Gene therapies for SMA and DMD

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1 Gene therapies for SMA and DMD Annemieke Aartsma-Rus September 2018

2 Disclosures Employed by LUMC, which has patents on exon skipping technology, some of which has been licensed to BioMarin and subsequently sublicensed to Sarepta. As co-inventor of some of these patents I am entitled to a share of royalties Ad hoc consultant for PTC Therapeutics, BioMarin Pharmaceuticals Inc., Alpha Anomeric, Global Guidepoint, GLG consultancy, Grunenthal, Wave, Sarepta, Eisai and BioClinica Member of the scientific advisory boards of ProQR, MirrX therapeutics and Philae Pharmaceuticals. Remuneration for these activities paid to LUMC. LUMC received speaker honoraria from PTC Therapeutics and BioMarin Pharmaceuticals.

3 Outline Introduction Genes and proteins What goes wrong with DMD and SMA Gene therapy approaches Gene addition, genome editing & splice modulation How do they work? Current state of the art for DMD and SMA Outstanding questions and challenges

4 Some basic biology: genes & proteins Proteins are the building blocks of our body Genes contain blueprint for proteins Mistake in gene mistake in protein Pathology depends on protein function and location Dystrophin: expression in muscle and brain SMN: expression everywhere

5 Muscles and neurons Skeletal muscles make up 30-40% of our body >750 different muscles Muscle can contract when receiving a signal from the brain Each muscle is innervated, each muscle fiber has its own motorneuron When muscle tissue is lost: paralysis When motor neurons are lost: paralysis

6 DMD: no functional dystrophin Healthy Duchenne Becker 6 Department of Human Genetics Annemieke Aartsma-Rus

7 DMD: no dystrophin Dystrophin stabilizes muscle fibers during contraction Without dystrophin muscle fibers are continously damaged This leads to chronic inflammation, fibrosis and reduced regeneration and eventually loss of muscle mass and function

8 SMA: less functional SMN protein 8 Department of Human Genetics Annemieke Aartsma-Rus

9 SMA: low amounts of SMN protein SMN protein has many functions Having no SMN is lethal for cells and organisms Having reduced amounts of SMN is especially damaging for motorneurons

10 SMA types and SMN amounts SMN protein produced by SMN1 & SMN2 genes SMN1 produces SMN at normal levels Each SMN2 gene produces only 10% SMN The SMN1 gene is not functional for SMA patients They rely on SMN2 gene for SMN protein The number of SMN2 genes varies per person Each SMN2 gene makes ~10% SMN amount needed More copies: less severe disease / later onset

11 Varying amounts of SMN protein

12 Genetic therapies Targeting DNA Gene addition Genome editing Targeting DNA transcripts Splice modulation Stop codon readthrough

13 Gene addition Add functional gene to muscle fibers or motorneurons This copy is then used to produce dystrophin/smn Genes are located in cell nucleus You need to target as many muscle fibers/motorneurons as possible How?

14 Viral vectors Virus can deliver genetic code to cells efficiently Hijack viruses to deliver dystrophin/smn gene Which virus to use? Adeno-associated virus Only virus that efficiently infects muscle/neurons Not pathogenic in humans Remove viral genes, add transgenes

15 Dystrophin is rather big AAV is very small (20 nm, mm) It has limited capacity for DNA SMN gene fits Dystrophin gene is too large (about 3 times ) Make microdystrophin Dystrophin with only most crucial domains Will this be functional?

16 Current state of the art SMA Intravenous delivery of AAV vectors with SMN genetic code in SMA type 1 patients with 2 SMN copies Low dose (67,000 billion/kg) and high dose (240,000 billion particles/kg) Trial ended but patients are still being followed up

17 Current state of the art SMA

18 Future plans (Avexis) Continue follow up of patients Confirmatory trials all open label 30 patients intravenous delivery Type 1, 2 SMN copies; Europe (NCT ) 20 patients intravenous delivery (NCT ) 1 or 2 SMN 2 copies 27 patients intrathecal delivery (NCT ) Compare 2 doses, 3 SMN2 copies

19 Current state of the art DMD 3 trials with micro-dystrophin ongoing Pfizer (12 pat, 2 doses) Ongoing no results reported yet Solid (16 pat, 4 doses) Ongoing temporary on hold Sarepta/Ohio (12 pat, 1 dose) 3 patients treated Results presented

20 Sarepta/Ohio DMD gene therapy trial 3 patients treated (3-4 year old) 200,000 billion viral particles/kg Pretreatment with high dose steroids Biopsy taken after 90 days Micro-dystrophin observed >70% fibers positive ~40% of levels healthy control muscle Follow up ongoing Currently on hold impurity during manufacturing

21 Outstanding questions Manufacturing (especially for DMD) Immunity against AAV Longevity (especially for muscle) Repeat injections possible? Functionality micro-dystrophin Dilution (motorneurons) Repeat injections possible? Long term safety?

22 Genetic therapies Targeting DNA Gene addition Genome editing Targeting DNA transcripts Splice modulation Stop codon readthrough

23 Splicing Exons Introns 7 Gene (DNA) Splicing messenger RNA RNA copy (pre mrna) dystrophin protein

24 Dystrophin exons

25 Duchenne: reading frame disrupted

26 Exon deletion Exon 46 Exon 47? Exon 51 Exon 52 Disrupted reading frame Protein translation truncated prematurely Dystrophin not functional

27 Becker: frame maintained

28 Becker: reading frame maintained Exon 46 Exon 47 Exon 52 Exon 53 Reading frame not disrupted Protein translation continues Dystrophin partly functional

29 Exon skipping to restore reading frame AON Exon 47 Intron 47/50 Exon 51 Intron 51 Exon 52 Intron 52 Reading frame restored Exon 46 Exon 47 Exon 52 Partially functional dystrophin

30 Mutation specific approach Exon All mutations Deletions 51 14% 21% % 13% % 12% % 11% % 5.6% % 4.5% 8 2.0% 2.9% Bladen et al, Hum Mut Department of Human Genetics Annemieke Aartsma-Rus

31 Current state of the art Clinical development exon 51 skipping AONs most advanced Drisapersen and Eteplirsen Drisapersen tested in >300 DMD patients Maybe slower disease progression (6MWT) Safety concerns Not approved Development stopped (Prosensa, GSK, BioMarin)

32 Eteplirsen is approved in the USA Based on very minor increases in dystrophin (<1%) No functional efficacy shown yet Company needs to confirm functional effect by 2021 Not approved in Europe 32 Department of Human Genetics Annemieke Aartsma-Rus

33 Current state of the art Confirmatory eteplirsen trials ongoing (Sarepta) Improved exon 51 skipping AONs WaVe Sarepta Exon 53 trials ongoing Sarepta and NS Pharma Dystrophin restoration 1-5% observed in biopsies Functional effects not yet tested Exon 45 skipping trials (Sarepta, Daichi Sankyo)

34 Outstanding issues DMD exon skipping Mutation specific approach Repeated treatment needed Not approved in Europe $$$$$ in USA Controversial approval in USA

35 SMN1 splicing SMN1 Exon 1-6 Exon 7 Exon 8 Exon 1-6 Exon 7 Exon 8 Functional SMN protein

36 SMN2 splicing SMN2 Exon 1-6 Exon 7 Exon 8 90% 10% Exon 1-6 Exon 8 Exon 1-6 Exon 7 Exon 8 No functional SMN protein Functional SMN protein

37 SMN2 splicing modulation SMN2 AON Exon 1-6 Exon 7 Exon 8 Exon 7 more recognizable Exon 1-6 Exon 8 Exon 1-6 Exon 7 Exon 8 More functional SMN protein

38 Nusinersen Delivery to motorneurons is not feasilbe with systemic treatment Intrathecal treatment is required Advantages Lower doses needed Less frequent delivery needed Lower load for liver and kidney

39 Nusinersen trial: type 1 SMA

40 Nusinersen trial: type 2 SMA

41 Nusinersen current status Approved for all SMA types by EMA and FDA Market access varies per country Which SMA types have access $$$$$ Not everyone responds Earlier treatment appears to have larger impact More people respond Larger response

42 Genetic therapies Targeting DNA Gene addition Genome editing Targeting DNA transcripts Splice modulation Stop codon readthrough

43 Genome editing and CRISPR/Cas9

44 DNA damage repair systems DNA repair mechanisms Repair Exchange (dividing cells) Error free repair using paired chromosome Glue (non dividing cells) Glue breakpoints together Some information will be lost

45 CRISPR/Cas9: exon skipping on DNA level?

46 Why the hype about CRISPR/Cas for DMD? Technique offers a lot of potential Targeted modification of DNA Generate model systems Therapy Many examples that it works for DMD In model systems. Media attention Crispr cure 46 Aartsma-Rus June 2018

47 What is not mentioned? Delivery How to deliver Cas9 and CRISPRs in muscles Viral Vectors (AAV) Currently tested for gene addition in trials Two component system Two step process Safety How specific are the Cas9s? 47 Aartsma-Rus June 2018

48 CRISPR Cure Not for Duchenne Leads to generation of partially functional dystrophins (like found in Becker MDs) Effect also depends on time of intervention 48 Aartsma-Rus June 2018

49 Genome editing for SMA Converted SMN2 gene into SMN1 gene in ips cells Can only be done in dividing cells May be possible to target the inhibitor for exon 7 inclusion But two step approach less efficient than gene addition SMN2 Exon 1-6 Exon 7 Exon 8 49 Aartsma-Rus June 2018

50 Genetic therapies Targeting DNA Gene addition Genome editing Targeting DNA transcripts Splice modulation Stop codon readthrough

51 Stop codon read through compounds 1 79 Cell ignores new stop signal Complete protein is made

52 Ataluren/translarna: approved for DMD Cell ignores new stop signal Complete protein is made

53 Stop codon read through summary Approved for DMD in Europe for ambulant patients 2 years and older Oral delivery Only applicable to nonsense mutations (13% DMD) Access varies per country ($$$$) Applicable for nonsense mutations in SMA in theory Crosses blood brain barrier BUT not very effective (low protein recovery)

54 Genetic therapies summary There are approved drugs Nusinersen, Translarna, Eteplirsen (USA) Many trials are ongoing Gene therapy (SMA and DMD) Exon skipping (DMD) Access to approved drugs is an issue