חידושים בתיקון הגנטי (דילוג אקסונים, מוטציות נקודתיות ועוד) בניוון שרירים דושן ובקר

Correction approaches for DMD May 28 2014

Some basic biology: genes & proteins • Proteins are building blocks of our body • Genes contain blueprint for proteins

Annemieke Aartsma-Rus

Gene Protein Cel nucleus DNA

Temporary gene copy(RNA) Cell body (cytoplasm)

Protein factory

Annemieke Aartsma-Rus

Some basic biology: genes & proteins • Proteins are building blocks of our body • Genes contain blueprint for proteins Mistake in gene

Mistake in RNA transcript

Mistake in Protein Annemieke Aartsma-Rus

Dystrophin

Dystrophin

Annemieke Aartsma-Rus

Duchenne: no functional dystrophin

Annemieke Aartsma-Rus

No functional dystrophin Muscle damage

Repair

Inflammation

Less blood flow

Too much Ca2+

Oxidative stress

Energy production impaired

Fibrosis

Loss of muscle tissue Loss of muscle function Annemieke Aartsma-Rus

Correction approaches Mistake in gene

Mistake in RNA transcript

Correct for mistake in Protein Annemieke Aartsma-Rus

Utrophin upregulation Nerve

Muscle

Dystrophin

Annemieke Aartsma-Rus

Utrophin

Utrophin upregulation • Utrophin resembles dystrophin • Utrophin can partly take over dystrophin function • Expressed in nerve cells, hardly in muscle • Find ways to get more utrophin in muscle

Annemieke Aartsma-Rus

Utrophin upregulation • Find compound to switch on utrophin gene volume • High throughput screening in cell models • Potential drugs screened further in patient-derived cell cultures and mouse models • Candidate drug tested in healthy volunteers (BMN195/SMNT C1100) by Biomarine • Uptake not sufficient Annemieke Aartsma-Rus

Utrophin upregulation • Summit made new formulation • Tested in healthy volunteers • Uptake much improved • Levels in blood now sufficient to increase utrophin • Tests in DMD patients being prepared • Safety dose finding trial complete • Phase 2 trial (2014) Annemieke Aartsma-Rus

Utrophin upregulation PTC • Finding compounds to improve translation from gene to protein Tivorsan • Biglycan protein delivery • Recruits utrophin and other dystrophin associated proteins

Annemieke Aartsma-Rus

Utrophin vs. Dystrophin • Similar proteins, but not identical • Utrophin can take over many dystrophin functions • Utrophin does not recruit nNOS (important to have sufficient blood flow to contracting muslce) • Utrophin does not recruit energy producing organels (mitochondria)

• Approach applies to ALL Duchenne patients (mutation independent) Annemieke Aartsma-Rus

Correction approaches Mistake in gene

Correct for mistake in RNA transcript

Correct for mistake in Protein Annemieke Aartsma-Rus

Exon skipping

Annemieke Aartsma-Rus

Duchenne: genetic code disrupted

Annemieke Aartsma-Rus

Duchenne: genetic code disrupted Exon 46

Exon 47

?

Exon 51

Exon 52

Protein translation stops prematurely

Dystrophin not functional Annemieke Aartsma-Rus

Becker: genetic code maintained

Annemieke Aartsma-Rus

Becker: genetic code maintained Exon 46

Exon 47

Exon 52

Exon 53

Protein translation continues

Dystrophin partly functional Less damage Annemieke Aartsma-Rus

Exon skipping: restore genetic code

Annemieke Aartsma-Rus

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

Annemieke Aartsma-Rus

Systemic study Sarepta II AVI-4658 (30 and 50 mg/kg) • Intravenous delivery • Earlier trial:20 mg/kg (12 weeks) is not sufficient • Weekly 12 weeks 50 mg/kg: biopsy • Weekly 24 weeks 30 mg/kg: biopsy • Weekly placebo 12 or 24 weeks: biopsy • After 24 weeks treatment: all 30 or 50 mg/kg/week • Patients have been treated for >90 weeks now Annemieke Aartsma-Rus

Dystrophin staining

Annemieke AartsmaRus

Department of Human Genetics

Annemieke Aartsma-Rus

24

Dystrophin results

Annemieke AartsmaRus

Department of Human Genetics

Annemieke Aartsma-Rus

25

Eteplirsen summary • Dystrophin restored for all patients 24 weeks after treatment • 6 minute walk test stabilized in 10/12 treated patients • No real placebo group • Sarepta plans confirmatory study for 2014 • Also preclinical work ongoing for exon 50, 45, 53 and 44) Annemieke Aartsma-Rus

Systemic trial (GSK/Prosensa) • Following dose finding study, all patients enrolled in an open label extension study • 6 mg/kg drisapirsen per week for 72 weeks • Then 8 weeks break • Then cycles of 8 weekly doses, 4 week break • All patients still in trial • Treated now for almost 4 years

Annemieke Aartsma-Rus

Goemans et al, NEJM 2011, 364: 1513-22

Open Label Extension Trial Drisapersen • Side effects observed • Local injection site reactions • Proteinuria (reversible during breaks) • Thrombocytopenia seen for some patients • Seen in all phase 2/3 trials drisapersen • 6 minute walk distance maintained for >3.5 years in 8/10 ambulant patients • Very encouraging results but no placebo group Annemieke Aartsma-Rus

Primary Endpoint: Change from Baseline (95% CI) in 6MWD (m), ITT Population

Visit

Comparison

Week 25 (Primary)

Week 49 (Secondary)

Treatment Difference

P-value

Weekly vs Placebo

35.09

0.014

Intermittent vs Placebo

3.51

0.801

Weekly vs Placebo

35.84

0.051

Intermittent vs Placebo

27.08

0.147

Study Design large placebo controlled trial

n = planned number randomized Numbers randomized (ITT and safety population) 6 mg/kg/week = 125 Placebo = 61

Primary Endpoint: 6MWD Adjusted Mean Change from Baseline (95% CI) in 6MWD (m) – Primary MMRM analysis ITT Population

Treatment difference = 10.3m, p-value = 0.415 at Week 48

- 42.3 m 4.7m

4.8m

1.8m

- 52.7 m Curves offset for visualization

Drisapersen most recent news • GSK stops drisapersen development • Prosensa obtained all rights and data

• Further analysis done • Phase 3 population more advanced disease • For many patients 96 week information available Annemieke Aartsma-Rus

Drisapersen preliminary further analysis

Annemieke Aartsma-Rus

Drisapersen preliminary further analysis

Annemieke Aartsma-Rus

Other trials Prosensa/GSK Drisapersen •Redosing patients anticipated to start Q3 2014 •Discussion with regulators ongoing

Exon 44: •Study completed, extension anticipated Q3/4 2014

Exon 45 and Exon 53: •Dose finding studies ongoing •Anticipate 2nd phase 2015

Preclinical work: •Exon 55 & exon 52 and rare mutations Annemieke Aartsma-Rus

Stop codon readthrough

1

79

Annemieke Aartsma-Rus

PTC124/ataluren PTC

1

79 Cell ignores new stop signal Complete protein is made

Annemieke Aartsma-Rus

PTC124/Ataluren • Tested in healthy controls: safe • Tested in 28 patients (dose finding) • Safe • Increased dystrophin expression

• Tested in 174 patients in 48 week trial • Placebo, high dose and low dose • Safe!

• No significant difference in primary outcome (6MWT) • Dystrophin levels? (analysis pending) Annemieke Aartsma-Rus

PTC124/Ataluren • Tested in healthy controls: safe • Tested in 28 patients (dose finding) • Safe • Increased dystrophin expression

• Tested in 174 patients in 48 week trial • Placebo, high dose and low dose • Safe!

• No significant different in primary outcome (6MWT) • Dystrophin levels? (analysis pending) Annemieke Aartsma-Rus

PTC124/Ataluren dosing • Dosing not optimal • Research done • Optimized dosing

effect

• Bell shaped curve

concentration

• May be responders and non-responders • Extension studies re-initiated in USA and Europe • New placebo-controlled trial started in USA and Europe in 220 patients • Requested conditional approval with EMA Annemieke Aartsma-Rus

Stop codon readthrough summary

Annemieke Aartsma-Rus

Correction approaches Correct for mistake in gene

Correct for mistake in RNA transcript

Correct for mistake in Protein Annemieke Aartsma-Rus

DNA editing • 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) Annemieke Aartsma-Rus

DNA editing • 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

Annemieke Aartsma-Rus

DNA scissor system • DNA scissors can cut DNA at specific location

Scissor cuts at/near mutation

Repair break with template (Correct small mutations)

Annemieke Aartsma-Rus

Repair break without template Small mutations will be introduced (can correct genetic code like exon skipping)

DNA scissor system • 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

Annemieke Aartsma-Rus

49 | TREAT-NMD Outcome Patient Care Joint EuroBioBank Standards Action & Website 2007-2011 Registries measures Trial research TACT SOPs Plan Site of & EU fundedRegistry Network Diagnosis Communication & Care

Three year work plan

www.treat-nmd.eu

Standardized genetic & Unique network of 18 Tests to decide whether TREAT-NMD Unified Regular experimental meetings 3 year Advisory plan to clinical2012 core data for trial members treatment being tested in onwards consolidate protocols Committee improve efforts and the for International consensus Information Extensive about website each recruitment a comparability trial is Alliance having any effect funded jointly Milestone-driven tackle Therapeutics, ofcommon studies publication recommended registered trial site kept in Stores & distributes problems approach through multiple 250,000 annualfor page hits standards of care Interface can vary one location ease of 440,000 Vital to quality use the DNA, correct cell Drawn Expert multidisciplinary up by a group of streams with global between countries whilst comparison outcome and tissue measure samples to 70,000 visitors annually Topics independent Maintains on researchers necessity, network body DMD-SMA-CMD-LGMD stillbased able to share core partners & membership prove if a in treatment works (listed momentum hosting each to & be protocol) establish rotated data Addresses organisational Monthly newsletter sent Independent between and new objective partners goals Family guides difficulties of identifying Governance Working harmonise the toto 3,500 recipients Ethical & governance best Approx guidance 40 sops on advancing updated in 25 different languages appropriate sites when Chair –of Annemieke Aartsmause most appropriate practice new therapies regularly for translations verified Rus setting up a trial Proven communication outcome measures for neuromuscular diseases Vice Chair – Ericplatform Hoffman >10,000 DMD diseases patients different Printed booklets or across countries Leads --30 Eric Hoffman Executive Committee Leads Marina Mora download from Lead Eugenio Mercuri Annemieke Aartsma-Rus Supported by academic Lucia Monaco Chair- Jan – Dominic Wells Secretariat - Verschuuren Katewebsite Bushby Leads Filippo Buccella

advisory boardVolker (“task force”) Marco Crimi Straub Coordinated Hugh by University Dawkins Funding - Telethon & Parent of NMD leaders Funding - US Sejersen (Dept of Leads - Thomas Medical Center Freiburg Organizations Funding COST Funding -Kathy Fondazione Funding – EC Defense) Funding -(operating AFM North & EC Telethon grant) (operating grant)

Summary • Some therapeutic approaches are mutation specific: good diagnostics crucial • Much is known about Duchenne – mainly through research funded by patient organisations • No treatment available yet • Improved care has had huge impact on disease progression and quality of life

Annemieke Aartsma-Rus