Gene editing and Targeted Integration Using Zinc Finger Nucleases for Subjects with Hemophilia B NIH Recombinant DNA Advisory Committee September 9, 2015 Nadia Ewing, MD, City of Hope John A. Zaia, MD, City of Hope Dale Ando, MD, Sangamo BioSciences, Inc. Michael Holmes, PhD, Sangamo BioSciences, Inc. Kathleen Meyer, MPH, PhD, DABT, Sangamo BioSciences, Inc.
Engineered ZFN ZFP ZFP • Contains two domains: – Nuclease domain of FokI restriction enzyme – Designed zinc finger protein (ZFP) • Cleaves as a dimer – cleavage requires the ZFNs to be spaced by 5-6 bp • May be engineered to cleave virtually any sequence • Effectively a “designer restriction enzyme”
In Vivo Genome Editing – Modifying Liver Hepatocytes to Correct Monogenic Disease ZFN-Driven Targeted Gene Addition ZFN-Driven Targeted Gene Addition / Gene Correction Beumer et al PNAS 2008 (Dana Carroll)
Systemic Delivery of ZFP Therapeutics via AAV Allows In Vivo Gene Correction of Monogenic Disease Peripheral IV Human albumin Nucleic Acids Coding for Administration ZFN Pair and ZFNs and Corrective Gene Corrective Gene in AAV Vectors AAV Traffics to Liver And ZFNs Permanently Modify the Genome ZFN AAV Vectors ZFN Liver Cells Secrete Corrected Protein Homology Corrective Gene Homology In Vivo Protein Replacement (e.g. Factor IX) 4 9/16/2015 Sangamo Confidential
Albumin Provides an Ideal Safe Harbor Locus in Liver • Targeted addition to a “Safe Harbor” locus – Integration of a therapeutic transgene into a “safe harbor” – Albumin gene as proposed “safe harbor” locus: Safe, tissue-specific, and highly expressed – Targeting intronic sequence alleviates concerns of knocking out a large fraction of alleles by NHEJ – Single reagent set can be used for multiple indications • Albumin — the most abundant blood plasma protein Safe to co-opt a small percentage of Albumin Tissue specific and only produced in the liver Very highly expressed relative to protein replacement therapies Normal Albumin levels in blood - 40,000-50,000ug/ml; Normal F9 levels – 4-5ug/ml - synthesis rate of albumin from liver – 105,000mg/week
ZFN-Based Approaches to Correct Hemophilia Using Albumin as a “Safe Harbor” Locus ZFN E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13 E14 E15 h Albumin : (Safe Harbor) Stop Donor: Homology hF9 Exons 2-8 Homology ITR ITR Therapeutic Transgene Cassette
Expression of hFIX Protein from the Albumin Locus
Overview of Mouse Studies 2-60 weeks wild type Analyze circulating hF.IX F9 ex2-8 IV delivery ZFN donor 2 weeks 1x10 11 5x10 11 hemophilia B Analyze clotting time AAV8 AAV8 week 8 adult model E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13 E14 E15 E1 ZFN Albumin gene ZFN F9 d onor: Homology Homology w.t. Exons 2-8 8
Stable Circulating hF.IX Following mALB ZFN and Donor Delivery in WT Mice (Data through 60 weeks) hF.IX Levels ALT Levels 100 6000 Plasma hF.IX (ng/ml -1 ) 80 4000 ALT IU/L 60 40 2000 20 0 0 0 4 8 1230 4050 60 0 4 8 1230 4050 60 Time (weeks after Injection) Time (weeks after Injection) Therapeutic hF.IX levels observed for 60 weeks (longest time point assayed) No change in ALT levels Katherine High Lab 9 9/16/2015 Children's Hospital of Philadelphia
Correction of Prolonged Clotting Times in HB Mice ** 1500 100 Plasma hF.IX (ng ml -1 ) 80 1000 (s) aPTT (s) 60 500 40 50 20 25 n.s. 0 Baseline Week 2 0 Baseline Week 2 Wild Type 100 % of Normal 50 0 Antigen Activity Katherine High Lab 10 Children's Hospital of Philadelphia
Hemophilia B • Inherited coagulation factor deficiency resulting from reduced levels or absence of FIX • Can routinely be distinguished from hemophilia A by measuring FIX and FVIII activity • Hemophilia A: 20.6 per 100,000 males – Severe: 50-60% • Hemophilia B: 5.3 per 100,000 males (1 in 30,000 males world wide) – Severe: 44%
Factor IX • Factor IX gene (F9) – On the long arm of the X chromosome – First cloned in 1982-1983, 34 kb long, contains 8 exons • Serine protease (requires FVIII as cofactor to form the Xase complex to cleave FX to generate Xa) • Biosynthesis: liver • Plasma concentration ~5 m g/mL (5,000 ng/mL) • Normal FIX level: 50 - 150%; severity of disease correlates with FIX levels (Severe - <1%, Moderate – 1-5%, Mild – 5-40%)
Control and Prevention of Bleeding in Hemophilia B Patients – On demand (frequency depending on bleeding severity) vs prophylaxis (scheduled infusions twice/week to once/1-2 weeks) • Factor replacement products – Fresh Frozen Plasma (when nothing else is available!) – Plasma derived FIX concentrates – Recombinant FIX concentrates – Extended half-life recombinant FIX • For patients with inhibitory antibodies: – Recombinant activated FVII, or activated prothrombin complex concentrates –
Economic Impact of Current Rx – Direct cost • Clotting factors account for ~98% of hemophilia care cost • On demand: $200,000 to 300,000 for the average 60 kg adult/year • Prophylaxis: $500,000 to $900,000 /year • Surgery or major trauma: ~$150,000 (FIX only) – Indirect cost • Disability • Unemployment • Absenteeism • Early retirement
A Phase I, Open-Label, Ascending Dose Study to Assess the Safety and Tolerability of AAV Factor IX Gene Therapy via Zinc Finger Nuclease (ZFN) mediated targeted integration of SB-FIX in Adult Subjects with Hemophilia B Investigational SB-FIX is composed of three AAV2/6 vectors that encode-- Products ZFN #1 that targets position 447-461 of the albumin locus ZFN #2 that targets position 468-485 of the albumin locus AAV 2/6 FIX Donor cDNA Study Design Phase 1, multi- site, open-label, dose escalation study Objectives Primary: Evaluate the safety and tolerability of SB- FIX Secondary: 1. Change from baseline in FIX levels 2. Change from baseline in coagulation parameters (aPTT) 3. Change from baseline in the frequency and severity of bleeding episodes 4. Immune response to AAV 2/6 and FIX inhibitors 5. Detection of SB-FIX in blood, saliva, urine, stool and semen
A Phase I, Open-Label, Ascending Dose Study to Assess the Safety and Tolerability of AAV Factor IX Gene Therapy via Zinc Finger Nuclease (ZFN) mediated targeted integration of SB-FIX in Adult Subjects with Hemophilia B Main Inclusion 1. Signed informed consent Criteria 2. Male >18 years of age 3. Severe hemophilia B (native circulating FIX activity <1%) resulting from a missense mutation or a nonsense mutation that has not been associated with an inhibitor 4. Receiving prophylactic FIX replacement therapy as per current guidelines for the management of hemophilia, or on demand treatment of bleeding episodes 5. Received >20 doses of FIX replacement therapy with absent FIX inhibitor 6. No history of an allergic reaction or anaphylaxis to FIX products 7. No contraindication to the use of corticosteroids for immunosuppression 8. Agreement to use barrier contraceptive until at least 3 consecutive semen samples after SB-FIX are negative for AAV2/6
Study Schema Study Period ~ 15 months Long Term Follow Up Infusion and Safety Evaluations - 12 months 36 months Hospitalization Screen for SB-FIX Infusion 3x/week ALT/AST BL Day 0 -1 1 2 3 4 5 6 7 8 9 10 11 12 16 20 24 28 32 36 40 44 48 52 M18, 24, 36, 48 Week
Mitigation of risk • Exclusion of subjects with mutations associated with inhibitor formation, or who have inhibitors at screening: monitoring for FIX inhibitors by (at least) monthly Bethesda assays • Exclusion criteria for active hepatitis and liver disease • Careful monitoring for transaminitis in weeks 2-12 and treatment with corticosteroids if 2-fold rise above ULN • Routine monitoring by liver elastography (Fibroscan)
Nonclinical Safety Evaluation Program for SB-FIX • In Vitro Pharmacology Studies – Proof-of-concept in mouse, nonhuman primate (NHP) and human hepatocytes • In Vitro Safety Studies – Soft agar transformation assay with human fibroblast cell line – Off-target analysis: SELEX-guided assessment (mouse, NHP and human genomes) – Off-target analysis (unbiased): Integration site assay • In Vivo Pharmacology, Biodistribution and Toxicology Studies (discussed today) – Mouse and NHP studies
In Vivo Pharmacology/Toxicology Studies • Surrogate ZFNs and hF9 donor components required due to species-specific differences in DNA sequences at the albumin intron 1 target locus – Mouse – Nonhuman primate: cynomolgus and rhesus monkeys • Study design – Single-Dose IV administration – 1:1:8 ratio for ZFN1:ZFN2:hF9 donor – Co-administration of three AAV vectors on Day 1 • Pharmacology/Toxicology studies – 3-month pilot pharmacodynamics (PD), biodistribution (BD) and toxicology study of AAV2/6 vectors in C57BL/6 mice – GLP 6-month PD, BD and toxicity study of AAV2/6 vectors in C57BL/6 mice – A sequential-dose PD, BD and toxicity study evaluating single- or co-administration of AAV2/8 vectors in rhesus monkeys; cynomolgus monkey bridging study – A dose range-finding and dose-ratio study of AAV2/6 vectors in cynomolgus monkeys – A 60-day study to evaluate PD, BD and toxicity of AAV2/6 vectors in cynomolgus monkeys
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