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295. Hematopoietic Stem Cell Gene Therapy (2.0) Based on Purified CD34+CD38- Cells
Stem Cell Engineering and Therapies II In summary, higher dose of TBI increased gene marking levels in transplanted rhesus macaques, while lower dose of TBI resulted in anti-GFP antibody production. Additional immunosuppressive therapy might be required in RIC to induce immunological tolerance for transgene products. Our findings should be valuable to consider conditioning regimens for clinical gene therapy.
Stem Cell Engineering and Therapies II 295. Hematopoietic Stem Cell Gene Therapy (2.0) Based on Purified CD34+CD38- Cells
Erika Zonari,1 Francesco Boccalatte,1 Tiziana Plati,1 Alessandro Aiuti,1 Giuliana Ferrari,1 Eugenio Montini,1 Luigi Naldini,1 Bernhard Gentner.1 1 S.Raffaele Telethon Institute for Gene Therapy, Milan, Italy. Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. To potentially improve the efficacy, safety and economic sustainability of HSPC transduction, we reasoned to genetically manipulate only the more potent CD34+CD38- HSPC, thereby improving HSPC maintenance in culture in the absence of differentiating cells and downscaling the cell therapy product by a factor of ten without compromising long-term engraftment. This approach would also decrease the total load of vector integration infused in the patients, thus improving its overall safety. First, we determined the engraftment kinetics of CD34+ mobilized peripheral blood (mPB) subpopulations over a 24wk xenotransplantation period. We sorted CD34+ mPB into 4 fractions with increasing expression levels of CD38 and marked each fraction with a specific fluorescent protein allowing to track the population of origin driving hematopoietic reconstitution. Differentially labeled fractions were mixed, and various combinations of CD38-, CD38int and CD38hi HSPC were injected into NSG mice (2 exp, n=30). Almost all long-term repopulating capacity (>90%) was contained within CD34+CD38- cells, and these cells took over hematopoiesis by 9wks. Instead, early reconstitution was mainly driven by CD34+CD38int progenitor cells. In the prospect of a clinical translation, we then modeled the co-administration of gene-modified CD34+CD38- mPB cells with uncultured CD34+CD38int/+ supporter cells aimed to drive fast hematopoietic recovery (3 exp, n=38). Repopulation by genemodified CD34+CD38- cells was slower (15wks) and incomplete (<60% at 24wks) in this setting. Additional, non-competitive transplants confirmed that freshly isolated CD34+CD38int cells possess long-term reconstituting capacity, which is lost after 2 days of standard culture. To improve the engraftment of the gene-modified CD34+CD38- cells when administered with uncultured CD38int/+ supporter cells, we optimized culture conditions of the CD38- and cell doses of the CD38int/+ population. We achieved accelerated engraftment kinetics of gene-modified CD38- cells by >5 wks and Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
re-established long-term (24wks) gene marking up to 85%, thus allowing to benefit from prompt hematopoietic recovery driven by transiently repopulating CD38int/+ supporter cells. Last, we optimized LV transduction in the framework of an improved culture protocol. Exposing CD34+ or CD34+CD38- mPB cells to prostaglandin E2 (PGE2) increased transduction efficiency 1.5-2.5x, allowing to markedly reduce pre-stimulation and LV exposure times better preserving HSC functions. Importantly, the higher gene-transfer efficiency was maintained for up to 24 wks following xenotransplantation (n=33), suggesting that PGE2 facilitated LV transduction in long-term HSC. In summary, these results support the clinical development of novel HSPC gene therapy protocols based on the modification of highly purified HSC subsets, with the prospect to improve the efficacy, safety and feasibility of future ex vivo gene therapy studies.
296. Pre-Selection of Anti-HIV Lentiviral Vector Gene Modified Hematopoietic Stem Cells Significantly Improves Protection from HIV Infection: The Basis for a Future Clinical Trial
Sharlie L. Barclay,1 Yimin Yang,1 Sirou Zhang,1 Ryan Fong,1 Alfonso Barraza,1 Jan A. Nolta,1 Mehrdad Abedi,1 Joseph S. Anderson.1 1 Internal Medicine, University of California Davis, Sacramento, CA. The successful treatment and cure of HIV in the Berlin Patient has highlighted the utility of HIV-resistant hematopoietic stem cells (HSC) to offer a potential functional cure for HIV infected individuals. Stem cell gene therapy for HIV can mimic this result by genetically engineering a patient’s own HSC with lentiviral vectors encoding HIV resistance genes. Clinical trials of HIV stem cell gene therapy have been unsuccessful at demonstrating complete efficacy due to the transplantation of a low percentage of anti-HIV gene expressing cells. Therefore, to bridge the gap between these previous approaches and the Berlin patient, we have developed a pre-selection process which allows for the purification of vector transduced cells prior to transplantation into patients. This pre-selective lentiviral vector (1TAX) encodes a triple combination of anti-HIV genes (a human/ rhesus macaque TRIM5alpha, a CCR5 shRNA, and a TAR decoy) in addition to a truncated/mutated form of human CD25 which is expressed on the cell surface of transduced HSC and is used to enrich for the HIV-resistant cell population. Purity levels of 1TAX transduced human HSC obtained after selection for CD25+ were >94% as determined by flow cytometry. Safety of the 1TAX vector transduced/purified human HSC was demonstrated in vivo in NOD-SCID-Rag-/-IL2rgamma-/(NRG) mice with the detection of engraftment and multi-lineage hematopoiesis into human T cells, B cells, and macrophages in the peripheral blood and lymphoid organs. Upon infection of 1TAX mice with a CCR5-tropic BaL-1 or a CXCR4-tropic NL43 strain of HIV-1, maintenance of normal human CD4+ cell levels were observed in the peripheral blood. A significant decrease (>4 logs) in HIV-1 plasma viremia was also demonstrated in 1TAX mice as compared to mice transplanted with nontransduced human HSC. Together, this work has demonstrated the safety and efficacy of this 1TAX pre-selective lentiviral vector in HSC and is the basis for a future human clinical trial of HIV stem cell gene therapy.
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Stem Cell Engineering and Therapies II 295. Hematopoietic Stem Cell Gene Therapy (2.0) Based on Purified CD34+CD38- Cells
Erika Zonari,1 Francesco Boccalatte,1 Tiziana Plati,1 Alessandro Aiuti,1 Giuliana Ferrari,1 Eugenio Montini,1 Luigi Naldini,1 Bernhard Gentner.1 1 S.Raffaele Telethon Institute for Gene Therapy, Milan, Italy. Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. To potentially improve the efficacy, safety and economic sustainability of HSPC transduction, we reasoned to genetically manipulate only the more potent CD34+CD38- HSPC, thereby improving HSPC maintenance in culture in the absence of differentiating cells and downscaling the cell therapy product by a factor of ten without compromising long-term engraftment. This approach would also decrease the total load of vector integration infused in the patients, thus improving its overall safety. First, we determined the engraftment kinetics of CD34+ mobilized peripheral blood (mPB) subpopulations over a 24wk xenotransplantation period. We sorted CD34+ mPB into 4 fractions with increasing expression levels of CD38 and marked each fraction with a specific fluorescent protein allowing to track the population of origin driving hematopoietic reconstitution. Differentially labeled fractions were mixed, and various combinations of CD38-, CD38int and CD38hi HSPC were injected into NSG mice (2 exp, n=30). Almost all long-term repopulating capacity (>90%) was contained within CD34+CD38- cells, and these cells took over hematopoiesis by 9wks. Instead, early reconstitution was mainly driven by CD34+CD38int progenitor cells. In the prospect of a clinical translation, we then modeled the co-administration of gene-modified CD34+CD38- mPB cells with uncultured CD34+CD38int/+ supporter cells aimed to drive fast hematopoietic recovery (3 exp, n=38). Repopulation by genemodified CD34+CD38- cells was slower (15wks) and incomplete (<60% at 24wks) in this setting. Additional, non-competitive transplants confirmed that freshly isolated CD34+CD38int cells possess long-term reconstituting capacity, which is lost after 2 days of standard culture. To improve the engraftment of the gene-modified CD34+CD38- cells when administered with uncultured CD38int/+ supporter cells, we optimized culture conditions of the CD38- and cell doses of the CD38int/+ population. We achieved accelerated engraftment kinetics of gene-modified CD38- cells by >5 wks and Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
re-established long-term (24wks) gene marking up to 85%, thus allowing to benefit from prompt hematopoietic recovery driven by transiently repopulating CD38int/+ supporter cells. Last, we optimized LV transduction in the framework of an improved culture protocol. Exposing CD34+ or CD34+CD38- mPB cells to prostaglandin E2 (PGE2) increased transduction efficiency 1.5-2.5x, allowing to markedly reduce pre-stimulation and LV exposure times better preserving HSC functions. Importantly, the higher gene-transfer efficiency was maintained for up to 24 wks following xenotransplantation (n=33), suggesting that PGE2 facilitated LV transduction in long-term HSC. In summary, these results support the clinical development of novel HSPC gene therapy protocols based on the modification of highly purified HSC subsets, with the prospect to improve the efficacy, safety and feasibility of future ex vivo gene therapy studies.
296. Pre-Selection of Anti-HIV Lentiviral Vector Gene Modified Hematopoietic Stem Cells Significantly Improves Protection from HIV Infection: The Basis for a Future Clinical Trial
Sharlie L. Barclay,1 Yimin Yang,1 Sirou Zhang,1 Ryan Fong,1 Alfonso Barraza,1 Jan A. Nolta,1 Mehrdad Abedi,1 Joseph S. Anderson.1 1 Internal Medicine, University of California Davis, Sacramento, CA. The successful treatment and cure of HIV in the Berlin Patient has highlighted the utility of HIV-resistant hematopoietic stem cells (HSC) to offer a potential functional cure for HIV infected individuals. Stem cell gene therapy for HIV can mimic this result by genetically engineering a patient’s own HSC with lentiviral vectors encoding HIV resistance genes. Clinical trials of HIV stem cell gene therapy have been unsuccessful at demonstrating complete efficacy due to the transplantation of a low percentage of anti-HIV gene expressing cells. Therefore, to bridge the gap between these previous approaches and the Berlin patient, we have developed a pre-selection process which allows for the purification of vector transduced cells prior to transplantation into patients. This pre-selective lentiviral vector (1TAX) encodes a triple combination of anti-HIV genes (a human/ rhesus macaque TRIM5alpha, a CCR5 shRNA, and a TAR decoy) in addition to a truncated/mutated form of human CD25 which is expressed on the cell surface of transduced HSC and is used to enrich for the HIV-resistant cell population. Purity levels of 1TAX transduced human HSC obtained after selection for CD25+ were >94% as determined by flow cytometry. Safety of the 1TAX vector transduced/purified human HSC was demonstrated in vivo in NOD-SCID-Rag-/-IL2rgamma-/(NRG) mice with the detection of engraftment and multi-lineage hematopoiesis into human T cells, B cells, and macrophages in the peripheral blood and lymphoid organs. Upon infection of 1TAX mice with a CCR5-tropic BaL-1 or a CXCR4-tropic NL43 strain of HIV-1, maintenance of normal human CD4+ cell levels were observed in the peripheral blood. A significant decrease (>4 logs) in HIV-1 plasma viremia was also demonstrated in 1TAX mice as compared to mice transplanted with nontransduced human HSC. Together, this work has demonstrated the safety and efficacy of this 1TAX pre-selective lentiviral vector in HSC and is the basis for a future human clinical trial of HIV stem cell gene therapy.
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