Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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 Disclosure: Jennifer Gori and Co-Authors are Full-time Employees of Editas Medicine Gene-Modified Autologous Hematopoietic Stem and Progenitor Cell Therapy ex vivo approach to gene correction of hematopoietic diseases CD34+ HSC erythroid myeloid lymphoid © 2016 Editas Medicine 2 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 Cas9/gRNA treated Human CD34+ cells D10A Nickase 24h 48h 72h 5’ PAM Blunt Effective for NHEJ © 2016 Editas Medicine PAM PAM 5’ Overhang Effective for HDR 3 a-Cas9 a-Actin Efficient and Reproducible Editing in HSCs Fold change in live CD34+ cells (7-AAD-AnnexinV-) Comparison of Wild-Type and D10A SpCas9 at b-hemoglobin locus (HBB) % Gene editing 100 75 50 25 0 CB-HSC mPB-HSC CB-HSC mPB-HSC WT Cas9 4 3 2 1 0 RNP D10A Nickase Reproducible gene editing across 20 donors Maintenance of viability of RNP treated HSCs © 2016 Editas Medicine 4 Control Gene-Edited HSCs Maintain Erythroid and Myeloid Multipotency ex vivo Analysis of gene editing in clonal derivatives of edited HSCs GEMM E GM M 100 G % Gene edited colonies # Hematopoietic colonies 200 150 100 50 Erythroid Myeloid 75 50 25 0 0 RNP control Monoallelic Biallelic RNP treated HSCs - Differentiate into erythroid and myeloid colonies - Monoallelic and biallelic gene disruption detected in HSC clones © 2016 Editas Medicine 5 Long-Term Engraftment of Cas9/gRNA RNP Treated Human HSCs Compare engraftment of RNP treated and control human CD34+ cells in mouse xenograft model Busulfan ± D10A RNP targeting HBB -4d CD34+ cells -2d Prestimulation -1d 0d Conditioning Infusion Parameter n Busulfan (mg/kg) 25 Control HSC mice 6 RNP HSC mice 7 CD34+ cell dose 570,000 4m Endpoint Reconstitution of human hematopoiesis in vivo (4+ months) Gene editing in marrow, spleen, blood (human subsets) © 2016 Editas Medicine 6 Gene Edited Cells Reconstitute Peripheral Blood Human CD45+ lymphoid and myeloid cells at 4 months 85% human CD45+ Mouse CD45 Subset within human CD45+ gate Human CD45 © 2016 Editas Medicine 7 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 100 70% human CD45+ Spleen 100 RNP Control 50 18% HSC 25 50 13% HSC 25 0 0 Subset within human CD45+ gate © 2016 Editas Medicine RNP Control 75 % Positive % Positive 75 73% human CD45+ Subset within human CD45+ gate 8 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 75 50 25 100 RNP Control % Gene editing % Gene editing 100 Spleen 0 RNP Control 75 50 25 0 50% gene editing HSC before transplantation 50% gene editing in engrafted cells in the blood, marrow, and spleen 4 months after transplantation © 2016 Editas Medicine 9 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 Erythroid (CD235) % Gene editing 100 10 75 50 25 0 HSC Myeloid 10 10 5 11% 4 3 12% 0 Erythroid -10 3 -10 3 0 10 3 10 HSCs (CD34) © 2016 Editas Medicine 10 4 10 5 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 (50% editing and 85% human blood reconstitution) © 2016 Editas Medicine 11 Electroporation of D10A RNP with Donor Supports Homology Directed Repair in HSCs 80 % Gene editing NHEJ 60 40 HDR 20 0 12% homology directed repair achieved after co-delivery of D10A RNP and single strand oligonucleotide donor © 2016 Editas Medicine 12 Jack Heath Aditi Chalishazar Christina Lee Will Selleck Tanushree Phadke Erik Corcoran Cecilia Cotta-Ramusino David Bumcrot