732. CRISPR/Cas9 Mediates Highly Efficient Gene Editing in Long-Term Engrafting Human Hematopoietic Stem/Progenitor Cells

Abstract

Transplantation of genetically corrected autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for patients with inherited hematologic disease. Genome editing with CRISPR/Cas9 ribonucleoprotein (RNP) precisely modifies gene targets in human cells, including HSPCs. In order to translate this technology to a clinical setting, gene editing must be reproducible and efficient across multiple patient donors without compromising HSPC viability, multipotency, and long-term engraftment capability. To determine the reproducibility of Cas9 RNP mediated gene editing in HSPCs, human CD34+ cells obtained from 15 different patient donors (cord blood n=12, mobilized peripheral blood [mPB] n=3) were electroporated with S. pyogenes Cas9 RNP targeting the β-hemoglobin (HBB) or AAVS1 genetic locus. DNA sequence analysis of 15 separate experiments demonstrated that Cas9 supported up to 72% gene editing in cord blood CD34+ cells and up to 61% gene editing in adult mPB CD34+ cells (mean % editing by DNA sequencing: 57% ± 8%). Cas9 induced multiple modifications that comprise insertions and deletions at the HBB locus, and some of the lesions were repaired through an HDR repair mechanism that used the homologous sequences from the endogenous HBD gene as a template (Gene Conversion). Gene edited CD34+ cells retained viability and hematopoietic colony forming cell (CFC) activity ex vivo, with no significant differences between treatment groups or across donors. Indels were detected in one (BFU-E/GEMM: 50%-75%, CFU-GM/M: 50-66%) or both (BFU-E/GEMM: 12-38%, CFU-GM/M: 12-50%) alleles, at high frequencies in clonal erythroid and myeloid colonies (range of 63-100% of individual colonies assayed across experiments). To evaluate engraftment potential, both Cas9 modified (unsorted) and untreated control human CD34+ cells were transplanted into immunodeficient mice. Twelve weeks after transplantation, up to 34% human CD45+ cell engraftment was detected in the peripheral blood of recipients of Cas9 RNP treated CD34+ cells. There was no significant difference in the engraftment of Cas9 RNP treated and untreated control CD34+ cells which were detected in blood, spleen, and bone marrow. Human lymphoid, myeloid, and erythroid cells were detected in blood and hematopoietic organs with no difference in lineage distribution between cohorts. Analysis of the bone marrow from both groups revealed an average of 20% human CD45+ and 13% CD34+CD45+ cell engraftment long-term. Analysis of human gDNA revealed that up to 20% gene editing in the marrow of treated mice and edited cells were also detected in the blood and spleen. In summary, these data show that Cas9 RNP gene edited primary human CD34+ HSPCs retained the ability to engraft long-term and reconstitute hematopoiesis in vivo at levels higher than reported previously. These findings also show that electroporation of HSPCs with Cas9 RNP mediated highly reproducible gene editing levels across multiple donors and did not alter hematopoietic reconstitution or differentiation properties in comparison to untreated control CD34+ cells.