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100. Development of an Integrase Deficient FIV Vector for Transient Gene Expression
RNA VIRUS VECTORS: GENE EXPRESSION previously (Cell Transplantation 13:197-211, 2004) were transduced with this lentivirus at different time points after the six-day old embryoid bodies were plated. Undifferentiated hESC were transduced at day 7. Flow cytometry analysis was performed for detection of GFP-positive cells after the transductions. Results and Conclusions: The relative GFP expression by quantitative RTPCR in hepatoma cells was 1.55 ± 0.54 and in non-hepatoma cells was 0.12 ± 0.01. These results indicated that GFP expression driven by the α1AT promoter was significantly higher in hepatoma cells than in non-hepatoma cells. Quantitative PCR showed that the relative copy number of HIV gag sequence from proviral DNA integration into the host genome was 2.6 ± 0.96 in hepatoma cells and was 15.7 ± 11.7 in non-hepatoma cells. These data demonstrate that high GFP expression in the hepatoma cells was not due to higher transduction efficiency. They suggest that transduction with this lentivirus vector containing the GFP marker gene can be employed to demonstrate liver-specific gene expression in transduced cells. Flow cytometry analysis showed that the percentage of GFP-positive cells was 1.37% in transduced, undifferentiated hESC, and 19.7 ± 5.03% in transduced, differentiated hESC at different time points in differentiation culture, and 0.68 ± 0.18% in untransduced, differentiated hESC. The results suggest that the use of the liverspecific lentivirus vector may well be effective in enhancing the purity of our hepatocyte-like, differentiated hESC.
100. Development of an Integrase Deficient FIV Vector for Transient Gene Expression Marisa Banasik,1 Melissa Hickey,2 Paul McCray Jr.1,2 Interdisciplinary Program in Genetics, University of Iowa, Iowa City, IA; 2Department of Pediatrics, University of Iowa, Iowa City, IA. 1
Lentiviral vectors mediate long term transgene expression through integration into host genomic DNA. However, integration comes with potential risks of insertional mutagenesis. In cases where transient gene expression is desired, an integrase (IN) deficient retroviral vector might be a useful alternative. Furthermore, IN deficient vectors may provide some advantages over alternative methods of transient gene expression due to the ability to pseudotype these vectors with heterologous envelopes that target specific cell types. Class I IN mutants have point mutations in the catalytic triad amino acids (DX39-58 DX35 E) of IN that disable this protein but leave other viral proteins functional. Transient gene expression from IN deficient vectors is mediated from episomal forms of vector DNA. 1-LTR and 2-LTR circles form through homologous recombination and non-homologous end joining, respectively, of the linear form of the reverse transcribed viral DNA. Although 1-LTR and 2-LTR circles are unable to efficiently integrate in the presence of functional IN protein, they can mediate transgene expression. It was previously reported that IN deficient HIV and FIV vectors could mediate transient gene transfer. In growth arrested cells, transgene expression was maintained. However, in rapidly dividing cells transgene expression was lost, and duration of expression was related to the rate of cell division. Because some gene transfer targets are mitotically quiescent and slowly dividing cells, IN deficient mutants may promote long term, though ultimately transient, gene expression. We hypothesized that IN deficient FIV vectors can be used to maintain transient gene expression in slowly dividing cell types, such as airway epithelia. We developed IN deficient FIV vectors with single (D66V, D118A, E154G), double (D66V/D118A, D66V/E154G, D118A/E154G), and triple (D66V/D118A/E154G) mutations that disrupt the catalytic triad of the IN protein. Preliminary data indicate that the FIV D66V IN mutant has titers similar to wild type IN vectors. Furthermore, we documented the production of 1-LTR and 2-LTR circles in human cell line cultures transduced with FIV D66V IN deficient vector. Ongoing studies are Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
comparing the persistence of transgene expression in IN deficient versus wild type IN FIV in dividing and non-dividing cells. Furthermore, to ensure that transgene expression seen in IN deficient vectors represents episomal and not integrated vector DNA, Southern blotting will be used. IN deficient lentiviral vectors may have applications for transient gene expression, long term expression in slowly dividing cells, and can be used as controls in studies with integration competent vectors.
101. Development of Lentiviral Vectors with Respiratory Epithelial Specific Transgene Expression Dinithi Senadheera,1 Benjamin Hendrickson,1 Denise Petersen,1 Karen Pepper,1 Carolyn Lutzko.1 1 Research Immunology & Bone Marrow Transplantation, Childrens Hopsital Los Angeles/ University of Southern California, Los Angeles, CA. Long-term gene therapy for respiratory disorders requires stable gene transfer to progenitor or stem cells and efficient transgene expression in their differentiated progeny. Constitutive transgene expression may be suitable for some gene therapy applications; however, regulated transgene expression in specific lineages will be required in others. In this study we developed and evaluated a series of lentivectors for respiratory epithelial lineage specific transgene expression. Specifically, we incorporated promoters from the surfactant protein C (SPC), Clara cell 10kd protein (CC10) or Jaagskiete sheep retrovirus (JSRV) promoters for expression in alveolar epithelial II cells (AECII; SPC), Clara cells (CC10) or both AECII and Clara cells (JSRV) into SIN lentiviral vectors. Vectors were evaluated by the expression of an eGFP reporter gene by flow cytometry. The lentivectors were tested in alveolar epithelial type II (AECII; MLE-12 and MLE-15), Clara cell (MTCC1-2, H441), kidney, muscle, blood and brain cell lines. In the first experiment, each cell line was transduced with a serial dilution of positive control lentivector supernatant (with the constitutive MND promoter), to determine the optimal virus concentration for single proviral integrants. This virus concentration producing 5-10% positive cells was used in subsequent experiments to minimize the proportion of cells containing multiple integrants. Four experiments were performed with each of the respiratory epitehlial promoters. In each experiment, cells were transduced by a single addition of viral supernatant and expression was evaluated by FACS for 8-weeks for the level of eGFP expression. The SPC promoted vector expressed eGFP with high specificity, in MLE-12 and -15 and H441 cells (mean fluorescent intensity [MFI]: 56-100), but not other cell types (<30) (p<0.05). The CC10 lentivector expressed the highest level of eGFP in MTCC1-2 and H441 (MFI: 113-148), at lower levels in MLE-12 and -15 (MFI: 48-56), and at background levels (<30) in all other cell types and the no promoter control (p<0.05). Surprisingly, expression from the JSRV promoter was observed in all epithelial and hematopoietic cell lines tested (MFI: 40-101), indicative of disregulated expression from this promoter in a lentiviral backbone. To confirm that all samples contained provirus, all transduced cell lines were subject to semi-quantitative eGFP PCR analysis. This analysis showed similar levels of gene transfer in all transduced cells samples, indicating that differences in transgene expression were due to differences is levels of expression from each promoter and not due to differences in the level of gene transfer. In on-going experiments we are evaluating the lineage and tissue expression profile from these vectors following intra-venous administration of vector supernatant to neonatal mice. The development of lentiviral vectors with respiratory epithelial specific expression patterns will provide a useful tool for the study of the biology of the respiratory epithelium and the development of gene therapy for respiratory disease. S41
100. Development of an Integrase Deficient FIV Vector for Transient Gene Expression Marisa Banasik,1 Melissa Hickey,2 Paul McCray Jr.1,2 Interdisciplinary Program in Genetics, University of Iowa, Iowa City, IA; 2Department of Pediatrics, University of Iowa, Iowa City, IA. 1
Lentiviral vectors mediate long term transgene expression through integration into host genomic DNA. However, integration comes with potential risks of insertional mutagenesis. In cases where transient gene expression is desired, an integrase (IN) deficient retroviral vector might be a useful alternative. Furthermore, IN deficient vectors may provide some advantages over alternative methods of transient gene expression due to the ability to pseudotype these vectors with heterologous envelopes that target specific cell types. Class I IN mutants have point mutations in the catalytic triad amino acids (DX39-58 DX35 E) of IN that disable this protein but leave other viral proteins functional. Transient gene expression from IN deficient vectors is mediated from episomal forms of vector DNA. 1-LTR and 2-LTR circles form through homologous recombination and non-homologous end joining, respectively, of the linear form of the reverse transcribed viral DNA. Although 1-LTR and 2-LTR circles are unable to efficiently integrate in the presence of functional IN protein, they can mediate transgene expression. It was previously reported that IN deficient HIV and FIV vectors could mediate transient gene transfer. In growth arrested cells, transgene expression was maintained. However, in rapidly dividing cells transgene expression was lost, and duration of expression was related to the rate of cell division. Because some gene transfer targets are mitotically quiescent and slowly dividing cells, IN deficient mutants may promote long term, though ultimately transient, gene expression. We hypothesized that IN deficient FIV vectors can be used to maintain transient gene expression in slowly dividing cell types, such as airway epithelia. We developed IN deficient FIV vectors with single (D66V, D118A, E154G), double (D66V/D118A, D66V/E154G, D118A/E154G), and triple (D66V/D118A/E154G) mutations that disrupt the catalytic triad of the IN protein. Preliminary data indicate that the FIV D66V IN mutant has titers similar to wild type IN vectors. Furthermore, we documented the production of 1-LTR and 2-LTR circles in human cell line cultures transduced with FIV D66V IN deficient vector. Ongoing studies are Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
comparing the persistence of transgene expression in IN deficient versus wild type IN FIV in dividing and non-dividing cells. Furthermore, to ensure that transgene expression seen in IN deficient vectors represents episomal and not integrated vector DNA, Southern blotting will be used. IN deficient lentiviral vectors may have applications for transient gene expression, long term expression in slowly dividing cells, and can be used as controls in studies with integration competent vectors.
101. Development of Lentiviral Vectors with Respiratory Epithelial Specific Transgene Expression Dinithi Senadheera,1 Benjamin Hendrickson,1 Denise Petersen,1 Karen Pepper,1 Carolyn Lutzko.1 1 Research Immunology & Bone Marrow Transplantation, Childrens Hopsital Los Angeles/ University of Southern California, Los Angeles, CA. Long-term gene therapy for respiratory disorders requires stable gene transfer to progenitor or stem cells and efficient transgene expression in their differentiated progeny. Constitutive transgene expression may be suitable for some gene therapy applications; however, regulated transgene expression in specific lineages will be required in others. In this study we developed and evaluated a series of lentivectors for respiratory epithelial lineage specific transgene expression. Specifically, we incorporated promoters from the surfactant protein C (SPC), Clara cell 10kd protein (CC10) or Jaagskiete sheep retrovirus (JSRV) promoters for expression in alveolar epithelial II cells (AECII; SPC), Clara cells (CC10) or both AECII and Clara cells (JSRV) into SIN lentiviral vectors. Vectors were evaluated by the expression of an eGFP reporter gene by flow cytometry. The lentivectors were tested in alveolar epithelial type II (AECII; MLE-12 and MLE-15), Clara cell (MTCC1-2, H441), kidney, muscle, blood and brain cell lines. In the first experiment, each cell line was transduced with a serial dilution of positive control lentivector supernatant (with the constitutive MND promoter), to determine the optimal virus concentration for single proviral integrants. This virus concentration producing 5-10% positive cells was used in subsequent experiments to minimize the proportion of cells containing multiple integrants. Four experiments were performed with each of the respiratory epitehlial promoters. In each experiment, cells were transduced by a single addition of viral supernatant and expression was evaluated by FACS for 8-weeks for the level of eGFP expression. The SPC promoted vector expressed eGFP with high specificity, in MLE-12 and -15 and H441 cells (mean fluorescent intensity [MFI]: 56-100), but not other cell types (<30) (p<0.05). The CC10 lentivector expressed the highest level of eGFP in MTCC1-2 and H441 (MFI: 113-148), at lower levels in MLE-12 and -15 (MFI: 48-56), and at background levels (<30) in all other cell types and the no promoter control (p<0.05). Surprisingly, expression from the JSRV promoter was observed in all epithelial and hematopoietic cell lines tested (MFI: 40-101), indicative of disregulated expression from this promoter in a lentiviral backbone. To confirm that all samples contained provirus, all transduced cell lines were subject to semi-quantitative eGFP PCR analysis. This analysis showed similar levels of gene transfer in all transduced cells samples, indicating that differences in transgene expression were due to differences is levels of expression from each promoter and not due to differences in the level of gene transfer. In on-going experiments we are evaluating the lineage and tissue expression profile from these vectors following intra-venous administration of vector supernatant to neonatal mice. The development of lentiviral vectors with respiratory epithelial specific expression patterns will provide a useful tool for the study of the biology of the respiratory epithelium and the development of gene therapy for respiratory disease. S41