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Gene Therapy Vectors (gene + therapy_vector)
Selected AbstractsRational development of a HIV-1 gene therapy vectorTHE JOURNAL OF GENE MEDICINE, Issue 10 2003D. S. Anson Abstract Background HIV-1 provides an attractive option as the basis for gene transfer vectors due to its ability to stably transduce non-cycling cell populations. In order to fully utilise the promise of HIV-1 as a vector it is important that the effects of viral cis sequence elements on vector function are carefully delineated. Methods In this study we have systematically evaluated the effect of various cis elements from the HIV-1 YU-2 genome that have been implicated as either affecting vector performance, or HIV-1 replication, on the efficiency of vector production (titre and infectivity). As a measure of the relative safety of vectors their propensity to inadvertently transfer the gagpol gene to transduced cells was assessed. Results Sequences that were found to increase vector titre were from the 5, end of the gag gene, from the 5, and 3, ends of the env gene, from immediately upstream of the polypurine tract, and the central polypurine tract. The substitution of the HIV-1 RRE with heterologous RNA transport elements, or the deletion of the RRE, resulted in greatly reduced vector titres. RNA analysis suggested that the role of the Rev/RRE system extends beyond simply acting as an RNA nuclear export signal. The relative safety of different vector designs was compared and an optimal construct selected. Conclusions Based on our results we have constructed a vector that is both more efficient, and has better safety characteristics, than the widely used pHR, HIV-1 vector construct. Copyright © 2003 John Wiley & Sons, Ltd. [source] Immune responses to gene therapy vectors in the context of corneal transplantationACTA OPHTHALMOLOGICA, Issue 2009T RITTER Purpose The genetic engineering of grafts or cells prior to transplantation is an attractive approach to protect the graft from allogeneic rejection. Virus vector-based gene therapy is a promising method for successful ex-vivo gene transfer however, the induction of an immune response against gene-modified tissues raises concern. Methods Different virus families (Adenovirus, Retrovirus, Adeno-associated virus, Herpesvirus) have been studied as gene therapy vehicles for the delivery of therapeutic molecules. Moreover, different serotypes or envelope proteins have been used to modulate transduction efficiencies of target cells or to evade pre-existing immunity. Results Here we review gene therapeutic applications using viral vectors in the context of cornea transplantation. Both local and systemic expression of immunomodulatory molecules have led to the prevention of corneal graft rejection. However, different results have been obtained with regard to the induction of immune responses after local or systemic expression of the gene therapy vector. Not surprisingly over-expression of anti-inflammatory molecules not only modulated allograft rejection but also influenced the immune response against the viral vector and virally transduced cells. Conclusion Recent clinical trials indicate that the application of viral vectors in ophthalmology is promising however, the generation of immune responses against the viral vector or virally transduced cells are still a serious obstacle for a broader application of gene therapy. Supported by Deutsche Forschungsgemeinschaft (DFG Pl 150/14-1 and Ri 764/10-1) and Science Foundation of Ireland (SFI 06/RFP/BIC056 and SFI 07/IN.1/B925) [source] Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugateFEBS JOURNAL, Issue 18 2007Sandra E. Geden Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments. [source] Redirecting lentiviral vectors by insertion of integrin-tageting peptides into envelope proteinsTHE JOURNAL OF GENE MEDICINE, Issue 7 2009Kouki Morizono Abstract Background Targeting gene therapy vectors that can home in on desired cell and tissue types in vivo comprise the ultimate gene delivery system. We have previously developed targeting lentiviral vectors by pseudotyping vectors with modified Sindbis virus envelope proteins. The envelope protein contains the Fc-binding region of protein A (ZZ domain), so the virus can be conjugated with antibodies. The conjugated antibody mediates specific transduction of the cells and tissues expressing the target antigens, both in vitro and in vivo. However, more stable conjugation of targeting molecules would be optimal for use in immunocompetent animals, as well as in humans. Methods We inserted integrin-targeting peptides into two sites of the targeting envelope proteins and determined whether the peptides serve as receptor-binding regions of the envelope proteins and redirect the pseudotyped viruses. Results The integrin-targeting peptides can mediate binding to cells via the interaction with integrins on target cells and transduction. Peptides with a higher binding affinity increase titers of pseudotyped virus. We found two regions on the envelope protein that can accommodate insertion and serve as receptor-binding regions. Combining the peptides in two distinct regions increased the titers of the virus. Conclusions Successful incorporation of targeting molecules into the envelope protein will broaden the application of targeting vectors for a wide variety of experimental and clinical settings. Copyright © 2009 John Wiley & Sons, Ltd. [source] Recent developments in adeno-associated virus vector technologyTHE JOURNAL OF GENE MEDICINE, Issue 7 2008Hildegard Büning Abstract Adeno-associated virus (AAV), a single-stranded DNA parvovirus, is emerging as one of the leading gene therapy vectors owing to its nonpathogenicity and low immunogenicity, stability and the potential to integrate site-specifically without known side-effects. A portfolio of recombinant AAV vector types has been developed with the aim of optimizing efficiency, specificity and thereby also the safety of in vitro and in vivo gene transfer. More and more information is now becoming available about the mechanism of AAV/host cell interaction improving the efficacy of recombinant AAV vector (rAAV) mediated gene delivery. This review summarizes the current knowledge of the infectious biology of AAV, provides an overview of the latest developments in the field of AAV vector technology and discusses remaining challenges. Copyright © 2008 John Wiley & Sons, Ltd. [source] Delivery of an Adenoviral Vector to the Crushed Recurrent Laryngeal Nerve,THE LARYNGOSCOPE, Issue 6 2003Adam Rubin MD Abstract Objectives Objectives were to create a model of recurrent laryngeal nerve injury for testing the efficacy of potential therapeutic viral gene therapy vectors and to demonstrate that remote injection of a viral vector does not cause significant additional neuronal injury. Study Design Animal model. Methods Rats were randomly assigned to three groups of 10 animals each. In group I, the recurrent laryngeal nerve was crushed. In group II, the nerve was crushed and then injected with an adenoviral vector containing no transgene. In group III, the nerve was identified but was not crushed. Rats were killed at 1 week, and their larynges and brainstems were cryosectioned in 15-,m sections. Laryngeal cryosections were processed for acetylcholine histochemical analysis (motor endplates) followed by neurofilament immunoperoxidase (nerve fibers). Percentage of nerve,endplate contact was determined and compared between groups. Fluorescent in situ hybridization was performed on brainstem sections from rats in group II to confirm the presence of virus. Results No significant difference in percentage of nerve,endplate contact exists between the two crushed-nerve groups (groups I and II) (P = .88). The difference between both crushed-nerve groups and the group with noncrushed nerves (group III) was highly significant (P <.0001). The presence of virus was confirmed in group II rats. Conclusions Crush provides a significant measurable injury to the recurrent laryngeal nerve and may be used as a model to explore therapeutic interventions for nerve injury. The remote injection of viral vector did not cause significant additional neuronal injury. Remote delivery of viral vectors to the central nervous system holds promise in the treatment of recurrent laryngeal nerve injury and central nervous system diseases. [source] A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatographyBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005María de las Mercedes Segura Abstract Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000. © 2005 Wiley Periodicals, Inc. [source] Fluid Mechanics, Cell Distribution, and Environment in Cell Cube BioreactorsBIOTECHNOLOGY PROGRESS, Issue 1 2003John G. Auni Cultivation of MRC-5 cells and attenuated hepatitis A virus (HAV) for the production of VAQTA, an inactivated HAV vaccine ( 1), is performed in the Cell Cube reactor, a laminar flow fixed-bed bioreactor with an unusual diamond-shaped, diverging-converging flow geometry. These disposable bioreactors have found some popularity for the production of cells and gene therapy vectors at intermediate scales of operation ( 2, 3). Early testing of the Cell Cube revealed that the fluid mechanical environment played a significant role in nonuniform cell distribution patterns generated during the cell growth phase. Specifically, the reactor geometry and manufacturing artifacts, in combination with certain inoculum practices and circulation flow rates, can create cell growth behavior that is not simply explained. Via experimentation and computational fluid dynamics simulations we can account for practically all of the observed cell growth behavior, which appears to be due to a complex mixture of flow distribution, particle deposition under gravity, fluid shear, and possibly nutritional microenvironment. [source] Immune responses to gene therapy vectors in the context of corneal transplantationACTA OPHTHALMOLOGICA, Issue 2009T RITTER Purpose The genetic engineering of grafts or cells prior to transplantation is an attractive approach to protect the graft from allogeneic rejection. Virus vector-based gene therapy is a promising method for successful ex-vivo gene transfer however, the induction of an immune response against gene-modified tissues raises concern. Methods Different virus families (Adenovirus, Retrovirus, Adeno-associated virus, Herpesvirus) have been studied as gene therapy vehicles for the delivery of therapeutic molecules. Moreover, different serotypes or envelope proteins have been used to modulate transduction efficiencies of target cells or to evade pre-existing immunity. Results Here we review gene therapeutic applications using viral vectors in the context of cornea transplantation. Both local and systemic expression of immunomodulatory molecules have led to the prevention of corneal graft rejection. However, different results have been obtained with regard to the induction of immune responses after local or systemic expression of the gene therapy vector. Not surprisingly over-expression of anti-inflammatory molecules not only modulated allograft rejection but also influenced the immune response against the viral vector and virally transduced cells. Conclusion Recent clinical trials indicate that the application of viral vectors in ophthalmology is promising however, the generation of immune responses against the viral vector or virally transduced cells are still a serious obstacle for a broader application of gene therapy. Supported by Deutsche Forschungsgemeinschaft (DFG Pl 150/14-1 and Ri 764/10-1) and Science Foundation of Ireland (SFI 06/RFP/BIC056 and SFI 07/IN.1/B925) [source] | |||||||||||||