Precise and Efficient Crispr/Cas9 Mediated Gene Editing in Long-Term Engrafting Human Hematopoietic Stem/Progenitor Cells

Transplantation of gene-modified autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for several hematologic diseases. However, a number of blood disorders may not be amenable to gene augmentation-based therapeutics. Targeted genome editing in human HSPCs could provide a therapeutic approach for these otherwise untreatable diseases. Here we demonstrate that CRISPR/Cas9 ribonucleoprotein (RNP) edits target genes in human HSPCs with high efficiency and precision. Human adult and umbilical cord blood (CB) CD34 + cells from 20 donors were electroporated with S. pyogenes or S. aureus Cas9 RNP targeting HBB , AAVS1 , or CXCR4 . Sequence analysis demonstrated up to 80% editing in CB CD34 + cells (mean±s.d: 61%±9%) and up to 57% in adult CD34 + cells (39%±13%). Delivery of Cas9 RNP and a single-stranded oligodeoxynucleotide donor (ssODN) led to up to 12% ssODN-mediated homology directed repair (HDR) and also led to a 20% increase in total gene editing (HDR+NHEJ)(RNP: 48%±15%; RNP+ssODN: 69%±8%). Both Cas9 RNP gene-edited CD34 + cells and donor-matched untreated control CD34 + cells reconstituted human hematopoiesis in primary and secondary recipient immunodeficient mice, with ~85% human CD45 + cell peripheral blood reconstitution 4 months after primary transplantation. Human T and B lymphoid, erythroid, and myeloid cells were detected in the spleen, thymus, and bone marrow with 20% CD34 + cell engraftment in the marrow of mice transplanted with RNP gene-edited or control CD34 + cells. The level of targeted gene editing in human erythroid, myeloid, and CD34 + cells that were recovered and enriched from the hematopoietic organs of primary recipients (~50%) was similar to the level of gene editing detected in the pre-infusion product (~60%). In summary, these results indicate that Cas9 gene-edited human HSPCs retain long-term engraftment potential and support multilineage blood reconstitution in vivo , thus supporting further investigation of CRISPR/Cas9 mediated gene-edited hematopoietic stem/progenitor cell therapies. Disclosures Heath: Editas Medicine: Employment. Chalishazar: Editas Medicine: Employment. Lee: Editas Medicine: Employment. Selleck: Editas Medicine: Employment. Cotta-Ramusino: Editas Medicine: Employment. Bumcrot: Editas Medicine: Employment. Gori: Editas Medicine: Employment.