The Science Behind Annapurna R. Crystal Department of Genetic Medicine Weill Cornell Medical College 2-17-16 Exhibit 99.1
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Why Gene Therapy ? Gene Modify gene expression Modify phenotype Advantages Versatile protein delivery system Persistent expression Local delivery
Basic Concept of Gene Therapy Therapeutic gene Human genome m m m m m m m m m m m m m m m m m m Delivery vehicle (vector) Target organ m m m m m m m m m m m m m m m m m m
Challenges of Gene Therapy Challenges Target choice – need, feasibility Targeting to site of disease Level of expression required Robust phenotype, clear demonstration of efficacy using FDA-acceptable parameters Safety – risk/benefit, dose-limiting immune related toxicity, lack of control of expression and inability to reverse Manufacturing Registration
Annapurna Programs Alpha 1-antitrypsin deficiency Hereditary angioedema Friedreich’s ataxia cardiac disease Severe allergy
AAVrh.10 Vectors ITR y ITR ITR CAG promoter Therapeutic gene (cDNA) Genome Capsid serotype AAVrh.10 AAV2 AAV2 Therapeutic genes Alpha 1-antitrypsin C1-esterase inhibitor Frataxin Anti-IgE
Why Gene Therapy? Alpha 1-antitrypsin deficiency, hereditary angioedema, severe allergy Reduced treatment burden Ideal pharmacokinetics - constant levels For hereditary angioedema and severe allergy, eliminate risk for attacks without need for immediate medical care Friedreich’s ataxia cardiac disease Intracellular delivery of the deficient protein in the target organ Unmet medical need for a fatal disease
Alpha 1-Antitrypsin Deficiency Common autosomal recessive disorder characterized by a marked reduction in serum a1AT levels Estimated 90,000 affected individuals in US Emphysema develops at ages 35-45 in cigarette smokers, 55-65 in non-smokers Small % develop liver cirrhosis a1AT normally protects the lung from the destructive potential of neutrophil elastase; if a1AT levels are low, neutrophil elastase slowly destroys the lung parenchyma Current therapy to protect the lung - weekly intravenous infusions of 4 gm of human a1AT purified from pooled plasma
Pathogenesis of α1-Antitrypsin Deficiency α1-antitrypsin Neutrophil elastase Lungs Liver Neutrophils Bone marrow Glu342GAG Lys342AAG Z mutation Liver hepatocyte
Augmentation Therapy with Purified Human a1-Antitrypsin Developed by Gadek and Crystal in 1981 FDA approved in 1988 Administration IV 60 mg/kg (~4 g), once weekly Safe - minimal adverse reactions Cost ~$100,000/yr
Lungs Liver Bone marrow Neutrophils Neutrophil elastase α1-antitrypsin A. Pathogenesis D.Epithelial lining fluid anti-neutrophil elastase capacity before and after augmentation therapy Normal ZZ pre-therapy ZZ 6 days post- therapy 1 2 3 4 5 ELF anti-neutrophil elastase capacity (mM) C.Serum α1AT following augmentation therapy with intravenous human α1AT pathogenesis Pulmonary capillary Interstitium Air Plasma proteins Endothelial cell Epithelial cell B. Alveolar endothelial-epithelial “barriers” ELF Logic Underlying “Biochemical Efficacy” for the Lung Manifestations of α1-antitrypsin Deficiency Serum α1-antitrypsin level (mg/dl) Serum α1-antitrypsin level (μM) Days
Association of a1-Antitrypsin Serum Levels and Risk for Emphysema MM MS SS MZ SZ ZZ Threshold 11 mM Null 0 10 20 30 40 50 a1AT serum level (mM) Phenotype At risk for emphysema Background risk
unidirectional valve parietal extrapleural interstitium parietal lymphatic pleural space stoma microvilli basal lamina parietal pleural visceral pleural capillary pulmonary interstitum alveolus pulmonary lymphatic A. Anatomy of the human pleura AAV vector coding for normal human a1AT B.Intrapleural gene therapy strategy Local production of α1AT AAVha1AT a1AT Mesothelial cells Lung Intravascular AAVα1AT AAVα1AT from the pleura via lymphatics to the venous circulation Hepatocyte expression of α1AT α1AT secreted into blood diffuses across the alveoli Logic for Intrapleural Gene Transfer to Treat α1-antitrypsin Deficiency
Time post-injection (wk) Serum human a1-antitrypsin levels (% of maximum) 0.1 1 10 100 0 1 2 3 4 Undetectable AAVch.5 AAVhu.11 AAVhu.41 AAVhu.47 AAVrh.34 AAVhu.1 AAVhu.13 AAVrh.16 AAVrh.24 AAVrh.22 AAVrh.21 AAVrh.13 AAVbb.2 AAV2 AAVcy.5 AAV7 AAVrh.2 AAVrh.8 AAVrh.43 AAVrh.20 AAVhu.37 AAV5 AAV9 AAV8 AAVrh.10 Vector Clade/ clone Species of origin E Rhesus macaque E Rhesus macaque E-D Rhesus macaque E-D Rhesus macaque E-D Rhesus macaque Serum Human a1AT Levels Following Intrapleural Administration to C57Bl/6 Mice of 25 Different Serotypes of AAV Expressing a1AT E Rhesus macaque F Human AAV5 Human E Human E Rhesus macaque E Rhesus macaque Rh.8 Rhesus macaque E Rhesus macaque D Rhesus macaque D Cynomolgus macaque B Human B Baboon B Human D Human C Human rh.34 Rhesus macaque B Human E Human C Human Ch.5 Chimpanzee
0 1 2 4 6 12 1 10 102 103 104 24 Time post-injection (wk) Human a1AT level in serum (mg/ml) Naive AAVrh.10hα1AT 0-24 wk Evaluate AAVrh.10hα1AT 1011 genome copies (gc) Intrapleural Human α1AT level in serum (ELISA) C57BL/6 mice n = 4/group Time Course of Serum Human α1AT Levels Following Intrapleural Administration of AAVrh.10hα1AT Therapeutic target 11 μM
12 wk Evaluate Serum and lavage human α1AT (ELISA) C57BL/6 mice n = 4/group Human α1AT Levels in Bronchoalveolar Lavage Fluid Compared to Serum Following Intrapleural Administration of AAVrh.10hα1AT Human a1AT (mg/mg protein) Naive AAVrh.10hα1AT Serum Serum Lavage Lavage 0 10 20 30 40 AAVrh.10hα1AT 1011 gc, intrapleural
Ultrasonography-guided Intrapleural Administration of AAVrh.10hα1AT to African Green Monkeys Single intrapleural injection of 1012 or 1013 genome copies of AAVrh.10ha1AT to African Green Monkeys (n=36) Quantify human α1AT mRNA in chest wall up to 1 yr All safety / toxicology parameters normal at all time points Days after AAVrh.10hα1AT administration 107 105 104 103 102 28 90 360 0 Limit of detection 1013 gc AAVrh.10hα1AT 1012 gc AAVrh.10hα1AT PBS 106 hα1AT copies/mg of total RNA
Hereditary Angioedema Autosomal dominant disorder associated with episodic attacks of swelling of face, extremities, genitals, GI tract and upper airways Airway edema can be life threatening Triggered by trauma, surgery, dental work, menstruation, medications, viral illness, stress Affects 1 in 10,000-50,000 15,000-30,000 emergency room visits/yr in the US
Hereditary Angioedema (2) Caused by mutations of the SERPING1 gene, coding for C1 esterase inhibitor (C1EI) that regulates the complement system C1EI - single chain, 510 residue, 105 kDa, glycosylated serine anti-protease expressed 10 by hepatocytes SERPING1 mutations result in reduced serum levels in C1EI (80-85% cases) or functional C1EI deficiency (15-20%) C1EI deficiency results in up-regulation of bradykinin, causing edema via leaky vessels 6-8,000 patients in the US Approved/reimbursed prophylactic recombinant C1EI therapy reduces number and severity of attacks, but requires 2x/week infusion and costs $500,000/yr
Gene Therapy for Hereditary Angioedema Proof-of-concept studies cannot be discussed at this time due to patent filings in progress
Friedreich Ataxia Approximately 5,000 patients in the US, 5,000-10,000 in Europe Autosomal recessive, results from variants in the frataxin (FXN) gene, a nuclear gene coding for a mitochondrial protein associated with iron sulfur clusters Impairs dorsal root ganglia, spinal cord and cerebellum resulting in gait ataxia, gradually worsening and spreading to the arms and the trunk, slurred and slowed speech Hypertrophic cardiomyopathy, >60% of patients die from cardiac events No effective therapy
MCK Mouse Model Recapitulating the Cardiomyopathy of Friedrich Ataxia Absence of the frataxin protein in myocardium and skeletal muscle MCK 4 5 7 11 0 Death 8 Progressive left ventricle (LV) systolic dysfunction Cell death and fibrosis Progressive LV hypertrophy and dilation Progressive cardiomyopathy Iron sulfur cluster deficit in heart Mitochondrial anomalies and cardiac iron deposits Biochemical features Cardiac hypertrophy (10 wk) Cardiac fibrosis (10 wk) Time (wk) Mutant Control Age (wk) CII/CIV activity (% WT) Succinate dehydrogenase activity 2 3 4 5 6 7 8 9 10 11 25 125 100 75 50 0 *
3 MCK Evaluation of survival and multiple cardiac phenotypes 5 7 Intravenous AAVrh10.hFXN 5.4x1013 vg/kg) 22 35 60 Time (wk) MCK Mice in Heart Failure Treated with AAVrh.10.hFXN Advanced heart failure
Untreated AAVrh10hFXN-treated Normal A. Correction of hypertrophy B. Correction of cardiac function C. Survival Some treated mice die from late myopathy (MCK mice, not patients, have myopathy) AAVrh10hFXN Treatment of MCK Mice with Heart Failure at 7 wk - Rapid Normalization 22 18 14 10 20 16 12 8 0 80 60 40 20 Wk Left ventricle mass (mg) 40 30 20 10 0 22 18 14 10 20 16 12 8 Wk Shortening fraction (%) 22 18 14 6 10 2 Wk 100 80 40 20 0 60 Survival (%) Untreated AAVrh10hFXN-treated Normal Untreated AAVrh10hFXN-treated Normal
Identifying a Robust Cardiac Phenotype for Cardiac Freidreich’s Ataxia Gene Therapy Studies to identify cardiac parameters Department of Genetic Medicine, Weill Cornell Hôpital Universitaire Pitié-Salpêtrière, Paris Parameters Genetic Neurologic Cardiac Serology Cardiac echo Cardiac mri Exercise CDG PET CT
Severe Allergy – Example Peanut Allergy Common food allergy, manifests with itchiness, urticaria, swelling, eczema, asthma, abdominal pain, hypotension and anaphylaxis Can be fatal 0.4–0.6% US population, 4,000 diagnosed/yr (11/day); most common cause of anaphylaxis in children presenting to the emergency ward Significant adverse effect on the quality of life No definitive therapy Strict avoidance Epinephrine Desensitization Omalizumab (Xolair) anti-IgE
Anti-IgE Therapy of Peanut Allergy Omalizumab (Xolair) is a humanized IgG1 anti-IgE monoclonal that binds to Fc portion of circulating IgE, preventing the IgE from binding to, and triggering, mast cells Mast cell mediators Y Y Y Y Peanut-specific IgE Y Y Mast cell Exposure to peanuts + Peanut antigens IgE anti-peanut-peanut antigen immune complex Y Y Y Y Therapy with anti-IgE (Xolair) IgG-anti-IgE Y Y Y Y Y Y Mast cell Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Characterization of NOD-scid IL2 Rgammanull Immunodeficient Mice Humanized with Immune Cells from Peanut Allergic and Control Donors PBMC from donor with no peanut allergy D. Post-peanut challenge PBMC from donor with peanut allergy PBMC from donor with no peanut allergy IgE response pre-/post-peanut sensitization (wk) A. Serum peanut-specific IgE Serum human peanut-specific IgE (IU/ml) 0 1 2 3 4 5 6 7 1 2 3 PBMC from donor with peanut allergy PBMC from donor with no peanut allergy B. Histamine C. Anaphylaxis score PBMC from donor with peanut allergy PBMC from donor with no peanut allergy PBMC from donor with peanut allergy 30 min post-peanut challenge Plasma histamine level ng/ml 0 500 1000 1500 2000 2500 3000 Anaphylaxis score p<0.02 p<0.04 * 0 2 3 4 1 * undetectable 5
Expression of AAVrh.10Anti-hIgE in NOD-scid IL2 Rgammanull Mice 2-44 wk Evaluate AAVrh.10anti-hIgE AAVrh.10IgGcontrol 1011 gc, intravenous Serum anti-hIgE antibody titer (every 2 wk) Nodscid IL2rγnull (n=4F/group) (6-8 wk) Time post-injection (wk) Serum anti-hIgE (μg/ml) AAVrh.10anti-hIgE AAVrh.10IgGcontrol PBS 0 50 100 150 200 250 300 350 0 2 4 6 8 10 12 14 16 18 20 22 24 44
Treatment of Peanut Antigen-Induced Systemic Anaphylaxis by Treatment with AAVrh.10anti-hIgE; Clinical and Molecular Phenotype A. Post-peanut (PN) challenge clinical phenotype Donor with PN allergy + omalizumab Donor with PN allergy + AAVrh.10anti-hIgE wk 10 (5 wk after therapy) wk 10 (5 wk after therapy) wk 10 (5 wk after therapy) B. Locomotor activity Cumulative distance traveled (cm) AAVrh.10IgGcontrol AAVrh.10anti-hIgE Omalizumab 0 100 200 300 400 500 wk 7 (2 wk after therapy) All AAVrh.10- IgGcontrol mice died * * p<0.001 p<0.001 p<0.5 p<0.001 C. Anaphylaxis score Anaphylaxis score (1-5) All AAVrh.10IgGcontrol mice died 0 1 2 3 4 5 AAVrh.10- IgGcontrol AAVrh.10-anti-hIgE Omali-zumab AAVrh.10- IgGcontrol AAVrh.10-anti-hIgE Omali-zumab * wk 7 (2 wk after therapy) wk 10 (5 wk after therapy) * p<0.001 p<0.001 p>0.5 p<0.001 p<0.001 p>0.5 0 1 2 AAVrh.10- IgGcontrol AAVrh.10-anti-hIgE Omali-zumab AAVrh.10- IgGcontrol AAVrh.10-anti-hIgE Omali-zumab * wk 6 (1 wk after therapy) wk 9 (4 wk after therapy) D. Histamine Anaphylaxis score (1-5) All AAVrh.10IgGcontrol mice died 3 4 5 * p<0.001 p<0.001 p<0.5 p<0.01 p<0.5 p<0.01 E. Passive cutaneous anaphylaxis Mouse serum: PBMC donor PN allergy + omalizumab Mouse serum: PBMC donor PN allergy + AAVrh.10anti-hIgE Human serum: PN allergic donor Human serum: non-PN allergic donor wk 7 (2 wk after therapy) wk 10 (5 wk after therapy)
Mouse Survival Following Treatment with AAVrh.10anti-hIgE, Xolair Alone or Control Vector p<0.03 p<0.01 p<0.01 AAVrh.10IgGcontrol Omalizumab AAVrh.10anti-hIgE 100 60 40 20 0 80 0 30 20 10 40 Survival (%) Days after therapy
Gene Therapy Gene Modify gene expression Modify phenotype
