1. Promising new drug candidates for Duchenne Muscular Dystrophy Dominic Wells Department of Comparative Biomedical Sciences Royal Veterinary College London

2. Disclosures Member of the Scientific Advisory Board for Akashi Therapeutics. Grant funding for DMD work from a range of charities, DoH and research councils. Have conducted DMD mouse model studies for Proximagen and AstraZeneca and have consulted for a number of other companies. Member of MDUK, Telethon, AFM grant committees.

3. © Imperial College London Pa ge 3 Duchenne muscular dystrophy

5. Large deletions and duplications Splice site mutations Small deletions and insertions Nonsense mutations Missense mutations From Roberts at al, 1994 Types of mutations associated with DMD Most mutations disrupt the open reading frame leading to a failure to fully translate the mRNA and produce a functional protein

6. Absence of dystrophin Activity induced damage Loss of cell signaling Hypercontraction Mitochondrial damage Activation of proteases Influx of calcium Necrosis Inflammation and fibrosis Oxidative stress Nitrosylation Loss of strength Failure of regeneration DMD pathology

7. http://neuromuscular.wustl.edu/pathol/dmdpath.htm

8. Longevity/QoL increased with: Respiratory assistance with positive pressure ventilation. Appropriate drug treatment of any developing cardiomyopathy and potential prevention with pre- symptomatic treatment. Corticosteroids can slow the progress of DMD, maintain ambulation beyond 12, reduce scoliosis and maintain FVC However progressive decline in muscle function reduces independence – so goal of experimental therapies is to halt or reverse this progressive decline

9. X Loss of reading frame leads to unstable protein In frame mutation generates an internally deleted protein Duchenne muscular dystrophyBecker muscular dystrophy

10. DNA RNA Protein Gene Addition e.g. microdystrophin via viral vector. Cell therapy. Modify RNA e.g. exon-skipping Modify translation e.g. gentamycin PTC 124 (ataluren) “Genetic” therapies

11. Gene therapy

12. Clinical trials in muscular dystrophy - AAV 2006-2009 Mendell (Ohio)/Asklepios Biopharmaceutical (Xiao Xiao and Jude Samulski) – AAV2.5-microdystrophin (Biostrophin) for DMD Intramuscular injection in 6 patients at two dose levels, no serious adverse events. But also no significant microdystrophin expression and evidence of autoreactive T cells (Mendell et al., 2010). However was a safe clinical trial ! (Bowles et al., 2011). Several other studies have used AAV to transfer sarcoglycans – e.g. Mendell et al 2010

13. Mendell et al, 2010 LGMD2D Gene therapy clinical trial

14. Follistatin gene transfer Mendell lab showed local AAV delivery of follistatin (antagonist to myostatin) increased muscle mass in non-human primates (Kota et al., 2009). Gene therapy trial run in BMD and IBM patients with evidence of safety and some functional improvement (Mendell et al., Mol Ther. 2014).

15. New gene therapy trials Follow on from the previous trials: Milo Therapeutics. rAAV1.CMV.huFollistatin344. Treatment for DMD, Phase 1/2, injections into gluteals, quadriceps and tibialis anterior. SOLID ventures. rAAVrh74MCKmicroDystrophin. Phase 1 intramuscular injections into the EDB.

16. Cell therapies

18. Cell therapy human clinical trials Potential to improve muscle regeneration and restore dystrophin but will not make new muscle per se 1990s – myoblast transplants – unsuccessful Trembley (Canada) continuing with a Phase 1/2 clinical trials with local delivery to a specific muscle. Mesoangioblast trial run by Guilio Cossu in Italy Stem cell trials (Phase 1/2) ongoing in Turkey and India. Cardiosphere derived cells delivered via intracoronary infusion (Capricor, USA, Phase 1/2)

19. Exon-skipping

20. Exon skipping as a treatment for DMD

21. Exon skipping – clinical trials Two chemistries taken to clinical trial – 2OmePS and PMO. Evidence of clinical benefit – arresting the progression of the disease for boys eligible for exon 51 skipping. Other exon targets current in clinical trial (44, 45, 53). Other antisense reagents: –Cell penetrating peptides linked to PMO –TricycloDNA oligonucleotides

22. Read-through of premature stop mutations

23. Approximately 15% of DMD patients have a premature stop mutation. Aminoglycoside antibiotics can “read-through” these mutations but are too toxic for long-term human use. High-throughput screening identified a much less toxic compound with similar activity (PTC124 – Ataluren). EU conditional approval as Translarna for a restricted age range.

25. Pharmacological approaches to DMD therapy Up-regulation of utrophin to treat DMD. Increasing muscle mass. Inhibiting the pathological process. Improving the bllod supply to muscles Nutritional supplements. All the above use compounds that can be delivered systemically thus offering the promise of treating all affected muscles. Many compounds are already in use in man.

26. Utrophin similar to dystrophin but does not localise nNOS Expression of utrophin developmentally precedes dystrophin Can correct mdx mouse Utrophin as a substitute for dystrophin SMT C1100 : Utrophin Inducer for DMD Currently in Phase 1b clinical trial (Summit). Biglycan: Stabilises utrophin at the muscle membrane (Tivorsan)

27. Anti-inflammatory / anti-fibrotics Corticosteroids – current standard of care where tolerated but have significant side effects Potential alternatives (most act by inhibiting NF  ): Halofugionone (HT-100 Phase ½, Akashi Therapeutics) CAT-1000 (Phase ½, Catabasis) VBP-15 (about to enter trial, ReveraGen) Nemo-binding domain (NBD) peptide And others……..

28. Increasing muscle mass Myostatin is a negative regulator of muscle mass Release of soluble form of the Activin IIB receptor to block myostatin signaling (Acceleron - ACE-031). Trial stopped because of bleeding. Antibody to block myostatin binding. Pfizer PF-06252616 (phase 1/2). Adnectin to block myostatin. Bristol-Myers Squib BMS-986089 (Phase 1/2) Other strategies: Propeptide blocker, Inhibition of myostatin production (siRNA)

29. Mitochondria are a significant calcium store but get overwhelmed in DMD. Mitochondrial biogenesis stimulated via PGC1a. A number of different drugs (approved and experimental) can do this. A variety of drugs will also improve function of existing mitochondria. Phase III Double-Blind, Randomised, Placebo-Controlled Study of the Efficacy, Safety and Tolerability of Idebenone (Catena®) in 10-18 Year Old Patients With Duchenne Muscular Dystrophy showed prevention of respiratory decline (Buyse et al., 2015) but only in patients not taking corticosteroids Improving mitochondrial function +/- biogenesis

30. Selected other mitochondrial drugs DeBio 025 (Solid Biosciences). Metformin (Clinical trials in Basel) PGC1alpha activators

31. Improving blood flow in muscle and heart The loss of the membrane associated nNOS means that muscle blood flow during exercise is impaired. Tadalafil and Sildenafil (Cialis and Viagra) – PDE5 inhibitors – have been trialled in DMD. Tadalafil has shown the most promising results

32. A new candidate therapeutic Simvastatin has shown remarkable effectiveness in reducing dystrophic pathology and increasing muscle force in mdx mice treated from 3, 12 and 52 weeks old (Whitehead et al., Oct 2015 PNAS).

33. Summary Exciting time for those working on or affected by a neuromuscular disease. Increasing interest from big and small pharma/biotech. Wide range of possibilities, many in or about to enter clinical trial. Vital to have good translational studies in pre-clinical models Clinical trial design is critical for good go/no-go decisions Combination therapies are the likely long-term outcome