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69. Selection of Hematopoietic Progenitor and Stem Cells in Transplanted Rhesus Macaques Using MGMT and HOXB4 Lentiviral Vectors
HEMATOLOGIC 68. Evidence for Correction of Mutations in the c-Kit Gene by rAAV Mediated Gene Targeting in W/Wv Mice
69. Selection of Hematopoietic Progenitor and Stem Cells in Transplanted Rhesus Macaques Using MGMT and HOXB4 Lentiviral Vectors
Arthur W. Nienhuis,1 John T. Gray,1 Serena Vayda,2 Amanda Cline,2 Marguerite V. Evans-Galea,1 David M. Bodine.2 1 Hematology, St. Jude Children’s Reserach Hospital, Memphis, TN; 2Genetics and Molecular Biology, National Human Genome Research Institute, Bethesda, MD.
Yan Shou,1 John Gray,1 Brian Agricola,1 Zhijun Ma,1 Geeta K. Nair,2 Clinton F. Stewart,2 Derek A. Persons,1 Brian P. Sorrentino.1 1 Experimental Hematology, St. Jude Children’s Research Hospital, Memphis, TN; 2Pharmaceutical Sciences.
Recombinant adeno-associated viral vectors (rAAV) correct genetic lesions by gene targeting via interactions between homologous sequences in the vector and a chromosomal gene at a frequency of up to 1% in cultured and primary cells (reviewed in Hendrie PC, Russell DW, Mol Ther. 12:9-17, 2005). Although gene addition with retroviral vectors has proven therapeutic for Severe Combined Immunodeficiency (SCID), integration carries the risk for insertional mutagenesis and proto-oncogene activation (Science 302: 415-419, 2003) so that alternative strategies are needed. Mice with mutations in both c-Kit genes (W locus) are sterile, have defects in pigmentation and have mild macrocytic anemia. Useful assays for gene correction are available since bone marrow cells from W/Wv mice do not form 14 day spleen colonies or achieve long-term engraftment of lethally irradiated mice. A single wild-type cell is able to reconstitute unirradiated W/Wv mice. The W mutation, a G to A substitution in the splice donor site at the exon 10 - intron 10 boundary, eliminates an AarI site and the Wv mutation, a C to T transition in the last codon of exon 13, creates a NsiI site. The targeting vector, rAAV-cKit, was derived by subcloning a 4.6 kb EcoR1-BglII fragment from the c-Kit locus, recovered from a BAC clone (RP23-2), into a plasmid containing rAAV2 ITRs. Using a self-complementary rAAV2 vector genome encoding Green Fluorescent Protein (GFP), we found that vector particles packaged with AAV serotype 1 capsid proteins were 10 fold more efficient at transducing lineage depleted, c-Kit+, ScaI+ murine bone marrow cells than particles packaged in serotype 2 capsid proteins. At a MOI of 105 serotype 1 vector particles/ target cell, more than 95% of the c-Kit+, ScaI+ cells were transduced. Using optimized conditions, we transduced 1.2 x 107 lineage depleted W/Wv bone marrow cells with 1.2 x 1012 rAAV-c-Kit, serotype 1 vector particles in serum-free medium containing 100 ng/ml of SCF, TPO, IL-6 and Flt-3L and 10 ng/ml of IL-3. Many spleen colonies were present in irradiated recipients 14 days after injection with transduced cells but none were found in controls which received mock transduced W/Wv cells. Southern blot analysis provided evidence that the AarI and NsiI sites reflecting the W and Wv mutations, respectively, had been completely or partially changed (AarI gained and NsiI lost) in the majority (4 of 5) colonies suggesting biallelic correction during colony development. In an initial experiment designed to evaluate the ability of rAAV-c-Kit to achieve gene correction in long-term repopulating cells, we found that 2 of 5 irradiated wildtype mice injected with transduced W/Wv lineage depleted cells survived at 6 weeks with normal hematopoiesis. Overall, our data suggest that rAAV vectors can be used to achieve gene correction in primitive, primary hematopoietic cells at a detectable frequency which may be useful for gene therapy of disorders such as SCID in which there is a positive selective advantage for gene corrected cells.
Gene transfer of O6-methylguanine-DNA-methyltransferase (MGMT) into HSCs results in efficient in vivo selection of murine HSCs after treatment with temozolomide (TMZ) and O 6 benzylguanine (BG). In addition, HOXB4 coexpression is associated with an increase in HSC selection efficiency using a DHFR drug selection system in mice. Based on these results, we have studied whether the MGMT and HOXB4 selection systems are efficacious in a rhesus macaque autologous transplantation model. SIV vectors were constructed incorporating either MGMT alone or MGMT together with HOXB4 in a GFP or YFP backbone. In our first transplant case, we purified CD34+ cells from mobilized bone marrow cells and split the graft into two parts: half of the cells were transduced with a SIV MGMT-GFP vector and half were transduced with a SIV MGMT-HOXB4-GFP vector. In our second and third transplant cases, half of the CD34+ cells were transduced with a SIV MGMTYFP vector and the other half with a SIV MGMT-HOXB4-GFP vector. During the early engraftment period, there was a slight advantage (2-fold maximal) for HOXB4 transduced cells relative to the “MGMT-only” vector. However, by 1 month posttransplantation, there was no difference in marking between the two vectors, suggesting HOXB4 had little effect on long-term repopulating cells. The first animal has been treated with 7 courses of TMZ/BG, starting with a fixed dose of 120 mg/m2 for BG and 170 mg/m2/day for 5 days for TMZ. The dose of TMZ was escalated on each course, and well tolerated by the animal. For the 7th course, the animal received 450 mg/m2 of TMZ with BG, a significant dose increase relative to the pediatric maximum tolerated dose (185 mg/ m2 without BG in irradiated patients). This increase in dose intensity was verified by formal pharmacokinetic measurements. After each course of drug treatment, there was a rapid increase in GFP marking in all lineages. The highest marking achieved after last course of TMZ/BG treatment was 91% for myeloid cells and 50% for lymphoid lineages. However, the proportion of GFP + cells subsequently declined and leveled off at 13% for granulocytes, 17% for B cells and 36% for T cells. The second animal had been treated with two courses of TMZ/BG and there was about 7-fold increase in GFP marking in granulocytes and a 3 and 8-fold increase in B, T cells respectively. Altogether, we have shown that TMZ selection was occurring mainly in progenitor cells with limited self-renewal capacity and to a lesser degree at the HSC level as evidence by increased baseline GFP marking. We were also able to achieve a significant increase in dose intensity in the first animal, and to successfully administer multiple courses of drug treatment. These results suggest a clinical strategy for hematopoietic protection in cancer treatment. We are now treating these animals with BCNU plus BG to determine if HSC selection efficiency is increased relative to TMZ. Lastly, we did not see evidence of HSC amplification with HOXB4 expression, suggesting that the robust selection seen in mice is relatively species specific.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
S29
69. Selection of Hematopoietic Progenitor and Stem Cells in Transplanted Rhesus Macaques Using MGMT and HOXB4 Lentiviral Vectors
Arthur W. Nienhuis,1 John T. Gray,1 Serena Vayda,2 Amanda Cline,2 Marguerite V. Evans-Galea,1 David M. Bodine.2 1 Hematology, St. Jude Children’s Reserach Hospital, Memphis, TN; 2Genetics and Molecular Biology, National Human Genome Research Institute, Bethesda, MD.
Yan Shou,1 John Gray,1 Brian Agricola,1 Zhijun Ma,1 Geeta K. Nair,2 Clinton F. Stewart,2 Derek A. Persons,1 Brian P. Sorrentino.1 1 Experimental Hematology, St. Jude Children’s Research Hospital, Memphis, TN; 2Pharmaceutical Sciences.
Recombinant adeno-associated viral vectors (rAAV) correct genetic lesions by gene targeting via interactions between homologous sequences in the vector and a chromosomal gene at a frequency of up to 1% in cultured and primary cells (reviewed in Hendrie PC, Russell DW, Mol Ther. 12:9-17, 2005). Although gene addition with retroviral vectors has proven therapeutic for Severe Combined Immunodeficiency (SCID), integration carries the risk for insertional mutagenesis and proto-oncogene activation (Science 302: 415-419, 2003) so that alternative strategies are needed. Mice with mutations in both c-Kit genes (W locus) are sterile, have defects in pigmentation and have mild macrocytic anemia. Useful assays for gene correction are available since bone marrow cells from W/Wv mice do not form 14 day spleen colonies or achieve long-term engraftment of lethally irradiated mice. A single wild-type cell is able to reconstitute unirradiated W/Wv mice. The W mutation, a G to A substitution in the splice donor site at the exon 10 - intron 10 boundary, eliminates an AarI site and the Wv mutation, a C to T transition in the last codon of exon 13, creates a NsiI site. The targeting vector, rAAV-cKit, was derived by subcloning a 4.6 kb EcoR1-BglII fragment from the c-Kit locus, recovered from a BAC clone (RP23-2), into a plasmid containing rAAV2 ITRs. Using a self-complementary rAAV2 vector genome encoding Green Fluorescent Protein (GFP), we found that vector particles packaged with AAV serotype 1 capsid proteins were 10 fold more efficient at transducing lineage depleted, c-Kit+, ScaI+ murine bone marrow cells than particles packaged in serotype 2 capsid proteins. At a MOI of 105 serotype 1 vector particles/ target cell, more than 95% of the c-Kit+, ScaI+ cells were transduced. Using optimized conditions, we transduced 1.2 x 107 lineage depleted W/Wv bone marrow cells with 1.2 x 1012 rAAV-c-Kit, serotype 1 vector particles in serum-free medium containing 100 ng/ml of SCF, TPO, IL-6 and Flt-3L and 10 ng/ml of IL-3. Many spleen colonies were present in irradiated recipients 14 days after injection with transduced cells but none were found in controls which received mock transduced W/Wv cells. Southern blot analysis provided evidence that the AarI and NsiI sites reflecting the W and Wv mutations, respectively, had been completely or partially changed (AarI gained and NsiI lost) in the majority (4 of 5) colonies suggesting biallelic correction during colony development. In an initial experiment designed to evaluate the ability of rAAV-c-Kit to achieve gene correction in long-term repopulating cells, we found that 2 of 5 irradiated wildtype mice injected with transduced W/Wv lineage depleted cells survived at 6 weeks with normal hematopoiesis. Overall, our data suggest that rAAV vectors can be used to achieve gene correction in primitive, primary hematopoietic cells at a detectable frequency which may be useful for gene therapy of disorders such as SCID in which there is a positive selective advantage for gene corrected cells.
Gene transfer of O6-methylguanine-DNA-methyltransferase (MGMT) into HSCs results in efficient in vivo selection of murine HSCs after treatment with temozolomide (TMZ) and O 6 benzylguanine (BG). In addition, HOXB4 coexpression is associated with an increase in HSC selection efficiency using a DHFR drug selection system in mice. Based on these results, we have studied whether the MGMT and HOXB4 selection systems are efficacious in a rhesus macaque autologous transplantation model. SIV vectors were constructed incorporating either MGMT alone or MGMT together with HOXB4 in a GFP or YFP backbone. In our first transplant case, we purified CD34+ cells from mobilized bone marrow cells and split the graft into two parts: half of the cells were transduced with a SIV MGMT-GFP vector and half were transduced with a SIV MGMT-HOXB4-GFP vector. In our second and third transplant cases, half of the CD34+ cells were transduced with a SIV MGMTYFP vector and the other half with a SIV MGMT-HOXB4-GFP vector. During the early engraftment period, there was a slight advantage (2-fold maximal) for HOXB4 transduced cells relative to the “MGMT-only” vector. However, by 1 month posttransplantation, there was no difference in marking between the two vectors, suggesting HOXB4 had little effect on long-term repopulating cells. The first animal has been treated with 7 courses of TMZ/BG, starting with a fixed dose of 120 mg/m2 for BG and 170 mg/m2/day for 5 days for TMZ. The dose of TMZ was escalated on each course, and well tolerated by the animal. For the 7th course, the animal received 450 mg/m2 of TMZ with BG, a significant dose increase relative to the pediatric maximum tolerated dose (185 mg/ m2 without BG in irradiated patients). This increase in dose intensity was verified by formal pharmacokinetic measurements. After each course of drug treatment, there was a rapid increase in GFP marking in all lineages. The highest marking achieved after last course of TMZ/BG treatment was 91% for myeloid cells and 50% for lymphoid lineages. However, the proportion of GFP + cells subsequently declined and leveled off at 13% for granulocytes, 17% for B cells and 36% for T cells. The second animal had been treated with two courses of TMZ/BG and there was about 7-fold increase in GFP marking in granulocytes and a 3 and 8-fold increase in B, T cells respectively. Altogether, we have shown that TMZ selection was occurring mainly in progenitor cells with limited self-renewal capacity and to a lesser degree at the HSC level as evidence by increased baseline GFP marking. We were also able to achieve a significant increase in dose intensity in the first animal, and to successfully administer multiple courses of drug treatment. These results suggest a clinical strategy for hematopoietic protection in cancer treatment. We are now treating these animals with BCNU plus BG to determine if HSC selection efficiency is increased relative to TMZ. Lastly, we did not see evidence of HSC amplification with HOXB4 expression, suggesting that the robust selection seen in mice is relatively species specific.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
S29