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305. Analysis of Sleeping Beauty Ransposons for Correction of Fanconi Anemia in Lymphoblastoid Cell Lines
HEMATOLOGIC-NOVEL TECHNOLOGIES, LINEAGE SPECIFIC REGULATION mediate stable and efficient gene transfer in human HPCs without reducing their differentiation potential and is promising for future SB-based HPC therapy.
305. Analysis of Sleeping Beauty Ransposons for Correction of Fanconi Anemia in Lymphoblastoid Cell Lines
Kendra A. Hyland,1 Erik R. Olson,1 Elena L. Aronovich,2 Perry B. Hackett,2 Bruce R. Blazar,3 Jakub Tolar,3 R. Scott McIvor.1 1 Discovery Genomics, Inc., Minneapolis, MN; 2Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 3Pediatrics, Medical School, University of Minnesota, Minneapolis, MN. Sleeping Beauty (SB) is a transposon vector system that can insert sequences into chromosomes to direct extended expression of therapeutic genes. Our goal is to develop the SB system for the treatment of Fanconi anemia (FA), a rare autosomal recessive disorder often accompanied by progressive bone marrow failure. Mutations in one of 13 genes that encode proteins in the FA DNArepair pathway can result in the disease. Approximately 85% of FA patients have mutations in the FA-A, FA-C, or FA-G genes. Using a green fluorescence protein (GFP) reporter gene and the CytoPulse electroporation system, electroporation conditions, including voltage, pulse width, and pulse number, were varied methodically in order to achieve the greatest possible loading of SB transposon-containing plasmid DNA into human lymphoblastoid cells derived from FA-C defective (FA-LCL) and normal patients (LCL). Culture of both types of cells with the antioxidant N-acetyl-L-cysteine after electroporation improved cell viability. Electroporation alone reduced cell viability of both types by 10-20% compared to untreated cells. Expression of the FA complementation group C (FA-C) or GFP transgenes in SB transposons, co-delivered with DNA or RNA-encoding transposase, was determined in normal LCL and in FA-LCL. Co-delivery of transposase-encoding DNA in normal LCL supported a 10- to 50fold increase in stable, long-term expression of GFP. In contrast, co-delivery of transposase-encoding DNA along with GFP-transposon did not support an increase in long-term GFP expression in FA-LCL. Co-electrotransfected FA-LCL also lacked SB-mediated excision products, which were detected by excision PCR assay in normal LCL. Transposase-mediated stable integration of individual transgenes in normal LCL is being confirmed by sequence analysis of transposonchromosome junctions recovered by linear amplification-mediated PCR. Our results suggest that Sleeping Beauty-mediated gene transfer may require complementation of FA-C deficiency in order to achieve effective transposition in FA-C deficient LCL and other therapeutically relevant cellular targets such as hematopoietic stem cells. These studies are ongoing in development of the SB system for treatment of Fanconi anemia in human patients.
306. Lentiviral Vectors Containing Internal Cellular Promoters Fail To Express Therapeutic Levels of CD18 in Canine Leukocyte Adhesion Deficiency
Everette J. R. Nelson,1 Michael J. Hunter,1 Laura M. Tuschong,1 Cedar J. Fowler,2 Thomas R. Bauer, Jr.,1 Dennis D. Hickstein.1 1 Eperimental Transplantation and Immunology, National Cancer Institute, Bethesda, MD; 2Howard Hughes Medical Scholars, Howard Hughes Medical Insitute, Chevy Chase, MD. In leukocyte adhesion deficiency (LAD), mutations in the ITGB2 gene encoding the leukocyte integrin CD18 result in defective leukocyte adhesion and migration leading to life-threatening bacterial infections. Canine leukocyte adhesion deficiency (CLAD), the homologue of LAD in children, represents a disease-specific large animal model of LAD in which new therapeutic approaches can be Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
assessed. In previous studies, we demonstrated that ex vivo gene transfer of canine CD18 into CLAD CD34+ cells using retroviral or foamy viral vectors resulted in reversal of the CLAD phenotype when the infusion of the autologous cells was preceded by 200 cGy total body irradiation (TBI). Both vectors used the MSCV promoter with the promoter located in the LTR of the γ-retroviral vector and located internally promoter in the foamy viral vector. Because of concerns related to potential genotoxicity from the MSCV promoter, we tested three different internal cellular promoters in a selfinactivating lentiviral vector in CLAD CD34+ cells. Results with the three cellular promoters– the human phosphoglycerate kinase (PGK) promoter and two different lengths of the human elongation factor 1α (EF1α) promoter, designated long (1169 bp) and short (248 bp) – were compared to the results with an internal MSCV promoter driving expression of canine CD18. The lentiviral vector backbone was pRRLSIN.cPPT.WPRE. Transduction efficiency was determined using a human LAD EBV B-cell line that lacks human CD18. The percentage of CD18+ cells following an overnight transduction of this cell line were, PGK 98.3%, EF1α short 96.2%, EF1α long 86.1%, and MSCV 95.4%. When comparable titers of each vector were used in an overnight transduction of CLAD CD34+ cells, the percentage of CD18+ cells 5 days after transduction were, PGK 19.4%, EF1α short 13.4%, EF1α long 7.1%, and MSCV 18.3%. In vitro results with the PGK and EF1α short promoter-based vectors led to in vivo testing in CLAD dogs. Autologous CLAD CD34+ cells were transduced overnight with the vector and infused after 200 cGy TBI. None of the CLAD treated dogs had greater than 0.4% CD18+ neutrophils in vivo, and only one survived the first two months of treatment (PGK vector, n=4; EF1α short vector, n=4). We are currently investigating the potential reasons for the low percentages of CD18+ neurophils in vivo in these animals. These results indicate that in the pRRL SIN lentiviral vector, alternative cellular promoters/enhancers, additional regulatory elements, or modified lentiviral vector backbones or packaging methods, will likely be required before these vectors could be efficacious in treating dogs with CLAD. γγα.
307. Tissue-Specific Lentiviral Vectors as a Tool for the Gene Therapy of the Erythroid Pyruvate Kinase Deficiency
Maria Garcia-Gomez,1,2,4 Nestor W. Meza,3 Susana Navarro,1,2,4 Maria E. Alonso-Ferrero,1,2,4 Juan A. Bueren,2,4 Jose C. Segovia.1,2,4 1 Differentiation and Cytometry Unit. Hematopoiesis Division., CIEMAT, Madrid, Spain; 2Hematopoiesis Division, CIEMAT, Madrid, Spain; 3LABIEMET, School of Medicine of Táchira, Universidad de los Andes, San Cristóbal, Venezuela; 4CIBERER, Madrid, Spain.
Human erythrocyte pyruvate kinase deficiency (PKD) is an autosomal recessive disorder produced by mutations in the PKLR gene, which encodes the erythrocyte pyruvate kinase (RPK). PKD is the most common cause of chronic nonspherocytic haemolytic anaemia. In severe cases, periodical blood transfusions and splenectomy can be clinically useful. However up to the date, there is no curative treatment for severe PKD. In these patients, haematopoietic stem cell gene therapy could be an effective and less invasive alternative. With this aim, we have developed lentiviral vectors harbouring the human RPK cDNA under the control of the erythroid tissue-specific sequences of the human PKLR promoter. As control, vectors expressing the eGFP under the erythroid tissue-specific or under the CMV ubiquitous promoters and human RPK cDNA under the CMV promoter were also developed. To test the specificity of the pklr promoter, human erythroid and non-erythroid cell lines were transduced with the different vectors. Significantly, pklr vectors mediated an erythroidspecific expression of the transgene. This specificity was confirmed in transduced cord blood CD34+ cells which were either differentiated in vitro with erythropoietin or transplanted into NOD/SCID mice. To S119
305. Analysis of Sleeping Beauty Ransposons for Correction of Fanconi Anemia in Lymphoblastoid Cell Lines
Kendra A. Hyland,1 Erik R. Olson,1 Elena L. Aronovich,2 Perry B. Hackett,2 Bruce R. Blazar,3 Jakub Tolar,3 R. Scott McIvor.1 1 Discovery Genomics, Inc., Minneapolis, MN; 2Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 3Pediatrics, Medical School, University of Minnesota, Minneapolis, MN. Sleeping Beauty (SB) is a transposon vector system that can insert sequences into chromosomes to direct extended expression of therapeutic genes. Our goal is to develop the SB system for the treatment of Fanconi anemia (FA), a rare autosomal recessive disorder often accompanied by progressive bone marrow failure. Mutations in one of 13 genes that encode proteins in the FA DNArepair pathway can result in the disease. Approximately 85% of FA patients have mutations in the FA-A, FA-C, or FA-G genes. Using a green fluorescence protein (GFP) reporter gene and the CytoPulse electroporation system, electroporation conditions, including voltage, pulse width, and pulse number, were varied methodically in order to achieve the greatest possible loading of SB transposon-containing plasmid DNA into human lymphoblastoid cells derived from FA-C defective (FA-LCL) and normal patients (LCL). Culture of both types of cells with the antioxidant N-acetyl-L-cysteine after electroporation improved cell viability. Electroporation alone reduced cell viability of both types by 10-20% compared to untreated cells. Expression of the FA complementation group C (FA-C) or GFP transgenes in SB transposons, co-delivered with DNA or RNA-encoding transposase, was determined in normal LCL and in FA-LCL. Co-delivery of transposase-encoding DNA in normal LCL supported a 10- to 50fold increase in stable, long-term expression of GFP. In contrast, co-delivery of transposase-encoding DNA along with GFP-transposon did not support an increase in long-term GFP expression in FA-LCL. Co-electrotransfected FA-LCL also lacked SB-mediated excision products, which were detected by excision PCR assay in normal LCL. Transposase-mediated stable integration of individual transgenes in normal LCL is being confirmed by sequence analysis of transposonchromosome junctions recovered by linear amplification-mediated PCR. Our results suggest that Sleeping Beauty-mediated gene transfer may require complementation of FA-C deficiency in order to achieve effective transposition in FA-C deficient LCL and other therapeutically relevant cellular targets such as hematopoietic stem cells. These studies are ongoing in development of the SB system for treatment of Fanconi anemia in human patients.
306. Lentiviral Vectors Containing Internal Cellular Promoters Fail To Express Therapeutic Levels of CD18 in Canine Leukocyte Adhesion Deficiency
Everette J. R. Nelson,1 Michael J. Hunter,1 Laura M. Tuschong,1 Cedar J. Fowler,2 Thomas R. Bauer, Jr.,1 Dennis D. Hickstein.1 1 Eperimental Transplantation and Immunology, National Cancer Institute, Bethesda, MD; 2Howard Hughes Medical Scholars, Howard Hughes Medical Insitute, Chevy Chase, MD. In leukocyte adhesion deficiency (LAD), mutations in the ITGB2 gene encoding the leukocyte integrin CD18 result in defective leukocyte adhesion and migration leading to life-threatening bacterial infections. Canine leukocyte adhesion deficiency (CLAD), the homologue of LAD in children, represents a disease-specific large animal model of LAD in which new therapeutic approaches can be Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
assessed. In previous studies, we demonstrated that ex vivo gene transfer of canine CD18 into CLAD CD34+ cells using retroviral or foamy viral vectors resulted in reversal of the CLAD phenotype when the infusion of the autologous cells was preceded by 200 cGy total body irradiation (TBI). Both vectors used the MSCV promoter with the promoter located in the LTR of the γ-retroviral vector and located internally promoter in the foamy viral vector. Because of concerns related to potential genotoxicity from the MSCV promoter, we tested three different internal cellular promoters in a selfinactivating lentiviral vector in CLAD CD34+ cells. Results with the three cellular promoters– the human phosphoglycerate kinase (PGK) promoter and two different lengths of the human elongation factor 1α (EF1α) promoter, designated long (1169 bp) and short (248 bp) – were compared to the results with an internal MSCV promoter driving expression of canine CD18. The lentiviral vector backbone was pRRLSIN.cPPT.WPRE. Transduction efficiency was determined using a human LAD EBV B-cell line that lacks human CD18. The percentage of CD18+ cells following an overnight transduction of this cell line were, PGK 98.3%, EF1α short 96.2%, EF1α long 86.1%, and MSCV 95.4%. When comparable titers of each vector were used in an overnight transduction of CLAD CD34+ cells, the percentage of CD18+ cells 5 days after transduction were, PGK 19.4%, EF1α short 13.4%, EF1α long 7.1%, and MSCV 18.3%. In vitro results with the PGK and EF1α short promoter-based vectors led to in vivo testing in CLAD dogs. Autologous CLAD CD34+ cells were transduced overnight with the vector and infused after 200 cGy TBI. None of the CLAD treated dogs had greater than 0.4% CD18+ neutrophils in vivo, and only one survived the first two months of treatment (PGK vector, n=4; EF1α short vector, n=4). We are currently investigating the potential reasons for the low percentages of CD18+ neurophils in vivo in these animals. These results indicate that in the pRRL SIN lentiviral vector, alternative cellular promoters/enhancers, additional regulatory elements, or modified lentiviral vector backbones or packaging methods, will likely be required before these vectors could be efficacious in treating dogs with CLAD. γγα.
307. Tissue-Specific Lentiviral Vectors as a Tool for the Gene Therapy of the Erythroid Pyruvate Kinase Deficiency
Maria Garcia-Gomez,1,2,4 Nestor W. Meza,3 Susana Navarro,1,2,4 Maria E. Alonso-Ferrero,1,2,4 Juan A. Bueren,2,4 Jose C. Segovia.1,2,4 1 Differentiation and Cytometry Unit. Hematopoiesis Division., CIEMAT, Madrid, Spain; 2Hematopoiesis Division, CIEMAT, Madrid, Spain; 3LABIEMET, School of Medicine of Táchira, Universidad de los Andes, San Cristóbal, Venezuela; 4CIBERER, Madrid, Spain.
Human erythrocyte pyruvate kinase deficiency (PKD) is an autosomal recessive disorder produced by mutations in the PKLR gene, which encodes the erythrocyte pyruvate kinase (RPK). PKD is the most common cause of chronic nonspherocytic haemolytic anaemia. In severe cases, periodical blood transfusions and splenectomy can be clinically useful. However up to the date, there is no curative treatment for severe PKD. In these patients, haematopoietic stem cell gene therapy could be an effective and less invasive alternative. With this aim, we have developed lentiviral vectors harbouring the human RPK cDNA under the control of the erythroid tissue-specific sequences of the human PKLR promoter. As control, vectors expressing the eGFP under the erythroid tissue-specific or under the CMV ubiquitous promoters and human RPK cDNA under the CMV promoter were also developed. To test the specificity of the pklr promoter, human erythroid and non-erythroid cell lines were transduced with the different vectors. Significantly, pklr vectors mediated an erythroidspecific expression of the transgene. This specificity was confirmed in transduced cord blood CD34+ cells which were either differentiated in vitro with erythropoietin or transplanted into NOD/SCID mice. To S119