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June 13, 2019 Jack Heath, Et Al. Genome Editing

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June 13, 2019 Jack Heath, Et Al. Genome Editing Genome Editing of HBG1/2 Promoter Leads to Robust HbF Induction In Vivo, While Editing of BCL11A Erythroid Enhancer Results in Erythroid Defects PS1518 Jack Heath*, Edouard de Dreuzy*, Minerva Sanchez, Scott Haskett, Tongyao Wang, Patricia Sousa, Gregory Gotta, Jamaica Siwak, Ramya Viswanathan, Frederick Ta, Emily Brennan, Diana Tabbaa, Eugenio Marco, John Zuris, Meltem Isik, Terence Ta, Georgia Giannoukos, Sandra Teixeira, Christopher Wilson, Charlie Albright, Kai-Hsin Chang Hurley Street, Cambridge, MA 02141 *Equal contributions 11 ׀ .Editas Medicine, Inc Introduction and Methods Results: Downregulation of BCL11A Expression Results: Editing Cis-regulatory Elements in the β-globin Locus Etiology of Sickle Cell Disease HBG1/2 Promoter-editing Displayed Normal HBG1/2 Promoter Editing Demonstrated BCL11A Erythroid Enhancer-editing Displayed Reduced Sickle cell disease (SCD) is caused by a single mutation E6V of the β-globin chain, leading to polymerization of Erythroid Output in BM of NBSGW Mice Long-term HbF Induction Erythroid Output in BM of NBSGW Mice hemoglobin (Hb) and formation of sickle hemoglobin (HbS) fibers when deoxygenated. Symptoms include anemia, acute chest syndrome, pain crises, and an array of other complications. Patients suffer significant morbidity and * p<0.05 *** p<0.001 1 40 early mortality. 90 90 *** *** 85 85 * CRISPR-based 80 Control 80 Control disruption of 75 BCL11A 75 HbF-repressing HBG1/2 30 Study Objective machinery 70 70 *** p<0.001 15 Engineered 15 like) (%) like) patient - The aim of this study was to assess two Patient β CD34+ cells* with / CD34+ cells* γ 20 different genome editing strategies switching reversed ( targeting the BCL11A and β-globin loci to 10 10 HbF Population (%) reverse hemoglobin switching for the Population (%) 10 treatment of sickle cell disease using 5 5 CRISPR/Cas. *** α α α α α α 0 0 0 hCD45 CD235a CD14 CD15 CD19 CD34 βs βs γ γ βs βs hCD45 CD235a CD14 CD15 CD19 CD34 Control HBG1/2 Control HBG1/2 Control HBG1/2 Leukocyte Erythroid Monocyte Neutrophil B-cell HSPC Leukocyte Erythroid Monocyte Neutrophil B-cell HSPC Cultured Erythroid CD235a Cultured Erythroid HbS HbF HbS from Pre-infusion Erythroid from BM *CD34+ hematopoietic stem and progenitor cells (HSPCs) CD34+ cells Approach 1: Approach 2: Reduced BCL11A Erythroid Output Coincided HBG1/2 Promoter Editing Demonstrated Optimization of Nuclease and gRNA Pairs Downregulation of BCL11A Expression by Targeting Editing Cis-regulatory Elements with Increased Non-productive Indels and No Erythroid Defect Increased HbF Induction to ~40% While in the β-Globin Locus its Erythroid Enhancer Increased Apoptosis Maintaining Editing Efficiency Adult Fetus Embryo BM Sorted Erythroid Cells Cultured Erythroids BM Sorted Erythroid Cells Cultured Erythroids HBB HBD HBG1 HBG2 HBE Indels HbF β-globin locus Reduced Erythroid Less Enrichment of Increased Similar Levels of Similar Levels of Indels in Similar Levels 50 Insulator Enhancer Insulator Output Editing Non-productive Edits Apoptosis Erythroid Output Erythroid compared to BM of Apoptosis 100 Intron2 GATAA (3’HS1) (HS1-4) (5’HS) chr2 LCR 80 40 120 *** 120 *** 300 *** 6 *** 120 120 300 6 BCL11A Erythroid-specific (%) - enhancer - 100 100 100 100 60 30 like) 80 80 200 4 80 80 200 4 - β / 2 γ BCL11A 60 60 40 ( 20 60 60 (%) Indels 1 40 40 100 2 40 40 100 2 HbF 20 10 HBB-HBG HBE LCR 20 20 Normalized Non 0 Normalized Non Log2 Enrichment 20 20 Caspase+ Cells % % Caspase+ Cells % chr11 HBE HBG2 HBG1 HBD HBB productive Indels (%) % Control (Erythroid) % productive productive Indels (%) % Control (Erythroid) % Normalized Indels (%) Normalized Indels 5120K 5140K 5160K 5180K 5200K 5220K 5240K 5260K 5280K 5300K 5320K 5340K 5360K 5380K 5400K 5420K 5440K (%) Normalized Indels 0 0 0 0 0 0 0 0 0 0 β-globin locus Insulator Enhance Insulator Mock Condition Condition Mock Condition Condition (5’HS) r (3’HS1) BM BM α α α α BM BM (HS1-4) • ~26,000 gRNAs were tested covering 320kb genomic region. 1 2 1 2 LCR γ γ s s Control β β Control HBG1/2 HBG1/2 Control Control BCL11A BCL11A Erythroid • ~300 HbF-inducing gRNA were identified. Erythroid Erythroid Erythroid HbF HbS • Most were mapped to β-globin locus including HBG, HBD, and HBB genes. *** p<0.001 Conclusions References Only Erythroid Progeny Displayed Reduced Total Indels or Intravenous (IV) Bone marrow infusion (BM) collection Enriched Non-Productive Indels Following Transplantation . Long-term engraftment was observed in immunocompromised NBSGW 1Bender MA. In: Gene Reviews® [Internet]. Seattle (WA): Methods used in this study tested for specific of BCL11A-edited CD34+ HSPCs mice with both BCL11A erythroid enhancer-edited and HBG1/2 promoter- University of Washington, Seattle; 1993-2018. preclinical target criteria: Edited or mock 8-16 weeks edited CD34+ HSPCs. transfected 2Chang et al. Presented at American Society of Hematology CD34+ cells NBSGW Mice 1. Successful editing of long-term HSCs Total Indels Non-productive Indels . In this study, BCL11A-edited CD34+ HSPCs had an erythroid (ASH 2018), San Diego, CA 2. Maintenance of normal HSPC function 120 300 differentiation defect in the NBSGW mouse model that was not observed 3. Robust, long-term induction of HbF 1 Lineage reconstitution by flow cytometry 100 in HBG1/2-edited CD34+ HSPCs. Human 80 200 Productive Acknowledgements Chimerism Erythroid B Cell Monocyte Neutrophils HSPCs - . In vivo-derived erythroid cells from BCL11A-edited CD34+ HSPCs had 60 hCD45/WBC CD235a/total hCD19/hCD45 hCD14/hCD45 hCD15/hCD45 hCD34/hCD45 reduced total indels and increased non-productive indels compared to 40 100 We’d like to thank Abigail Vogelaar, Kiran Gogi, Jen DaSilva, , 2 Editing analysis by Next-Gen Sequencing (NGS) (%) Indels other tested lineages, a phenomenon not observed with HBG1/2 promoter 20 Andrew Sadowski, Katherine Loveluck, Hoson Chao, Eric • Unfractionated BM (%) Normalized Indels editing. Tillotson, Aditi Chalishazar, Abhishek Dass, Deepak Reyon, Ari 0 0 • Flow sorted erythroids, B cells, neutrophils, and Lin-HSPCs Friedland, Fred Harbinski, Haiyan Jiang for scientific Normalized Non BM BM . There was robust induction of HbF in HBG1/2 promoter-edited erythroid contributions; Mrudula Donepudi for support in preparing this 3 Analysis of HbF (γ/β-like) expression by reverse phase UPLC HSPC HSPC 2 presentation, and Bob Brown for graphic design support. B Cells B Cells cells from long-term (8-16 week) in vivo studies. Erythroid • Flow sorted CD235a+ erythroid cells Erythroid Neutrophil Neutrophil • Ex vivo cultured erythroid cells from chimeric BM . Further optimization of nuclease and gRNA combinations led to HbF Disclosures: 4 Apoptosis assessment of cultured erythroid cells by flow cytometry expression of ~40%. Employees and Shareholders of Editas Medicine: Although editing of long-term HSCs was observed, normal HSPC JH, ED, TW, PS, GG, JS, RV, FT, EB, DT, EM, JZ, MI, TT, GG, ST, CW, CA, KHC. function was not maintained. Therefore, BCL11A erythroid enhancer- . IND-enabling activities have been initiated. Former employees of Editas Medicine Inc.: MS, SH. editing did not meet the preclinical target criteria. Presented at the 24th Congress of the European Hematology Association, 2019, Amsterdam editasmedicine.com © 2019 Editas Medicine 11 Hurley Street | Cambridge, MA 02141.
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