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Gene therapies for SMA and DMD
Annemieke Aartsma-Rus
September 2018
Disclosures
Sept 2018
• 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
• 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
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
Annemieke Aartsma-RusDepartment of Human Genetics6
DMD: no functional dystrophin
Healthy Duchenne Becker
Sept 2018
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
Annemieke Aartsma-RusDepartment of Human Genetics8
SMA: less functional SMN protein
Sept 2018
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
13
5
6
7Gene (DNA)
RNA copy (pre mRNA)
messenger RNA
1 - - - - - - - - - 79
dystrophin protein
Splicing
2
4
3 4 56 7
1 21 2 3 4 5 6 7 8
Sept 2018
Dystrophin exons
Sept 2018
Duchenne: reading frame disrupted
Sept 2018
Exon 48-50 deletion
Disrupted reading frame
Exon 46 Exon 47 Exon 51 Exon 52?
Protein translation truncated prematurely
Dystrophin not functional
Sept 2018
Becker: frame maintained
Sept 2018
Becker: reading frame maintained
Protein translation continues
Dystrophin partly functional
Exon 46 Exon 47 Exon 52 Exon 53
Reading frame not disrupted
Sept 2018
Exon skipping to restore reading frame
Exon 52Intron 51Intron 47/50Exon 47 Exon 51 Intron 52AON
Exon 46 Exon 47 Exon 52
Reading frame restored
Partially functional dystrophin
Annemieke Aartsma-RusDepartment of Human Genetics30
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
Sept 2018
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)
Annemieke Aartsma-RusDepartment of Human Genetics32
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
Sept 2018
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
• $$$$$ in USA
• Controversial approval in USA
Sept 2018
SMN1 splicing
Exon 1-6 Exon 7 Exon 8SMN1
Exon 1-6 Exon 7 Exon 8
Functional SMN protein
Sept 2018
SMN2 splicing
Exon 1-6 Exon 7 Exon 8SMN2
Exon 1-6 Exon 8
Functional SMN protein
Exon 1-6 Exon 7 Exon 8
No functional SMN protein
90% 10%
Sept 2018
SMN2 splicing modulation
Exon 1-6 Exon 7 Exon 8SMN2
Exon 1-6 Exon 8
More functional SMN protein
Exon 1-6 Exon 7 Exon 8
AON
Exon 7 more recognizable
Sept 2018
Nusinersen
• Delivery to motorneurons is not feasilbe with systemic treatment
• Intrathecal treatment is required
• Advantages
• Lower doses needed
• Less frequent delivery needed
• Lower load for liver and kidney
Sept 2018
Nusinersen trial: type 1 SMA
Sept 2018
Nusinersen trial: type 2 SMA
Sept 2018
Nusinersen current status
• Approved for all SMA types by EMA and FDA
• Market access varies per country
• Which SMA types have access
• $$$$$
• 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
Genome editing and CRISPR/Cas9
DNA repair mechanisms
Exchange (dividing cells)
Error free repair using paired
chromosome
Glue (non dividing cells)
Glue breakpoints together
Some information will be lost
Repair
DNA damage repair systems
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
June 201846 Aartsma-Rus
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?
June 201847 Aartsma-Rus
“CRISPR Cure”
• Not for Duchenne
• Leads to generation of partially functional dystrophins
(like found in Becker MDs)
• Effect also depends on time of intervention
June 201848 Aartsma-Rus
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
June 201849 Aartsma-Rus
Exon 1-6 Exon 7 Exon 8SMN2
Sept 2018
Genetic therapies
• Targeting DNA
• Gene addition
• Genome editing
• Targeting DNA transcripts
• Splice modulation
• Stop codon readthrough
Stop codon read through compounds
1 79
Cell ignores new stop signal
Complete protein is made
Ataluren/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)
Sept 2018
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
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