The Experts below are selected from a list of 27999 Experts worldwide ranked by ideXlab platform
Richard A Morgan - One of the best experts on this subject based on the ideXlab platform.
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use of the piggybac transposon to create stable packaging cell lines for the production of clinical grade self inactivating γ Retroviral Vectors
Human Gene Therapy Methods, 2014Co-Authors: Steven A Feldman, Richard A Morgan, Mary A Black, Tristen S Park, Paul F Robbins, James N Kochenderfer, Steven A RosenbergAbstract:Efforts to improve the biosafety of γ-Retroviral-mediated gene therapy have resulted in a shift toward the use of self-inactivating (SIN) γ-Retroviral Vectors. However, scale-up and manufacturing of such Vectors requires significant optimization of transient transfection-based processes or development of novel platforms for the generation of stable producer cell clones. To that end, we describe the use of the piggybac transposon to generate stable producer cell clones for the production of SIN γ-Retroviral Vectors. The piggybac transposon is a universal tool allowing for the stable integration of SIN γ-Retroviral constructs into murine (PG13) and human 293-based Phoenix (GALV and RD114, respectively) packaging cell lines without reverse transcription. Following transposition, a high-titer clone is selected for manufacture of a master cell bank and subsequent γ-Retroviral vector supernatant production. Packaging cell clones created using the piggybac transposon have comparable titers to non-SIN Vectors generated via conventional methods. We describe herein the use of the piggybac transposon for the production of stable packaging cell clones for the manufacture of clinical-grade SIN γ-Retroviral Vectors for ex vivo gene therapy clinical trials.
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development of improved adenosine deaminase Retroviral Vectors
Journal of Virology, 1998Co-Authors: Masafumi Onodera, Richard A Morgan, David M Nelson, Akihiro Yachie, Jayashree G Jagadeesh, Bruce A Bunnell, Michael R BlaeseAbstract:A series of adenosine deaminase (ADA) Retroviral Vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a "simplified" vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA-) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA- severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA Retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.
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development and analysis of Retroviral Vectors expressing human factor viii as a potential gene therapy for hemophilia a
Human Gene Therapy, 1995Co-Authors: Marinee Chuah, Thierry Vandendriessche, Richard A MorganAbstract:ABSTRACT To develop a potential gene therapy strategy for the treatment of hemophilia A, we constructed several Retroviral Vectors expressing a B-domain-deleted factor VIII (FVIII) cDNA. We confirm...
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Retroviral Vectors containing putative internal ribosome entry sites development of a polycistronic gene transfer system and applications to human gene therapy
Nucleic Acids Research, 1992Co-Authors: Richard A Morgan, Larry A Couture, Orna Elroystein, Jack A Ragheb, Bernard Moss, French W AndersonAbstract:Recombinant Retroviral Vectors producing multicistronic mRNAs were constructed. Picornavirus putative internal ribosome entry sites (IRES) were used to confer cap-independent translation of an internal cistron. Internal cistrons were engineered by ligation of various lengths of the IRES of encephalomyocarditis (EMC) virus or polio virus to the E. coli chloramphenicol acetyltransferase (CAT) gene. The IRES/CAT fusions were introduced into Retroviral Vectors 3' to the translation stop codon of the neomycin phosphotransferase (NEO) gene, and the molecular constructs transfected into Retroviral vector packaging lines. Retroviral vector producer cells efficiently express the internal CAT gene product only when the full length IRES is used. Both the EMC/CAT and polio/CAT Retroviral Vectors produced high titer vector supernatant capable of productive transduction of target cells. To test the generality of this gene transfer system, a Retroviral vector containing an IRES fusion to the human adenosine deaminase (ADA) gene was constructed. Producer cell supernatant was used to transduce NIH/3T3 cells, and transduced cells were shown to express NEO, and ADA. Novel three-gene-containing Retroviral Vectors were constructed by introducing the EMC/ADA fusion into either an existing internal-promoter-containing vector, or a polio/CAT bicistronic vector. Producer cell clones of the three-gene Vectors synthesize all three gene products, were of high titer, and could productively transduce NIH/3T3 cells. By utilizing cap-independent translation units, IRES Vectors can produce polycistronic mRNAs which enhance the ability of Retroviral-mediated gene transfer to engineer cells to produce multiple foreign proteins.
Taiki Higashimoto - One of the best experts on this subject based on the ideXlab platform.
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the woodchuck hepatitis virus post transcriptional regulatory element reduces readthrough transcription from Retroviral Vectors
Gene Therapy, 2007Co-Authors: Taiki Higashimoto, Fabrizia Urbinati, Ajay Perumbeti, A Zarzuela, Lung Ji Chang, Donald B Kohn, G Jiang, Punam MalikAbstract:The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from Retroviral Vectors
Donald B Kohn - One of the best experts on this subject based on the ideXlab platform.
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the woodchuck hepatitis virus post transcriptional regulatory element reduces readthrough transcription from Retroviral Vectors
Gene Therapy, 2007Co-Authors: Taiki Higashimoto, Fabrizia Urbinati, Ajay Perumbeti, A Zarzuela, Lung Ji Chang, Donald B Kohn, G Jiang, Punam MalikAbstract:The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from Retroviral Vectors
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consistent persistent expression from modified Retroviral Vectors in murine hematopoietic stem cells
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Paul B Robbins, Dianne C Skelton, Stephanie Halene, Earl H Leonard, Donald B KohnAbstract:Retroviral Vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo. MoMuLV-based Vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified Retroviral Vectors and showed that they function significantly better than MoMuLV-based Vectors in vitro. To test the efficacy of the modified Vectors in hematopoietic stem cells in vivo, we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2°CFU-S). We found a significant increase in the frequency of expression with our modified Vectors (>90% expression in vector DNA containing 2°CFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2°CFU-S). Expression from the modified Vectors was highly consistent, with expression in >50% of the vector-containing 2°CFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0–10% of the vector containing 2°CFU-S from 8 recipients and expression in >50% of the vector-containing 2°CFU-S from 4 other recipients. In addition, we established that the modified Vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective Retroviral Vectors for application in vivo.
Punam Malik - One of the best experts on this subject based on the ideXlab platform.
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the woodchuck hepatitis virus post transcriptional regulatory element reduces readthrough transcription from Retroviral Vectors
Gene Therapy, 2007Co-Authors: Taiki Higashimoto, Fabrizia Urbinati, Ajay Perumbeti, A Zarzuela, Lung Ji Chang, Donald B Kohn, G Jiang, Punam MalikAbstract:The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from Retroviral Vectors
Thomas J Hope - One of the best experts on this subject based on the ideXlab platform.
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woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by Retroviral Vectors
Journal of Virology, 1999Co-Authors: Romain Zufferey, John Edward Donello, Didier Trono, Thomas J HopeAbstract:The expression of genes delivered by Retroviral Vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy. Here, we explored the possibility that the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) might help resolve this problem. Insertion of the WPRE in the 3′ untranslated region of coding sequences carried by either oncoRetroviral or lentiviral Vectors substantially increased their levels of expression in a transgene-, promoter- and vector-independent manner. The WPRE thus increased either luciferase or green fluorescent protein production five- to eightfold, and effects of a comparable magnitude were observed with either the immediate-early cytomegalovirus or the herpesvirus thymidine kinase promoter and with both human immunodeficiency virus- and murine leukemia virus-based Vectors. The WPRE exerted this influence only when placed in the sense orientation, consistent with its predicted posttranscriptional mechanism of action. These results demonstrate that the WPRE significantly improves the performance of Retroviral Vectors and emphasize that posttranscriptional regulation of gene expression should be taken into account in the design of gene delivery systems.
