734. Pre-Clinical Development and Qualification of ZFN-Mediated Disruption of CCR5 in Human Hematopoietic Stem and Progenitor Cells
Authors: Rodica Stan, David DiGiusto, Paula Cannon, Colin Exline, Lijing Li, Anitha Rao, Jianbin Wang, Gary Lee, Philip D. Gregory, Nancy Gonzalez, Victoria Bedell, Michael C. Holmes, John A. Zaia
Journal: Molecular Therapy (2016)

INTRODUCTION: Gene therapy for HIV-1 infection is a promising alternative to lifelong combination anti-viral drug treatment. When deleted, chemokine receptor 5 (CCR5), the co-receptor required for R5-tropic HIV-1 infection of human cells, renders cells resistant to HIV-1 infection. This has formed a strategy for the potential cure of HIV-1 infection by disrupting CCR5 genomic sequences in hematopoietic stem/progenitor cells developed using Zinc Finger Nucleases (ZFN). We demonstrate that delivery of CCR5-specific ZFN mRNA (SB-728mR) to HSPC via electroporation results in efficient disruption of CCR5 resulting in a high percentage of cells possessing biallelic modification and yields clinical-scale numbers of CCR5-modified HSPC with minimal cellular cytotoxicity, while maintaining their hematopoietic potential both in vitro and in vivo. METHODS: Pre-clinical experiments were performed at scale using donor HSPC with different concentrations of SB-728mR to optimize the electroporation-mediated CCR5 disruption in CD34+ HSPC for clinical studies in HIV. SB728mR-HSPC were evaluated in vitro for: a) mono- vs bi-allelic disruption and off-target ZFN cutting using MiSeq deep sequencing and the surveyor nuclease assay; b) total yield and viability; and c) hematopoietic function in colony forming unit (CFU) assays. Tumorigenicity studies were performed in NOD-scid-IL2Rgammanull (NSG) mice irradiated with 150 cGy and IV injected with 1e6 SB-728mR-HSPC. equivalent of one clinical dose (150e6 CD34+ cells).Engraftment efficiency was analyzed by %CD45 cells in PBMC at intervals for 5 months. RESULTS: We have demonstrated the specificity and efficacy of ZFN-based CCR5 disruption in HSPC: a) 40-60% CCR5 disruption with 50 to 150 µg/mL mRNA, with off-target modification observed at CCR2 and in 3 additional sites that occur in non-coding (either at intergenic or intronic) sequences; and b) effective disruption of ≤70% CFU from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. The tumorigenicity study demonstrated the safety of the gene modified cell product. SB-728mR-HSPC and untransfected control HSPC successfully engrafted as shown by human hematopoietic progeny measured in blood and bone marrow in all animals. Gene modified cells were observed in blood and bone marrow during the course of the study and were generally well tolerated. No ZFN treatment related tumor formation was observed in any animals. CONCLUSIONS: We have developed a method to genetically modify the CCR5 locus in human CD34+ HSPC. This approach is safe, efficient and reproducible. A combination of in vitro and in vivo studies evaluated ZFN specificity and potential tumorigenicity of ZFN modified cells in NSG mice. The established process complies with the regulatory requirements and results of the preclinical studies were used to support the filing of an IND with the FDA. The clinical trial testing SB-728mR-HSPC ([email protected]) is currently enrolling patients with the support of the California Institute for Regenerative Medicine.