Highly Efficient CRISPR/Cas9 Gene Editing and Long-Term Engraftment of Human Hematopoietic Stem and Progenitor Cells

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1 Highly Efficient CRISPR/Cas9 Gene Editing and Long-Term Engraftment of Human Hematopoietic Stem and Progenitor Cells J. M. Heath, A. Chalishazar, C.S. Lee, W. Selleck, C. Cotta-Ramusino, D. Bumcrot, J.L. Gori

2 Disclosure: Jennifer Gori and Co-Authors are Full-time Employees of Editas Medicine

3 Gene-Modified Autologous Hematopoietic Stem and Progenitor Cell Therapy ex vivo approach to gene correction of hematopoietic diseases CD34 + HSC erythroid myeloid lymphoid 216 Editas Medicine 2

4 Rationale for Delivery of Cas9 RNP for Gene Editing in HSCs Effective gene editing and transient nuclease expression Hypothesis - Cas9 RNP would support gene editing in HSCs without impacting viability or functionality in vivo Electroporation of Cas9/gRNA ribonucleoprotein (RNP) - High efficiency - Limited exposure WT Cas9 D1A Nickase Cas9/gRNA treated Human CD34 + cells 5 Blunt PAM PAM PAM 5 Overhang a-cas9 24h 48h 72h Effective for NHEJ Effective for HDR a-actin 216 Editas Medicine 3

5 % Gene editing Fold change in live CD34 + cells (7-AAD-AnnexinV-) Efficient and Reproducible Editing in HSCs Comparison of Wild-Type and D1A SpCas9 at b-hemoglobin locus (HBB) CB-HSC mpb-hsc CB-HSC mpb-hsc RNP Control WT Cas9 D1A Nickase Reproducible gene editing across 2 donors Maintenance of viability of RNP treated HSCs 216 Editas Medicine 4

6 # Hematopoietic colonies % Gene edited colonies Gene-Edited HSCs Maintain Erythroid and Myeloid Multipotency ex vivo Analysis of gene editing in clonal derivatives of edited HSCs 2 GEMM E GM M G 1 Erythroid Myeloid RNP control Monoallelic Biallelic RNP treated HSCs - Differentiate into erythroid and myeloid colonies - Monoallelic and biallelic gene disruption detected in HSC clones 216 Editas Medicine 5

7 Long-Term Engraftment of Cas9/gRNA RNP Treated Human HSCs Compare engraftment of RNP treated and control human CD34 + cells in mouse xenograft model ± D1A RNP targeting HBB Busulfan -4d -2d -1d CD34 + cells Prestimulation Conditioning Parameter Busulfan (mg/kg) 25 Control HSC mice 6 RNP HSC mice 7 CD34 + cell dose 57, n d Infusion 4m Endpoint Reconstitution of human hematopoiesis in vivo (4+ months) Gene editing in marrow, spleen, blood (human subsets) 216 Editas Medicine 6

8 Mouse CD45 Gene Edited Cells Reconstitute Peripheral Blood Human CD45 + lymphoid and myeloid cells at 4 months 85% human CD45 + Subset within human CD45 + gate Human CD Editas Medicine 7

9 % Positive % Positive Gene Edited Human Blood Cells and HSCs Repopulate the Bone Marrow and Spleen 5 million HSCs recovered from bone marrow of each recipient Bone Marrow Spleen % human CD45 + RNP Control % human CD45 + RNP Control % HSC 25 13% HSC Subset within human CD45 + gate Subset within human CD45 + gate 216 Editas Medicine 8

10 % Gene editing % Gene editing Efficient Gene Editing Detected in Human Blood Cells in the Bone Marrow and Spleen Gene editing in vivo equal to editing in pre-infusion product Bone Marrow Spleen 1 75 RNP Control 1 75 RNP Control % gene editing HSC before transplantation 5% gene editing in engrafted cells in the blood, marrow, and spleen 4 months after transplantation 216 Editas Medicine 9

11 % Gene editing Erythroid (CD235) Gene Editing is Maintained in HSC Progeny Differentiated in vivo Gene editing in engrafted HSCs is maintained in progeny in vivo Sorted Bone Marrow Fractions % HSC Myeloid Erythroid % HSCs (CD34) 216 Editas Medicine 1

12 Summary and Conclusions Cas9/gRNA RNP supports efficient and reproducible gene editing in human HSCs across donors (57% ± 8) Gene edited HSCs retain phenotype, viability, and differentiation potential ex vivo Gene edited human HSCs retain long-term engraftment and multipotency in vivo (5% editing and 85% human blood reconstitution) 216 Editas Medicine 11

13 % Gene editing Electroporation of D1A RNP with Donor Supports Homology Directed Repair in HSCs 8 NHEJ HDR 12% homology directed repair achieved after co-delivery of D1A RNP and single strand oligonucleotide donor 216 Editas Medicine 12

14 Jack Heath Aditi Chalishazar Christina Lee Will Selleck Tanushree Phadke Erik Corcoran Cecilia Cotta-Ramusino David Bumcrot