The Experts below are selected from a list of 2823 Experts worldwide ranked by ideXlab platform
Alan J Kingsman - One of the best experts on this subject based on the ideXlab platform.
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minimal requirement for a Lentivirus Vector based on human immunodeficiency virus type 1
Journal of Virology, 1998Co-Authors: Kyriacos A Mitrophanous, Susan M Kingsman, Alan J KingsmanAbstract:The use of human immunodeficiency virus Vectors for gene therapy is hampered by concern over their safety. This concern might be ameliorated, in part, if the viral accessory genes and proteins could be eliminated from the Vector genomes and particles. Here we describe a minimal Vector system that is capable of transducing nondividing cells and which does not contain tat, vif, vpr, vpu, and nef.
Didier Trono - One of the best experts on this subject based on the ideXlab platform.
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conditional suppression of cellular genes Lentivirus Vector mediated drug inducible rna interference
Journal of Virology, 2003Co-Authors: Maciej Wiznerowicz, Didier TronoAbstract:RNA interference has emerged as a powerful technique to downregulate the expression of specific genes in cells and in animals, thus opening new perspectives in fields ranging from developmental genetics to molecular therapeutics. Here, we describe a method that significantly expands the potential of RNA interference by permitting the conditional suppression of genes in mammalian cells. Within a Lentivirus Vector background, we subjected the polymerase III promoter-dependent production of small interfering RNAs to doxycycline-controllable transcriptional repression. The resulting system can achieve the highly efficient and completely drug-inducible knockdown of cellular genes. As Lentivirus Vectors can stably transduce a wide variety of targets both in vitro and in vivo and can be used to generate transgenic animals, the present system should have broad applications.
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Lentivirus Vector gene expression during es cell derived hematopoietic development in vitro
Journal of Virology, 2000Co-Authors: Isao Hamaguchi, Niels Bjarne Woods, Elisabet Andersson, Cecilia Fahlman, Leif Carlsson, Rachel Zufferey, Ioannis Panagopoulos, Hanna K A Mikkola, Didier Trono, Stefan KarlssonAbstract:The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based Lentivirus Vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus Vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped Lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus Vectors leads to efficient transduction of ES cells. Lentivirus Vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many Lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze Lentivirus Vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.
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Self-inactivating Lentivirus Vector for safe and efficient in vivo gene delivery.
Journal of virology, 1998Co-Authors: Rachel Zufferey, T Dull, Dorys Quiroz, Anatoly Bukovsky, Ronald J Mandel, Luigi Naldini, Didier TronoAbstract:In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based Vectors results in transgene expression that is stable over several months. However, the use of HIV-1 Vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 Vector with a 400-nucleotide deletion in the 3' long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect Vector titers or transgene expression in vitro. The self-inactivating Vector transduced neurons in vivo as efficiently as a Vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived Vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the Vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the Vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable Vectors.
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A third-generation Lentivirus Vector with a conditional packaging system.
Journal of virology, 1998Co-Authors: T Dull, Rachel Zufferey, M Nguyen, Didier Trono, Ronald J Mandel, M. Kelly, Luigi NaldiniAbstract:Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide Lentivirus Vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type 1 accessory open reading frames are dispensable for efficient gene transduction by a Lentivirus Vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the Vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5' LTR chimeric transfer construct, we achieved optimal yields of Vector of high transducing efficiency (up to 10(7) transducing units [TU]/ml and 10(4) TU/ng of p24). This third-generation Lentivirus Vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the Vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the Vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of Lentivirus Vectors.
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highly efficient and sustained gene transfer in adult neurons with a Lentivirus Vector
Journal of Virology, 1997Co-Authors: Ulrike Blomer, Tal Kafri, Didier Trono, Luigi Naldini, Inder M. Verma, Fred H GageAbstract:The identification of monogenic and complex genes responsible for neurological disorders requires new approaches for delivering therapeutic protein genes to significant numbers of cells in the central nervous system. A Lentivirus-based Vector capable of infecting dividing and quiescent cells was investigated in vivo by injecting highly concentrated viral Vector stock into the striatum and hippocampus of adult rats. Control brains were injected with a Moloney murine leukemia virus, adenovirus, or adeno-associated virus Vector. The volumes of the areas containing transduced cells and the transduced-cell densities were stereologically determined to provide a basis for comparison among different viral Vectors and variants of the viral Vector stocks. The efficiency of infection by the Lentivirus Vector was improved by deoxynucleoside triphosphate pretreatment of the Vector and was reduced following mutation of integrase and the Vpr-matrix protein complex involved in the nuclear translocation of the preintegration complex. The Lentivirus Vector system was able to efficiently and stably infect quiescent cells in the primary injection site with transgene expression for over 6 months. Triple labeling showed that 88.7% of striatal cells transduced by the Lentivirus Vector were terminally differentiated neurons.
Inder M. Verma - One of the best experts on this subject based on the ideXlab platform.
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targeted delivery of proteins across the blood brain barrier
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Brian Spencer, Inder M. VermaAbstract:Treatment of many neuronal degenerative disorders will require delivery of a therapeutic protein to neurons or glial cells across the whole CNS. The presence of the blood–brain barrier hampers the delivery of these proteins from the blood, thus necessitating a new method for delivery. Receptors on the blood–brain barrier bind ligands to facilitate their transport to the CNS; therefore, we hypothesized that by targeting these receptors, we may be able to deliver proteins to the CNS for therapy. Here, we report the use of the Lentivirus Vector system to deliver the lysosomal enzyme glucocerebrosidase and a secreted form of GFP to the neurons and astrocytes in the CNS. We fused the low-density lipoprotein receptor-binding domain of the apolipoprotein B to the targeted protein. This approach proved to be feasible for delivery of the protein and could possibly be used as a general method for delivery of therapeutic proteins to the CNS.
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generation of a stable cell line producing high titer self inactivating lentiviral Vectors
Molecular Therapy, 2001Co-Authors: Kailin Xu, Thomas J. Mccown, Inder M. Verma, Tal KafriAbstract:Abstract To facilitate the generation of SIN Lentivirus Vector-producer cell lines, we have developed a novel conditional SIN (cSIN) Lentivirus Vector, which retains its SIN properties in normal target cells yet can be produced at high titers from tetracycline-regulated packaging cell lines. The design of the cSIN Vector is based on replacing the Vector U3 transcription regulatory elements with the Tet-responsive element, which allows Vector production exclusively in cells expressing the synthetic Tet-regulated transactivator (tTA). In contrast minimal Vector production (∼200 IU/ml) is obtained in target cells that do not express the tTA, even in the presence of all HIV-1 proteins. Following transduction of the Tet-regulated SODk1 Lentivirus Vector-packaging cell line with the cSIN Vector, high titers of cSIN recombinant Vector (>10 6 IU/ml) could be generated, which efficiently transduced terminally differentiated neurons in normal rat brain.
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development of a self inactivating Lentivirus Vector
Journal of Virology, 1998Co-Authors: Hiroyuki Miyoshi, Ulrike Blomer, Fred H Gage, Masayo Takahashi, Inder M. VermaAbstract:We have constructed a new series of Lentivirus Vectors based on human immunodeficiency virus type 1 (HIV-1) that can transduce nondividing cells. The U3 region of the 5′ long terminal repeat (LTR) in Vector constructs was replaced with the cytomegalovirus (CMV) promoter, resulting in Tat-independent transcription but still maintaining high levels of expression. A self-inactivating (SIN) Vector was constructed by deleting 133 bp in the U3 region of the 3′ LTR, including the TATA box and binding sites for transcription factors Sp1 and NF-κB. The deletion is transferred to the 5′ LTR after reverse transcription and integration in infected cells, resulting in the transcriptional inactivation of the LTR in the proviruses. SIN viruses can be generated with no significant decreases in titer. Injection of viruses into the rat brain showed that a SIN Vector containing the green fluorescent protein gene under the control of the internal CMV promoter transduced neurons as efficiently as a wild-type Vector. Interestingly, a wild-type Vector without an internal promoter also successfully transduced neurons in the brain, indicating that the HIV-1 LTR promoter is transcriptionally active in neurons even in the absence of Tat. Furthermore, injection of viruses into the subretinal space of the rat eye showed that wild-type Vector transduced predominantly retinal pigment epithelium and photoreceptor cells, while SIN Vector was able to transduce other types of retinal cells, including bipolar, Muller, horizontal, and amacrine cells. This finding suggests that the HIV-1 LTR can negatively influence the internal CMV promoter in some cell types. SIN HIV Vectors should be safer for gene therapy, and they also have broader applicability as a means of high-level gene transfer and expression in nondividing cells.
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highly efficient and sustained gene transfer in adult neurons with a Lentivirus Vector
Journal of Virology, 1997Co-Authors: Ulrike Blomer, Tal Kafri, Didier Trono, Luigi Naldini, Inder M. Verma, Fred H GageAbstract:The identification of monogenic and complex genes responsible for neurological disorders requires new approaches for delivering therapeutic protein genes to significant numbers of cells in the central nervous system. A Lentivirus-based Vector capable of infecting dividing and quiescent cells was investigated in vivo by injecting highly concentrated viral Vector stock into the striatum and hippocampus of adult rats. Control brains were injected with a Moloney murine leukemia virus, adenovirus, or adeno-associated virus Vector. The volumes of the areas containing transduced cells and the transduced-cell densities were stereologically determined to provide a basis for comparison among different viral Vectors and variants of the viral Vector stocks. The efficiency of infection by the Lentivirus Vector was improved by deoxynucleoside triphosphate pretreatment of the Vector and was reduced following mutation of integrase and the Vpr-matrix protein complex involved in the nuclear translocation of the preintegration complex. The Lentivirus Vector system was able to efficiently and stably infect quiescent cells in the primary injection site with transgene expression for over 6 months. Triple labeling showed that 88.7% of striatal cells transduced by the Lentivirus Vector were terminally differentiated neurons.
Kenneth Cornetta - One of the best experts on this subject based on the ideXlab platform.
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Lentivirus mediated correction of artemis deficient severe combined immunodeficiency
Human Gene Therapy, 2017Co-Authors: Divya Punwani, Misako Kawahara, Ukina Sanford, Kiran Patel, Denise A Carbonaro, Andrea D Karlen, Sara Khan, Kenneth Cornetta, Jason Yu, Michael RotheAbstract:During B and T lymphocyte maturation, V(D)J recombination is initiated by creation of DNA double-strand breaks. Artemis is an exonuclease essential for their subsequent repair by nonhomologous end-joining. Mutations in DCLRE1C, the gene encoding Artemis, cause T−B−NK+ severe combined immunodeficiency (ART-SCID) and also confer heightened sensitivity to ionizing radiation and alkylating chemotherapy. Although allogeneic hematopoietic cell transplantation can treat ART-SCID, conditioning regimens are poorly tolerated, leading to early mortality and/or late complications, including short stature, endocrinopathies, and dental aplasia. However, without alkylating chemotherapy as preconditioning, patients usually have graft rejection or limited T cell and no B cell recovery. Thus, addition of normal DCLRE1C cDNA to autologous hematopoietic stem cells is an attractive strategy to treat ART-SCID. We designed a self-inactivating Lentivirus Vector containing human Artemis cDNA under transcriptional regulation of th...
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human immunodeficiency virus type 1 derived Lentivirus Vectors pseudotyped with envelope glycoproteins derived from ross river virus and semliki forest virus
Journal of Virology, 2004Co-Authors: Christoph Kahl, Jon Marsh, Joanne Fyffe, David Avram Sanders, Kenneth CornettaAbstract:Ross River virus (RRV) and Semliki Forest virus (SFV) are two alphaviruses that have a high degree of amino acid homology, as well as a very broad host range. We show here that envelope glycoproteins derived from both viruses can pseudotype human immunodeficiency virus type 1 (HIV-1)-derived Lentivirus Vectors. Both RRV and SFV glycoproteins considerably expand the host range of the Lentivirus Vector, and Vectors can be efficiently concentrated by ultracentrifugation. A systematic analysis comparing the alphaviral glycoproteins to the vesicular stomatitis virus glycoprotein (VSV-G) revealed that Lentivirus Vectors incorporate RRV glycoproteins with an efficiency comparable to that of VSV-G. Both pseudotypes have comparable physical titers, but infectious titers with the RRV pseudotype are lower than with VSV-G. Incorporation of SFV glycoproteins into Lentivirus Vector is less efficient, leading to decreased physical and infectious titers. The transduction rates with VSV-G-, RRV-, and SFV-pseudotyped Lentivirus Vectors into adherent cell lines can be significantly increased by using a combination of Polybrene and plates coated with CH-296 recombinant fibronectin fragments. Together, our data suggest that RRV and SFV glycoproteins might be suitable as alternatives to VSV-G for pseudotyping Lentivirus Vectors.
Robert E Donahue - One of the best experts on this subject based on the ideXlab platform.
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fibronectin fragment ch 296 inhibits apoptosis and enhances ex vivo gene transfer by murine retrovirus and human Lentivirus Vectors independent of viral tropism in nonhuman primate cd34 cells
Molecular Therapy, 2001Co-Authors: Robert E Donahue, Irvin S Y Chen, Brian P Sorrentino, Robert G Hawley, Robert P WerstoAbstract:The fibronectin fragment CH-296 improved gene transfer to cytokine-mobilized nonhuman primate CD34+ cells irrespective of tropism to the MoMLV, GaLV, and VSV-G envelope proteins using murine stem cell virus (MSCV) and human immunodeficiency virus-1 (HIV-1)-based retrovirus Vectors. For the HIV-1 Lentivirus Vector, CH-296 enhanced gene transfer in the absence of added hematopoietic growth factors necessary for ex vivo stem cell expansion. In the presence of CH-296, apoptosis of CD34+ cells was inhibited, and in mobilized peripheral blood CD34+ cells, cell division was stimulated as measured by cell history/tracking experiments.
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marking and gene expression by a Lentivirus Vector in transplanted human and nonhuman primate cd34 cells
Journal of Virology, 2000Co-Authors: Dong Sung An, Robert P Wersto, Brian A Agricola, Mark E Metzger, Stephanie Lu, Rafael G Amado, Irvin S Y Chen, Robert E DonahueAbstract:Recently, gene delivery Vectors based on human immunodeficiency virus (HIV) have been developed as an alternative mode of gene delivery. These Vectors have a number of advantages, particularly in regard to the ability to infect cells which are not actively dividing. However, the use of Vectors based on human immunodeficiency virus raises a number of issues, not the least of which is safety; therefore, further characterization of marking and gene expression in different hematopoietic lineages in primate animal model systems is desirable. We use two animal model systems for gene therapy to test the efficiency of transduction and marking, as well as the safety of these Vectors. The first utilizes the rhesus animal model for cytokine-mobilized autologous peripheral blood CD34+ cell transplantation. The second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal model useful specifically for human T-lymphoid progenitor cell reconstitution. In the rhesus macaques, detectable levels of Vector were observed in granulocytes, lymphocytes, monocytes, and, in one animal with the highest levels of marking, erythrocytes and platelets. In transplanted SCID-hu mice, we directly compared marking and gene expression of the Lentivirus Vector and a murine leukemia virus-derived Vector in thymocytes. Marking was observed at comparable levels, but the Lentivirus Vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral Vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in either animal model system. Thus, these results indicate that while Lentivirus Vectors have no apparent deleterious effects and may have advantages over murine retroviral Vectors, further study of the requirements for optimal use are warranted.