Gene Therapy for Hemoglobinopathies

Janet L. Kwiatkowski, MD, MSCE

Cellicon Valley ‘21: Future of Cell and Gene Therapies May 6, 2021 Disclosures

• Consultant: bluebird bio, (), Imara, Agios

• Site principal investigator: bluebird bio, Sangamo, Bioverativ, CRISPR, ApoPharma (Chiesi), Terumo BCT Overview

• Background

• methods – Gene addition – Gene editing – Post-transcriptional silencing of BCL11A for SCD

• Efficacy of gene therapy

• Safety of gene therapy Sickle Cell Anemia and β-Thalassemia

Adult Hemoglobin Sickling Hb Mutations that alter the structure and red cells a b (Single mutation, β6 gluàval ) Heme b a Mutations that reduce the synthesis Alpha/Heme Aggregates a a or hemichromes (>200 mutations aHeme β0-severe; β+,HbE -milder)

Consequences of Abnormal β-Globin Consequences of Reduced β-Globin Chain Structure Chain Production Vaso-occlusion Ineffective erythropoiesis Hemolysis Hemolysis Anemia Anemia

Similar approaches to gene therapy for both disorders with disease-specific modifications in treatment and assessment of efficacy Courtesy of S. Rivella Gene Addition • Self-inactivating lentiviral vector

• Beta globin gene addition – Lentiglobin (beti-cel, bb1111): Single amino acid substitution T87Q that has anti-sickling properties and can be distinguished from HbA by HPLC – GLOBE – Others in earlier phase of study

• Gamma globin gene addition – RVT-1801: Modified gamma globin with anti-sickling properties – Also utilizes non-myeloablative conditioning Gene Editing

• Rationale: raise HbF levels by reducing BCL11A, a HbF TALEN repressor, or editing gamma CRISPR/Cas globin promoter region Double strand break ZFN • Improves alpha:beta-like imbalance • Reduces Hb S polymerization Non- homologous end rejoining Homologous recombination

• Ongoing clinical trials • CRISPR Insertions or • Zinc finger deletions Donor DNA “indels”

Non functional gene Gene replacement/correction Lentiviral vector targeting BCL11A (BCH-BB694)

• Gene therapy approach using a lentiviral vector which encodes a short hairpin RNA (shRNA) targeting BCL11A mRNA embedded in a microRNA • Downregulation of BCL11A • Regulated erythroid expression: avoids off-target toxicity in HSCs and B Cells

HSC, hematopoietic stem cells Guda S et al, Mol Ther 2015;23(9):1465–74; Brendel C et al, JCI, 2016;126(10):3868–78; Esrick, et al NEJM 2021 384;3:205. NCT 03282656. Ex-Vivo Gene therapy: Schema

HSC Collection: HSCs harvested from bone marrow or by mobilization & apheresis

Hematopoietic stem cells

Informed Consent Transduction or Gene Editing: HSC modified by transduction (gene addition) or gene editing

Modified hematopoietic stem cells

Reinfusion: Conditioning (partial or full myeloablation) and reinfusion of genetically modified HSC Efficacy of Gene Therapy HGB-207 and HGB-212 interim results: transfusion status following Beti-cel infusion

HGB-207 Hb (g/dL) Patient receiving RBC transfusions at last study visit Patient not receiving RBC transfusions 0 0 1 11.8 34.4 Non-β β 2 9.9* 11.4 Transfusion independence achieved 3 12.1 29.3 (weighted average Hb ≥9 g/dL without 4 12.8 28.8 any RBC transfusions for ≥12 months) 5 13.0 28.9 6 14.3 28.6 IVSI-110/IVSI-110 genotype 0 7 13.1 28.1 β /IVSI-110 genotype 8 12.5 22.4 β+/IVSI-110 genotype 9 12.0 22.5 10 10.9 24.3 11 7.2† 21.6 12 13.1 18.2 13 10.1 18.6 14 11.5 17.5 15 9.5 17.8 16 11.2 17.6 17 11.6 14.9 HGB-207 18 9.7 14.1 19 10.3 14.3 • 91% (20/22) with > 3mo follow-up 20 9.2* 21 11.4 9.2 stopped transfusion HGB-212 22 10.0 6.6 β0β0 & 1 14.0 24.1 HGB-212 IVS I-110 2 10.2 19.9 3 13.0 16.4 • 85% (11/13) have been off 4 8.5‡ 1.7 5 9.8 15.0 transfusions for > 6mo 6 12.7 15.3 7 9.5 14.5 8 7.7 0.0 9 11.3 13.4 10 10.8 11.4 11 8.8 10.8 12 10.2 7.7 13 9.4 7.4 14 9.0 2.4 Patient <12 years old at screening 0 3 6 9 12 15 18 21 24 0 6 12 18 24 30 36 Patient 12–18 years old at screening Months post beti-cel infusion Patient >18 years old at screening Months post beti-cel infusion

*Supported by transfusions; †Patient’s total Hb level at Month 22 was 13.2 g/dL. Following a planned orthopedic surgery, the patient had blood loss, which required 1 packed RBC transfusion;‡Transfusion within 60 days. Hb, haemoglobin; RBC, red blood cell; TDT, transfusion-dependent β-thalassemia.. Data as of 3 March 2020 Porter, EHA 2020; Yannaki, EHA 2020 Sickle Cell Disease (HGB-206 Group C) Improvement in Hemoglobin; HbAT87Q ≥ 40%

Median total Hb (g/dL) 8.9 11.7 11.8 11.8 11.7 11.7 11.5 11.0 11.3 11.5

(min–max) (g/dL) (6.4–12.5) (8.1–14.8) (9.1–14.4) (9.5–15.1) (9.3–15.4) (9.7–15.0) (9.6–14.9) (10.7–15.2) (10.5–16.2) (10.4–15.0) 20

) HbA (transfused) L

d HbS / g ( 15 HbA2 n o

i HbF t

a T87Q

r HbA

t 10 21%

n Baseline total Hb e 39% 50% 53% 54% 55% 52% 50% 47% 46% c n

o 5 c

b 35% 46% 44% 43% 43% 44% 48% 50% 52% H 0 Base0line 3 6 9 12 15 18 21 24 30 Months post-LentiGlobin infusion N* 15 25 22 22 17 13 10 8 7 3

Improvement in markers of hemolysis (LDH, bilirubin, reticulocyte count)

Data as of 20 August 2020 % represents median Hb fraction as % of total Hb; *Number of patients with data available. Hb, hemoglobin; max, maximum; min, minimum. HGB-206 Group C: Complete resolution of VOEs ≥6 months post-LentiGlobin treatment

24m HbAT87Q 24m stabilization† 0 DP IC - - 7 0 17 0 17 0 29 * 0 7 0 5 1 23 0 10 0 VOEs classified 11 0 as severe VOEs 4 0 8 0 All other VOEs 6 0 5 0 N=19 5 1 17 3 21 0 7

Total number of eventsofnumber Total overyears 2 pre 0

21 0 eventsyearsof number Total 2 to up post 19

24 Months Prior to Informed Consent Post-LentiGlobin Follow-up (months) Median (min–max) annualized VOE rate 5 (2.0–14.5) 0 (0–4.3) Data as of 20 August 2020 Protocol VOE are shown; Patients with ≥ 4 sVOE at baseline before IC and with ≥ 6 months of follow-up post-DP infusion are included. A VOE includes episodes of acute pain with no medically determined cause other than a vaso-occlusion, lasting more than 2 hours and severe enough to require care at a medical facility, a VOE includes acute episodes of pain, acute chest syndrome, acute hepatic sequestration, and acute splenic sequestration; †HbAT87Q expression stabilizes within 6 months; *One death, unlikely related to LentiGlobin, > 18 months post treatment in a patient with significant baseline SCD-related cardiopulmonary disease. Note: In the last datacut, one patient had a non-serious VOC at Day 107. The event is recorded as an investigator reported VOE but does not meet the definition of a protocol VOE DP, drug product; IC, informed consent; max, maximum; min, minimum; sVOEs, severe VOEs; VOE, vaso-occlusive event; VOC, vaso-occlusive crisis. CRISPR in Transfusion Dependent Thalassemia (CTX001): Rapid improvement in HbF, Total Hb

Franjoul, et al, ASH 2020 CRISPR in Sickle Cell Disease (CTX001): Improvement in Fetal and Total Hemoglobin

Fetal hemoglobin expression is pancellular Franjoul, et al, ASH 2020 CRISPR in Sickle Cell Disease (CTX001): Resolution of Vasoocclusive Pain Episodes following Treatment

Franjoul, et al, ASH 2020 Post-transcriptional Genetic Silencing of BCL11A (BCH-BB694)

Patient Follow-up % Hb F F-cells (%) Recent Hb (mo)

2 24 22.7 71 11.4

3* 18 20.4 58.9 9.5 4 21 31.9 81.9 11.1 6 15 38.8 65.3 11.0 7 12 29 70.6 11.0 8 6 41.3 93.6 9.3

*Remains on transfusions • No vaso-occlusive pain events or acute chest • 1 episode recurrent priapism >5 months post gene tx

Esrick, et al NEJM 2021 384;3:205. NCT 03282656 Safety of Gene Therapy

• Adverse effects typical of myeloablative chemotherapy – Beticel: VOD in 5/41 subjects with thalassemia; bb1111: no VOD in SCD – HLH reported in 1 patient with TDT treated with CTX011 - though secondary to conditioning – Long-term: infertility risk

• Lentiglobin: slow platelet engraftment

• No vector mediated replication competent LV – One subject with thalassemia treated with beticel acquired HIV infection, 23 mo after drug product infusion – Wildtype HIV-1 documented

• Sudden death in one subject with SCD, Group C, 20 months after treatment, thought unrelated to the gene therapy MDS/AML in Lentiglobin Studies

Patient: SCD Pt 1 SCD Pt 2 SCD Pt 3 Year of Diagnosis: 2018 2021 2021 Age 45 year old Adult Adult Study/Group HGB-206/Group A HGB-206/Group C HGB-206/Group A Time from dosing to 3 years 6 months 5.5 years diagnosis MDS (progressed to Presenting diagnosis MDS? AML AML) Trisomy 8 Monosomy 7 No blasts/dysplasia Vector detected in blasts inserted No vector in blast identified therefore MDS in VAMP4 cells cannot be confirmed VAMP4 with no known role in Relevant findings Follow-up BMA without leukemia/oncogenesis Pre-tx BM negative for genetic abnormality No impact on gene monosomy 7 and Diagnosis revised to expression/regulation NGS for 54 mutations transfusion-dependent Mutations in RUNX1, PTPN11 associated with AML anemia

Lentiglobin SCD trials placed on hold. Clinical use of Zynteglo for thalassemia (Europe) on hold Summary/Conclusions

• Beta globin gene addition trials have achieved hemoglobin levels that enable transfusion independence in β-thalassemia and improvement in anemia and vasoocclusive events in SCD

• CRISPR gene editing targeting BCL11a also with early results showing robust HbF production, transfusion independence in thalassemia and improvement in anemia and vasoocclusive events

• Safety profile generally consistent with myeloablative conditioning, autologous transplant, and the underlying blood disorder – Cases of MDS/AML in SCD have resulted in clinical trial hold

• Gene therapy may offer an alternative curative/disease modifying treatment option but a better understanding of leukemia risk especially for patients with SCD is needed