294. Myeloablative Conditioning Is Required for Efficient Engraftment of Gene-Modified Cells and Prevention of Antibody Production Against Transgene Products in a Rhesus Stem Cell Gene Therapy Model

Abstract

Reduced intensity conditioning (RIC) regimens are desirable for hematopoietic stem cell (HSC)-targeted gene therapy. However, in previous gene therapy trials, low gene marking was reported in peripheral blood cells, raising the question that RIC might be insufficient for (1) opening niches for efficient engraftment and (2) inducing immunological tolerance to transgenes. Therefore, we sought to evaluate both engraftment and tolerance for genetically modified cells using our rhesus HSC gene therapy model following RIC.We performed a dose de-escalation of total body irradiation (TBI) (10, 8, 6, and 4Gy) as RIC transplantation (total 19 animals), in which mobilized rhesus CD34+ cells were transduced with a VSV-G-pseudotyped chimeric HIV-1 vector encoding GFP (or YFP) and these cells were transplanted into autologous animals following TBI. We evaluated GFP-positive rates (%GFP), average vector copy number per cell (VCN), and antibody titers against GFP and VSV-G in peripheral blood 6 months after transplantation.When we evaluated in vitro %GFP in transduced rhesus CD34+ cells, efficient transduction (22-71%) was observed among all TBI groups (without correlation). After transplantation of the transduced CD34+ cells, GFP-positive cells were detected in peripheral blood cells in all animals. Increasing doses of TBI resulted in higher %GFP with logarithmic regression in both granulocytes (R2=0.64, p<0.01) and lymphocytes (R2=0.67, p<0.01)(Figure). Similar results were observed between TBI dose and VCNs in both granulocytes (R2=0.54, p<0.01) and lymphocytes (R2=0.52, p<0.01). These data suggest that higher doses of TBI improve engraftment of gene-modified hematopoietic repopulating cells.To evaluate immunological tolerance for gene-modified cells, we measured both anti-GFP and anti-VSV-G antibody titers using serum samples from transplanted animals. Lower dose TBI allowed anti-GFP antibody production with logarithmic regression (R2=0.84, p<0.01)(Figure), while no significant effect of anti-VSV-G antibody was observed among all TBI groups (R2=0.003, p=0.87). These data demonstrate that higher dose TBI is important to induce immunological tolerance for gene-modified cells in a rhesus transplantation model.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. View Large Image | Download PowerPoint Slide