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436. Novel SIN Lentiviral Vector Producer Cell Lines Constructed with SIN γ-Retroviral Vectors Yield High Titer Product Which Efficiently Transduces Human CD34+ NOG Repopulating Stem Cells
Cell Processing and Vector Production This observed toxicity was dependent on delivery of specific shRNA expression cassettes, since vectors expressing a reporter gene alone displayed no toxicity. We attempted to eliminate toxicity by shortening the shRNA recognition sequence from 21 to 19 nucleotides and observed improved cell survival in the liver and heart by histological examination at 2 and 6-week timepoints with no fatalities at 3 months weeks after IV injection of the AAV6 lacZ 19mer shRNA. We applied the AAV6 shRNA delivery approach to a mouse model of myotonic dystrophy (DM1). DM1 is caused by expression of an expanded CAG repeat from the 3’ UTR of the dystrophia myotonica protein kinase gene, DMPK. Trangenic mice expressing 250 repeats from the 3’ UTR of the α-skeletal actin gene (HSALR) display myotonia (delayed release following contraction) in their skeletal muscles and have similar features of the human disease including changes in splicing of many genes such as the chloride channel gene 1 (CLCN1) and the skeletal muscle Ca2+ pump (SERCA). We designed shRNAs targeting the human transgene HSA mRNA to reduce expression of the disease-causing allele in the HSALR mouse. Two HSA shRNA vectors tested by intramuscular injection decreased the frequency of missplicing events, a hallmark of the disease. Splicing studies of mRNA from quadriceps muscle following systemic delivery of one of these AAV6 HSA shRNAs resulted in a decrease in missplicing events in the quadriceps muscle. The splicing changes occurred even with a low level of transduction as indicated by histological staining for reporter gene expression and further studies are in progress. The ability to affect mRNA levels with efficient, widespread delivery of sequences that can direct RNAi is a potentially powerful gene therapy approach for treatment of dominant genetic disease.
Cell Processing and Vector Production 436. Novel SIN Lentiviral Vector Producer Cell Lines Constructed with SIN γ-Retroviral Vectors Yield High Titer Product Which Efficiently Transduces Human CD34+ NOG Repopulating Stem Cells
Robert E. Throm,1 Annastasia Ouma,1 Anantharaman Chandrasekaran,1 Phillip W. Hargrove,1 Yoon Sang Kim,1 Arthur W. Nienhuis,1 Derek A. Persons,1 John T. Gray.1 1 Hematology, St. Jude Children’s Research Hospital, Memphis, TN. Lentiviral vectors have shown significant promise in pre-clinical gene therapy applications. However, currently available producer cell lines typically do not provide titers comparable to those obtained using transient transfection of 293T cells, requiring clinical investigators to use the more cumbersome transfection method. We endeavored to construct HIV-based producer cell lines that would provide broad utility for both research purposes and clinical applications. The HIV gagpol, rev, and tat genes were codon optimized and inserted into self-inactivating (SIN) MLV-based vectors. The gagpol vector used a CMV promoter, while the rev, tat, and a VSV-G expressing vector all used doxycycline repressible promoters. Beginning with a well characterized 293T master cell bank, cell lines were derived with integrated HIV gagpol and rev genes (GPR), gagpol, rev and tat genes (GPRT), or gagpol, rev and VSV glycoprotein G genes (GPRG). GFP expressing vector genomes were introduced by transduction with HIV vectors with intact 3’ U3 regions, or calcium phosphate transfection of SIN HIV vector fragments excised from plasmids and ligated to selectable marker cassettes. These cell lines all produced vector with unconcentrated titers comparable to those produced by 4 plasmid transfection of 293T cells, (2-5 x 107 HeLa transducing units/ml, tu/ml), either after transfection of the remaining genetic components, or in the case of full producer cell lines, upon simple induction by removal of doxycycline. Under optimized conditions, several lines produced unconcentrated vector titers near 108 tu/ml, including a producer clone for a non-mobilizable SIN vector. Vector Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
produced from GPR based cell lines showed a 300-fold reduction in contaminating gag sequences relative to 293T transfection products, as determined by real time PCR. The yield of infectious vector particles from the GPR cell line was not affected by over one year of continuous passage in the repressed state, demonstrating robust stability. For the VSV-G containing lines, passage for 2-3 months did reduce potency, although with careful maintenance induced titers could be maintained above 2 x 107 tu/ml. As a validation of the suitability of these cells for production of clinical vector preparations, human CD34+ peripheral blood cells were transduced and transplanted into non-obese, diabetic IL2RG -/- (NOG) mice. Unconcentrated vector with Hela titer of ~7 x 107 tu/ml was produced in serum free medium by transfection of a VSV-G expression plasmid into a GPRT cell line containing an integrated GFP expressing vector genome. Human CD34+ cells transduced at a range of MOIs (from 10-68) engrafted with similar efficiency (1-20%) as those of mock transduced cells, and up to 60% of the engrafted human cells expressed GFP 5 months after transplantation. The assortment of cell lines generated should greatly facilitate large scale production of clinical lentiviral vectors, both by transfection methods and for the creation of stable producer cell lines.
437. Production of High Titer GMP-Grade Retroviral Vectors by Transfection in a Closed System Bioreactor
Johannes C. M. van der Loo,1 William P. Swaney,1 Travis E. Zeigler,1 Ami R. Terwilliger,1 Diana L. Nordling,1 Axel Schambach,2 Christopher Baum,2 David A. Williams,3 Punam Malik,1 Lilith Reeves.1 1 Division of Experimental Hematology, Cincinnati Children’s Research Foundation, Cincinnati, OH; 2Department of Experimental Hematology, Hannover Medical School, Hannover, Germany; 3Division of Hematology/Oncology, Children’s Hospital Boston, Boston, MA. The adverse events of insertional mutagenesis in a SCID-X1 clinical trial have led to the development of safety-enhanced gammaretroviral vectors for clinical use. One of the modifications being studied is the use of a self-inactivating (SIN) long-terminal repeat (LTR). This vector configuration has prompted a shift in the method with which large scale clinical-grade vectors are produced, from the use of stable producer lines to transient transfection-based techniques. The main challenge of instituting this methodology was to develop SIN vector plasmids that produce high amounts of genomic vector RNA in packaging cells, and to design a scalable process with closed system processing compatible with large scale transfection and virus harvest. Using improved expression plasmids, the Cincinnati Children’s Research Foundation (CCRF) Vector Production Facility (VPF), an academic GMP manufacturing laboratory, has developed such a method based on the Wave Bioreactor® production platform. In this modified protocol, cells from a certified 293T master cell bank are expanded, mixed with transfection reagents, and pumped into a 2, 10 or 20 Liter Wave CellBag containing FibraCel® discs. Cells are cultured in DMEM with GlutaMax® and 10% FBS at 37°C, 5% CO2 at a rocking speed of 22 rpm and 6° angle. At 16-20 hrs post-transfection, the media is changed and virus is harvested at three 12-hour intervals, filtered through a leukocyte reduction filter, aliquoted and frozen at -70°C in Cryocyte freezing containers. Of the parameters tested, the timing of transfection, the amount of plasmid DNA, and the temperature all greatly affected vector titer. Mixing cells with plasmid and transfection mixture prior to seeding onto FibraCel, as compared to transfecting cells 1-day post-seeding as is standard in tissue culture plastic, increased the titer at least 40-fold from < 104 to 4 x 105 IU/mL. Similarly, increasing the amount of plasmid DNA per mL from 4.6 to 9.2 µg doubled the titer in the Wave, while it reduced titer by 20-40% in tissue culture flasks (Figure S165
Cell Processing and Vector Production 436. Novel SIN Lentiviral Vector Producer Cell Lines Constructed with SIN γ-Retroviral Vectors Yield High Titer Product Which Efficiently Transduces Human CD34+ NOG Repopulating Stem Cells
Robert E. Throm,1 Annastasia Ouma,1 Anantharaman Chandrasekaran,1 Phillip W. Hargrove,1 Yoon Sang Kim,1 Arthur W. Nienhuis,1 Derek A. Persons,1 John T. Gray.1 1 Hematology, St. Jude Children’s Research Hospital, Memphis, TN. Lentiviral vectors have shown significant promise in pre-clinical gene therapy applications. However, currently available producer cell lines typically do not provide titers comparable to those obtained using transient transfection of 293T cells, requiring clinical investigators to use the more cumbersome transfection method. We endeavored to construct HIV-based producer cell lines that would provide broad utility for both research purposes and clinical applications. The HIV gagpol, rev, and tat genes were codon optimized and inserted into self-inactivating (SIN) MLV-based vectors. The gagpol vector used a CMV promoter, while the rev, tat, and a VSV-G expressing vector all used doxycycline repressible promoters. Beginning with a well characterized 293T master cell bank, cell lines were derived with integrated HIV gagpol and rev genes (GPR), gagpol, rev and tat genes (GPRT), or gagpol, rev and VSV glycoprotein G genes (GPRG). GFP expressing vector genomes were introduced by transduction with HIV vectors with intact 3’ U3 regions, or calcium phosphate transfection of SIN HIV vector fragments excised from plasmids and ligated to selectable marker cassettes. These cell lines all produced vector with unconcentrated titers comparable to those produced by 4 plasmid transfection of 293T cells, (2-5 x 107 HeLa transducing units/ml, tu/ml), either after transfection of the remaining genetic components, or in the case of full producer cell lines, upon simple induction by removal of doxycycline. Under optimized conditions, several lines produced unconcentrated vector titers near 108 tu/ml, including a producer clone for a non-mobilizable SIN vector. Vector Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
produced from GPR based cell lines showed a 300-fold reduction in contaminating gag sequences relative to 293T transfection products, as determined by real time PCR. The yield of infectious vector particles from the GPR cell line was not affected by over one year of continuous passage in the repressed state, demonstrating robust stability. For the VSV-G containing lines, passage for 2-3 months did reduce potency, although with careful maintenance induced titers could be maintained above 2 x 107 tu/ml. As a validation of the suitability of these cells for production of clinical vector preparations, human CD34+ peripheral blood cells were transduced and transplanted into non-obese, diabetic IL2RG -/- (NOG) mice. Unconcentrated vector with Hela titer of ~7 x 107 tu/ml was produced in serum free medium by transfection of a VSV-G expression plasmid into a GPRT cell line containing an integrated GFP expressing vector genome. Human CD34+ cells transduced at a range of MOIs (from 10-68) engrafted with similar efficiency (1-20%) as those of mock transduced cells, and up to 60% of the engrafted human cells expressed GFP 5 months after transplantation. The assortment of cell lines generated should greatly facilitate large scale production of clinical lentiviral vectors, both by transfection methods and for the creation of stable producer cell lines.
437. Production of High Titer GMP-Grade Retroviral Vectors by Transfection in a Closed System Bioreactor
Johannes C. M. van der Loo,1 William P. Swaney,1 Travis E. Zeigler,1 Ami R. Terwilliger,1 Diana L. Nordling,1 Axel Schambach,2 Christopher Baum,2 David A. Williams,3 Punam Malik,1 Lilith Reeves.1 1 Division of Experimental Hematology, Cincinnati Children’s Research Foundation, Cincinnati, OH; 2Department of Experimental Hematology, Hannover Medical School, Hannover, Germany; 3Division of Hematology/Oncology, Children’s Hospital Boston, Boston, MA. The adverse events of insertional mutagenesis in a SCID-X1 clinical trial have led to the development of safety-enhanced gammaretroviral vectors for clinical use. One of the modifications being studied is the use of a self-inactivating (SIN) long-terminal repeat (LTR). This vector configuration has prompted a shift in the method with which large scale clinical-grade vectors are produced, from the use of stable producer lines to transient transfection-based techniques. The main challenge of instituting this methodology was to develop SIN vector plasmids that produce high amounts of genomic vector RNA in packaging cells, and to design a scalable process with closed system processing compatible with large scale transfection and virus harvest. Using improved expression plasmids, the Cincinnati Children’s Research Foundation (CCRF) Vector Production Facility (VPF), an academic GMP manufacturing laboratory, has developed such a method based on the Wave Bioreactor® production platform. In this modified protocol, cells from a certified 293T master cell bank are expanded, mixed with transfection reagents, and pumped into a 2, 10 or 20 Liter Wave CellBag containing FibraCel® discs. Cells are cultured in DMEM with GlutaMax® and 10% FBS at 37°C, 5% CO2 at a rocking speed of 22 rpm and 6° angle. At 16-20 hrs post-transfection, the media is changed and virus is harvested at three 12-hour intervals, filtered through a leukocyte reduction filter, aliquoted and frozen at -70°C in Cryocyte freezing containers. Of the parameters tested, the timing of transfection, the amount of plasmid DNA, and the temperature all greatly affected vector titer. Mixing cells with plasmid and transfection mixture prior to seeding onto FibraCel, as compared to transfecting cells 1-day post-seeding as is standard in tissue culture plastic, increased the titer at least 40-fold from < 104 to 4 x 105 IU/mL. Similarly, increasing the amount of plasmid DNA per mL from 4.6 to 9.2 µg doubled the titer in the Wave, while it reduced titer by 20-40% in tissue culture flasks (Figure S165