1. Understanding Gene and Cell Therapy Approaches for DMD November 6 2015

2. Overview Annemieke Aartsma-Rus What does dystrophin do? What happens when there is no dystrophin? How does drug development work What genetic approaches are in development to replace dystrophin? How do they work Challenges/opportunities

3. Some basic biology: genes & proteins Annemieke Aartsma-Rus Proteins are building blocks of our body Genes contain blueprint for proteins Mistake in gene  mistake in protein Genes have a volume switch (protein only produced in proper tissue) Dystrophin protein has a function in muscle Mistake in dystrophin  Problems in muscle

4. Muscles Annemieke Aartsma-Rus 30-40% of our body is muscle >750 different muscles Muscles can grow bigger or smaller Muscles use a lot of energy Only maintained when needed Muscles are damaged when used too much Muscles have efficient system to repair damage and prevent future damage (grow bigger)

5. Muscle contraction Annemieke Aartsma-Rus

6. Muscle fibers Annemieke Aartsma-Rus Skeleton Connective tissue

7. Dystrophin Annemieke Aartsma-Rus Dystrophin provides stability to muscle fibers during contraction Connects skeleton of muscle fibers to connective tissues surrounding muscle fibers No dystrophin  Connection lost Muscle more sensitive to damage Chronic damage: repair system cannot keep up Loss of muscle tissue and function

8. Dystrophin Annemieke Aartsma-Rus Dystrophin

9. Duchenne: no functional dystrophin Annemieke Aartsma-Rus

10. What happens without dystrophin Annemieke Aartsma-Rus InflammationRepair Muscle damage Fibrosis Less blood flowToo much Ca 2+ Oxidative stressMitochondria damaged Loss of muscle tissue Loss of muscle function

11. What are muscle cross sections? Muscle cross sections Annemieke Aartsma-Rus H&E staining Fibers pink Nuclei blue

12. Dystrophin staining Dystrophin immune staining Annemieke Aartsma-Rus HealthyMdx mouseDuchenne

13. Revertant fibers & trace amounts Annemieke Aartsma-Rus Revertant Fibers Trace amounts Untreated DMD muscle

14. Western blotting Western blot Proteins isolated from muscle Proteins isolated from muscle Separated by size Separated by size Stain for dystrophin Stain for dystrophin Annemieke Aartsma-Rus DMD Becker (dilutions) Control (diluted) LongShort

15. Therapy development Annemieke Aartsma-Rus Dystrophin is missing Trying to replace dystrophin Also many other therapies aiming at improving muscle quality/slowing down disease processes

16. What are cell models Annemieke Aartsma-Rus Cells derived from patients Muscle biopsies Skin biopsies  converted into muscle cells (lab) Expanded in the lab (limited!!) “Immortalized” cells Muscle stem cells treated with viruses Keep expanding, but have been modified Cell cultures are model systems Valuable tool for early stages of research

17. What are mdx mice? Annemieke Aartsma-Rus Mdx mouse Mutation in mouse dystrophin gene No dystrophin protein But….disease not very severe Very efficient muscle regeneration Turns up volume switch utrophin gene in muscle No dsytrophin & utrophin: severe disease (Double knockout mouse)

18. Other animal models Annemieke Aartsma-Rus Golden retriever muscular dystrophy (GRMD) Mutation in dog dystrophin gene No dystrophin protein Mutation beginning gene Severe disease (muscle & heart) Muscle weakness, remain ambulant Most dogs do not live > year Severity is variable Rare mild individuals

19. Other animal models Annemieke Aartsma-Rus Pig model Mutation in pig dystrophin gene No dystrophin protein Deletion exon 52 Severe disease (muscle & heart)

20. Drug testing in patients Annemieke Aartsma-Rus Test compound properties Taken up by tissues efficiently? How quickly cleared from the body? Test compound for efficacy Does it work? At which dose? Test compound for safety Are there side effects? At which dose? Are they tolerable?

21. Development of therapies Annemieke Aartsma-Rus Tests from cell and animal models to clinical trials All steps are important to show proof-of-concept (does it work in a model system ?) Next steps are always more complicated Success in one step is no guarantee for success in subsequent steps Clinical trials are experiments in humans May not work, may not be safe

22. Cell therapy Annemieke Aartsma-Rus Muscle stem cells Isolate muscle stem cells from healthy donor Expand outside the body (culture in lab) Transplant into patients Transplanted cells repair muscle Transplanted cells make dystrophin

23. Muscle stem cells (myoblasts) Annemieke Aartsma-Rus Immune response (suppress) Do not exit circulation after injection Local injection: stay close to injection site Tremblay (Canada): multiple local injections Local dystrophin restoration Not feasible for larger muscles

24. Stem cell therapy Annemieke Aartsma-Rus Stem cells from fat, bone and bloodvessel walls Can exit bloodstream and migrate into muscle Very low efficiency Mesangioblasts most promising Encouraging results in dog model Safety trial ongoing in Italy (Giulio Cossu) 3 patients received stem cells Some side effects Preparing for injection 2 more patients

25. Niche Annemieke Aartsma-Rus InflammationRepair Muscle damage Fibrosis Dystrophic muscle is damaged (scar tissue/fibrosis) The few transplanted stem cells that reach muscle Do not receive proper signals to become muscle Receive signals from scar tissue: more fibrosis

26. Immunity Annemieke Aartsma-Rus Transplanting cells from one person to another will elicit an immune response Need chronic immune suppression Side effects Isolate patient stem cells, expand in the lab, correct mutation with gene therapy & transplant No immune response Gene therapy more efficient in cultured cells than in muscles

27. Cell therapy summary Annemieke Aartsma-Rus Opportunities Applicable to all patients Deliver dystrophin gene and repair muscle Currently in very early stage clinical development Challenges Efficiency very low Damaged muscle gives wrong signals to cells Immunity (only with allogenic transplantation

28. Gene Therapy Annemieke Aartsma-Rus Add functional gene to muscle cells patients Dystrophin protein made from new gene Applicable to ALL patients Genes located in nucleus cells How to get gene into (majority) nuclei of muscle cells?

29. Gene Therapy Annemieke Aartsma-Rus Maaike van Putten

30. Gene Therapy Annemieke Aartsma-Rus Virus Small organism that injects genetic information into cells Use to deliver dystrophin gene Adapt Remove virus genes (pathogenic) Add new gene (dystrophin)

31. Gene Therapy Annemieke Aartsma-Rus Which virus? Most viruses do not infect muscle tissue Muscle cells do not divide often Lot of connective tissue (filters out viruses) Exception: adeno-associated virus (AAV) Preference for muscle Not pathogenic in man

32. Gene Therapy Annemieke Aartsma-Rus Very small (20 nm, 0.00002 mm) Capacity: 4.500 DNA subunits Dystrophin gene: 2.200.000 DNA subunits Genetic code gene: 14.000 subunits Remove part from genetic code Only essential parts remain

33. Gene Therapy Annemieke Aartsma-Rus Microdystrophin Only crucial domains Fits in AAV particle

34. Gene Therapy Annemieke Aartsma-Rus Clinical trials Safety study in 6 Duchenne patients 2006/7, USA: local injection biceps (Mendell,Samulski, Xiao Xiao) Immune response! Dystrophin in 2/6 patients (very low levels) Prepare for bigger trial (whole muscle treatment)

35. Upscaling Annemieke Aartsma-Rus Mouse12 gram muscle Monkey 4 kg muscle Human boy10-25 kg muscle Monkeys and humans much larger than mice Need much more viruses Manufacturing systems optimized to allow production of sufficient amounts for treating human limbs at clinical grade 333x 2-6x

36. Delivery Annemieke Aartsma-Rus Whole animal delivery possible for mouse Not feasible (yet) for large animals Limited by amount of virus Produced Injected Whole limb delivery in development for human Hydrodynamic limb perfusion (most efficient) Regional limb perfusion (less damage)

37. Limb perfusion Annemieke Aartsma-Rus Tested in monkeys and dogs with ‘color gene’ Delivery to multiple muscles feasible Tested in adult MD patients with saline Possible for lower leg or arm (less efficient) Not yet tested in humans to deliver gene

38. Gene Therapy Summary Annemieke Aartsma-Rus Opportunities Applicable to all patients Currently in early clincial development (safety/tolerability tests) Challenges Microdystrophin only partially functional Delivery Immunity

39. Gene/cell therapy: DNA editing Annemieke Aartsma-Rus DNA has a repair system Activated upon DNA damage Use this system to correct for DNA mistakes? Mutation Template with correct DNA information DNA repair system Mistake corrected (in one cell)

40. DNA editing Annemieke Aartsma-Rus Challenge: DNA repair system very inefficient (1 in 1,000,000 – 1,000,000,000 cells) Much more efficient when DNA is ‘broken’ (1 in 10-1000 cells)  Have to generate DNA breaks at/close to mutation

41. DNA scissor system Annemieke Aartsma-Rus DNA scissors can cut DNA at specific location Scissor cuts at/near mutation Repair break with template (Correct small mutations) Repair break without template Small mutations will be introduced (can correct genetic code like exon skipping)

42. DNA scissor system Annemieke Aartsma-Rus Combination of DNA scissors to restore genetic code Scissors cut around exon  break is repaired Exon is deleted  Genetic code restored (like exon skipping)

43. DNA scissor system Annemieke Aartsma-Rus DNA scissors can make breaks in DNA Different types of scissors in development Zinc Fingers, TALENs and RGNs Challenge: deliver scissors and templates to muscles

44. Exon skipping Annemieke Aartsma-Rus Normal Duchenne

45. Dystrophin gene Annemieke Aartsma-Rus

46. Splicing Annemieke Aartsma-Rus Exons Introns 1 3 5 6 7 Gene (DNA) RNA copy (pre mRNA) messenger RNA 1 - - - - - - - - - 79 dystrophin protein dystrophin protein Splicing 2 4 3 4 5 6 7 1 2 12345678

47. Duchenne: genetic code disrupted Annemieke Aartsma-Rus

48. Duchenne: genetic code disrupted Annemieke Aartsma-Rus Protein translation stops prematurely Dystrophin not functional Exon 46Exon 47Exon 51 Exon 52 ?

49. Becker: genetic code maintained Annemieke Aartsma-Rus

50. Becker: genetic code maintained Annemieke Aartsma-Rus Protein translation continues Dystrophin partly functional Less damage Exon 46Exon 47 Exon 52 Exon 53

51. Exon skipping: restore genetic code Annemieke Aartsma-Rus

52. Applicability Annemieke Aartsma-Rus Aartsma-Rus Hum Mutat 2009, 30:293-9 hotspot

53. Exon skip chemistries Annemieke Aartsma-Rus Two chemistries in clinical development GSK/Prosensa: 2’-O-methyl phosphorothioate (drisapirsen) AVI-Biopharma/Sarepta: phosphorodiamidate morpholino oligomers (eteplirsen) Exon 51

54. Exon skipping summary Annemieke Aartsma-Rus Opportunities AON delivery easier than genes/cells Clinical trial results encouraging Challenges Mutation specific approach Need to develop many AONs to treat majority of patients Repeated treatment needed (also opportunity)

55. Stop codon readthrough Annemieke Aartsma-Rus 179

56. PTC124/ataluren Annemieke Aartsma-Rus 179 PTC Cell ignores new stop signal Complete protein is made

57. Stop codon readthrough summary Annemieke Aartsma-Rus Opportunities Oral delivery Applicable to multiple diseases Challenges Mutation specific approach (15%)