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1054. Hematopoietic and Non-Hematopoietic Engraftment after Bone Marrow Transplantation in Newborn Mice
STEM CELL GENE THERAPY irradiation showed that the transferred p53 trans-activated the p53related gene, endogenous mouse p21. Further, radiosensitivity in the p53-/-mGS(p53HAC) had a significantly higher after irradiation as compared to parental p53-/-mGS cells. These data suggest that the HAC vector containing a p53 genomic insert mediates the production of functional human p53 protein and the HAC was able to rescue the p53-/-phenotypes in the p53-/-mGS cells. Next, the p53-/-mGS(p53HAC) cells were injected into nude mice to assess the capacity of differentiation. The transplanted p53-/mGS(p53HAC) cells gave rise to typical teratomas and some differentiated cell types were detected. Thus, we demonstrated that HAC vector could be transferable to mGS cells and HAC vector containing desired genome corrected a genetic defect in mGS cells by MMCT approach. In future, a combination of mGS cells and HAC-mediated gene delivery may provide a useful treatment for genetic defects, when efficient methods for differentiation in stem cells including mGS cells are developed.
1054. Hematopoietic and Non-Hematopoietic Engraftment after Bone Marrow Transplantation in Newborn Mice Emanuela M. Bruscia,1 Joanna E. Price,1 Elizabeth Ziegler,2 Diane S. Krause.1 1 Laboratory Medicine, Yale University School of Medicine, New Haven, CT; 2Section of Immunobiology and Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT. Classical gene therapy protocols have employed different vehicles (e.g. viruses, liposomes, peptides) for tissue specific gene delivery. An alternative approach for delivery of therapeutic genes to specific tissues is to use adult stem cells as the ‘vector’ for a combination of cell and gene therapy. Ideally, bone marrow derived cells (BMDC) could be taken from a patient, genetically corrected in vitro via gene therapy, and then infused back to the patient to repair damaged tissue as fully functional genetically corrected cells. Recent discoveries show that, in addition to hematopoietic stem cells (HSC) that differentiate into all mature cells of the blood, there are BMDC that can engraft as epithelial cells throughout the body including the lung, liver and GI tract following transplantation into lethally irradiated recipients. Thus, autologous BM transplantation (BMT) based gene therapy could be useful for diseases associated with dysfunctions of these tissues. Treatments for several genetic diseases, to be effective, should be performed at the time of birth or in early childhood. BMT preparative regimens are well described for adult mice, but protocols for myelosuppression in newborn murine pups are few. Here, we compared different preparative regimens for newborn mice including: a) two doses of busulfan (B) in utero (15gr/ kg each), b) one dose of busulfan (15gr/kg) in utero plus 400 cGy total body irradiation (TBI) on day 1 of life, c) 400 or 750cGy TBI on day 1 of life, or d) no pretreatment. For each regimen, the pups were transplanted with BM within 24h of birth (after irradiation). In order to determine which method is least toxic to the newborn mice while allowing a high level of hematopoietic engraftment, we assessed peripheral blood counts as well as donor hematopoietic and nonhematopoietic (epithelial) engraftment were evaluated at multiple times post-transplant. The level of toxicity was from highest to lowest: TBI 750 > 1B+ TBI400 > TBI400 > 2xB > no myelosuppression, and TBI 750 mice were sacrificed prior to 1 month post-BMT. The scale of hematopoietic chimerism was from best to worst: 1B +TBI400 > TBI750=TBI400 > 2xB > non-
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myeloablation, with peripheral blood chimerism up to 86% in 1B+ TBI400 treated mice and just 1.0% for non-myeloablated mice. BM derived (BMD) epithelial cells were found in lung and GI but not in the liver of transplanted recipients. The percentage of BMD epithelial cells ranged from 0-0.02% with the highest levels in the 2xB groups and 1B+ TBI400 (1B+ TBI400 = 2xB > 400TBI > no myeloablation). This level of BMD epithelial cells is similar to the percentage found after BM transplantation into lethally irradiated adult mice. In conclusion, our data suggest that the best myelosuppressive preparative regimens for the hematopoietic and nonhematopoietic engraftment include in utero exposure to busulfan, and that BMT in newborn mice leads to levels of epithelial engraftment that are similar to those in adult recipients.
1055. High Levels of Human Factor IX Expression in Hemophilia B Mice after Adult Stem Cell-Based Gene Therapy Combined with NonMyeloablative Conditioning Alex H. Chang,1 Leszek Lisowski,1 Michel Sadelain.1 1 Laboratory of Gene Transfer and Gene Expression, SloanKettering Research Institute, New York, NY. Various cell types have been targeted for expression of clotting factors in experimental models of hemophilia, with muscle and hepatocyte-directed gene transfer advancing to clinical trials. Hematopoietic stem cells (HSCs) are attractive alternative targets because of their self-renewing properties, their ability to generate blood cells over extended periods of time, and their ease of manipulation ex vivo. However, several hurdles must be overcome to make this approach safe and practical. These include the need to express sufficient amount of therapeutic gene products and minimize the risk of insertional mutagenesis and the toxicity of host conditioning to facilitate HSC engraftment. We have previously developed an erythoid cell-specific platform for long-term and systemic therapeutic protein delivery in vivo using an erythroidspecific lentiviral vector. We demonstrated in an immune competent C57BL/6 hemophilia B mouse model, that therapeutic levels of human factor IX (hFIX) can be expressed at low average vector copy (VC) number (250-350 ng/mL with under 0.5 VC per cell). In this study, we examined the level of erythroid-specific protein delivery obtained after engraftment following minimal conditioning, eventually in the presence of in vivo methylguanine methyltransferase (MGMT)-mediated drug resistance. Low to moderate levels of hFIX expression were achieved in control, lethally irradiated mice (average 100-200 ng/ml), 4-weeks post-transplantation. In non-myeloablated recipients conditioned with 400 cGy or Busulfan (20 mg/kg, given twice at 48 h and 24 h before engraftment), the levels of hFIX expression were ranged from negligible to low (0-20 ng/ml and 0-100 ng/ml, respectively). Four weeks after the first round of BG/BCNU (30 mg/kg and 5 mg/kg, respectively), a wide range of hFIX expression levels were achieved, reaching in some mice levels equivalent to 20% of physiological levels in humans. We are currently monitoring longterm hFIX expression, as well as the average VC number in blood cells and CFUs. Further rounds of drug selection are also in progress. In conclusion, our data suggest that it is possible to express high levels of a secreted protein from HSC-derived erythroid cells engrafted under minimal conditioning. The combined features of reduced intensity conditioning, reduced insertional mutagenesis due to low vector copy number requirement and erythroid-specific transgene expression, as well as long-term protein expression at therapeutic levels, increase the potential applicability of adult stem cell-based gene therapy in non-lethal disorders such as hemophilia.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
1054. Hematopoietic and Non-Hematopoietic Engraftment after Bone Marrow Transplantation in Newborn Mice Emanuela M. Bruscia,1 Joanna E. Price,1 Elizabeth Ziegler,2 Diane S. Krause.1 1 Laboratory Medicine, Yale University School of Medicine, New Haven, CT; 2Section of Immunobiology and Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT. Classical gene therapy protocols have employed different vehicles (e.g. viruses, liposomes, peptides) for tissue specific gene delivery. An alternative approach for delivery of therapeutic genes to specific tissues is to use adult stem cells as the ‘vector’ for a combination of cell and gene therapy. Ideally, bone marrow derived cells (BMDC) could be taken from a patient, genetically corrected in vitro via gene therapy, and then infused back to the patient to repair damaged tissue as fully functional genetically corrected cells. Recent discoveries show that, in addition to hematopoietic stem cells (HSC) that differentiate into all mature cells of the blood, there are BMDC that can engraft as epithelial cells throughout the body including the lung, liver and GI tract following transplantation into lethally irradiated recipients. Thus, autologous BM transplantation (BMT) based gene therapy could be useful for diseases associated with dysfunctions of these tissues. Treatments for several genetic diseases, to be effective, should be performed at the time of birth or in early childhood. BMT preparative regimens are well described for adult mice, but protocols for myelosuppression in newborn murine pups are few. Here, we compared different preparative regimens for newborn mice including: a) two doses of busulfan (B) in utero (15gr/ kg each), b) one dose of busulfan (15gr/kg) in utero plus 400 cGy total body irradiation (TBI) on day 1 of life, c) 400 or 750cGy TBI on day 1 of life, or d) no pretreatment. For each regimen, the pups were transplanted with BM within 24h of birth (after irradiation). In order to determine which method is least toxic to the newborn mice while allowing a high level of hematopoietic engraftment, we assessed peripheral blood counts as well as donor hematopoietic and nonhematopoietic (epithelial) engraftment were evaluated at multiple times post-transplant. The level of toxicity was from highest to lowest: TBI 750 > 1B+ TBI400 > TBI400 > 2xB > no myelosuppression, and TBI 750 mice were sacrificed prior to 1 month post-BMT. The scale of hematopoietic chimerism was from best to worst: 1B +TBI400 > TBI750=TBI400 > 2xB > non-
S404
myeloablation, with peripheral blood chimerism up to 86% in 1B+ TBI400 treated mice and just 1.0% for non-myeloablated mice. BM derived (BMD) epithelial cells were found in lung and GI but not in the liver of transplanted recipients. The percentage of BMD epithelial cells ranged from 0-0.02% with the highest levels in the 2xB groups and 1B+ TBI400 (1B+ TBI400 = 2xB > 400TBI > no myeloablation). This level of BMD epithelial cells is similar to the percentage found after BM transplantation into lethally irradiated adult mice. In conclusion, our data suggest that the best myelosuppressive preparative regimens for the hematopoietic and nonhematopoietic engraftment include in utero exposure to busulfan, and that BMT in newborn mice leads to levels of epithelial engraftment that are similar to those in adult recipients.
1055. High Levels of Human Factor IX Expression in Hemophilia B Mice after Adult Stem Cell-Based Gene Therapy Combined with NonMyeloablative Conditioning Alex H. Chang,1 Leszek Lisowski,1 Michel Sadelain.1 1 Laboratory of Gene Transfer and Gene Expression, SloanKettering Research Institute, New York, NY. Various cell types have been targeted for expression of clotting factors in experimental models of hemophilia, with muscle and hepatocyte-directed gene transfer advancing to clinical trials. Hematopoietic stem cells (HSCs) are attractive alternative targets because of their self-renewing properties, their ability to generate blood cells over extended periods of time, and their ease of manipulation ex vivo. However, several hurdles must be overcome to make this approach safe and practical. These include the need to express sufficient amount of therapeutic gene products and minimize the risk of insertional mutagenesis and the toxicity of host conditioning to facilitate HSC engraftment. We have previously developed an erythoid cell-specific platform for long-term and systemic therapeutic protein delivery in vivo using an erythroidspecific lentiviral vector. We demonstrated in an immune competent C57BL/6 hemophilia B mouse model, that therapeutic levels of human factor IX (hFIX) can be expressed at low average vector copy (VC) number (250-350 ng/mL with under 0.5 VC per cell). In this study, we examined the level of erythroid-specific protein delivery obtained after engraftment following minimal conditioning, eventually in the presence of in vivo methylguanine methyltransferase (MGMT)-mediated drug resistance. Low to moderate levels of hFIX expression were achieved in control, lethally irradiated mice (average 100-200 ng/ml), 4-weeks post-transplantation. In non-myeloablated recipients conditioned with 400 cGy or Busulfan (20 mg/kg, given twice at 48 h and 24 h before engraftment), the levels of hFIX expression were ranged from negligible to low (0-20 ng/ml and 0-100 ng/ml, respectively). Four weeks after the first round of BG/BCNU (30 mg/kg and 5 mg/kg, respectively), a wide range of hFIX expression levels were achieved, reaching in some mice levels equivalent to 20% of physiological levels in humans. We are currently monitoring longterm hFIX expression, as well as the average VC number in blood cells and CFUs. Further rounds of drug selection are also in progress. In conclusion, our data suggest that it is possible to express high levels of a secreted protein from HSC-derived erythroid cells engrafted under minimal conditioning. The combined features of reduced intensity conditioning, reduced insertional mutagenesis due to low vector copy number requirement and erythroid-specific transgene expression, as well as long-term protein expression at therapeutic levels, increase the potential applicability of adult stem cell-based gene therapy in non-lethal disorders such as hemophilia.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy