285. Foamy Viral Vector Expressing Human CD18 Results in High Levels of Transduction and Multilineage Engraftment with CD18+ LAD-1 Cells in NSG Mice

Compared to other classes of retroviral vectors used for HSPC gene therapy, foamy viral vectors (FVV) have distinct advantages, including their non-pathogenicity, a safer integration profile, and a high-efficiency transduction of quiescent cells. Proof-of principle of its therapeutic safety and efficacy in HSPCs was provided with the correction of canine Leukocyte Adhesion Deficiency (CLAD). Dogs with CLAD and individuals with LAD type 1 (LAD-1) suffer from recurrent and life-threatening infections caused by mutations in the β2 integrin CD18 subunit. In this study, G-CSF-mobilized CD34+ HSPCs were isolated from a 19 YO male with severe LAD-1 due to homozygous deletions in CD18. CD34+ cells were transduced for 16 hours at MOI of 0, 5, 10, and 20 with a FVV expressing a human codon optimized CD18 transgene. Flow cytometry of CD34+ cells cultured for 3 days after transduction demonstrated CD18+ cell surface expression in 40-45% of cells. Thirty NSG mice were transplanted with CD18-FVV-transduced human LAD-1 HSPCs (~1.2 × 105 cells/mouse), and human cell engraftment was measured in murine BM 5 months after transplantation using flow cytometry. Human CD45+ cells were detected in all mice (average ~1%). Mice transplanted with mock-transduced (MOI=0) LAD-1 CD34+ cells showed a 3.4-fold, 2-fold and 1.4-fold lower engraftment compared to mice injected with CD34+ cells transduced with FVV at MOI 5 (p 0.05), respectively, suggesting a selective homing/engraftment or survival/proliferative advantage of CD18+ cells. The inverse relationship between engraftment levels and MOI correlated with a gradual decrease in cell survival with increasing MOI, most likely due to toxicity from DMSO (required for FVV cryopreservation) during transduction; cell viabilities of 91%, 84%, 82% and 69% were obtained at MOI 0, 5, 10 and 20, respectively, indicating that further increase in MOI would lead to increasing toxicity. High-level, clinically relevant gene marking levels were obtained; the percentages of human cells expressing CD18 in the murine BM 5 months post-transplantation were 36.0 ± 3.9%, 33.9 ± 5.1%, and 44.5 ± 1.6% at MOI 5, 10 and 20, respectively. Quantitative PCR analysis of vector integrants within engrafted human cells indicated a single integration event occurred in the majority of long-term repopulating HSPCs at all MOI tested. Flow cytometry-based lineage analysis of bone marrow from mice transplanted with FVV-transduced LAD-1 CD34+ cells revealed human CD18+ cells in all lineages, with a predominance in the CD13+ myeloid compartment (88.3 ± 4.5%) compared to other lineages, including CD20+ lymphoid (9.1 ± 3.9%), CD235a+ erythroid (0.2 ± 0.1%) and CD41+ megakaryocytic (0.6 ± 0.2%) lineages. Interestingly, human myeloid engraftment was superior in recipient mice engrafted with human CD18+ cells (81.5 ± 4.3%) compared to animals transplanted with non-transduced (CD18-) LAD-1 cells (65.3 ± 11.3%). Integration site analysis of engrafted human cells is ongoing. Thus, FVV-mediated transduction of human LAD-1 CD34+ cells leads to clinically significant levels of CD18 expression, supporting the use of this CD18-expressing FVV in a human clinical trial.