Gene therapies for SMA and DMD

Annemieke Aartsma-Rus September 2018 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.

Sept 2018 Outline

• Introduction • Genes and proteins • What goes wrong with DMD and SMA

• approaches • Gene addition, genome editing & splice modulation • How do they work? • Current state of the art for DMD and SMA • Outstanding questions and challenges Sept 2018 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

Sept 2018 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

Sept 2018 DMD: no functional dystrophin

Healthy Duchenne Becker

6 Department of Human Genetics Annemieke Aartsma-Rus 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

Sept 2018 SMA: less functional SMN protein

8 Department of Human Genetics Annemieke Aartsma-Rus 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

Sept 2018 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 Sept 2018 Varying amounts of SMN protein

Sept 2018 Genetic therapies

• Targeting DNA • Gene addition • Genome editing • Targeting DNA transcripts • Splice modulation • Stop codon readthrough

Sept 2018 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?

Sept 2018 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’

Sept 2018 Dystrophin is rather big

• AAV is very small (20 nm, 0.00002 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?

Sept 2018 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

Sept 2018 Current state of the art SMA

Sept 2018 Future plans (Avexis)

• Continue follow up of patients • Confirmatory trials – all open label • 30 patients intravenous delivery • Type 1, 2 SMN copies; Europe (NCT03461289) • 20 patients intravenous delivery (NCT03306277) • 1 or 2 SMN 2 copies • 27 patients intrathecal delivery (NCT03381729) • Compare 2 doses, 3 SMN2 copies

Sept 2018 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

Sept 2018 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

Sept 2018 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?

Sept 2018 Genetic therapies

• Targeting DNA • Gene addition • Genome editing • Targeting DNA transcripts • Splice modulation • Stop codon readthrough

Sept 2018 Splicing

Exons Introns 3 6 1 2 5 7 Gene (DNA) 4

Splicing messenger RNA 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 1 ------79

RNA copy (pre mRNA) dystrophin protein

Sept 2018 Dystrophin exons

Sept 2018 Duchenne: reading frame disrupted

Sept 2018 Exon 48-50 deletion

Exon 46 Exon 47 ? Exon 51 Exon 52 Disrupted reading frame

Protein translation truncated prematurely

Dystrophin not functional

Sept 2018 Becker: frame maintained

Sept 2018 Becker: reading frame maintained

Exon 46 Exon 47 Exon 52 Exon 53 Reading frame not disrupted

Protein translation continues

Dystrophin partly functional

Sept 2018 Exon skipping to restore reading frame

Exon 47 Intron 47/50 ExonAON 51 Intron 51 Exon 52 Intron 52

Reading frame restored

Exon 46 Exon 47 Exon 52

Partially functional dystrophin

Sept 2018 Mutation specific approach

Exon All mutations Deletions 51 14% 21% 45 9.0% 13% 53 8.1% 12% 44 7.6% 11% 50 3.8% 5.6% 43 3.1% 4.5% 8 2.0% 2.9%

Bladen et al, Hum Mut 2015 30 Department of Human Genetics Annemieke Aartsma-Rus Current state of the art • Clinical development exon 51 skipping AONs most advanced • and • Drisapersen tested in >300 DMD patients • Maybe slower disease progression (6MWT) • Safety concerns • Not approved • Development stopped (Prosensa, GSK, BioMarin)

Sept 2018 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 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) Sept 2018 Outstanding issues DMD exon skipping • Mutation specific approach • Repeated treatment needed

• Not approved in Europe • $$$$$ in USA • Controversial approval in USA

Sept 2018 SMN1 splicing

SMN1 Exon 1-6 Exon 7 Exon 8

Exon 1-6 Exon 7 Exon 8

Functional SMN protein

Sept 2018 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

Sept 2018 SMN2 splicing modulation

SMN2 Exon 1-6 Exon 7 AON Exon 8

Exon 7 more recognizable

Exon 1-6 Exon 8 Exon 1-6 Exon 7 Exon 8

More functional SMN protein

Sept 2018 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

Sept 2018 Nusinersen trial: type 1 SMA

Sept 2018 Nusinersen trial: type 2 SMA

Sept 2018 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

Sept 2018 Genetic therapies

• Targeting DNA • Gene addition • Genome editing • Targeting DNA transcripts • Splice modulation • Stop codon readthrough

Sept 2018 Genome editing and CRISPR/Cas9 DNA damage repair systems

DNA repair mechanisms

Repair

Exchange (dividing cells) Glue (non dividing cells) Error free repair using paired Glue breakpoints together chromosome Some information will be lost CRISPR/Cas9: exon skipping on DNA level? 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 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 “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 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 Genetic therapies

• Targeting DNA • Gene addition • Genome editing • Targeting DNA transcripts • Splice modulation • Stop codon readthrough

Sept 2018 Stop codon read through compounds

1 79 Cell ignores new stop signal Complete protein is made /translarna: approved for DMD

Cell ignores new stop signal Complete protein is made 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) 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

Sept 2018