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825. Immune Modulation in Hemophilia A Mice Using Anti-CD20 and Liver-Directed Gene Transfer
HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY II 824. Recombinant AAV2-Mediated β-Globin Expression in Human Fetal Hematopoietic Cells from the Aborted Fetus with β-Thalassemia
Jing Tian,1,2 Feng Wang,1,2 Jin-feng Xue,1,2 Fei Zhou,1,2 Liu-jiang Song,1,2 Meng-qun Tan.1,2 1 Physiology, Central South University Xiang-Ya School of Medicine, Changsha, Hunan, China; 2Xiang-Ya Bio-Medicine Institute, Shenzhen, Guangdong, China. Background: The most severe form of β-Thalassemia, β°Thalassemia major, is characterized by complete absence of normal β-globin chain, and is often lethal. Autologous transplantation of genetically-modified hematopoietic stem cells (HSCs) using lentiviral vectors is currently being pursued as a possible treatment. As an alternative, recombinant adeno-associated virus (rAAV) vectors offer promise due to their low immunogenicity, broad host-range, and efficient infection of a wide variety of cell types. We have previously documented rAAV2-mediated β-globin gene transfer and expression in human fetal liver cells in vivo. In this study, we investigated whether rAAV2 could also mediate expression of normal β-globin gene in human HSCs from β-Thalassemia patients. Methods: Human HSCs were isolated from two aborted fetuses at 20 and 24 weeks of gestation. One fetus was heterozygous for β41/42(-TCTT)/ β71/72(+A), and the other was β41/42(-TCTT) /βE. The isolated HSCs were mock-transduced or transduced with rAAV2-β-globin at 5×104 v/cell, followed by i.v. injection into sub-lethally irradiated BALB/c nude mice. Recipient mice were analyzed longitudinally on days 14, 21 for β41/42/β71/72 fetus, and days 21, 35 for β41/42/βE fetus. Human β-globin gene expression in recipient mice was characterized by RT-PCR and allele-specific PCR (ASPCR). The levels of human globin expression were determined by HPLC. Results: Human β-actin and β-globin transcripts were detected by RT-PCR in bone marrow cells from all recipient mice, indicating that human hematopoietic cells were successfully transplanted in these mice and that human β-globin gene was transcriptionally active. ASPCR analyses revealed that whereas the mutant β41-42/β71-72 b-chain gene transcripts were detected in all recipients, the normal b-chain gene transcripts were only detected in mice transplanted with rAAV2-b-globin transduced human HSCs. For β41/42/βE fetus, the mutant β41/42 gene transcripts were detected using the mutant primers, while the mutant βE and normal b-chain gene transcripts were detected using normal primers. However, only normal b-chain fragments, but not βE mutant, were sensitive to MnLI restriction enzyme, consistent with the loss of MnLI cleavage site in the βE mutant allele. The level of human hemoglobin expressed in red blood cells of recipient mice was measured using a highly sensitive HPLC. Expression of human β-globin was confirmed in recipient mice by mass spectrometry. The b-chain to a-chain ratio in recipient mice transplanted with rAAV2-b-globin-transduced human HSCs was improved over that in mice that received mocktransduced cells. Conclusion: Our results indicate that human HSCs from β-Thalassemic fetuses can be efficiently transduced by rAAV2-β-globin vectors followed by expression of normal human β-globin protein. These studies provide the proof-of-principle that rAAV2-mediated gene transfer into human HSCs may be a feasible approach for gene therapy of β-Thalassemia.
825. Immune Modulation in Hemophilia A Mice Using Anti-CD20 and Liver-Directed Gene Transfer Brandon K. Sack,1 Babak Moghimi,1 Roland W. Herzog.1 1 Pediatrics, University of Florida, Gainesville, FL.
B cell depleting antibodies are gaining popularity as a solution to antibody-mediated diseases including neutralizing antibodies to FVIII in hemophilia. We used an anti-murine CD20 IgG2a antibody (kindly provided by Biogen Idec) to deplete B cells in two strains of hemophilia A mice and investigated the potential of this treatment Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
to induce tolerance to FVIII when combined with gene therapy. To deplete CD20+ B cells, either BALB/c or mixed-background BL/6-129/sv mice were given 2 doses of 10mg/kg αCD20 (3 weeks apart). Depletion of B cells was confirmed by flow cytometry of peripheral blood and secondary lymphoid organs. Mice in the group “AAV8+αCD20” (n=5 each strain) were given 1011vg/mouse of an AAV8 vector expressing FVIII under a liver-specific promoter one week after the first αCD20 injection. A second group (n=5 for each strain) was given AAV8-FVIII only. Ten weeks after AAV8-FVIII treatment (8 weeks after last αCD20 treatment), the mice were challenged iv with 1U/mouse/week of FVIII protein for 4 weeks. BL/6-129/sv mice showed mild and transient decrease in clotting times with the AAV8+αCD20 group reaching 0.5-1.25% of normal FVIII levels compared to >0.5% in AAV8-only mice. All mice returned to untreated levels after protein challenge. However, the two groups differed dramatically in the magnitude of their antibody responses to FVIII challenge. BL/6-129/sv mice in the AAV8-only group had a mean anti-FVIII IgG1 titer of 7001ng/ml (±867), corresponding to 336 BU (±88). AAV8+αCD20 mice were hypo-responsive with anti-FVIII IgG1 titers of 1609ng/mL (±868) and Bethesda titers of 22 BU (±11). BALB/c mice treated in the same manner showed better efficacy of gene therapy, with clotting activities reaching 1-2% in both AAV8-only and AAV8+αCD20 groups. Following challenge as above, 1/3 mice in the AAV8-only developed an antibody response (544 ng IgG1/mL, 5.3 BU). None of the 5 mice treated with both αCD20 and AAV8-FVIII developed a detectable antibody response. Seven weeks after the last FVIII injection, mice were challenged for a second time. One week later, mice were tested for the ability to have their clotting times corrected with exogenous F.VIII protein. Indeed, AAV8+αCD20 and the 2 tolerant AAV8-only mice showed correction of clotting similar to that in naïve mice receiving F.VIII for the first time. Control mice formed inhibitors in response to protein therapy, and therefore their coagulation times could not be corrected. Importantly, the antibody response of mice initially receiving only αCD20 did not differ in any respect from untreated HA mice, indicating that immune competence had been regained over time as B cells repopulated the immune system. Cytokine RNA analysis of splenocytes stimulated with F.VIII showed a reduction in the expression of IL-2, IL-4 and IFN-gamma in AAV8+αCD20 mice but little change in expression of markers for Tregs (FoxP3, CTLA-4 and CD25), suggesting that a robust Treg response may not be responsible for the long-term tolerance observed. In summary, hepatic AAV gene transfer during transient B cell depletion may be an optimal immune modulatory therapy to achieve tolerance to conventional protein therapy for hemophilia A.
826. Differential Lineage Transduction Results in Limited FVIII Expression after Ex Vivo Modification of Hematopoietic Progenitor Cells
Jennifer M. Johnston,1 Christopher B. Doering,1 H. Trent Spencer.1 Emory University, Atlanta, GA.
1
We recently engineered a high expression factor VIII transgene (HP-fVIII) and successfully treated hemophilia A mice using genemodified hematopoietic stem cells. We have optimized several aspects of the viral vector including 1) comparison of HIV and SIVbased vectors, 2) comparison of internal promoters in SIN vectors, specifically eF1alpha, CMV, PGK and the MSCV oncoretroviral promoter, 3) the value of the WPRE sequence, and 4) various HP-fVIII sequences. A final optimized construct contains a B-domain deleted chimeric human/porcine transgene containing only 10% porcine fVIII sequence, which is required to confer high level expression. The transgene is driven by an eF1-alpha internal promoter, and the WPRE element was shown to be inconsequential as it provided no benefit to fVIII expression or viral titer and did not limit transcription through the 3’ LTR. In addition to vector optimization, we tested the S315
Jing Tian,1,2 Feng Wang,1,2 Jin-feng Xue,1,2 Fei Zhou,1,2 Liu-jiang Song,1,2 Meng-qun Tan.1,2 1 Physiology, Central South University Xiang-Ya School of Medicine, Changsha, Hunan, China; 2Xiang-Ya Bio-Medicine Institute, Shenzhen, Guangdong, China. Background: The most severe form of β-Thalassemia, β°Thalassemia major, is characterized by complete absence of normal β-globin chain, and is often lethal. Autologous transplantation of genetically-modified hematopoietic stem cells (HSCs) using lentiviral vectors is currently being pursued as a possible treatment. As an alternative, recombinant adeno-associated virus (rAAV) vectors offer promise due to their low immunogenicity, broad host-range, and efficient infection of a wide variety of cell types. We have previously documented rAAV2-mediated β-globin gene transfer and expression in human fetal liver cells in vivo. In this study, we investigated whether rAAV2 could also mediate expression of normal β-globin gene in human HSCs from β-Thalassemia patients. Methods: Human HSCs were isolated from two aborted fetuses at 20 and 24 weeks of gestation. One fetus was heterozygous for β41/42(-TCTT)/ β71/72(+A), and the other was β41/42(-TCTT) /βE. The isolated HSCs were mock-transduced or transduced with rAAV2-β-globin at 5×104 v/cell, followed by i.v. injection into sub-lethally irradiated BALB/c nude mice. Recipient mice were analyzed longitudinally on days 14, 21 for β41/42/β71/72 fetus, and days 21, 35 for β41/42/βE fetus. Human β-globin gene expression in recipient mice was characterized by RT-PCR and allele-specific PCR (ASPCR). The levels of human globin expression were determined by HPLC. Results: Human β-actin and β-globin transcripts were detected by RT-PCR in bone marrow cells from all recipient mice, indicating that human hematopoietic cells were successfully transplanted in these mice and that human β-globin gene was transcriptionally active. ASPCR analyses revealed that whereas the mutant β41-42/β71-72 b-chain gene transcripts were detected in all recipients, the normal b-chain gene transcripts were only detected in mice transplanted with rAAV2-b-globin transduced human HSCs. For β41/42/βE fetus, the mutant β41/42 gene transcripts were detected using the mutant primers, while the mutant βE and normal b-chain gene transcripts were detected using normal primers. However, only normal b-chain fragments, but not βE mutant, were sensitive to MnLI restriction enzyme, consistent with the loss of MnLI cleavage site in the βE mutant allele. The level of human hemoglobin expressed in red blood cells of recipient mice was measured using a highly sensitive HPLC. Expression of human β-globin was confirmed in recipient mice by mass spectrometry. The b-chain to a-chain ratio in recipient mice transplanted with rAAV2-b-globin-transduced human HSCs was improved over that in mice that received mocktransduced cells. Conclusion: Our results indicate that human HSCs from β-Thalassemic fetuses can be efficiently transduced by rAAV2-β-globin vectors followed by expression of normal human β-globin protein. These studies provide the proof-of-principle that rAAV2-mediated gene transfer into human HSCs may be a feasible approach for gene therapy of β-Thalassemia.
825. Immune Modulation in Hemophilia A Mice Using Anti-CD20 and Liver-Directed Gene Transfer Brandon K. Sack,1 Babak Moghimi,1 Roland W. Herzog.1 1 Pediatrics, University of Florida, Gainesville, FL.
B cell depleting antibodies are gaining popularity as a solution to antibody-mediated diseases including neutralizing antibodies to FVIII in hemophilia. We used an anti-murine CD20 IgG2a antibody (kindly provided by Biogen Idec) to deplete B cells in two strains of hemophilia A mice and investigated the potential of this treatment Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
to induce tolerance to FVIII when combined with gene therapy. To deplete CD20+ B cells, either BALB/c or mixed-background BL/6-129/sv mice were given 2 doses of 10mg/kg αCD20 (3 weeks apart). Depletion of B cells was confirmed by flow cytometry of peripheral blood and secondary lymphoid organs. Mice in the group “AAV8+αCD20” (n=5 each strain) were given 1011vg/mouse of an AAV8 vector expressing FVIII under a liver-specific promoter one week after the first αCD20 injection. A second group (n=5 for each strain) was given AAV8-FVIII only. Ten weeks after AAV8-FVIII treatment (8 weeks after last αCD20 treatment), the mice were challenged iv with 1U/mouse/week of FVIII protein for 4 weeks. BL/6-129/sv mice showed mild and transient decrease in clotting times with the AAV8+αCD20 group reaching 0.5-1.25% of normal FVIII levels compared to >0.5% in AAV8-only mice. All mice returned to untreated levels after protein challenge. However, the two groups differed dramatically in the magnitude of their antibody responses to FVIII challenge. BL/6-129/sv mice in the AAV8-only group had a mean anti-FVIII IgG1 titer of 7001ng/ml (±867), corresponding to 336 BU (±88). AAV8+αCD20 mice were hypo-responsive with anti-FVIII IgG1 titers of 1609ng/mL (±868) and Bethesda titers of 22 BU (±11). BALB/c mice treated in the same manner showed better efficacy of gene therapy, with clotting activities reaching 1-2% in both AAV8-only and AAV8+αCD20 groups. Following challenge as above, 1/3 mice in the AAV8-only developed an antibody response (544 ng IgG1/mL, 5.3 BU). None of the 5 mice treated with both αCD20 and AAV8-FVIII developed a detectable antibody response. Seven weeks after the last FVIII injection, mice were challenged for a second time. One week later, mice were tested for the ability to have their clotting times corrected with exogenous F.VIII protein. Indeed, AAV8+αCD20 and the 2 tolerant AAV8-only mice showed correction of clotting similar to that in naïve mice receiving F.VIII for the first time. Control mice formed inhibitors in response to protein therapy, and therefore their coagulation times could not be corrected. Importantly, the antibody response of mice initially receiving only αCD20 did not differ in any respect from untreated HA mice, indicating that immune competence had been regained over time as B cells repopulated the immune system. Cytokine RNA analysis of splenocytes stimulated with F.VIII showed a reduction in the expression of IL-2, IL-4 and IFN-gamma in AAV8+αCD20 mice but little change in expression of markers for Tregs (FoxP3, CTLA-4 and CD25), suggesting that a robust Treg response may not be responsible for the long-term tolerance observed. In summary, hepatic AAV gene transfer during transient B cell depletion may be an optimal immune modulatory therapy to achieve tolerance to conventional protein therapy for hemophilia A.
826. Differential Lineage Transduction Results in Limited FVIII Expression after Ex Vivo Modification of Hematopoietic Progenitor Cells
Jennifer M. Johnston,1 Christopher B. Doering,1 H. Trent Spencer.1 Emory University, Atlanta, GA.
1
We recently engineered a high expression factor VIII transgene (HP-fVIII) and successfully treated hemophilia A mice using genemodified hematopoietic stem cells. We have optimized several aspects of the viral vector including 1) comparison of HIV and SIVbased vectors, 2) comparison of internal promoters in SIN vectors, specifically eF1alpha, CMV, PGK and the MSCV oncoretroviral promoter, 3) the value of the WPRE sequence, and 4) various HP-fVIII sequences. A final optimized construct contains a B-domain deleted chimeric human/porcine transgene containing only 10% porcine fVIII sequence, which is required to confer high level expression. The transgene is driven by an eF1-alpha internal promoter, and the WPRE element was shown to be inconsequential as it provided no benefit to fVIII expression or viral titer and did not limit transcription through the 3’ LTR. In addition to vector optimization, we tested the S315