, reproducibility and consistency of the PEI-based transfection method that is often used in preparation of recombinant lentiviral or retroviral vectors. The contributions to transfection efficiency of multi-factors including concentration of PEI or DNA, dilution buffer for PEI/DNA, manner to prepare PEI/DNA complexes, influence of serum, incubation time for PEI/DNA complexes, and transfection time were studied. Gentle mixing during the preparation of PEI/DNA transfection complexes is critical for a high transfection efficiency. PEI could be stored at room temperature or 4 °C, and most importantly, multigelation should be avoided. The transfection efficiency of the PEI-based new method in different types of cells, such as 293T, Cos-7, HeLa, HepG2, Hep3B, Huh7 and L02, was also higher than that of the previous method. After optimization, the titer of our lentiviral system or retroviral system produced by PEI-based new method was about 10- or 3-times greater than that produced by PEI-based previous method, respectively. We provide a rapid and efficient PEI-based method for preparation of recombinant lentiviral or retroviral vectors which is useful for making iPS cells as well as transduction of primary cell cultures.

  19. Low-Affinity Neurotrophin Receptor p75 Promotes the Transduction of Targeted Lentiviral Vectors to Cholinergic Neurons of Rat Basal Forebrain.

    PubMed

    Antyborzec, Inga; O'Leary, Valerie B; Dolly, James O; Ovsepian, Saak V

    2016-10-01

    Basal forebrain cholinergic neurons (BFCNs) are one of the most affected neuronal types in Alzheimer's disease (AD), with their extensive loss documented at late stages of the pathology. While discriminatory provision of neuroprotective agents and trophic factors to these cells is thought to be of substantial therapeutic potential, the intricate topography and structure of the forebrain cholinergic system imposes a major challenge. To overcome this, we took advantage of the physiological enrichment of BFCNs with a low-affinity p75 neurotrophin receptor (p75(NTR)) for their targeting by lentiviral vectors within the intact brain of adult rat. Herein, a method is described that affords selective and effective transduction of BFCNs with a green fluorescence protein (GFP) reporter, which combines streptavidin-biotin technology with anti-p75(NTR) antibody-coated lentiviral vectors. Specific GFP expression in cholinergic neurons was attained in the medial septum and nuclei of the diagonal band Broca after a single intraventricular administration of such targeted vectors. Bioelectrical activity of GFP-labeled neurons was proven to be unchanged. Thus, proof of principle is obtained for the utility of the low-affinity p75(NTR) for targeted transduction of vectors to BFCNs in vivo.

  20. The β-globin locus control region in combination with the EF1α short promoter allows enhanced lentiviral vector-mediated erythroid gene expression with conserved multilineage activity.

    PubMed

    Montiel-Equihua, Claudia A; Zhang, Lin; Knight, Sean; Saadeh, Heba; Scholz, Simone; Carmo, Marlene; Alonso-Ferrero, Maria E; Blundell, Michael P; Monkeviciute, Aiste; Schulz, Reiner; Collins, Mary; Takeuchi, Yasuhiro; Schmidt, Manfred; Fairbanks, Lynette; Antoniou, Michael; Thrasher, Adrian J; Gaspar, H Bobby

    2012-07-01

    Some gene therapy strategies are compromised by the levels of gene expression required for therapeutic benefit, and also by the breadth of cell types that require correction. We designed a lentiviral vector system in which a transgene is under the transcriptional control of the short form of constitutively acting elongation factor 1α promoter (EFS) combined with essential elements of the locus control region of the β-globin gene (β-LCR). We show that the β-LCR can upregulate EFS activity specifically in erythroid cells but does not alter EFS activity in myeloid or lymphoid cells. Experiments using the green fluorescent protein (GFP) reporter or the human adenosine deaminase (ADA) gene demonstrate 3-7 times upregulation in vitro but >20 times erythroid-specific upregulation in vivo, the effects of which were sustained for 1 year. The addition of the β-LCR did not alter the mutagenic potential of the vector in in vitro mutagenesis (IM) assays although microarray analysis showed that the β-LCR upregulates ~9% of neighboring genes. This vector design therefore combines the benefits of multilineage gene expression with high-level erythroid expression, and has considerable potential for correction of multisystem diseases including certain lysosomal storage diseases through a hematopoietic stem cell (HSC) gene therapy approach.

  1. 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.

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

    PubMed Central

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

    2015-01-01

    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

  3. Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized, purified hematopoietic stem cells.

    PubMed

    Levasseur, Dana N; Ryan, Thomas M; Pawlik, Kevin M; Townes, Tim M

    2003-12-15

    Although sickle cell anemia was the first hereditary disease to be understood at the molecular level, there is still no adequate long-term treatment. Allogeneic bone marrow transplantation is the only available cure, but this procedure is limited to a minority of patients with an available, histocompatible donor. Autologous transplantation of bone marrow stem cells that are transduced with a stably expressed, antisickling globin gene would benefit a majority of patients with sickle cell disease. Therefore, the development of a gene therapy protocol that corrects the disease in an animal model and is directly translatable to human patients is critical. A method is described in which unmobilized, highly purified bone marrow stem cells are transduced with a minimum amount of self-inactivating (SIN) lentiviral vector containing a potent antisickling beta-globin gene. These cells, which were transduced in the absence of cytokine stimulation, fully reconstitute irradiated recipients and correct the hemolytic anemia and organ pathology that characterize the disease in humans. The mean increase of hemoglobin concentration was 46 g/L (4.6 g/dL) and the average lentiviral copy number was 2.2; therefore, a 21-g/L /vector copy increase (2.1-g/dL) was achieved. This transduction protocol may be directly translatable to patients with sickle cell disease who cannot tolerate current bone marrow mobilization procedures and may not safely be exposed to large viral loads.

  4. Generation of β cell-specific human cytotoxic T cells by lentiviral transduction and their survival in immunodeficient human leucocyte antigen-transgenic mice

    PubMed Central

    Babad, J; Mukherjee, G; Follenzi, A; Ali, R; Roep, B O; Shultz, L D; Santamaria, P; Yang, O O; Goldstein, H; Greiner, D L; DiLorenzo, T P

    2015-01-01

    Several β cell antigens recognized by T cells in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D) are also T cell targets in the human disease. While numerous antigen-specific therapies prevent diabetes in NOD mice, successful translation of rodent findings to patients has been difficult. A human leucocyte antigen (HLA)-transgenic mouse model incorporating human β cell-specific T cells might provide a better platform for evaluating antigen-specific therapies. The ability to study such T cells is limited by their low frequency in peripheral blood and the difficulty in obtaining islet-infiltrating T cells from patients. We have worked to overcome this limitation by using lentiviral transduction to ‘reprogram’ primary human CD8 T cells to express three T cell receptors (TCRs) specific for a peptide derived from the β cell antigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP265–273) and recognized in the context of the human class I major histocompatibility complex (MHC) molecule HLA-A2. The TCRs bound peptide/MHC multimers with a range of avidities, but all bound with at least 10-fold lower avidity than the anti-viral TCR used for comparison. One exhibited antigenic recognition promiscuity. The β cell-specific human CD8 T cells generated by lentiviral transduction with one of the TCRs released interferon (IFN)-γ in response to antigen and exhibited cytotoxic activity against peptide-pulsed target cells. The cells engrafted in HLA-A2-transgenic NOD-scid IL2rγnull mice and could be detected in the blood, spleen and pancreas up to 5 weeks post-transfer, suggesting the utility of this approach for the evaluation of T cell-modulatory therapies for T1D and other T cell-mediated autoimmune diseases. PMID:25302633

  5. Efficient Transduction of Human and Rhesus Macaque Primary T Cells by a Modified Human Immunodeficiency Virus Type 1-Based Lentiviral Vector.

    PubMed

    He, Huan; Xue, Jing; Wang, Weiming; Liu, Lihong; Ye, Chaobaihui; Cong, Zhe; Kimata, Jason T; Qin, Chuan; Zhou, Paul

    2017-03-01

    Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors efficiently transduce genes to human, but not rhesus, primary T cells and hematopoietic stem cells (HSCs). The poor transduction of HIV-1 vectors to rhesus cells is mainly due to species-specific restriction factors such as rhesus TRIM5α. Previously, several strategies to modify HIV-1 vectors were developed to overcome rhesus TRIM5α restriction. While the modified HIV-1 vectors efficiently transduce rhesus HSCs, they remain suboptimal for rhesus primary T cells. Recently, HIV-1 variants that encode combinations of LNEIE mutations in capsid (CA) protein and SIVmac239 Vif were found to replicate efficiently in rhesus primary T cells. Thus, the present study tested whether HIV-1 vectors packaged by a packaging construct containing these CA substitutions could efficiently transduce both human and rhesus primary CD4 T cells. To accomplish this, LNEIE mutations were made in the packaging construct CEMΔ8.9, and recombinant HIV-1 vectors packaged by Δ8.9 WT or Δ8.9 LNEIE were generated. Transduction rates, CA stability, and vector integration in CEMss-CCR5 and CEMss-CCR5-rhTRIM5α/green fluorescent protein cells, as well as transduction rates in human and rhesus primary CD4 T cells by Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors, were compared. Finally, the influence of rhesus TRIM5α variations in transduction rates to primary CD4 T cells from a cohort of 37 Chinese rhesus macaques was studied. While it maintains efficient transduction for human T-cell line and primary CD4 T cells, Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α-mediated CA degradation, resulting in significantly higher transduction efficiency of rhesus primary CD4 T cells than Δ8.9 WT-packaged HIV-1 vector. Rhesus TRIM5α variations strongly influence transduction efficiency of rhesus primary CD4 T cells by both Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors. Thus, it is concluded that Δ8.9 LNEIE-packaged HIV-1

  6. Fetal gene transfer using lentiviral vectors and the potential for germ cell transduction in rhesus monkeys (Macaca mulatta).

    PubMed

    Lee, C Chang I; Jimenez, Daniel F; Kohn, Donald B; Tarantal, Alice F

    2005-04-01

    Genetic modification of germ cells in somatic gene therapy protocols is a concern, particularly with fetal approaches. This study focused on the potential for germ cell gene transfer post-fetal gene delivery using a human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vector pseudotyped with the vesicular stomatitis virus-glycoprotein (VSV-G). Rhesus monkey fetuses (n = 47) were administered vector supernatant (10(7) infectious particles per fetus) via the intraperitoneal (IP), intrapulmonary (Ipu), or intracardiac routes (Ica) under ultrasound guidance. Tissue harvests were performed near term or 3 months postnatal age, and genomic DNA obtained to analyze for vector sequences from collected sections of gonads and gonadal cells obtained by laser capture microdissection (germ cells, stroma, epithelium). Results indicated no evidence of germ cell gene transfer in fetuses or infants with Ipu or Ica routes of administration. However, evidence of the transgene (1.33 +/- 0.78 enhanced green fluorescent protein [EGFP] copies per copy epsilon-globin) was found in females, but not males, when using the IP administration approach (p < 0.05). The highest EGFP copies were detected on the surface epithelium (p < 0.05). The results of these studies suggest that the HIV-1-derived lentiviral vector pseudotyped with VSV-G may transduce a subpopulation of gonadal cells in female fetuses with IP administration, whereas no evidence of gene transfer was shown to occur in males or with organ-targeting approaches.

  7. Immunization of Mice with Lentiviral Vectors Targeted to MHC Class II+ Cells Is Due to Preferential Transduction of Dendritic Cells In Vivo

    PubMed Central

    Ciré, Séverine; Da Rocha, Sylvie; Yao, Roseline; Fisson, Sylvain; Buchholz, Christian J.; Collins, Mary K.; Galy, Anne

    2014-01-01

    Gene transfer vectors such as lentiviral vectors offer versatile possibilities to express transgenic antigens for vaccination purposes. However, viral vaccines leading to broad transduction and transgene expression in vivo, are undesirable. Therefore, strategies capable of directing gene transfer only to professional antigen-presenting cells would increase the specific activity and safety of genetic vaccines. A lentiviral vector pseudotype specific for murine major histocompatibilty complex class II (LV-MHCII) was recently developed and the present study aims to characterize the in vivo biodistribution profile and immunization potential of this vector in mice. Whereas the systemic administration of a vector pseudotyped with a ubiquitously-interacting envelope led to prominent detection of vector copies in the liver of animals, the injection of an equivalent amount of LV-MHCII resulted in a more specific biodistribution of vector and transgene. Copies of LV-MHCII were found only in secondary lymphoid organs, essentially in CD11c+ dendritic cells expressing the transgene whereas B cells were not efficiently targeted in vivo, contrary to expectations based on in vitro testing. Upon a single injection of LV-MHCII, naive mice mounted specific effector CD4 and CD8 T cell responses against the intracelllular transgene product with the generation of Th1 cytokines, development of in vivo cytotoxic activity and establishment of T cell immune memory. The targeting of dendritic cells by recombinant viral vaccines must therefore be assessed in vivo but this strategy is feasible, effective for immunization and cross-presentation and constitutes a potentially safe alternative to limit off-target gene expression in gene-based vaccination strategies with integrative vectors. PMID:25058148

  8. Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection

    PubMed Central

    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 CD8pos and also CD4pos 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

  9. Lentiviral transduction of primary myeloma cells with CD80 and CD154 generates antimyeloma effector T cells.

    PubMed

    Cignetti, Alessandro; Vallario, Antonella; Follenzi, Antonia; Circosta, Paola; Capaldi, Antonio; Gottardi, Daniela; Naldini, Luigi; Caligaris-Cappio, Federico

    2005-04-01

    The development of immunotherapy approaches designed to obtain tumor-specific T cells might help eradicate residual malignant cells in multiple myeloma (MM) patients. To this end, we used autologous primary MM cells as antigen-presenting cells (APC). Gene transfer of both CD80 and CD154 by lentiviral vectors was necessary to significantly improve the APC function of human MM cells. Simultaneous CD80/CD154 expression on MM cells allowed the generation of CD8+ T cells that recognized unmodified MM cells in 11 of 16 cases, specifically in six of six patients with low-stage disease, but only in five of ten patients with advanced disease. The activity of CD8+ T cells was MHC restricted and MM specific. In seven of seven cases, CD8+ T cell activity was inhibited by monoclonal antibodies against HLA class I, and in four of four cases, CD8+ T cells recognized autologous MM cells but not autologous normal B and T lymphocytes nor bone marrow stromal cells. In addition, the activity of CD8+ T cells was directed against allogeneic MM cells that shared at least one MHC allele with the autologous counterpart, but not against MHC mismatched MM cells. These data lay the ground for the isolation of new MM antigens and for the design of vaccination protocols with primary MM cells genetically engineered to express immunostimulatory molecules.

  10. 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.

  11. Lentiviral vectors displaying modified measles virus gp overcome pre-existing immunity in in vivo-like transduction of human T and B cells.

    PubMed

    Lévy, Camille; Amirache, Fouzia; Costa, Caroline; Frecha, Cecilia; Muller, Claude P; Kweder, Hasan; Buckland, Robin; Cosset, François-Loïc; Verhoeyen, Els

    2012-09-01

    Gene transfer into quiescent T and B cells is important for gene therapy and immunotherapy approaches. Previously, we generated lentiviral vectors (LVs) pseudotyped with Edmonston (Ed) measles virus (MV) hemagglutinin (H) and fusion (F) glycoproteins (H/F-LVs), which allowed efficient transduction of quiescent human T and B cells. However, a major obstacle in the use of H/F-LVs in vivo is that most of the human population is vaccinated against measles. As the MV humoral immune response is exclusively directed against the H protein of MV, we mutated the two dominant epitopes in H, Noose, and NE. LVs pseudotyped with these mutant H-glycoproteins escaped inactivation by monoclonal antibodies (mAbs) but were still neutralized by human serum. Consequently, we took advantage of newly emerged MV-D genotypes that were less sensitive to MV vaccination due to a different glycosylation pattern. The mutation responsible was introduced into the H/F-LVs, already mutated for Noose and NE epitopes. We found that these mutant H/F-LVs could efficiently transduce quiescent lymphocytes in the presence of high concentrations of MV antibody-positive human serum. Finally, upon incubation with total blood, mimicking the in vivo situation, the mutant H/F-LVs escaped MV antibody neutralization, where the original H/F-LVs failed. Thus, these novel H/F-LVs offer perspectives for in vivo lymphocyte-based gene therapy and immunotherapy.

  12. Novel lentiviral vectors displaying "early-acting cytokines" selectively promote survival and transduction of NOD/SCID repopulating human hematopoietic stem cells.

    PubMed

    Verhoeyen, Els; Wiznerowicz, Maciej; Olivier, Delphine; Izac, Brigitte; Trono, Didier; Dubart-Kupperschmitt, Anne; Cosset, François-Loïc

    2005-11-15

    A major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells, such as human CD34(+) cells, that reside in the G(0) phase of the cell cycle and that are highly enriched in hematopoietic stem cells. This hampers their application for gene therapy of hematopoietic cells. Here, we designed novel LVs that overcome this restriction by displaying "early-acting cytokines" on their surface. Display of thrombopoietin, stem cell factor, or both cytokines on the LV surface allowed efficient gene delivery into quiescent cord blood CD34(+) cells. Moreover, these surface-engineered LVs preferentially transduced and promoted survival of resting CD34(+) cells rather than cycling cells. Finally, and most importantly, these novel LVs allowed superior gene transfer in the most immature CD34(+) cells as compared to conventional LVs, even when the latter vectors were used to transduce cells in the presence of recombinant cytokines. This was demonstrated by their capacity to promote selective transduction of CD34(+) cell in in vitro derived long-term culture-initiating cell (LTC-IC) colonies and of long-term NOD/SCID repopulating cells (SRCs) in vivo.

  13. Transduction of photoreceptors with equine infectious anemia virus lentiviral vectors: safety and biodistribution of StarGen for Stargardt disease.

    PubMed

    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

    2013-06-12

    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. 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. 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. In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.

  14. 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

  15. Further reduction in adenovirus vector-mediated liver transduction without largely affecting transgene expression in target organ by exploiting microrna-mediated regulation and the Cre-loxP recombination system.

    PubMed

    Bennett, David; Sakurai, Fuminori; Shimizu, Kahori; Matsui, Hayato; Tomita, Kyoko; Suzuki, Takayuki; Katayama, Kazufumi; Kawabata, Kenji; Mizuguchi, Hiroyuki

    2012-12-03

    In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into the 3'-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3'-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen.

  16. Lentiviral vectors pseudotyped with a modified RD114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and CD34+ cells derived from human and nonhuman primates.

    PubMed

    Sandrin, Virginie; Boson, Bertrand; Salmon, Patrick; Gay, Wilfried; Nègre, Didier; Le Grand, Roger; Trono, Didier; Cosset, François-Loïc

    2002-08-01

    Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors, their interaction with the host immune system, and their host range. We have investigated the capacity of a panel of GPs of both retroviral (amphotropic murine leukemia virus [MLV-A]; gibbon ape leukemia virus [GALV]; RD114, feline endogenous virus) and nonretroviral (fowl plague virus [FPV]; Ebola virus [EboV]; vesicular stomatitis virus [VSV]; lymphocytic choriomeningitis virus [LCMV]) origins to pseudotype lentiviral vectors derived from simian immunodeficiency virus (SIVmac251). SIV vectors were efficiently pseudotyped with the FPV hemagglutinin, VSV-G, LCMV, and MLV-A GPs. In contrast, the GALV and RD114 GPs conferred much lower infectivity to the vectors. Capitalizing on the conservation of some structural features in the transmembrane domains and cytoplasmic tails of the incorporation-competent MLV-A GP and in RD114 and GALV GPs, we generated chimeric GPs encoding the extracellular and transmembrane domains of GALV or RD114 GPs fused to the cytoplasmic tail (designated TR) of MLV-A GP. Importantly, SIV-derived vectors pseudotyped with these GALV/TR and RD114/TR GP chimeras had significantly higher titers than vectors coated with the parental GPs. Additionally, RD114/TR-pseudotyped vectors were efficiently concentrated and were resistant to inactivation induced by the complement of both human and macaque sera, indicating that modified RD114 GP-pseudotyped lentiviral vectors may be of particular interest for in vivo gene transfer applications. Furthermore, as compared to vectors pseudotyped with other retroviral GPs or with VSV-G, RD114/TR-pseudotyped vectors showed augmented transduction of human and macaque primary blood lymphocytes and CD34+ cells.

  17. In vivo transduction by intravenous injection of a lentiviral vector expressing human ADA into neonatal ADA gene knockout mice: a novel form of enzyme replacement therapy for ADA deficiency.

    PubMed

    Carbonaro, Denise A; Jin, Xiangyang; Petersen, Denise; Wang, Xingchao; Dorey, Fred; Kil, Ki Soo; Aldrich, Melissa; Blackburn, Michael R; Kellems, Rodney E; Kohn, Donald B

    2006-06-01

    Using a mouse model of adenosine deaminase-deficient severe combined immune deficiency syndrome (ADA-deficient SCID), we have developed a noninvasive method of gene transfer for the sustained systemic expression of human ADA as enzyme replacement therapy. The method of delivery is a human immunodeficiency virus 1-based lentiviral vector given systemically by intravenous injection on day 1 to 2 of life. In this article we characterize the biodistribution of the integrated vector, the expression levels of ADA enzyme activity in various tissues, as well as the efficacy of systemic ADA expression to correct the ADA-deficient phenotype in this mouse model. The long-term expression of enzymatically active ADA achieved by this method, primarily from transduction of liver and lung, restored immunologic function and significantly extended survival. These studies illustrate the potential for sustained in vivo production of enzymatically active ADA, as an alternative to therapy by frequent injection of exogenous ADA protein.

  18. TRIM5α variations influence transduction efficiency with lentiviral vectors in both human and rhesus CD34(+) cells in vitro and in vivo.

    PubMed

    Evans, Molly E; Kumkhaek, Chutima; Hsieh, Matthew M; Donahue, Robert E; Tisdale, John F; Uchida, Naoya

    2014-02-01

    Human immunodeficiency virus type 1 (HIV-1) vectors can transduce human hematopoietic stem cells (HSC), but transduction efficiency varies among individuals. The innate immune factor tripartite motif-containing protein 5α (TRIM5α) plays an important role for restriction of retroviral infection. In this study, we examined whether TRIM5α could account for variations in transduction efficiency using both an established rhesus gene therapy model and human CD34(+) cell culture. Evaluation of TRIM5α genotypes (Mamu-1, -2, -3, -4, -5, and TrimCyp) in 16 rhesus macaques that were transplanted with transduced CD34(+) cells showed a significant correlation between TRIM5α Mamu-4 and high gene marking in both lymphocytes and granulocytes 6 months after transplantation. Since significant human TRIM5α coding polymorphisms were not known, we evaluated TRIM5α expression levels in human CD34(+) cells from 14 donors. Three days after HIV-1 vector transduction, measured transduction efficiency varied significantly among donors and was negatively correlated with TRIM5α expression levels. In summary, transduction efficiency in both rhesus and human CD34(+) cells was influenced by TRIM5α variations (genotypes and expression levels). Our findings are important for both understanding and mitigating the variability of transduction efficiency for rhesus and human CD34(+) cells.

  19. Transduction of Human Antigen-Presenting Cells with Integrase-Defective Lentiviral Vector Enables Functional Expansion of Primed Antigen-Specific CD8+ T Cells

    PubMed Central

    Bona, Roberta; Michelini, Zuleika; Leone, Pasqualina; Macchia, Iole; Klotman, Mary E.; Salvatore, Mirella

    2010-01-01

    Abstract Nonintegrating lentiviral vectors are being developed as a efficient and safe delivery system for both gene therapy and vaccine purposes. Several reports have demonstrated that a single immunization with integration-defective lentiviral vectors (IDLVs) delivering viral or tumor model antigens in mice was able to elicit broad and long-lasting specific immune responses in the absence of vector integration. At present, no evidence has been reported showing that IDLVs are able to expand preexisting immune responses in the human context. In the present study, we demonstrate that infection of human antigen-presenting cells (APCs), such as monocyte-derived dendritic cells (DCs) and macrophages with IDLVs expressing influenza matrix M1 protein resulted in effective induction of in vitro expansion of M1-primed CD8+ T cells, as evaluated by both pentamer staining and cytokine production. This is the first demonstration that IDLVs represent an efficient delivery system for gene transfer and expression in human APCs, useful for immunotherapeutic applications. PMID:20210625

  20. Imaging herpes simplex virus type 1 amplicon vector-mediated gene expression in human glioma spheroids.

    PubMed

    Kaestle, Christine; Winkeler, Alexandra; Richter, Raphaela; Sauer, Heinrich; Hescheler, Jürgen; Fraefel, Cornel; Wartenberg, Maria; Jacobs, Andreas H

    2011-06-01

    Vectors derived from herpes simplex virus type 1 (HSV-1) have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector-mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fluorescent protein (HSV-GFP). After infection or microscopy-guided vector injection of glioma spheroids at various spheroid sizes, injection pressures and injection times, the extent of HSV-1 vector-mediated gene expression was investigated via laser scanning microscopy. Infection of spheroids with HSV-GFP demonstrated a maximal depth of vector-mediated GFP expression at 70 to 80 μm. A > 80% transduction efficiency was reached only in small spheroids with a diameter of < 150 μm. Guided vector injection into the spheroids showed transduction efficiencies ranging between < 10 and > 90%. The results demonstrated that vector-mediated gene expression in glioma spheroids was strongly dependent on the mode of vector application-injection pressure and injection time being the most important parameters. The assessment of these vector application parameters in tissue models will contribute to the development of safe and efficient gene therapy protocols for clinical application.

  1. High Efficiency Gene Transfer to Airways of Mice Using Influenza Hemagglutinin Pseudotyped Lentiviral Vectors

    PubMed Central

    Patel, Manij; Giddings, Angela M.; Sechelski, John; Olsen, John C.

    2014-01-01

    Background A limitation to efficient lentivirus-mediated airway gene transfer is the lack of receptors to commonly used viral envelopes on the luminal surface of airway epithelia. The use of viral envelopes with natural tropism to the airway could be useful for overcoming this limitation. Methods We investigated influenza hemagglutinin (HA) pseudotyped EIAV-derived lentiviral vector-mediated gene transfer to the airway epithelium of adult and newborn mice. For these studies high-titer vectors were delivered by intranasal administration. In addition, we tested the feasibility of vector re-dosing to the nasal airway. Results Delivery of high-titer HA pseudotyped lentiviral vectors by nasal administration to newborn mouse pups or adult mice results in efficient transduction of airway epithelial cells in the nose, trachea, and lungs. In the nose vector expression was predominant in the respiratory epithelium and was not observed in the olfactory epithelium. In the trachea and large airways of the lung approximately 46% and 40%, respectively, of surface epithelial cells could be transduced. The efficiency of re-dosing to the nasal airway of mice was found to be dependent upon the age of the animal when the first dose is administered and the length of time between doses. Conclusions A single intranasal dose of concentrated influenza HA-pseudotyped lentiviral vector is sufficient for efficient gene transfer to the airways of mice. This is a promising result that could lead to the development of effective gene transfer reagents for the treatment of cystic fibrosis and other human lung diseases. PMID:23319179

  2. Vector-Mediated In Vivo Antibody Expression.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis.

  3. Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors

    PubMed Central

    Oakland, Mayumi; Maury, Wendy; McCray, Paul B; Sinn, Patrick L

    2013-01-01

    Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN) responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV) in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction. PMID:23360952

  4. 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.

  5. Vector-mediated antibody gene transfer for infectious diseases.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2015-01-01

    This chapter discusses the emerging field of vector-mediated antibody gene transfer as an alternative vaccine for infectious disease, with a specific focus on HIV. However, this methodology need not be confined to HIV-1; the general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets like hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. This approach is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. With vector-mediated gene transfer, the antibody gene is delivered to the host, via a recombinant adeno-associated virus (rAAV) vector; this in turn results in long-term endogenous antibody expression from the injected muscle that confers protective immunity. Vector-mediated antibody gene transfer can rapidly move existing, potent broadly cross-neutralizing HIV-1-specific antibodies into the clinic. The gene transfer products demonstrate a potency and breadth identical to the original product. This strategy eliminates the need for immunogen design and interaction with the adaptive immune system to generate protection, a strategy that so far has shown limited promise.

  6. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector.

    PubMed

    Nanba, Daisuke; Matsushita, Natsuki; Toki, Fujio; Higashiyama, Shigeki

    2013-10-18

    The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine.

  7. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

    PubMed Central

    2013-01-01

    Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

  8. Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions.

    PubMed

    ter Brake, Olivier; Berkhout, Ben

    2007-09-01

    HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production.

  9. Five recombinant simian immunodeficiency virus pseudotypes lead to exclusive transduction of retinal pigmented epithelium in rat.

    PubMed

    Duisit, Ghislaine; Conrath, Hervé; Saleun, Sylvie; Folliot, Sebastien; Provost, Nathalie; Cosset, François-Loïc; Sandrin, Virginie; Moullier, Philippe; Rolling, Fabienne

    2002-10-01

    The purpose of our study was to evaluate lentiviral vector-mediated rat retinal transduction using simian immunodeficiency virus (SIV) pseudotyped with envelope proteins from vesicular stomatitis virus G glycoprotein (VSV-G), Mokola virus G protein (MK-G), amphotropic murine leukemia virus envelope (4070A-Env), influenza A virus hemagglutinin (HA), lymphocytic choriomeningitis virus G protein (LCMV-G), and RD114 retrovirus envelope (RD114-Env). The six pseudotyped lentivirus vectors carried CMV-driven green fluorescent protein (GFP) or beta-galactosidase (beta-gal) reporter genes. Intravitreal and subretinal injections of each pseudotyped recombinant SIV were performed in cohorts of Wistar rats. Our results showed that no transgene expression was detected after intravitreal injection of each pseudotyped SIV vector. Also, no transduction could be detected following subretinal injection of RD114 pseudotyped SIV vectors. However, selective transduction of retinal pigment epithelium (RPE) cells was repeatedly obtained after subretinal delivery of VSV-G, MK-G, 4070A-Env, HA, and LCMV-G pseudotyped SIV. GFP expression was maximum as soon as 4 days postadministration for VSV-G, MK-G, 4070A-Env, and HA pseudotypes, with no evidence of pseudotransduction for VSV-G. Maximum transgene expression was observed 3 weeks postinjection for LCMV-6. Importantly, HA and VSV-G pseudotyped SIV lead to such a high level of transgene expression that GFP-related toxicity occurred. Therefore, when a high level of GFP synthesis is achieved, replacement of enhanced GFP (egfp, Aequorea victoria) by a low-toxicity GFP (Renilla reniformis) cDNA is necessary to allow long-term expression.

  10. Computational model of a vector-mediated epidemic

    NASA Astrophysics Data System (ADS)

    Dickman, Adriana Gomes; Dickman, Ronald

    2015-05-01

    We discuss a lattice model of vector-mediated transmission of a disease to illustrate how simulations can be applied in epidemiology. The population consists of two species, human hosts and vectors, which contract the disease from one another. Hosts are sedentary, while vectors (mosquitoes) diffuse in space. Examples of such diseases are malaria, dengue fever, and Pierce's disease in vineyards. The model exhibits a phase transition between an absorbing (infection free) phase and an active one as parameters such as infection rates and vector density are varied.

  11. 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

  12. 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

  13. Immune response to lentiviral bilirubin UDP-glucuronosyltransferase gene transfer in fetal and neonatal rats.

    PubMed

    Seppen, J; van Til, N P; van der Rijt, R; Hiralall, J K; Kunne, C; Elferink, R P J Oude

    2006-04-01

    Gene therapy for inherited disorders might cause an immune response to the therapeutic protein. A solution would be to introduce the gene in the fetal or neonatal period, which should lead to tolerization. Lentiviral vectors mediate long-term gene expression, and are well suited for gene therapy early in development. A model for fetal or neonatal gene therapy is the inherited disorder of bilirubin metabolism, Crigler-Najjar disease (CN). The absence of bilirubin UDP-glucoronyltransferase (UGT1A1) activity in CN patients causes high serum levels of unconjugated bilirubin and brain damage in infancy. CN is attractive for the development of gene therapy because the mutant Gunn rat closely mimics the human disease. Injection of UGT1A1 lentiviral vectors corrected the hyperbilirubinemia for more than a year in rats injected as fetuses and for up to 18 weeks in rats injected the day of birth. UGT1A1 gene transfer was confirmed by the presence of bilirubin glucuronides in bile. All animals injected with UGT1A1 lentiviral vectors developed antibodies to UGT1A1. Animals injected with green fluorescent protein (GFP) lentiviral vectors did not develop antibodies to GFP. Our results indicate that fetal and neonatal gene therapy with immunogenic proteins such as UGT1A1 does not necessarily lead to tolerization.

  14. 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

  15. Impaired clearance of accumulated lysosomal glycogen in advanced Pompe disease despite high-level vector-mediated transgene expression

    PubMed Central

    Sun, Baodong; Zhang, Haoyue; Bird, Andrew; Li, Songtao; Young, Sarah P.; Koeberl, Dwight D.

    2013-01-01

    Background Infantile-onset glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) causes death early in childhood from cardiorespiratory failure in absence of effective treatment, whereas late-onset Pompe disease causes a progressive skeletal myopathy. The limitations of enzyme replacement therapy could potentially be addressed with adeno-associated virus (AAV) vector-mediated gene therapy. Methods AAV vectors containing tissue-specific regulatory cassettes, either liver-specific or muscle-specific, were administered to 12 and 17 month old Pompe disease mice to evaluate the efficacy of gene therapy in advanced Pompe disease. Biochemical correction was evaluated through GAA activity and glycogen content analyses of the heart and skeletal muscle. Western blotting, urinary biomarker, and Rotarod performance were evaluated following vector administration. Results The AAV vector containing the liver-specific regulatory cassette secreted high-level hGAA into the blood and corrected glycogen storage in the heart and diaphragm. The biochemical correction of the heart and diaphragm was associated with efficacy, as reflected by increased Rotarod performance; however, the clearance of glycogen from skeletal muscles was relatively impaired, in comparison with younger Pompe disease mice. An alternative vector containing a muscle-specific regulatory cassette transduced skeletal muscle with high efficiency, but also failed to achieve complete clearance of accumulated glycogen. Decreased transduction of the heart and liver in older mice, especially in females, was implicated as a cause for reduced efficacy in advanced Pompe disease. Conclusion The impaired efficacy of AAV vector-mediated gene therapy in old Pompe disease mice emphasized the need for early treatment to achieve full efficacy. PMID:19621331

  16. Biosafety Features of Lentiviral Vectors

    PubMed Central

    Schambach, Axel; Zychlinski, Daniela; Ehrnstroem, Birgitta

    2013-01-01

    Abstract Over the past decades, lentiviral vectors have evolved as a benchmark tool for stable gene transfer into cells with a high replicative potential. Their relatively flexible genome and ability to transduce many forms of nondividing cells, combined with the potential for cell-specific pseudotyping, provides a rich resource for numerous applications in experimental platforms and therapeutic settings. Here, we give an overview of important biosafety features of lentiviral vectors, with detailed discussion of (i) the principles of the lentiviral split-genome design used for the construction of packaging cells; (ii) the relevance of modifications introduced into the lentiviral long terminal repeat (deletion of enhancer/promoter sequences and introduction of insulators); (iii) the basic features of mRNA processing, including the Rev/Rev-responsive element (RRE) interaction and the modifications of the 3′ untranslated region of lentiviral vectors with various post-transcriptional regulatory elements affecting transcriptional termination, polyadenylation, and differentiation-specific degradation of mRNA; and (iv) the characteristic integration pattern with the associated risk of transcriptional interference with cellular genes. We conclude with considerations regarding the importance of cell targeting via envelope modifications. Along this course, we address canonical biosafety issues encountered with any type of viral vector: the risks of shedding, mobilization, germline transmission, immunogenicity, and insertional mutagenesis. PMID:23311447

  17. Biosafety features of lentiviral vectors.

    PubMed

    Schambach, Axel; Zychlinski, Daniela; Ehrnstroem, Birgitta; Baum, Christopher

    2013-02-01

    Over the past decades, lentiviral vectors have evolved as a benchmark tool for stable gene transfer into cells with a high replicative potential. Their relatively flexible genome and ability to transduce many forms of nondividing cells, combined with the potential for cell-specific pseudotyping, provides a rich resource for numerous applications in experimental platforms and therapeutic settings. Here, we give an overview of important biosafety features of lentiviral vectors, with detailed discussion of (i) the principles of the lentiviral split-genome design used for the construction of packaging cells; (ii) the relevance of modifications introduced into the lentiviral long terminal repeat (deletion of enhancer/promoter sequences and introduction of insulators); (iii) the basic features of mRNA processing, including the Rev/Rev-responsive element (RRE) interaction and the modifications of the 3' untranslated region of lentiviral vectors with various post-transcriptional regulatory elements affecting transcriptional termination, polyadenylation, and differentiation-specific degradation of mRNA; and (iv) the characteristic integration pattern with the associated risk of transcriptional interference with cellular genes. We conclude with considerations regarding the importance of cell targeting via envelope modifications. Along this course, we address canonical biosafety issues encountered with any type of viral vector: the risks of shedding, mobilization, germline transmission, immunogenicity, and insertional mutagenesis.

  18. Intrastriatal Delivery of Integration-Deficient Lentiviral Vectors in a Rat Model of Parkinson's Disease.

    PubMed

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

    2016-01-01

    Standard integration-proficient lentiviral vectors (IPLVs) are effective at much lower doses than other vector systems and have shown promise in several gene therapy approaches. Their main drawback is the potential risk of insertional mutagenesis. Novel biosafety-enhanced integration-deficient lentiviral vectors (IDLVs) offer a significant improvement and comparable transduction efficacy to their integrating counterparts in some central nervous system applications. We describe here methods for (1) production of IDLVs (and IPLVs), (2) IDLV/IPLV delivery into the striatum of a rat model of Parkinson's disease, and (3) postmortem brain processing.

  19. Lentiviral vector engineering for anti-HIV RNAi gene therapy.

    PubMed

    ter Brake, Olivier; Westerink, Jan-Tinus; Berkhout, Ben

    2010-01-01

    RNA interference or RNAi-based gene therapy for the treatment of HIV-1 infection has recently emerged as a highly effective antiviral approach. The lentiviral vector system is a good candidate for the expression of antiviral short hairpin RNAs (shRNA) in HIV-susceptible cells. However, this strategy can give rise to vector problems because the anti-HIV shRNAs can also target the HIV-based lentiviral vector system. In addition, there may be self-targeting of the shRNA-encoding sequences within the vector RNA genome in the producer cell. The insertion of microRNA (miRNA) cassettes in the vector may introduce Drosha cleavage sites that will also result in the destruction of the vector genome during the production and/or the transduction process. Here, we describe possible solutions to these lentiviral-RNAi problems. We also describe a strategy for multiple shRNA expression to establish a combinatorial RNAi therapy.

  20. Analysis of Partial Recombinants in Lentiviral Vector Preparations

    PubMed Central

    Kuate, Seraphin; Marino, Michael P.

    2014-01-01

    Abstract The presence of replication-competent lentivirus (RCL) in lentiviral vector preparations is a major safety concern for clinical applications of such vectors. RCL are believed to emerge from rare recombinant vector genomes that are referred to as partial recombinants or Psi-Gag recombinants. To quantitatively determine the fraction of partial recombinants in lentiviral vector preparations and to analyze them at the DNA sequence level, we established a drug selection assay involving a lentiviral packaging construct containing a drug-resistance gene encoding blasticidin (BSD) resistance. Upon transduction of target cells, the BSD resistance gene confers BSD resistance to the transduced cells. The results obtained indicate that there were up to 156 BSD-resistant colonies in a total of 106 transducing vector particles. The predicted recombination events were verified by polymerase chain reaction using genomic DNA obtained from BSD-resistant cell clones and by DNA sequence analysis. In an attempt to reduce the emergence of partial recombinants, sequence overlaps between the packaging and the vector constructs were reduced by substituting the Rev response element (RRE) present in the vector construct using a heterologous RRE element derived from simian immunodeficiency virus (SIVmac239). The results obtained showed that a reduction of sequence overlaps resulted in an up to sevenfold reduction of the frequency of BSD-resistant colonies, indicating that the capacity to form partial recombinants was diminished. PMID:24367910

  1. 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

  2. New protocol for lentiviral vector mass production.

    PubMed

    Segura, María Mercedes; Garnier, Alain; Durocher, Yves; Ansorge, Sven; Kamen, Amine

    2010-01-01

    Multiplasmid transient transfection is the most widely used technique for the generation of lentiviral vectors. However, traditional transient transfection protocols using 293 T adherent cells and calcium phosphate/DNA co-precipitation followed by ultracentrifugation are tedious, time-consuming, and difficult to scale up. This chapter describes a streamlined protocol for the fast mass production of lentiviral vectors and their purification by affinity chromatography. Lentiviral particles are generated by transient transfection of suspension growing HEK 293 cells in serum-free medium using polyethylenimine (PEI) as transfection reagent. Lentiviral vector production is carried out in Erlenmeyer flasks agitated on orbital shakers requiring minimum supplementary laboratory equipment. Alternatively, the method can be easily scaled up to generate larger volumes of vector stocks in bioreactors. Heparin affinity chromatography allows for selective concentration and purification of lentiviral particles in a singlestep directly from vector supernatants. The method is suitable for the production and purification of different vector pseudotypes.

  3. IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes.

    PubMed

    Verhoeyen, Els; Dardalhon, Valerie; Ducrey-Rundquist, Odile; Trono, Didier; Taylor, Naomi; Cosset, François-Loïc

    2003-03-15

    Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription, nuclear import, and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell-based gene therapy trials performed to date, cells have been activated via their cognate antigen receptor. To couple activation with gene transfer, we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However, transduction of highly purified resting T cells with these anti-CD3-displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here, we describe interleukin-7 (IL-7)-displaying HIV-1-derived vectors. Like recombinant IL-7, these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore, a single exposure to the IL-7-displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells, preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether, these results suggest that IL-7-displaying vectors could constitute interesting tools for T-cell-targeted gene therapy.

  4. Lentiviral-mediated gene correction of mucopolysaccharidosis type IIIA

    PubMed Central

    Anson, Donald S; McIntyre, Chantelle; Thomas, Belinda; Koldej, Rachel; Ranieri, Enzo; Roberts, Ainslie; Clements, Peter R; Dunning, Kylie; Byers, Sharon

    2007-01-01

    Background Mucopolysaccharidosis type IIIA (MPS IIIA) is the most common of the mucopolysaccharidoses. The disease is caused by a deficiency of the lysosomal enzyme sulphamidase and results in the storage of the glycosaminoglycan (GAG), heparan sulphate. MPS IIIA is characterised by widespread storage and urinary excretion of heparan sulphate, and a progressive and eventually profound neurological course. Gene therapy is one of the few avenues of treatment that hold promise of a sustainable treatment for this disorder. Methods The murine sulphamidase gene cDNA was cloned into a lentiviral vector and high-titre virus produced. Human MPS IIIA fibroblast cultures were transduced with the sulphamidase vector and analysed using molecular, enzymatic and metabolic assays. High-titre virus was intravenously injected into six 5-week old MPS IIIA mice. Three of these mice were pre-treated with hyperosmotic mannitol. The weight of animals was monitored and GAG content in urine samples was analysed by polyacrylamide gel electrophoresis. Results Transduction of cultured MPS IIIA fibroblasts with the sulphamidase gene corrected both the enzymatic and metabolic defects. Sulphamidase secreted by gene-corrected cells was able to cross correct untransduced MPS IIIA cells. Urinary GAG was found to be greatly reduced in samples from mice receiving the vector compared to untreated MPS IIIA controls. In addition, the weight of treated mice became progressively normalised over the 6-months post-treatment. Conclusion Lentiviral vectors appear promising vehicles for the development of gene therapy for MPS IIIA. PMID:17227588

  5. Viral vector mediated continuous expression of interleukin-10 in DRG alleviates pain in type 1 diabetic animals.

    PubMed

    Thakur, Vikram; Gonzalez, Mayra; Pennington, Kristen; Chattopadhyay, Munmun

    2016-04-01

    Painful diabetic neuropathy is a common and difficult to treat complication of diabetes. A growing body of evidence implicates the role of inflammatory mediators in the damage to the peripheral axons and in the pathogenesis of neuropathic pain. Increased expression of pro-inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the peripheral nervous system suggests the possibility of change in pain perception in diabetes. In this study we investigated that continuous delivery of IL10 in the nerve fibers achieved by HSV vector mediated transduction of dorsal root ganglion (DRG) in animals with Type 1 diabetes, blocks the nociceptive and stress responses in the DRG neurons by reducing IL1β expression along with inhibition of phosphorylation of p38 MAPK (mitogen-activated protein kinase) and protein kinase C (PKC). The continuous expression of IL10 also alters Toll like receptor (TLR)-4 expression in the DRG with increased expression of heat shock protein (HSP)-70 in conjunction with the reduction of pain. Taken together, this study suggests that macrophage activation in the peripheral nervous system may be involved in the pathogenesis of pain in Type 1 diabetes and therapeutic benefits of HSV mediated local expression of IL10 in the DRG with the reduction of a number of proinflammatory cytokines, subsequently inhibits the development of painful neuropathy along with a decrease in stress associated markers in the DRG. This basic and preclinical study provides an important evidence for a novel treatment strategy that could lead to a clinical trial for what is currently a treatment resistant complication of diabetes. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Characterising dark matter searches at colliders and direct detection experiments: Vector mediators

    DOE PAGES

    Buchmueller, Oliver; Dolan, Matthew J.; Malik, Sarah A.; ...

    2015-01-09

    We introduce a Minimal Simplified Dark Matter (MSDM) framework to quantitatively characterise dark matter (DM) searches at the LHC. We study two MSDM models where the DM is a Dirac fermion which interacts with a vector and axial-vector mediator. The models are characterised by four parameters: mDM, Mmed , gDM and gq, the DM and mediator masses, and the mediator couplings to DM and quarks respectively. The MSDM models accurately capture the full event kinematics, and the dependence on all masses and couplings can be systematically studied. The interpretation of mono-jet searches in this framework can be used to establishmore » an equal-footing comparison with direct detection experiments. For theories with a vector mediator, LHC mono-jet searches possess better sensitivity than direct detection searches for light DM masses (≲5 GeV). For axial-vector mediators, LHC and direct detection searches generally probe orthogonal directions in the parameter space. We explore the projected limits of these searches from the ultimate reach of the LHC and multi-ton xenon direct detection experiments, and find that the complementarity of the searches remains. In conclusion, we provide a comparison of limits in the MSDM and effective field theory (EFT) frameworks to highlight the deficiencies of the EFT framework, particularly when exploring the complementarity of mono-jet and direct detection searches.« less

  7. Characterising dark matter searches at colliders and direct detection experiments: Vector mediators

    SciTech Connect

    Buchmueller, Oliver; Dolan, Matthew J.; Malik, Sarah A.; McCabe, Christopher

    2015-01-09

    We introduce a Minimal Simplified Dark Matter (MSDM) framework to quantitatively characterise dark matter (DM) searches at the LHC. We study two MSDM models where the DM is a Dirac fermion which interacts with a vector and axial-vector mediator. The models are characterised by four parameters: mDM, Mmed , gDM and gq, the DM and mediator masses, and the mediator couplings to DM and quarks respectively. The MSDM models accurately capture the full event kinematics, and the dependence on all masses and couplings can be systematically studied. The interpretation of mono-jet searches in this framework can be used to establish an equal-footing comparison with direct detection experiments. For theories with a vector mediator, LHC mono-jet searches possess better sensitivity than direct detection searches for light DM masses (≲5 GeV). For axial-vector mediators, LHC and direct detection searches generally probe orthogonal directions in the parameter space. We explore the projected limits of these searches from the ultimate reach of the LHC and multi-ton xenon direct detection experiments, and find that the complementarity of the searches remains. In conclusion, we provide a comparison of limits in the MSDM and effective field theory (EFT) frameworks to highlight the deficiencies of the EFT framework, particularly when exploring the complementarity of mono-jet and direct detection searches.

  8. Constraining flavor changing interactions from LHC Run-2 dilepton bounds with vector mediators

    NASA Astrophysics Data System (ADS)

    Queiroz, Farinaldo S.; Siqueira, Clarissa; Valle, José W. F.

    2016-12-01

    Within the context of vector mediators, is a new signal observed in flavor changing interactions, particularly in the neutral mesons systems K0 -Kbar0, D0 -Dbar0 and B0 -B0 bar , consistent with dilepton resonance searches at the LHC? In the attempt to address this very simple question, we discuss the complementarity between flavor changing neutral current (FCNC) and dilepton resonance searches at the LHC run 2 at 13 TeV with 3.2 fb-1 of integrated luminosity, in the context of vector mediators at tree level. Vector mediators, are often studied in the flavor changing framework, specially in the light of the recent LHCb anomaly observed at the rare B decay. However, the existence of stringent dilepton bound severely constrains flavor changing interactions, due to restrictive limits on the Z‧ mass. We discuss this interplay explicitly in the well motivated framework of a 3-3-1 scheme, where fermions and scalars are arranged in the fundamental representation of the weak SU(3) gauge group. Due to the paucity of relevant parameters, we conclude that dilepton data leave little room for a possible new physics signal stemming from these systems, unless a very peculiar texture parametrization is used in the diagonalization of the CKM matrix. In other words, if a signal is observed in such flavor changing interactions, it unlikely comes from a 3-3-1 model.

  9. Herpes simplex virus vector-mediated delivery of neurturin rescues erectile dysfunction of cavernous nerve injury

    PubMed Central

    Kato, Ryuichi; Wolfe, Darren; Coyle, Christian H.; Wechuck, James B.; Tyagi, Pradeep; Tsukamoto, Taiji; Nelson, Joel B.; Glorioso, Joseph C.; Chancellor, Michael B.; Yoshimura, Naoki

    2008-01-01

    Summary Neurturin (NTN), a member of glial cell line-derived neurotrophic factor (GDNF) family, is known as an important neurotrphic factor for penis-projecting neurons. We recently demonstrated significant protection from erectile dysfunction (ED) following a replication defective herpes simplex virus (HSV) vector-mediated GDNF delivery to the injured cavernous nerve. Herein we applied HSV vector-mediated delivery of NTN to this ED model. Rat cavernous nerve was injured bilaterally using a clamp and dry ice. For HSV-treated groups, 20μl of vector stock was administered directly to the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein (GFP) and lacZ (HSV-LacZ) was used as a control. Intracavernous pressure along with systemic arterial pressure (ICP/AP) was measured 2 and 4 weeks after the nerve injury. Fluorogold (FG) was injected into the penile crus 7 days before sacrifice to assess neuronal survival. Four weeks after nerve injury, rats treated with HSV-NTN exhibited significantly higher ICP/AP compared to untreated or control vector treated groups. The HSV-NTN group had more FG-positive MPG neurons than control group following injury. HSV vector-mediated delivery of NTN could be a viable approach for improvement of erectile dysfunction following cavernous nerve injury. PMID:18668142

  10. 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.

  11. Treatment of pulmonary metastatic tumors in mice using lentiviral vector-engineered stem cells

    PubMed Central

    Zhang, X; Zhao, P; Kennedy, C; Chen, K; Wiegand, J; Washington, G; Marrero, L; Cui, Y

    2008-01-01

    Active cancer immunotherapy relies on functional tumor-specific effector T lymphocytes for tumor elimination. Dendritic cells (DCs), as most potent antigen-presenting cells, have been popularly employed in clinical and experimental tumor treatments. We have previously demonstrated that lentiviral vector-mediated transgene delivery to DC progenitors, including bone marrow cells and hematopoietic stem cells, followed by transplantation supports systemic generation of great numbers of tumor antigen-presenting DCs. These DCs subsequently stimulate marked and systemic immune activation. Here, we examined whether this level of immune activation is sufficient to overcome tumor-induced tolerogenic environment for treating an established aggressive epithelial tumor. We showed that a combination treatment of granulocyte macrophage-colony stimulating factor and cytosine-phosphate-guanine-containing oligonucleotide stimulated large numbers of tumor antigen-presenting DCs in situ from transgene-modified stem cells. Moreover, these in situ generated and activated DCs markedly stimulated activation of antigen-specific CD4 and CD8 T cells by augmenting their numbers, as well as function, even in a tumor-bearing tolerogenic environment. This leads to significant improvement in the therapeutic efficacy of established pulmonary metastases. This study suggests that lentiviral vector-modified stem cells as DC progenitors may be used as an effective therapeutic regimen for treating metastatic epithelial tumors. PMID:18084244

  12. Targeting retroviral and lentiviral vectors.

    PubMed

    Sandrin, V; Russell, S J; Cosset, F L

    2003-01-01

    Retroviral vectors capable of efficient in vivo gene delivery to specific target cell types or to specific locations of disease pathology would greatly facilitate many gene therapy applications. The surface glycoproteins of membrane-enveloped viruses stand among the choice candidates to control the target cell receptor recognition and host range of retroviral vectors onto which they are incorporated. This can be achieved in many ways, such as the exchange of glycoprotein by pseudotyping, their biochemical modifications, their conjugation with virus-cell bridging agents or their structural modifications. Understanding the fundamental properties of the viral glycoproteins and the molecular mechanism of virus entry into cells has been instrumental in the functional alteration of their tropism. Here we briefly review the current state of our understanding of the structure and function of viral envelope glycoproteins and we discuss the emerging targeting strategies based on retroviral and lentiviral vector systems.

  13. 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.

  14. Sustained knockdown of a disease-causing gene in patient-specific induced pluripotent stem cells using lentiviral vector-based gene therapy.

    PubMed

    Eggenschwiler, Reto; Loya, Komal; Wu, Guangming; Sharma, Amar Deep; Sgodda, Malte; Zychlinski, Daniela; Herr, Christian; Steinemann, Doris; Teckman, Jeffrey; Bals, Robert; Ott, Michael; Schambach, Axel; Schöler, Hans Robert; Cantz, Tobias

    2013-09-01

    Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for studies on disease-related developmental processes and may serve as an autologous cell source for future treatment of many hereditary diseases. New genetic engineering tools such as zinc finger nucleases and transcription activator-like effector nuclease allow targeted correction of monogenetic disorders but are very cumbersome to establish. Aiming at studies on the knockdown of a disease-causing gene, lentiviral vector-mediated expression of short hairpin RNAs (shRNAs) is a valuable option, but it is limited by silencing of the knockdown construct upon epigenetic remodeling during differentiation. Here, we propose an approach for the expression of a therapeutic shRNA in disease-specific iPSCs using third-generation lentiviral vectors. Targeting severe α-1-antitrypsin (A1AT) deficiency, we overexpressed a human microRNA 30 (miR30)-styled shRNA directed against the PiZ variant of A1AT, which is known to cause chronic liver damage in affected patients. This knockdown cassette is traceable from clonal iPSC lines to differentiated hepatic progeny via an enhanced green fluorescence protein reporter expressed from the same RNA-polymerase II promoter. Importantly, the cytomegalovirus i/e enhancer chicken β actin (CAG) promoter-driven expression of this construct is sustained without transgene silencing during hepatic differentiation in vitro and in vivo. At low lentiviral copy numbers per genome we confirmed a functional relevant reduction (-66%) of intracellular PiZ protein in hepatic cells after differentiation of patient-specific iPSCs. In conclusion, we have demonstrated that lentiviral vector-mediated expression of shRNAs can be efficiently used to knock down and functionally evaluate disease-related genes in patient-specific iPSCs.

  15. Rabies virus envelope glycoprotein targets lentiviral vectors to the axonal retrograde pathway in motor neurons.

    PubMed

    Hislop, James N; Islam, Tarin A; Eleftheriadou, Ioanna; Carpentier, David C J; Trabalza, Antonio; Parkinson, Michael; Schiavo, Giampietro; Mazarakis, Nicholas D

    2014-06-06

    Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.

  16. Feasibility of retroviral vector-mediated in utero gene transfer to the fetal rabbit.

    PubMed

    Moreno, Rafael; Rosal, Marta; Cabero, Lluis; Gratacós, Eduard; Aran, Josep M

    2005-01-01

    Successful treatment or prevention of severe hereditary diseases could conceivably be achieved by genetic intervention early in development. Viral vector-mediated fetal gene transfer is proving a valuable tool to test the above concept in relevant animal models. Although the pregnant rabbit is a well-recognized model for fetal therapy, few preclinical assays have used it to validate fetal gene transfer approaches. In this preliminary study we assessed for the first time the feasibility of retroviral vector-mediated in utero gene transfer in the fetal rabbit. Different amounts of the vesicular stomatitis virus G pseudotyped MFG(nls)LacZ retroviral vector, expressing a nuclear-localized beta-galactosidase reporter protein were injected intraperitoneally and -hepatically into 20- to 22-day-old fetuses. At 8-9 days post-treatment, the pups were sacrificed and the tissues harvested for analysis. Evidence of gene transfer was obtained by PCR amplification of proviral sequences within genomic DNA isolated from the treated samples. Transgenic beta-galactosidase expression was assessed by X-gal histochemical staining. By intraperitoneal injection 43% of the viable fetuses treated (3/7) showed evidence of successful LacZ gene transfer and low-level beta-galactosidase expression into liver and heart, whereas by intrahepatic injection roughly 38% (3/8) of the livers were positive for LacZ gene transfer and expression. The success rate for the viable fetuses rose to 67% positive livers (4/6) when a near double amount of recombinant virus was injected using a 10-fold concentrated virus stock. In terms of short-term safety, fetal and maternal survival rates approached 80% of treated fetuses, and 100% of treated does. The pregnant rabbit is a useful and reliable model allowing the design of further studies to optimize the conditions for effective, safer, and persistent retroviral vector-mediated fetal gene transfer. Copyright (c) 2005 S. Karger AG, Basel.

  17. Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells.

    PubMed

    Aarbiou, Jamil; Copreni, Elena; Buijs-Offerman, Ruvalic M; van der Wegen, Pascal; Castellani, Stefano; Carbone, Annalucia; Tilesi, Francesca; Fradiani, Piera; Hiemstra, Pieter S; Yueksekdag, Guelnihal; Diana, Anna; Rosenecker, Joseph; Ascenzioni, Fiorentina; Conese, Massimo; Scholte, Bob J

    2012-12-01

    Epithelial sodium channel (ENaC) hyperactivity has been implicated in the pathogenesis of cystic fibrosis (CF) by dysregulation of fluid and electrolytes in the airways. In the present study, we show proof-of-principle for ENaC inhibition by lentiviral-mediated RNA interference. Immortalized normal (H441) and CF mutant (CFBE) airway cells, and differentiated human bronchial epithelial cells in air liquid interface culture (HBEC-ALI) were transduced with a vesicular stomatitis virus G glycoprotein pseudotyped lentiviral (LV) vector expressing a short hairpin RNA (shRNA) targeting the α subunit of ENaC (ENaCα), and a marker gene. Efficacy of ENaCα down-regulation was assayed by the real-time polymerase chain reaction (PCR), membrane potential assay, western blotting, short-circuit currents and fluid absorption. Off-target effects were investigated by a lab-on-a-chip quantitative PCR array. Transduction to near one hundred percentage efficiency of H441, CFBE and HBEC-ALI was achieved by the addition of the LV vector before differentiation and polarization. Transduction resulted in the inhibition of ENaCα mRNA and antigen expression, and a proportional decrease in ENaC-dependent short circuit current and fluid transport. No effect on transepithelial resistance or cAMP-induced secretion responses was observed in HBEC-ALI. The production of interferon α and pro-inflammatory cytokine mRNA, indicating Toll-like receptor 3 or RNA-induced silencing complex mediated off-target effects, was not observed in HBEC-ALI transduced with this vector. We have established a generic method for studying the effect of RNA interference in HBEC-ALI using standard lentiviral vectors. Down-regulation of ENaCα by lentiviral shRNA expression vectors as shown in the absence off-target effects has potential therapeutic value in the treatment of cystic fibrosis. Copyright © 2012 John Wiley & Sons, Ltd.

  18. Viral vector-mediated transgenic cell therapy in regenerative medicine: safety of the process.

    PubMed

    Liu, Yang; Wang, Dong-An

    2015-04-01

    There are safety concerns regarding viral vectors in regenerative medicine research because of adverse experiences in conventional gene therapy with systemic delivery of recombinant virus. Transgenic cell therapy emerges as an attractive strategy, in which the genes of interest are delivered in vitro into isolated cells first; instead of transgene vectors, these transgenic cells are then implanted back to the host. This ex vivo strategy enables the examination of cell viability and phenotype before subsequent transplantation and prevents to the most extent the potential delivery-related hazards caused by exposure of viral components to the host. The transgenic implants are often localized, thus traceable for safety monitoring except those cases involving systemic distribution of transgenic cells. The safety of ex vivo process used in viral vector-mediated transgenic cell therapy for regenerative medicine purpose. Safety concerns related to viral vector delivery can be dispelled in the majority of regenerative medicine applications by transgenic cell therapy. The ex vivo process executes in vitro transfection before subsequent transplantation of transgenic cells so that it avoids the exposure of viral components (particularly capsids or envelops) to the host, while this exposure is inevitable in conventional in vivo gene therapy. Besides, the practice of localized cell implantation and in vitro manipulation also reinforce the safety of transgenic cell therapy. Given the significantly reduced delivery-related hazard, viral vector-mediated transgenic cell therapy can be generally considered as a safe approach for most regenerative medicine applications.

  19. Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways.

    PubMed

    Kushwah, R; Oliver, J R; Cao, H; Hu, J

    2007-08-01

    Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors.

  20. 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

  1. Surface-engineering of lentiviral vectors.

    PubMed

    Verhoeyen, Els; Cosset, François-Loïc

    2004-02-01

    Vectors derived from retroviridae offer particularly flexible properties in gene transfer applications given the numerous possible associations of various viral surface glycoproteins (determining cell tropism) with different types of retroviral cores (determining genome replication and integration). Lentiviral vectors should be preferred gene delivery vehicles over vectors derived from onco-retroviruses such as murine leukemia viruses (MLVs) that cannot transduce non-proliferating target cells. Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors, their interaction with the host immune system and their host range. There are however important gene transfer restrictions to some non-proliferative tissues or cell types and recent studies have shown that progenitor hematopoietic stem cells in G(0), non-activated primary blood lymphocytes or monocytes were not transducible by lentiviral vectors. Moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer applications in vivo. Several innovative approaches have been explored to overcome such problems that have given rise to novel concepts in the field and have provided promising results in preliminary evaluations in vivo. Here we review the different approaches explored to upgrade lentiviral vectors, aiming at developing vectors suitable for in vivo gene delivery.

  2. Distribution properties of lentiviral vectors administered into the striatum by convection-enhanced delivery.

    PubMed

    White, Edward; Bienemann, Alison; Megraw, Lisa; Bunnun, Chotirote; Wyatt, Marcella; Taylor, Hannah; Gill, Steven

    2012-01-01

    Before the successful use of lentiviral vectors in clinical trials it is essential that strategies for direct vector delivery into the brain be evaluated in vivo, particularly as these vectors are significantly larger than the brain extracellular space. To date no such studies have been undertaken. In this study, convection-enhanced delivery (CED) was employed in an attempt to achieve widespread lentiviral delivery in the striatum. Infusions of equine infectious anemia virus (EIAV) and HIV vector constructs expressing the reporter gene β-galactosidase (β-Gal) were undertaken into the striatum at a range of flow rates and viral titers. In rats, all EIAV and HIV infusions led to the extensive transduction of cells in perivascular spaces throughout the brain. Although infusions were performed under standardized conditions, the number and volume of distribution of transduced cells were highly variable, with approximately one-third of EIAV infusions leading to no concentrated cell transduction in the striatum. Heparin coinfusion had no effect on EIAV distribution, although coinfusion of nimodipine resulted in a significant reduction in the number and volume of distribution of transduced cells. Intrastriatal EIAV delivery in pigs led to extensive transduction of mainly neurons, which could be effectively visualized in real time by T(2)-weighted magnetic resonance imaging. No infusions were associated with a significant inflammatory response. Therefore, despite its large size, lentiviral vectors can be administered by CED to the striatum in both small and large animal models. However, the variability in vector distribution under standardized conditions and widespread vector distribution through the perivascular spaces raise serious concerns regarding the practicality of lentivirus-mediated gene therapy in the brain in clinical practice.

  3. Introducing a cleavable signal peptide enhances the packaging efficiency of lentiviral vectors pseudotyped with Japanese encephalitis virus envelope proteins.

    PubMed

    Liu, Hanyang; Wu, Rui; Yuan, Lei; Tian, Geng; Huang, Xiaobo; Wen, Yiping; Ma, Xiaoping; Huang, Yong; Yan, Qigui; Zhao, Qin; Cao, Sanjie; Wen, Xintian

    2017-02-02

    Research into the properties of Japanese encephalitis virus (JEV) has been facilitated by use of pseudotyped viruses. The signal peptide is a key determinant for membrane targeting and membrane insertion, which could affect packaging of pseudotyped viruses. In this study, we generated three lentiviral vectors pseudotyped with JEV envelope proteins that co-express either a strong signal peptide from vesicular stomatitis virus (VSV)-G (VSVMEpv) or a weak signal peptide of JEV (SPMEpv), or a virus without a signal peptide in front of the JEV prM/E (MEpv). Western blot demonstrated that JEV E protein and HIV p24 were present in the same particles of the three pseudotyped JEV-E based lentiviral vectors. Electron microscopy revealed that the three pseudotyped JEV-E based lentiviral vectors were 120-180nm in diameter. Real-time quantitative reverse transcriptase polymerase chain reaction showed that the titer of VSVMEpv was 17-fold higher than that of MEpv, while the titer of SPMEpv was six-fold higher than that of MEpv. Inclusion of a signal peptide enhanced packaging efficiency of pseudotyped JEV-E based lentiviral vectors. With a strong signal peptide helping they generate a higher number of viral particles. Green fluorescent protein and luciferase expression showed that the transduction titer or relative fluorescence units of VSVMEpv, SPMEpv and MEpv were not significantly different. We suggest that the signal peptide does not influence the infectivity of pseudotyped JEV-E based lentiviral vectors. In addition, our findings indicated that pseudotyped JEVs show preferential tropism for BHK-21 cells, supporting the mimic function displayed by parental JEV. Therefore, our study provided a cost-effective method to generate pseudotyped JEV-E based lentiviral vectors, which may represent a valid model to investigate some of the infectious properties of JEV. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

    PubMed

    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.

  5. 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

  6. 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.

  7. 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.

  8. Dendritic cell functional improvement in a preclinical model of lentiviral-mediated gene therapy for Wiskott-Aldrich syndrome

    PubMed Central

    Catucci, Marco; Prete, Francesca; Bosticardo, Marita; Castiello, Maria Carmina; Draghici, Elena; Locci, Michela; Roncarolo, Maria Grazia; Aiuti, Alessandro; Benvenuti, Federica; Villa, Anna

    2011-01-01

    Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by the defective expression of the WAS protein (WASP) in hematopoietic cells. It has been shown that dendritic cells (DCs) are functionally impaired in WAS patients and was−/− mice. We have previously demonstrated the efficacy and safety of a murine model of WAS gene therapy (GT), using stem cells transduced with a lentiviral vector. The aim of this study was to investigate whether GT can correct DC defects in was−/− mice. As DCs expressing WASP were detected in the secondary lymphoid organs of the treated mice, we tested the in vitro and in vivo function of bone marrow-derived DCs (BMDCs). The BMDCs showed efficient in vitro uptake of latex beads and Salmonella typhimurium. When BMDCs from the treated mice (GT BMDCs) and the was−/− mice were injected into wild type hosts, we found a higher number of cells that had migrated to the draining lymph nodes compared to mice injected with was−/− BMDCs. Finally, we found that OVA-pulsed GT BMDCs or vaccination with anti-DEC205 OVA fusion protein can efficiently induce antigen-specific T cell activation in vivo. These findings show that WAS GT significantly improves DC function, thus adding new evidence of the preclinical efficacy of lentiviral vector-mediated WAS GT. PMID:22189416

  9. Targeting of Magnetic Nanoparticle-coated Microbubbles to the Vascular Wall Empowers Site-specific Lentiviral Gene Delivery in vivo

    PubMed Central

    Heun, Yvonn; Hildebrand, Staffan; Heidsieck, Alexandra; Gleich, Bernhard; Anton, Martina; Pircher, Joachim; Ribeiro, Andrea; Mykhaylyk, Olga; Eberbeck, Dietmar; Wenzel, Daniela; Pfeifer, Alexander; Woernle, Markus; Krötz, Florian; Pohl, Ulrich; Mannell, Hanna

    2017-01-01

    In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro. Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo, we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy. PMID:28042335

  10. Restriction of transgene expression to the B-lymphoid progeny of human lentivirally transduced CD34+ cells.

    PubMed

    Moreau, Thomas; Bardin, Florence; Imbert, Jean; Chabannon, Christian; Tonnelle, Cécile

    2004-07-01

    Development of gene transfer strategies will necessitate improved efficiency and control of transduction and transgene expression. We here provide evidence that targeting expression of the GFP reporter gene to the B-lymphoid progeny of genetically modified human hematopoietic progenitor cells can be achieved through the insertion of regulatory sequences from the human CD19 gene promoter into a lentiviral vector. Based on a bioinformatics approach, three human CD19-derived sequences were designed and inserted into a self-inactivated lentiviral vector backbone upstream of the GFP gene: S.CD19 (230 bp), M.CD19 (464 bp), and L.CD19 (1274 bp). These new lentiviral vectors efficiently transduced cord blood CD34(+) cells. The M.CD19 and especially L.CD19 sequences preferentially targeted GFP expression to in vitro and in vivo differentiated CD19(+) progeny; moreover, transgene expression was detected from the CD34(+) pro/pre-B cell to the mature peripheral IgM(+) B cell stage. In contrast, GFP expression was weak or absent in primary T-lymphoid and uncommitted progenitor cells or in erythroid, natural killer, or myeloid differentiated cells. Such B-lineage-specific lentiviral vectors may be useful for correcting inherited disorders that affect B-lymphoid cells or for deciphering the transcriptional program that controls B cell commitment and differentiation.

  11. 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

  12. Targeting of Magnetic Nanoparticle-coated Microbubbles to the Vascular Wall Empowers Site-specific Lentiviral Gene Delivery in vivo.

    PubMed

    Heun, Yvonn; Hildebrand, Staffan; Heidsieck, Alexandra; Gleich, Bernhard; Anton, Martina; Pircher, Joachim; Ribeiro, Andrea; Mykhaylyk, Olga; Eberbeck, Dietmar; Wenzel, Daniela; Pfeifer, Alexander; Woernle, Markus; Krötz, Florian; Pohl, Ulrich; Mannell, Hanna

    2017-01-01

    In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro. Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo, we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy.

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

    PubMed

    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

    2009-06-01

    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).

  14. Lentiviral-mediated genetic correction of hematopoietic and mesenchymal progenitor cells from Fanconi anemia patients.

    PubMed

    Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Lozano, M Luz; 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

    2009-06-01

    Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-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).

  15. 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

  16. Molecular Determinants of Vectofusin-1 and Its Derivatives for the Enhancement of Lentivirally Mediated Gene Transfer into Hematopoietic Stem/Progenitor Cells*

    PubMed Central

    Majdoul, Saliha; Seye, Ababacar K.; Kichler, Antoine; Holic, Nathalie; Galy, Anne; Bechinger, Burkhard; Fenard, David

    2016-01-01

    Gene delivery into hCD34+ hematopoietic stem/progenitor cells (HSPCs) using human immunodeficiency virus, type 1-derived lentiviral vectors (LVs) has several promising therapeutic applications. Numerous clinical trials are currently underway. However, the 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 or cationic compounds. Recently, we discovered Vectofusin-1, a new transduction enhancer, also called LAH4-A4, a short histidine-rich amphipathic peptide derived from the LAH4 family of DNA transfection agents. Vectofusin-1 enhances the infectivity of lentiviral and γ-retroviral vectors pseudotyped with various envelope glycoproteins. In this study, we compared a family of Vectofusin-1 isomers and showed that Vectofusin-1 remains the lead peptide for HSPC transduction enhancement with LVs pseudotyped with vesicular stomatitis virus glycoproteins and also with modified gibbon ape leukemia virus glycoproteins. By comparing the capacity of numerous Vectofusin-1 variants to promote the modified gibbon ape leukemia virus glycoprotein-pseudotyped lentiviral vector infectivity of HSPCs, the lysine residues on the N-terminal extremity of Vectofusin-1, a hydrophilic angle of 140° formed by the histidine residues in the Schiffer-Edmundson helical wheel representation, hydrophobic residues consisting of leucine were all found to be essential and helped to define a minimal active sequence. The data also show that the critical determinants necessary for lentiviral transduction enhancement are partially different from those necessary for efficient antibiotic or DNA transfection activity of LAH4 derivatives. In conclusion, these results help to decipher the action mechanism of Vectofusin-1 in the context of hCD34+ cell-based gene therapy. PMID:26668323

  17. Dystrophin Delivery to Muscles of mdx Mice Using Lentiviral Vectors Leads to Myogenic Progenitor Targeting and Stable Gene Expression

    PubMed Central

    Kimura, En; Li, Sheng; Gregorevic, Paul; Fall, Brent M; Chamberlain, Jeffrey S

    2009-01-01

    To explore whether stable transduction of myogenic stem cells using lentiviral vectors could be of benefit for treating dystrophic muscles, we generated vectors expressing a functional microdystrophin/enhanced green fluorescence protein fusion (µDys/eGFP) gene. Lentiviral vector injection into neonatal mdx4cv muscles resulted in widespread and stable expression of dystrophin for at least 2 years. This expression resulted in a significant amelioration of muscle pathophysiology as assessed by a variety of histological and functional assays. To assess whether this long-term expression was accompanied by stable transduction of satellite cells, we harvested muscle mononuclear cells 1 year after vector injection. Up to 20% of the cultured myoblast colonies expressed the µDys/eGFP transgene following myotube formation. Furthermore, transplantation of the muscle mononuclear cells into secondary mdx4cv recipients showed their ability to regenerate dystrophin-expressing myofibers in vivo. The ability to isolate myogenic cells able to form dystrophin-positive myotubes or myofibers in vitro and in vivo >1 year postinjection indicates that the vectors stably transduced muscle satellite cells, or a progenitor of such cells, in neonatal mdx4cv muscles. These studies suggest that integrating lentiviral vectors have potential utility for gene therapy of muscular dystrophy. PMID:19888194

  18. Uncovering and Dissecting the Genotoxicity of Self-inactivating Lentiviral Vectors In Vivo

    PubMed Central

    Cesana, Daniela; Ranzani, Marco; Volpin, Monica; Bartholomae, Cynthia; Duros, Caroline; Artus, Alexandre; Merella, Stefania; Benedicenti, Fabrizio; Sergi Sergi, Lucia; Sanvito, Francesca; Brombin, Chiara; Nonis, Alessandro; Serio, Clelia Di; Doglioni, Claudio; von Kalle, Christof; Schmidt, Manfred; Cohen-Haguenauer, Odile; Naldini, Luigi; Montini, Eugenio

    2014-01-01

    Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety. PMID:24441399

  19. You can hide but you have to run: direct detection with vector mediators

    NASA Astrophysics Data System (ADS)

    D'Eramo, Francesco; Kavanagh, Bradley J.; Panci, Paolo

    2016-08-01

    We study direct detection in simplified models of Dark Matter (DM) in which interactions with Standard Model (SM) fermions are mediated by a heavy vector boson. We consider fully general, gauge-invariant couplings between the SM, the mediator and both scalar and fermion DM. We account for the evolution of the couplings between the energy scale of the mediator mass and the nuclear energy scale. This running arises from virtual effects of SM particles and its inclusion is not optional. We compare bounds on the mediator mass from direct detection experiments with and without accounting for the running. In some cases the inclusion of these effects changes the bounds by several orders of magnitude, as a consequence of operator mixing which generates new interactions at low energy. We also highlight the importance of these effects when translating LHC limits on the mediator mass into bounds on the direct detection cross section. For an axial-vector mediator, the running can alter the derived bounds on the spin-dependent DM-nucleon cross section by a factor of two or more. Finally, we provide tools to facilitate the inclusion of these effects in future studies: general approximate expressions for the low energy couplings and a public code runDM to evolve the couplings between arbitrary energy scales.

  20. You can hide but you have to run: Direct detection with vector mediators

    SciTech Connect

    D’Eramo, Francesco; Kavanagh, Bradley J.; Panci, Paolo

    2016-08-18

    We study direct detection in simplified models of Dark Matter (DM) in which interactions with Standard Model (SM) fermions are mediated by a heavy vector boson. We consider fully general, gauge-invariant couplings between the SM, the mediator and both scalar and fermion DM. We account for the evolution of the couplings between the energy scale of the mediator mass and the nuclear energy scale. This running arises from virtual effects of SM particles and its inclusion is not optional. We compare bounds on the mediator mass from direct detection experiments with and without accounting for the running. In some cases the inclusion of these effects changes the bounds by several orders of magnitude, as a consequence of operator mixing which generates new interactions at low energy. We also highlight the importance of these effects when translating LHC limits on the mediator mass into bounds on the direct detection cross section. For an axial-vector mediator, the running can alter the derived bounds on the spin-dependent DM-nucleon cross section by a factor of two or more. Lastly, we provide tools to facilitate the inclusion of these effects in future studies: general approximate expressions for the low energy couplings and a public code runDM to evolve the couplings between arbitrary energy scales.

  1. Apparent vector-mediated parent-to-offspring transmission in an avian malaria-like parasite.

    PubMed

    Chakarov, Nayden; Linke, Burkhard; Boerner, Martina; Goesmann, Alexander; Krüger, Oliver; Hoffman, Joseph I

    2015-03-01

    Parasite transmission strategies strongly impact host-parasite co-evolution and virulence. However, studies of vector-borne parasites such as avian malaria have neglected the potential effects of host relatedness on the exchange of parasites. To test whether extended parental care in the presence of vectors increases the probability of transmission from parents to offspring, we used high-throughput sequencing to develop microsatellites for malaria-like Leucocytozoon parasites of a wild raptor population. We show that host siblings carry genetically more similar parasites than unrelated chicks both within and across years. Moreover, chicks of mothers of the same plumage morph carried more similar parasites than nestlings whose mothers were of different morphs, consistent with matrilineal transmission of morph-specific parasite strains. Ours is the first evidence of an association between host relatedness and parasite genetic similarity, consistent with vector-mediated parent-to-offspring transmission. The conditions for such 'quasi-vertical' transmission may be common and could suppress the evolution of pathogen virulence.

  2. You can hide but you have to run: Direct detection with vector mediators

    DOE PAGES

    D’Eramo, Francesco; Kavanagh, Bradley J.; Panci, Paolo

    2016-08-18

    We study direct detection in simplified models of Dark Matter (DM) in which interactions with Standard Model (SM) fermions are mediated by a heavy vector boson. We consider fully general, gauge-invariant couplings between the SM, the mediator and both scalar and fermion DM. We account for the evolution of the couplings between the energy scale of the mediator mass and the nuclear energy scale. This running arises from virtual effects of SM particles and its inclusion is not optional. We compare bounds on the mediator mass from direct detection experiments with and without accounting for the running. In some casesmore » the inclusion of these effects changes the bounds by several orders of magnitude, as a consequence of operator mixing which generates new interactions at low energy. We also highlight the importance of these effects when translating LHC limits on the mediator mass into bounds on the direct detection cross section. For an axial-vector mediator, the running can alter the derived bounds on the spin-dependent DM-nucleon cross section by a factor of two or more. Lastly, we provide tools to facilitate the inclusion of these effects in future studies: general approximate expressions for the low energy couplings and a public code runDM to evolve the couplings between arbitrary energy scales.« less

  3. Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A.

    PubMed

    Johnston, J M; Denning, G; Doering, C B; Spencer, H T

    2013-06-01

    We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain-deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino-acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: (1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), (2) HIV versus SIV viral vector systems and (3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha, cytomegalovirus (CMV) or phosphoglycerate kinase promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.

  4. Generation of an Optimized Lentiviral Vector Encoding a High-Expression Factor VIII Transgene for Gene Therapy of Hemophilia A

    PubMed Central

    Johnston, Jennifer M.; Denning, Gabriela; Doering, Christopher B.; Spencer, H. Trent

    2012-01-01

    We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: 1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), 2) HIV versus SIV viral vector systems, and 3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha (EF1α), cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE. PMID:22996197

  5. AAV vector-mediated reversal of hypoglycemia in canine and murine glycogen storage disease type Ia.

    PubMed

    Koeberl, Dwight D; Pinto, Carlos; Sun, Baodong; Li, Songtao; Kozink, Daniel M; Benjamin, Daniel K; Demaster, Amanda K; Kruse, Meghan A; Vaughn, Valerie; Hillman, Steven; Bird, Andrew; Jackson, Mark; Brown, Talmage; Kishnani, Priya S; Chen, Yuan-Tsong

    2008-04-01

    Glycogen storage disease type Ia (GSD-Ia) profoundly impairs glucose release by the liver due to glucose-6-phosphatase (G6Pase) deficiency. An adeno-associated virus (AAV) containing a small human G6Pase transgene was pseudotyped with AAV8 (AAV2/8) to optimize liver tropism. Survival was prolonged in 2-week-old G6Pase (-/-) mice by 600-fold fewer AAV2/8 vector particles (vp), in comparison to previous experiments involving this model (2 x 10(9) vp; 3 x 10(11) vp/kg). When the vector was pseudotyped with AAV1, survival was prolonged only at a higher dose (3 x 10(13) vp/kg). The AAV2/8 vector uniquely prevented hypoglycemia during fasting and fully corrected liver G6Pase deficiency in GSD-Ia mice and dogs. The AAV2/8 vector has prolonged survival in three GSD-Ia dogs to >11 months, which validated this strategy in the large animal model for GSD-Ia. Urinary biomarkers, including lactate and 3-hydroxybutyrate, were corrected by G6Pase expression solely in the liver. Glycogen accumulation in the liver was reduced almost to the normal level in vector-treated GSD-Ia mice and dogs, as was the hepatocyte growth factor (HGF) in GSD-Ia mice. These preclinical data demonstrated the efficacy of correcting hepatic G6Pase deficiency, and support the further preclinical development of AAV vector-mediated gene therapy for GSD-Ia.

  6. A translatable, closed recirculation system for AAV6 vector-mediated myocardial gene delivery in the large animal.

    PubMed

    Swain, JaBaris D; Katz, Michael G; White, Jennifer D; Thesier, Danielle M; Henderson, Armen; Stedman, Hansell H; Bridges, Charles R

    2011-01-01

    Current strategies for managing congestive heart failure are limited, validating the search for an alternative treatment modality. Gene therapy holds tremendous promise as both a practical and translatable technology platform. Its effectiveness is evidenced by the improvements in cardiac function observed in vector-mediated therapeutic transgene delivery to the murine myocardium. A large animal model validating these results is the likely segue into clinical application. However, controversy still exists regarding a suitable method of vector-mediated cardiac gene delivery that provides for efficient, global gene transfer to the large animal myocardium that is also clinically translatable and practical. Intramyocardial injection and catheter-based coronary delivery techniques are attractive alternatives with respect to their clinical applicability; yet, they are fraught with numerous challenges, including concerns regarding collateral gene expression in other organs, low efficiency of vector delivery to the myocardium, inhomogeneous expression, and untoward immune response secondary to gene delivery. Cardiopulmonary bypass (CPB) delivery with dual systemic and isolated cardiac circuitry precludes these drawbacks and has the added advantage of allowing for control of the pharmacological milieu, multiple pass recirculation through the coronary circulation, the selective addition of endothelial permeabilizing agents, and an increase in vector residence time. Collectively, these mechanics significantly improve the efficiency of global, vector-mediated cardiac gene delivery to the large animal myocardium, highlighting a potential therapeutic strategy to be extended to some heart failure patients.

  7. Query by transduction.

    PubMed

    Ho, Shen-Shyang; Wechsler, Harry

    2008-09-01

    There has been recently a growing interest in the use of transductive inference for learning. We expand here the scope of transductive inference to active learning in a stream-based setting. Towards that end this paper proposes Query-by-Transduction (QBT) as a novel active learning algorithm. QBT queries the label of an example based on the p-values obtained using transduction. We show that QBT is closely related to Query-by-Committee (QBC) using relations between transduction, Bayesian statistical testing, Kullback-Leibler divergence, and Shannon information. The feasibility and utility of QBT is shown on both binary and multi-class classification tasks using SVM as the choice classifier. Our experimental results show that QBT compares favorably, in terms of mean generalization, against random sampling, committee-based active learning, margin-based active learning, and QBC in the stream-based setting.

  8. Lentiviral Effector Pathways of TRIM Proteins.

    PubMed

    Turrini, Filippo; Di Pietro, Andrea; Vicenzi, Elisa

    2014-04-01

    The human tripartite motif (TRIM) family, composed of more than 77 members, encompasses an emerging group of innate antiviral factors. Most TRIM proteins are characterized by being E3 ubiquitin ligases, but also engage in specific interactions with a variety of cellular and viral partners. They are involved in many cellular processes, including cell differentiation, transcriptional regulation, cytoskeleton remodeling, intracellular trafficking, membrane repair, and oncogenesis. In regard to antiviral immunity, they restrict both retroviruses and lentiviruses as well as other DNA and RNA viruses. This review will focus on the TRIM members endowed with anti-retroviral and anti-lentiviral activities and, in particular, human immunodeficiency virus.

  9. 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

  10. Shedding of clinical-grade lentiviral vectors is not detected in a gene therapy setting.

    PubMed

    Cesani, M; Plati, T; Lorioli, L; Benedicenti, F; Redaelli, D; Dionisio, F; Biasco, L; Montini, E; Naldini, L; Biffi, A

    2015-06-01

    Gene therapy using viral vectors that stably integrate into ex vivo cultured cells holds great promises for the treatment of monogenic diseases as well as cancer. However, carry-over of infectious vector particles has been described to occur upon ex vivo transduction of target cells. This, in turn, may lead to inadvertent spreading of viral particles to off-target cells in vivo, raising concerns for potential adverse effects, such as toxicity of ectopic transgene expression, immunogenicity from in vivo transduced antigen-presenting cells and, possibly, gene transfer to germline cells. Here, we have investigated factors influencing the extent of lentiviral vector (LV) shedding upon ex vivo transduction of human hematopoietic stem and progenitor cells. Our results indicate that, although vector carry-over is detectable when using laboratory-grade vector stocks, the use of clinical-grade vector stocks strongly decreases the extent of inadvertent transduction of secondary targets, likely because of the higher degree of purification. These data provide supportive evidence for the safe use of the LV platform in clinical settings.

  11. Baculoviral vector-mediated transient and stable transgene expression in human embryonic stem cells.

    PubMed

    Zeng, Jieming; Du, Juan; Zhao, Ying; Palanisamy, Nallasivam; Wang, Shu

    2007-04-01

    Human embryonic stem (hES) cells as a renewable cell source have great prospective applications in both developmental biology research and regenerative medicine. To realize these potentials, the development of effective and safe genetic manipulation methods in hES cells is an obvious demand. We report here that baculoviral vectors were able to transduce hES cells efficiently. In transient transduction experiments, a recombinant baculoviral vector equipped with a human elongation factor 1-alpha promoter and a woodchuck hepatitis post-transcriptional regulatory element transduced up to 80% of cells in hES cell clumps and embryoid bodies. For prolonged transgene expression, hybrid baculoviral vectors that have incorporated a rep gene and inverted terminal repeat sequences from adeno-associated virus were produced. These hybrid vectors yielded stable transgene expression during the prolonged undifferentiated proliferation of hES cells and after differentiation. Baculoviral transduction did not affect the normal growth, phenotype, and pluripotency of hES cells. Thus, baculoviral vectors suitable for both transient overexpression and long-term stable expression are an attractive option for genetic manipulation of hES cells.

  12. T cell receptor (TCR) gene transfer with lentiviral vectors allows efficient redirection of tumor specificity in naive and memory T cells without prior stimulation of endogenous TCR.

    PubMed

    Circosta, Paola; Granziero, Luisa; Follenzi, Antonia; Vigna, Elisa; Stella, Stefania; Vallario, Antonella; Elia, Angela Rita; Gammaitoni, Loretta; Vitaggio, Katiuscia; Orso, Francesca; Geuna, Massimo; Sangiolo, Dario; Todorovic, Maja; Giachino, Claudia; Cignetti, Alessandro

    2009-12-01

    We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

  13. 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.

  14. Lentiviral Vector-based Insertional Mutagenesis Identifies Genes Involved in the Resistance to Targeted Anticancer Therapies

    PubMed Central

    Ranzani, Marco; Annunziato, Stefano; Calabria, Andrea; Brasca, Stefano; Benedicenti, Fabrizio; Gallina, Pierangela; Naldini, Luigi; Montini, Eugenio

    2014-01-01

    The high transduction efficiency of lentiviral vectors in a wide variety of cells makes them an ideal tool for forward genetics screenings addressing issues of cancer research. Although molecular targeted therapies have provided significant advances in tumor treatment, relapses often occur by the expansion of tumor cell clones carrying mutations that confer resistance. Identification of the culprits of anticancer drug resistance is fundamental for the achievement of long-term response. Here, we developed a new lentiviral vector-based insertional mutagenesis screening to identify genes that confer resistance to clinically relevant targeted anticancer therapies. By applying this genome-wide approach to cell lines representing two subtypes of HER2+ breast cancer, we identified 62 candidate lapatinib resistance genes. We validated the top ranking genes, i.e., PIK3CA and PIK3CB, by showing that their forced expression confers resistance to lapatinib in vitro and found that their mutation/overexpression is associated to poor prognosis in human breast tumors. Then, we successfully applied this approach to the identification of erlotinib resistance genes in pancreatic cancer, thus showing the intrinsic versatility of the approach. The acquired knowledge can help identifying combinations of targeted drugs to overcome the occurrence of resistance, thus opening new horizons for more effective treatment of tumors. PMID:25195596

  15. Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming.

    PubMed

    Warlich, Eva; Kuehle, Johannes; Cantz, Tobias; Brugman, Martijn H; Maetzig, Tobias; Galla, Melanie; Filipczyk, Adam A; Halle, Stephan; Klump, Hannes; Schöler, Hans R; Baum, Christopher; Schroeder, Timm; Schambach, Axel

    2011-04-01

    Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell-derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events.

  16. Muscle Fiber Type-Predominant Promoter Activity in Lentiviral-Mediated Transgenic Mouse

    PubMed Central

    Suga, Tomohiro; Kimura, En; Morioka, Yuka; Ikawa, Masahito; Li, Sheng; Uchino, Katsuhisa; Uchida, Yuji; Yamashita, Satoshi; Maeda, Yasushi; Chamberlain, Jeffrey S.; Uchino, Makoto

    2011-01-01

    Variations in gene promoter/enhancer activity in different muscle fiber types after gene transduction was noticed previously, but poorly analyzed. The murine stem cell virus (MSCV) promoter drives strong, stable gene expression in hematopoietic stem cells and several other cells, including cerebellar Purkinje cells, but it has not been studied in muscle. We injected a lentiviral vector carrying an MSCV-EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and observed strong EGFP expression in muscle fibers, primary cultured myoblasts, and myotubes isolated from injected muscles. We also generated lentiviral-mediated transgenic mice carrying the MSCV-EGFP cassette and detected transgene expression in striated muscles. LvMSCV-EGFP transgenic mice showed fiber type-dependent variations in expression: highest in types I and IIA, intermediate in type IID/X, and lowest in type IIB fibers. The soleus and diaphragm muscles, consisting mainly of types I and IIA, are most severely affected in the mdx mouse model of muscular dystrophy. Further analysis of this promoter may have the potential to achieve certain gene expression in severely affected muscles of mdx mice. The Lv-mediated transgenic mouse may prove a useful tool for assessing the enhancer/promoter activities of a variety of different regulatory cassettes. PMID:21445245

  17. Lentiviral Vector Design and Imaging Approaches to Visualize the Early Stages of Cellular Reprogramming

    PubMed Central

    Warlich, Eva; Kuehle, Johannes; Cantz, Tobias; Brugman, Martijn H; Maetzig, Tobias; Galla, Melanie; Filipczyk, Adam A; Halle, Stephan; Klump, Hannes; Schöler, Hans R; Baum, Christopher; Schroeder, Timm; Schambach, Axel

    2011-01-01

    Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell–derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events. PMID:21285961

  18. Transient increase in intrahepatic pressure mediates successful treatment of the Gunn rat with reduced doses of lentiviral vector.

    PubMed

    Schmitt, Françoise; Flageul, Maude; Dariel, Anne; Pichard, Virginie; Pontes, Cecilia Abarrategui; Boni, Sébastien; Podevin, Guillaume; Myara, Anne; Ferry, Nicolas; Nguyen, Tuan Huy

    2010-10-01

    Lentiviral vectors can stably transduce hepatocytes and are promising tools for gene therapy of hepatic diseases. Although hepatocytes are accessible to blood-borne viral vectors through fenestrations of the hepatic endothelium, improved liver transduction after delivery of vectors to the blood stream is needed. As the normal endothelial fenestration and lentiviral vectors are similar in size (150 nm), we hypothesized that a transient increase in hepatic blood pressure may enhance in vivo gene transfer to hepatocytes. We designed a simple surgical procedure, by which the liver is temporarily excluded from blood flow. Lentiviral vectors were injected in a large volume to increase intrahepatic pressure. We demonstrated that in the Gunn rat, a model of Crigler-Najjar disease, the administration of low vector doses (corresponding to a multiplicity of infection of 0.2) by this procedure resulted in therapeutic correction of hyperbilirubinemia, without toxicity. The correction was sustained for 10 months (end of study). The same vector amounts yielded only partial correction after intraportal delivery. We believe that this new and clinically applicable strategy may broaden the range of genetic liver diseases accessible to gene therapy.

  19. Long-term correction of hemophilia A mice following lentiviral mediated delivery of an optimized canine factor VIII gene.

    PubMed

    Staber, J M; Pollpeter, M J; Anderson, C-G; Burrascano, M; Cooney, A L; Sinn, P L; Rutkowski, D T; Raschke, W C; McCray, P B

    2017-09-14

    Current therapies for hemophilia A include frequent prophylactic or on-demand intravenous factor treatments which are costly, inconvenient and may lead to inhibitor formation. Viral vector delivery of factor VIII (FVIII) cDNA has the potential to alleviate the debilitating clotting defects. Lentiviral-based vectors delivered to murine models of hemophilia A mediate phenotypic correction. However, a limitation of lentiviral-mediated FVIII delivery is inefficient transduction of target cells. Here, we engineer a feline immunodeficiency virus (FIV) -based lentiviral vector pseudotyped with the baculovirus GP64 envelope glycoprotein to mediate efficient gene transfer to mouse hepatocytes. In anticipation of future studies in FVIII-deficient dogs, we investigated the efficacy of FIV-delivered canine FVIII (cFVIII). Codon-optimization of the cFVIII sequence increased activity and decreased blood loss as compared to the native sequence. Further, we compared a standard B-domain deleted FVIII cDNA to a cDNA including 256 amino acids of the B-domain with 11 potential asparagine-linked oligosaccharide linkages. Restoring a partial B-domain resulted in modest reduction of endoplasmic reticulum (ER) stress markers. Importantly, our optimized vectors achieved wild-type levels of phenotypic correction with minimal inhibitor formation. These studies provide insights into optimal design of a therapeutically relevant gene therapy vector for a devastating bleeding disorder.Gene Therapy advance online publication, 14 September 2017; doi:10.1038/gt.2017.67.

  20. Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy.

    PubMed

    Vink, Conrad A; Counsell, John R; Perocheau, Dany P; Karda, Rajvinder; Buckley, Suzanne M K; Brugman, Martijn H; Galla, Melanie; Schambach, Axel; McKay, Tristan R; Waddington, Simon N; Howe, Steven J

    2017-08-02

    Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Intrapulmonary and intramyocardial gene transfer in rhesus monkeys (Macaca mulatta): safety and efficiency of HIV-1-derived lentiviral vectors for fetal gene delivery.

    PubMed

    Tarantal, Alice F; McDonald, Ruth J; Jimenez, Daniel F; Lee, C Chang I; O'Shea, Cristin E; Leapley, Alyssa C; Won, Rosa H; Plopper, Charles G; Lutzko, Carolyn; Kohn, Donald B

    2005-07-01

    Fetal gene transfer was studied using intrapulmonary and intramyocardial transfer of SIN HIV-1-derived lentiviral vectors expressing EGFP in rhesus monkeys. Fetuses were monitored sonographically during gestation and tissue analyses performed at term or 3 months postnatal age. Animals remained healthy during the study period as evidenced by normal growth, development, hematology, clinical chemistry, echocardiography, and pulmonary function tests. Strong pulmonary fluorescence was observed postnatally after fetal intrapulmonary delivery of lenti-CMV, but not lenti-SP-C, and compared to nontransferred controls. High EGFP copy numbers were found by quantitative PCR with both vector constructs in lung lobes (lentiviral vector-mediated gene delivery.

  2. Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

    PubMed Central

    Link, Nils; Aubel, Corinne; Kelm, Jens M.; Marty, René R.; Greber, David; Djonov, Valentin; Bourhis, Jean; Weber, Wilfried; Fussenegger, Martin

    2006-01-01

    The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide. PMID:16449199

  3. Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles.

    PubMed

    Link, Nils; Aubel, Corinne; Kelm, Jens M; Marty, René R; Greber, David; Djonov, Valentin; Bourhis, Jean; Weber, Wilfried; Fussenegger, Martin

    2006-01-30

    The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide.

  4. A supramolecular assembly mediates lentiviral DNA integration

    PubMed Central

    Serrao, Erik; Locke, Julia; Swuec, Paolo; Jónsson, Stefán R.; Kotecha, Abhay; Cook, Nicola J.; Pye, Valerie E.; Taylor, Ian A.; Andrésdóttir, Valgerdur; Engelman, Alan N.; Costa, Alessandro; Cherepanov, Peter

    2017-01-01

    Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 Å resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors. PMID:28059770

  5. Envelope Determinants of Equine Lentiviral Vaccine Protection

    PubMed Central

    Craigo, Jodi K.; Ezzelarab, Corin; Cook, Sheila J.; Chong, Liu; Horohov, David; Issel, Charles J.; Montelaro, Ronald C.

    2013-01-01

    Lentiviral envelope (Env) antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV) vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy. The study identified a significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the challenge virus Env gp90 protein compared to the vaccine virus gp90. The report demonstrated approximately 100% protection of immunized ponies from disease after challenge by virus with a homologous gp90 (EV0), and roughly 40% protection against challenge by virus (EV13) with a gp90 13% divergent from the vaccine strain. In the current study we examine whether the protection observed when challenging with the EV0 strain could be conferred to animals via chimeric challenge viruses between the EV0 and EV13 strains, allowing for mapping of protection to specific Env sequences. Viruses containing the EV13 proviral backbone and selected domains of the EV0 gp90 were constructed and in vitro and in vivo infectivity examined. Vaccine efficacy studies indicated that homology between the vaccine strain gp90 and the N-terminus of the challenge strain gp90 was capable of inducing immunity that resulted in significantly lower levels of post-challenge virus and significantly delayed the onset of disease. However, a homologous N-terminal region alone inserted in the EV13 backbone could not impart the 100% protection observed with the EV0 strain. Data presented here denote the complicated and potentially contradictory relationship between in vitro virulence and in vivo pathogenicity. The study highlights the importance of structural conformation for immunogens and emphasizes the need for

  6. Transcriptional activity of novel ALDH1L1 promoters in the rat brain following AAV vector-mediated gene transfer

    PubMed Central

    Mudannayake, Janitha M; Mouravlev, Alexandre; Fong, Dahna M; Young, Deborah

    2016-01-01

    Aldehyde dehydrogenase family 1, member L1 (ALDH1L1) is a recently characterized pan-astrocytic marker that is more homogenously expressed throughout the brain than the classic astrocytic marker, glial fibrillary acidic protein. We generated putative promoter sequence variants of the rat ALDH1L1 gene for use in adeno-associated viral vector-mediated gene transfer, with an aim to achieve selective regulation of transgene expression in astrocytes in the rat brain. Unexpectedly, ALDH1L1 promoter variants mediated transcriptional activity exclusively in neurons in the substantia nigra pars compacta as assessed by luciferase reporter expression at 3 weeks postvector infusion. This selectivity for neurons in the substantia nigra pars compacta also persisted in the context of adeno-associated viral serotype 5, 8 or 9 vector-mediated gene delivery. An in vivo promoter comparison showed the highest performing ALDH1L1 promoter variant mediated higher transgene expression than the neuronal-specific synapsin 1 and tyrosine hydroxylase promoters. The ALDH1L1 promoter was also transcriptionally active in dentate granule neurons following intrahippocampal adeno-associated viral vector infusion, whereas transgene expression was detected in both striatal neurons and astrocytes following vector infusion into the striatum. Our results demonstrate the potential suitability of the ALDH1L1 promoter as a new tool in the development of gene therapy and disease modelling applications. PMID:27990448

  7. Rescue from photoreceptor degeneration in the rd mouse by human immunodeficiency virus vector-mediated gene transfer.

    PubMed

    Takahashi, M; Miyoshi, H; Verma, I M; Gage, F H

    1999-09-01

    Retinitis pigmentosa (RP) is the most common inherited retinal disease, in which photoreceptor cells degenerate, leading to blindness. Mutations in the rod photoreceptor cGMP phosphodiesterase beta subunit (PDEbeta) gene are found in patients with autosomal recessive RP as well as in the rd mouse. We have recently shown that lentivirus vectors based on human immunodeficiency virus (HIV) type 1 achieve stable and efficient gene transfer into retinal cells. In this study, we evaluated the potential of HIV vector-mediated gene therapy for RP in the rd mouse. HIV vectors containing a gene encoding a hemagglutinin (HA)-tagged PDEbeta were injected into the subretinal spaces of newborn rd mouse eyes. One to three rows of photoreceptor nuclei were observed in the eyes for at least 24 weeks postinjection, whereas no photoreceptor cells remained in the eyes of control animals at 6 weeks postinjection. Expression of HA-tagged PDEbeta in the rescued photoreceptor cells was confirmed by two-color confocal immunofluorescence analysis using anti-HA and anti-opsin antibodies. HIV vector-mediated gene therapy appears to be a promising means for the treatment of recessive forms of inherited retinal degeneration.

  8. Long-Term Follow-up of Foamy Viral Vector-Mediated Gene Therapy for Canine Leukocyte Adhesion Deficiency

    PubMed Central

    Bauer, Thomas R; Tuschong, Laura M; Calvo, Katherine R; Shive, Heather R; Burkholder, Tanya H; Karlsson, Eleanor K; West, Robert R; Russell, David W; Hickstein, Dennis D

    2013-01-01

    The development of leukemia following gammaretroviral vector-mediated gene therapy for X-linked severe combined immunodeficiency disease and chronic granulomatous disease (CGD) has emphasized the need for long-term follow-up in animals treated with hematopoietic stem cell gene therapy. In this study, we report the long-term follow-up (4–7 years) of four dogs with canine leukocyte adhesion deficiency (CLAD) treated with foamy viral (FV) vector-mediated gene therapy. All four CLAD dogs previously received nonmyeloablative conditioning with 200 cGy total body irradiation followed by infusion of autologous, CD34+ hematopoietic stem cells transduced by a FV vector expressing canine CD18 from an internal Murine Stem Cell Virus (MSCV) promoter. CD18+ leukocyte levels were >2% following infusion of vector-transduced cells leading to ongoing reversal of the CLAD phenotype for >4 years. There was no clinical development of lymphoid or myeloid leukemia in any of the four dogs and integration site analysis did not reveal insertional oncogenesis. These results showing disease correction/amelioration of disease in CLAD without significant adverse events provide support for the use of a FV vector to treat children with leukocyte adhesion deficiency type 1 (LAD-1) in a human gene therapy clinical trial. PMID:23531552

  9. Quantitation of signal transduction.

    PubMed

    Krauss, S; Brand, M D

    2000-12-01

    Conventional qualitative approaches to signal transduction provide powerful ways to explore the architecture and function of signaling pathways. However, at the level of the complete system, they do not fully depict the interactions between signaling and metabolic pathways and fail to give a manageable overview of the complexity that is often a feature of cellular signal transduction. Here, we introduce a quantitative experimental approach to signal transduction that helps to overcome these difficulties. We present a quantitative analysis of signal transduction during early mitogen stimulation of lymphocytes, with steady-state respiration rate as a convenient marker of metabolic stimulation. First, by inhibiting various key signaling pathways, we measure their relative importance in regulating respiration. About 80% of the input signal is conveyed via identifiable routes: 50% through pathways sensitive to inhibitors of protein kinase C and MAP kinase and 30% through pathways sensitive to an inhibitor of calcineurin. Second, we quantify how each of these pathways differentially stimulates functional units of reactions that produce and consume a key intermediate in respiration: the mitochondrial membrane potential. Both the PKC and calcineurin routes stimulate consumption more strongly than production, whereas the unidentified signaling routes stimulate production more than consumption, leading to no change in membrane potential despite increased respiration rate. The approach allows a quantitative description of the relative importance of signal transduction pathways and the routes by which they activate a specific cellular process. It should be widely applicable.

  10. Transduction in Bacillus subtilis.

    PubMed

    THORNE, C B

    1962-01-01

    Thorne, Curtis B. (Fort Detrick, Frederick, Md.). Transduction in Bacillus subtilis. J. Bacteriol. 83:106-111. 1962.-A bacteriophage, SP-10, isolated from soil carries out general transduction in Bacillus subtilis. Phage propagated on a streptomycin-resistant mutant of the wild-type strain W-23 was capable of transducing to prototrophy strain 168 (indole(-)), as well as all of the auxotrophic mutants of W-23-S(r) tested, which included mutants requiring arginine, histidine, adenine, guanine, thiamine, leucine, or methionine. Although strain 168 was transduced by phage SP-10, lytic activity on this strain could not be detected and attempts to propagate the phage on it failed. Transductions occurred at frequencies in the range of 10(-6) to 10(-5) per plaque-forming unit. Homologous phage was ineffective, deoxyribonuclease had no effect on the frequency of transduction, and transduction was prevented by the addition of phage antiserum. Phage SP-10 was capable of lysogenizing strain W-23-S(r), and this condition was maintained through repeated growth and sporulation cycles in potato-extract medium. Although heating at 65 C for 60 min inactivated free phage particles, spores retained their lysogenic condition after such heat treatment. When heat-treated spores of the lysogenic cultures were used as inocula for growth in a nutrient broth-yeast extract-glucose medium, filtrates contained 10(9), or more, phage particles per ml.

  11. Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy.

    PubMed

    Kong, J; Kim, S-R; Binley, K; Pata, I; Doi, K; Mannik, J; Zernant-Rajang, J; Kan, O; Iqball, S; Naylor, S; Sparrow, J R; Gouras, P; Allikmets, R

    2008-10-01

    Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4-/- mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4 -/- mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E. Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters. Abca4-/- mice were injected subretinally with 1 microl ( approximately 5.0 x 10(5) TU) of each EIAV vector in one eye at postnatal days 4 and 5. An injection of saline, an EIAV-null vector, or an uninjected contralateral eye served as a control. Mice were killed at various times after injection to determine photoreceptor (PR) transduction efficiency and A2E concentrations. EIAV-LacZ vectors transduced from 5 to 20% of the PRs in the injected area in mice. Most importantly, a single subretinal injection of EIAV-CMV-ABCA4 to Abca4-/- mouse eyes substantially reduced disease-associated A2E accumulation compared to untreated and mock-treated control eyes. Treated eyes of Abca4-/- mice accumulated 8-12 pmol per eye (s.d.=2.7) of A2E 1 year after treatment, amounts comparable to wt controls, whereas mock-treated or untreated eyes had 3-5 times more A2E (27-39 pmol per eye, s.d.=1.5; P=0.001-0.005). Although extrapolation to humans requires caution, the high transduction efficiency of both rod and cone photoreceptors and the statistically significant reduction of A2E accumulation in the mouse model of STGD1 suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases.

  12. Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy

    PubMed Central

    Kong, J; Kim, S-R; Binley, K; Pata, I; Doi, K; Mannik, J; Zernant-Rajang, J; Kan, O; Iqball, S; Naylor, S; Sparrow, JR; Gouras, P; Allikmets, R

    2011-01-01

    Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4−/− mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4−/− mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E. Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters. Abca4−/− mice were injected subretinally with 1 µl (~5.0 × 105 TU) of each EIAV vector in one eye at postnatal days 4 and 5. An injection of saline, an EIAV-null vector, or an uninjected contralateral eye served as a control. Mice were killed at various times after injection to determine photoreceptor (PR) transduction efficiency and A2E concentrations. EIAV-LacZ vectors transduced from 5 to 20% of the PRs in the injected area in mice. Most importantly, a single subretinal injection of EIAV-CMV-ABCA4 to Abca4−/− mouse eyes substantially reduced disease-associated A2E accumulation compared to untreated and mock-treated control eyes. Treated eyes of Abca4−/− mice accumulated 8–12 pmol per eye (s.d. = 2.7) of A2E 1 year after treatment, amounts comparable to wt controls, whereas mock-treated or untreated eyes had 3–5 times more A2E (27–39 pmol per eye, s.d. = 1.5; P = 0.001–0.005). Although extrapolation to humans requires caution, the high transduction efficiency of both rod and cone photoreceptors and the statistically significant reduction of A2E accumulation in the mouse model of STGD1 suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases

  13. 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

  14. Adenoviral Vector-Mediated Gene Therapy for Gliomas: Coming of Age

    PubMed Central

    Castro, Maria G.; Candolfi, Marianela; Wilson, Thomas J.; Calinescu, Alexandra; Paran, Christopher; Kamran, Neha; Koschmann, Carl; Moreno-Ayala, Mariela A.; Assi, Hikmat; Lowenstein, Pedro R.

    2014-01-01

    Introduction Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults; it carries a dismal prognosis. Adenoviral vector (Ad)-mediated gene transfer is being developed as a promising therapeutic strategy for GBM. Preclinical studies have demonstrated safety and efficacy of adenovirus administration into the brain and tumor mass in rodents and into the non-human primates’ brain. Importantly Ads have been safely administered within the tumor resection cavity in humans. Areas Covered Background on GBM and Ad vectors; we describe gene therapy strategies for GBM and discuss the value of combination approaches. Finally we discuss the results of the human clinical trials for GBM that have used adenoviral vectors. Expert Opinion The transduction characteristics of Ad vectors, and their safety profile, added to their capacity to achieve high levels of transgene expression have made them powerful vectors for the treatment of GBM. Recent gene therapy successes in the treatment of retinal diseases and systemic brain metabolic diseases, encourages the development of gene therapy for malignant glioma. Exciting clinical trials are currently recruiting patients; although it is large randomized phase III controlled clinical trials that will provide the final decision on the success of gene therapy for the treatment of GBM. PMID:24773178

  15. [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.

  16. Simian immunodeficiency virus-Vpx for improving integrase defective lentiviral vector-based vaccines

    PubMed Central

    2012-01-01

    Background Integrase defective lentiviral vectors (IDLV) represent a promising delivery system for immunization purposes. Human dendritic cells (DC) are the main cell types mediating the immune response and are readily transduced by IDLV, allowing effective triggering of in vitro expansion of antigen-specific primed CD8+ T cells. However, IDLV expression in transduced DC is at lower levels than those of the integrase (IN) competent counterpart, thus requiring further improvement of IDLV for future use in the clinic. Results In this paper we show that the addition of simian immunodeficiency (SIV)-Vpx protein in the vector preparation greatly improves transduction of human and simian DC, but not of murine DC, thus increasing the ability of transduced DC to act as functional antigen presenting cells, in the absence of integrated vector sequences. Importantly, the presence of SIV-Vpx allows for using lower dose of input IDLV during in vitro transduction, thus further improving the IDLV safety profile. Conclusions These results have significant implications for the development of IDLV-based vaccines. PMID:22913641

  17. DNA Methylation and Histone Modifications Are the Molecular Lock in Lentivirally Transduced Hematopoietic Progenitor Cells

    PubMed Central

    Ngai, Siew Ching; Rosli, Rozita; Al Abbar, Akram

    2015-01-01

    Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV. PMID:25961011

  18. 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

  19. 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.

  20. Restoration of normal lysosomal function in mucopolysaccharidosis type VII cells by retroviral vector-mediated gene transfer.

    PubMed Central

    Wolfe, J H; Schuchman, E H; Stramm, L E; Concaugh, E A; Haskins, M E; Aguirre, G D; Patterson, D F; Desnick, R J; Gilboa, E

    1990-01-01

    Retroviral vectors were constructed containing a rat beta-glucuronidase cDNA driven by heterologous promoters. Vector-mediated gene transfer into human and canine beta-glucuronidase-deficient mucopolysaccharidosis type VII fibroblasts completely corrected the deficiency in beta-glucuronidase enzymatic activity. In primary cultures of canine mucopolysaccharidosis type VII retinal pigment epithelial cells, which contain large amounts of undegraded glycosaminoglycan substrates, vector correction restored normal processing of specific glycosaminoglycans in the lysosomal compartment. In canine mucopolysaccharidosis type VII bone marrow cells, beta-glucuronidase was expressed at high levels in transduced cells. Thus, the vector-encoded beta-glucuronidase was expressed at therapeutic levels in the appropriate organelle and corrected the metabolic defect in cells exhibiting the characteristic pathology of this lysosomal storage disorder. Images PMID:2158095

  1. An AAV Vector-Mediated Gene Delivery Approach Facilitates Reconstitution of Functional Human CD8+ T Cells in Mice

    PubMed Central

    Wilson, James M.; Tsuji, Moriya

    2014-01-01

    In the present study, a novel adeno-associated virus (AAV) vector-mediated gene delivery approach was taken to improve the reconstitution of functional CD8+ T cells in humanized mice, thereby mimicking the human immune system (HIS). Human genes encoding HLA-A2 and selected human cytokines (A2/hucytokines) were introduced to an immune-deficient mouse model [NOD/SCID/IL2rγnull (NSG) mice] using AAV serotype 9 (AAV9) vectors, followed by transplantation of human hematopoietic stem cells. NSG mice transduced with AAV9 encoding A2/hucytokines resulted in higher levels of reconstitution of human CD45+ cells compared to NSG mice transduced with AAV9 encoding HLA-A2 alone or HLA-A2-transgenic NSG mice. Furthermore, this group of HIS mice also mounted the highest level of antigen-specific A2-restricted human CD8+ T-cell response upon vaccination with recombinant adenoviruses expressing human malaria and HIV antigens. Finally, the human CD8+ T-cell response induced in human malaria vaccine-immunized HIS mice was shown to be functional by displaying cytotoxic activity against hepatocytes that express the human malaria antigen in the context of A2 molecules. Taken together, our data show that AAV vector-mediated gene delivery is a simple and efficient method to transfer multiple human genes to immune-deficient mice, thus facilitating successful reconstitution of HIS in mice. The HIS mice generated in this study should ultimately allow us to swiftly evaluate the T-cell immunogenicity of various human vaccine candidates in a pre-clinical setting. PMID:24516613

  2. 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

  3. 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

    2016-05-01

    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. © 2016

  4. Highly efficient lentiviral gene transfer in CD34+ and CD34+/38-/lin- cells from mobilized peripheral blood after cytokine prestimulation.

    PubMed

    Géronimi, Fabien; Richard, Emmanuel; Redonnet-Vernhet, Isabelle; Lamrissi-Garcia, Isabelle; Lalanne, Magalie; Ged, Cécile; Moreau-Gaudry, François; De Verneuil, Hubert

    2003-01-01

    Because mobilized peripheral blood (mPB) represents an attractive source of cells for gene therapy, we investigated lentiviral gene transfer in CD34(+) cells and the stem/progenitor-cell-enriched CD34(+)/38(-)/lin(-) cell subset isolated from mPB. In this study, we used an optimized third-generation self-inactivating lentiviral vector containing both the central polypurine tract and the woodchuck hepatitis posttranscriptional regulatory element sequences and encoding enhanced green fluorescent protein (EGFP) under the control of the elongation factor lalpha promoter. This lentivector was first used to compare multiplicity of infection (MOI)-dependent gene transfer efficiency in cord blood (CB) versus mPB CD34(+)-derived cells, colony-forming cells (CFCs), and long-term culture-initiating cells (LTC-ICs). Results showed a difference in the percentage of transduced cells particularly significant at low MOIs. A plateau was reached where 15% and 25% of CB and mPB cells, respectively, remained refractory to lentiviral trans-duction. Effects of a cytokine prestimulation period (18 hours) with interleukin-3, stem cell factor, Flt-3 ligand, and thrombopoietin were then analyzed in total cells, CFCs, and LTC-ICs derived from mPB CD34(+) cells. Transduction levels in those conditions demonstrated a two- and fourfold increase in CFCs and LTC-ICs, respectively, compared with unstimulated (<3 hours) control cells. Moreover, using the same transduction protocol, we were able to efficiently transduce CD34(+)/38(-)/lin(-) cells isolated from mPB, with up to >85% of colonies derived from LTC-ICs expressing EGFP and gene transfer levels remaining stable for 10 weeks in liquid culture. We therefore demonstrate a highly efficient gene transfer in this therapeutically relevant target cell population.

  5. 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.

  6. Optimized production and concentration of lentiviral vectors containing large inserts.

    PubMed

    al Yacoub, Nadya; Romanowska, Malgorzata; Haritonova, Natalie; Foerster, John

    2007-07-01

    Generation of high titer lentiviral stocks and efficient virus concentration are central to maximize the utility of lentiviral technology. Here we evaluate published protocols for lentivirus production on a range of transfer vectors differing in size (7.5-13.2 kb). We present a modified virus production protocol robustly yielding useful titers (up to 10(7)/ml) for a range of different transfer vectors containing packaging inserts up to 7.5 kb. Moreover, we find that virus recovery after concentration by ultracentrifugation depends on the size of the packaged inserts, heavily decreasing for large packaged inserts. We describe a fast (4 h) centrifugation protocol at reduced speed allowing high virus recovery even for large and fragile lentivirus vectors. The protocols outlined in the current report should be useful for many labs interested in producing and concentrating high titer lentiviral stocks.

  7. Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy.

    PubMed

    Baldwin, Kismet; Urbinati, Fabrizia; Romero, Zulema; Campo-Fernandez, Beatriz; Kaufman, Michael L; Cooper, Aaron R; Masiuk, Katelyn; Hollis, Roger P; Kohn, Donald B

    2015-05-01

    Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising ∼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-β(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy. © 2015 AlphaMed Press.

  8. 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.

  9. Lentivirally overexpressed T-bet regulates T-helper cell lineage commitment in chronic hepatitis B patients.

    PubMed

    Liu, Xueni; Tang, Zhenghao; Zhang, Yi; Hu, Jianjun; Li, Dan; Zang, Guoqing; Yu, Yongsheng

    2012-08-01

    Chronic hepatitis B virus (HBV) infection is commonly considered to occur as a result of disturbance of the immune system. T-box expressed in T cells (T-bet) is an essential transcription factor for T helper (Th) cell differentiation and function. The aim of this study was to investigate the effect of T-bet overexpression on Th cell differentiation and the possible mechanism in chronic hepatitis B (CHB) patients. CD4+ T cells from the peripheral blood of 23 CHB patients, 8 acute hepatitis B (AHB) patients and 10 healthy controls were isolated. T-bet mRNA expression of CD4+ T cells was detected by quantitative real-time polymerase chain reaction (PCR). The T-bet DNA fragment was subcloned into the pGC-FU vector containing GFP to generate a recombinant lentiviral vector, pGC-FU-T-bet, while a no-load pGC-FU vector was used as the negative control. After transduction into CD4+ T cells from another 22 CHB patients, the induction of Th1- and Th2-type cytokines was assayed by an enzyme-linked immunosorbent assay (ELISA), and RT-PCR and western blot analysis were used to measure the mRNA and transcription levels of H2.0-like homeobox (HLX1), GATA-3 and STAT-6. T-bet mRNA expression in CD4+ T cells from AHB patients was enhanced compared with CHB patients and healthy controls. Th1-type cytokines and HLX1 expression was upregulated, while Th2-type cytokines and GATA-3 and STAT-6 expression was repressed after lentiviral introduction of T-bet. In conclusion, lentivirally overexpressed T-bet regulates Th cell lineage commitment in CHB patients, which may be mediated by regulating HLX1, GATA-3 and STAT-6 expression.

  10. Lentiviral-Transduced Human Mesenchymal Stem Cells Persistently Express Therapeutic Levels of Enzyme in a Xenotransplantation Model of Human Disease

    PubMed Central

    Meyerrose, Todd E.; Roberts, Marie; Ohlemiller, Kevin K.; Vogler, Carole A.; Wirthlin, Louisa; Nolta, Jan A.; Sands, Mark S.

    2009-01-01

    Bone marrow-derived mesenchymal stem cells (MSCs) are a promising platform for cell- and gene-based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD-SCID MPSVII]). Primary human bone marrow-derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme β-glucuronidase (MSCs-GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD-SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs-GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs-GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease. PMID:18436861

  11. Efficient transfer of HTLV-1 tax gene in various primary and immortalized cells using a flap lentiviral vector.

    PubMed

    Royer-Leveau, Christelle; Mordelet, Elodie; Delebecque, Frédéric; Gessain, Antoine; Charneau, Pierre; Ozden, Simona

    2002-08-01

    Human T cell leukemia virus type 1 (HTLV-1) causes two major diseases: adult T-cell leukemia-lymphoma and tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). In order to understand the involvement of Tax protein in HTLV-1 pathogenesis, we constructed a HIV-1 based lentiviral vector containing the central DNA flap sequence and either the green fluorescent protein (GFP) or the HTLV-1 tax genes. Using these vectors, GFP and tax genes were introduced in several primary and immortalized cells of endothelial, lymphoid, astrocytic or macrophagic origin. As assessed by GFP expression, up to 100% efficiency of transduction was obtained for all cell types tested. Tax expression was detected by Western blot and immuno-fluorescence in the transduced cells. After transduction, the Tax transcriptional activity was confirmed by the transactivation of HTLV-1 LTR-lacZ or HTLV-1 LTR-GFP reporter genes. Increased CD25 and HLA DR expression was observed in human peripheral blood lymphocytes transduced with the Tax vector. These results indicate that both pathways of Tax transactivation, CREB (viral LTR) and NF-kappa B (CD25 and HLA DR), are functional after transduction by TRIP Tax vector. Therefore, this vector provides a useful tool for investigating the role of the Tax viral protein in the pathogenesis of diseases linked to HTLV-1 infection.

  12. 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

  13. Auxin signal transduction.

    PubMed

    Hagen, Gretchen

    2015-01-01

    The plant hormone auxin (indole-3-acetic acid, IAA) controls growth and developmental responses throughout the life of a plant. A combination of molecular, genetic and biochemical approaches has identified several key components involved in auxin signal transduction. Rapid auxin responses in the nucleus include transcriptional activation of auxin-regulated genes and degradation of transcriptional repressor proteins. The nuclear auxin receptor is an integral component of the protein degradation machinery. Although auxin signalling in the nucleus appears to be short and simple, recent studies indicate that there is a high degree of diversity and complexity, largely due to the existence of multigene families for each of the major molecular components. Current studies are attempting to identify interacting partners among these families, and to define the molecular mechanisms involved in the interactions. Future goals are to determine the levels of regulation of the key components of the transcriptional complex, to identify higher-order complexes and to integrate this pathway with other auxin signal transduction pathways, such as the pathway that is activated by auxin binding to a different receptor at the outer surface of the plasma membrane. In this case, auxin binding triggers a signal cascade that affects a number of rapid cytoplasmic responses. Details of this pathway are currently under investigation. © 2015 Authors; published by Portland Press Limited.

  14. Exponential enhancement of oncolytic vesicular stomatitis virus potency by vector-mediated suppression of inflammatory responses in vivo.

    PubMed

    Altomonte, Jennifer; Wu, Lan; Chen, Li; Meseck, Marcia; Ebert, Oliver; García-Sastre, Adolfo; Fallon, John; Woo, Savio L C

    2008-01-01

    Oncolytic virotherapy is a promising strategy for treatment of malignancy, although its effectiveness is hampered by host antiviral inflammatory responses. The efficacy of treatment of oncolytic vesicular stomatitis virus (VSV) in rats bearing multifocal hepatocellular carcinoma (HCC) can be substantially elevated by antibody-mediated depletion of natural killer (NK) cells. In order to test the hypothesis that the oncotyic potency of VSV can be exponentially elevated by evasion of inflammatory responses in vivo, we constructed a recombinant VSV vector expressing equine herpes virus-1 glycoprotein G, which is a broad-spectrum viral chemokine binding protein (rVSV-gG). Infusion of rVSV-gG via the hepatic artery into immune-competent rats bearing syngeneic and multifocal HCC in their livers, resulted in a reduction of NK and NKT cells in the tumors and a 1-log enhancement in intratumoral virus titer in comparison with a reference rVSV vector. The treatment led to increased tumor necrosis and substantially prolonged animal survival without toxicities. These results indicate that rVSV-gG has the potential to be developed as an effective and safe oncolytic agent to treat patients with advanced HCC. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host antiviral inflammatory responses could have general applicability in the field of oncolytic virotherapy for cancer.

  15. S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection.

    PubMed

    Verghese, Santhosh Chakkaramakkil; Goloviznina, Natalya A; Skinner, Amy M; Lipps, Hans J; Kurre, Peter

    2014-04-01

    Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting 'anchoring' non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.

  16. Retargeting vesicular stomatitis virus glycoprotein pseudotyped lentiviral vectors with enhanced stability by in situ synthesized polymer shell.

    PubMed

    Liang, Min; Yan, Ming; Lu, Yunfeng; Chen, Irvin S Y

    2013-02-01

    The ability to introduce transgenes with precise specificity to the desired target cells or tissues is key to a more facile application of genetic therapy. Here, we describe a novel method using nanotechnology to generate lentiviral vectors with altered recognition of host cell receptor specificity. Briefly, the infectivity of the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors was shielded by a thin polymer shell synthesized in situ onto the viral envelope, and new binding ability was conferred to the shielded virus by introducing acrylamide-tailored cyclic arginine-glycine-aspartic acid (cRGD) peptide to the polymer shell. We termed the resulting virus "targeting nanovirus." The targeting nanovirus had similar titer with VSV-G pseudotypes and specifically transduced Hela cells with high transduction efficiency. In addition, the encapsulation of the VSV-G pseudotyped lentivirus by the polymer shell did not change the pathway that VSV-G pseudotypes enter and fuse with cells, as well as later events such as reverse transcription and gene expression. Furthermore, the targeting nanovirus possessed enhanced stability in the presence of human serum, indicating protection of the virus by the polymer shell from human serum complement inactivation. This novel use of nanotechnology demonstrates proof of concept for an approach that could be more generally applied for redirecting viral vectors for laboratory and clinical purposes.

  17. Construction of stable producer cells to make high-titer lentiviral vectors for dendritic cell-based vaccination.

    PubMed

    Lee, Chi-Lin; Chou, Michael; Dai, Bingbing; Xiao, Liang; Wang, Pin

    2012-06-01

    Lentiviral vectors (LVs) enveloped with an engineered Sindbis virus glycoprotein can specifically bind to dendritic cells (DCs) through the surface receptor DC-SIGN and induce antigen expression, thus providing an efficient method for delivering DC-directed vaccines. In this study, we constructed a stable producer line (LV-MGFP) for synthesizing DC-SIGN-targeted HIV-1-based LVs (DC-LVs) encoding green fluorescent protein (GFP) by a concatemeric array transfection technique. We demonstrated that the established stable clones could routinely produce vector supernatants with titers above 10(7) transduction units per milliliter (TU/mL) during a continuous 3-month cell passage. The producer cells were also capable of generating similar titers of DC-LVs in serum-free medium. Moreover, the addition of 1-deoxymannojirimycin (DMJ) enabled the producer cells to manufacture DC-LVs with both improved titers and enhanced potency to evoke antigen-specific CD8(+) T cell responses in mice. The stable lines could accommodate the replacement of the internal murine stem cell virus (MSCV) promoter with the human ubiquitin-C (Ubi) promoter in the lentiviral backbone. The resulting DC-LVs bearing Ubi exhibited the enhanced potency to elicit vaccine-specific immunity. Based on accumulated evidence, our studies support the application of this production method in manufacturing DC-LVs for preclinical and clinical testing of novel DC-based immunization. Copyright © 2011 Wiley Periodicals, Inc.

  18. Retargeting Vesicular Stomatitis Virus Glycoprotein Pseudotyped Lentiviral Vectors with Enhanced Stability by In Situ Synthesized Polymer Shell

    PubMed Central

    Liang, Min; Yan, Ming; Lu, Yunfeng

    2013-01-01

    Abstract The ability to introduce transgenes with precise specificity to the desired target cells or tissues is key to a more facile application of genetic therapy. Here, we describe a novel method using nanotechnology to generate lentiviral vectors with altered recognition of host cell receptor specificity. Briefly, the infectivity of the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors was shielded by a thin polymer shell synthesized in situ onto the viral envelope, and new binding ability was conferred to the shielded virus by introducing acrylamide-tailored cyclic arginine-glycine-aspartic acid (cRGD) peptide to the polymer shell. We termed the resulting virus “targeting nanovirus.” The targeting nanovirus had similar titer with VSV-G pseudotypes and specifically transduced Hela cells with high transduction efficiency. In addition, the encapsulation of the VSV-G pseudotyped lentivirus by the polymer shell did not change the pathway that VSV-G pseudotypes enter and fuse with cells, as well as later events such as reverse transcription and gene expression. Furthermore, the targeting nanovirus possessed enhanced stability in the presence of human serum, indicating protection of the virus by the polymer shell from human serum complement inactivation. This novel use of nanotechnology demonstrates proof of concept for an approach that could be more generally applied for redirecting viral vectors for laboratory and clinical purposes. PMID:23327104

  19. Lentiviral hematopoietic stem cell gene therapy in inherited metabolic disorders.

    PubMed

    Wagemaker, Gerard

    2014-10-01

    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.

  20. 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

  1. CD30 Receptor-Targeted Lentiviral Vectors for Human Induced Pluripotent Stem Cell-Specific Gene Modification.

    PubMed

    Friedel, Thorsten; Jung-Klawitter, Sabine; Sebe, Attila; Schenk, Franziska; Modlich, Ute; Ivics, Zoltán; Schumann, Gerald G; Buchholz, Christian J; Schneider, Irene C

    2016-05-01

    Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4(high) cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation, efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved, while retaining their pluripotency. When added during the reprogramming process, CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus, CD30-LV may serve as novel tool for the selective gene transfer into PSCs with broad applications in basic and therapeutic research.

  2. The use of an optimized chimeric envelope glycoprotein enhances the efficiency of retrograde gene transfer of a pseudotyped lentiviral vector in the primate brain.

    PubMed

    Tanabe, Soshi; Inoue, Ken-Ichi; Tsuge, Hitomi; Uezono, Shiori; Nagaya, Kiyomi; Fijiwara, Maki; Kato, Shigeki; Kobayashi, Kazuto; Takada, Masahiko

    2017-02-28

    Lentiviral vectors have been used not only for various basic research experiments, but also for a wide range of gene therapy trials in animal models. The development of a pseudotyped lentiviral vector with the property of retrograde infection allows us to introduce foreign genes into neurons that are localized in regions innervating the site of vector injection. Here, we report the efficiency of retrograde gene transfer of a recently developed FuG-E pseudotyped lentiviral vector in the primate brain by comparing its transduction pattern with that of the parental FuG-C pseudotyped vector. After injection of the FuG-E vector encoding green fluorescent protein (GFP) into the striatum of macaque monkeys, many GFP-immunoreactive neurons were found in regions projecting to the striatum, such as the cerebral cortex, thalamus, and substantia nigra. Quantitative analysis revealed that in all regions, the number of neurons retrogradely transduced with the FuG-E vector was larger than in the FuG-C vector injection case. It was also confirmed that the FuG-E vector displayed explicit neuronal specificity to the same extent as the FuG-C vector. This vector might promote approaches to pathway-selective gene manipulation and provide a powerful tool for effective gene therapy trials against neurological disorders through enhanced retrograde delivery.

  3. Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.

    PubMed

    Throm, Robert E; Ouma, Annastasia A; Zhou, Sheng; Chandrasekaran, Anantharaman; Lockey, Timothy; Greene, Michael; De Ravin, Suk See; Moayeri, Morvarid; Malech, Harry L; Sorrentino, Brian P; Gray, John T

    2009-05-21

    Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 10(7) transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common gamma chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 x 10(7) TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

  4. New-generation multicistronic expression platform: pTRIDENT vectors containing size-optimized IRES elements enable homing endonuclease-based cistron swapping into lentiviral expression vectors.

    PubMed

    Fux, Cornelia; Langer, Dominik; Kelm, Jens M; Weber, Wilfried; Fussenegger, Martin

    2004-04-20

    Capitalizing on a proven multicistronic expression vector platform we have designed novel pTRIDENT vectors which (1). enable coordinated expression of three desired transgenes, (2). are size-optimized, (3). take advantage of small highly efficient internal ribosome entry sites of the GTX or Rbm3 type, (4). harbor various sites specific for homing endonucleases facilitating promoter/multicistronic expression unit/polyadenylation site swapping as well as (5). straightforward integration into human HIV-l-based lentiviral expression vectors tailored to contain compatible homing endonucleases. Multicistronic expression profiles of novel pTRIDENT vectors engineered for different tricistronic expression configurations encoding human low-molecular-weight urokinase-type plasminogen activator (u-PA(LMW)) or Bacillus stearothermophilus-derived alpha-amylase (SAMY), human vascular endothelial growth factor (hVEGF), and human placental secreted alkaline phosphatase (SEAP) have been quantified in Chinese hamster ovary cells (CHO-K1), mouse fibroblasts (NIH/3T3), and/or human fibrosarcoma (HT-1080) cells. In addition, a pTRIDENT-derived SAMY-VEGF-SEAP expression cassette transferred into a compatible lentiviral expression vector enabled simultaneous high-level transgene expression following transduction of transgenic lentiviral particles into primary human chondrocytes. Copyright 2004 Wiley Periodicals, Inc.

  5. A novel method for banking stem cells from human exfoliated deciduous teeth: lentiviral TERT immortalization and phenotypical analysis.

    PubMed

    Yin, Zhanhai; Wang, Qi; Li, Ye; Wei, Hong; Shi, Jianfeng; Li, Ang

    2016-04-04

    Stem cells from human exfoliated deciduous teeth (SHED) have recently attracted attention as novel multipotential stem cell sources. However, their application is limited due to in vitro replicative senescence. Ectopic expression of telomerase reverse transcriptase (TERT) is a promising strategy for overcoming this replicative senescence. Nevertheless, its potential application and the phenotype as well as tumorigenicity have never been assessed in SHED. TERT expression was stably restored in SHED (TERT-SHED) isolated from healthy children aged 6-8 years using lentiviral transduction with a puromycin selection marker. The expression of TERT was detected using reverse transcription polymerase chain reaction, Western blot and immunofluorescence. Surface markers of SHED were detected by flow cytometry. Enzyme-linked immunosorbent assay was used to assess senescence-associated β-galactosidase, while CCK-8 methods were used to examine the proliferation capacity of SHED and TERT-SHED at different passages. Moreover, multilineage differentiation, karyotype, colony formation in soft agar, and tumor formation in nude mice of SHED and TERT-SHED were also examined. Lentiviral transduction induced stable TERT expression even in SHED at the 40th passage. TERT-SHED showed robust proliferation capacity and low concentration of β-galactosidase. Although they had some different biomarkers than early passage SHED, TERT-SHED at late passage showed similar mutilineage differentiation as TERT at early passage. Moreover, TERT-SHED at late passage showed normal karyotype, no soft agar colony formation, and no tumor formation in nude mice. TERT-immortalized SHED may be a promising resource for stem-cell therapy, although attention should be paid to the biological behavior of the cells.

  6. Characterization of retroviral infectivity and superinfection resistance during retrovirus-mediated transduction of mammalian cells.

    PubMed

    Liao, J; Wei, Q; Fan, J; Zou, Y; Song, D; Liu, J; Liu, F; Ma, C; Hu, X; Li, L; Yu, Y; Qu, X; Chen, L; Yu, X; Zhang, Z; Zhao, C; Zeng, Z; Zhang, R; Yan, S; Wu, T; Wu, X; Shu, Y; Lei, J; Li, Y; Zhang, W; Wang, J; Reid, R R; Lee, M J; Huang, W; Wolf, J M; He, T-C; Wang, J

    2017-04-07

    Retroviral vectors including lentiviral vectors are commonly-used tools to stably express transgenes or RNA molecules in mammalian cells. Their utilities are roughly divided into two categories, stable overexpression of transgenes and RNA molecules, which requires maximal transduction efficiency, or functional selection with retrovirus-based libraries, which takes advantage of retroviral superinfection resistance. However, the dynamic features of retrovirus-mediated transduction are not well-characterized. Here, we engineered two MSCV-based retroviral vectors expressing dual fluorescence proteins and antibiotic markers and analyzed virion production efficiency and virion stability, dynamic infectivity and superinfection resistance in different cell types, and strategies to improve transduction efficiency. We found that the highest virion production occurred between 60 and 72 h after transfection. The stability of the harvested virion supernatant decreased by >60% after three days in storage. We found that retrovirus infectivity varied drastically in the tested human cancer lines, while low transduction efficiency was partially overcome with increased virus titer, prolonged infection duration, and/or repeated infections. Furthermore, we demonstrated that retrovirus receptors PIT1 and PIT2 were lowly expressed in the analyzed cells, and that PIT1 and/or PIT2 overexpression significantly improved transduction efficiency in certain cell lines. Thus, our findings provide resourceful information for the optimal conditions of retroviral-mediated gene delivery.Gene Therapy accepted article preview online, 07 April 2017. doi:10.1038/gt.2017.24.

  7. Improved Hepatocyte Engraftment After Portal Vein Occlusion in LDL Receptor-Deficient WHHL Rabbits and Lentiviral-Mediated Phenotypic Correction In Vitro

    PubMed Central

    Goulinet-Mainot, Sylvie; Tranchart, Hadrien; Groyer-Picard, Marie-Thérèse; Lainas, Panagiotis; Saloum Diop, Papa; Holopherne, Delphine; Gonin, Patrick; Benihoud, Karim; Ba, Nathalie; Gauthier, Olivier; Franco, Dominique; Guettier, Catherine; Pariente, Danièle; Weber, Anne; Dagher, Ibrahim; Huy Nguyen, Tuan

    2012-01-01

    Innovative cell-based therapies are considered as alternatives to liver transplantation. Recent progress in lentivirus-mediated hepatocyte transduction has renewed interest in cell therapy for the treatment of inherited liver diseases. However, hepatocyte transplantation is still hampered by inefficient hepatocyte engraftment. We previously showed that partial portal vein embolization (PVE) improved hepatocyte engraftment in a nonhuman primate model. We developed here an ex vivo approach based on PVE and lentiviral-mediated transduction of hepatocytes from normal (New Zealand White, NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits: the large animal model of familial hypercholesterolemia type IIa (FH). FH is a life-threatening human inherited autosomal disease caused by a mutation in the low-density lipoprotein receptor (LDLR) gene, which leads to severe hypercholesterolemia and premature coronary heart disease. Rabbit hepatocytes were isolated from the resected left liver lobe, and the portal branches of the median lobes were embolized with Histoacryl® glue under radiologic guidance. NZW and WHHL hepatocytes were each labeled with Hoechst dye or transduced with lentivirus expressing GFP under the control of a liver-specific promoter (mTTR, a modified murine transthyretin promoter) and were then immediately transplanted back into donor animals. In our conditions, 65–70% of the NZW and WHHL hepatocytes were transduced. Liver repopulation after transplantation with the Hoechst-labeled hepatocytes was 3.5 ± 2%. It was 1.4 ± 0.6% after transplantation with either the transduced NZW hepatocytes or the transduced WHHL hepatocytes, which was close to that obtained with Hoechst-labeled cells, given the mean transduction efficacy. Transgene expression persisted for at least 8 weeks posttransplantation. Transduction of WHHL hepatocytes with an LDLR-encoding vector resulted in phenotypic correction in vitro as assessed by internalization of fluorescent LDL

  8. Sustaining expression of B domain-deleted human factor VIII mediated by using lentiviral vectors in NOD/SCID mouse.

    PubMed

    Li, Yan-Jie; Chen, Chong; Zeng, Ling-Yu; Cao, Jiang; Xu, Kai-Lin

    2012-06-01

    Recently, gene therapy has been become a promising approach to cure hemophilia A, a most common recessive bleeding disease. The aim of this study was to determine the perspective of lentiviral vector in hemophilia A gene therapy in vitro and in NOD/SCID mice. Lentivirus transfer vector pXZ9/BDDFVIII containing human B-domain-deleted Factor VIII-IRES-eGFP coding sequence and mock control pXZ9 were constructed. Lentivirus was prepared by co-transfecting 3 plasmids into 293FT cells. 293FT, HLF, human bone marrow mesenchymal stem cells and Chang-liver cells were transfected with the prepared virus. Coagulant activity of human FVIII, human FVIII antigen, human FVIII mRNA transcription and genomic integration were assayed by ELISA, one-step method, RT-PCR and PCR after infection. Lentiviral particles were concentrated by ultracentrifugation and NOD/SCID mice were transfected via portal vein injection. Human FVIII antigen in mouse blood plasma was analyzed by ELISA. eGFP expression was observed by fluorescent microscopy and human FVIII transcription in mouse liver was analyzed by RT-PCR at one month after transduction. The results showed that the high titer of recombinant virus was prepared and used to efficiently transduce the target cells in vitro. At 72 h after transfection, high levels of FVIII activity and FVIII antigen were detected. Human FVIII gene transcription could be detected in the liver of NOD/SCID mice received lentiviral particles carrying FVIII gene. Mouse hepatocytes were transfected with recombinant lentivirus efficiently in vivo. Human FVIII level in mouse blood plasma reached to (49 ± 6) mU, (54 ± 8) mU and (23 ± 4) mU at 72 h, one week and one month after transfection respectively. It is concluded that the lentiviral particles carrying BDDhFVIII gene can high efficiently transfect the target cells both in vitro and in vivo, and the transfected target cells can secrete hFVIII efficiently. The sustained expression of human FVIII in NOD/SCID mice is

  9. Vector-Mediated Delivery of a Polyamide ("Peptide") Nucleic Acid Analogue through the Blood-Brain Barrier in vivo

    NASA Astrophysics Data System (ADS)

    Pardridge, William M.; Boado, Ruben J.; Kang, Young-Sook

    1995-06-01

    Polyamide ("peptide") nucleic acids (PNAs) are molecules with antigene and antisense effects that may prove to be effective neuropharmaceuticals if these molecules are enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier in vivo. The model PNA used in the present studies is an 18-mer that is antisense to the rev gene of human immunodeficiency virus type 1 and is biotinylated at the amino terminus and iodinated at a tyrosine residue near the carboxyl terminus. The biotinylated PNA was linked to a conjugate of streptavidin (SA) and the OX26 murine monoclonal antibody to the rat transferrin receptor. The blood-brain barrier is endowed with high transferrin receptor concentrations, enabling the OX26-SA conjugate to deliver the biotinylated PNA to the brain. Although the brain uptake of the free PNA was negligible following intravenous administration, the brain uptake of the PNA was increased at least 28-fold when the PNA was bound to the OX26-SA vector. The brain uptake of the PNA bound to the OX26-SA vector was 0.1% of the injected dose per gram of brain at 60 min after an intravenous injection, approximating the brain uptake of intravenously injected morphine. The PNA bound to the OX26-SA vector retained the ability to bind to synthetic rev mRNA as shown by RNase protection assays. In summary, the present studies show that while the transport of PNAs across the blood-brain barrier is negligible, delivery of these potential neuropharmaceutical drugs to the brain may be achieved by coupling them to vector-mediated peptide-drug delivery systems.

  10. Early, sustained efficacy of adeno-associated virus vector-mediated gene therapy in glycogen storage disease type Ia.

    PubMed

    Koeberl, D D; Sun, B D; Damodaran, T V; Brown, T; Millington, D S; Benjamin, D K; Bird, A; Schneider, A; Hillman, S; Jackson, M; Beaty, R M; Chen, Y T

    2006-09-01

    The deficiency of glucose-6-phosphatase (G6Pase) underlies life-threatening hypoglycemia and growth retardation in glycogen storage disease type Ia (GSD-Ia). An adeno-associated virus (AAV) vector encoding G6Pase was pseudotyped as AAV8 and administered to 2-week-old GSD-Ia mice (n = 9). Median survival was prolonged to 7 months following vector administration, in contrast to untreated GSD-Ia mice that survived for only 2 weeks. Although GSD-Ia mice were initially growth-retarded, treated mice increased fourfold in weight to normal size. Blood glucose was partially corrected by 2 weeks following treatment, whereas blood cholesterol normalized. Glucose-6-phosphatase activity was partially corrected to 25% of the normal level at 7 months of age in treated mice, and blood glucose during fasting remained lower in treated, affected mice than in normal mice. Glycogen storage was partially corrected in the liver by 2 weeks following treatment, but reaccumulated to pre-treatment levels by 7 months old (m.o.). Vector genome DNA decreased between 3 days and 3 weeks in the liver following vector administration, mainly through the loss of single-stranded genomes; however, double-stranded vector genomes were more stable. Although CD8+ lymphocytic infiltrates were present in the liver, partial biochemical correction was sustained at 7 m.o. The development of efficacious AAV vector-mediated gene therapy could significantly reduce the impact of long-term complications in GSD-Ia, including hypoglycemia, hyperlipidemia and growth failure.

  11. 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.

  12. Stable genetic modification of human embryonic stem cells by lentiviral vectors.

    PubMed

    Gropp, Michal; Itsykson, Pavel; Singer, Orna; Ben-Hur, Tamir; Reinhartz, Etti; Galun, Eithan; Reubinoff, Benjamin E

    2003-02-01

    Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of the early preimplantation embryo. An efficient strategy for stable genetic modification of hES cells may be highly valuable for manipulating the cells in vitro and may promote the study of hES cell biology, human embryogenesis, and the development of cell-based therapies. Here, we demonstrate that vectors derived from self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1) are efficient tools for stable genetic modification of hES cells. Transduction of hES cells by a modified vector derived from SIN HIV-1 and containing the woodchuck hepatitis regulatory element (WPRE) and the central polypurine tract (cPPT) sequence facilitated stable transgene expression during prolonged (38 weeks) undifferentiated proliferation in vitro. Southern blot analysis revealed that the viral vector had integrated into the host cells' DNA. Transgene expression was maintained throughout differentiation into progeny of all three germ layers both in vitro and in vivo in teratomas. Thus, the transduced hES cells retained the capability for self-renewal and their pluripotent potential. Genetic modification of hES cells by lentiviral vectors provides a powerful tool for basic and applied research in the area of human ES cells.

  13. 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

  14. CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

    PubMed Central

    Liu, Jingjing; Zhang, Di; Kimata, Jason T.; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. PMID:25541967

  15. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

    PubMed

    Wang, Weiming; Ye, Chaobaihui; Liu, Jingjing; Zhang, Di; Kimata, Jason T; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.

  16. Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-activating polypeptide.

    PubMed

    Maurice, Marielle; Verhoeyen, Els; Salmon, Patrick; Trono, Didier; Russell, Stephen J; Cosset, François-Loïc

    2002-04-01

    In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1-derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G(0) to G(1) phases of the cell cycle. Compared to unmodified HIV-1-based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

  17. NIH oversight of human gene transfer research involving retroviral, lentiviral, and adeno-associated virus vectors and the role of the NIH recombinant DNA advisory committee.

    PubMed

    O'Reilly, Marina; Shipp, Allan; Rosenthal, Eugene; Jambou, Robert; Shih, Tom; Montgomery, Maureen; Gargiulo, Linda; Patterson, Amy; Corrigan-Curay, Jacqueline

    2012-01-01

    In response to public and scientific concerns regarding human gene transfer research, the National Institutes of Health (NIH) developed a transparent oversight system that extends to human gene transfer protocols that are either conducted with NIH funding or conducted at institutions that receive NIH funding for recombinant DNA research. The NIH Recombinant DNA Advisory Committee (RAC) has been the primary advisory body to NIH regarding the conduct of this research. Human gene transfer research proposals that are subject to the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) must be submitted to the NIH Office of Biotechnology Activities (OBA), and protocols that raise novel scientific, safety, medical, ethical, or social issues are publicly discussed at the RAC's quarterly public meetings. OBA also convenes gene transfer safety symposia and policy conferences to provide a public forum for scientific experts to discuss emerging issues in the field. This transparent system of review promotes the rapid exchange of important scientific information and dissemination of data. The goal is to optimize the conduct of individual research protocols and to advance gene transfer research generally. This process has fostered the development of retroviral, lentiviral, and adeno-associated viral vector mediated gene delivery.

  18. 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.

  19. Overview of the HIV-1 Lentiviral Vector System.

    PubMed

    Ramezani, Ali; Hawley, Robert G

    2002-11-01

    Replication-defective oncoretroviral vectors have been the most widely used vehicles for gene-transfer studies because of their capacity to efficiently introduce and stably express transgenes in mammalian cells. A limitation of oncoretroviral vectors is that cell division is required for proviral integration into the host genome. By comparison, lentiviruses such as human immunodeficiency virus type 1 (HIV-1) have evolved a nuclear-import machinery that allows them to infect nondividing as well as dividing cells. This unique property has led to the development of lentiviral vectors for gene delivery to a variety of nondividing or slowly dividing cells including neurons and glial cells of the central nervous system and others. This unit is intended to provide an overview of HIV-1 molecular biology and an introduction to successive generations of HIV-1-based lentiviral vectors.

  20. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.

    PubMed

    Biffi, Alessandra; Montini, Eugenio; Lorioli, Laura; Cesani, Martina; Fumagalli, Francesca; Plati, Tiziana; Baldoli, Cristina; Martino, Sabata; Calabria, Andrea; Canale, Sabrina; Benedicenti, Fabrizio; Vallanti, Giuliana; Biasco, Luca; Leo, Simone; Kabbara, Nabil; Zanetti, Gianluigi; Rizzo, William B; Mehta, Nalini A L; Cicalese, Maria Pia; Casiraghi, Miriam; Boelens, Jaap J; Del Carro, Ubaldo; Dow, David J; Schmidt, Manfred; Assanelli, Andrea; Neduva, Victor; Di Serio, Clelia; Stupka, Elia; Gardner, Jason; von Kalle, Christof; Bordignon, Claudio; Ciceri, Fabio; Rovelli, Attilio; Roncarolo, Maria Grazia; Aiuti, Alessandro; Sessa, Maria; Naldini, Luigi

    2013-08-23

    Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.

  1. Lentiviral vectors: turning a deadly foe into a therapeutic agent.

    PubMed

    Trono, D

    2000-01-01

    The past 3 years have witnessed the spectacular irruption of lentiviral vectors into the limelight of the gene therapy scene. Owing to their ability to deliver transgenes in tissues that had long appeared irremediably refractory to stable genetic manipulation, lentivectors have opened fresh perspectives for the genetic treatment of a wide array of hereditary as well as acquired disorders, and a concrete proposal for their clinical use seems imminent. This article traces the path that has led to this rapid development and describes the current state of the art in the design and production of lentiviral vectors. The important question of biosafety is discussed. This system seems to have the edge over other gene delivery tools for particular targets, however, there remain several issues to be resolved before lentivectors make it to the bedside. Gene Therapy (2000) 7, 20-23.

  2. 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.

  3. Prolonged Integration Site Selection of a Lentiviral Vector in the Genome of Human Keratinocytes

    PubMed Central

    Qian, Wei; Wang, Yong; Li, Rui-fu; Zhou, Xin; Liu, Jing; Peng, Dai-zhi

    2017-01-01

    Background Lentiviral vectors have been successfully used for human skin cell gene transfer studies. Defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. However, genome-wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood. Material/Methods In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software. Results The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions. Conclusions This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases. PMID:28255155

  4. Pheromone Transduction in Moths

    PubMed Central

    Stengl, Monika

    2010-01-01

    Calling female moths attract their mates late at night with intermittent release of a species-specific sex-pheromone blend. Mean frequency of pheromone filaments encodes distance to the calling female. In their zig-zagging upwind search male moths encounter turbulent pheromone blend filaments at highly variable concentrations and frequencies. The male moth antennae are delicately designed to detect and distinguish even traces of these sex pheromones amongst the abundance of other odors. Its olfactory receptor neurons sense even single pheromone molecules and track intermittent pheromone filaments of highly variable frequencies up to about 30 Hz over a wide concentration range. In the hawkmoth Manduca sexta brief, weak pheromone stimuli as encountered during flight are detected via a metabotropic PLCβ-dependent signal transduction cascade which leads to transient changes in intracellular Ca2+ concentrations. Strong or long pheromone stimuli, which are possibly perceived in direct contact with the female, activate receptor-guanylyl cyclases causing long-term adaptation. In addition, depending on endogenous rhythms of the moth's physiological state, hormones such as the stress hormone octopamine modulate second messenger levels in sensory neurons. High octopamine levels during the activity phase maximize temporal resolution cAMP-dependently as a prerequisite to mate location. Thus, I suggest that sliding adjustment of odor response threshold and kinetics is based upon relative concentration ratios of intracellular Ca2+ and cyclic nucleotide levels which gate different ion channels synergistically. In addition, I propose a new hypothesis for the cyclic nucleotide-dependent ion channel formed by insect olfactory receptor/coreceptor complexes. Instead of being employed for an ionotropic mechanism of odor detection it is proposed to control subthreshold membrane potential oscillation of sensory neurons, as a basis for temporal encoding of odors. PMID:21228914

  5. A Drosophila mechanosensory transduction channel.

    PubMed

    Walker, R G; Willingham, A T; Zuker, C S

    2000-03-24

    Mechanosensory transduction underlies a wide range of senses, including proprioception, touch, balance, and hearing. The pivotal element of these senses is a mechanically gated ion channel that transduces sound, pressure, or movement into changes in excitability of specialized sensory cells. Despite the prevalence of mechanosensory systems, little is known about the molecular nature of the transduction channels. To identify such a channel, we analyzed Drosophila melanogaster mechanoreceptive mutants for defects in mechanosensory physiology. Loss-of-function mutations in the no mechanoreceptor potential C (nompC) gene virtually abolished mechanosensory signaling. nompC encodes a new ion channel that is essential for mechanosensory transduction. As expected for a transduction channel, D. melanogaster NOMPC and a Caenorhabditis elegans homolog were selectively expressed in mechanosensory organs.

  6. EIAV vector-mediated delivery of endostatin or angiostatin inhibits angiogenesis and vascular hyperpermeability in experimental CNV.

    PubMed

    Balaggan, K S; Binley, K; Esapa, M; MacLaren, R E; Iqball, S; Duran, Y; Pearson, R A; Kan, O; Barker, S E; Smith, A J; Bainbridge, J W B; Naylor, S; Ali, R R

    2006-08-01

    We evaluated the efficacy of equine infectious anaemia virus (EIAV)-based lentiviral vectors encoding endostatin (EIAV.endostatin) or angiostatin (EIAV.angiostatin) in inhibiting angiogenesis and vascular hyperpermeability in the laser-induced model of choroidal neovascularisation (CNV). Equine infectious anaemia virus.endostatin, EIAV.angiostatin or control (EIAV.null) vectors were administered into the subretinal space of C57Bl/6J mice. Two weeks after laser injury CNV areas and the degree of vascular hyperpermeability were measured by image analysis of in vivo fluorescein angiograms. Compared with EIAV.null-injected eyes, EIAV.endostatin resulted in a 59.5% (P<0.001) reduction in CNV area and a reduction in hyperpermeability of 25.6% (P<0.05). Equine infectious anaemia virus.angiostatin resulted in a 50.0% (P<0.05) reduction in CNV area and a 23.9% (P<0.05) reduction in hyperpermeability. Equine infectious anaemia virus.endostatin, but not EIAV.angiostatin significantly augmented the frequency of apoptosis within the induced CNV as compared with injected controls. TdT-dUTP terminal nick end labeling analysis 5 weeks post-injection, and histological and retinal flatmount analysis 12 months post-injection revealed no evidence of vector- or transgene expression-related deleterious effects on neurosensory retinal cells, or mature retinal vasculature in non-lasered eyes. Highly expressing EIAV-based vectors encoding endostatin or angiostatin effectively control angiogenesis and hyperpermeability in experimental CNV without long-term deleterious effects, supporting the use of such a strategy in the management of patients with exudative age-related macular degeneration.

  7. An Efficient Large-Scale Retroviral Transduction Method Involving Preloading the Vector into a RetroNectin-Coated Bag with Low-Temperature Shaking

    PubMed Central

    Dodo, Katsuyuki; Chono, Hideto; Saito, Naoki; Tanaka, Yoshinori; Tahara, Kenichi; Nukaya, Ikuei; Mineno, Junichi

    2014-01-01

    In retroviral vector-mediated gene transfer, transduction efficiency can be hampered by inhibitory molecules derived from the culture fluid of virus producer cell lines. To remove these inhibitory molecules to enable better gene transduction, we had previously developed a transduction method using a fibronectin fragment-coated vessel (i.e., the RetroNectin-bound virus transduction method). In the present study, we developed a method that combined RetroNectin-bound virus transduction with low-temperature shaking and applied this method in manufacturing autologous retroviral-engineered T cells for adoptive transfer gene therapy in a large-scale closed system. Retroviral vector was preloaded into a RetroNectin-coated bag and incubated at 4°C for 16 h on a reciprocating shaker at 50 rounds per minute. After the supernatant was removed, activated T cells were added to the bag. The bag transduction method has the advantage of increasing transduction efficiency, as simply flipping over the bag during gene transduction facilitates more efficient utilization of the retroviral vector adsorbed on the top and bottom surfaces of the bag. Finally, we performed validation runs of endoribonuclease MazF-modified CD4+ T cell manufacturing for HIV-1 gene therapy and T cell receptor-modified T cell manufacturing for MAGE-A4 antigen-expressing cancer gene therapy and achieved over 200-fold (≥1010) and 100-fold (≥5×109) expansion, respectively. In conclusion, we demonstrated that the large-scale closed transduction system is highly efficient for retroviral vector-based T cell manufacturing for adoptive transfer gene therapy, and this technology is expected to be amenable to automation and improve current clinical gene therapy protocols. PMID:24454964

  8. Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex

    PubMed Central

    Gerits, Annelies; Vancraeyenest, Pascaline; Vreysen, Samme; Laramée, Marie-Eve; Michiels, Annelies; Gijsbers, Rik; Van den Haute, Chris; Moons, Lieve; Debyser, Zeger; Baekelandt, Veerle; Arckens, Lutgarde; Vanduffel, Wim

    2015-01-01

    Abstract. Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area. PMID:26839901

  9. Lentiviral-Human Heme Oxygenase Targeting Endothelium Improved Vascular Function in Angiotensin II Animal Model of Hypertension

    PubMed Central

    Cao, Jian; Sodhi, Komal; Inoue, Kazuyoshi; Quilley, John; Rezzani, Rita; Rodella, Luigi; Vanella, Luca; Germinario, Lucrezia; Stec, David E.; Kappas, Attallah

    2011-01-01

    Abstract We examined the hypothesis that vascular and renal dysfunction caused by angiotensin II (Ang II) through increased levels of blood pressure, inflammatory cytokines, and oxidative stress in Sprague–Dawley rats can be prevented by lentiviral-mediated delivery of endothelial heme oxygenase (HO)-1. We targeted the vascular endothelium using a lentiviral construct expressing human HO-1 under the control of the endothelium-specific promoter VE-cadherin (VECAD-HO-1) and examined the effect of long-term human HO-1 expression on blood pressure in Ang II-mediated increases in blood pressure and oxidant stress. A bolus injection of VECAD-HO-1 into the renal artery resulted in expression of human HO-1 for up to 6–9 weeks. Sprague–Dawley rats were implanted with Ang II minipumps and treated with lentivirus carrying either the HO-1 or green fluorescent protein. Renal tissue from VECAD-HO-1-transduced rats expresses human HO-1 mRNA and proteins without an effect on endogenous HO-1. Infusion of Ang II increased blood pressure (p < 0.001) but decreased vascular relaxation in response to acetylcholine, endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (peNOS) levels, and renal and plasma levels of adiponectin (p < 0.05); in contrast, plasma tumor necrosis factor-α and monocyte chemoattractant protein-1 levels increased. Ang II-treated animals had higher levels of superoxide anion and inducible nitric oxide synthase and increased urinary protein and plasma creatinine levels. Lentiviral transduction with the VECAD-HO-1 construct attenuated the increase in blood pressure (p < 0.05), improved vascular relaxation, increased plasma adiponectin, and prevented the elevation in urinary protein and plasma creatinine in Ang II-treated rats. Endothelial-specific expression of HO-1 also reduced oxidative stress and levels of inflammatory cytokines resulting in increased expression of the anti-apoptotic proteins phosphorylated AKT, phosphorylated AMP

  10. Molecular basis of mechanosensory transduction

    NASA Astrophysics Data System (ADS)

    Gillespie, Peter G.; Walker, Richard G.

    2001-09-01

    Mechanotransduction - a cell's conversion of a mechanical stimulus into an electrical signal - reveals vital features of an organism's environment. From hair cells and skin mechanoreceptors in vertebrates, to bristle receptors in flies and touch receptors in worms, mechanically sensitive cells are essential in the life of an organism. The scarcity of these cells and the uniqueness of their transduction mechanisms have conspired to slow molecular characterization of the ensembles that carry out mechanotransduction. But recent progress in both invertebrates and vertebrates is beginning to reveal the identities of proteins essential for transduction.

  11. Meeting Report: Teaching Signal Transduction

    ERIC Educational Resources Information Center

    Kramer, IJsbrand; Thomas, Geraint

    2006-01-01

    In July, 2005, the European Institute of Chemistry and Biology at the campus of the University of Bordeaux, France, hosted a focused week of seminars, workshops, and discussions around the theme of "teaching signal transduction." The purpose of the summer school was to offer both junior and senior university instructors a chance to…

  12. Meeting Report: Teaching Signal Transduction

    ERIC Educational Resources Information Center

    Kramer, IJsbrand; Thomas, Geraint

    2006-01-01

    In July, 2005, the European Institute of Chemistry and Biology at the campus of the University of Bordeaux, France, hosted a focused week of seminars, workshops, and discussions around the theme of "teaching signal transduction." The purpose of the summer school was to offer both junior and senior university instructors a chance to…

  13. 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.

  14. 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

  15. In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice.

    PubMed

    Jimenez, Veronica; Muñoz, Sergio; Casana, Estefania; Mallol, Cristina; Elias, Ivet; Jambrina, Claudia; Ribera, Albert; Ferre, Tura; Franckhauser, Sylvie; Bosch, Fatima

    2013-12-01

    Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes.

  16. Adenoviral and adeno-associated viral vectors-mediated neuronal gene transfer to cardiovascular control regions of the rat brain.

    PubMed

    Zhang, Yanling; Gao, Yongxin; Speth, Robert C; Jiang, Nan; Mao, Yingying; Sumners, Colin; Li, Hongwei

    2013-01-01

    Viral vectors have been utilized extensively to introduce genetic material into the central nervous system. In order to investigate gene functions in cardiovascular control regions of rat brain, we applied WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) enhanced-adenoviral (Ad) and adeno-assoicated virus (AAV) type 2 vectors to mediate neuronal gene delivery to the paraventricular nucleus of the hypothalamus, the nucleus tractus solitarius and the rostral ventrolateral medulla, three important cardiovascular control regions known to express renin-angiotensin system (RAS) genes. Ad or AAV2 harboring an enhanced green fluorescent protein (EGFP) reporter gene or the angiotensin type 2 receptor gene were microinjected into these brain regions in adult rats. Our results demonstrated that both AAV2 and Ad vectors elicited long-term neuronal transduction in these regions. Interestingly, we found that the WPRE caused expression of GFP driven by the synapsin1 promoter in pure glial cultures or co-cultures of neurons and glia derived from rat hypothalamus and brainstem. However, in rat paraventricular nucleus WPRE did not cause expression of GFP in glia. This demonstrates the potential use of these vectors in studies of physiological functions of certain genes in the cardiovascular control regions of the brain.

  17. 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

  18. Fetal gene transfer using lentiviral vectors: in vivo detection of gene expression by microPET and optical imaging in fetal and infant monkeys.

    PubMed

    Tarantal, Alice F; Lee, C Chang I; Jimenez, Daniel F; Cherry, Simon R

    2006-12-01

    Fetal intraperitoneal administration of human immunodeficiency virus (HIV)-l-derived lentiviral vectors (10(7) infectious particles/fetus) has consistently shown high levels of transduction and gene expression in the omentum, peritoneum, and diaphragm when assessed by polymerase chain reaction (PCR) and whole tissue fluorescence. In vivo imaging techniques were explored with early-gestation long-tailed macaques that were administered the vesicular stomatitis virus-glycoprotein (VSV-G)-pseudotyped HIV-1-derived lentiviral vector expressing a mutant herpes simplex virus type 1 thymidine kinase (HSV-1-sr39tk) and firefly luciferase under the control of the cytomegalovirus (CMV) promoter. Fetuses were monitored sonographically and twice during gestation 9-[4-[18F]Fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) was injected into the fetal circulation under ultrasound guidance in preparation for microPET imaging. All newborns were delivered at term by cesarean section and raised in the nursery for postnatal studies. At 2 months postnatal age, animals were imaged and biodistribution was assessed. Optical imaging for firefly luciferase expression was also performed every 2 months postnatal age. Under all imaging conditions gene expression was observed in the abdominal region, and closely paralleled findings from prior studies based on whole tissue fluorescence. These investigations have shown that HSV-1-sr39tk and firefly luciferase can be used to safely detect transgene expression at multiple time points in fetal and infant monkeys in vivo and without evidence of adverse effects.

  19. Lentivirus Gene Transfer in Murine Hematopoietic Progenitor Cells Is Compromised by a Delay in Proviral Integration and Results in Transduction Mosaicism and Heterogeneous Gene Expression in Progeny Cells

    PubMed Central

    Mikkola, Hanna; Woods, Niels-Bjarne; Sjögren, Marketa; Helgadottir, Hildur; Hamaguchi, Isao; Jacobsen, Sten-Eirik; Trono, Didier; Karlsson, Stefan

    2000-01-01

    Human immunodeficiency virus type 1-based lentivirus vectors containing the green fluorescent protein (GFP) gene were used to transduce murine Lin− c-kit+ Sca1+ primitive hematopoietic progenitor cells. Following transduction, the cells were plated into hematopoietic progenitor cell assays in methylcellulose and the colonies were scored for GFP positivity. After incubation for 20 h, lentivirus vectors transduced 27.3% ± 6.7% of the colonies derived from unstimulated target cells, but transduction was more efficient when the cells were supported with stem cell factor (SCF) alone (42.0% ± 5.5%) or SCF, interleukin-3 (IL-3), and IL-6 (53.3 ± 1.8%) during transduction. The, vesicular stomatitis virus glycoprotein-pseudotyped MGIN oncoretrovirus control vector required IL-3, IL-6, and SCF for significant transduction (39.3 ± 9.4%). Interestingly, only a portion of the progeny cells within the lentivirus-transduced methylcellulose colonies expressed GFP, in contrast to the homogeneous expression in oncoretrovirus-transduced colonies. Secondary plating of the primary GFP+ lentivirus vector-transduced colonies revealed vector PCR+ GFP+ (42%), vector PCR− GFP− (46%), and vector PCR+ GFP− (13%) secondary colonies, indicating true genetic mosaicism with respect to the viral genome in the progeny cells. The degree of vector mosaicism in individual colonies could be reduced by extending the culture time after transduction and before plating into the clonal progenitor cell assay, indicating a delay in the lentiviral integration process. Furthermore, supplementation with exogenous deoxynucleoside triphosphates during transduction decreased mosaicism within the colonies. Although cytokine stimulation during transduction correlates with higher transduction efficiency, rapid cell division after transduction may result in loss of the viral genome in the progeny cells. Therefore, optimal transduction may require activation without promoting intense cell proliferation prior

  20. Gene transfer in ovarian cancer cells: a comparison between retroviral and lentiviral vectors.

    PubMed

    Indraccolo, Stefano; Habeler, Walter; Tisato, Veronica; Stievano, Laura; Piovan, Erich; Tosello, Valeria; Esposito, Giovanni; Wagner, Ralf; Uberla, Klaus; Chieco-Bianchi, Luigi; Amadori, Alberto

    2002-11-01

    Local gene therapy could be a therapeutic option for ovarian carcinoma, a life-threatening malignancy, because of disease containment within the peritoneal cavity in most patients. Lentiviral vectors, which are potentially capable of stable transgene expression, may be useful to vehicle therapeutic molecules requiring long-term production in these tumors. To investigate this concept, we used lentiviral vectors to deliver the enhanced green fluorescent protein (EGFP) gene to ovarian cancer cells. Their efficiency of gene transfer was compared with that of a retroviral vector carrying the same envelope. In vitro, both vectors infected ovarian cancer cells with comparable efficiency under standard culture conditions; however, the lentiviral vector was much more efficient in transducing growth-arrested cells when compared with the retroviral vector. Gene transfer was fully neutralized by an anti-VSV-G antibody, and in vitro stability was similar. In vivo, the lentiviral vector delivered the transgene 10-fold more efficiently to ovarian cancer cells growing i.p. in SCID mice, as evaluated by real-time PCR analysis of the tumors. Confocal microscopy analysis of tumor sections showed a dramatic difference at the level of transgene expression, because abundant EGFP(+) cells were detected only in mice receiving the lentiviral vector. Quantitative analysis by flow cytometry confirmed this and indicated 0.05 and 5.6% EGFP(+) tumor cells after administration of the retroviral and lentiviral vector, respectively. Injection of ex vivo transduced tumor cells, sorted for EGFP expression, indicated that the lentiviral vector was considerably more resistant to in vivo silencing in comparison with the retroviral vector. Finally, multiple administrations of a murine IFN-alpha(1)-lentiviral vector to ovarian carcinoma-bearing mice significantly prolonged the animals' survival, indicating the therapeutic efficacy of this approach. These findings indicate that lentiviral vectors deserve

  1. Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses.

    PubMed

    Kuate, Seraphin; Stefanou, Daniela; Hoffmann, Dennis; Wildner, Oliver; Uberla, Klaus

    2004-11-01

    The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.

  2. Phosphorylation in halobacterial signal transduction.

    PubMed Central

    Rudolph, J; Tolliday, N; Schmitt, C; Schuster, S C; Oesterhelt, D

    1995-01-01

    Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli. Images PMID:7556066

  3. Bacteriophage Transduction in Staphylococcus epidermidis

    PubMed Central

    Olson, Michael E.; Horswill, Alexander R.

    2016-01-01

    The genetic manipulation of Staphylococcus epidermidis for molecular experimentation has long been an area of difficulty. Many of the traditional laboratory techniques for strain construction are laborious and hampered by poor efficiency. The ability to move chromosomal genetic markers and plasmids using bacteriophage transduction has greatly increased the speed and ease of S. epidermidis studies. These molecular genetic advances have advanced the S. epidermidis research field beyond a select few genetically tractable strains and facilitated investigations of clinically relevant isolates. PMID:24222465

  4. Optimized adeno-associated viral vector-mediated striatal DOPA delivery restores sensorimotor function and prevents dyskinesias in a model of advanced Parkinson's disease.

    PubMed

    Björklund, Tomas; Carlsson, Thomas; Cederfjäll, Erik Ahlm; Carta, Manolo; Kirik, Deniz

    2010-02-01

    Viral vector-mediated gene transfer utilizing adeno-associated viral vectors has recently entered clinical testing as a novel tool for delivery of therapeutic agents to the brain. Clinical trials in Parkinson's disease using adeno-associated viral vector-based gene therapy have shown the safety of the approach. Further efforts in this area will show if gene-based approaches can rival the therapeutic efficacy achieved with the best pharmacological therapy or other, already established, surgical interventions. One of the strategies under development for clinical application is continuous 3,4-dihydroxyphenylalanine delivery. This approach has been shown to be efficient in restoring motor function and reducing established dyskinesias in rats with a partial lesion of the nigrostriatal dopamine projection. Here we utilized high purity recombinant adeno-associated viral vectors serotype 5 coding for tyrosine hydroxylase and its co-factor synthesizing enzyme guanosine-5'-triphosphate cyclohydrolase-1, delivered at an optimal ratio of 5 : 1, to show that the enhanced 3,4-dihydroxyphenylalanine production obtained with this optimized delivery system results in robust recovery of function in spontaneous motor tests after complete dopamine denervation. We found that the therapeutic efficacy was substantial and could be maintained for at least 6 months. The tyrosine hydroxylase plus guanosine-5'-triphosphate cyclohydrolase-1 treated animals were resistant to developing dyskinesias upon peripheral l-3,4-dihydroxyphenylalanine drug challenge, which is consistent with the interpretation that continuous dopamine stimulation resulted in a normalization of the post-synaptic response. Interestingly, recovery of forelimb use in the stepping test observed here was maintained even after a second lesion depleting the serotonin input to the forebrain, suggesting that the therapeutic efficacy was not solely dependent on dopamine synthesis and release from striatal serotonergic terminals

  5. Vector-mediated release of GABA attenuates pain-related behaviors and reduces NaV1.7 in DRG neurons

    PubMed Central

    Chattopadhyay, Munmun; Mata, Marina; Fink, David J.

    2012-01-01

    Pain is a common and debilitating accompaniment of neuropathy that occurs as a complication of diabetes. In the current study, we examined the effect of continuous release of gamma amino butyric acid (GABA), achieved by gene transfer of glutamic acid decarboxylase (GAD67) to dorsal root ganglia (DRG) in vivo using a nonreplicating herpes simplex virus (HSV)-based vector (vG) in a rat model of painful diabetic neuropathy (PDN). Subcutaneous inoculation of vG reduced mechanical hyperalgesia, thermal hyperalgesia and cold allodynia in rats with PDN. Continuous release of GABA from vector transduced cells in vivo prevented the increase in the voltage gated sodium channel isoform 1.7 (NaV1.7) protein that is characteristic of PDN. In vitro, infection of primary DRG neurons with vG prevented the increase in NaV1.7 resulting from exposure to hyperglycemia. The effect of vector-mediated GABA on NaV1.7 levels in vitro was blocked by phaclofen but not by bicuculline, a GABAB receptor effect that was blocked by pertussis toxin-(PTX) interference with Gα(i/o) function. Taken in conjunction with our previous observation that continuous activation of delta opioid receptors by vector-mediated release of enkephalin also prevents the increase in NaV1.7 in DRG exposed to hyperglycemia in vitro or in vivo, the observations in this report suggest a novel common mechanism through which activation of G protein coupled receptors (GPCR) in DRG neurons regulate the phenotype of the primary afferent. PMID:21486703

  6. Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors

    PubMed Central

    Hanayama, Hiroyuki; Ohashi, Kazuo; Utoh, Rie; Shimizu, Hirofumi; Ise, Kazuya; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Tsuchiya, Hiroyuki; Okano, Teruo; Gotoh, Mitsukazu

    2015-01-01

    To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells. PMID:26858906

  7. Risks Associated With Lentiviral Vector Exposures and Prevention Strategies

    PubMed Central

    Schlimgen, Ryan; Howard, John; Wooley, Dawn; Thompson, Maureen; Baden, Lindsey R.; Yang, Otto O.; Christiani, David C.; Mostoslavsky, Gustavo; Diamond, David V.; Duane, Elizabeth Gilman; Byers, Karen; Winters, Thomas; Gelfand, Jeffrey A.; Fujimoto, Gary; Hudson, T. Warner; Vyas, Jatin M.

    2016-01-01

    Lentiviral vectors (LVVs) are powerful genetic tools that are being used with greater frequency in biomedical laboratories and clinical trials. Adverse events reported from initial clinical studies provide a basis for risk assessment of occupational exposures, yet many questions remain about the potential harm that LVVs may cause. We review those risks and provide a framework for principal investigators, Institutional Biosafety Committees, and occupational health professionals to assess and communicate the risks of exposure to staff. We also provide recommendations to federal research and regulatory agencies for tracking LVV exposures to evaluate long-term outcomes. U.S. Food and Drug Administration approved antiviral drugs for HIV have theoretical benefits in LVV exposures, although evidence to support their use is currently limited. If treatment is appropriate, we recommend a 7-day treatment with an integrase inhibitor with or without a reverse transcriptase inhibitor within 72 hours of exposure. PMID:27930472

  8. Risks Associated With Lentiviral Vector Exposures and Prevention Strategies.

    PubMed

    Schlimgen, Ryan; Howard, John; Wooley, Dawn; Thompson, Maureen; Baden, Lindsey R; Yang, Otto O; Christiani, David C; Mostoslavsky, Gustavo; Diamond, David V; Duane, Elizabeth Gilman; Byers, Karen; Winters, Thomas; Gelfand, Jeffrey A; Fujimoto, Gary; Hudson, T Warner; Vyas, Jatin M

    2016-12-01

    Lentiviral vectors (LVVs) are powerful genetic tools that are being used with greater frequency in biomedical laboratories and clinical trials. Adverse events reported from initial clinical studies provide a basis for risk assessment of occupational exposures, yet many questions remain about the potential harm that LVVs may cause. We review those risks and provide a framework for principal investigators, Institutional Biosafety Committees, and occupational health professionals to assess and communicate the risks of exposure to staff. We also provide recommendations to federal research and regulatory agencies for tracking LVV exposures to evaluate long-term outcomes. U.S. Food and Drug Administration approved antiviral drugs for HIV have theoretical benefits in LVV exposures, although evidence to support their use is currently limited. If treatment is appropriate, we recommend a 7-day treatment with an integrase inhibitor with or without a reverse transcriptase inhibitor within 72 hours of exposure.

  9. Conserved Interaction of Lentiviral Vif Molecules with HIV-1 Gag and Differential Effects of Species-Specific Vif on Virus Production.

    PubMed

    Zheng, Wenwen; Ling, Limian; Li, Zhaolong; Wang, Hong; Rui, Yajuan; Gao, Wenying; Wang, Shaohua; Su, Xing; Wei, Wei; Yu, Xiao-Fang

    2017-04-01

    The virion infectivity factor (Vif) open reading frame is conserved among most lentiviruses. Vif molecules contribute to viral replication by inactivating host antiviral factors, the APOBEC3 cytidine deaminases. However, various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC3. Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported. Here, we show for the first time that diverse lentiviral Vif molecules maintain the ability to interact with the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55(Gag)) polyprotein. Surprisingly, bovine immunodeficiency virus (BIV) Vif, but not HIV-1 Vif, interfered with HIV-1 production and viral infectivity even in the absence of APOBEC3. Further analysis revealed that BIV Vif demonstrated an enhanced interaction with Pr55(Gag) compared to that of HIV-1 Vif, and BIV Vif defective for the Pr55(Gag) interaction lost its ability to inhibit HIV-1. The C-terminal region of capsid (CA) and the p2 region of Pr55(Gag), which are important for virus assembly and maturation, were involved in the interaction. Transduction of CD4(+) T cells with BIV Vif blocked HIV-1 replication. Thus, the conserved Vif-Pr55(Gag) interaction provides a potential target for the future development of antiviral strategies.IMPORTANCE The conserved Vif accessory proteins of primate lentiviruses HIV-1, simian immunodeficiency virus (SIV), and BIV all form ubiquitin ligase complexes to target host antiviral APOBEC3 proteins for degradation, with different cellular requirements and using different molecular mechanisms. Here, we demonstrate that BIV Vif can interfere with HIV-1 Gag maturation and suppress HIV-1 replication through interaction with the precursor of the Gag (Pr55(Gag)) of HIV-1 in virus-producing cells. Moreover, the HIV-1 and SIV Vif proteins are conserved in terms of their interactions with HIV-1 Pr55(Gag) although HIV-1 Vif proteins

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

    PubMed Central

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

    2014-01-01

    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. PMID:24463623

  11. 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

  12. Dendritic cell-targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and cGAS activation.

    PubMed

    Kim, Jocelyn T; Liu, Yarong; Kulkarni, Rajan P; Lee, Kevin K; Dai, Bingbing; Lovely, Geoffrey; Ouyang, Yong; Wang, Pin; Yang, Lili; Baltimore, David

    2017-07-21

    Dendritic cell (DC) activation and antigen presentation are critical for efficient priming of T cell responses. Here, we study how lentiviral vectors (LVs) deliver antigen and activate DCs to generate T cell immunization in vivo. We report that antigenic proteins delivered in vector particles via pseudotransduction were sufficient to stimulate an antigen-specific immune response. The delivery of the viral genome encoding the antigen increased the magnitude of this response in vivo but was irrelevant in vitro. Activation of DCs by LVs was independent of MyD88, TRIF, and MAVS, ruling out an involvement of Toll-like receptor or RIG-I-like receptor signaling. Cellular DNA packaged in LV preparations induced DC activation by the host STING (stimulator of interferon genes) and cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase) pathway. Envelope-mediated viral fusion also activated DCs in a phosphoinositide 3-kinase-dependent but STING-independent process. Pseudotransduction, transduction, viral fusion, and delivery of cellular DNA collaborate to make the DC-targeted LV preparation an effective immunogen. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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

    PubMed

    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 (~10(7) 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 10(8) IU/mL, which upon concentration increased to 10(10) 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.

  14. Differentiation of Human Mesenchymal Stem Cells into Insulin Producing Cells by Using A Lentiviral Vector Carrying PDX1

    PubMed Central

    Allahverdi, Amir; Abroun, Saied; Jafarian, Arefeh; Soleimani, Masoud; Taghikhani, Mohammad; Eskandari, Fatemeh

    2015-01-01

    Objective Type I diabetes is an immunologically-mediated devastation of insulin producing cells (IPCs) in the pancreatic islet. Stem cells that produce β-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells (MSCs) are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 (PDX1) is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults. Materials and Methods We induced the over-expression of the PDX1 gene in human bone marrow MSCs (BM-MSCs) by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions. Results After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction (RT-PCR) and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations. Conclusion MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation. PMID:26199902

  15. Notable reduction in illegitimate integration mediated by a PPT-deleted, nonintegrating lentiviral vector.

    PubMed

    Kantor, Boris; Bayer, Matthew; Ma, Hong; Samulski, Jude; Li, Chengwen; McCown, Thomas; Kafri, Tal

    2011-03-01

    Nonintegrating lentiviral vectors present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling short-term expression of potentially hazardous gene products. However, residual, integrase-independent integration raises a concern that may limit the usefulness of this system. Here we present a novel 3' polypurine tract (PPT)-deleted lentiviral vector that demonstrates impaired integration efficiency and, when packaged into integrase-deficient particles, significantly reduced illegitimate integration. Cells transduced with PPT-deleted vectors exhibited predominantly 1-long terminal repeat (LTR) circles and a low level of linear genomes after reverse transcription (RT). Importantly, the PPT-deleted vector exhibited titers and in vitro and in vivo expression levels matching those of conventional nonintegrating lentiviral vectors. This safer nonintegrating lentiviral vector system will support emerging technologies, such as those based on transient expression of zinc-finger nucleases (ZFNs) for gene editing, as well as reprogramming factors for inducing pluripotency.

  16. Notable Reduction in Illegitimate Integration Mediated by a PPT-deleted, Nonintegrating Lentiviral Vector

    PubMed Central

    Kantor, Boris; Bayer, Matthew; Ma, Hong; Samulski, Jude; Li, Chengwen; McCown, Thomas; Kafri, Tal

    2011-01-01

    Nonintegrating lentiviral vectors present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling short-term expression of potentially hazardous gene products. However, residual, integrase-independent integration raises a concern that may limit the usefulness of this system. Here we present a novel 3′ polypurine tract (PPT)-deleted lentiviral vector that demonstrates impaired integration efficiency and, when packaged into integrase-deficient particles, significantly reduced illegitimate integration. Cells transduced with PPT-deleted vectors exhibited predominantly 1–long terminal repeat (LTR) circles and a low level of linear genomes after reverse transcription (RT). Importantly, the PPT-deleted vector exhibited titers and in vitro and in vivo expression levels matching those of conventional nonintegrating lentiviral vectors. This safer nonintegrating lentiviral vector system will support emerging technologies, such as those based on transient expression of zinc–finger nucleases (ZFNs) for gene editing, as well as reprogramming factors for inducing pluripotency. PMID:21157436

  17. Sensory Transduction in Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Brown, Austin L.; Ramot, Daniel; Goodman, Miriam B.

    The roundworm Caenorhabditis elegans has a well-defined and comparatively simple repertoire of sensory-guided behaviors, all of which rely on its ability to detect chemical, mechanical or thermal stimuli. In this chapter, we review what is known about the ion channels that mediate sensation in this remarkable model organism. Genetic screens for mutants defective in sensory-guided behaviors have identified genes encoding channel proteins, which are likely transducers of chemical, thermal, and mechanical stimuli. Such classical genetic approaches are now being coupled with molecular genetics and in vivo cellular physiology to elucidate how these channels are activated in specific sensory neurons. The ion channel superfamilies implicated in sensory transduction in C. elegans - CNG, TRP, and DEG/ENaC - are conserved across phyla and also appear to contribute to sensory transduction in other organisms, including vertebrates. What we learn about the role of these ion channels in C. elegans sensation is likely to illuminate analogous processes in other animals, including humans.

  18. Lentiviral Vector Gene Transfer of Endostatin/Angiostatin for Macular Degeneration (GEM) Study

    PubMed Central

    Campochiaro, Peter A.; Lauer, Andreas K.; Sohn, Elliott H.; Mir, Tahreem A.; Naylor, Stuart; Anderton, Matthew C.; Kelleher, Michelle; Harrop, Richard; Ellis, Scott; Mitrophanous, Kyriacos A.

    2017-01-01

    Neovascular age-related macular degeneration (NVAMD) is a prevalent cause of vision loss. Intraocular injections of VEGF-neutralizing proteins provide benefit, but many patients require frequent injections for a prolonged period. Benefits are often lost over time due to lapses in treatment. New treatments that sustain anti-angiogenic activity are needed. This study tested the safety and expression profile of a lentiviral Equine Infectious Anemia Virus (EIAV) vector expressing endostatin and angiostatin (RetinoStat®). Patients with advanced NVAMD were enrolled at three centers in the United States, and the study eye received a subretinal injection of 2.4 × 104 (n = 3), 2.4 × 105 (n = 3), or 8.0 × 105 transduction units (TU; n = 15). Each of the doses was well-tolerated with no dose-limiting toxicities. There was little or no ocular inflammation. There was one procedure-related serious adverse event (AE), a macular hole, which was managed without difficulty and resolved. There was a vector dose-related increase in aqueous humor levels of endostatin and angiostatin with high reproducibility among subjects within cohorts. Mean levels of endostatin and angiostatin peaked between 12 and 24 weeks after injection of 2.4 × 105 TU or 8.0 × 105 TU at 57–81 ng/mL for endostatin and 15–27 ng/mL for angiostatin, and remained stable through the last measurement at week 48. Long-term follow-up demonstrated expression was maintained at last measurement (2.5 years in eight subjects and >4 years in two subjects). Despite an apparent reduction in fluorescein angiographic leakage that broadly correlated with the expression levels in the majority of patients, only one subject showed convincing evidence of anti-permeability activity in these late-stage patients. There was no significant change in mean lesion size in subjects injected with 8.0 × 105 TU. These data demonstrate that EIAV vectors provide a safe platform with robust and

  19. Lentiviral Vector Gene Transfer of Endostatin/Angiostatin for Macular Degeneration (GEM) Study.

    PubMed

    Campochiaro, Peter A; Lauer, Andreas K; Sohn, Elliott H; Mir, Tahreem A; Naylor, Stuart; Anderton, Matthew C; Kelleher, Michelle; Harrop, Richard; Ellis, Scott; Mitrophanous, Kyriacos A

    2017-01-01

    Neovascular age-related macular degeneration (NVAMD) is a prevalent cause of vision loss. Intraocular injections of VEGF-neutralizing proteins provide benefit, but many patients require frequent injections for a prolonged period. Benefits are often lost over time due to lapses in treatment. New treatments that sustain anti-angiogenic activity are needed. This study tested the safety and expression profile of a lentiviral Equine Infectious Anemia Virus (EIAV) vector expressing endostatin and angiostatin (RetinoStat(®)). Patients with advanced NVAMD were enrolled at three centers in the United States, and the study eye received a subretinal injection of 2.4 × 10(4) (n = 3), 2.4 × 10(5) (n = 3), or 8.0 × 10(5) transduction units (TU; n = 15). Each of the doses was well-tolerated with no dose-limiting toxicities. There was little or no ocular inflammation. There was one procedure-related serious adverse event (AE), a macular hole, which was managed without difficulty and resolved. There was a vector dose-related increase in aqueous humor levels of endostatin and angiostatin with high reproducibility among subjects within cohorts. Mean levels of endostatin and angiostatin peaked between 12 and 24 weeks after injection of 2.4 × 10(5) TU or 8.0 × 10(5) TU at 57-81 ng/mL for endostatin and 15-27 ng/mL for angiostatin, and remained stable through the last measurement at week 48. Long-term follow-up demonstrated expression was maintained at last measurement (2.5 years in eight subjects and >4 years in two subjects). Despite an apparent reduction in fluorescein angiographic leakage that broadly correlated with the expression levels in the majority of patients, only one subject showed convincing evidence of anti-permeability activity in these late-stage patients. There was no significant change in mean lesion size in subjects injected with 8.0 × 10(5) TU. These data demonstrate that EIAV vectors provide a safe platform with robust

  20. Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors.

    PubMed

    Miao, Carol H

    2016-01-01

    Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1bα promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α-granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable

  1. rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

    PubMed Central

    2013-01-01

    Background Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra. Results We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical

  2. rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration.

    PubMed

    Oliveras-Salvá, Marusela; Van der Perren, Anke; Casadei, Nicolas; Stroobants, Stijn; Nuber, Silke; D'Hooge, Rudi; Van den Haute, Chris; Baekelandt, Veerle

    2013-11-25

    Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra. We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical analysis that showed

  3. Preclinical evaluation of efficacy and safety of an improved lentiviral vector for the treatment of β-thalassemia and sickle cell disease.

    PubMed

    Negre, Olivier; Bartholomae, Cynthia; Beuzard, Yves; Cavazzana, Marina; Christiansen, Lauryn; Courne, Céline; Deichmann, Annette; Denaro, Maria; de Dreuzy, Edouard; Finer, Mitchell; Fronza, Raffaele; Gillet-Legrand, Béatrix; Joubert, Christophe; Kutner, Robert; Leboulch, Philippe; Maouche, Leïla; Paulard, Anaïs; Pierciey, Francis J; Rothe, Michael; Ryu, Byoung; Schmidt, Manfred; von Kalle, Christof; Payen, Emmanuel; Veres, Gabor

    2015-01-01

    A previously published clinical trial demonstrated the benefit of autologous CD34(+) cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered β-globin gene (β(A-T87Q)-globin) in a subject with β thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease.

  4. Desmin-regulated Lentiviral Vectors for Skeletal Muscle Gene Transfer

    PubMed Central

    Talbot, Gillian E; Waddington, Simon N; Bales, Olivia; Tchen, Rose C; Antoniou, Michael N

    2009-01-01

    Lentiviral vectors (LVs) are highly attractive as a gene therapy agent as they are able to stably integrate their genomes in both dividing and nondividing cells and, in principle, provide long-term therapeutic benefit. However, their performance in skeletal muscle in adult animals has, to date, been disappointing. In order to gain clearer insight into their utility in this tissue type, we have conducted an extensive quantitative comparison of constitutive and muscle-specific promoter activities in skeletal muscle and nonmuscle systems following LV delivery in cell lines and neonatal mice. Our data show that LV delivery to hind leg skeletal muscle of neonatal mouse results in long-term transgene expression in adulthood. We find that the human desmin (DES) promoter/enhancer is the first muscle-specific control region to match the activity of the highly active constitutive human cytomegalovirus (hCMV) promoter/enhancer in skeletal muscle within a LV context both in vitro and in vivo. Furthermore, the DES promoter/enhancer provides six- to eightfold greater expression per viral copy than the muscle-specific human muscle creatine kinase (CKM) promoter/enhancer. DES also confers a more reproducible and tissue-specific transgene expression profile compared to CKM and is therefore a highly attractive regulatory element for use in muscle gene therapy vectors. PMID:19935780

  5. Lentiviral vectors for the treatment of primary immunodeficiencies.

    PubMed

    Farinelli, Giada; Capo, Valentina; Scaramuzza, Samantha; Aiuti, Alessandro

    2014-07-01

    In the last years important progress has been made in the treatment of several primary immunodeficiency disorders (PIDs) with gene therapy. Hematopoietic stem cell (HSC) gene therapy indeed represents a valid alternative to conventional transplantation when a compatible donor is not available and recent success confirmed the great potential of this approach. First clinical trials performed with gamma retroviral vectors were promising and guaranteed clinical benefits to the patients. On the other hand, the outcome of severe adverse events as the development of hematological abnormalities highlighted the necessity to develop a safer platform to deliver the therapeutic gene. Self-inactivating (SIN) lentiviral vectors (LVVs) were studied to overcome this hurdle through their preferable integration pattern into the host genome. In this review, we describe the recent advancements achieved both in vitro and at preclinical level with LVVs for the treatment of Wiskott-Aldrich syndrome (WAS), chronic granulomatous disease (CGD), ADA deficiency (ADA-SCID), Artemis deficiency, RAG1/2 deficiency, X-linked severe combined immunodeficiency (γchain deficiency, SCIDX1), X-linked lymphoproliferative disease (XLP) and immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.

  6. Nigrostriatal α-synucleinopathy induced by viral vector-mediated overexpression of human α-synuclein: A new primate model of Parkinson's disease

    PubMed Central

    Kirik, Deniz; Annett, Lucy E.; Burger, Corinna; Muzyczka, Nicholas; Mandel, Ronald J.; Björklund, Anders

    2003-01-01

    We used a high-titer recombinant adeno-associated virus (rAAV) vector to express WT or mutant human α-synuclein in the substantia nigra of adult marmosets. The α-synuclein protein was expressed in 90–95% of all nigral dopamine neurons and distributed by anterograde transport throughout their axonal and dendritic projections. The transduced neurons developed severe neuronal pathology, including α-synuclein-positive cytoplasmic inclusions and granular deposits; swollen, dystrophic, and fragmented neuritis; and shrunken and pyknotic, densely α-synuclein-positive perikarya. By 16 wk posttransduction, 30–60% of the tyrosine hydroxylase-positive neurons were lost, and the tyrosine hydroxylase-positive innervation of the caudate nucleus and putamen was reduced to a similar extent. The rAAV-α-synuclein-treated monkeys developed a type of motor impairment, i.e., head position bias, compatible with this magnitude of nigrostriatal damage. rAAV vector-mediated α-synuclein gene transfer provides a transgenic primate model of nigrostriatal α-synucleinopathy that is of particular interest because it develops slowly over time, like human Parkinson's disease (PD), and expresses neuropathological features (α-synuclein-positive inclusions and dystrophic neurites, in particular) that are similar to those seen in idiopathic PD. This model offers new opportunities for the study of pathogenetic mechanisms and exploration of new therapeutic targets of particular relevance to human PD. PMID:12601150

  7. Meeting Report: Teaching Signal Transduction

    PubMed Central

    Kramer, IJsbrand; Thomas, Geraint

    2006-01-01

    In July, 2005, the European Institute of Chemistry and Biology at the campus of the University of Bordeaux, France, hosted a focused week of seminars, workshops, and discussions around the theme of “teaching signal transduction.” The purpose of the summer school was to offer both junior and senior university instructors a chance to reflect on the development and delivery of their teaching activities in this area. This was achieved by combining open seminars with restricted access workshops and discussion events. The results suggest ways in which systems biology, information and communication technology, Web-based investigations, and high standard illustrations might be more effectively and efficiently incorporated into modern cell biology courses. PMID:17012185

  8. Electromagnetic transduction of ultrasonic waves

    NASA Astrophysics Data System (ADS)

    Passarelli, Frank; Alers, George; Alers, Ron

    2012-05-01

    Excitation and detection of ultrasonic vibrations without physical contact has proven to be of great commercial value. First used to excite the resonant vibration of bar shaped laboratory specimens in the 1930's, it was Bruce Thompson's contributions in 1973-5 that launched their practical application to a wide range of difficult NDE problems. As a fresh PhD, he championed the use of mathematical models for the electromagnetic transduction process in order to guide the design and construction of practical transducers. His early papers presented both theoretical and experimental results that exposed the wide range of wave types that could be generated along with the environmental conditions that could be overcome. Several laboratories around the world established research programs to apply the electromagnetic transducer (EMAT) to specific NDE problems. This paper will summarize those applications made by the authors.

  9. Mimicking photosynthetic solar energy transduction.

    PubMed

    Gust, D; Moore, T A; Moore, A L

    2001-01-01

    Increased understanding of photosynthetic energy conversion and advances in chemical synthesis and instrumentation have made it possible to create artificial nanoscale devices and semibiological hybrids that carry out many of the functions of the natural process. Artificial light-harvesting antennas can be synthesized and linked to artificial reaction centers that convert excitation energy to chemical potential in the form of long-lived charge separation. Artificial reaction centers can form the basis for molecular-level optoelectronic devices. In addition, they may be incorporated into the lipid bilayer membranes of artificial vesicles, where they function as components of light-driven proton pumps that generate transmembrane proton motive force. The proton gradient may be used to synthesize adenosine triphosphate via an ATP synthase enzyme. The overall energy transduction process in the liposomal system mimics the solar energy conversion system of a photosynthetic bacterium. The results of this research illustrate the advantages of designing functional nanoscale devices based on biological paradigms.

  10. Application of lentiviral vectors for development of production cell lines and safety testing of lentiviral-derived cells or products.

    PubMed

    Plewa, Cherylene

    2010-01-01

    Lentiviral vectors (LVs) are frequently used to engineer cell lines for preclinical research purposes including assay development and target validation. Development of production cell lines for manufacturing recombinant protein therapeutics may also benefit from the use of LVs because they may reduce timelines and generate more uniform or higher expressing stable pools and clones. In addition, LVs could be advantageous for engineering new, alternative host cell substrates due to their ability to efficiently transduce most cell types. We demonstrate here that NS0 mouse myeloma cells, a host cell frequently used for protein production, can be transduced with LVs to greater than 80% efficiency and with no cytotoxic effects. The use of LVs for engineering of production cell lines will require additional testing procedures. Since LVs have previously been used in human gene therapy clinical trials, safety testing assays and procedures have been developed that could easily be applied to the development process for manufacturing cell lines to ensure the absence of unwanted viral material in cell banks and biologic products.

  11. Sentra, a database of signal transduction proteins.

    SciTech Connect

    Maltsev, N.; Marland, E.; Yu, G. X.; Bhatnagar, S.; Lusk, R.; Mathematics and Computer Science

    2002-01-01

    Sentra (http://www-wit.mcs.anl.gov/sentra) is a database of signal transduction proteins with the emphasis on microbial signal transduction. The database was updated to include classes of signal transduction systems modulated by either phosphorylation or methylation reactions such as PAS proteins and serine/threonine kinases, as well as the classical two-component histidine kinases and methyl-accepting chemotaxis proteins. Currently, Sentra contains signal transduction proteins from 43 completely sequenced prokaryotic genomes as well as sequences from SWISS-PROT and TrEMBL. Signal transduction proteins are annotated with information describing conserved domains, paralogous and orthologous sequences, and conserved chromosomal gene clusters. The newly developed user interface supports flexible search capabilities and extensive visualization of the data.

  12. SENTRA, a database of signal transduction proteins.

    SciTech Connect

    D'Souza, M.; Romine, M. F.; Maltsev, N.; Mathematics and Computer Science; PNNL

    2000-01-01

    SENTRA, available via URL http://wit.mcs.anl.gov/WIT2/Sentra/, is a database of proteins associated with microbial signal transduction. The database currently includes the classical two-component signal transduction pathway proteins and methyl-accepting chemotaxis proteins, but will be expanded to also include other classes of signal transduction systems that are modulated by phosphorylation or methylation reactions. Although the majority of database entries are from prokaryotic systems, eukaroytic proteins with bacterial-like signal transduction domains are also included. Currently SENTRA contains signal transduction proteins in 34 complete and almost completely sequenced prokaryotic genomes, as well as sequences from 243 organisms available in public databases (SWISS-PROT and EMBL). The analysis was carried out within the framework of the WIT2 system, which is designed and implemented to support genetic sequence analysis and comparative analysis of sequenced genomes.

  13. Lentiviral vectors can be used for full-length dystrophin gene therapy.

    PubMed

    Counsell, John R; Asgarian, Zeinab; Meng, Jinhong; Ferrer, Veronica; Vink, Conrad A; Howe, Steven J; Waddington, Simon N; Thrasher, Adrian J; Muntoni, Francesco; Morgan, Jennifer E; Danos, Olivier

    2017-12-01

    Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

  14. Lentiviral vectors can be used for full-length dystrophin gene therapy

    PubMed Central

    Counsell, John R.; Asgarian, Zeinab; Meng, Jinhong; Ferrer, Veronica; Vink, Conrad A.; Howe, Steven J.; Waddington, Simon N.; Thrasher, Adrian J.; Muntoni, Francesco; Morgan, Jennifer E.; Danos, Olivier

    2017-01-01

    Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a ‘template-switching’ lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD. PMID:28303972

  15. 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.

  16. The promotion of functional recovery and nerve regeneration after spinal cord injury by lentiviral vectors encoding Lingo-1 shRNA delivered by Pluronic F-127.

    PubMed

    Wu, Hong-Fu; Cen, Jing-Sheng; Zhong, Qian; Chen, Luming; Wang, Jue; Deng, David Y B; Wan, Yong

    2013-02-01

    Lingo-1 is selectively expressed on both oligodendrocytes and neurons in the central nervous system (CNS) and serves as a key negative regulator of nerve regeneration, implying a therapeutic target for spinal cord injury (SCI). Here we described a strategy to knock-down Lingo-1 expression in vivo using lentiviral vectors encoding Lingo-1 short harpin interfering RNA (shRNA) delivered by Pluronic F-127 (PF-127) gel, a non-cytotoxic scaffold and gene delivery carrier, after the complete transection of the T10 spinal cord in adult rats. We showed administration of PF-127 encapsulating Lingo-1 shRNA lentiviral vectors efficiently down-regulated the expression of Lingo-1, and exhibited transduction efficiency comparable to using vectors alone in oligodendrocyte culture in vitro. Furthermore, similar silencing effects and higher transfection efficiency were observed in vivo when Lingo-1 shRNA was co-delivered to the injured site by PF-127 gel with lower viral concentrations. Cografting of gel and Lingo-1 RNAi significantly promoted functional recovery and nerve regeneration, enhanced neurite outgrowth and synapses formation, preserved myelinated axons, and induced the proliferation of glial cells. In addition, the combined implantation also improved neuronal survival and inhibited cell apoptosis, which may be associated with the attenuation of endoplasmic reticulum (ER) stress after SCI. Together, our data indicated that delivering Lingo-1 shRNA by gel scaffold was a valuable treatment approach to SCI and PF-127 delivery of viral vectors to the spinal cord may provide strategy to study and develop therapies for SCI. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.

    PubMed

    Aiuti, Alessandro; Biasco, Luca; Scaramuzza, Samantha; Ferrua, Francesca; Cicalese, Maria Pia; Baricordi, Cristina; Dionisio, Francesca; Calabria, Andrea; Giannelli, Stefania; Castiello, Maria Carmina; Bosticardo, Marita; Evangelio, Costanza; Assanelli, Andrea; Casiraghi, Miriam; Di Nunzio, Sara; Callegaro, Luciano; Benati, Claudia; Rizzardi, Paolo; Pellin, Danilo; Di Serio, Clelia; Schmidt, Manfred; Von Kalle, Christof; Gardner, Jason; Mehta, Nalini; Neduva, Victor; Dow, David J; Galy, Anne; Miniero, Roberto; Finocchi, Andrea; Metin, Ayse; Banerjee, Pinaki P; Orange, Jordan S; Galimberti, Stefania; Valsecchi, Maria Grazia; Biffi, Alessandra; Montini, Eugenio; Villa, Anna; Ciceri, Fabio; Roncarolo, Maria Grazia; Naldini, Luigi

    2013-08-23

    Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.

  18. Gravitational Effects on Signal Transduction

    NASA Technical Reports Server (NTRS)

    Sytkowski, Arthur J.

    1999-01-01

    An understanding of the mechanisms by which individual cells perceive gravity and how these cells transduce and respond to gravitational stimuli is critical for the development of long-term manned space flight experiments. We now propose to use a well-characterized model erythroid cell system and to investigate gravitational perturbations of its erythropoietin (Epo) signaling pathway and gene regulation. Cells will be grown at 1-G and in simulated microgravity in the NASA Rotating Wall Vessel bioreactor (RWV). Cell growth and differentiation, the Epo-receptor, the protein kinase C pathway to the c-myc gene, and the protein phosphatase pathway to the c-myb gene will be studied and evaluated as reporters of gravitational stimuli. The results of these experiments will have impact on the problems of 1) gravitational sensing by individual cells, and 2) the anemia of space flight. This ground-based study also will serve as a Space Station Development Study in gravitational effects on intracellular signal transduction.

  19. Intratumoral delivery of vector mediated IL-2 in combination with vaccine results in enhanced T cell avidity and anti-tumor activity.

    PubMed

    Kudo-Saito, Chie; Garnett, Charlie T; Wansley, Elizabeth K; Schlom, Jeffrey; Hodge, James W

    2007-12-01

    Systemic IL-2 is currently employed in the therapy of several tumor types, but at the price of often severe toxicities. Local vector mediated delivery of IL-2 at the tumor site may enhance local effector cell activity while reducing toxicity. To examine this, a model using CEA-transgenic mice bearing established CEA expressing tumors was employed. The vaccine regimen was a s.c. prime vaccination with recombinant vaccinia (rV) expressing transgenes for CEA and a triad of costimulatory molecules (TRICOM) followed by i.t. boosting with rF-CEA/TRICOM. The addition of intratumoral (i.t.) delivery of IL-2 via a recombinant fowlpox (rF) IL-2 vector greatly enhanced anti-tumor activity of a recombinant vaccine, resulting in complete tumor regression in 70-80% of mice. The anti-tumor activity was shown to be dependent on CD8(+) cells and NK1.1(+). Cellular immune assays revealed that the addition of rF-IL-2 to the vaccination therapy enhanced CEA-specific tetramer(+) cell numbers, cytokine release and CTL lysis of CEA(+) targets. Moreover, tumor-bearing mice vaccinated with the CEA/TRICOM displayed an antigen cascade, i.e., CD8(+) T cell responses to two other antigens expressed on the tumor and not the vaccine: wild-type p53 and endogenous retroviral antigen gp70. Mice receiving rF-IL-2 during vaccination demonstrated higher avidity CEA-specific, as well as higher avidity gp70-specific, CD8(+) T cells when compared with mice vaccinated without rF-IL-2. These studies demonstrate for the first time that the level and avidity of antigen specific CTL, as well as the therapeutic outcome can be improved with the use of i.t. rF-IL-2 with vaccine regimens.

  20. Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography

    PubMed Central

    Kutner, Robert H; Puthli, Sharon; Marino, Michael P; Reiser, Jakob

    2009-01-01

    Background During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly. Methods Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography. Results Our results show that unconcentrated LV vector stocks with titers in excess of 108 transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm2 tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 × 1010 TU were recovered from a single HYPERFlask vessel. Conclusion The protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols. PMID:19220915

  1. 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.

  2. Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography.

    PubMed

    Kutner, Robert H; Puthli, Sharon; Marino, Michael P; Reiser, Jakob

    2009-02-16

    During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly. Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography. Our results show that unconcentrated LV vector stocks with titers in excess of 108 transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm2 tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 x 1010 TU were recovered from a single HYPERFlask vessel. The protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols.

  3. Histone Deacetylase Inhibition Activates Transgene Expression from Integration-Defective Lentiviral Vectors in Dividing and Non-Dividing Cells

    PubMed Central

    Pelascini, Laetitia P.L.; Janssen, Josephine M.

    2013-01-01

    Abstract Integration-defective lentiviral vectors (IDLVs) are being increasingly deployed in both basic and preclinical gene transfer settings. Often, however, the IDLV transgene expression profile is muted when compared to that of their integration-proficient counterparts. We hypothesized that the episomal nature of IDLVs turns them into preferential targets for epigenetic silencing involving chromatin-remodeling histone deacetylation. Therefore, vectors carrying an array of cis-acting elements and transcriptional unit components were assembled with the aid of packaging constructs encoding either the wild-type or the class I mutant D116N integrase moieties. The transduction levels and transgene-product yields provided by each vector class were assessed in the presence and absence of the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A. To investigate the role of the target cell replication status, we performed experiments in growth-arrested human mesenchymal stem cells and in post-mitotic syncytial myotubes. We found that IDLVs are acutely affected by HDACs regardless of their genetic makeup or target cell replication rate. Interestingly, the magnitude of IDLV transgene expression rescue due to HDAC inhibition varied in a vector backbone– and cell type–dependent manner. Finally, investigation of histone modifications by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) revealed a paucity of euchromatin marks distributed along IDLV genomes when compared to those measured on isogenic integration-competent vector templates. These findings support the view that IDLVs constitute preferential targets for epigenetic silencing involving histone deacetylation, which contributes to dampening their full transcriptional potential. Our data provide leads on how to most optimally titrate and deploy these promising episomal gene delivery vehicles. PMID:23140481

  4. In Vivo Stable Transduction of Humanized Liver Tissue in Chimeric Mice via High-Capacity Adenovirus–Lentivirus Hybrid Vector

    PubMed Central

    Kataoka, Miho; Tateno, Chise; Yoshizato, Katsutoshi; Kawasaki, Yoshiko; Kimura, Takahiro; Faure-Kumar, Emmanuelle; Palmer, Donna J.; Ng, Philip; Okamura, Haruki; Kasahara, Noriyuki

    2010-01-01

    Abstract We developed hybrid vectors employing high-capacity adenovirus as a first-stage carrier encoding all the components required for in situ production of a second-stage lentivirus, thereby achieving stable transgene expression in secondary target cells. Such vectors have never previously been tested in normal tissues, because of the scarcity of suitable in vivo systems permissive for second-stage lentivirus assembly. Here we employed a novel murine model in which endogenous liver tissue is extensively reconstituted with engrafted human hepatocytes, and successfully achieved stable transduction by the second-stage lentivirus produced in situ from first-stage adenovirus. This represents the first demonstration of the functionality of adenoviral-lentiviral hybrid vectors in a normal parenchymal organ in vivo. PMID:19725756

  5. Transduced CD34+ cells from adrenoleukodystrophy patients with HIV-derived vector mediate long-term engraftment of NOD/SCID mice.

    PubMed

    Benhamida, Sonia; Pflumio, Françoise; Dubart-Kupperschmitt, Anne; Zhao-Emonet, Jing Chao; Cavazzana-Calvo, Marina; Rocchiccioli, Francis; Fichelson, Serge; Aubourg, Patrick; Charneau, Pierre; Cartier, Nathalie

    2003-03-01

    X-linked adrenoleukodystrophy (ALD), an inherited demyelinating disorder of the central nervous system, can be corrected by allogeneic bone marrow transplantation, likely due to the turnover of brain macrophages that are bone marrow derived. ALD is characterized by an accumulation of very long chain fatty acids (VLCFA) due to the deficiency of an ATP binding cassette transporter that imports these fatty acids in peroxisomes. Murine retroviral transduction results in metabolic correction of ALD CD34(+) cells in vitro but reinfusion of these cells into ALD patients would not provide clinical benefit owing to the absence of selective advantage conferred by transgene expression. High-efficiency transduction of ALD CD34(+) peripheral blood mobilized cells was achieved using an HIV-based vector driving ALD gene expression under the elongation factor 1 alpha promoter and a protocol without prestimulation of CD34(+) cells with cytokines prior to transduction to preserve their stem cell properties. Efficient expression of the ALD gene was demonstrated in monocytes/macrophages derived from cultures of transduced ALD CD34(+) cells and in long-term culture initiating cells. VLCFA metabolism was corrected in transduced CD34(+), CFU-derived, and LTC-derived cells, indicating that the vector-encoded ALD protein was fully functional. Transplantation of transduced ALD CD34(+) cells into NOD/SCID mice resulted in long-term expression of ALD protein in monocytes/macrophages derived from engrafted stem cells.

  6. Umami taste transduction mechanisms1234

    PubMed Central

    2009-01-01

    l-Glutamate elicits the umami taste sensation, now recognized as a fifth distinct taste quality. A characteristic feature of umami taste is its potentiation by 5′-ribonucleotides such as guanosine-5'-monophosphate and inosine 5′-monophosphate, which also elicit the umami taste on their own. Recent data suggest that multiple G protein–coupled receptors contribute to umami taste. This review will focus on events downstream of the umami taste receptors. Ligand binding leads to Gβγ activation of phospholipase C β2, which produces the second messengers inositol trisphosphate and diacylglycerol. Inositol trisphosphate binds to the type III inositol trisphosphate receptor, which causes the release of Ca2+ from intracellular stores and Ca2+-dependent activation of a monovalent-selective cation channel, TRPM5. TRPM5 is believed to depolarize taste cells, which leads to the release of ATP, which activates ionotropic purinergic receptors on gustatory afferent nerve fibers. This model is supported by knockout of the relevant signaling effectors as well as physiologic studies of isolated taste cells. Concomitant with the molecular studies, physiologic studies show that l-glutamate elicits increases in intracellular Ca2+ in isolated taste cells and that the source of the Ca2+ is release from intracellular stores. Both Gα gustducin and Gα transducin are involved in umami signaling, because the knockout of either subunit compromises responses to umami stimuli. Both α-gustducin and α-transducin activate phosphodiesterases to decrease intracellular cAMP. The target of cAMP in umami transduction is not known, but membrane-permeant analogs of cAMP antagonize electrophysiologic responses to umami stimuli in isolated taste cells, which suggests that cAMP may have a modulatory role in umami signaling. PMID:19571214

  7. Pseudotyped AAV Vector-Mediated Gene Transfer in a Human Fetal Trachea Xenograft Model: Implications for In Utero Gene Therapy for Cystic Fibrosis

    PubMed Central

    Leung, Alice; Katz, Anna B.; Lim, Foong-Yen; Habli, Mounira; Jones, Helen N.; Wilson, James M.; Crombleholme, Timothy M.

    2012-01-01

    Background Lung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. Our previous work in a human-fetal trachea xenograft model suggests the potential benefit for treating CF in utero. In this study, we aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls. Methodology/Principal Findings Human fetal trachea or postnatal bronchus controls were xenografted onto immunocompromised SCID mice for a four-week engraftment period. After injection of AAV2/2, 2/1, 2/5, 2/7 or 2/8 with a LacZ reporter into both types of xenografts, we analyzed for transgene expression in the respiratory epithelium and SMGs. At 1 month, transduction by AAV2/2 and AAV2/8 in respiratory epithelium and SMG cells was significantly greater than that of AAV2/1, 2/5, and 2/7 in xenograft tracheas. Efficiency in SMG transduction was significantly greater in AAV2/8 than AAV2/2. At 3 months, AAV2/2 and AAV2/8 transgene expression was >99% of respiratory epithelium and SMG. At 1 month, transduction efficiency of AAV2/2 and AAV2/8 was significantly less in adult postnatal bronchial xenografts than in fetal tracheal xenografts. Conclusions/Significance Based on the effectiveness of AAV vectors in SMG transduction, our findings suggest the potential utility of pseudotyped AAV vectors for treatment of cystic fibrosis. The human fetal trachea xenograft model may serve as an effective tool for further development of fetal gene therapy strategies for the in utero treatment of cystic fibrosis. PMID:22937069

  8. Transduction of chemical into electrical energy.

    PubMed

    Nachmansohn, D

    1976-01-01

    The paper recalls some fundamental notions, developed by Otto Meyerhof, which were used in the analysis of the transduction of chemical into mechanical energy during muscular contraction. These notions formed the basis of the approach to the analysis of the transduction of chemical into electrical energy, i.e., the very principle underlying nerve and muscle excitability and bioelectricity. Instrumental for this purpose was the use, since 1937, of electric organs of fish, a tissue highly specialized for bioelectrogenesis.

  9. Transduction of chemical into electrical energy.

    PubMed Central

    Nachmansohn, D

    1976-01-01

    The paper recalls some fundamental notions, developed by Otto Meyerhof, which were used in the analysis of the transduction of chemical into mechanical energy during muscular contraction. These notions formed the basis of the approach to the analysis of the transduction of chemical into electrical energy, i.e., the very principle underlying nerve and muscle excitability and bioelectricity. Instrumental for this purpose was the use, since 1937, of electric organs of fish, a tissue highly specialized for bioelectrogenesis. Images PMID:1061129

  10. On the calculation of signal transduction ability of signaling transduction pathways in intracellular communication: systematic approach.

    PubMed

    Chen, Bor-Sen; Wu, Chia-Chou

    2012-06-15

    The major function of signal transduction pathways in cells is to sense signals from the environment and process the information through signaling molecules in order to regulate the activity of transcription factors. On the molecular level, the information transmitted by a small number of signal molecules is amplified in the internal signaling pathway through enzyme catalysis, molecular modification and via the activation or inhibition of interactions. However, the dynamic system behavior of a signaling pathway can be complex and, despite knowledge of the pathway components and interactions, it is still a challenge to interpret the pathways behavior. Therefore, a systematic method is proposed in this study to quantify the signal transduction ability. Based on the non-linear signal transduction system, signal transduction ability can be investigated by solving a Hamilton-Jacobi inequality (HJI)-constrained optimization problem. To avoid difficulties associated with solving a complex HJI-constrained optimization problem for signal transduction ability, the Takagi-Sugeno fuzzy model is introduced to approximate the non-linear signal transduction system by interpolating several local linear systems so that the HJI-constrained optimization problem can be replaced by a linear matrix inequality (LMI)-constrained optimization problem. The LMI problem can then be efficiently solved for measuring signal transduction ability. Finally, the signal transduction ability of two important signal transduction pathways was measured by the proposed method and confirmed using experimental data, which is useful for biotechnological and therapeutic application and drug design.

  11. 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

  12. Development and use of SIV-based Integrase defective lentiviral vector for immunization

    PubMed Central

    Michelini, Zuleika; Negri, Donatella RM; Baroncelli, Silvia; Spada, Massimo; Leone, Pasqualina; Bona, Roberta; Klotman, Mary E.; Cara, Andrea

    2009-01-01

    Integrase (IN) defective lentiviral vectors have a high safety profile and might prove useful as immunizing agents especially against HIV-1. However, IN defective SIV-based vectors must be developed in order to test their potential in the non human primate models (NHP) of AIDS. To this aim we tested a novel SIV-based IN defective lentiviral vector for its ability to induce sustained immune responses in mice. BALB/c mice were immunized once intramuscularly with a SIV-based IN defective lentiviral vector expressing the model antigen enhanced green fluorescence protein (eGFP). Immune responses were evaluated 90 days after the injection and compared with those elicited with the IN competent counterpart. The IN defective vector was able to efficiently elicit specific and long-lasting polyfunctional immune responses as evaluated by enzyme-linked immunospot (ELISPOT) assays for interferon-γ (IFN-γ) in spleens, bone marrow (BM) and draining lymph nodes, and by intracellular staining (ICS) for IFN-γ, Interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) in both splenocytes and BM cells without integration of the vector into the host genome. This is the first demonstration that an IN defective SIV-based lentiviral vector provides effective immunization, thus paving the way for the construction of IN defective vectors expressing SIV antigen(s) and test their efficacy against a SIV virus challenge in the NHP model of AIDS. PMID:19523909

  13. 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

  14. 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

  15. Equine infectious anemia viral vector-mediated codelivery of endostatin and angiostatin driven by retinal pigmented epithelium-specific VMD2 promoter inhibits choroidal neovascularization.

    PubMed

    Kachi, Shu; Binley, Katie; Yokoi, Katsutoshi; Umeda, Naoyasu; Akiyama, Hideo; Muramatu, Daisuke; Iqball, Sharifah; Kan, On; Naylor, Stuart; Campochiaro, Peter A

    2009-01-01

    Equine infectious anemia virus (EIAV) is a nonprimate lentivirus that does not cause human disease. Subretinal injection into mice of a recombinant EIAV lentiviral vector in which lacZ is driven by a CMV promoter (EIAV CMV LacZ) resulted in rapid and strong expression of LacZ in retinal pigmented epithelial (RPE) cells and some other cells including ganglion cells, resulting in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside within the optic nerve. Substitution of the RPE-specific promoter from the vitelliform macular dystrophy (VMD2) gene for the CMV promoter resulted in prolonged (at least 1 year) expression of LacZ that was restricted to RPE cells, albeit reduced 6- to 10-fold compared with the CMV promoter. Similarly, the amount of FLAG-tagged endostatin detected in eyes injected with the EIAV VMD2 Endo(FLAG) vector was similar to that seen in eyes injected with a vector that expressed both endostatin and angiostatin [EIAV VMD2 Endo(FLAG)/Angio]; expression was approximately 6-fold lower than with identical vectors in which the CMV promoter drove expression. Compared with murine eyes treated with a control EIAV vector, subretinal injection of EIAV vectors expressing murine endostatin alone or in combination with angiostatin driven by either the CMV or VMD2 promoter caused significant suppression of choroidal neovascularization (NV) at laser-induced rupture sites in Bruch's membrane. These data support proceeding toward clinical studies with EIAV-based gene therapy for choroidal NV, using the VMD2 promoter to selectively drive expression of a combination of endostatin and angiostatin in RPE cells.

  16. The Cornucopia of Intestinal Chemosensory Transduction

    PubMed Central

    Bertrand, Paul P.

    2009-01-01

    The chemosensory transduction mechanisms that the gastrointestinal (GI) tract uses to detect chemical and nutrient stimuli are poorly understood. The GI tract is presented with a wide variety of stimuli including potentially harmful chemicals or toxins as well as ‘normal’ stimuli including nutrients, bacteria and mechanical forces. Sensory transduction is at its simplest the conversion of these stimuli into a neural code in afferent nerves. Much of the information encoded is used by the enteric nervous system to generate local reflexes while complementary information is sent to the central nervous system via afferents or by release of hormones to affect behaviour. This review focuses on the chemosensory transduction mechanisms present in the GI tract. It examines the expression and localisation of the machinery for chemosensory transduction. It summarises the types of cells which might be involved in detecting stimuli and releasing neuroactive transmitters. Finally, it highlights the idea that chemosensory transduction mechanisms in the GI tract utilise many overlapping and complementary mechanisms for detecting and transducing stimuli into reflex action. PMID:20582275

  17. A self-inactivating lentiviral vector for SCID-X1 gene therapy that does not activate LMO2 expression in human T cells.

    PubMed

    Zhou, Sheng; Mody, Disha; DeRavin, Suk See; Hauer, Julia; Lu, Taihe; Ma, Zhijun; Hacein-Bey Abina, Salima; Gray, John T; Greene, Michael R; Cavazzana-Calvo, Marina; Malech, Harry L; Sorrentino, Brian P

    2010-08-12

    To develop safer and more effective vectors for gene therapy of X-linked severe combined immunodeficiency (SCID-X1), we have evaluated new self-inactivating lentiviral vectors based on the HIV virus. The CL20i4-hgamma(c)-Revgen vector contains the entire human common gamma chain (gamma(c)) genomic sequence driven by the gamma(c) promoter. The CL20i4-EF1alpha-hgamma(c)OPT vector uses a promoter fragment from the eukaryotic elongation factor alpha (EF1alpha) gene to express a codon-optimized human gamma(c) cDNA. Both vectors contain a 400-bp insulator fragment from the chicken beta-globin locus within the self-inactivating long-terminal repeat. Transduction of bone marrow cells using either of these vectors restored T, B, and natural killer lymphocyte development and function in a mouse SCID-X1 transplantation model. Transduction of human CD34(+) bone marrow cells from SCID-X1 patients with either vector restored T-cell development in an in vitro assay. In safety studies using a Jurkat LMO2 activation assay, only the CL20i4-EF1alpha-hgamma(c)OPT vector lacked the ability to transactivate LMO2 protein expression, whereas the CL20i4-hgamma(c)-Revgen vector significantly activated LMO2 protein expression. In addition, the CL20i4-EF1alpha-hgamma(c)OPT vector has not caused any tumors in transplanted mice. We conclude that the CL20i4-EF1alpha-hgamma(c)OPT vector may be suitable for testing in a clinical trial based on these preclinical demonstrations of efficacy and safety.

  18. 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

  19. A SIN lentiviral vector containing PIGA cDNA allows long-term phenotypic correction of CD34+-derived cells from patients with paroxysmal nocturnal hemoglobinuria.

    PubMed

    Robert, David; Mahon, François-Xavier; Richard, Emmanuel; Etienne, Gabriel; de Verneuil, Hubert; Moreau-Gaudry, François

    2003-03-01

    Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell (HSC) disorder in which an acquired somatic mutation of the X-linked PIGA gene results in a deficiency in GPI-anchored surface proteins. Clinically, PNH is dominated by a chronic hemolytic anemia, often associated with recurrent nocturnal exacerbations, neutropenia, thrombocytopenia, and thrombotic tendency. Allogenic bone marrow transplantation is the only potentially curative treatment for severe forms of PNH but is associated with a high treatment-related morbidity and mortality. HSC gene therapy could provide a new therapeutic option, especially when an HLA-matched donor is not available. To develop an efficient gene transfer approach, we have designed a new SIN lentiviral vector (TEPW) that contains the PIGA cDNA driven by the human elongation factor 1 alpha promoter, the central DNA flap of HIV-1, and the WPRE cassette. TEPW transduction led to a complete surface expression of the GPI anchor and CD59 in PIGA-deficient cell lines without any selection procedure. Moreover, efficient gene transfer was achieved in bone marrow and mobilized peripheral blood CD34(+) cells derived from two patients with severe PNH disease. This expression was stable during erythroid, myeloid, and megakaryocytic liquid culture differentiation. CD59 surface cell expression was fully restored during 5 weeks of long-term culture.

  20. 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

  1. Purinergic mechanosensory transduction and visceral pain.

    PubMed

    Burnstock, Geoffrey

    2009-11-30

    In this review, evidence is presented to support the hypothesis that mechanosensory transduction occurs in tubes and sacs and can initiate visceral pain. Experimental evidence for this mechanism in urinary bladder, ureter, gut, lung, uterus, tooth-pulp and tongue is reviewed. Potential therapeutic strategies are considered for the treatment of visceral pain in such conditions as renal colic, interstitial cystitis and inflammatory bowel disease by agents that interfere with mechanosensory transduction in the organs considered, including P2X3 and P2X2/3 receptor antagonists that are orally bioavailable and stable in vivo and agents that inhibit or enhance ATP release and breakdown.

  2. Purinergic mechanosensory transduction and visceral pain

    PubMed Central

    2009-01-01

    In this review, evidence is presented to support the hypothesis that mechanosensory transduction occurs in tubes and sacs and can initiate visceral pain. Experimental evidence for this mechanism in urinary bladder, ureter, gut, lung, uterus, tooth-pulp and tongue is reviewed. Potential therapeutic strategies are considered for the treatment of visceral pain in such conditions as renal colic, interstitial cystitis and inflammatory bowel disease by agents that interfere with mechanosensory transduction in the organs considered, including P2X3 and P2X2/3 receptor antagonists that are orally bioavailable and stable in vivo and agents that inhibit or enhance ATP release and breakdown. PMID:19948030

  3. A soluble CAR-SCF fusion protein improves adenoviral vector-mediated gene transfer to c-Kit-positive hematopoietic cells.

    PubMed

    Itoh, Akira; Okada, Takashi; Mizuguchi, Hiroyuki; Hayakawa, Takao; Mizukami, Hiroaki; Kume, Akihiro; Takatoku, Masaaki; Komatsu, Norio; Hanazono, Yutaka; Ozawa, Keiya

    2003-11-01

    Although adenoviral vectors primarily derived from the adenovirus serotype 5 (Ad5) are widely used for many gene transfer applications, they cannot efficiently infect hematopoietic cells, since these cells do not express the coxsackie-adenoviral receptor (CAR). We have developed a soluble fusion protein that bridges adenoviral fibers and the c-Kit receptor to alter Ad5 tropism to immature hematopoietic cells. The CAR-SCF fusion protein consists of the extracellular domains of CAR and stem cell factor (SCF). The human megakaryoblastic leukemia cell lines UT-7 and M07e, human chronic myelogenous leukemia cell line K-562, and erythroleukemia cell line TF-1 were used to assess CAR-SCF-assisted Ad5-mediated gene transfer. Hematopoietic cell lines were infected with an Ad5 vector (Ad5-eGFP) or a fiber-mutant Ad5/F35 (Ad5/F35-eGFP) expressing the enhanced green fluorescent protein gene in the presence or absence of CAR-SCF. Twenty-four hours after infection, more than 80% of M07e cells infected in the presence of CAR-SCF were eGFP-positive, compared with very few eGFP-positive cells following Ad5-eGFP infection in the absence of CAR-SCF. The enhancement of Ad5-eGFP infection by CAR-SCF was greater than that caused by Ad5/F35-eGFP (50%). The ability of CAR-SCF to enhance Ad5-eGFP infectivity was highly dependent on cellular c-Kit expression levels. Furthermore, CAR-SCF also enhanced Ad5-mediated gene transfer into human primary CD34(+) cells. The CAR-SCF fusion protein assists Ad5-mediated transduction to c-Kit(+) CAR(-) hematopoietic cells. The use of this fusion protein would enhance a utility of Ad5-mediated hematopoietic cell transduction strategies. Copyright 2003 John Wiley & Sons, Ltd.

  4. Inhibitors targeting two-component signal transduction.

    PubMed

    Watanabe, Takafumi; Okada, Ario; Gotoh, Yasuhiro; Utsumi, Ryutaro

    2008-01-01

    A two-component signal transduction system (TCS) is an attractive target for antibacterial agents. In this chapter, we review the TCS inhibitors developed during the past decade and introduce novel drug discovery systems to isolate the inhibitors of the YycG/YycF system, an essential TCS for bacterial growth, in an effort to develop a new class of antibacterial agents.

  5. 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.

  6. Genomic integration occurs in the packaging cell via unexported lentiviral precursors.

    PubMed

    Mosabbir, Abdullah Al; Truong, Kevin

    2016-10-01

    To use HIV-1 based lentivirus components to produce gene integration and the formation of a stable cell line in the packaging cell line without viral infection. A co-transfection of a Human Embryonic Kidney (HEK) 293 packaging cell line with Gag-pol (GP) and a transfer vector, without the envelope vector, produces a stable cell line after 2 weeks of selection. Furthermore, a matrix protein deficient GP in the packaging vector enhances this integration. This supports that, in theory, unexported lentiviral cores produced within the packaging cell can infect itself without requiring the release of any lentiviral particles. If the packaging cell is also the target cell, then gene integration leading to a stable cell line can be accomplished without viral particle infection.

  7. 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.

  8. Transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector.

    PubMed

    Hiripi, L; Negre, D; Cosset, F-L; Kvell, K; Czömpöly, T; Baranyi, M; Gócza, E; Hoffmann, O; Bender, B; Bosze, Zs

    2010-10-01

    Transgenic rabbit is the preferred disease model of atherosclerosis, lipoprotein metabolism and cardiovascular diseases since upon introducing genetic mutations of human genes, rabbit models reflect human physiological and pathological states more accurately than mouse models. Beyond that, transgenic rabbits are also used as bioreactors to produce pharmaceutical proteins in their milk. Since in the laboratory rabbit the conventional transgenesis has worked with the same low efficiency in the last twenty five years and truly pluripotent embryonic stem cells are not available to perform targeted mutagenesis, our aim was to adapt lentiviral transgenesis to this species. A simian immunodeficiency virus based replication defective lentiviral vector was used to create transgenic rabbit through perivitelline space injection of fertilized oocytes. The enhanced green fluorescent protein (GFP) gene was placed under the ubiquitous CAG promoter. Transgenic founder rabbits showed mosaic pattern of GFP expression. Transgene integration and expression was revealed in tissues derived from all three primary germ layers. Transgene expression was detected in the developing sperm cells and could get through the germ line without epigenetic silencing, albeit with very low frequency. Our data show for the first time, that lentiviral transgenesis could be a feasible and viable alternative method to create genetically modified laboratory rabbit.

  9. Lentiviral vectors and cardiovascular diseases: a genetic tool for manipulating cardiomyocyte differentiation and function.

    PubMed

    Di Pasquale, E; Latronico, M V G; Jotti, G S; Condorelli, G

    2012-06-01

    Engineered recombinant viral vectors are a powerful tool for vehiculating genetic information into mammalian cells. Because of their ability to infect both dividing and non-dividing cells with high efficiency, lentiviral vectors have gained particular interest for basic research and preclinical studies in the cardiovascular field. We review here the major applications for lentiviral-vector technology in the cardiovascular field: we will discuss their use in trailing gene expression during the induction of differentiation, in protocols for the isolation of cardiac cells and in the tracking of cardiac cells after transplantation in vivo; we will also describe lentivirally-mediated gene delivery uses, such as the induction of a phenotype of interest in a target cell or the treatment of cardiovascular diseases. In addition, a section of the review will be dedicated to reprogramming approaches, focusing attention on the generation of pluripotent stem cells and on transdifferentiation, two emerging strategies for the production of cardiac myocytes from human cells and for the investigation of human diseases. Finally, in order to give a perspective on their future clinical use we will critically discuss advantages and disadvantages of lentivirus-based strategies for the treatment of cardiovascular diseases.

  10. Assessment of selected media supplements to improve F/HN lentiviral vector production yields.

    PubMed

    Gélinas, Jean-François; Davies, Lee A; Gill, Deborah R; Hyde, Stephen C

    2017-08-31

    The development of lentiviral-based therapeutics is challenged by the high cost of current Good Manufacturing Practices (cGMP) production. Lentiviruses are enveloped viruses that capture a portion of the host cell membrane during budding, which then constitutes part of the virus particle. This process might lead to lipid and protein depletion in the cell membrane and affect cell viability. Furthermore, growth in suspension also causes stresses that can affect virus production yields. To assess the impact of these issues, selected supplements (Cholesterol Lipid Concentrate, Chemically Defined Lipid Concentrate, Lipid Mixture 1, Gelatin Peptone N3, N-Acetyl-L-Cysteine and Pluronic F-68) were assayed in order to improve production yields in a transient transfection production of a Sendai virus F/HN-pseudotyped HIV-1-based third generation lentiviral vector in FreeStyle 293 (serum-free media) in suspension. None of the supplements tested had a significant positive impact on lentiviral vector yields, but small non-significant improvements could be combined to increase vector production in a cell line where other conditions have been optimised.

  11. A guide to approaching regulatory considerations for lentiviral-mediated gene therapies.

    PubMed

    White, Michael; Whittaker, Roger; Stoll, Elizabeth Ann

    2017-06-12

    Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well-characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is non-pathogenic and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy. The focus is on European regulations, and how they are administered in the United Kingdom, although many of the principles will be similar for other regions including the United States. The report justifies the rationale for using third-generation lentiviral vectors to achieve gene delivery for in vivo and ex vivo applications; briefly summarises the extant regulatory guidance for gene therapies, categorised as advanced therapeutic medicinal products (ATMPs); provides guidance on specific regulatory issues regarding gene therapies; presents an overview of the key stakeholders to be approached when pursuing clinical trials authorization for an ATMP; and includes a brief catalogue of the documentation required to submit an application for regulatory approval of a new gene therapy.

  12. A Guide to Approaching Regulatory Considerations for Lentiviral-Mediated Gene Therapies.

    PubMed

    White, Michael; Whittaker, Roger; Gándara, Carolina; Stoll, Elizabeth A

    2017-08-01

    Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is nonpathogenic, and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy. The focus is on European regulations and how they are administered in the United Kingdom, although many of the principles will be similar for other regions, including the United States. The report justifies the rationale for using third-generation lentiviral vectors to achieve gene delivery for in vivo and ex vivo applications; briefly summarizes the extant regulatory guidance for gene therapies, categorized as advanced therapeutic medicinal products (ATMPs); provides guidance on specific regulatory issues regarding gene therapies; presents an overview of the key stakeholders to be approached when pursuing clinical trials authorization for an ATMP; and includes a brief catalogue of the documentation required to submit an application for regulatory approval of a new gene therapy.

  13. A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

    PubMed

    Ebina, Hirotaka; Kanemura, Yuka; Misawa, Naoko; Sakuma, Tetsushi; Kobayashi, Tomoko; Yamamoto, Takashi; Koyanagi, Yoshio

    2015-01-01

    DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV) provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease system targeting HIV long terminal repeats (LTR). Here, we generated a more efficient strategy that enables the excision of HIV proviral DNA using customized transcription activator-like effector nucleases (TALENs) targeting the same HIV LTR site. A single transfection of TALEN-encoding mRNA, prepared from in vitro transcription, resulted in more than 80% of lentiviral vector DNA being successfully removed from the T cell lines. Furthermore, we developed a lentiviral vector system that takes advantage of the efficient proviral excision with TALENs and permits the simple selection of gene-transduced and excised cells in T cell lines.

  14. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26612671

  16. 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

  17. Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors

    PubMed Central

    Farley, Daniel C; McCloskey, Laura; Thorne, Barbara A; Tareen, Semih U; Nicolai, Christopher J; Campbell, David J; Bannister, Richard; Stewart, Hannah J; Pearson, Laura JE; Moyer, Bentley J; Robbins, Scott H; Zielinski, Leah; Kim, Tae; Radcliffe, Pippa A; Mitrophanous, Kyriacos A; Gombotz, Wayne R; Miskin, James E; Kelley-Clarke, Brenna

    2015-01-01

    It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector. PMID:26029728

  18. A Guide to Approaching Regulatory Considerations for Lentiviral-Mediated Gene Therapies

    PubMed Central

    White, Michael; Whittaker, Roger; Gándara, Carolina; Stoll, Elizabeth A.

    2017-01-01

    Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is nonpathogenic, and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy. The focus is on European regulations and how they are administered in the United Kingdom, although many of the principles will be similar for other regions, including the United States. The report justifies the rationale for using third-generation lentiviral vectors to achieve gene delivery for in vivo and ex vivo applications; briefly summarizes the extant regulatory guidance for gene therapies, categorized as advanced therapeutic medicinal products (ATMPs); provides guidance on specific regulatory issues regarding gene therapies; presents an overview of the key stakeholders to be approached when pursuing clinical trials authorization for an ATMP; and includes a brief catalogue of the documentation required to submit an application for regulatory approval of a new gene therapy. PMID:28817344

  19. 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.

  20. Neuropathological and behavioral consequences of adeno-associated viral vector-mediated continuous intrastriatal neurotrophin delivery in a focal ischemia model in rats.

    PubMed

    Andsberg, Gunnar; Kokaia, Zaal; Klein, Ronald L; Muzyczka, Nicholas; Lindvall, Olle; Mandel, Ronald J

    2002-03-01

    Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were continuously delivered to the striatum at biologically active levels via recombinant adeno-associated viral (rAAV) gene transfer 4-5 weeks prior to 30 min of middle cerebral artery occlusion (MCAO). The magnitude of the deficits in a battery of behavioral tests designed to assess striatal function was highly correlated to the extent of ischemic damage determined by unbiased stereological estimations of striatal neuron numbers. The delivery of neurotrophins lead to mild functional improvements in the ischemia-induced motor impairments assessed 3-5 weeks after the insult, in agreement with a small but significant increase of the survival of dorsolateral striatal neurons. Detailed phenotypic analysis demonstrated that the parvalbumin-containing interneurons were spared to a greater extent by the neurotrophin treatment as compared to the projection neurons, which agreed with the specificity for interneuron transduction by the rAAV vector. These data show the advantage of the never previously performed combination of precise quantification of the ischemia-induced neuropathology along with detailed behavioural analysis for assessing neuroprotection after stroke. We observe that intrastriatal delivery of NGF and BDNF using a viral vector system can mitigate, albeit only moderately, neuronal death following stroke, which leads to detectable functional sparing.

  1. Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria.

    PubMed

    Harding, C O; Gillingham, M B; Hamman, K; Clark, H; Goebel-Daghighi, E; Bird, A; Koeberl, D D

    2006-03-01

    Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pah(enu2) mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pah(enu2) mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5+/-2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism.

  2. Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria

    PubMed Central

    Harding, CO; Gillingham, MB; Hamman, K; Clark, H; Goebel-Daghighi, E; Bird, A; Koeberl, DD

    2009-01-01

    Novel recombinant adeno-associated virus vectors pseudo-typed with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pahenu2 mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pahenu2 mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5±2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism. PMID:16319949

  3. [Construction of recombinant lentiviral vector of Tie2-RNAi and its influence on malignant melanoma cells in vitro].

    PubMed

    Shan, Xiu-ying; Liu, Zhao-liang; Wang, Biao; Guo, Guo-xiang; Wang, Mei-shui; Zhuang, Fu-lian; Cai, Chuan-shu; Zhang, Ming-feng; Zhang, Yan-ding

    2011-07-01

    To construct lentivector carrying Tie2-Small interfering RNA (SiRNA), so as to study its influence on malignant melanoma cells. Recombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with XbaI, ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-I or pNL-EGFP-U6-Tie2-II, and then the electrophoresis clones was sequenced. Plasmids of pNL-EGFP-U6-Tie2-I and pNL-EGFP-U6-Tie2-II were constructed and combined with pVSVG and pHelper, respectively, to constitute lentiviral vector system of three plasmids. The Lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Tie2- I and pNL-EGFP-U6-Tie2-II lentivirus. Then the supernatant was collected to determine the titer. Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency. The recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing. And the titer of lentiviral vector was 8.8 x 10(3)/ml, which was determined by 293T cell. The results of Realtime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells (P<0.01). There was no significant difference in the expression level (P>0.05) between the two lentiviral vectors of Tie2-RNAi. Lentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly. The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.

  4. [Protein transduction, from technology to physiology].

    PubMed

    Prochiantz, Alain

    2006-01-01

    In the early 90s, we found that the DNA-binding domain (homeodomain) of Antennapedia, a homeoprotein transcription factor, was internalized by live cells gaining access to their cytoplasm and nuclei. It was soon revealed that internalization is due to the third helix of the homeodomain, composed of sixteen amino acids. This short peptide baptized Penetratin is the first of a large series of transduction peptides widely used for the internalization of all sorts of cargoes in vitro and in vivo. Although transduction peptides are being developed with the latter practical goal, the most intriguing outcome of our initial observation is that full-length homeoproteins are transferred between cells and have non-cell autonomous transcriptional and translational activities. This new signaling mechanism requires that homeoproteins be internalized and secreted. Secretion is Golgi independent and requires a small sequence also present in the homeodomain but distinct from the Penetratin sequence. The consequences of this novel signaling mechanism are briefly discussed.

  5. Advances in Targeting Signal Transduction Pathways

    PubMed Central

    McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Sun, Lin; Davis, Nicole M.; Abrams, Stephen L.; Franklin, Richard A.; Cocco, Lucio; Evangelisti, Camilla; Chiarini, Francesca; Martelli, Alberto M.; Libra, Massimo; Candido, Saverio; Ligresti, Giovanni; Malaponte, Grazia; Mazzarino, Maria C.; Fagone, Paolo; Donia, Marco; Nicoletti, Ferdinando; Polesel, Jerry; Talamini, Renato; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Dulińska-Litewka, Joanna; Laidler, Piotr; D'Assoro, Antonio B.; Drobot, Lyudmyla; Umezawa, Kazuo; Montalto, Giuseppe; Cervello, Melchiorre; Demidenko, Zoya N.

    2012-01-01

    Over the past few years, significant advances have occurred in both our understanding of the complexity of signal transduction pathways as well as the isolation of specific inhibitors which target key components in those pathways. Furthermore critical information is being accrued regarding how genetic mutations can affect the sensitivity of various types of patients to targeted therapy. Finally, genetic mechanisms responsible for the development of resistance after targeted therapy are being discovered which may allow the creation of alternative therapies to overcome resistance. This review will discuss some of the highlights over the past few years on the roles of key signaling pathways in various diseases, the targeting of signal transduction pathways and the genetic mechanisms governing sensitivity and resistance to targeted therapies. PMID:23455493

  6. The ethylene signal transduction pathway in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Kieber, J. J.; Evans, M. L. (Principal Investigator)

    1997-01-01

    The gaseous hormone ethylene is an important regulator of plant growth and development. Using a simple response of etiolated seedlings to ethylene as a genetic screen, genes involved in ethylene signal transduction have been identified in Arabidopsis. Analysis of two of these genes that have been cloned reveals that ethylene signalling involves a combination of a protein (ETR1) with similarity to bacterial histidine kinases and a protein (CTR1) with similarity to Raf-1, a protein kinase involved in multiple signalling cascades in eukaryotic cells. Several lines of investigation provide compelling evidence that ETR1 encodes an ethylene receptor. For the first time there is a glimpse of the molecular circuitry underlying the signal transduction pathway for a plant hormone.

  7. The ethylene signal transduction pathway in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Kieber, J. J.; Evans, M. L. (Principal Investigator)

    1997-01-01

    The gaseous hormone ethylene is an important regulator of plant growth and development. Using a simple response of etiolated seedlings to ethylene as a genetic screen, genes involved in ethylene signal transduction have been identified in Arabidopsis. Analysis of two of these genes that have been cloned reveals that ethylene signalling involves a combination of a protein (ETR1) with similarity to bacterial histidine kinases and a protein (CTR1) with similarity to Raf-1, a protein kinase involved in multiple signalling cascades in eukaryotic cells. Several lines of investigation provide compelling evidence that ETR1 encodes an ethylene receptor. For the first time there is a glimpse of the molecular circuitry underlying the signal transduction pathway for a plant hormone.

  8. Signal transduction in T lymphocytes in microgravity

    NASA Technical Reports Server (NTRS)

    Cogoli, A.

    1997-01-01

    More than 120 experiments conducted in space in the last 15 years have shown that dramatic changes are occurring in several types of single cells during their exposure to microgravity. One focus of today's research on cells in space is on signal transduction, especially those steps involving the cytoskeleton and cell-cell interactions. Signal transduction is often altered in microgravity as well as in hypergravity. This leads to changes in cell proliferation, genetic expression and differentiation. Interesting examples are leukocytes, HeLa cells, epidermoid cells and osteoblastic cells. Signalling pathways were studied in T lymphocytes in microgravity by several investigators after the discovery that mitogenic activation in vitro is virtually nil at 0g. T cells are a good model to study signal transduction because three extracellular signals (mitogen, IL-1 and IL-2) are required for full activation, and two classical pathways (via proteins G and PKC) are activated within the cell. In addition, low molecular weight GTP-binding proteins (Ras and Rap) are interacting with the cytoskeleton. The data at 0g support the notion that the expression of IL-2 receptor is inhibited at 0g, while mitogen binding and the transmission of IL-1 by accessory cells occur normally. In addition, alterations of the cytoskeleton suggest that the interaction with Rap proteins is disturbed. Data obtained with phorbol esters indicate that the function of PKC is changed in microgravity. Similar conclusions are drawn from the results with epidermoid cells A431.

  9. Mechanotransduction and auditory transduction in Drosophila.

    PubMed

    Kernan, Maurice J

    2007-08-01

    Insects are utterly reliant on sensory mechanotransduction, the process of converting physical stimuli into neuronal receptor potentials. The senses of proprioception, touch, and hearing are involved in almost every aspect of an adult insect's complex behavioral repertoire and are mediated by a diverse array of specialized sensilla and sensory neurons. The physiology and morphology of several of these have been described in detail; genetic approaches in Drosophila, combining behavioral screens and sensory electrophysiology with forward and reverse genetic techniques, have now revealed specific proteins involved in their differentiation and operation. These include three different TRP superfamily ion channels that are required for transduction in tactile bristles, chordotonal stretch receptors, and polymodal nociceptors. Transduction also depends on the normal differentiation and mechanical integrity of the modified cilia that form the neuronal sensory endings, the accessory structures that transmit stimuli to them and, in bristles, a specialized receptor lymph and transepithelial potential. Flies hear near-field sounds with a vibration-sensitive, antennal chordotonal organ. Biomechanical analyses of wild-type antennae reveal non-linear, active mechanical properties that increase their sensitivity to weak stimuli. The effects of mechanosensory and ciliary mutations on antennal mechanics show that the sensory cilia are the active motor elements and indicate distinct roles for TRPN and TRPV channels in auditory transduction and amplification.

  10. Signal transduction in T lymphocytes in microgravity

    NASA Technical Reports Server (NTRS)

    Cogoli, A.

    1997-01-01

    More than 120 experiments conducted in space in the last 15 years have shown that dramatic changes are occurring in several types of single cells during their exposure to microgravity. One focus of today's research on cells in space is on signal transduction, especially those steps involving the cytoskeleton and cell-cell interactions. Signal transduction is often altered in microgravity as well as in hypergravity. This leads to changes in cell proliferation, genetic expression and differentiation. Interesting examples are leukocytes, HeLa cells, epidermoid cells and osteoblastic cells. Signalling pathways were studied in T lymphocytes in microgravity by several investigators after the discovery that mitogenic activation in vitro is virtually nil at 0g. T cells are a good model to study signal transduction because three extracellular signals (mitogen, IL-1 and IL-2) are required for full activation, and two classical pathways (via proteins G and PKC) are activated within the cell. In addition, low molecular weight GTP-binding proteins (Ras and Rap) are interacting with the cytoskeleton. The data at 0g support the notion that the expression of IL-2 receptor is inhibited at 0g, while mitogen binding and the transmission of IL-1 by accessory cells occur normally. In addition, alterations of the cytoskeleton suggest that the interaction with Rap proteins is disturbed. Data obtained with phorbol esters indicate that the function of PKC is changed in microgravity. Similar conclusions are drawn from the results with epidermoid cells A431.

  11. In vivo protein transduction to the CNS.

    PubMed

    Loftus, L T; Li, H-F; Gray, A J; Hirata-Fukae, C; Stoica, B A; Futami, J; Yamada, H; Aisen, P S; Matsuoka, Y

    2006-01-01

    Proteins and peptides are useful research and therapeutic tools, however applications are limited because delivery to the desired location is not easily achievable. There are two hurdles in protein/peptide delivery to the brain: the blood-brain barrier and intracellular penetration. Penetration to both brain and the intracellular space can be achieved by adjusting hydrophilicity, and small molecule pharmacological agents have been successfully developed using this approach. But with proteins and peptides, it is difficult to modify the hydrophilicity without influencing biological functions. Trans-acting factor protein from the human immunodeficiency virus contains a highly conserved cationic peptide sequence necessary for transduction across the cell membrane. While trans-acting factor peptide has been used for in vitro protein transduction, its in vivo application is very limited because it is rapidly degraded by proteolysis. Polyethylenimine is a chemically synthesized small molecule cationization agent; the charge density is greater than a peptide-based cationic cluster such as trans-acting factor, and it is resistant to proteolysis in vivo. We first tested intracellular protein transduction following direct brain injection in mice using polyethylenimine-conjugated green fluorescence protein and beta-galactosidase (molecular weights 29 and 540 kDa, respectively). Polyethylenimine-conjugates penetrated to the intracellular space immediately surrounding the injection site within one hour. We further tested polyethylenimine-mediated protein transduction following intranasal administration, which bypasses the blood-brain barrier. Polyethylenimine-conjugates in pH 7.5 solution did not reach the brain, probably because the polyethylenimine-conjugates penetrated into the intracellular space where first exposed to the tissue, i.e. at the nasal mucosae. We temporarily reduced the electrostatic interaction between cationized polyethylenimine-conjugates and cellular

  12. Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion

    PubMed Central

    Zheng, Yongxiang; Yu, Fei; Wu, Yiming; Si, Longlong; Xu, Huan; Zhang, Chuanling; Xia, Qing; Xiao, Sulong; Wang, Qi; He, Qiuchen; Chen, Peng; Wang, Jiangyun; Taira, Kazunari; Zhang, Lihe; Zhou, Demin

    2015-01-01

    With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure–function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus–host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications. PMID:25765642

  13. Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion.

    PubMed

    Zheng, Yongxiang; Yu, Fei; Wu, Yiming; Si, Longlong; Xu, Huan; Zhang, Chuanling; Xia, Qing; Xiao, Sulong; Wang, Qi; He, Qiuchen; Chen, Peng; Wang, Jiangyun; Taira, Kazunari; Zhang, Lihe; Zhou, Demin

    2015-06-23

    With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure-function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus-host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.

  14. Feline Immunodeficiency Virus Cross-Species Transmission: Implications for Emergence of New Lentiviral Infections.

    PubMed

    Lee, Justin; Malmberg, Jennifer L; Wood, Britta A; Hladky, Sahaja; Troyer, Ryan; Roelke, Melody; Cunningham, Mark; McBride, Roy; Vickers, Winston; Boyce, Walter; Boydston, Erin; Serieys, Laurel; Riley, Seth; Crooks, Kevin; VandeWoude, Sue

    2017-03-01

    Owing to a complex history of host-parasite coevolution, lentiviruses exhibit a high degree of species specificity. Given the well-documented viral archeology of human immunodeficiency virus (HIV) emergence following human exposures to simian immunodeficiency virus (SIV), an understanding of processes that promote successful cross-species lentiviral transmissions is highly relevant. We previously reported natural cross-species transmission of a subtype of feline immunodeficiency virus, puma lentivirus A (PLVA), between bobcats (Lynx rufus) and mountain lions (Puma concolor) for a small number of animals in California and Florida. In this study, we investigate host-specific selection pressures, within-host viral fitness, and inter- versus intraspecies transmission patterns among a larger collection of PLV isolates from free-ranging bobcats and mountain lions. Analyses of proviral and viral RNA levels demonstrate that PLVA fitness is severely restricted in mountain lions compared to that in bobcats. We document evidence of diversifying selection in three of six PLVA genomes from mountain lions, but we did not detect selection among 20 PLVA isolates from bobcats. These findings support the hypothesis that PLVA is a bobcat-adapted virus which is less fit in mountain lions and under intense selection pressure in the novel host. Ancestral reconstruction of transmission events reveals that intraspecific PLVA transmission has occurred among panthers (Puma concolor coryi) in Florida following the initial cross-species infection from bobcats. In contrast, interspecific transmission from bobcats to mountain lions predominates in California. These findings document outcomes of cross-species lentiviral transmission events among felids that compare to the emergence of HIV from nonhuman primates.IMPORTANCE Cross-species transmission episodes can be singular, dead-end events or can result in viral replication and spread in the new species. The factors that determine which outcome

  15. Feline Immunodeficiency Virus Cross-Species Transmission: Implications for Emergence of New Lentiviral Infections

    PubMed Central

    Lee, Justin; Malmberg, Jennifer L.; Wood, Britta A.; Hladky, Sahaja; Troyer, Ryan; Roelke, Melody; Cunningham, Mark; McBride, Roy; Vickers, Winston; Boyce, Walter; Boydston, Erin; Serieys, Laurel; Riley, Seth; Crooks, Kevin

    2016-01-01

    ABSTRACT Owing to a complex history of host-parasite coevolution, lentiviruses exhibit a high degree of species specificity. Given the well-documented viral archeology of human immunodeficiency virus (HIV) emergence following human exposures to simian immunodeficiency virus (SIV), an understanding of processes that promote successful cross-species lentiviral transmissions is highly relevant. We previously reported natural cross-species transmission of a subtype of feline immunodeficiency virus, puma lentivirus A (PLVA), between bobcats (Lynx rufus) and mountain lions (Puma concolor) for a small number of animals in California and Florida. In this study, we investigate host-specific selection pressures, within-host viral fitness, and inter- versus intraspecies transmission patterns among a larger collection of PLV isolates from free-ranging bobcats and mountain lions. Analyses of proviral and viral RNA levels demonstrate that PLVA fitness is severely restricted in mountain lions compared to that in bobcats. We document evidence of diversifying selection in three of six PLVA genomes from mountain lions, but we did not detect selection among 20 PLVA isolates from bobcats. These findings support the hypothesis that PLVA is a bobcat-adapted virus which is less fit in mountain lions and under intense selection pressure in the novel host. Ancestral reconstruction of transmission events reveals that intraspecific PLVA transmission has occurred among panthers (Puma concolor coryi) in Florida following the initial cross-species infection from bobcats. In contrast, interspecific transmission from bobcats to mountain lions predominates in California. These findings document outcomes of cross-species lentiviral transmission events among felids that compare to the emergence of HIV from nonhuman primates. IMPORTANCE Cross-species transmission episodes can be singular, dead-end events or can result in viral replication and spread in the new species. The factors that determine

  16. Feline immunodeficiency virus cross-species transmission: Implications for emergence of new lentiviral infections

    USGS Publications Warehouse

    Lee, Justin; Malmberg, Jennifer L.; Wood, Britta A.; Hladky, Sahaja; Troyer, Ryan; Roelke, Melody; Cunningham, Mark W.; McBride, Roy; Vickers, Winston; Boyce, Walter; Boydston, Erin E.; Serieys, Laurel E.K.; Riley, Seth P D; Crooks, Kevin R.; VandeWoude, Sue

    2016-01-01

    Owing to a complex history of host-parasite coevolution, lentiviruses exhibit a high degree of species specificity. Given the well-documented viral archeology of HIV emergence following human exposures to SIV, understanding processes that promote successful cross-species lentiviral transmissions is highly relevant. We have previously reported natural cross-species transmission of a subtype of feline immunodeficiency virus, puma lentivirus A (PLVA), between bobcats (Lynx rufus) and mountain lions (Puma concolor) in a small number of animals in California and Florida. In this study we investigate host-specific selection pressures, within-host viral fitness, and inter- vs. intra-species transmission patterns among a larger collection of PLV isolates from free-ranging bobcats and mountain lions. Analysis of proviral and viral RNA levels demonstrates that PLVA fitness is severely restricted in mountain lions compared to bobcats. We document evidence of diversifying selection in three of six PLVA genomes from mountain lions, but did not detect selection among twenty PLVA isolates from bobcats. These findings support that PLVA is a bobcat-adapted virus, which is less fit in mountain lions and under intense selection pressure in the novel host. Ancestral reconstruction of transmission events reveals intraspecific PLVA transmission has occurred among panthers (Puma concolor coryi) in Florida following initial cross-species infection from bobcats. In contrast, interspecific transmission from bobcats to mountain lions predominates in California. These findings document outcomes of cross-species lentiviral transmission events among felids that compare to emergence of HIV from nonhuman primates.IMPORTANCE Cross-species transmission episodes can be singular, dead-end events or can result in viral replication and spread in the new species. The factors that determine which outcome will occur are complex, and the risk of new virus emergence is therefore difficult to predict. Here

  17. Tracing retinal vessel trees by transductive inference

    PubMed Central

    2014-01-01

    Background Structural study of retinal blood vessels provides an early indication of diseases such as diabetic retinopathy, glaucoma, and hypertensive retinopathy. These studies require accurate tracing of retinal vessel tree structure from fundus images in an automated manner. However, the existing work encounters great difficulties when dealing with the crossover issue commonly-seen in vessel networks. Results In this paper, we consider a novel graph-based approach to address this tracing with crossover problem: After initial steps of segmentation and skeleton extraction, its graph representation can be established, where each segment in the skeleton map becomes a node, and a direct contact between two adjacent segments is translated to an undirected edge of the two corresponding nodes. The segments in the skeleton map touching the optical disk area are considered as root nodes. This determines the number of trees to-be-found in the vessel network, which is always equal to the number of root nodes. Based on this undirected graph representation, the tracing problem is further connected to the well-studied transductive inference in machine learning, where the goal becomes that of properly propagating the tree labels from those known root nodes to the rest of the graph, such that the graph is partitioned into disjoint sub-graphs, or equivalently, each of the trees is traced and separated from the rest of the vessel network. This connection enables us to address the tracing problem by exploiting established development in transductive inference. Empirical experiments on public available fundus image datasets demonstrate the applicability of our approach. Conclusions We provide a novel and systematic approach to trace retinal vessel trees with the present of crossovers by solving a transductive learning problem on induced undirected graphs. PMID:24438151

  18. Mechano-Transduction: From Molecules to Tissues

    PubMed Central

    Pruitt, Beth L.; Dunn, Alexander R.; Weis, William I.; Nelson, W. James

    2014-01-01

    External forces play complex roles in cell organization, fate, and homeostasis. Changes in these forces, or how cells respond to them, can result in abnormal embryonic development and diseases in adults. How cells sense and respond to these mechanical stimuli requires an understanding of the biophysical principles that underlie changes in protein conformation and result in alterations in the organization and function of cells and tissues. Here, we discuss mechano-transduction as it applies to protein conformation, cellular organization, and multi-cell (tissue) function. PMID:25405923

  19. Mechanisms of sensory transduction in the skin.

    PubMed

    Lumpkin, Ellen A; Caterina, Michael J

    2007-02-22

    Sensory neurons innervating the skin encode the familiar sensations of temperature, touch and pain. An explosion of progress has revealed unanticipated cellular and molecular complexity in these senses. It is now clear that perception of a single stimulus, such as heat, requires several transduction mechanisms. Conversely, a given protein may contribute to multiple senses, such as heat and touch. Recent studies have also led to the surprising insight that skin cells might transduce temperature and touch. To break the code underlying somatosensation, we must therefore understand how the skin's sensory functions are divided among signalling molecules and cell types.

  20. Signal transduction mechanisms in plants: an overview

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Thompson, G. Jr; Roux, S. J.

    2001-01-01

    This article provides an overview on recent advances in some of the basic signalling mechanisms that participate in a wide variety of stimulus-response pathways. The mechanisms include calcium-based signalling, G-protein-mediated-signalling and signalling involving inositol phospholipids, with discussion on the role of protein kinases and phosphatases interspersed. As a further defining feature, the article highlights recent exciting findings on three extracellular components that have not been given coverage in previous reviews of signal transduction in plants, extracellular calmodulin, extracellular ATP, and integrin-like receptors, all of which affect plant growth and development.

  1. Auditory neuroscience: Development, transduction, and integration

    PubMed Central

    Hudspeth, A. J.; Konishi, Masakazu

    2000-01-01

    Hearing underlies our ability to locate sound sources in the environment, our appreciation of music, and our ability to communicate. Participants in the National Academy of Sciences colloquium on Auditory Neuroscience: Development, Transduction, and Integration presented research results bearing on four key issues in auditory research. How does the complex inner ear develop? How does the cochlea transduce sounds into electrical signals? How does the brain's ability to compute the location of a sound source develop? How does the forebrain analyze complex sounds, particularly species-specific communications? This article provides an introduction to the papers stemming from the meeting. PMID:11050196

  2. The Molecular Basis of Mechanosensory Transduction

    PubMed Central

    Marshall, Kara L.; Lumpkin, Ellen A.

    2014-01-01

    Multiple senses including hearing, touch, and osmotic regulation, require the ability to convert force into an electrical signal: a process called mechanotransduction. Mechanotransduction occurs through specialized proteins that open an ion channel pore in response to a mechanical stimulus. Many of these proteins remain unidentified in vertebrates, but known mechanotransduction channels in lower organisms provide clues into their identity and mechanism. Bacteria, fruit flies, and nematodes have all been used to elucidate the molecules necessary for force transduction. This chapter discusses many different mechanical senses and takes an evolutionary approach to review the proteins responsible for mechanotransduction in various biological kingdoms. PMID:22399400

  3. Signal transduction mechanisms in plants: an overview

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Thompson, G. Jr; Roux, S. J.

    2001-01-01

    This article provides an overview on recent advances in some of the basic signalling mechanisms that participate in a wide variety of stimulus-response pathways. The mechanisms include calcium-based signalling, G-protein-mediated-signalling and signalling involving inositol phospholipids, with discussion on the role of protein kinases and phosphatases interspersed. As a further defining feature, the article highlights recent exciting findings on three extracellular components that have not been given coverage in previous reviews of signal transduction in plants, extracellular calmodulin, extracellular ATP, and integrin-like receptors, all of which affect plant growth and development.

  4. Cochlear transduction: an integrative model and review

    PubMed Central

    Brownell, William E.

    2009-01-01

    A model for cochlear transduction is presented that is based on considerations of the cell biology of its receptor cells, particularly the mechanisms of transmitter release at recepto-neural synapses. Two new interrelated hypotheses on the functional organization of the organ of Corti result from these considerations, one dealing with the possibility of electrotonic interaction between inner and outer hair cells and the other with a possible contributing source to acoustic emissions of cochlear origin that results from vesicular membrane turnover. PMID:6282796

  5. Viral vector mediated expression of mutant huntingtin in the dorsal raphe produces disease-related neuropathology but not depressive-like behaviors in wildtype mice.

    PubMed

    Pitzer, Mark; Lueras, Jordan; Warden, Anna; Weber, Sydney; McBride, Jodi

    2015-05-22

    depressive-like behaviors. Wildtype mice were injected with an adeno-associated virus (AAV2/1) encoding HTT containing either a pathogenic (N171-82Q) or control (N171-16Q) CAG repeat length into the ventral DRN and depressive-like behaviors and motor behaviors were assessed for 12 weeks post-surgery. Quantitative PCR and immunohistochemistry (IHC) verified positive transduction in the ventral aspects of the DRN, including the ventral sub-nucleus (DRv) and interfascicular sub-nucleus (DRif). IHC demonstrated microgliosis in and around the injection site and mHTT-positive inclusions in serotonin-producing neurons and a small percentage of astrocytes in animals injected with N171-82Q compared to controls. Moreover, N171-82Q injected mice showed a 75% reduction in cells that stained positive for the serotonin synthesis enzyme, tryptophan hydroxylase-2 (TPH2) compared to controls (p<0.05). Despite mHTT-mediated pathology in the DRv and DRif, no significant changes in depressive-like behavior were detected. Consequently, we conclude that 12 weeks of N171-82Q expression in the ventral sub-nuclei of the DRN of wildtype mice causes characteristic disease-related cellular neuropathology but is not sufficient to elicit depressive-like behaviors. Ongoing studies are investigating whether a larger injection volume that transfects a larger percentage of the DRN and/or a longer time course of mHTT expression might elicit depressive-like behaviors. Moreover, mHTT expression in other regions of the brain, such as the hippocampal dentate gyrus and/or the frontal cortex might be necessary to elicit HD depression. Together, these results may prove helpful in addressing which therapeutic and/or pharmacological strategies might be most efficacious when treating depressive symptomology in patients suffering from HD.

  6. Viral vector mediated expression of mutant huntingtin in the dorsal raphe produces disease-related neuropathology but not depressive-like behaviors in wildtype mice

    PubMed Central

    Pitzer, Mark; Lueras, Jordan; Warden, Anna; Weber, Sydney; McBride, Jodi

    2017-01-01

    depressive-like behaviors. Wildtype mice were injected with an adeno-associated virus (AAV2/1) encoding HTT containing either a pathogenic (N171-82Q) or control (N171-16Q) CAG repeat length into the ventral DRN and depressive-like behaviors and motor behaviors were assessed for 12 weeks post-surgery. Quantitative PCR and immunohistochemistry (IHC) verified positive transduction in the ventral aspects of the DRN, including the ventral sub-nucleus (DRv) and interfascicular sub-nucleus (DRif). IHC demonstrated microgliosis in and around the injection site and mHTT-positive inclusions in serotonin-producing neurons and a small percentage of astrocytes in animals injected with N171-82Q compared to controls. Moreover, N171-82Q injected mice showed a 75% reduction in cells that stained positive for the serotonin synthesis enzyme, tryptophan hydroxylase-2 (TPH2) compared to controls (p <0.05). Despite mHTT-mediated pathology in the DRv and DRif, no significant changes in depressive-like behavior were detected. Consequently, we conclude that 12 weeks of N171-82Q expression in the ventral sub-nuclei of the DRN of wildtype mice causes characteristic disease-related cellular neuropathology but is not sufficient to elicit depressive-like behaviors. Ongoing studies are investigating whether a larger injection volume that transfects a larger percentage of the DRN and/or a longer time course of mHTT expression might elicit depressive-like behaviors. Moreover, mHTT expression in other regions of the brain, such as the hippocampal dentate gyrus and/or the frontal cortex might be necessary to elicit HD depression. Together, these results may prove helpful in addressing which therapeutic and/or pharmacological strategies might be most efficacious when treating depressive symptomology in patients suffering from HD. PMID:25732261

  7. Signal transduction during cold stress in plants.

    PubMed

    Solanke, Amolkumar U; Sharma, Arun K

    2008-04-01

    Cold stress signal transduction is a complex process. Many physiological changes like tissue break down and senescence occur due to cold stress. Low temperature is initially perceived by plasma membrane either due to change in membrane fluidity or with the help of sensors like Ca(2+) permeable channels, histidine kinases, receptor kinases and phospholipases. Subsequently, cytoskeleton reorganization and cytosolic Ca(2+) influx takes place. Increase in cytosolic Ca(2+) is sensed by CDPKs, phosphatase and MAPKs, which transduce the signals to switch on transcriptional cascades. Photosynthetic apparatus have also been thought to be responsible for low temperature perception and signal transduction. Many cold induced pathways are activated to protect plants from deleterious effects of cold stress, but till date, most studied pathway is ICE-CBF-COR signaling pathway. However, the importance of CBF independent pathways in cold acclimation is supported by few Arabidopsis mutants' studies. Cold stress signaling has certain pathways common with other abiotic and biotic stress signaling which suggest cross-talks among these. Most of the economically important crops are sensitive to low temperature, but very few studies are available on cold susceptible crop plants. Therefore, it is necessary to understand signal transducing components from model plants and utilize that knowledge to improve survival of cold sensitive crop plants at low temperature.

  8. Transductive face sketch-photo synthesis.

    PubMed

    Wang, Nannan; Tao, Dacheng; Gao, Xinbo; Li, Xuelong; Li, Jie

    2013-09-01

    Face sketch-photo synthesis plays a critical role in many applications, such as law enforcement and digital entertainment. Recently, many face sketch-photo synthesis methods have been proposed under the framework of inductive learning, and these have obtained promising performance. However, these inductive learning-based face sketch-photo synthesis methods may result in high losses for test samples, because inductive learning minimizes the empirical loss for training samples. This paper presents a novel transductive face sketch-photo synthesis method that incorporates the given test samples into the learning process and optimizes the performance on these test samples. In particular, it defines a probabilistic model to optimize both the reconstruction fidelity of the input photo (sketch) and the synthesis fidelity of the target output sketch (photo), and efficiently optimizes this probabilistic model by alternating optimization. The proposed transductive method significantly reduces the expected high loss and improves the synthesis performance for test samples. Experimental results on the Chinese University of Hong Kong face sketch data set demonstrate the effectiveness of the proposed method by comparing it with representative inductive learning-based face sketch-photo synthesis methods.

  9. Activity Dependent Signal Transduction in Skeletal Muscle

    NASA Technical Reports Server (NTRS)

    Hamilton, Susan L.

    1999-01-01

    The overall goals of this project are: 1) to define the initial signal transduction events whereby the removal of gravitational load from antigravity muscles, such as the soleus, triggers muscle atrophy, and 2) to develop countermeasures to prevent this from happening. Our rationale for this approach is that, if countermeasures can be developed to regulate these early events, we could avoid having to deal with the multiple cascades of events that occur downstream from the initial event. One of our major findings is that hind limb suspension causes an early and sustained increase in intracellular Ca(2+) concentration ([Ca (2+)](sub i)). In most cells the consequences of changes in ([Ca (2+)](sub i))depend on the amplitude, frequency and duration of the Ca(2+) signal and on other factors in the intracellular environment. We propose that muscle remodeling in microgravity represents a change in the balance among several CA(2+) regulated signal transduction pathways, in particular those involving the transcription factors NFAT and NFkB and the pro-apoptotic protein BAD. Other Ca(2+) sensitive pathways involving PKC, ras, rac, and CaM kinase II may also contribute to muscle remodeling.

  10. Activity Dependent Signal Transduction in Skeletal Muscle

    NASA Technical Reports Server (NTRS)

    Hamilton, Susan L.

    1999-01-01

    The overall goals of this project are: 1) to define the initial signal transduction events whereby the removal of gravitational load from antigravity muscles, such as the soleus, triggers muscle atrophy, and 2) to develop countermeasures to prevent this from happening. Our rationale for this approach is that, if countermeasures can be developed to regulate these early events, we could avoid having to deal with the multiple cascades of events that occur downstream from the initial event. One of our major findings is that hind limb suspension causes an early and sustained increase in intracellular Ca(2+) concentration ([Ca (2+)](sub i)). In most cells the consequences of changes in ([Ca (2+)](sub i))depend on the amplitude, frequency and duration of the Ca(2+) signal and on other factors in the intracellular environment. We propose that muscle remodeling in microgravity represents a change in the balance among several CA(2+) regulated signal transduction pathways, in particular those involving the transcription factors NFAT and NFkB and the pro-apoptotic protein BAD. Other Ca(2+) sensitive pathways involving PKC, ras, rac, and CaM kinase II may also contribute to muscle remodeling.

  11. Lentiviral vector system for coordinated constitutive and drug controlled tetracycline-regulated gene co-expression.

    PubMed

    Stahlhut, Maike; Schwarzer, Adrian; Eder, Matthias; Yang, Min; Li, Zhixiong; Morgan, Michael; Schambach, Axel; Kustikova, Olga S

    2015-09-01

    Constitutive co-expression of cooperating transgenes using retroviral integrating vectors is frequently used for genetic modification of different cell types to establish therapeutic or cancer models. However, such approaches are unable to dissect the influence of dose, order and reversibility of transgene expression on the fate of newly developed therapeutic/malignant phenotypes. We present a modular lentiviral vector system, which provides expression of constitutive and inducible components. To demonstrate its functionality, we constitutively expressed the well-described transcription factor Meis1 followed by inducible co-expression of collaborating partner Hoxa9 under the control of tetracycline responsive promoters in murine fibroblasts and primary hematopoietic progenitor cells (HPCs). Fluorescent markers to track transgene co-expression revealed tightly controlled, efficiently inducible and reversible but cell type dependent gene transfer over time. We demonstrated dose-dependent blockade of myeloid differentiation when both Meis1/Hoxa9 were concomitantly overexpressed in primary HPCs in vitro, but the absence of the transformed phenotype in non-induced samples or when Hoxa9 expression was down-regulated. This system combines the advantages of lentiviral gene transfer and the opportunity for drug-controlled co-expression of multiple transgenes to dissect, among others, gene networks governing complex cell behavior, such as proto-oncogene dose-dependent leukemogenic pathways or collaborating mechanisms of genes enhancing competitive fitness of hematopoietic cells.

  12. Correction of Methylmalonic Aciduria In Vivo Using a Codon-Optimized Lentiviral Vector

    PubMed Central

    Wong, Edward S.Y.; McIntyre, Chantelle; Peters, Heidi L.; Ranieri, Enzo; Anson, Donald S.

    2014-01-01

    Abstract Methylmalonic aciduria is a rare disorder of organic acid metabolism with limited therapeutic options, resulting in high morbidity and mortality. Positive results from combined liver/kidney transplantation suggest, however, that metabolic sink therapy may be efficacious. Gene therapy offers a more accessible approach for the treatment of methylmalonic aciduria than organ transplantation. Accordingly, we have evaluated a lentiviral vector–mediated gene transfer approach in an in vivo mouse model of methylmalonic aciduria. A mouse model of methylmalonic aciduria (Mut−/−MUTh2) was injected intravenously at 8 weeks of age with a lentiviral vector that expressed a codon-optimized human methylmalonyl coenzyme A mutase transgene, HIV-1SDmEF1αmurSigHutMCM. Untreated Mut−/−MUTh2 and normal mice were used as controls. HIV-1SDmEF1αmurSigHutMCM-treated mice achieved near-normal weight for age, and Western blot analysis demonstrated significant methylmalonyl coenzyme A enzyme expression in their livers. Normalization of liver methylmalonyl coenzyme A enzyme activity in the treated group was associated with a reduction in plasma and urine methylmalonic acid levels, and a reduction in the hepatic methylmalonic acid concentration. Administration of the HIV-1SDmEF1αmurSigHutMCM vector provided significant, although incomplete, biochemical correction of methylmalonic aciduria in a mouse model, suggesting that gene therapy is a potential treatment for this disorder. PMID:24568291

  13. 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.

  14. 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

  15. 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

  16. Extensive Methylation of Promoter Sequences Silences Lentiviral Transgene Expression During Stem Cell Differentiation In Vivo

    PubMed Central

    Herbst, Friederike; Ball, Claudia R; Tuorto, Francesca; Nowrouzi, Ali; Wang, Wei; Zavidij, Oksana; Dieter, Sebastian M; Fessler, Sylvia; van der Hoeven, Franciscus; Kloz, Ulrich; Lyko, Frank; Schmidt, Manfred; von Kalle, Christof; Glimm, Hanno

    2012-01-01

    Lentiviral vectors (LV) are widely used to stably transfer genes into target cells investigating or treating gene functions. In addition, gene transfer into early murine embryos may be improved to efficiently generate transgenic mice. We applied lentiviral gene transfer to generate a mouse model transgenic for SET binding protein-1 (Setbp1) and enhanced green fluorescent protein (eGFP). Neither transgenic founders nor their vector-positive offspring transcribed or expressed the transgenes. Bisulfite sequencing of the internal spleen focus-forming virus (SFFV) promoter demonstrated extensive methylation of all analyzed CpGs in the transgenic mice. To analyze the impact of Setbp1 on epigenetic silencing, embryonic stem cells (ESC) were differentiated into cardiomyocytes (CM) in vitro. In contrast to human promoters in LV, virally derived promoter sequences were strongly methylated during differentiation, independent of the transgene. Moreover, the commonly used SFFV promoter (SFFVp) was highly methylated with remarkable strength and frequency during hematopoietic differentiation in vivo in LV but less in γ-retroviral (γ-RV) backbones. In summary, we conclude that LV using an internal SFFVp are not suitable to generate transgenic mice or perform constitutive expression studies in differentiating cells. Choosing the appropriate promoter is also crucial to allow stable transgene expression in clinical gene therapy. PMID:22434137

  17. A multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors for functional gene analysis.

    PubMed

    Weber, Kristoffer; Bartsch, Udo; Stocking, Carol; Fehse, Boris

    2008-04-01

    Functional gene analysis requires the possibility of overexpression, as well as downregulation of one, or ideally several, potentially interacting genes. Lentiviral vectors are well suited for this purpose as they ensure stable expression of complementary DNAs (cDNAs), as well as short-hairpin RNAs (shRNAs), and can efficiently transduce a wide spectrum of cell targets when packaged within the coat proteins of other viruses. Here we introduce a multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors designed according to the "building blocks" principle. Using a wide spectrum of different fluorescent markers, including drug-selectable enhanced green fluorescent protein (eGFP)- and dTomato-blasticidin-S resistance fusion proteins, LeGO vectors allow simultaneous analysis of multiple genes and shRNAs of interest within single, easily identifiable cells. Furthermore, each functional module is flanked by unique cloning sites, ensuring flexibility and individual optimization. The efficacy of these vectors for analyzing multiple genes in a single cell was demonstrated in several different cell types, including hematopoietic, endothelial, and neural stem and progenitor cells, as well as hepatocytes. LeGO vectors thus represent a valuable tool for investigating gene networks using conditional ectopic expression and knock-down approaches simultaneously.

  18. Influenza M2 envelope protein augments avian influenza hemagglutinin pseudotyping of lentiviral vectors.

    PubMed

    McKay, T; Patel, M; Pickles, R J; Johnson, L G; Olsen, J C

    2006-04-01

    Lentivirus-based gene transfer has the potential to efficiently deliver DNA-based therapies into non-dividing epithelial cells of the airway for the treatment of lung diseases such as cystic fibrosis. However, significant barriers both to lung-specific gene transfer and to production of lentivirus vectors must be overcome before these vectors can be routinely used for applications to the lung. In this study, we investigated whether the ability to produce lentiviral vectors pseudotyped with fowl plague virus hemagglutinin (HA) could be improved by co-expression of influenza virus M2 in vector-producing cells. We found that M2 expression led to a 10-30-fold increase in production of HA-pseudotyped lentivirus vectors based upon equine infectious anemia virus (EIAV) or human immunodeficiency virus type 1 (HIV-1). Experiments using the M2 inhibitor amantadine and a drug-resistant mutant of M2 established that the ion channel activity of M2 was important for M2-dependent augmentation of vector production. Furthermore, the neuraminidase activity necessary for particle release from producer cells could also be incorporated into producer cells by co-expression of influenza NA cDNA. Lentiviral vectors pseudotyped with influenza envelope proteins were able to efficiently transduce via the apical membrane of polarized mouse tracheal cultures in vitro as well as mouse tracheal epithelia in vivo.

  19. Lentiviral-mediated RNAi inhibition of Sbds in murine hematopoietic progenitors impairs their hematopoietic potential

    PubMed Central

    Rawls, Amy S.; Gregory, Alyssa D.; Woloszynek, Jill R.; Liu, Fulu

    2007-01-01

    Shwachman-Diamond syndrome (SDS) is a rare multisystem disorder characterized by exocrine pancreatic insufficiency, multilineage hematopoietic dysfunction, and metaphyseal chondrodysplasia. Bone marrow dysfunction is present in nearly all patients with SDS, with neutropenia being the most common abnormality. The majority of patients with SDS have mutations in the Shwachman Bodian Diamond syndrome (SBDS) gene. We have developed a strategy to examine the consequences of lentiviral-mediated RNA interference (RNAi) of Sbds on hematopoiesis. Here, we report that both Sbds RNA and protein expression can be efficiently inhibited in primary murine hematopoietic cells using lentiviral-mediated RNAi. Inhibition of Sbds results in a defect in granulocytic differentiation in vitro and impairs myeloid progenitor generation in vivo. In addition, short-term hematopoietic engraftment was impaired, which is due in part to reduced homing of hematopoietic progenitors to the bone marrow. Finally, we show that inhibition of Sbds is associated with a decrease in circulating B lymphocytes, despite evidence of normal B lymphopoiesis. These data provide the first evidence that loss of Sbds is sufficient to induce abnormalities in hematopoiesis. PMID:17638857

  20. Lack of evidence of conserved lentiviral sequences in pigs with post weaning multisystemic wasting syndrome.

    PubMed Central

    Bratanich, A; Lairmore, M; Heneine, W; Konoby, C; Harding, J; West, K; Vasquez, G; Allan, G; Ellis, J

    1999-01-01

    In order to investigate the role of retroviruses in the recently described porcine postweaning multisystemic wasting syndrome (PMWS) serum and leukocytes were screened for reverse transcriptase (RT) activity, and tissues were examined for the presence of conserved lentiviral sequences using degenerate primers in a polymerase chain reaction (PCR). Serum and stimulated leukocytes from the blood and lymph nodes from pigs with PMWS, as well as from control pigs had RT activity that was detected by the sensitive Amp-RT assay. A 257-bp fragment was amplified from DNA from the blood and bone marrow of pigs with PMWS. This fragment was identical in size to conserved lentiviral sequences that were amplified from plasmids containing DNA from several lentiviruses. Cloning and sequencing of the fragment from affected pigs, however, did not reveal homology with the recognized lentiviruses. Together the results of these analyses suggest that the RT activity present in tissues from control and affected pigs is the result of endogenous retrovirus expression, and that a lentivirus is not a primary pathogen in PMWS. Images Figure 1. Figure 2. PMID:10480463

  1. Prolonged expansion of human nucleus pulposus cells expressing human telomerase reverse transcriptase mediated by lentiviral vector.

    PubMed

    Wu, Jianhong; Wang, Deli; Ruan, Dike; He, Qing; Zhang, Yan; Wang, Chaofeng; Xin, Hongkui; Xu, Cheng; Liu, Yue

    2014-01-01

    Human degenerative disc disease (DDD) is characterized by progressive loss of human nucleus pulposus (HNP) cells and extracellular matrix, in which the massive deposition are secreted by HNP cells. Cell therapy to supplement HNP cells to degenerated discs has been thought to be a promising strategy to treat DDD. However, obtaining a large quality of fully functional HNP cells has been severely hampered by limited proliferation capacity of HNP cells in vitro. Previous studies have used lipofectamine or recombinant adeno-associated viral (rAAV) vectors to deliver human telomerase reverse transcriptase (hTERT) into ovine or HNP cells to prolong the activity of nucleus pulposus cells with limited success. Here we developed a lentiviral vector bearing both hTERT and a gene encoding green fluorescence protein (L-hTERT/EGFP). This vector efficiently mediated both hTERT and EGFP into freshly isolated HNP cells. The expressions of both transgenes in L-hTERT/EGFP transduced HNP cells were detected up to day 210 post viral infection, which was twice as long as rAAV vector did. Furthermore, we observed restored telomerase activity, maintained telomere length, delayed cell senescence, and increased cell proliferation rate in those L-hTERT/EGFP transduced HNP cells. Our study suggests that lentiviral vector might be a useful gene delivery vehicle for HNP cell therapy to treat DDD.

  2. 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

  3. Nonintegrating Lentiviral Vectors Can Effectively Deliver Ovalbumin Antigen for Induction of Antitumor Immunity

    PubMed Central

    Hu, Biliang; Yang, Haiguang; Dai, Bingbing; Tai, April

    2009-01-01

    Abstract It has been demonstrated that nonintegrating lentiviral vectors (NILVs) are efficient in maintaining transgene expression in vitro and in vivo. Gene delivery by NILVs can significantly reduce nonspecific vector integration, which has been shown to cause malignant transformation in patients receiving gene therapy for X-linked severe combined immunodeficiency. Strong and sustained immune responses were observed after a single immunization with NILVs carrying viral antigens. However, there is no report to date that evaluates the efficacy of NILVs in inducing antigen-specific antitumor immunity. Using a well-characterized tumor model, we tested in vivo immunization with a self-inactivating lentiviral vector harboring a defective integrase. A high frequency of ovalbumin peptide (OVAp1)-specific CD8+ T cells and a substantial antibody response were detected in naive mice immunized with an NILV encoding an OVA transgene. Furthermore, this immunization method completely protected the mice against the growth of E.G7 tumor cells expressing the OVA antigen. Thus, this study provides evidence that immunization using NILVs can be a safe and promising approach for exploring cancer immunotherapy. PMID:19663564

  4. Lentiviral vectors as tools to understand central nervous system biology in mammalian model organisms.

    PubMed

    Parr-Brownlie, Louise C; Bosch-Bouju, Clémentine; Schoderboeck, Lucia; Sizemore, Rachel J; Abraham, Wickliffe C; Hughes, Stephanie M

    2015-01-01

    Lentiviruses have been extensively used as gene delivery vectors since the mid-1990s. Usually derived from the human immunodeficiency virus genome, they mediate efficient gene transfer to non-dividing cells, including neurons and glia in the adult mammalian brain. In addition, integration of the recombinant lentiviral construct into the host genome provides permanent expression, including the progeny of dividing neural precursors. In this review, we describe targeted vectors with modified envelope glycoproteins and expression of transgenes under the regulation of cell-selective and inducible promoters. This technology has broad utility to address fundamental questions in neuroscience and we outline how this has been used in rodents and primates. Combining viral tract tracing with immunohistochemistry and confocal or electron microscopy, lentiviral vectors provide a tool to selectively label and trace specific neuronal populations at gross or ultrastructural levels. Additionally, new generation optogenetic technologies can be readily utilized to analyze neuronal circuit and gene functions in the mature mammalian brain. Examples of these applications, limitations of current systems and prospects for future developments to enhance neuroscience knowledge will be reviewed. Finally, we will discuss how these vectors may be translated from gene therapy trials into the clinical setting.

  5. Comparative analysis of molecular strategies attenuating positional effects in lentiviral vectors carrying multiple genes.

    PubMed

    Osti, Daniela; Marras, Emanuela; Ceriani, Isabella; Grassini, Greta; Rubino, Tiziana; Viganò, Daniela; Parolaro, Daniela; Perletti, Gianpaolo

    2006-09-01

    Efficient, high-level expression of multiple genes is often difficult to achieve in retroviral vectors, due to positional effects affecting transcription of adjacent sequences. Here we describe the comparative analysis of different strategies for co-expressing two model cDNA sequences in the context of a second generation lentiviral vector system. A first option was based on the generation of a polycistronic construct by subcloning an internal ribosome entry site (IRES) sequence between tandem cDNAs. IRES-dependent translation of the cDNA placed downstream (3') of the first transgene was poor, and the protein was barely detectable in transduced cells. A similar result was obtained when both transgenes were placed under the transcriptional control of two independent internal promoters. When these independent transcription units were separated by the 5'HS4 chromatin insulator of the chicken beta-globin locus, a marked increase of the expression of the downstream protein was observed. Similarly, insertion of a polyadenylation sequence between the tandem transcription units fully restored - in transfection experiments - the expression of the downstream sequence, whose protein pattern was identical to the single-gene control, suggesting that in this specific construct transcriptional interference was the likely cause of the observed positional effects. These results indicate that chromatin insulator sequences can be useful molecular tools to overcome positional effects in the context of lentiviral vectors.

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

    PubMed Central

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

    2015-01-01

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

  7. 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.

  8. Development of a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR).

    PubMed

    Sigl, Reinhard; Ploner, Christian; 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.

  9. Differences in Vector Genome Processing and Illegitimate Integration of Non-Integrating Lentiviral Vectors

    PubMed Central

    Shaw, Aaron M.; Joseph, Guiandre L.; Jasti, Aparna C.; Sastry-Dent, Lakshmi; Witting, Scott; Cornetta, Kenneth

    2016-01-01

    A variety of mutations in lentiviral vector expression systems have been shown to generate a non-integrating phenotype. We studied a novel 12 base-pair U3-LTR integrase attachment site deletion (U3-LTR att site) mutant and found similar physical titers to the previously reported integrase catalytic core mutant IN/D116N. Both mutations led to a greater than two log reduction in vector integration; with IN/D116N providing lower illegitimate integration frequency, while the U3-LTR att site mutant provided a higher level of transgene expression. The improved expression of the U3-LTR att site mutant could not be explained solely based on an observed modest increase in integration frequency. In evaluating processing, we noted significant differences in unintegrated vector forms, with the U3-LTR att site mutant leading to a predominance of 1-LTR circles. The mutations also differed in the manner of illegitimate integration. The U3-LTR att site mutant vector demonstrated integrase-mediated integration at the intact U5-LTR att site and non-integrase mediated integration at the mutated U3-LTR att site. Finally, we combined a variety of mutations and modifications and assessed transgene expression and integration frequency to show that combining modifications can improve the potential clinical utility of non-integrating lentiviral vectors. PMID:27682478

  10. Sox2 transduction enhances cardiovascular repair capacity of blood-derived mesoangioblasts.

    PubMed

    Koyanagi, Masamichi; Iwasaki, Masayoshi; Rupp, Stefan; Tedesco, Francesco Saverio; Yoon, Chang-Hwan; Boeckel, Jes-Niels; Trauth, Janina; Schütz, Corina; Ohtani, Kisho; Goetz, Rebekka; Iekushi, Kazuma; Bushoven, Philipp; Momma, Stefan; Mummery, Christine; Passier, Robert; Henschler, Reinhard; Akintuerk, Hakan; Schranz, Dietmar; Urbich, Carmen; Galvez, Beatriz G; Cossu, Giulio; Zeiher, Andreas M; Dimmeler, Stefanie

    2010-04-16

    Complementation of pluripotency genes may improve adult stem cell functions. Here we show that clonally expandable, telomerase expressing progenitor cells can be isolated from peripheral blood of children. The surface marker profile of the clonally expanded cells is distinct from hematopoietic or mesenchymal stromal cells, and resembles that of embryonic multipotent mesoangioblasts. Cell numbers and proliferative capacity correlated with donor age. Isolated circulating mesoangioblasts (cMABs) express the pluripotency markers Klf4, c-Myc, as well as low levels of Oct3/4, but lack Sox2. Therefore, we tested whether overexpression of Sox2 enhances pluripotency and facilitates differentiation of cMABs in cardiovascular lineages. Lentiviral transduction of Sox2 (Sox-MABs) enhanced the capacity of cMABs to differentiate into endothelial cells and cardiomyocytes in vitro. Furthermore, the number of smooth muscle actin positive cells was higher in Sox-MABs. In addition, pluripotency of Sox-MABs was shown by demonstrating the generation of endodermal and ectodermal progenies. To test whether Sox-MABs may exhibit improved therapeutic potential, we injected Sox-MABs into nude mice after acute myocardial infarction. Four weeks after cell therapy with Sox-MABs, cardiac function was significantly improved compared to mice treated with control cMABs. Furthermore, cell therapy with Sox-MABs resulted in increased number of differentiated cardiomyocytes, endothelial cells, and smooth muscle cells in vivo. The complementation of Sox2 in Oct3/4-, Klf4-, and c-Myc-expressing cMABs enhanced the differentiation into all 3 cardiovascular lineages and improved the functional recovery after acute myocardial infarction.

  11. Analysis of cellular signal transduction from an information theoretic approach.

    PubMed

    Uda, Shinsuke; Kuroda, Shinya

    2016-03-01

    Signal transduction processes the information of various cellular functions, including cell proliferation, differentiation, and death. The information for controlling cell fate is transmitted by concentrations of cellular signaling molecules. However, how much information is transmitted in signaling pathways has thus far not been investigated. Shannon's information theory paves the way to quantitatively analyze information transmission in signaling pathways. The theory has recently been applied to signal transduction, and mutual information of signal transduction has been determined to be a measure of information transmission. We review this work and provide an overview of how signal transduction transmits informational input and exerts biological output. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells.

    PubMed

    Xu, Zhen; Chen, Feng; Zhang, Lingling; Lu, Jing; Xu, Peng; Liu, Guang; Xie, Xuemin; Mu, Wenli; Wang, Yajun; Liu, Depei

    2016-10-01

    Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.

  13. 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.

  14. Functional correction of fanconi anemia group C hematopoietic cells by the use of a novel lentiviral vector.

    PubMed

    Yamada, K; Olsen, J C; Patel, M; Rao, K W; Walsh, C E

    2001-04-01

    Lentiviral vectors transduce nondividing hematopoietic cells more efficiently than other currently available vector systems. Here we report the results of human hematopoietic cell gene transfer using lentiviral vectors based upon human immunodeficiency virus (HIV-1) and equine infectious anemia virus (EIAV). EIAV is a nonprimate lentivirus and is severely restricted in its host range to horses and closely related equines. We employed the EIAV vector system to test for gene transfer to human Fanconi anemia (FA) hematopoietic cells by comparison with HIV-1- and Moloney murine leukemia virus-based systems. Fanconi anemia is characterized by bone marrow failure secondary to stem cell dysfunction. Fanconi anemia group C EBV-transformed lymphoblasts were transduced with all three viral vectors. Phenotypic correction of FA cells, as measured by mitomycin C drug resistance, was observed with a similar efficiency in all vector systems. This is the first description of lentiviral correction of FA cells and suggests that lentiviral vectors may be useful for FA gene transfer.

  15. Cocal-pseudotyped lentiviral vectors resist inactivation by human serum and efficiently transduce primate hematopoietic repopulating cells.

    PubMed

    Trobridge, Grant D; Wu, Robert A; Hansen, Michael; Ironside, Christina; Watts, Korashon L; Olsen, Philip; Beard, Brian C; Kiem, Hans-Peter

    2010-04-01

    Lentiviral vectors are established as efficient and convenient vehicles for gene transfer. They are almost always pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) due to the high titers that can be achieved, their stability, and broad tropism. We generated a novel cocal vesiculovirus envelope glycoprotein plasmid and compared the properties of lentiviral vectors pseudotyped with cocal, VSV-G, and a modified feline endogenous retrovirus envelope glycoprotein (RD114/TR). Cocal-pseudotyped lentiviral vectors can be produced at titers as high as with VSV-G, have a broad tropism, and are stable, allowing for efficient concentration by centrifugation. Additionally, cocal vectors are more resistant to inactivation by human serum than VSV-G-pseudotyped vectors, and efficiently transduce human CD34(+) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating cells (SRCs), and long-term primate hematopoietic repopulating cells. These studies establish the potential of cocal-pseudotyped lentiviral vectors for a variety of scientific and therapeutic gene transfer applications, including in vivo gene delivery and hematopoietic stem cell (HSC) gene therapy.

  16. Protein transduction domain delivery of therapeutic macromolecules.

    PubMed

    van den Berg, Arjen; Dowdy, Steven F

    2011-12-01

    Owing to their unprecedented selectivity, specific activity and potential for 1000+ fold amplification of signal, macromolecules, such as peptides, catalytic protein domains, complete proteins, and oligonucleotides, offer great potential as therapeutic molecules. However, therapeutic use of macromolecules is limited by their poor penetration in tissues and their inability to cross the cellular membrane. The discovery of small cationic peptides that cross the membrane, called Protein Transduction Domains (PTDs) or Cell Penetrating Peptides (CPPs), in the late 1980s opened the door to cellular delivery of large, bioactive molecules. Now, PTDs are widely used as research tools, and impressively, multiple clinical trials are testing PTD-mediated delivery of macromolecular drug conjugates in patients with a variety of diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Green Light to Illuminate Signal Transduction Events

    PubMed Central

    Balla, Tamas

    2009-01-01

    When cells are exposed to hormones that act on cell surface receptors, information is processed through the plasma membrane into the cell interior via second messengers generated in the inner leaflet of the plasma membrane. Individual biochemical steps along this cascade, starting with ligand binding to receptors to activation of guanine nucleotide binding proteins and their downstream effectors such as adenylate cyclase or phospholipase C, have been biochemically characterized. However, the complexity of temporal and spatial integration of these molecular events requires that they be studied in intact cells. The great expansion of fluorescent techniques and improved imaging technologies such as confocal- and TIRF microscopy combined with genetically engineered protein modules has provided a completely new approach to signal transduction research. Spatial definition of biochemical events followed with real-time temporal resolution has become a standard goal and we are breaking the resolution barrier of light microscopes with several new techniques. PMID:19818623

  18. Transduction of mechanical strain in bone

    NASA Technical Reports Server (NTRS)

    Duncan, R. L.

    1995-01-01

    One physiologic consequence of extended periods of weightlessness is the rapid loss of bone mass associated with skeletal unloading. Conversely, mechanical loading has been shown to increase bone formation and stimulate osteoblastic function. The mechanisms underlying mechanotransduction, or how the osteoblast senses and converts biophysical stimuli into cellular responses has yet to be determined. For non-innervated mechanosensitive cells like the osteoblast, mechanotransduction can be divided into four distinct phases: 1) mechanocoupling, or the characteristics of the mechanical force applied to the osteoblast, 2) biochemical coupling, or the mechanism through which mechanical strain is transduced into a cellular biochemical signal, 3) transmission of signal from sensor to effector cell and 4) the effector cell response. This review examines the characteristics of the mechanical strain encountered by osteoblasts, possible biochemical coupling mechanisms, and how the osteoblast responds to mechanical strain. Differences in osteoblastic responses to mechanical strain are discussed in relation to the types of strain encountered and the possible transduction pathways involved.

  19. Transduction of mechanical strain in bone

    NASA Technical Reports Server (NTRS)

    Duncan, R. L.

    1995-01-01

    One physiologic consequence of extended periods of weightlessness is the rapid loss of bone mass associated with skeletal unloading. Conversely, mechanical loading has been shown to increase bone formation and stimulate osteoblastic function. The mechanisms underlying mechanotransduction, or how the osteoblast senses and converts biophysical stimuli into cellular responses has yet to be determined. For non-innervated mechanosensitive cells like the osteoblast, mechanotransduction can be divided into four distinct phases: 1) mechanocoupling, or the characteristics of the mechanical force applied to the osteoblast, 2) biochemical coupling, or the mechanism through which mechanical strain is transduced into a cellular biochemical signal, 3) transmission of signal from sensor to effector cell and 4) the effector cell response. This review examines the characteristics of the mechanical strain encountered by osteoblasts, possible biochemical coupling mechanisms, and how the osteoblast responds to mechanical strain. Differences in osteoblastic responses to mechanical strain are discussed in relation to the types of strain encountered and the possible transduction pathways involved.

  20. [ALPHA-ACTININS AND SIGNAL TRANSDUCTION PATHWAYS].

    PubMed

    Panyushev, N V; Tentler, D G

    2015-01-01

    Involvement of actin cytoskeleton proteins in signal transduction from cell surface to the nucleus, including regulation of transcription factors activity, has now been supported by a lot of experimental data. Here-with, cytoskeletal proteins may have different functions than ones they execute in the cytoplasm. Particularly, alpha-actinin 4 stabilizing actin microfilaments in the cytoplasm can translocate to the nucleus and change the activity of several transcription factors. Despite the lack of nuclear import signal and DNA binding domain, alpha-actinin 4 can bind to promoter sequences, and co-activate NF-κB-dependent transcription. Selective regulation of NF-κB gene targets may indicate involvement of alpha-actinin 4 in determining the specificity of cell response to NF-κB activation in cells of different types.

  1. Studying Cellular Signal Transduction with OMIC Technologies

    PubMed Central

    Landry, Benjamin D.; Clarke, David C.; Lee, Michael J.

    2016-01-01

    In the gulf between genotype and phenotype exists proteins and, in particular, protein signal transduction systems. These systems use a relatively limited parts list to respond to a much longer list of extracellular, environmental, and/or mechanical cues with rapidity and specificity. Most signaling networks function in a highly nonlinear and often contextual manner. Furthermore, these processes occur dynamically across space and time. Because of these complexities, systems and “OMIC” approaches are essential for the study of signal transduction. One challenge in using OMIC-scale approaches to study signaling is that the “signal” can take different forms in different situations. Signals are encoded in diverse ways such as protein-protein interactions, enzyme activities, localizations, or post-translational modifications to proteins. Furthermore, in some cases signals may be encoded only in the dynamics, duration, or rates of change of these features. Accordingly, systems-level analyses of signaling may need to integrate multiple experimental and/or computational approaches. As the field has progressed, the non-triviality of integrating experimental and computational analyses has become apparent. Successful use of OMIC methods to study signaling will require the “right” experiments and the “right” modeling approaches, and it is critical to consider both in the design phase of the project. In this review, we discuss common OMIC and modeling approaches for studying signaling, emphasizing the philosophical and practical considerations for effectively merging these two types of approaches to maximize the probability of obtaining reliable and novel insights into signaling biology. PMID:26244521

  2. Striatal Signal Transduction and Drug Addiction

    PubMed Central

    Philibin, Scott D.; Hernandez, Adan; Self, David W.; Bibb, James A.

    2011-01-01

    Drug addiction is a severe neuropsychiatric disorder characterized by loss of control over motivated behavior. The need for effective treatments mandates a greater understanding of the causes and identification of new therapeutic targets for drug development. Drugs of abuse subjugate normal reward-related behavior to uncontrollable drug-seeking and -taking. Contributions of brain reward circuitry are being mapped with increasing precision. The role of synaptic plasticity in addiction and underlying molecular mechanisms contributing to the formation of the addicted state are being delineated. Thus we may now consider the role of striatal signal transduction in addiction from a more integrative neurobiological perspective. Drugs of abuse alter dopaminergic and glutamatergic neurotransmission in medium spiny neurons of the striatum. Dopamine receptors important for reward serve as principle targets of drugs abuse, which interact with glutamate receptor signaling critical for reward learning. Complex networks of intracellular signal transduction mechanisms underlying these receptors are strongly stimulated by addictive drugs. Through these mechanisms, repeated drug exposure alters functional and structural neuroplasticity, resulting in transition to the addicted biological state and behavioral outcomes that typify addiction. Ca2+ and cAMP represent key second messengers that initiate signaling cascades, which regulate synaptic strength and neuronal excitability. Protein phosphorylation and dephosphorylation are fundamental mechanisms underlying synaptic plasticity that are dysregulated by drugs of abuse. Increased understanding of the regulatory mechanisms by which protein kinases and phosphatases exert their effects during normal reward learning and the addiction process may lead to novel targets and pharmacotherapeutics with increased efficacy in promoting abstinence and decreased side effects, such as interference with natural reward, for drug addiction. PMID

  3. Calcium and signal transduction in plants

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Reddy, A. S.

    1993-01-01

    Environmental and hormonal signals control diverse physiological processes in plants. The mechanisms by which plant cells perceive and transduce these signals are poorly understood. Understanding biochemical and molecular events involved in signal transduction pathways has become one of the most active areas of plant research. Research during the last 15 years has established that Ca2+ acts as a messenger in transducing external signals. The evidence in support of Ca2+ as a messenger is unequivocal and fulfills all the requirements of a messenger. The role of Ca2+ becomes even more important because it is the only messenger known so far in plants. Since our last review on the Ca2+ messenger system in 1987, there has been tremendous progress in elucidating various aspects of Ca(2+) -signaling pathways in plants. These include demonstration of signal-induced changes in cytosolic Ca2+, calmodulin and calmodulin-like proteins, identification of different Ca2+ channels, characterization of Ca(2+) -dependent protein kinases (CDPKs) both at the biochemical and molecular levels, evidence for the presence of calmodulin-dependent protein kinases, and increased evidence in support of the role of inositol phospholipids in the Ca(2+) -signaling system. Despite the progress in Ca2+ research in plants, it is still in its infancy and much more needs to be done to understand the precise mechanisms by which Ca2+ regulates a wide variety of physiological processes. The purpose of this review is to summarize some of these recent developments in Ca2+ research as it relates to signal transduction in plants.

  4. Studying Cellular Signal Transduction with OMIC Technologies.

    PubMed

    Landry, Benjamin D; Clarke, David C; Lee, Michael J

    2015-10-23

    In the gulf between genotype and phenotype exists proteins and, in particular, protein signal transduction systems. These systems use a relatively limited parts list to respond to a much longer list of extracellular, environmental, and/or mechanical cues with rapidity and specificity. Most signaling networks function in a highly non-linear and often contextual manner. Furthermore, these processes occur dynamically across space and time. Because of these complexities, systems and "OMIC" approaches are essential for the study of signal transduction. One challenge in using OMIC-scale approaches to study signaling is that the "signal" can take different forms in different situations. Signals are encoded in diverse ways such as protein-protein interactions, enzyme activities, localizations, or post-translational modifications to proteins. Furthermore, in some cases, signals may be encoded only in the dynamics, duration, or rates of change of these features. Accordingly, systems-level analyses of signaling may need to integrate multiple experimental and/or computational approaches. As the field has progressed, the non-triviality of integrating experimental and computational analyses has become apparent. Successful use of OMIC methods to study signaling will require the "right" experiments and the "right" modeling approaches, and it is critical to consider both in the design phase of the project. In this review, we discuss common OMIC and modeling approaches for studying signaling, emphasizing the philosophical and practical considerations for effectively merging these two types of approaches to maximize the probability of obtaining reliable and novel insights into signaling biology. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Calcium and signal transduction in plants

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Reddy, A. S.

    1993-01-01

    Environmental and hormonal signals control diverse physiological processes in plants. The mechanisms by which plant cells perceive and transduce these signals are poorly understood. Understanding biochemical and molecular events involved in signal transduction pathways has become one of the most active areas of plant research. Research during the last 15 years has established that Ca2+ acts as a messenger in transducing external signals. The evidence in support of Ca2+ as a messenger is unequivocal and fulfills all the requirements of a messenger. The role of Ca2+ becomes even more important because it is the only messenger known so far in plants. Since our last review on the Ca2+ messenger system in 1987, there has been tremendous progress in elucidating various aspects of Ca(2+) -signaling pathways in plants. These include demonstration of signal-induced changes in cytosolic Ca2+, calmodulin and calmodulin-like proteins, identification of different Ca2+ channels, characterization of Ca(2+) -dependent protein kinases (CDPKs) both at the biochemical and molecular levels, evidence for the presence of calmodulin-dependent protein kinases, and increased evidence in support of the role of inositol phospholipids in the Ca(2+) -signaling system. Despite the progress in Ca2+ research in plants, it is still in its infancy and much more needs to be done to understand the precise mechanisms by which Ca2+ regulates a wide variety of physiological processes. The purpose of this review is to summarize some of these recent developments in Ca2+ research as it relates to signal transduction in plants.

  6. A novel transgenic mouse model produced from lentiviral germline integration for the study of beta-thalassemia gene therapy.

    PubMed

    Li, Wei; Xie, Shuyang; Guo, Xinbing; Gong, Xiuli; Wang, Shu; Lin, Dan; Zhang, Jingzhi; Ren, Zhaorui; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao

    2008-03-01

    beta-thalassemia is one of the most common genetic diseases in the world and requires extensive therapy. Lentiviral-mediated gene therapy has been successfully exploited in the treatment of beta-thalassemia and showed promise in clinical application. Using a human beta-globin transgenic mouse line in a beta-thalassemia diseased model generated with a lentiviral-mediated approach, we investigate the stable therapeutic effect on a common thalassemia syndrome. Human beta-globin gene lentiviral vector was constr ucted, followed by subzonal microinjection into single-cell embryos of beta(IVS-2-654)-thalassemia mice to generate a transgenic line. Human beta-globin gene expression was examined with RT-PCR, Western-blotting and ELISA. The hematologic parameters and tissue pathology were investigated over time in founder mice and their off-spring. Transgenic mice with stable expression of the lentivirus carrying human beta-globin gene were obtained. A marked improvement in red blood cell indices and a dramatic reduction in red blood cell anisocytosis, poikilocytosis and target cells were observed. Nucleated cell proportion was greatly decreased in bone marrow, and splenomegaly with extramedullary hematopoiesis was ameliorated. Iron deposition in liver was also reduced. There was a two-fold increase in the survival rate of the beta(IVS-2-654) mice carrying human beta-globin transgene. Significantly, the germline integration of the lentiviral construct was obtained and stable hematologic phenotype correction was observed over the next two generations of the transgenic mice. The generation of human beta-globin transgenic mice in a beta(IVS-2-654)-thalassemia mouse mediated with lentiviral vectors provides a useful model and offers an attractive means to investigate the transgenic stable therapeutic effect in beta-thalassemia.

  7. Genetic Analysis in Bacillus pumilus by PBS1-Mediated Transduction

    PubMed Central

    Lovett, Paul S.; Young, Frank E.

    1970-01-01

    Bacteriophage PBS1 mediates generalized transduction in Bacillus pumilus NRRL B-3275 (BpB1). Transduction frequencies for single auxotrophic markers are of the order of 10−4 transductants per plaque-forming unit in crude phage lysates. The characteristics of PBS1 propagated on BpB1 and the properties of the system of transduction are similar to those reported for PBS1 propagated on Bacillus subtilis. By transduction, eight amino acid auxotrophic markers in BpB1 have been oriented into two linkage groups. One group contains the auxotrophic markers arginine A, leucine, and phenylalanine, and the other group contains the markers lysine, serine, tryptophan, isoleucine-valine, and isoleucine. The nature and relative order of the markers within each linkage group suggest that the arrangement of genes in these areas of the chromosome of BpB1 is similar to the arrangement of phenotypically comparable genes in two linkage groups (defined by PBS1 transduction) in B. subtilis. However, transduction of any of the above cited markers in BpB1 to prototrophy with PBS1 propagated on B. subtilis 168 could not be demonstrated. In addition to BpB1, seven other strains of B. pumilus can be infected with PBS1. Transduction has been demonstrated in three of these strains. Images PMID:5413829

  8. Serotonin Signal Transduction in Two Groups of Autistic Patients

    DTIC Science & Technology

    2012-10-01

    AD_________________ Award Number: TITLE: Serotonin Signal Transduction in Two Groups of Autistic ...Serotonin Signal Transduction in Two Groups of Autistic Patients 5a. CONTRACT NUMBER W81XWH-11-1-0820 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER...Furthermore, while treatment with selective serotonin uptake inhibitors (SSRI) is routine for autistic patients, therapeutic benefit is variable and

  9. Plasmid transfer via transduction from Streptococcus thermophilus to Lactococcus lactis.

    PubMed

    Ammann, Andreas; Neve, Horst; Geis, Arnold; Heller, Knut J

    2008-04-01

    Using Streptococcus thermophilus phages, plasmid transduction in Lactococcus lactis was demonstrated. The transduction frequencies were 4 orders of magnitude lower in L. lactis than in S. thermophilus. These results are the first evidence that there is phage-mediated direct transfer of DNA from S. thermophilus to L. lactis. The implications of these results for phage evolution are discussed.

  10. Report of an Army Workshop on Convergence Forecasting: Mechanochemical Transduction

    DTIC Science & Technology

    2012-07-01

    Breakout Session 1, Group 1.........................................................................10  Figure 3. Potential Ultrasound -Mediated...Capabilities in Mechanochemical Transduction ..........10  Figure 4. Factors that Limit Potential Ultrasound -Mediated Mechanochemical Transduction... ultrasound as a mechanism to induce mechanochemical reactions. If ultrasound is to be used to provide the mechanical energy for subsequent chemical

  11. Optogenetics - Bringing light into the darkness of mammalian signal transduction.

    PubMed

    Mühlhäuser, Wignand W D; Fischer, Adrian; Weber, Wilfried; Radziwill, Gerald

    2017-02-01

    Cells receive many different environmental clues to which they must adapt accordingly. Therefore, a complex signal transduction network has evolved. Cellular signal transduction is a highly dynamic process, in which the specific outcome is a result of the exact spatial and temporal resolution of single sub-events. While conventional techniques, like chemical inducer systems, have led to a sound understanding of the architecture of signal transduction pathways, the spatiotemporal aspects were often impossible to resolve. Optogenetics, based on genetically encoded light-responsive proteins, has the potential to revolutionize manipulation of signal transduction processes. Light can be easily applied with highest precision and minimal invasiveness. This review focuses on examples of optogenetic systems which were generated and applied to manipulate non-neuronal mammalian signaling processes at various stages of signal transduction, from cell membrane through cytoplasm to nucleus. Further, the future of optogenetic signaling will be discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Separate TRP channels mediate amplification and transduction in drosophila

    NASA Astrophysics Data System (ADS)

    Lehnert, Brendan P.; Baker, Allison E.; Wilson, Rachel I.

    2015-12-01

    Auditory receptor cells rely on mechanically-gated channels to transform sound stimuli into neural activity. Several TRP channels have been implicated in Drosophila auditory transduction, but mechanistic studies have been hampered by the inability to record subthreshold signals from receptor neurons. We developed a non-invasive method for measuring these signals by recording from a central neuron that is electrically coupled to a genetically-defined population of auditory receptors. We find that the TRPN family member NompC, which is necessary for the active amplification of motion by the auditory organ, is not required for transduction. Instead, NompC sensitizes the transduction complex to movement and precisely regulates the static forces on the complex. In contrast, the TRPV channels Nanchung and Inactive are required for responses to sound, suggesting they are components of the transduction complex. Thus, transduction and active amplification are genetically separable processes in Drosophila hearing.

  13. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Direct gene transfer in the Gottingen minipig CNS using stereotaxic lentiviral microinjections.

    PubMed

    Norgaard Glud, Andreas; Hedegaard, Claus; Nielsen, Mette Slot; Sørensen, Jens Christian; Bendixen, Christian; Jensen, Poul Henning; Larsen, Knud; Bjarkam, Carsten Reidies

    2010-01-01

    We aim to induce direct viral mediated gene transfer in the substantia nigra (SN) of the Gottingen minipig using MRI guided stereotaxic injections of lentiviral vectors encoding enhanced green fluorescent protein (EGFP). Nine female Gottingen minipigs were injected unilaterally into the SN with 6 per 2.5 microliters lentivirus capable of transducing cells and mediating expression of recombinant EGFP. The animals were euthanized after four (n=3) or twenty weeks (n=6). Fresh brain tissue from three animals was used for PCR. The remaining six brains were cryo- or paraffin-sectioned for fluorescence, Nissl-, and immunohistochemical EGFP visualization. EGFP was seen in nigral neurons, axons, glial cells, endothelial cells, and in nigral fibers targeting the striatum. PCR-based detection confirmed the presence of the transgene in SN, whereas all other examined brain areas were negative, indicating that the immunohistochemically detected EGFP in the striatum derived from transfected nigral cells.

  15. Reversal of senescence in mouse fibroblasts through lentiviral suppression of p53.

    PubMed

    Dirac, Annette M G; Bernards, René

    2003-04-04

    Senescence is generally defined as an irreversible state of G(1) cell cycle arrest in which cells are refractory to growth factor stimulation. In mouse embryo fibroblasts (MEFs), induction of senescence requires the presence of p19(ARF) and p53, as genetic ablation of either of these genes allows escape from senescence and leads to immortalization. We have developed a lentiviral vector that directs the synthesis of a p53-specific short hairpin transcript, which mediates stable suppression of p53 expression through RNA interference. We show that suppression of p53 expression in senescent MEFs leads to rapid cell cycle re-entry, is associated with loss of expression of senescence-associated genes, and leads to immortalization. These data indicate that senescence in MEFs is reversible and demonstrate that both initiation and maintenance of senescence is p53-dependent.

  16. 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.

  17. Resistance to human immunodeficiency virus type 1 (HIV-1) generated by lentivirus vector-mediated delivery of the CCR5{Delta}32 gene despite detectable expression of the HIV-1 co-receptors.

    PubMed

    Jin, Qingwen; Marsh, Jon; Cornetta, Kenneth; Alkhatib, Ghalib

    2008-10-01

    It has previously been demonstrated that there are two distinct mechanisms for genetic resistance to human immunodeficiency virus type 1 (HIV-1) conferred by the CCR5Delta32 gene: the loss of wild-type CCR5 surface expression and the generation of CCR5Delta32 protein, which interacts with CXCR4. To analyse the protective effects of long-term expression of the CCR5Delta32 protein, recombinant lentiviral vectors were used to deliver the CCR5Delta32 gene into human cell lines and primary peripheral blood mononuclear cells that had been immortalized by human T-cell leukemia virus type 1. Blasticidin S-resistant cell lines expressing the lentivirus-encoded CCR5Delta32 showed a significant reduction in HIV-1 Env-mediated fusion assays. It was shown that CD4(+) T lymphocytes expressing the lentivirus-encoded CCR5Delta32 gene were highly resistant to infection by a primary but not by a laboratory-adapted X4 strain, suggesting different infectivity requirements. In contrast to previous studies that analysed the CCR5Delta32 protective effects in a transient expression system, this study showed that long-term expression of CCR5Delta32 conferred resistance to HIV-1 despite cell-surface expression of the HIV co-receptors. The results suggest an additional unknown mechanism for generating the CCR5Delta32 resistance phenotype and support the hypothesis that the CCR5Delta32 protein acts as an HIV-suppressive factor by altering the stoichiometry of the molecules involved in HIV-1 entry. The lentiviral-CCR5Delta32 vectors offer a method of generating HIV-resistant cells by delivery of the CCR5Delta32 gene that may be useful for stem cell- or T-cell-based gene therapy for HIV-1 infection.

  18. A Nonintegrative Lentiviral Vector-Based Vaccine Provides Long-Term Sterile Protection against Malaria

    PubMed Central

    Coutant, Frédéric; Sanchez David, Raul Yusef; Félix, Tristan; Boulay, Aude; Caleechurn, Laxmee; Souque, Philippe; Thouvenot, Catherine; Bourgouin, Catherine

    2012-01-01

    Trials testing the RTS,S candidate malaria vaccine and radiation-attenuated sporozoites (RAS) have shown that protective immunity against malaria can be induced and that an effective vaccine is not out of reach. However, longer-term protection and higher protection rates are required to eradicate malaria from the endemic regions. It implies that there is still a need to explore new vaccine strategies. Lentiviral vectors are very potent at inducing strong immunological memory. However their integrative status challenges their safety profile. Eliminating the integration step obviates the risk of insertional oncogenesis. Providing they confer sterile immunity, nonintegrative lentiviral vectors (NILV) hold promise as mass pediatric vaccine by meeting high safety standards. In this study, we have assessed the protective efficacy of NILV against malaria in a robust pre-clinical model. Mice were immunized with NILV encoding Plasmodium yoelii Circumsporozoite Protein (Py CSP) and challenged with sporozoites one month later. In two independent protective efficacy studies, 50% (37.5–62.5) of the animals were fully protected (p = 0.0072 and p = 0.0008 respectively when compared to naive mice). The remaining mice with detectable parasitized red blood cells exhibited a prolonged patency and reduced parasitemia. Moreover, protection was long-lasting with 42.8% sterile protection six months after the last immunization (p = 0.0042). Post-challenge CD8+ T cells to CSP, in contrast to anti-CSP antibodies, were associated with protection (r = −0.6615 and p = 0.0004 between the frequency of IFN-g secreting specific T cells in spleen and parasitemia). However, while NILV and RAS immunizations elicited comparable immunity to CSP, only RAS conferred 100% of sterile protection. Given that a better protection can be anticipated from a multi-antigen vaccine and an optimized vector design, NILV appear as a promising malaria vaccine. PMID:23133649

  19. Establishment of mouse leukemia cell lines expressing human CD4/CCR5 using lentiviral vectors.

    PubMed

    Li, Ya-Jing; ZhuGe, Fu-Yan; Zeng, Chang-Chun; He, Jin-Yang; Tan, Ning; Liang, Juan

    2017-04-01

    A low-cost rodent model of HIV infection and which presents high application value is an effective tool to investigate HIV infection and pathogenesis. However, development of such a small animal model has been hampered by the unsuitability of rodent cells for HIV-1 replication given that the retrovirus HIV-1 has high selectivity to its host cell. Our study used the mouse leukemia cell lines L615 and L1210 that were induced by murine leukemia virus and transfected with hCD4/CCR5 loaded-lentiviral vector. Lentiviral vectors containing the genes hCD4/CCR5 under the transcriptional control of cytomegalovirus promoter were designed. Transfection efficiencies of human CD4 and CCR5 in L615 and L1210 cells were analyzed by quantitative real-time polymerase chain reaction (RT-PCR) and Western blot assay. Results showed that hCD4 and CCR5 proteins were expressed on the cell surface, demonstrating that the L615 and L1210 cells were humanized and that they possess the characteristics necessary for HIV infection of human host cells. Moreover, the sensitivity of human CD4/CCR5 transgenic mouse cells to HIV infection was confirmed by RT-PCR and ELISA. Mouse leukemia cell lines that could express hCD4 and CCR5 were thus established to facilitate normal entry of HIV-1 so that a human CD4/CCR5 transgenic mice cell model can be used to investigate the transmission and pathogenesis of HIV/AIDS and potential antiviral drugs against this disease.

  20. Production of germline transgenic pigs co-expressing double fluorescent proteins by lentiviral vector.

    PubMed

    Chen, Xiao-Yu; Zhu, Zhi-Wei; Yu, Fu-Xian; Huang, Jing; Hu, Xiao-Rui; Pan, Jian-Zhi

    2016-11-01

    Genomic integration of transgene by lentiviral vector has been proved an efficient method to produce single-transgenic animals. But it failed to create multi-gene transgenic offspring. Here, we have exploited lentivirus to generate the double-transgenic piglets through the female germline. The recombinant lentivirus containing fluorescent proteins genes (DsRed1 and Venus) were injected into the perivitelline space of 2-cell stage in vitro porcine embryos. Compared to control group, there was no significantly decreased in the proportion of blastocysts, and the two fluorescent protein genes were co-expressed in almost all the injected embryos. Total of 32 injected in vitro embryos were transferred to 2 recipients. One recipient gave birth of three live offspring, and one female piglet was identified as genomic transgene integration by PCR analysis. Subsequently, the female transgenic founder was mated naturally with a wild-type boar and gave birth of two litters of total 23 F(1) generation piglets, among which Venus and DsRed1 genes were detected in 11 piglets and 10 kinds of organs by PCR and RT-PCR respectively. The co-expression of two fluorescent proteins was visible in four different frozen tissue sections from the RT-PCR positive piglets, and 3 to 5 copies of the transgenes were detected to be integrated into the second generation genome by southern blotting analysis. The transgenes were heritable and stably integrated in the F(1) generation. The results indicated for the first time that lentiviral vector combined with natural mating has the potential to become a simple and practical technology to create germline double-transgenic livestock or biomedical animals.

  1. The Membrane and Lipids as Integral Participants in Signal Transduction: Lipid Signal Transduction for the Non-Lipid Biochemist

    ERIC Educational Resources Information Center

    Eyster, Kathleen M.

    2007-01-01

    Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction.…

  2. The Membrane and Lipids as Integral Participants in Signal Transduction: Lipid Signal Transduction for the Non-Lipid Biochemist

    ERIC Educational Resources Information Center

    Eyster, Kathleen M.

    2007-01-01

    Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction.…

  3. First-in-Human Treatment With a Dendritic Cell-targeting Lentiviral Vector-expressing NY-ESO-1, LV305, Induces Deep, Durable Response in Refractory Metastatic Synovial Sarcoma Patient.

    PubMed

    Pollack, Seth M; Lu, Hailing; Gnjatic, Sacha; Somaiah, Neeta; O'Malley, Ryan B; Jones, Robin L; Hsu, Frank J; Ter Meulen, Jan

    2017-10-01

    Effective induction of antitumor T cells is a pivotal goal of cancer immunotherapy. To this end, lentiviral vectors (LV) are uniquely poised to directly prime CD8 T-cell responses via transduction of dendritic cells in vivo and have shown promise as active cancer therapeutics in preclinical tumor models. However, until now, significant barriers related to production and regulation have prevented their widespread use in the clinic. We developed LV305, a dendritic cell-targeting, integration-deficient, replication incompetent LV from the ZVex platform, encoding the full-length cancer-testis antigen NY-ESO-1. LV305 is currently being evaluated in phase 1 and 2 trials in metastatic recurrent cancer patients with NY-ESO-1 positive solid tumors as a single agent and in combination with anti-PD-L1. Here we report on the first patient treated with LV305, a young woman with metastatic, recurrent, therapy-refractive NY-ESO-1 synovial sarcoma. The patient developed a robust NY-ESO-1-specific CD4 and CD8 T-cell response after 3 intradermal injections with LV305, and subsequently over 85% disease regression that is continuing for >2.5 years posttherapy. No adverse events >grade 2 occurred. This case demonstrates that LV305 can be safely administered and has the potential to induce a significant clinical benefit and immunologic response in a patient with advanced stage cancer.

  4. Gene expression profiling of common signal transduction pathways affected by rBMSCs/F92A-Cav1 in the lungs of rat with pulmonary arterial hypertension.

    PubMed

    Chen, Haiying; Yang, Hongli; Xu, Chong; Yue, Hongmei; Xia, Peng; Strappe, Pádraig Michael; Wang, Lei; Pan, Li; Tang, Wenqiang; Chen, Shuangfeng; Wang, Lexin

    2016-10-01

    Pulmonary arterial hypertension (PAH) is associated with sustained vasoconstriction, inflammation and suppressed apoptosis of smooth muscle cells. Our previous studies have found that rat bone marrow-derived mesenchymal stem cells (rBMSCs) transduced with a mutant caveolin-1(F92A-Cav1) could enhance endothelial nitric oxide synthase (eNOS) activity and improve pulmonary vascular remodeling, but the potential mechanism is not yet fully explored. The present study was to investigate the gene expression profile upon rBMSCs/F92A-Cav1delivered to PAH rat to evaluate the role of F92A-Cav1 in its regulation. PAH was induced with monocrotaline (MCT, 60mg/kg) prior to delivery of lentiviral vector transduced rBMSCs expressing Cav1 or F92A-Cav1. Gene expression profiling was performed using Rat Signal Transduction PathwayFinder array. The expression changes of 84 key genes representing 10 signal transduction pathways in rat following rBMSCs/F92A-Cav1 treatment was examined. Screening with the Rat Signal Transduction PathwayFinder R(2) PCR Array system and subsequent western blot, immunohistochemistry or real time PCR analysis revealed that F92A-Cav1 modified rBMSCs can inhibit the inflammation factors (TNF-alpha, Icam1 and C/EBPdelta), pro-proliferation genes (c-Myc, Bcl2a1d, Notch1and Hey2), oxidative stress gene (Hmox1) and activate cell cycle arrested gene Cdkn1a, ameliorating inflammation and inhibiting cell proliferation in PAH rat. rBMSCs/F92A-Cav1 inhibits inflammation and cell proliferation by regulating signaling pathways that related to inflammation, proliferation, cell cycle and oxidative stress. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  5. Signal transduction by the Fat cytoplasmic domain

    PubMed Central

    Pan, Guohui; Feng, Yongqiang; Ambegaonkar, Abhijit A.; Sun, Gongping; Huff, Matthew; Rauskolb, Cordelia; Irvine, Kenneth D.

    2013-01-01

    The large atypical cadherin Fat is a receptor for both Hippo and planar cell polarity (PCP) pathways. Here we investigate the molecular basis for signal transduction downstream of Fat by creating targeted alterations within a genomic construct that contains the entire fat locus, and by monitoring and manipulating the membrane localization of the Fat pathway component Dachs. We establish that the human Fat homolog FAT4 lacks the ability to transduce Hippo signaling in Drosophila, but can transduce Drosophila PCP signaling. Targeted deletion of conserved motifs identifies a four amino acid C-terminal motif that is essential for aspects of Fat-mediated PCP, and other internal motifs that contribute to Fat-Hippo signaling. Fat-Hippo signaling requires the Drosophila Casein kinase 1ϵ encoded by discs overgrown (Dco), and we characterize candidate Dco phosphorylation sites in the Fat intracellular domain (ICD), the mutation of which impairs Fat-Hippo signaling. Through characterization of Dachs localization and directed membrane targeting of Dachs, we show that localization of Dachs influences both the Hippo and PCP pathways. Our results identify a conservation of Fat-PCP signaling mechanisms, establish distinct functions for different regions of the Fat ICD, support the correlation of Fat ICD phosphorylation with Fat-Hippo signaling, and confirm the importance of Dachs membrane localization to downstream signaling pathways. PMID:23318637

  6. Receptors and transduction of umami taste stimuli.

    PubMed

    Kinnamon, Sue C; Vandenbeuch, Aurelie

    2009-07-01

    L-glutamate and 5'-ribonucleotides, such as GMP and IMP, elicit the "umami" taste, also known as the fifth taste. This review will highlight recent advancements in our understanding of umami taste receptors and their downstream signaling effectors in taste receptor cells. Several G protein-coupled receptors that bind umami stimuli have been identified in taste buds, including the heterodimer T1R1/T1R3, truncated and brain forms of mGluR4 and mGluR1, brain mGluR2, and brain mGluR3. Further, ionotropic glutamate receptors are expressed in taste cells and may play a role in glutamate transduction or signaling between taste cells and/or nerve fibers. Knockout of T1R1 or T1R3 reduces, but does not eliminate, responses to umami stimuli, suggesting that multiple receptors contribute to umami taste. The signaling effectors downstream of umami G protein-coupled receptors involve Gbetagamma activation of PLCbeta2 to elicit Ca(2+) release from intracellular stores and activation of a cation channel, TRPM5. In fungiform and palatal taste buds, T1R1/T1R3 is co-expressed with Galpha gustducin and transducin, but the Galpha proteins involved in circumvallate taste buds have not been identified. In most taste fields, however, cAMP antagonizes responses to umami stimuli, suggesting that the Galpha subunit serves to modulate umami taste sensitivity.

  7. Melanin, Radiation, and Energy Transduction in Fungi.

    PubMed

    Casadevall, Arturo; Cordero, Radames J B; Bryan, Ruth; Nosanchuk, Joshua; Dadachova, Ekaterina

    2017-03-01

    Melanin pigments are found in many diverse fungal species, where they serve a variety of functions that promote fitness and cell survival. Melanotic fungi inhabit some of the most extreme habitats on earth such as the damaged nuclear reactor at Chernobyl and the highlands of Antarctica, both of which are high-radiation environments. Melanotic fungi migrate toward radioactive sources, which appear to enhance their growth. This phenomenon, combined with the known capacities of melanin to absorb a broad spectrum of electromagnetic radiation and transduce this radiation into other forms of energy, raises the possibility that melanin also functions in harvesting such energy for biological usage. The ability of melanotic fungi to harness electromagnetic radiation for physiological processes has enormous implications for biological energy flows in the biosphere and for exobiology, since it provides new mechanisms for survival in extraterrestrial conditions. Whereas some features of the way melanin-related energy transduction works can be discerned by linking various observations and circumstantial data, the mechanistic details remain to be discovered.

  8. Glycosphingolipid–Protein Interaction in Signal Transduction

    PubMed Central

    Russo, Domenico; Parashuraman, Seetharaman; D’Angelo, Giovanni

    2016-01-01

    Glycosphingolipids (GSLs) are a class of ceramide-based glycolipids essential for embryo development in mammals. The synthesis of specific GSLs depends on the expression of distinctive sets of GSL synthesizing enzymes that is tightly regulated during development. Several reports have described how cell surface receptors can be kept in a resting state or activate alternative signalling events as a consequence of their interaction with GSLs. Specific GSLs, indeed, interface with specific protein domains that are found in signalling molecules and which act as GSL sensors to modify signalling responses. The regulation exerted by GSLs on signal transduction is orthogonal to the ligand–receptor axis, as it usually does not directly interfere with the ligand binding to receptors. Due to their properties of adjustable production and orthogonal action on receptors, GSLs add a new dimension to the control of the signalling in development. GSLs can, indeed, dynamically influence progenitor cell response to morphogenetic stimuli, resulting in alternative differentiation fates. Here, we review the available literature on GSL–protein interactions and their effects on cell signalling and development. PMID:27754465

  9. Signal transduction activated by cannabinoid receptors.

    PubMed

    Díaz-Laviada, Inés; Ruiz-Llorente, Lidia

    2005-07-01

    Since the discovery that cannabinoids exert biological actions through binding to specific receptors, signal mechanisms triggered by these receptors have been focus of extensive study. This review summarizes the current knowledge of the signalling events produced by cannabinoids from membrane receptors to downstream regulators. Two types of cannabinoid receptors have been identified to date: CB(1) and CB(2) both belonging to the heptahelichoidal receptor family but with different tissue distribution and signalling mechanisms. Coupling to inhibitory guanine nucleotide-binding protein and thus inhibition of adenylyl cyclase has been observed in both receptors but other signal transduction pathways that are regulated or not by these G proteins are differently activated upon ligand-receptor binding including ion channels, sphingomyelin hydrolysis, ceramide generation, phospholipases activation and downstream targets as MAP kinase cascade, PI3K, FAK or NOS regulation. Cannabinoids may also act independently of CB(1)or CB(2) receptors. The existence of new unidentified putative cannabinoid receptors has been claimed by many investigators. Endocannabinoids activate vanilloid TRPV1 receptors that may mediate some of the cannabinoid effects. Other actions of cannabinoids can occur through non-receptor-mediated mechanisms.

  10. Confocal Scanner for Highly Sensitive Photonic Transduction of Nanomechanical Resonators

    NASA Astrophysics Data System (ADS)

    Diao, Zhu; Losby, Joseph E.; Sauer, Vincent T. K.; Westwood, Jocelyn N.; Freeman, Mark R.; Hiebert, Wayne K.

    2013-06-01

    We show that a simple confocal laser scanning system can be used to couple light through grating couplers into nanophotonic circuits. The coupling efficiency is better than 15% per coupler. Our technique avoids using multi-axis fibre stages and is especially advantageous when the nanophotonic circuit is kept in vacuum, e.g., for nanomechanical resonator displacement transduction. This was demonstrated by recording the resonant response of a nanomechanical doubly clamped beam embedded in a race-track optical cavity. The nanophotonic transduction offers an increase of two orders of magnitude in transduction responsivity compared with conventional free-space optical interferometry.

  11. Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies.

    PubMed

    Liu, Ying Poi; Vink, Monique A; Westerink, Jan-Tinus; Ramirez de Arellano, Eva; Konstantinova, Pavlina; Ter Brake, Olivier; Berkhout, Ben

    2010-07-01

    RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers. We show that shRNAs, which target the vector genomic RNA, strongly reduced lentiviral vector titers but inhibition of the RNAi pathway via saturation could rescue vector production. The presence of miRNAs in the vector RNA genome (sense orientation) results in a minor titer reduction due to Drosha processing. A major cause for titer reduction of miRNA vectors is due to incompatibility of the cytomegalovirus promoter with the lentiviral vector system. Replacement of this promoter with an inducible promoter resulted in an almost complete restoration of the vector titer. We also showed that antisense poly(A) signal sequences can have a dramatic effect on the vector titer. These results show that not all sequences are compatible with the lentiviral vector system and that care should be taken in the design of lentiviral vectors encoding RNAi inducers.

  12. Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

    PubMed Central

    Liu, Ying Poi; Vink, Monique A.; Westerink, Jan-Tinus; Ramirez de Arellano, Eva; Konstantinova, Pavlina; Ter Brake, Olivier; Berkhout, Ben

    2010-01-01

    RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers. We show that shRNAs, which target the vector genomic RNA, strongly reduced lentiviral vector titers but inhibition of the RNAi pathway via saturation could rescue vector production. The presence of miRNAs in the vector RNA genome (sense orientation) results in a minor titer reduction due to Drosha processing. A major cause for titer reduction of miRNA vectors is due to incompatibility of the cytomegalovirus promoter with the lentiviral vector system. Replacement of this promoter with an inducible promoter resulted in an almost complete restoration of the vector titer. We also showed that antisense poly(A) signal sequences can have a dramatic effect on the vector titer. These results show that not all sequences are compatible with the lentiviral vector system and that care should be taken in the design of lentiviral vectors encoding RNAi inducers. PMID:20498457

  13. The transduction properties of intercostal muscle mechanoreceptors

    PubMed Central

    Holt, Gregory A; Johnson, Richard D; Davenport, Paul W

    2002-01-01

    Background Intercostal muscles are richly innervated by mechanoreceptors. In vivo studies of cat intercostal muscle have shown that there are 3 populations of intercostal muscle mechanoreceptors: primary muscle spindles (1°), secondary muscle spindles (2°) and Golgi tendon organs (GTO). The purpose of this study was to determine the mechanical transduction properties of intercostal muscle mechanoreceptors in response to controlled length and velocity displacements of the intercostal space. Mechanoreceptors, recorded from dorsal root fibers, were localized within an isolated intercostal muscle space (ICS). Changes in ICS displacement and the velocity of ICS displacement were independently controlled with an electromagnetic motor. ICS velocity (0.5 – 100 μm/msec to a displacement of 2,000 μm) and displacement (50–2,000 μm at a constant velocity of 10 μm/msec) parameters encompassed the full range of rib motion. Results Both 1° and 2° muscle spindles were found evenly distributed within the ICS. GTOs were localized along the rib borders. The 1° spindles had the greatest discharge frequency in response to displacement amplitude followed by the 2° afferents and GTOs. The 1° muscle spindles also possessed the greatest discharge frequency in response to graded velocity changes, 3.0 spikes·sec-1/μm·msec-1. GTOs had a velocity response of 2.4 spikes·sec-1/μm·msec-1 followed by 2° muscle spindles at 0.6 spikes·sec-1/μm·msec-1. Conclusion The results of this study provide a systematic description of the mechanosenitivity of the 3 types of intercostal muscle mechanoreceptors. These mechanoreceptors have discharge properties that transduce the magnitude and velocity of intercostal muscle length. PMID:12392601

  14. The sensory transduction pathways in bacterial chemotaxis

    NASA Technical Reports Server (NTRS)

    Taylor, Barry L.

    1989-01-01

    Bacterial chemotaxis is a useful model for investigating in molecular detail the behavioral response of cells to changes in their environment. Peritrichously flagellated bacteria such as coli and typhimurium swim by rotating helical flagella in a counterclockwise direction. If flagellar rotation is briefly reversed, the bacteria tumble and change the direction of swimming. The bacteria continuously sample the environment and use a temporal sensing mechanism to compare the present and immediate past environments. Bacteria respond to a broad range of stimuli including changes in temperature, oxygen concentration, pH and osmotic strength. Bacteria are attracted to potential sources of nutrition such as sugars and amino acids and are repelled by other chemicals. In the methylation-dependent pathways for sensory transduction and adaptation in E. coli and S. typhimurium, chemoeffectors bind to transducing proteins that span the plasma membrane. The transducing proteins are postulated to control the rate of autophosphorylation of the CheA protein, which in turn phosphorylates the CheY protein. The phospho-CheY protein binds to the switch on the flagellar motor and is the signal for clockwise rotation of the motor. Adaptation to an attractant is achieved by increasing methylation of the transducing protein until the attractant stimulus is cancelled. Responses to oxygen and certain sugars involve methylation-independent pathways in which adaption occurs without methylation of a transducing protein. Taxis toward oxygen is mediated by the electron transport system and changes in the proton motive force. Recent studies have shown that the methylation-independent pathway converges with the methylation-dependent pathway at or before the CheA protein.

  15. Gravitational sensory transduction chain in flagellates

    NASA Astrophysics Data System (ADS)

    Häder, D.-P.; Richter, P.; Ntefidou, M.; Lebert, M.

    Earlier hypotheses have assumed that gravitactic orientation in flagellates, such as the photosynthetic unicell Euglena gracilis, is brought about by passive alignment of the cells in the water column by being tail heavy. A recent experiment on a sounding rocket (TEXUS 40) comparing immobilized cells with mobile cells demonstrated that the passive buoy effect can account for approximately 20% of the orientation of the cells in a gravity field. The cells show either positive or negative gravitaxis depending on other external or internal factors. Shortly after inoculation, the tendency of young cells to swim downward in the water column can be readily reverted by adding micromolar concentrations of some heavy metal ions including copper, cadmium or lead. The negative gravitaxis of older cells is converted into a positive one by stress factors such as increasing salinity or exposure to excessive visible or UV radiation. The mechanism for this switch seems to involve reactive oxygen species since the gravitactic sign change was suppressed when oxygen was removed by flushing the cell suspension with nitrogen. Also, the addition of radical scavengers (Trolox, ascorbic acid or potassium cyanide) abolished or reduced the gravitactic sign change. Addition of hydrogen peroxide induced a gravitactic sign change in the absence of external stress factors. The primary reception for the gravity vector seems to involve mechanosensitive ion channels which specifically gate calcium ions inward. We have identified several gene sequences for putative mechanosensory channels in Euglena and have applied RNAi to identify which of these channels are involved in graviperception. The influx of Ca 2+ activates calmodulin (CaM) which has been shown to be involved in the sensory transduction chain of graviorientation. It is known that an adenylyl cyclase is bound to the flagellar membrane in Euglena which is activated by CaM. This enzyme produces cAMP which has also been shown to be the key

  16. Pheromones cause disease: pheromone/odourant transduction.

    PubMed

    Nicholson, B

    2001-09-01

    This paper compares two models of the sense of smell and demonstrates that the new model has advantages over the accepted model with implications for medical research. The accepted transduction model had an odourant or pheromone contacting an aqueous sensory lymph then movement through it to a receptor membrane beneath. If the odourant or pheromone were non-soluble, the odourant/pheromone supposedly would be bound to a soluble protein in the lymph to be carried across. Thus, an odourant/carrier protein complex physically moved through the receptor lymph/mucus to interact with a membrane bound receptor. After the membranous receptor interaction, the molecule would be deactivated and any odourant/pheromone-binding protein recycled. This new electrical chemosensory model being proposed here has the pheromone or other odourant generating an electrical event in the extra-cellular mucus. Before the pheromone arrives, proteins of the 'carrier class' dissolved in the receptor mucus slowly and continuously sequester ions. A sensed pheromonal chemical species sorbs to the mucus and immediately binds to the now ion-holding dissolved protein. The binding of the pheromone to the protein causes a measurable conformational change in the pheromone/odourant-binding protein, desequestering ions. Releasing the bound ions changes the potential differences across a nearby super-sensitive dendritic membrane resulting in dendrite excitation. Pheromones will be implicated in the aetiology of the infectious, psychiatric and autoimmune diseases. This is the third article in a series of twelve to systematically explore this contention (see references 1-9). Copyright 2001 Harcourt Publishers Ltd.

  17. Signal Transduction in the Footsteps of Goethe and Schiller

    PubMed Central

    Friedrich, Karlheinz; Lindquist, Jonathan A; Entschladen, Frank; Serfling, Edgar; Thiel, Gerald; Kieser, Arnd; Giehl, Klaudia; Ehrhardt, Christina; Feller, Stephan M; Ullrich, Oliver; Schaper, Fred; Janssen, Ottmar; Hass, Ralf

    2009-01-01

    The historical town of Weimar in Thuringia, the "green heart of Germany" was the sphere of Goethe and Schiller, the two most famous representatives of German literature's classic era. Not yet entirely as influential as those two cultural icons, the Signal Transduction Society (STS) has nevertheless in the last decade established within the walls of Weimar an annual interdisciplinary Meeting on "Signal Transduction – Receptors, Mediators and Genes", which is well recognized as a most attractive opportunity to exchange results and ideas in the field. The 12th STS Meeting was held from October 28 to 31 and provided a state-of-the-art overview of various areas of signal transduction research in which progress is fast and discussion lively. This report is intended to share with the readers of CCS some highlights of the Meeting Workshops devoted to specific aspects of signal transduction. PMID:19193215

  18. Signal transduction in the footsteps of goethe and schiller.

    PubMed

    Friedrich, Karlheinz; Lindquist, Jonathan A; Entschladen, Frank; Serfling, Edgar; Thiel, Gerald; Kieser, Arnd; Giehl, Klaudia; Ehrhardt, Christina; Feller, Stephan M; Ullrich, Oliver; Schaper, Fred; Janssen, Ottmar; Hass, Ralf

    2009-02-04

    The historical town of Weimar in Thuringia, the "green heart of Germany" was the sphere of Goethe and Schiller, the two most famous representatives of German literature's classic era. Not yet entirely as influential as those two cultural icons, the Signal Transduction Society (STS) has nevertheless in the last decade established within the walls of Weimar an annual interdisciplinary Meeting on "Signal Transduction - Receptors, Mediators and Genes", which is well recognized as a most attractive opportunity to exchange results and ideas in the field.The 12th STS Meeting was held from October 28 to 31 and provided a state-of-the-art overview of various areas of signal transduction research in which progress is fast and discussion lively. This report is intended to share with the readers of CCS some highlights of the Meeting Workshops devoted to specific aspects of signal transduction.

  19. Inhibitory effect of adenoviral vector-mediated delivery of p21WAF1/CIP1 on retinal vascular endothelial cell proliferation and tube formation in cultured Rhesus monkey cells (RF/6A).

    PubMed

    Han, Jindong; Yuan, Zhigang; Yan, Hua

    2013-06-01

    To investigate the inhibitory effect(s) of adenovirus (Ad) vector-mediated delivery of p21(WAF1/CIP1) (Ad-p21) on proliferation and tube formation in Rhesus monkey choroid-retina vascular endothelial cells (RF/6A). In vitro-cultured RF/6A cells were divided into three groups: phosphate-buffered saline (PBS), Ad-p21-transfected, and negative control. Plasmid vectors were transfected via Ad-p21. The mRNA and protein expressions of p21 and cyclin-dependent kinase (CDK)2 in RF/6A cells were measured by reverse transcription-PCR (RT-PCR) and western blot analyses. Cell-cycle distributions were analyzed by flow cytometry. Matrigel was used as a matrix for endothelial cell tube formation. Expressions of p21 mRNA and protein were greater, and expressions of CDK2 mRNA and protein lower, in the Ad-p21-transfected group than in either the PBS or negative control groups. Cell-cycle distribution analysis indicated that the proportion of G0/G1 cells was significantly higher in the Ad-p21 transfected group than in either the PBS or negative control groups (p = 0.000). There were significantly fewer endothelial cell tubes in the Ad-p21-transfected group than in either the PBS or negative control groups (p = 0.004). Ad-p21 inhibits RF/6A cell proliferation and tube formation. The underlying mechanism to account for this may be that overexpression of p21 arrests the cell-cycle transition from the G1- to the S-phase via inhibition of CDK2 activity.

  20. Falsification of the ionic channel theory of hair cell transduction.

    PubMed

    Rossetto, Michelangelo

    2013-11-01

    The hair cell provides the transduction of mechanical vibrations in the balance and acoustic sense of all vertebrates that swim, walk, or fly. The current theory places hair cell transduction in a mechanically controlled ion channel. Although the theory of a mechanical input modulating the flow of ions through an ion pore has been a useful tool, it is falsified by experimental data in the literature and can be definitively falsified by a proposed experiment.

  1. 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.

  2. 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.

  3. Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer

    PubMed Central

    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

  4. 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

  5. Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo.

    PubMed

    Centlivre, M; Zhou, X; Pouw, S M; Weijer, K; Kleibeuker, W; Das, A T; Blom, B; Seppen, J; Berkhout, B; Legrand, N

    2010-01-01

    The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated 'human immune system' (HIS) mice by transplanting newborn BALB/c Rag2(-/-)IL-2Rgamma(c)(-/-) immunodeficient mice with human hematopoietic stem cells transduced with a doxycycline-inducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression.

  6. Development of an enhanced B-specific lentiviral vector expressing BTK: a tool for gene therapy of XLA.

    PubMed

    Moreau, T; Barlogis, V; Bardin, F; Nunes, J A; Calmels, B; Chabannon, C; Tonnelle, C

    2008-06-01

    Further development of haematopoietic stem cell (HSC) gene therapy will depend on enhancement of gene transfer safety: ad hoc improvement of vector design relating to each particular disease is thus a crucial issue for HSC gene therapy. We modified a previously described lentiviral vector by adding the Emumar B-specific enhancer to a human CD19 promoter-derived sequence (Mol Ther 2004;10:45-56). We thus significantly improved the level of expression of the green fluorescent protein (GFP) reporter gene while retaining the specificity of expression in B-cell progeny of transduced human CD34+ progenitor cells obtained from cord blood or adult bone marrow. Indeed, GFP was strongly expressed from early medullary pro-B cells to splenic mature B cells whereas transgene expression remained low in transduced immature progenitors as in myeloid and T-lymphoid progeny retrieved fr