Current Research Strategies and Therapeutic Approaches in Duchenne Muscular Dystrophy

Current Research Strategies and Therapeutic Approaches in Duchenne Muscular Dystrophy H. Lee Sweeney, Ph.D. Department of Physiology University of Pennsylvania Perelman School of Medicine

Current Research Strategies and Therapeutic Approaches in Duchenne Muscular Dystrophy Ultimately, a multi-drug target, multi-drug approach is the key to developing an effective treatment regimen for DMD.

By understanding the pathophysiology of Duchenne Muscular Dystrophy, therapeutic targets are unveiled. Current Duchenne muscular dystrophy therapeutic targets can be grouped into five categories. Only the first addresses the primary genetic defect (resulting in the loss of dystrophin protein). The rest address downstream aspects of the pathogenesis. 1) Replacement of dystrophin/utrophin 2) Increasing muscle mass and regeneration 3) Decreasing inflammation and fibrosis 4) Correcting blood flow regulation 5) Correcting perturbations in calcium handing

Duchenne Muscular Dystrophy: utrophin Functional Roles of Dystrophin: Molecular Basis and Possible Treatment Strategies Mechanical - transmits force from the contractile apparatus to connective tissue/tendon Organizer - positions a number of proteins at the muscle membrane (NOS, ion channels,etc.) Signaling - likely plays a number of signaling roles, including a key role in calcium homeostasis Contraction causes rupture of the muscle membrane, which allows calcium inflow. There also there may be increased flux through ion (TRP) channels and leakiness of the internal calcium storage compartment (SR) via the ryanadine receptor (SR-calcium release channel). Excessive calcium activates breakdown of muscle (via calpain and other proteases) and may trigger cell death program. Cell death triggers an inflammatory response. Activation of fibroblasts can lead to fibrosis, which prevents muscle regeneration (modulated by IGF-I and myostatin).

Possible Approaches to Therapy for DMD Gene Replacement Therapy - Stem cells Viral delivery of dystrophin/utrophin mini-gene Viral-directed exon skipping (U7-snRNA) RNA manipulation - Oligonucleotides ( oligos ) to alter RNA splicing Drugs that will cause read-through of a premature stop codon in the mrna Pharmacological agents - *Drugs/factors to promote muscle repair (IGF-I; anti-mstn) *Drugs/biologicals that will increase the amount of utrophin or integrin Drugs/biologicals to decrease membrane damage (e.g. poloxamers) or decrease damage from calcium overload (e.g. SERCA activator*, TRP channel inhibitor, etc.) *Project Catalyst Targets

Targeted Development of New Drugs for Muscular Dystrophy NINDS NIAMS

In the clinic: 1) Nonsense Suppression (PTC124): Phase 2b in DMD completed 2) Exon skipping using oligonucleotides: Prosensa/GSK and AVI BioPharma 3) AAV delivery of micro-dystrophin (Phase 1 completed) 4) Myostatin/Activin inhibition (soluble activin receptor/ myostatin antibody) 5) Mesangioblasts (cell therapy) 6) AAV delivery of follistatin (BMD) 7) PDE5 inhibition for skeletal and cardiac muscle

Nearing the clinic: 1) Utrophin up-regulation 2) Anti-fibrotic (halofuginone) 3) Anti-inflammatory drugs targeting NFκB 4) Drugs to normalize calcium leaks

A New Paradigm for Treating Genetic Disorders: Mutation-based, rather than Disease-based, Therapy Drug (PTC124) Nonsense (Premature Termination) Codon Normal Termination Codon mrna Full-length Protein

Eccentric contrac+ons reveal large deficit between healthy and diseased muscle Deficit from lack of dystrophin Petrof et al. PNAS, 1993

Low level dystrophin upregula+on provides modest improvement in fragility } ~5% dystrophin

Protec+on against contrac+le injury is dependent upon extent of dystrophin upregula+on } ~10% dystrophin } ~5% dystrophin

Protec+on against contrac+le injury is dependent upon extent of dystrophin upregula+on and number of repeated contrac+ons 2 nd ECC 5 th ECC

A Pivotal Phase 2b Randomized, Placebo-Controlled Study Was Designed to Evaluate Ataluren Safety and Efficacy in DBMD Eligibility Criteria: DBMD Nonsense mutation Males 5 years Able to walk 75 m Stable corticosteroid (if receiving) R/S N=~55 N=~55 High-Dose Ataluren* Low-Dose Ataluren** Placebo Open-Label Extension Study High-Dose Ataluren* Randomization & Stratification: Age Corticosteroid use Baseline 6MWD N=~55 48 Weeks Primary Endpoint: 6-minute walk distance * High-dose ataluren = 20 mg/kg (morning), 20 mg/kg (midday), 40 mg/kg (evening) ** Low-dose ataluren = 10 mg/kg (morning), 10 mg/kg (midday), 20 mg/kg (evening)

6MWD Results Showed Low-Dose Ataluren Slowed Loss of Walking Ability Compared to Placebo* Change in 6MWD, mean ± SE (m) 20 0-20 -40-60 Baseline Low Dose (N=57) High Dose (N=60) Placebo (N=57) High-Dose vs. Placebo Mean Difference at Week 48: + 0.8 m ITT p = 0.9788 PP p = 0.9412 Low-Dose vs. Placebo Mean Difference at Week 48: + 29.7 m ITT p = 0.0584 PP p = 0.0462 6 12 18 24 30 36 42 48 Time (weeks) -12.9 (±71) m Delta = 29.7 m -42.6 (±90) m -41.8 (±89) m * Post hoc analysis ITT: Intent to treat PP: Per protocol

Time to Persistent 10% 6MWD Worsening Indicated Slower Disease Progression in the Low-Dose Ataluren Group 100 90 Low-Dose vs placebo Log-rank p=0.039 (HR =0.52, 0.28-0.97) 80 Percent Not Progressed 70 60 50 40 30 20 High-Dose Ataluren (N=60) Low-Dose Ataluren (N=57) High-Dose vs Placebo Log-rank p=0.606 (HR=1.15, 0.67-1.98) 74% not progressed 56% not progressed 52% not progressed 10 Placebo (n=57) 0 0 48 96 144 192 240 288 336 Days

Human Myotube* Data Demonstrate a Bell-Shaped Ataluren Dose-Response Curve for Dystrophin Expression ataluren µg/ml 0 Control protein Dystrophin 15 Mean Increase in Full-Length Dystrophin Expression Relative to Control 2.5 5.0 7.5 10.0 15.0 Dystrophin Expression Fold Increase ± SEM 10 5 20.0 30.0 0 1 2.5 5 7.5 10 15 20 30 40 Ataluren Concentration (µg/ml) Immunofluorescence assay Patient enrolled in Phase 2a DMD trial * Pretreatment samples (n=35) from Phase 2a DMD trial patients

Clinical Progress Nonsense read-through PTC124 (Ataluren) has undergone a Phase 2b clinical trial in DMD patients. (Saturday: Research Session #4) The drug provided clinical benefit at the low dose in the trial. At the low does, patients were relatively stable (6 minute walk) over the course of a year. There likely were variable and low levels of dystrophin made in the patients as a result of the combined effects of nonsense-mediated decay, the type of premature stop codon and the overall RNA context of the mutation. Low level dystrophin expression will likely slow but not stop disease progression. This approach applies to ~15% of DMD patients.

RNA manipulation RNA splicing modification Oligonucleotides directed at splice junctions/enhancers to produce exon slipping -production of truncated dystrophin Essentially, the objective is to turn DMD into a mild BMD phenotype. Conceptually, this means that skipping has to be developed for a number of exons, and that the resulting protein and clinical benefit will depend upon the patient s specific deletion.

Phase of DMD exons showing mono-skippable exons M DP427 CH1 CH2 R1 R2 R3 1 1.2 2 2.1 3 7.1 4 1 5 1.4 6 1.2 7 0.6 8 4.7 9 0.3 10 1.1 11 0.2 12 1.2 13 0.9 14 0.5 Actin-binding Rod domain R3 H2 R4 R5 R6 R7 R8 15-16 0.5 17 0.9 18 1 19 0.8 20 0.5 21 0.3 22 0.6 23-24 0.1 25 0 26 - H1 Rod domain R8 R9 DP260 R10 R11 R12 R13 R14 27-28 0.2 29 0.1 30 0.2 31 0.1 32 0.1 33 0.1 34 0 35 0.1 36 0 37 0.1 38 0.3 Rod domain R14 R15 R16 DP140 R17 R18 R19 H3 39 0 40 0.2 Rod domain H3 79 0 41 0.2 42 0.6 43 1.1 44 4.6 45 25.7 46 7.2 47 3.6 48 8.9 49 4.5 50 3.7 R20 R21 R22 DP116 R23 R24 51 5.3 52 1.8 53 1.2 54 0.2 55 0.2 56 0.2 57 0.1 59 0.2 79 (3 UTR) 58 0.1 60 0.4 61 0.1 Rod domain End of Rod domain H4 DP71 62 0.1 65 0.1 66-67 - 68 0 69-70 - 71 0.1 72 0 74-75 0 76 - WW 77-78 - 64 0.2 CYS-rich ZZ Dystroglycan binding site C-ter 73 0.1 α1-syntrophin binding site β1-syntrophin binding site H4 C-ter mono-skippable exons Dystrophin domains

Phase of DMD exons showing deletion of exon 50 M DP427 CH1 CH2 R1 R2 R3 1 1.2 2 2.1 3 7.1 4 1 5 1.4 6 1.2 7 0.6 8 4.7 9 0.3 10 1.1 11 0.2 12 1.2 13 0.9 14 0.5 Actin-binding Rod domain R3 H2 R4 R5 R6 R7 R8 15-16 0.5 17 0.9 18 1 19 0.8 20 0.5 21 0.3 22 0.6 23-24 0.1 25 0 26 - H1 Rod domain R8 R9 DP260 R10 R11 R12 R13 R14 27-28 0.2 29 0.1 30 0.2 31 0.1 32 0.1 33 0.1 34 0 35 0.1 36 0 37 0.1 38 0.3 Rod domain R14 R15 R16 DP140 R17 R18 R19 H3 39 0 40 0.2 41 0.2 42 0.6 43 1.1 44 4.6 45 25.7 46 7.2 47 3.6 48 8.9 49 4.5 Rod domain H3 R20 R21 R22 DP116 R23 R24 H4 51 5.3 52 1.8 53 1.2 54 0.2 55 0.2 56 0.2 57 0.1 59 0.2 79 0 79 (3 UTR) 58 0.1 60 0.4 61 0.1 Rod domain End of Rod domain H4 DP71 62 0.1 65 0.1 66-67 - 68 0 69-70 - 71 0.1 72 0 74-75 0 76 - WW 77-78 - 64 0.2 CYS-rich ZZ Dystroglycan binding site C-ter 73 0.1 α1-syntrophin binding site β1-syntrophin binding site C-ter mono-skippable exons Dystrophin domains

Phase of DMD exons showing deletion of exon 50 and skipping of exon 51 M DP427 CH1 CH2 R1 R2 R3 1 1.2 2 2.1 3 7.1 4 1 5 1.4 6 1.2 7 0.6 8 4.7 9 0.3 10 1.1 11 0.2 12 1.2 13 0.9 14 0.5 Actin-binding Rod domain R3 H2 R4 R5 R6 R7 R8 15-16 0.5 17 0.9 18 1 19 0.8 20 0.5 21 0.3 22 0.6 23-24 0.1 25 0 26 - H1 Rod domain R8 R9 DP260 R10 R11 R12 R13 R14 27-28 0.2 29 0.1 30 0.2 31 0.1 32 0.1 33 0.1 34 0 35 0.1 36 0 37 0.1 38 0.3 Rod domain R14 R15 R16 DP140 R17 R18 R19 H3 39 0 40 0.2 41 0.2 42 0.6 43 1.1 44 4.6 45 25.7 46 7.2 47 3.6 48 8.9 49 4.5 Rod domain H3 R20 R21 R22 DP116 R23 R24 H4 79 0 52 1.8 53 1.2 54 0.2 55 0.2 56 0.2 57 0.1 59 0.2 79 (3 UTR) 58 0.1 60 0.4 61 0.1 Rod domain End of Rod domain H4 DP71 62 0.1 65 0.1 66-67 - 68 0 69-70 - 71 0.1 72 0 74-75 0 76 - WW 77-78 - 64 0.2 CYS-rich ZZ Dystroglycan binding site C-ter 73 0.1 α1-syntrophin binding site β1-syntrophin binding site C-ter mono-skippable exons Dystrophin domains

RNA splicing modifications Clinical Progress Clinical trials have occurred and are ongoing using either O-methyloligonucleotides (Prosensa/GSK) or morpholinos (AVI BioPharma). (Friday: Research Session #2) The heart does not take the oligos up to a great degree (likely because its membranes are not as leaky) and thus there may be no cardiac impact. Furthermore, not all truncated dystrophin molecules will be equally efficacious (as in the case of BMD). That is to say, while some skipping may produce mild BMD-like phenotypes, while others will produce more severe phenotypes. Lastly, many deletions in the rod will make the resulting dystrophin unable to localize NOS (nitric oxide synthase). A major unresolved problem is whether or not some of the resulting dystrophin proteins will cause an immune response in some patients.

In the clinic: 1) Nonsense Suppression (PTC124): Phase 2b in DMD completed 2) Exon skipping using oligonucleotides: Prosensa/GSK and AVI BioPharma 3) AAV delivery of micro-dystrophin (Phase 1 trial) 4) Myostatin/Activin inhibition (soluble activin receptor/ myostatin antibody) 5) Mesangioblasts (cell therapy) 6) AAV delivery of follistatin (BMD) 7) PDE5 inhibition for skeletal and cardiac muscle

Gene Replacement Therapy Viral gene therapy AAV / micro-dystrophin or micro-utrophin AAV / U7-directed exon skipping

Schematic of a Dystrophin Mini-gene for Viral (AAV)-Based Gene Therapy CMV or Muscle- Specific Promoter U7-exon mask or Human µ-dystrophin PolyA

Major Hurdles Facing Viral-Based Gene Therapy for Muscular Dystrophy Clinical Development: The completed AAV-µ-dystrophin trial was not designed for efficacy. The data that has been presented to date has not shown expression, possibly due to immune response(s) against either vector or transgene or both. At least in some patients, an immune response was detected against the µ-dystrophin. It will be necessary to develop immune suppression procedures to allow repeated virus administrations and better methods of preventing presentation of neoantigens in the transgene.

In the clinic: 1) Nonsense Suppression (PTC124): Phase 2b in DMD completed 2) Exon skipping using oligonucleotides: Prosensa/GSK and AVI BioPharma 3) AAV delivery of micro-dystrophin (Phase 1 trial) 4) Myostatin/Activin inhibition (soluble activin receptor/ myostatin antibody) 5) Mesangioblasts (cell therapy) 6) AAV delivery of follistatin (BMD) 7) PDE5 inhibition for skeletal and cardiac muscle

Skeletal Muscle Growth and Repair uses Satellite Cells (muscle stem cells) HGF leads to activation of satellite cells. IGF-I stimulates satellite cell proliferation and differentiation. Myostatin, Activin A and TGF-β can antagonize IGF-I actions.

Myostatin K.O. Mouse Wild Type Myostatin Null Specific Force Decreased

Signaling via the Activin Receptors

Strategies to Achieve Postnatal Myosatin Inhibition Neutralizing antibody against myostatin (MYO-29 clinical trial by Wyeth; Wagner et al. Ann Neurol 63: 561 571, 2008) Delivery of N-terminal myostatin propeptide Delivery of follistatin or FLRG (myostatin binding proteins) Delivery of soluble Activin IIB receptor (Acceleron/Shire clinical trial)

Inhibition via Soluble Activin IIB Receptor

Myostatin / Activin Inhibition-Based Therapy for Muscular Dystrophy Clinical Development: The Phase I activin IIB decoy receptor (Acceleron/Shire; ACE-031) was for safety in normal volunteers, but muscle hypertrophy was observed. The Phase 2 trial in DMD was stopped due to bleeding. A Phase I study with a myostatin neutralizing antibody has begun (Pfizer), with DMD as a possible target for later phase development. The expectation is that this approach will result in bigger and stronger muscles in DMD patients and may lessen fibrosis. The muscle will still be unstable, so satellite cell repair will continue, but will be more successful. Ultimately, humans (unlike mice) will be exhausted of satellite cells and the muscle function will be lost. However, it is thought that this approach will extend the number of years that the muscle can successfully repair. The heart will not benefit from this approach, so other drugs must be used to protect the heart. Furthermore, new drugs need to be developed to slow the destruction of the skeletal muscle.

Major Hurdles Facing Viral-Based Gene Therapy for Muscular Dystrophy Clinical Development: The idea is to use a molecule that inhibits myostatin and activin signaling within skeletal muscle (follistatin), but to have it made only in skeletal muscle that has been injected with recombinant AAV that is designed to produce follistatin. The main drawback of this approach is that while the muscle will become larger and stronger, the muscle will still be unstable, and as it is repaired by satellite cells, the viral transgenes will be lost (i.e. follistatin expression will decrease over time). This is why the trial is being done in BMD, where muscle turnover is slower and thus the effects will last longer. Since follistatin is not a neoantigen, an immune response is not expected.

In the clinic: 1) Nonsense Suppression (PTC124): Phase 2b in DMD completed 2) Exon skipping using oligonucleotides: Prosensa/GSK and AVI BioPharma 3) AAV delivery of micro-dystrophin (Phase 1 completed) 4) Myostatin/Activin inhibition (soluble activin receptor) 5) Mesangioblasts (cell therapy) 6) AAV delivery of follistatin (BMD) 7) PDE5 inhibition for skeletal and cardiac muscle Nearing the clinic: 1) Utrophin up-regulation

Major Hurdles Facing Stem Cell-Based Therapy for Muscular Dystrophy Clinical Development: While stem cells seem like the ultimate answer for treating DMD, the approach is a number of years away from being a treatment reality. The questions that must be answered include: 1) How do we get the stem cells to the right place and have them adopt the correct fate (skeletal muscle)? In diseased tissue, the cells may become fat or fibrosis and make the situation worse instead of better. 2) Will there be an immune response to donor cells, or even to modified patient cells if dystrophin is seen as a neoantigen? Will life-long immune suppression be needed? 3) Can enough cells be obtained and expanded to treat the patient s muscles without losing their stem cell properties.

Development of PDE5 Inhibition Therapy for DMD Clinical Development: PPMD is sponsoring a trial to compare the effects of sildenafil and tadalafil on activating blood flow in DMD skeletal muscle. In parallel, we are comparing their effects on cardiomyopathy in the dystrophic (GRMD) dog. (Friday: Research Session #1) Ongoing trial (sponsored by Charlie s Fund) at Johns Hopkins to examine impact of sildenafil on the cardiomyopathy in DMD. The goal is to ascertain whether either tadalafil or sildenafil have skeletal muscle and/or cardiac benefits in DMD.

Nearing the clinic: 1) Utrophin up-regulation 2) Anti-fibrotic (halofuginone) 3) Anti-inflammatory drugs targeting NFκB 4) Drugs to normalize calcium leaks

Nearing the clinic: 1) Utrophin up-regulation (Friday: Research Session #1) 2) Anti-fibrotic (halofuginone) 3) Anti-inflammatory drugs targeting NFκB 4) Drugs to normalize calcium leaks

Dystrophin and Utrophin structural similarities Dystrophin Actin Binding H1 H2 Rod domain H3 H4 CR CT Utrophin β-spectrin α-spectrin α-actinin

Nearing the clinic: 1) Utrophin up-regulation (Friday: Research Session #1) 2) Anti-fibrotic (halofuginone) (Saturday: Research Session #4) 3) Anti-inflammatory drugs targeting NFκB (Saturday: Research Session #4) 4) Drugs to normalize calcium leaks

Phases of skeletal muscle injury and repair

By understanding the pathophysiology of Duchenne Muscular Dystrophy, therapeutic targets are unveiled. 1) Replacement of dystrophin/utrophin 2) Increasing muscle mass and regeneration 3) Decreasing inflammation and fibrosis 4) Correcting blood flow regulation 5) Correcting perturbations in calcium handing