CRISPR-mediated Editing of Hematopoietic Stem Cells for the Treatment of β-hemoglobinopathies

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1 CRISPR-mediated Editing of Hematopoietic Stem Cells for the Treatment of β-hemoglobinopathies Jennifer Gori American Society of Gene & Cell Therapy May 11, 2017 editasmedicine.com 1

2 Highlights Developed high-throughput CRISPR-based screening method in human hematopoietic stem cells (HSCs) to identify sites that increase fetal hemoglobin (HbF) protein Novel editing approach at -globin locus supports potent induction of HbF protein in erythroid progeny of healthy adult human HSCs CRISPR-edited HSCs engraft efficiently in bone marrow and reconstitute blood production long term in vivo in mice Edited HSCs have the potential to provide a durable therapy for patients with -hemoglobinopathies 2

3 Presentation Overview Sickle Cell Disease, -globin Gene Regulation, and and Editing Editing Strategy Strategy Platform for CRISPR Medicines Editing of Adult Human HSCs to Increase HbF Protein Repopulating the Blood System with Edited HSCs 3

4 Mutation in -Hemoglobin Gene Causes Sickling Sickle Cell Disease, -globin Gene Regulation, and Editing Strategy DNA Protein Tetramer Low O 2 -globin G A G GLU HbA S-globin G T G VAL s s s s s s s s HbS Disease Symptoms and Complications Vaso-occlusion, pain crises, acute chest syndrome, stroke, embolism, hypertension, organ damage, anemia, premature mortality 4

5 Human -globin Locus: Regulation of Gene Expression Sickle Cell Disease, -globin Gene Regulation, and Editing Strategy Globin Switch % Globin synthesis Adapted from Canver and Orkin, Blood, e g g g d Months post-conception Onset of SCD symptoms -globin Locus Insulator (3 HS1) HbS Adult d Fetus Ag Gg e g g Embryo Enhancer (HS1-4) LCR Insulator (5 HS) HbA HbF 5

6 Globin Switching Model is Based on Chromatin Looping and LCR Interaction with Globin Promoters Sickle Cell Disease, -globin Gene Regulation, and Editing Strategy -globin Locus d Ag Gg e Insulator (3 HS1) Enhancer (HS1-4) LCR Insulator (5 HS) Chromatin Looping Model of -globin Gene Transcription e HbA d Ag Gg HbF Adapted from Kim & Dean, Mol Cell, 2012 and Wilber et al, Blood, 2011 Locus control region (LCR) regulates -like globin genes 6

7 Several Proteins Involved in Globin Switch Regulation Sickle Cell Disease, -globin Gene Regulation, and Editing Strategy Gamma Globin Expression à HbF Beta Globin Expression à HbA e e d Ag Gg d Ag Gg Adapted from Kim & Dean, Mol Cell, 2012 and Wilber et al, Blood, 2011 Complex process regulated by transcriptional activation and repression (e.g., BCL11A, SOX6) 7

8 Hereditary Persistence of Fetal Hemoglobin (HPFH): Inherited Mutations that Increase Fetal Hemoglobin Sickle Cell Disease, -globin Gene Regulation, and Editing Strategy -globin Locus d Ag Gg e Deletions Causing HPFH Adapted from Higgs, Engel and Stamatoyannopoulos (Review) Lancet, 2012 Lower Rate of Acute Chest Syndrome in SCD patient with elevated HbF Point Mutations Causing HPFH Enhanced Survival of SCD patients with HbF > ~8.6% Acute Chest Rate (episodes/year) Fetal Hemoglobin (%) Patients with elevated HbF have reduced disease severity, enhanced survival Castro et al., Blood, 1994, Platt et al., NEJM, 1994, Powars et al., Blood,

9 CRISPR Editing to Induce Fetal Hemoglobin Sickle Cell Disease, -globin Gene Regulation, and Editing Strategy Bone Marrow CD34 + HSC Genome Editing Strategy Disrupt regulatory elements in -globin locus that repress HbF 9

10 Presentation Overview Sickle Cell Disease, -globin Gene Regulation, and Editing Strategy Platform for CRISPR Medicines Editing of Adult Human HSCs to Increase HbF Protein Repopulating the Blood System with Edited HSCs 10

11 CRISPR Unlocks Genome Editing Platform for CRISPR Medicines Guide Sequence Cas9 DNA PAM Cut Sites ~100 nt DNA Guide Sequence Cpf1 PAM Cut Sites 42 nt Nuclease Guide RNA Editing machinery can be engineered to target many genomic locations 11

12 Unparalleled Platforms for CRISPR Medicines Platform for CRISPR Medicines # potential target sites Access to Cas9 and Cpf1 systems, species, and variants supports targeting of sites that are hard to target with most commonly used version of Cas9 12

13 Scalable, Consistent Engineered Cell Therapies Platform for CRISPR Medicines Electroporation + RNA Guided Endonuclease RNA Guide Ribonucleoprotein Particle (RNP) Patient Cell Engineered Patient Cell Engineer multiple components to HSC sensitivity (maintain cell viability, potency) 2017 Editas Medicine 13

14 Covalently-Coupled Dual grna Create Opportunities Platform for CRISPR Medicines Single grna 5 3 Heterogeneous product (full-length, truncated, errors) 5 3 Covalently-Coupled Dual grna (dgrna) Well-defined product (full-length only) 5 3 Length change (bases) Length change (bases) Targeting sequence Position (5 à 3 ) Targeting sequence 2e 04 2e 04 2e 04 3e 04 6e e e e e e 04 5e e e 04 3e 04 8e e e e e e 04 4e e e e 04 3e 04 6e e e Position Position (5 à 3 ) Frequency of error high low 5 3 Increased flexibility to positional and end modifications Fidelity, scale, and purity are potentially superior for making medicines 14

15 Orthogonal Specificity Approaches for Best grnas Platform for CRISPR Medicines Proprietary in silico Prediction of Cutting Sites Testing of On-Target Cutting (Cas9, Cpf1 WT, nickase) Targeted Panels for Detection of Sites from Biased & Unbiased Screens Unbiased Detection of Off-Target Cuts and Genomic Alterations (e.g., GUIDE-Seq, UDiTaS ) Combine computational with unbiased empirical cell-based methods to accurately and thoroughly identify potential off-target sites and select best grnas 15

16 Presentation Overview Sickle Cell Disease, -globin Gene Regulation, and Editing Strategy Platform for CRISPR Medicines Editing of Adult Human HSCs to Increase HbF Protein Repopulating the Blood System with Edited HSCs 16

17 Guide Screening in CD34 + Cells Supports Target Identification Based on HbF Protein Induction Editing of Adult Human HSCs to Increase HbF d Ag Gg e Example of grna Targets in Screen Adult CD34 + cells Erythroid differentiation HITS Hits with more potent HbF/edit ratio advance to optimization and validation studies 17

18 Published Results Show Potential of BCL11A Editing Editing of Editing Adult Human in Adult HSCs to Increase HbF BCL11A ZFN mrna dose response: Editing and HbF induction Estimation of correlation between HbF and editing provides benchmark %HbF Protein (background subtracted) BCL11Ae ZFN 1:4 HbF/Edit ratio Adapted from Chang et al., Molecular Therapy, % Editing ZFN editing of BCL11A erythroid enhancer yields HbF induction with 1:4 HbF/edit ratio 18

19 Screening grnas in CD34 + Cells Identifies Potent Hits Editing of Adult Human HSCs to Increase HbF Hit 1 Hit 2 Hit 3 HIt 4 Hit 5 Hit 6 BCL11Ae ZFN Based on ZFN disruption of BCL11A erythroid enhancer Chang et al., Molecular Therapy, 2016 Exclude grnas 1:4 HbF/Edit Ratio Hits with more potent HbF/edit ratio advance to optimization and validation studies HbF Protein Analysis by HPLC = Fetal hemoglobin /Total - like hemoglobin Background subtraction - For edited samples, HbF levels detected in donor matched untreated controls were subtracted 19

20 Potent HbF Protein Induction by New Editing Approach Editing of Adult Human HSCs to Increase HbF [RNP] 3.75 µm 2.5 µm 0 µm CRISPR targeting -globin locus 1:2 HbF/Edit Ratio 25% HbF Protein BCL11Ae ZFN 1:4 HbF/Edit Ratio Based on ZFN disruption of BCL11A erythroid enhancer Chang et al., Molecular Therapy, 2016 Low-dose RNP editing increases HbF protein to ~25% in erythroid progeny of healthy HSCs 0, 2.5, 3.75 µm RNP tested, 2 different HSC donors 20

21 Presentation Overview Sickle Cell Disease, -globin Gene Regulation, and Editing Strategy Platform for CRISPR Medicines Editing of Adult Human HSCs to Increase HbF Protein Repopulating the Blood System with Edited HSCs 21

22 Long-term Engrafted Hematopoietic Stem Cell Analysis is Required for Target Validation Repopulating the Blood System with Edited HSCs HSC(1%) CD34 + Committed Progenitors(50%) MPP (2%) Erythroid Myeloid Lymphoid <1% of bulk CD34 + cells are true HSCs (self-renewing and multipotent) Edits in bulk CD34 + cells may not represent edits in long-term functional HSCs 22

23 Long-Term Engraftment Analysis of Edited Human Hematopoietic Stem Cells Repopulating the Blood System with Edited HSCs Ex Vivo In Vivo Day -3 Thaw CD34 + Day -1 Edit Day 0 Freeze Day +1 Thaw Infuse (1x10 6 /mouse) 4 Months 1 Endpoint Engraftment Edits in HSC HbF/Edit (RBC) 2 Transplant Analyze long-term reconstitution of human hematopoiesis in vivo (4+ months) Evaluate editing in marrow, spleen, and blood (human subsets) 23

24 Adult Human Hematopoietic Stem Cells Edited at -Globin Locus Repopulate Bone Marrow Repopulating the Blood System with Edited HSCs % Human CD45 + cells (of all CD45) Human Blood Cell Repopulation of Bone Marrow at 4 months 0 >50% Human CD45 + Control RNP 1 RNP 2 %Lineage + within hcd Human HSCs, Lymphoid, Myeloid Cells in CD45 + Cells B cells T cells Myeloid 11% CD34 + HSC Edited LT-HSCs engraft, maintain multipotency (4 months post-transplant) Mouse CD45 + Do multiple edited HSCs contribute hematopoiesis in vivo? Human CD34 + Use indel diversity as a marker for HSC polyclonality Human CD45 + Human CD

25 Lineage Tracking of HSC Progeny in vivo Repopulating the Blood System with Edited HSCs Gene therapy results in multiple sites that support tracking HSC contribution based on the unique and common integration sites detected Gene editing should only result in one site modified Unique alleles that occur from differences in DNA repair at the target site are used to survey for edited HSC diversity and differentiation potential 25

26 Tracking Hematopoiesis Based on Edited Alleles Repopulating the Blood System with Edited HSCs DSB NHEJ Insertion Deletion barcodes Each unique edit provides barcode based on indel characteristics and position Multiple unique indels in HSCs/progeny suggests edited HSC diversity maintained 26

27 Polyclonal Hematopoiesis from CRISPR-Edited HSCs Evaluating CRISPR-edited HSC polyclonality: HBB as model locus for method development Repopulating the Blood System with Edited HSCs Low contributing edited alleles (grouped) Top 5 most abundant unique alleles (rank ordered) HBB RNP 4 months after transplantation of CD34 + cells edited at HBB locus Multiple edited HSCs are contributing to blood lineages with no dominant edited allele detected 27

28 Summary and Conclusions Developed a high throughput screening platform in HSCs to evaluate efficiency and potency of CRISPR nucleases and grnas in HSCs targeting HbF protein induction Identified potent hits that increase HbF protein ~25% in erythroid progeny of healthy donor CD34 + cells treated with low dose RNP Edited CD34 + cells reconstitute hematopoiesis in vivo and engraft long-term (>50% chimerism) CRISPR-edited HSCs for the treatment of -hemoglobinopathies have potential to provide superior clinical benefit to patients 28