Genome Editing of HBG1/2 Promoter Leads to Robust Hbf Induction in Vivo While Editing of BCL11A Erythroid Enhancer Shows Erythroid Defect

Genome Editing of HBG1/2 Promoter Leads to Robust HbF Induction In Vivo While Editing of BCL11A Erythroid Enhancer Shows Erythroid Defect

Kai-Hsin Chang, Minerva Sanchez, Jack Heath, Edouard de Dreuzy, Scott Haskett, Abigail Vogelaar, Kiran Gogi, Jen DaSilva, Tongyao Wang, Andrew Sadowski, Gregory Gotta, Jamaica Siwak, Ramya Viswanathan, Katherine Loveluck, Hoson Chao, Eric Tillotson, Aditi Chalishazar, Abhishek Dass, Frederick Ta, Emily Brennan, Diana Tabbaa, Eugenio Marco, John Zuris, Deepak Reyon, Meltem Isik, Ari Friedland, Terence Ta, Fred Harbinski, Georgia Giannoukos, Sandra Teixeira, Christopher Wilson, Charlie Albright, Haiyan Jiang

Editas Medicine, Inc., Cambridge, MA

60th Annual Meeting and Exposition of American Society of Hematology December 2, 2018, San Diego, CA

Etiology of Sickle Cell Disease

In Vivo Study Design to Evaluate Two Approaches to Increase Fetal Hemoglobin (HbF) Expression

Effect of Downregulating BCL11A Expression by Targeting its Erythroid Enhancer

Editing Cis-regulatory Elements in b-Globin Locus

Conclusion

Amino Hemoglobin Low DNA Acid Tetramer O2 a a a a b-globin GLU G A G b b b b HbA a a a a a a a a s s s VAL b b b -globin G T G bs bs bs bs bs bs HbS HbS Fiber

• Sickle cell disease (SCD) is caused by a single mutation E6V of the b-globin chain, leading to polymerization of 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 early mortality.

Bender MA. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Stephens K, Amemiya A, editors. Gene Reviews® [Internet]. © 2018 Editas Medicine Seattle (WA): University of Washington, Seattle; 1993-2018. Harnessing Natural Anti-sickling Hemoglobin to Treat Sickle Cell Disease

Embryo Fetus Adult b-globin locus HBE HBG2 HBG1 HBD HBB

Insulator Enhancer Insulator (5’HS) (HS1-4) (3’HS1) LCR a a a a g g HbS b b HbF HbA 100 a g 75 b a a a a g g b b Onset of Globin switch 50 e SCD 25 Symptoms % Globin % Synthesis Globin d 0 0 3 6 9 12 15 18 Months Post-conception

CRISPR-based disruption of HbF-repressing machinery

Engineered patient Patient CD34+ cells* CD34+ cells* with switching reversed

a a a a a a bs bs g g bs bs HbS HbF HbS

Successful Maintenance Robust editing of of normal long-term long-term HSPC induction of HSCs functions HbF

Intravenous (IV) Bone marrow infusion (BM) collection

Edited of mock 8-16 weeks transfected CD34+ cells NBSGW Mice 1 Lineage reconstitution by flow cytometry Human Chimerism Erythroid B Cell Monocyte Neutrophils HSPCs hCD45/WBC CD235a/total hCD19/hCD45 hCD14/hCD45 hCD15/hCD45 hCD34/hCD45

2 Editing analysis by Next-Gen Sequencing (NGS) • Unfractionated BM • Flow sorted erythroids, B cells, neutrophils, and Lin-HSPCs 3 Analysis of HbF (γ/β-like) expression by reverse phase UPLC • Flow sorted CD235a+ erythroid cells • Ex vivo cultured erythroid cells from chimeric BM 4 Apoptosis assessment of cultured erythroid cells by flow cytometry

Intron2 GATAA chr2

BCL11A Erythroid-specific enhancer

BCL11A

chr11 HBE HBG2 HBG1 HBD HBB b-globin locus Insulator Enhancer Insulator (5’HS) (HS1-4) a a (3’HS1) LCR a a g g bs bs HbF HbS

85 80 Control 75 BCL11A 70 p<0.001 15 ***

10 Population (%) Population

5 ***

0 hCD45 CD235a CD14 CD15 CD19 CD34 Leukocyte Erythroid Monocyte Neutrophil B-cell HSPC

BM Sorted erythroid cells Cultured erythroids Reduced Increased Less editing Enrichment Of erythroid output non-productive edits apoptosis

120 *** 120 *** 300 *** 6 ***

100 100 productive 80 80 - 200 4

60 60 Indels (%) Indels 40 40 100 2

20 20 Cells % Caspase+ % Control (Erythroid) % Control Normalized Indels (%) Normalized non 0 0 0 0 BM BM Control Control BCL11A BCL11A Erythroid Erythroid p<0.001 © 2018 Editas Medicine BCL11A Erythroid Enhancer-editing Failed to Meet Preclinical Target Criteria

Successful Maintenance Robust editing of of normal long-term long-term HSPC induction of HSCs functions HbF

Adult Fetus Embryo HBB HBD HBG1 HBG2 HBE b-globin locus Insulator Enhancer Insulator (3’HS1) (HS1-4) (5’HS) LCR

HBB HBG1-2 (2)

2 t n

e 2 m h c i r

n 1 e

v 1 a

. g v

A 0 HBE LCR 0 HBB-HBG Log2 Enrichment Log2 -1 5120K 5140K 5160K 5180K 5200K 5220K 5240K 5260K 5280K 5300K 5320K 5340K 5360K 5380K 5400K 5420K 5440K

coordinates • ~26,000 gRNAs were tested covering 320kb genomic region • ~300 HbF-inducing gRNA were identified • Most were mapped to b-globin locus including HBG, HBD, and HBB genes

30 like) (%) - b /

g 20 (

HbF 10

0 Control HBG1/2

Multiple enzymes including SpCas9, SaCas9, Cpf1 (Cas12a) and gRNA combinations were tested

90 85

80 Control 75 HBG1/2 70

10 Population (%) Population

0 hCD45 CD235a CD14 CD15 CD19 CD34 Leukocyte Erythroid Monocyte Neutrophil B-cell HSPC

BM Sorted erythroid cells Cultured erythroids

Similar levels of Similar levels of indels in Similar levels erythroid output erythroid compared to BM of apoptosis

120 120 300 6

100 100

80 80 productive 200 4 - 60 60

40 40 (%) Indels 100 2

20 20 Cells % Caspase+ % Control (Erythroid) % Control Normalized Indels (%)

0 0 Normalized non 0 0 BM BM Control Control HBG1/2 HBG1/2 Erythroid Erythroid

*** *** 30

20 like) (%) - b / g ( 10 HbF

0 Control HBG1/2 Control HBG1/2

Successful Maintenance Robust editing of of normal long-term long-term HSPC induction of HSCs functions HbF

Long-term engraftment observed in immunocompromised NBSGW mice with both BCL11A erythroid enhancer-edited and HBG1/2 promoter-edited CD34+ HSPCs

In this study, BCL11A-edited CD34+ HSPCs had an erythroid differentiation defect in the NBSGW mouse model that was not observed in HBG1/2-edited CD34+ HSPCs

Robust induction of HbF in HBG1/2 promoter-edited erythroid cells from long-term in vivo study

Pipeline

Platform

Program Management

CMC

Clinical

Lab Operations