- Email: [email protected]
465. In Vivo Selection of Genetically Modified Primary Human Hematopoietic Cells Using a Cell Growth Switch
GENE REGULATION: REGULATED SYSTEMS AND CELL ENGINEERING ml in H1, and 80% EGFP+ with hSF6. Higher viral concentrations generally did not increase the level of transduction, but in some experiments resulted in toxicity. We next optimized proviral transgene expression in hESC cells by comparing various vector backbones and cellular and viral promoters. The most efficient expression was observed from the minimal SMPU vector backbone compared to standard SIN lentiviral vector (CCL based). Moderate levels of EGFP expression (MFI: 250-400) were observed from most of the promoters tested (MND, ubiquitin, EF1a, and PGK), with the exception of the CMV promoter which had lower levels of expression (MFI<100). We next evaluated the stability of transgene expression over time post-transduction. Overall, the proportion of cells expressing EGFP decreased by 3-fold and the mean fluorescent intensity (MFI) per expressing cell decreased by half during culture. It is possible that the proportion of EGFP+ cells decreased due to their terminal differentiation or loss during culture; it is also possible that the decreased EGFP expression is due to vector gene silencing. Molecular studies to investigate these possibilities are on going. In the last set of experiments, we evaluated the potential of the Binterferon scaffold attachment region (SAR) to maintain high levels of transgene expression. Incorporation of the SAR did not result in a higher MFI of EGFP expression. However, at one month posttransduction, the proportion of EGFP+ was maintained, while the proportion of EGFP+ cells transduced with an identical vector without the SAR had decreased by approximately one-half. We are monitoring the long term effects of the B-SAR element on expression in hESC. In summary, we have optimized the conditions for efficient gene transfer into hESC, which will facilitate studies on the biology of hESC differentiation and development of stem cells as vehicles for gene delivery.
GENE REGULATION: REGULATED SYSTEMS AND CELL ENGINEERING 464. Development of a Novel Gene Regulation System Downstream of Promoter Hongzhe Duan,1 Chengwen Li,1 Richard Jude Samulski.1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.
1
Current regulation systems for vector delivered transgene has focused primarily on controlling promoter activity. In this study, we assayed the ability to regulate expression of transgene cassettes after vector delivery downstream of promoter activity through use of alternative splicing. A unique human beta globin intron carrying a point mutation causing aberrant splicing was engineered into the open reading frame of vector expressing reporter or therapeutic transgene cassettes under constitutively expressing CBA promoter. After introduction into target cells, vector derived mRNAs are aberrantly spliced and no functional proteins are produced. Administration of target antisense oligonucleotides (AON) specific for alternative splice site restores wild type splicing and functional protein expression. Using this approach, we transfected luciferase reporter transgene cassettes carrying IVS2-654 intron plasmids into 293 cells. 24 hr post transfection cell lysates were obtained and luciferase assays preformed. We observed 6-8 folds increase in luciferase activity after AON treatment equivalent to levels obtained with wile type intron. This restoration of correct splicing and protein expression was not dependent on location of IVS2-654 intron. Regulated expression in vivo was tested using AAV vector carry regulated transgene cassette in mouse model. One week after portal vein injection of 1010 genomes, 25mg/kg AON was administered IP. We observed an 8-10 folds increase over control that persisted for over 1 month before returning to base-line level. Long-term study shows AON regulation 1yr. post vector administration. To further S180
optimize this regulation system, we constructed a series of intron mutants that removed 250-400nt of 5’ sequences as well as engineered point mutations that would strengthen use of alternative splice sites. Results of these efforts resulted in mutant intron cassettes that reduced background expression dramatically and resulted in 160230 folds increase in protein expression after oligo treatment. In conclusion, we have successfully developed and tested in vitro and in vivo a novel regulation system dependent on alternative splicing for regulate gene expression. Obvious benefits are the fact that no foreign or additional proteins are required reducing the risk of immune response. More importantly, this cis-acting regulation system can be used in potentially any transgene or vector expression system to control protein expression with high efficiency.
465. In Vivo Selection of Genetically Modified Primary Human Hematopoietic Cells Using a Cell Growth Switch Linlin Wang,1 Yasuo Nagasawa,1 Brent Wood,2 Ingrid Lintmaer,1 Thalia Papayannopoulou,1 Michael Harkey,3 Cynthia Nougirat,3 C. Anthony Blau.1 1 Department of Medicine/Hematology, University of Washington, Seattle, WA; 2Department of Laboratory Medicine, University of Washington, Seattle, WA; 3Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA. Methods for regulating the growth of transplanted cells may have many applications in gene and cell therapy. One such method uses conditional signaling molecules that can be activated in response to artificial ligands called chemical inducers of dimerization (CIDs). Here we examine the response of human cord blood cells to a CIDtriggered growth signal, in vivo. CD34+ cells transduced with a lentivirus vector encoding a derivative of the thrombopoietin receptor (F36VMpl) and green fluorescent protein (GFP) were transplanted into immune deficient mice. CID treatment was associated with a >12 – fold expansion of GFP+ CD71+ erythroid cells that were localized mainly in the marrow, and a unique subset of multipotential and erythroid progenitors that were confined to the spleen. Both the CD71+ cells and the progenitor colonies were intensely GFP+. The CID response was oligoclonal, with most colonies containing a single transgene copy. Despite expressing F36Vmpl, GFP+ B lymphoid and myeloid cells showed no response. CID-responsive progenitors and CD71+ cells were detectable for up to 5 weeks following the end of CID; however these effects were not sustained in secondary transplant recipients. These findings establish CIDs as growth factors for genetically modified human hematopoietic cells. The University of Washington has submitted a patent application for this application. Any royalties accruing from this application may lead to income for Dr. Blau
466. Long-Term Rapamycin Control of Transgene Expression in Mouse Salivary Glands in a Single AAV Vector Jianghua Wang,1 Antonis Voutetakis,1 Milton Papa,2 Victor M. Rivera,3 Tim Clackson,3 Beatrijs M. Lodde,1 Fumi Mineshiba,1 Bruce J. Baum.1 1 Gene Therapy and Therapeutics Branch, NIDCR/National Institutes of Health, Bethesda, MD; 2ACU, NIDCR/National Institutes of Health, Bethesda, MD; 3Gene Therapeutics, ARIAD Pharmaceuticals, Cambridge, MA. Background: Salivary glands are a promising gene transfer platform for secretory therapeutic protein delivery (Baum et al., Trends Mol Med 2004; Zufferey and Aebischer, Gene Ther 2004). For ideal clinical application, therapeutic protein levels need to be controlled. We have shown, as proof-of-concept, that the rapamycinMolecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
GENE REGULATION: REGULATED SYSTEMS AND CELL ENGINEERING 464. Development of a Novel Gene Regulation System Downstream of Promoter Hongzhe Duan,1 Chengwen Li,1 Richard Jude Samulski.1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.
1
Current regulation systems for vector delivered transgene has focused primarily on controlling promoter activity. In this study, we assayed the ability to regulate expression of transgene cassettes after vector delivery downstream of promoter activity through use of alternative splicing. A unique human beta globin intron carrying a point mutation causing aberrant splicing was engineered into the open reading frame of vector expressing reporter or therapeutic transgene cassettes under constitutively expressing CBA promoter. After introduction into target cells, vector derived mRNAs are aberrantly spliced and no functional proteins are produced. Administration of target antisense oligonucleotides (AON) specific for alternative splice site restores wild type splicing and functional protein expression. Using this approach, we transfected luciferase reporter transgene cassettes carrying IVS2-654 intron plasmids into 293 cells. 24 hr post transfection cell lysates were obtained and luciferase assays preformed. We observed 6-8 folds increase in luciferase activity after AON treatment equivalent to levels obtained with wile type intron. This restoration of correct splicing and protein expression was not dependent on location of IVS2-654 intron. Regulated expression in vivo was tested using AAV vector carry regulated transgene cassette in mouse model. One week after portal vein injection of 1010 genomes, 25mg/kg AON was administered IP. We observed an 8-10 folds increase over control that persisted for over 1 month before returning to base-line level. Long-term study shows AON regulation 1yr. post vector administration. To further S180
optimize this regulation system, we constructed a series of intron mutants that removed 250-400nt of 5’ sequences as well as engineered point mutations that would strengthen use of alternative splice sites. Results of these efforts resulted in mutant intron cassettes that reduced background expression dramatically and resulted in 160230 folds increase in protein expression after oligo treatment. In conclusion, we have successfully developed and tested in vitro and in vivo a novel regulation system dependent on alternative splicing for regulate gene expression. Obvious benefits are the fact that no foreign or additional proteins are required reducing the risk of immune response. More importantly, this cis-acting regulation system can be used in potentially any transgene or vector expression system to control protein expression with high efficiency.
465. In Vivo Selection of Genetically Modified Primary Human Hematopoietic Cells Using a Cell Growth Switch Linlin Wang,1 Yasuo Nagasawa,1 Brent Wood,2 Ingrid Lintmaer,1 Thalia Papayannopoulou,1 Michael Harkey,3 Cynthia Nougirat,3 C. Anthony Blau.1 1 Department of Medicine/Hematology, University of Washington, Seattle, WA; 2Department of Laboratory Medicine, University of Washington, Seattle, WA; 3Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA. Methods for regulating the growth of transplanted cells may have many applications in gene and cell therapy. One such method uses conditional signaling molecules that can be activated in response to artificial ligands called chemical inducers of dimerization (CIDs). Here we examine the response of human cord blood cells to a CIDtriggered growth signal, in vivo. CD34+ cells transduced with a lentivirus vector encoding a derivative of the thrombopoietin receptor (F36VMpl) and green fluorescent protein (GFP) were transplanted into immune deficient mice. CID treatment was associated with a >12 – fold expansion of GFP+ CD71+ erythroid cells that were localized mainly in the marrow, and a unique subset of multipotential and erythroid progenitors that were confined to the spleen. Both the CD71+ cells and the progenitor colonies were intensely GFP+. The CID response was oligoclonal, with most colonies containing a single transgene copy. Despite expressing F36Vmpl, GFP+ B lymphoid and myeloid cells showed no response. CID-responsive progenitors and CD71+ cells were detectable for up to 5 weeks following the end of CID; however these effects were not sustained in secondary transplant recipients. These findings establish CIDs as growth factors for genetically modified human hematopoietic cells. The University of Washington has submitted a patent application for this application. Any royalties accruing from this application may lead to income for Dr. Blau
466. Long-Term Rapamycin Control of Transgene Expression in Mouse Salivary Glands in a Single AAV Vector Jianghua Wang,1 Antonis Voutetakis,1 Milton Papa,2 Victor M. Rivera,3 Tim Clackson,3 Beatrijs M. Lodde,1 Fumi Mineshiba,1 Bruce J. Baum.1 1 Gene Therapy and Therapeutics Branch, NIDCR/National Institutes of Health, Bethesda, MD; 2ACU, NIDCR/National Institutes of Health, Bethesda, MD; 3Gene Therapeutics, ARIAD Pharmaceuticals, Cambridge, MA. Background: Salivary glands are a promising gene transfer platform for secretory therapeutic protein delivery (Baum et al., Trends Mol Med 2004; Zufferey and Aebischer, Gene Ther 2004). For ideal clinical application, therapeutic protein levels need to be controlled. We have shown, as proof-of-concept, that the rapamycinMolecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy