Gene Editing and Targeted Integration Using Zinc Finger Nucleases - PowerPoint PPT Presentation

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

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

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

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

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

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 Modified after Neufeld Heparan Sulfates in the lysosome of all tissues leads to and Muenzer 2001 dysfunction in several tissues in MPS I patients 6

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

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

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

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

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

Cervical Spinal Compression in a Patient with MPS 12

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

Median Nerve Compression 14

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

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

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

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

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

• 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

Mucopolysaccharidosis: Poor Growth After HSCT 21

Glycosaminoglycan Before and After Bone Marrow Transplant Reference Citation Case At Diagnosis Reference Ratio Post-HSCT* S Range** Range** GAG/creatini GAG/creatini GAG/creatini GAG/creatini % Upper % Upper No. ne ne ne ne Limit Limit (mg/g) (mg/g) (mg/g) (mg/g) Herskhovitz et al, 1 7.0 5.0 140% 2009 2 8.0 5.0 160% 3 107 5.0 713% 22.0 15.0 147% 4 56 15.0 373% 15.0 15.0 100% Hite et al,1999 and 176 10.7 1,645% 7.6 6.5 117% current MEAN 911% 133% 22

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Mucopolysaccharidosis Type IH: Neurogeneration 38

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

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

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

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 130 Visual Reception 120 Transplant Alone Transplant + ERT 110 Standard Score (100 ± 15) 100 90 80 70 60 50 40 Baseline 2y post Baseline 2y post 42 Significantly different slopes 9.83 (5.57, 14.10) <0.001

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

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

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

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’ Genomic DNA 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 5’ Nuclease Heterodimeric Sequence specific Sequence specific designed Zinc FokI Nuclease designed Zinc Finger Protein Finger Protein (ZFP1) (ZFP2) ZFN = Designer restriction enzyme 46

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