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772. Efficient Transduction of Macaque Hematopoietic Repopulating Cells with HIV-Derived Lentiviral Vectors
771. Successful Gene Therapy of Canine Leukocyte Adhesion Deficiency Using Foamy Viral Vectors
Thomas R. Bauer, Jr,1 James M. Allen.' Laura M. Tuschong,' Erik M. Olson;' Tanya H. Burkholder! Mehreen Hai,' Yu-chen GU,I Dennis D. Hickstein,' David W. Russell.' 'Experimental Transplantation and Immunology Branch. National Cancer Institute. National Institutes ofHealth• Bethesda. AID; 2Department ofHematology. University ofWashington, Seattle, IVA; J Division ofVeterinary Services, Office ofResearch Services, National Institutes ofHealth, Bethesda. MD. The recent successes in treating the genetic immunodeficiencies X-linked severe combined immunodeficiency (X-SCID) and chronic granulomatous disease have demonstrated the therapeutic potential of hematopoietic stem cell gene therapy. However, the gammaretroviral vectors used in these trials have subsequently resulted in insertional activation of nearby oncogenes, oligoclonal hematopoiesis, and leukemia or gene silencing. These developments have prompted studies of modified or alternative vector systems. We previously reported results using a vector based on foamy virus (FV) to treat four dogs with canine leukocyte adhesion deficiency or CLAD, a lethal genetic immunodeficiency disease caused by defects in the Icukocyte integrin CD 18. The four CLAD dogs all received non-myeloablative conditioning followed by infusion ofFV transduced-autologous CD34+ hematopoietic stem cells. All four dogs currently have circulating CD 18+ neutrophil levels ranging from 3-6%, with CDI8+ lymphocyte levels ranging from 15-30%. In vitro testing ofleukocytes has shown the CDIl/CDI8 molecule present on FV vector transduced cells is appropriately regulated, with up-regulation and activation of the CDI8 molecule occurring only after stimulation, identical to results seen with normal cells . In vitro lymphocyte proliferation assays using Staphylococcal enterotoxin A and Concavalin A demonstrated that the increase in CD 18+ T-Iymphocyte levels compared to CD 18+ neutrophil levels is due to a selective proliferation of the CD 18+ T-Iymphocytes in response to mitogens. Clinically, all four FV vector treated dogs have had complete reversal ofthc CLAD phenotype, which has been sustained for greater than one and one-half years following treatment, and all have had normalization oftheir WBC counts following gene therapy. In contrast, four CLAD control dogs were euthanized by six months of age due to complications from CLAD, and all had persistently elevated WBC counts until the time of death. To date, there have been no genotoxic complications in the four FV vector treated CLAD dogs, and integration site analysis has continued to demonstrate polyclonal marking of the transduced cells. These results support the usc of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD.
772. Efficient Transduction of Macaque Hematopoietic Repopulating Cells with HIVDerived Lentiviral Vectors
Grant D. Trobridgc.P Brian C. Beard,' Philip Olsen,' Punam Malik! Hans-Peter Kiem.P 'Clinical Research Division , Fred Hutchinson Cancer Research Center; Seattle , H'A; 'Departmem ofMedicine. University of Washington, Seattle, H';I; J£rperimental Hematology. Cincinnati Children s Hospital Medical Center; Cincinnati, Off.
Lentiviral vectors arc attractive for hematopoietic stem cell gene therapy because they do not require mitosis for transduction, and can efficiently transduce hematopoietic repopulating cells. Lentiviral vectors also do not integrate as frequently near promoter regions as gammaretroviral vectors, and self-inactivating lentiviral vectors pscudotyped with VSV-G can be produced at high-titer and concentrated by centrifugation. Experiments to evaluate HIV-derived Molecular Therapy Volume 15.Supplement 1. ~by Cop yright © The Americ...m Society o t"Gene Therapy
2007
lentiviral vectors in nonhuman primates prior to clinical trials has been hampered by low transduction frequencies, due in part to host cell restriction by trim5a. We have studied transduction of various monkey species with HlV-derived lentiviral vectors ineluding baboons (Papio cynocephalus anubis) , cynomolgus macaques, (Macaca fascicularis), rhesus macaques (Macaca rnulatta), and pigtailed macaques (Maeaca nemestrina). Pigtailed macaque CD34+ cells were transduced at similar frequencies to human CD34+ cells and at much higher frequencies compared to the other species. Using our optimized transduction conditions we have now established efficient gene transfer to pigtailed macaque long-term repopulating cells. In three macaques we observed high level marking with I-IIV-derived lentiviral vectors that contain an EGFP reporter gene expressed from a Pgk promoter using relatively low MOIs (5-10). In these studies a rapid 2-day transduction protocol was used to preserve the engraftment potential of the ex vivo cultured cells. In the first animal marking is stable with over 27% ofgranulocytes and 23% of lymphocytes expressing EGFPat 450 days post-transplantation. The second animal has over 25% EGFP-positive granulocytes and over 10% in lymphocytes 98 days post-transplantation. The third animal has over 19% EGFP-positive granulocytes 12 days post-transplantation. LAM-PCR analysis of integration sites has demonstrated that repopulating cells arc highly polyelonal and transgene expression has been detected in all hematopoietic lineages (B-cells, T-cells , granulocytes, red blood cells as well as platelets). These data show that lentiviral vectors are highly effective for hematopoietic stem cell gene therapy, particularly for diseases in which maintaining the engraftrnent potential of stem cells by using a rapid transduction protocol is critical. Importantly, these data also show that the pigtailed macaque is an cxeellent model to evaluate the efficacy and safety of HIV-dcrived lentiviral vectors proposed for clinical gene therapy protocols.
773. Clinically Relevant Levels of Hematopoietic Stem Cell Gene Transfer in Rhesus Macaques Nearly Four Years Following Transplantation with Autologous Hematopoietic Cells Transduced with an Simian Immunodeficiency Virus-Based Lentiviral Vector Yoon Sang Kim,' Yoon-Jin Kim,' Peiman Hematti,' Bum Kee
Hong,' Donahue Robert,' Arthur W. Nienhuis,' Cynthia E. Dun-
bar,' Derek A. Persons. 1 'Hematology, St. Jude Children s Research Hospital, Memphis, TN; 2Hematology Branch, National Heart, Lung; and Blood Institute, National Institutes ofHealth, Bethesda, MD. We have previously reported that a lentiviral gene transfer vector derived from the simian immunodeficiency virus (SIV) was efficient at transducing rhesus CD34+cells, resulting in high-level in vivo marking of progeny blood cells in three transplanted rhcsus macaques up to 6 months post-transplantation (I-Ianawa et al., 103:4062-9, 2004). Marking levels , as assessed by OFP expression encoded by the vector genome, in granulocytes and monocytes averaged 18% ± 8% and 15% ± 7%, respectively. A comparison of vector integration sites in these animals compared to animals receiving MLV-transduced cells revealed a different general pattern of integration , with SIV integrants strongly favoring entire transcription units and gene -dense regions of the genome, while MLV favored regions surrounding transcription start sites (Hematti ct al., PLoS BioI. 2: Nov23 ; e423 , 2004). To evaluate long-term hematopoietic stem cell (HSC) marking and vector expression in the progeny blood cells of these animals almost 4 years post-transplantation, we performed a comprehensive analysis of the peripheral blood (PB) and bone marrow (BM) using multiparameter flow cytometry coupled with DNA analysis for vector copy numb er in various cell populations from the animals using quantitative PCR (qPCR). S297
Thomas R. Bauer, Jr,1 James M. Allen.' Laura M. Tuschong,' Erik M. Olson;' Tanya H. Burkholder! Mehreen Hai,' Yu-chen GU,I Dennis D. Hickstein,' David W. Russell.' 'Experimental Transplantation and Immunology Branch. National Cancer Institute. National Institutes ofHealth• Bethesda. AID; 2Department ofHematology. University ofWashington, Seattle, IVA; J Division ofVeterinary Services, Office ofResearch Services, National Institutes ofHealth, Bethesda. MD. The recent successes in treating the genetic immunodeficiencies X-linked severe combined immunodeficiency (X-SCID) and chronic granulomatous disease have demonstrated the therapeutic potential of hematopoietic stem cell gene therapy. However, the gammaretroviral vectors used in these trials have subsequently resulted in insertional activation of nearby oncogenes, oligoclonal hematopoiesis, and leukemia or gene silencing. These developments have prompted studies of modified or alternative vector systems. We previously reported results using a vector based on foamy virus (FV) to treat four dogs with canine leukocyte adhesion deficiency or CLAD, a lethal genetic immunodeficiency disease caused by defects in the Icukocyte integrin CD 18. The four CLAD dogs all received non-myeloablative conditioning followed by infusion ofFV transduced-autologous CD34+ hematopoietic stem cells. All four dogs currently have circulating CD 18+ neutrophil levels ranging from 3-6%, with CDI8+ lymphocyte levels ranging from 15-30%. In vitro testing ofleukocytes has shown the CDIl/CDI8 molecule present on FV vector transduced cells is appropriately regulated, with up-regulation and activation of the CDI8 molecule occurring only after stimulation, identical to results seen with normal cells . In vitro lymphocyte proliferation assays using Staphylococcal enterotoxin A and Concavalin A demonstrated that the increase in CD 18+ T-Iymphocyte levels compared to CD 18+ neutrophil levels is due to a selective proliferation of the CD 18+ T-Iymphocytes in response to mitogens. Clinically, all four FV vector treated dogs have had complete reversal ofthc CLAD phenotype, which has been sustained for greater than one and one-half years following treatment, and all have had normalization oftheir WBC counts following gene therapy. In contrast, four CLAD control dogs were euthanized by six months of age due to complications from CLAD, and all had persistently elevated WBC counts until the time of death. To date, there have been no genotoxic complications in the four FV vector treated CLAD dogs, and integration site analysis has continued to demonstrate polyclonal marking of the transduced cells. These results support the usc of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD.
772. Efficient Transduction of Macaque Hematopoietic Repopulating Cells with HIVDerived Lentiviral Vectors
Grant D. Trobridgc.P Brian C. Beard,' Philip Olsen,' Punam Malik! Hans-Peter Kiem.P 'Clinical Research Division , Fred Hutchinson Cancer Research Center; Seattle , H'A; 'Departmem ofMedicine. University of Washington, Seattle, H';I; J£rperimental Hematology. Cincinnati Children s Hospital Medical Center; Cincinnati, Off.
Lentiviral vectors arc attractive for hematopoietic stem cell gene therapy because they do not require mitosis for transduction, and can efficiently transduce hematopoietic repopulating cells. Lentiviral vectors also do not integrate as frequently near promoter regions as gammaretroviral vectors, and self-inactivating lentiviral vectors pscudotyped with VSV-G can be produced at high-titer and concentrated by centrifugation. Experiments to evaluate HIV-derived Molecular Therapy Volume 15.Supplement 1. ~by Cop yright © The Americ...m Society o t"Gene Therapy
2007
lentiviral vectors in nonhuman primates prior to clinical trials has been hampered by low transduction frequencies, due in part to host cell restriction by trim5a. We have studied transduction of various monkey species with HlV-derived lentiviral vectors ineluding baboons (Papio cynocephalus anubis) , cynomolgus macaques, (Macaca fascicularis), rhesus macaques (Macaca rnulatta), and pigtailed macaques (Maeaca nemestrina). Pigtailed macaque CD34+ cells were transduced at similar frequencies to human CD34+ cells and at much higher frequencies compared to the other species. Using our optimized transduction conditions we have now established efficient gene transfer to pigtailed macaque long-term repopulating cells. In three macaques we observed high level marking with I-IIV-derived lentiviral vectors that contain an EGFP reporter gene expressed from a Pgk promoter using relatively low MOIs (5-10). In these studies a rapid 2-day transduction protocol was used to preserve the engraftment potential of the ex vivo cultured cells. In the first animal marking is stable with over 27% ofgranulocytes and 23% of lymphocytes expressing EGFPat 450 days post-transplantation. The second animal has over 25% EGFP-positive granulocytes and over 10% in lymphocytes 98 days post-transplantation. The third animal has over 19% EGFP-positive granulocytes 12 days post-transplantation. LAM-PCR analysis of integration sites has demonstrated that repopulating cells arc highly polyelonal and transgene expression has been detected in all hematopoietic lineages (B-cells, T-cells , granulocytes, red blood cells as well as platelets). These data show that lentiviral vectors are highly effective for hematopoietic stem cell gene therapy, particularly for diseases in which maintaining the engraftrnent potential of stem cells by using a rapid transduction protocol is critical. Importantly, these data also show that the pigtailed macaque is an cxeellent model to evaluate the efficacy and safety of HIV-dcrived lentiviral vectors proposed for clinical gene therapy protocols.
773. Clinically Relevant Levels of Hematopoietic Stem Cell Gene Transfer in Rhesus Macaques Nearly Four Years Following Transplantation with Autologous Hematopoietic Cells Transduced with an Simian Immunodeficiency Virus-Based Lentiviral Vector Yoon Sang Kim,' Yoon-Jin Kim,' Peiman Hematti,' Bum Kee
Hong,' Donahue Robert,' Arthur W. Nienhuis,' Cynthia E. Dun-
bar,' Derek A. Persons. 1 'Hematology, St. Jude Children s Research Hospital, Memphis, TN; 2Hematology Branch, National Heart, Lung; and Blood Institute, National Institutes ofHealth, Bethesda, MD. We have previously reported that a lentiviral gene transfer vector derived from the simian immunodeficiency virus (SIV) was efficient at transducing rhesus CD34+cells, resulting in high-level in vivo marking of progeny blood cells in three transplanted rhcsus macaques up to 6 months post-transplantation (I-Ianawa et al., 103:4062-9, 2004). Marking levels , as assessed by OFP expression encoded by the vector genome, in granulocytes and monocytes averaged 18% ± 8% and 15% ± 7%, respectively. A comparison of vector integration sites in these animals compared to animals receiving MLV-transduced cells revealed a different general pattern of integration , with SIV integrants strongly favoring entire transcription units and gene -dense regions of the genome, while MLV favored regions surrounding transcription start sites (Hematti ct al., PLoS BioI. 2: Nov23 ; e423 , 2004). To evaluate long-term hematopoietic stem cell (HSC) marking and vector expression in the progeny blood cells of these animals almost 4 years post-transplantation, we performed a comprehensive analysis of the peripheral blood (PB) and bone marrow (BM) using multiparameter flow cytometry coupled with DNA analysis for vector copy numb er in various cell populations from the animals using quantitative PCR (qPCR). S297