Gene Editing and Targeted Integration Using Zinc Finger Nucleases for Subjects with Mucopolysaccharidosis I (MPS I)

Transcription

1 Gene Editing and Targeted Integration Using Zinc Finger Nucleases for Subjects with Mucopolysaccharidosis I (MPS I) NIH Recombinant DNA Advisory Committee December 4, 2015 Chester Whitley, PhD, MD, University of Minnesota Thomas Wechsler, PhD, Sangamo BioSciences, Inc. Scott McIvor, PhD, University of Minnesota

2 Agenda MPS I Clinical Background Preclinical Overview ZFN technology and the albumin safe harbor locus approach SB-318 IND-enabling nonclinical safety evaluation program In vitro Evaluation of SB-318 in HepG2 cells Clinical Protocol 2

3 Agenda MPS I Clinical Background Preclinical Overview ZFN technology and the albumin safe harbor locus approach SB-318 IND-enabling nonclinical safety evaluation program In vitro Evaluation of SB-318 in HepG2 cells Clinical Protocol 3

4 Mucopolysaccharidosis Type I: Clinical Background Lysosomal a-l-iduronidase enzyme metabolic defect Nosology Hurler syndrome Attenuated MPS I Scheie syndrome Hurler-Scheie syndrome Treated Hurler syndrome Outcome of current treatments Enzyme replacement therapy (ERT) Hematopoietic stem cell transplantation (HCST) Bone marrow transplantation (BMT) Umbilical cord transplantation (UCT) Response of combined therapies 4

5 Mucopolysaccharidosis Type I Lysosomal a-l-iduronidase enzyme metabolic defect Nosology Hurler syndrome Attenuated MPS I Scheie syndrome Hurler-Scheie syndrome Treated Hurler syndrome Outcome of current treatments Enzyme replacement therapy (ERT) Hematopoietic stem cell transplantation (HCST) Bone marrow transplantation (BMT) Umbilical cord transplantation (UCT) Response of combined therapies 5

6 Lysosomal Storage Diseases MPS I and MPS II Glycoaminoglycans (GAGs) Iduronate 2-Sulfatase (IDS) in MPS II a-l-iduronidase (IDUA) in MPS I Accumulation of GAGs like Dermatan and Heparan Sulfates in the lysosome of all tissues leads to dysfunction in several tissues in MPS I patients 6 Modified after Neufeld and Muenzer 2001

7 Mucopolysaccharidosis: Lysosomal inclusions Liver pathology in mucopolysaccharidosis (MPS) Disease MPS I Normal 7

8 Mucopolysaccharidosis Type I Lysosomal a-l-iduronidase enzyme metabolic defect Nosology Hurler syndrome Attenuated MPS I Scheie syndrome Hurler-Scheie syndrome Treated Hurler syndrome Outcome of current treatments Enzyme replacement therapy (ERT) Hematopoietic stem cell transplantation (HCST) Bone marrow transplantation (BMT) Umbilical cord transplantation (UCT) Response of combined therapies 8

9 Mucopolysaccharidosis Type I Scheie syndrome (MPS IS) Hurler-Scheie syndrome (MPS IHS) Hurler syndrome (MPS IH) Hurler syndrome (MPS IH) 9

10 Mucopolysaccharidosis Type I Lysosomal a-l-iduronidase enzyme metabolic defect Nosology Hurler syndrome Attenuated MPS I Scheie syndrome Hurler-Scheie syndrome Treated Hurler syndrome Outcome of current treatments Enzyme replacement therapy (ERT) Hematopoietic stem cell transplantation (HCST) Bone marrow transplantation (BMT) Umbilical cord transplantation (UCT) Response of combined therapies 10

11 Orthopaedic Problems in MPS Spine Kypophosis, scoliosis Cervical cored compression Upper extremity Carpal tunnel syndrome Trigger digits Lower extremity Hip dubluxations Genu varum Digital deformities 11

12 Cervical Spinal Compression in a Patient with MPS 12

13 Carpal Tunnel Syndrome Carpal Tunnel is a Fixed Size Deposits of GAGs in tendons/tissue around tendons Enlarged carpal bones 13

14 Median Nerve Compression 14

15 Trigger Digits Results in Joint Stiffness Loss of PIP flexion Loss of DIP extension 15

16 Hip Subluxation If untreated, will lead to osteoarthritis (OA) at early age OA in this age patient is difficult to treat X-ray of 18 yr old pt 16

17 Genu Valgus Deformities Knock-knees Previously: Proximal Tibial stapling procedures Staples: High Complication Rate 17

18 Mucopolysaccharidosis Type I: Outcome of Treatment Lysosomal a-l-iduronidase enzyme metabolic defect Nosology Hurler syndrome Attenuated MPS I Scheie syndrome Hurler-Scheie syndrome Treated Hurler syndrome Outcome of current treatments Enzyme replacement therapy (ERT) Hematopoietic stem cell transplantation (HCST) Bone marrow transplantation (BMT) Umbilical cord transplantation (UCT) Response of combined therapies 18

19 Mucopolysaccharidosis Type I Lysosomal a-l-iduronidase enzyme metabolic defect Nosology Hurler syndrome Attenuated MPS I Scheie syndrome Hurler-Scheie syndrome Treated Hurler syndrome Outcome of current treatments Enzyme replacement therapy (ERT) Hematopoietic stem cell transplantation (HCST) Bone marrow transplantation (BMT) Umbilical cord transplantation (UCT) Response of combined therapies 19

20 Twenty-two year old male Transplanted at 18 months old Twenty years later: -Full donor engraftment confirmed by molecular studies -Donor levels of enzyme in peripheral leukocytes 20

21 Mucopolysaccharidosis: Poor Growth After HSCT 21

22 Glycosaminoglycan Before and After Bone Marrow Transplant Citation Case At Diagnosis Reference Range** Ratio Post-HSCT* Reference Range** S No. GAG/creatini ne (mg/g) GAG/creatini ne (mg/g) % Upper Limit GAG/creatini ne (mg/g) GAG/creatini ne (mg/g) % Upper Limit Herskhovitz et al, % % % % % % Hite et al,1999 and current ,645% % MEAN 911% 133% 22

23 23

24 24

25 25

26 26

27 27

28 28

29 29

30 30

31 31

32 32

33 33

34 34

35 35

36 36

37 37

38 Mucopolysaccharidosis Type IH: Neurogeneration 38

39 Neurologic Deterioration in Hurler Syndrome The rate of decline in Hurler syndrome without hematopoietic stem cell transplant is about 20 points per year, i.e., IQ drops 1.6 points per month The earlier the transplant, the better the cognitive outcome 39

40 Developmental Quotient (DQ) Declines in Children with Hurler Syndrome during the First Year after Hematopoietic Stem Cell Transplantation During the initial post-transplant year, children with Hurler syndrome continue to decline at the same rate as before. After 1 year of donor engraftment, IQ remains stable. 40

41 Neurologic Deterioration in Hurler Syndrome The rate of decline in Hurler syndrome without hematopoietic stem cell transplant is about 20 points per year, i.e., IQ drops 1.6 points per month The earlier the transplant, the better the cognitive outcome Would combining intravenous enzyme replacement therapy (ERT) with hematopoietic stem cell transplant (HCT) improve the cognitive outcome? 41

42 Standard Score (100 ± 15) Would Combining Intravenous Enzyme Replacement Therapy (ERT) with Hematopoietic Stem Cell Transplant (HCT) Improve the Cognitive Outcome? Visual Problem Solving is better in the combined therapy (HCT + ERT) group compared to the HCT-alone group Visual Reception Transplant Alone Transplant + ERT Baseline 2y post Baseline 2y post 42

43 Mucopolysaccharidosis Type I Scheie syndrome (MPS IS) Hurler-Scheie syndrome (MPS IHS) Hurler syndrome (MPS IH) Hurler syndrome (MPS IH) 43

44 Attenuated Mucopolysaccharidosis Type I Scheie syndrome ERT Hurler-Scheie syndrome ERT Hurler syndrome ERT Hurler syndrome BMT 44

45 Agenda MPS I Clinical Background Preclinical Overview ZFN technology and the albumin safe harbor locus approach SB-318 IND-enabling nonclinical safety evaluation program In vitro Evaluation of SB-318 in primary and transformed Hepatocytes Clinical Protocol 45

46 Technology Overview Engineered ZFN technology 5 T A C C C A A C G C G A A T T A T G G C G G C G T G C G C T T A A C G C A T G G G T 3 A T G G G T T G C G C T T A A T A C C G C C G C A C G C G A A T T G C G T A C C C A 3 5 Genomic DNA Nuclease Sequence specific designed Zinc Finger Protein (ZFP1) Heterodimeric FokI Nuclease Sequence specific designed Zinc Finger Protein (ZFP2) ZFN = Designer restriction enzyme 46

47 ZFN mediated DNA double-strand break leads to targeted gene correction Targeted Gene Addition / Gene Correction 47

48 ZFN Technology and the Albumin Safe Harbor Locus Approach ZFN-Mediated Gene Therapy Therapeutic Gene of Choice ZFN2 5 T A C C C A A C G C G A A T T A T G G C G G C G T G C G C T T A A C G C A T G G G T 3 A T G G G T T G C G C T T A A T A C C G C C G C A C G C G A A T T G C G T A C C C A 3 5 Genomic DNA ZFN1 Nuclease 5 T A C C C A A C G C G A A T T A T G 3 A T G G G T T G C G C T T A A T A C C G C G C G T G C G C T T A A C G C A T G A C G C G A A T T G C G T A C G G T C C A 3 5 Albumin safe harbor locus 5 T A C CCAACGCGAAT T A T G 3 A T G G G T T G C G C T T A A T A C Therapeutic Gene of of Choice T G C G C T T A A C G C A T G G G T A C G C G A A T T G C G T A C C C A 3 5 Hemoph. B MPS I MPS II Gaucher / Fabry SB-FIX SB

49 Systemic Delivery of ZFP Therapeutics via AAV Allows in vivo Gene Correction of Monogenic Diseases Human albumin ZFN pair and therapeutic gene Packaged into AAV2/6 vectors for delivery into liver One-time intravenous (IV) administration ZFN ZFN AAV Vectors ZFN1 ZFN2 hidua Liver cells secrete therapeutic protein into bloodstream Homology Therapeutic Gene Homology Cross correction of secondary tissues like spleen, heart lung and kidney 49

50 Targeted Integration at the Albumin Locus Leads to High Transgene Expression Albumin (the most abundant blood plasma protein) has all properties of an ideal In vivo Protein Replacement Platform gene: Tissue specific and only produced in the liver Safe to co-opt a small percentage of albumin Very highly expressed relative to protein replacement therapies > Normal albumin levels in blood: mg/ml ; Synthesis rate from liver: ~105 g/week Disease: MPS I Gaucher Fabry ERT Dose: (mg/week 75kg patient) ERT Half life: ±49 (minutes) % of Albumin production rate: 0.054% 0.063% 0.047% A very small fraction of natural albumin synthesis is needed to drive therapeutic levels of the new protein 50

51 Targeted Integration at the Albumin Locus Leads to High Transgene Expression Albumin (the most abundant blood plasma protein) has all properties of an ideal In vivo Protein Replacement Platform gene: Tissue specific and only produced in the liver Safe to co-opt a small percentage of albumin Very highly expressed relative to protein replacement therapies > Normal albumin levels in blood: mg/ml ; Synthesis rate from liver: ~105 g /week 51

52 Expression of hidua Protein from the Albumin Locus 52

53 SB-318 IND-Enabling Nonclinical Safety Evaluation Program In Vitro Pharmacology Studies Proof-of-concept in mouse and human hepatocytes In Vitro Safety Studies GLP soft agar transformation assay with SB-318 in human fibroblast cell line No in vitro transformation potential evident Off-target analysis: SELEX-guided assessment (mouse and human genomes) Off-target analysis (unbiased): Oligo integration assay In Vivo Pharmacology, Biodistribution and Toxicology Studies Wild-type C57BL/6 mice Prevention and treatment models in MPS I mice Cynomolgus monkeys 53

54 In vivo Pharmacology/Toxicology Studies Surrogate ZFNs and hidua donor components required due to species-specific differences in DNA sequences at the albumin intron 1 target locus Mouse Cynomolgus monkey Study design Single-Dose IV administration 1:1:8 ratio for ZFN1:ZFN2:hIDUA donor Co-administration of three AAV vectors on Day 1 Pharmacology/Toxicology studies Completed Studies Proof-of-concept studies in wildtype and MPS I mice (prevention model) In-Progress Studies 4-month hybrid pharmacology and toxicology study in MPS I mice GLP 6-month PD, BD and toxicity study of AAV2/6 vectors in C57BL/6 mice A 90-day pilot study to evaluate pharmacology, biodistribution and potential toxicity of AAV2/6 vectors in cynomolgus monkeys 54

55 Evaluation of SB-318 Specificity ZFN candidate specificity was evaluated by monitoring off-target modification at sites identified through SELEX-guided bioinformatics analysis No off-target modification observed at the Top 80 predicted off-target sites in human transformed and primary hepatocytes Unbiased genome-wide off-target assessment via oligo integration analysis Only one off-target site (SMCHD1) among all candidate sites with more than 5 integration events has been confirmed in human transformed and primary hepatocytes 55

56 Oligo Capture Analysis Overview 56

57 Off-Target Site Identified Smchd1 - Off-target site in human SMCHD1 gene was tested by dose titration experiment in human hepatocytes - There is a clear dose relationship between albumin on-target and SMCHD1 off-target activity - Off-target activity is about 2 logs lower than on-target activity - There is no detectable off-target activity below 10-20% on-target activity - Levels of on-target modification in the clinical setting are predicted to be <10% at albumin % indels % indels 50% 40% 30% 20% Gene modification at halb locus 10% Expected clinical range 0.0 3e3 1e4 3e4 1e5 3e5 1e6 1e4 1e5 mock MOI 2.0% 1.5% 1.0% 0.5% 0.0 SB-FIX ZFNs (SBS47171, SBS47898) GFP Gene modification at hsmchd1 locus 3e3 1e4 3e4 1e5 3e5 1e6 1e4 1e5 mock SB-FIX ZFNs (SBS47171, SBS47898) GFP AAV2/6 MOI AAV2/6 57

58 SMCHD1 is a Weak Off-Target Site in Non-Human Primate Cells in vitro and in vivo A) SMCHD1 off-target site is completely conserved between human and NHP B) In vitro NHP surrogate reagents can cut SMCHD1 in a dose-dependent manner, similar to human SB-318 reagents. Dose titration experiments in rhesus monkey hepatocytes show that only at on-target activity of >10% very low levels of SMCHD1 (<0.02%) modification are detectable C) In vivo day 90 samples from an NHP study using SB-318 surrogate reagents show no detectable off-target activity at SMCHD1 at on-target activities of up to 2.1%. In other studies there was no detectable off-target activity at Smchd1 despite on-target activities of up to 7.4% or 8.7% 58

59 SMCHD1 Gene Function - Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) - Mouse model demonstrated that SMCHD1 is necessary for embryonic development in females. Homozygous male k.o. are initially normal, about half die early (unknown), the other half has normal lifespan/health > No phenotypes associated with liver were reported; > ZFN expression is restricted to liver through use of a hepatocyte-specific promoter; > Little to no off-target activity seen at on-target levels that model therapeutic setting; > Low levels of SMCHD1 modification should not affect liver cells given they are polyploidy - SMCHD1 has role in DNA damage signalling > We have not observed any increase in DNA Damage Response markers in vitro - Germline SMCHD1 mutations are associated with Facioscapulohumeral muscular dystrophy type 2 (FSHD2) > This disease is caused by muscle cell-specific misregulation of DUX4 transcript; > ZFN expression is restricted to liver through use of a hepatocyte-specific promoter; > No ZFN activity has been observed in tissues other than the liver 59

60 In vitro Evaluation of SB-318 in HepG2 cells 60

61 In vitro Evaluation of SB-318 in HepG2 cells hidua protein level and Enzymatic Activity hidua ELISA IDUA Enzymatic Activity hidua (ng/ml) SB-318 treated HepG2 pools IDUA (nmol/hr/ml) SB-318 treated HepG2 pools HepG2 cells clone 21 clone 25 clone 30 SB-318 halb ZFNs + hidua donor HepG2 cells clone 21 clone 25 clone 30 SB-318 halb ZFNs + hidua donor 61

62 In vitro Evaluation of SB-318 in HepG2 cells hidua protein level and Enzymatic Activity hidua ELISA IDUA Enzymatic Activity hidua (ng/ml) SB-318 treated HepG2 pools IDUA (nmol/hr/ml) SB-318 treated HepG2 pools HepG2 cells clone 21 clone 25 clone 30 SB-318 halb ZFNs + hidua donor HepG2 cells clone 21 clone 25 clone 30 SB-318 halb ZFNs + hidua donor Donor integration in allele 1: Genotyping at allele 2: HDR NHEJ HDR 3bp 56bp 10bp Del. Ins. Del. 62

63 In vitro Evaluation of SB-318 in HepG2 cells mrna Analysis at Albumin Locus of HepG2 Subclones Donor integration in allele 1: Genotyping at allele 2: HDR NHEJ HDR 3bp 56bp 10bp Del. Ins. Del. 63

64 In vitro Evaluation of SB-318 in HepG2 cells mrna Analysis at Albumin Locus of HepG2 Subclones 64

65 Summary of Preclinical Studies SB-318 ZFNs are highly sequence-specific chimeric nucleases Off-target activity was assessed by unbiased and biased genome-wide assays SMCHD1 was the only off-target locus detected by unbiased oligo capture assay Off-target activity at the SMCHD1 locus is dose dependent and ~2 logs lower than on-target activity in vitro and in vivo (NHP). No anchorage-independent cell growth observed after SB-318 treatment in GLP soft agar transformation assay Studies in clonal HepG2 cell lines with integrated hidua donor showed robust transgene expression independent of integration mechanism 65

66 AAV Mediated Delivery of ZFNs and hidua cdna in Wildtype and MPS I mice - 4-week Proof of concept study in wildtype mice - 60-day therapeutic MPS I model study in 4-5 month old MPS I mice day preventative MPS I model study in 1-2 month old MPS I mice Study analysis a) Genotyping at albumin locus (not shown) b) Enzymatic activity of hidua protein in liver, plasma and secondary tissues c) Urinary and tissue GAG analysis 66

67 AAV Mediated Delivery of ZFNs and hidua cdna results in Supraphysiological Levels of hidua activity in Wildtype mice IDUA activity Supraphysiological levels of hidua activity detected in liver (primary tissue), plasma and secondary tissues like spleen 67

68 AAV Mediated Delivery of ZFNs and hidua cdna Results in Correction of hidua Activity in 4-5 month old MPS I Mice IDUA activity Supraphysiological levels of hidua activity detected in liver (primary tissue), plasma and secondary tissues (spleen, kidney and lung) of MPS I mice 68

69 AAV Mediated Delivery of ZFNs and hidua cdna Corrects GAG Accumulation in 1-2 month old MPS I Mice Significant reduction of urinary and tissue GAG levels in MPS I mice after day 21/60 69

70 Summary of Preclinical Studies SB-318 ZFNs are highly sequence-specific chimeric nucleases Off-target activity was assessed by unbiased and biased genome-wide assays SMCHD1 was the only off-target locus detected by unbiased oligo capture assay Off-target activity at the SMCHD1 locus is dose dependent and ~2 logs lower than on-target activity in vitro and in vivo No anchorage-independent cell growth observed after SB-318 treatment in GLP soft agar transformation assay Studies in clonal HepG2 cell lines with integrated hidua donor showed robust transgene expression independent of integration mechanism Proof-of-Concept demonstrated in wild-type and MPS I mice 70

71 Agenda MPS I Clinical Background Preclinical Overview ZFN technology and the albumin safe harbor locus approach SB-318 IND-enabling nonclinical safety evaluation program In vitro Evaluation of SB-318 in HepG2 cells Clinical Protocol 71

72 Phase 1 SB-318 MPS I Study - Overview A Phase I, Multicenter, Open-label, Single-dose, Dose Ranging Study to Assess the Safety and Tolerability of SB-318, a raav2/6- based Gene Therapy in Subjects with Mucopolysaccharidosis I (MPS I) Multicenter, open-label, single dose, dose ranging study Investigational Agent SB-318 is a combination of three AAV2/6 vectors encoding - SB-47171: Left side-zinc finger nuclease vector (ZFN1) - SB-47898: Right-side zinc finger nuclease vector (ZFN2) - SB-IDUA: huidua donor vector Number of subjects: 9 subjects; 2 subjects/cohort with expansion to 5 at the third cohort Study Duration: Three years 72

73 Phase 1 SB-318 MPS I Study - Overview Objectives - Primary: Safety and tolerability of SB Secondary: Assess Immune response to AAV2/6 Exploratory Change from baseline in the following assessments 1. Liver biopsy for measurement of IDUA activity and GAGs at baseline, 6 months and one year 2. Leukocyte IDUA activity 3. Urine GAG/creatinine ratio (µg/mg) 4. Forced vital capacity (percent of predicted normal) 5. Six-Minute Walk Test (6MWT) 6. Joint ROM 7. Liver and spleen volume 73

74 Phase 1 SB-318 MPS I Study - Overview Inclusion Criteria 1. Written informed consent 2. Male or female >18 yrs of age 3. Clinical diagnosis of attenuated MPS I deficiency (Hurler-Scheie, Scheie, or Hurlers s/p BMT) confirmed by a) IDUA gene sequencing, or b) receiving treatment with laronidase,, or c) a history of an elevated urinary GAG/crt ratio, or d) documentation of tissue, or e) plasma IDUA < 10% of the normal range. 4. Forced vital capacity (FVC) of <80% of predicted 5. No history of infusion reactions or anaphylaxis to laronidase 6. Negative pregnancy test (female of childbearing potential) 7. Sexually mature subjects must agree to use barrier contraceptive as follows: females for three months; males, until at least 3 consecutive semen samples after SB-318 administration are negative for AAV 2/6 8. No contraindication to the use of corticosteroids for immunosuppression 74

75 Phase 1 SB-318 MPS I Study - Overview Exclusion Criteria 1. Neutralizing antibodies to AAV 2/6 at titers >1:4 2. Clinically significant organic disease (unless secondary to MPS I) such as cardiovascular, hepatic, pulmonary, neurologic, or renal disease 3. Serious intercurrent illness 4. Active hepatitis B (Hepatitis B DNA, or HBsAg positive) or hepatitis C (HCV RNA viral load). To be considered negative, a history of sustained virologic response (viral assays in two sample collected at least six months apart must be negative) 5. Abnormal baseline liver function tests (ALT or AST >2x upper limit of normal (ULN), albumin <3.5 g/dl, total bilirubin > 2x ULN, 6. History of liver disease such as hepatitis, steatosis, cholangitis, cirrhosis, biliary disease, etc. (Gilbert s syndrome is excluded) 7. Current treatment with systemic (iv or oral) immunomodulatory agent or steroid use (topical treatment is allowed, e.g., asthma or eczema) 8. Participation in prior investigational drug or medical device study within the previous 6 months 9. Prior treatment with a gene therapy product 75

76 Phase 1 SB-318 MPS I Study - Overview Dose of Study Drugs ZFN 1 (vg/kg) ZFN 2 (vg/kg) cdna Donor (vg/kg) Total raav Dose (vg/kg) 5.00E E E E E E E E E E E E+13 Dose Escalation 1. Dose escalation cannot occur until at least 4 weeks after the last subject in the preceding cohort has been dosed, and safety data from the prior cohort has been reviewed by the Safety Monitoring Committee 2. Three additional subjects will be enrolled and treated in a cohort if one subject within a cohort develops a dose limiting toxicity (DLT) defined as a two Grade 2 AE in the same organ system that dose not resolve in 14 days of treatment, and any grade 3,or 4 76

77 Follow Up 36 months Phase 1 SB-318 MPS I Study Schema Study Period ~ 15 months Infusion and Safety Evaluations - 12 months Screen Hospitalization for SB-318 Infusion BL Day Week M18, 24, 36, 48 77

78 Phase 1 SB-318 MPS I Study - Overview Safety Monitoring and Mitigation Plan Liver function (AST, ALT, bilirubin, alkaline phosphatase, albumin and PT) will be monitored three times weekly during the first 12 weeks after SB-318 infusion and then monthly thereafter Key Potential Anticipated Risks Transaminitis due to cell-mediated immunity to capsid and/or AAV gene product. Steroids (oral or intravenous) may be administered per PI Reduction in albumin synthesis. The fraction given of transduced cells with albumin locus disruption (<1%), and has not been observed in preclinical rodent and NHP animal studies 78