Polyclonal chemoprotection against temozolomide in a large-animal model of drug resistance gene therapy.

Incorporation of drug resistance genes into gene vectors has 2 important roles in stem cell gene therapy: increasing the proportion of gene-corrected cells in vivo (ie, in vivo selection) and marrow protection to permit higher or more tightly spaced doses of chemotherapy in the treatment of malignant diseases. We studied in a clinically relevant canine model of gene therapy the P140K mutant of the drug resistance gene methylguanine methyltransferase (MGMT), which encodes a DNA-repair enzyme that confers resistance to the combination of the MGMT inhibitor O(6)-benzylguanine (O(6)BG) and nitrosourea drugs such as carmustine and methylating agents such as temozolomide. Two dogs received MGMT(P140K)-transduced autologous CD34(+)-selected cells. After stable engraftment, gene marking in granulocytes was between 3% and 16% in the 2 animals, respectively. Repeated administration of O(6)BG and temozolomide resulted in a multilineage increase in gene-modified repopulating cells with marking levels of greater than 98% in granulocytes. MGMT(P140K) overexpression prevented the substantial myelosuppression normally associated with this drug combination. Importantly, hematopoiesis remained polyclonal throughout the course of the study. Extrahematopoietic toxicity was minimal, and no signs of myelodysplasia or leukemia were detected. These large-animal data support the evaluation of MGMT(P140K) in conjunction with O(6)BG and temozolomide in clinical trials.

Efficient marking of murine long-term repopulating stem cells targeting unseparated marrow cells at low lentiviral vector particle concentration.

HIV-1-derived lentivirus vectors offer unique biological properties for gene delivery to hematopoietic stem cells and, when used at high multiplicities of infection (m.o.i.), permit efficient gene transfer after minimal target cell stimulation. However, such a strategy has been shown to promote multicopy proviral integration, potentially increasing the risk of insertional mutagenesis. To minimize cell manipulation, we targeted unseparated marrow and demonstrated that transduction at an m.o.i. of 1 resulted in up to 12% vector-modified peripheral blood leukocytes and successful repopulation of secondary recipients with vector-marked cells. Real-time PCR showed on average 1.8 proviral integrants per GFP-marked cell. By comparison, a cohort of animals transplanted with cells transduced at m.o.i. of 10 under otherwise unchanged conditions showed up to 45% marking with an average of 7 copies per GFP-expressing cell. Both m.o.i. groups demonstrated sustained proviral expression with stable GFP fluorescence intensity. In summary, we have identified conditions for lentiviral gene transfer involving minimal ex vivo target cell manipulation and have shown that the m.o.i. is a critical determinant of proviral copy number in lentivirus-transduced murine long-term repopulating cells. Thus, gene transfer efficiencies may be limited when single-copy integration is desired and additional strategies such as in vivo selection may be required to improve the frequency of gene-modified cells.

The structural basis for methylmalonic aciduria. The crystal structure of archaeal ATP:cobalamin adenosyltransferase.

ATP:cobalamin adenosyltransferase MMAB was recently identified as the gene responsible for a disorder of cobalamin metabolism in humans (cblB complementation group). The crystal structure of the MMAB sequence homologue from Thermoplasma acidophilum (TA1434; GenBank identification number gi|16082403) was determined to a resolution of 1.5 A. TA1434 was confirmed to be an ATP:cobalamin adenosyltransferase, which depended absolutely on divalent metal ions (Mg2+ > Mn2+ > Co2+) and only used ATP or dATP as adenosyl donors. The apparent Km of TA1434 was 110 microM (kcat = 0.23 s(-1)) for ATP, 140 microM (kcat = 0.11 s(-1)) for dATP, and 3 microM (kcat = 0.18 s(-1)) for cobalamin. TA1434 is a trimer in solution and in the crystal structure, with each subunit composed of a five-helix bundle. The location of disease-related point mutations and other residues conserved among the homologues of TA1434 suggest that the active site lies at the junctions between the subunits. Mutations in TA1434 that correspond to the disease-related mutations resulted in proteins that were inactive for ATP:cobalamin adenosyltransferase activity in vitro, confirming that these mutations define the molecular basis of the human disease.

In vivo administration of interferon gamma does not cause marrow aplasia in mice with a targeted disruption of FANCC.

OBJECTIVE: Hematopoietic cells from patients with Fanconi anemia (FA) and mice carrying a targeted disruption of the gene encoding complementation group C protein (FANCC(-/-)) demonstrate an apoptotic phenotype in response to alkylating agents and cytokines including interferon gamma (IFN-gamma) in vitro. The aim of this study was to explore these apoptosis-inducing effects of IFN-gamma on the bone marrow of FANCC(-/-) mice as a potential strategy to select gene-corrected cells in vivo. Following pharmacokinetic studies to determine if serum concentrations effective in vitro can be achieved in vivo, we injected FANCC(-/-) mice with recombinant murine IFN-gamma. Hematopoietic effects were investigated by serial determinations of blood counts, progenitor colony formation, and marrow cellularity. RESULTS: Serial weekly intraperitoneal administrations of escalating doses of rmIFN-gamma did not affect peripheral blood counts in FANCC(-/-) mice, even after subsequent antibody-mediated fas ligation. Additionally, prolonged exposure after sequential daily administration of recombinant IFN-gamma did not impair progenitor cell clonogenicity in vitro. Pharmacokinetic data confirmed that the failure of IFN-gamma to induce marrow aplasia occurred in spite of peak serum levels greater than 100-fold in excess of those effective in vitro. CONCLUSION: We conclude that in spite of the well-documented in vitro apoptotic tendency of FA-phenotype hematopoietic cells, the in vivo administration of IFN-gamma with and without subsequent fas ligation does not induce bone marrow failure in FANCC(-/-) (129SvJ strain) mice. Additional selective pressure may be necessary to achieve targeted ablation of uncorrected, FA-phenotype, marrow cells.