High Levels of Multilineage Hematopoietic Cell Gene Marking Following Lentiviral Vector Transduction and Transplantation of Steady-State Bone Marrow CD34⁺ cells in a Non-Human Primate Model.

Abstract 2464

Poster Board II-441

Non-human primate autologous hematopoietic stem cell (HSC) transplantation models have been invaluable in developing the ex vivo transduction protocols used in human gene transfer clinical trials. Previous studies of HSC gene transfer using gamma-retroviral and lentiviral vectors in these models have almost exclusively utilized cytokine-mobilized peripheral blood (PB) or bone marrow (BM) CD34⁺ cells as target cells. The administration of the combination of granulocyte colony stimulating factor (G-CSF) plus stem cell factor (SCF) became standard as a priming method prior to HSC collection after it was shown that SCF coupled with G-CSF resulted in significantly improved long-term gene transfer levels, compared to mobilization with G-CSF alone (Hematti et al., Blood 2003 101:2199-2205). However, SCF is not available in the US for human gene transfer trials due to its history of causing severe anaphylactic reactions. For this reason and to define a clinically relevant gene transfer protocol for sickle cell disease, in which G-CSF administration is also contraindicated, we evaluated lentiviral stem cell gene transfer using steady-state BM CD34⁺ cells in the pigtail macaque transplantation model. Bone marrow was harvested from the long bones and CD34⁺ cells were enriched using an anti-CD34 antibody and immunomagetic beads (92% purity). Cells were prestimulated overnight in serum-free medium containing 100 ng/ml of FLT-3 ligand, thrombopoietin, and stem cell factor. Cells were then exposed for 14 hours to a VSV-G pseudotyped, HIV-based lentiviral vector encoding GFP at an MOI of 40. Cells were collected, washed, and placed back into culture in serum-free medium containing the above cytokines for an additional 24 hours. Subsequently, cells were collected (15 × 10⁶/kg) and transplanted into an animal which had been conditioned with 950 cGy. Marking of the graft, as assessed by flow cytometry for GFP expression, was 74% and all hematopoietic colonies (CFU) obtained in methylcellulose cultures were GFP positive. Neutrophil recovery (ANC > 500) was observed on day 20. One month following transplant, 36% of peripheral blood granulocytes (GRANS) were GFP positive. At two months post-transplant, marking levels were: GRANS 27%, RBC 43%, and platelets (PLT) 22%. These levels have remained steady out to 6 months post-transplantation: GRANS 21%, lymphocytes 20%, RBC 17%, and PLTS 16%. A BM aspirate was obtained and analysis of BM CFU showed that 186 of 508 (37%) were GFP positive. Southern blot analysis and q-PCR on DNA from peripheral blood leukocytes and BM showed a vector copy number ranging from 0.6 to 1.0 over time, suggesting that the positive cells contained several vector copies. No evidence of clonal dominance has been observed by Southern blot analysis using restriction enzymes which cut only once in the vector. Insertion site analysis using deep sequencing technology is in progress. A second animal has recently been transplanted (11 × 10⁶ cells/kg) with graft marking of greater than 50%. These data are encouraging as they suggest that therapeutic levels of lentiviral vector gene transfer can be obtained using CD34⁺ cells from steady-state bone marrow as the HSC cell source. This is important since SCF cannot be used in humans and for patients with sickle cell disease, administration of G-CSF is also contraindicated.