1. Objective 1.1: Development of a clinical trial for LAD-1. A clinical trial for gene therapy of LAD-1 using first-in-human foamy viral vectors (FVV) is in preparation and enrollment is expected to start in FY17. In preparation for this trial, we have performed: a) Process-development and scale-up of FVV production Process-development and scale-up was performed using the MSCV-GFP and MSCV-hCD18 FVV in collaboration with investigators at Cincinnati Childrens Hospital-Vector Production Facility. A considerable effort was needed to develop processes for large-scale concentration and purification of this extremely serum dependent virus. Since FV is a non-pathogenic virus, its isolation and purification techniques had to be developed. Assays required for assuring safety and potency of MSCV-hCD18 FVV were developed, and some of the final safety assays still need further development to a cGMP-grade assay. Production of clinical grade FVV stocks for gene therapy of LAD-1 is ongoing. The process-development and scale-up were published in FY16 (Nasimuzzaman et al. Mol Ther Meth & Clin Devel 2016). b) Pre-clinical testing of FVV transduction in CD34+ cells derived from a subject with severe LAD CD34+ cells were transduced for 16 hours at MOI of 0, 1, 2, and 5 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 33-45% of cells. Higher MOIs resulted in decreased cell survival with no significant benefit in transduction efficiency, suggesting an optimal MOI of 1-2. NSG mice were transplanted with MSCV-hCD18-transduced human LAD-1 HSPCs (1 x 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 MSCV-hCD18 at MOI=1 (p<0.01), 2 (p<0.05) and 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, 1, 2 and 5, 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 1, 2 and 5, 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 MSCV-hCD18-transduced LAD-1 CD34+ cells revealed human CD18+ cells in both CD13+ myeloid (35.5 6.9%) and CD20+ lymphoid (39.8 35.7%) compartments. 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 revealed a polyclonal pattern of integration with no evidence of insertional mutagenesis 5 months post-transplantation. Thus, MSCV-hCD18-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. This work was presented at the American Society of Cell and Gene Therapy in FY16. A manuscript is in preparation. 2. Objective 1.2: Optimization of the CRISPR/Cas9 genome editing technology in human HSCs. We have optimized genome editing via NHEJ pathways in primary CD34+ cells in FY16. Efficiencies of INDEL formation and gene knockout of 50-70% are routinely obtained. Based on a recent publication indicating that HSCs with CXCR4 haploinsufficiency have a competitive repopulating advantage, we have performed monoallelic knockout of CXCR4 in normal human CD34+ cells. The impact of CXCR4 haploinsufficiency on human HSC engraftment and proliferation was assessed using the gold-standard immuno-deficient (NSG) murine model. In contrast to the observation made with murine HSCs, no increase in engraftment was observed several months after transplantation of CXCR4-haploinsufficient human CD34+ cells compared to control cells. Disruption of CXCR4 did not negatively impact overall engraftment or skew lineage differentiation. These data suggest that CXCR4 may be of interest as a safe-harbor site for genome editing. Genetic perturbation of CXCR4, a co-receptor for HIV entry, may also be of interest to protect CD34-derived T cells from infection with CXCR4-trophic HIV-1 strains. In FY17, experiments will be conducted to optimize targeted introduction of a gene of interest (e.g. GFP) in the CXCR4 and other safe harbor loci via homologous recombination (HR) DNA repair pathways. Adeno-associated virus (AAV) serotype 6 will be used to deliver homology arms to CXCR4 and other genes to favor HR pathways. Search for novel serotypes of AAV capable of efficient transduction of human HSPCs is underway to further enhance HR-triggered genome editing in human CD34+ cells.

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