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Future treatments for patients with DMD Annemieke Aartsma‐Rus February 27 2011
Dystrophin
Dysferlin Caveolin Calpain‌
Annemieke Aartsma-Rus
Dystrophin
Dystrophin
Annemieke Aartsma-Rus
Duchenne: no functional dystrophin
Annemieke Aartsma-Rus
Pathology No dystrophin/DGC
Muscle damage
Increase calcium levels Activation proteases
Inflammation
Mitochondria damaged
More muscle damage
Fibrosis
Less regeneration
Loss of muscle tissue Loss of muscle function Annemieke Aartsma-Rus
Therapeutic approaches for DMD • Cell therapy • Gene therapy • Genetic therapy • Exon skipping (DMD specific) • Stop codon readthrough • Drug therapy • Utrophin upregulation (DMD specific) • Myostatin inhibition Annemieke Aartsma-Rus
Gene Therapy • DMD: mutation in dystrophin gene • Solution: add functional copy of gene to muscle cells • Need viral vector • Most vectors primarily infect dividing cells • Muscle is postmitotic • Epimysium, Perimysium and endomysium • AAV effective at infecting muscle Annemieke Aartsma-Rus
Gene Therapy AAV • Not pathogenic in humans • Initial tests used AAV serotype 2 (most common) • >10 serotypes now identified • Some better muscle transduction (e.g. AAV5, AAV6) • Very small virus (20 nm) • Small cloning capacity (max 4.5 kb) • Dystrophin cDNA is 14 kb… • cDNAs other NMDs smaller! Annemieke Aartsma-Rus
Gene Therapy for DMD
Actin binding Central Rod: 4 hinges, 24 repeats
• Remove all “redundant” domains • Maintain only essential domains • Multiple tested Optimal thus far: ABD, H1, R1‐ 2, H3, R22‐24, CR Annemieke Aartsma-Rus
Cysteine Rich C‐term
Gene Therapy for DMD • Tested in mdx mouse (using AAV, CMV promoter) • Partial protection against muscle damage • Tested in Duchenne dog model • Immune response to viral vectors • Immune suppression (transient) necessary
Annemieke Aartsma-Rus
Liu et al, Mol Ther 2005, 11: 245‐56 Yuasa et al, Gene Ther 2007; 14:1249‐60
Gene Therapy for DMD • Tested in patients (Jerry Mendell/Jude Samulski, USA) • Safety trial (2006‐2007) • 6 patients injected in arm muscle (locally)
• Muscle biopsy 42 or 90 days later • Dystrophin detected in 2 biopsies (very low levels) • Viral genomes detected in all biopsies • Immune response to dystrophin! Annemieke Aartsma-Rus
Gene Therapy Immunity problems • Vector •Precludes repeated injections • Different serotypes solution? • Transient immune suppression sufficient in human?
• Dystrophin • Used CMV promoter • Artificial dystrophin • Elispot assay (false positives) • Dystrophin present Annemieke Aartsma-Rus
Gene Therapy for DMD Future • Mendell/Samulski • Prepare for trial to test whole muscle treatment • Immune suppression • Chamberlain • IM study with other AAV serotype • Compare with and without immune suppression • Compare to utrophin delivery Annemieke Aartsma-Rus
Cell therapy for NMDs
Satelite cells
Muscle stem cells challenges • Do not home to muscle • Poor migration and survival • Immune suppression needed Annemieke Aartsma-Rus
Cell therapy for DMD Myoblasts • Tremblay (Canada): multiple local injections in muscle not realistic for whole body treatment Other stem cells (e.g. fat, bloodvessels, bone) • Home to muscle from bloodstream • Very low efficiency
Annemieke Aartsma-Rus
Skuk et al, J Neuropathol Exp Neurol 2006, 68: 629‐38
Cell therapy for DMD Mesangioblasts • Good results in dog model • Clinical trial initiated in Italy in 2011 (Giulio Cossu) Autologous stem cells (combination gene therapy) • Tests in mice Stamcell tourism • No evidence yet that stem cell treatment works in patients or that treatment is safe! Annemieke Aartsma-Rus
PTC124/ataluren
PTC
1
79 Cell ignores aberrant stop signal Complete protein is made
Annemieke Aartsma-Rus
PTC124/Ataluren in DMD • Tested in DMD patient‐derived cells • Tested in mdx mice • Dystrophin restoration • Increase specific force, decrease CK Control
Annemieke Aartsma-Rus
Mdx
Mdx + PTC124
Welch et al, Nature 2007, 447: 87‐91
PTC124/Ataluren • Tested in healthy controls: safe • Tested in 28 patients (dose finding) • Safe • Increased dystrophin expression by 11% (for 65%)
• 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 future • Dosing may not have been optimal • Bell shaped curve • Further investigation needed • Optimal dosing • Subset of responders? • May be new trials in future effect
• OLE reopens globally concentration Annemieke Aartsma-Rus
Exon skipping for DMD
Annemieke Aartsma-Rus
Splicing Exons 3
2
1
6 5
Introns 7
Gene (DNA)
4
1
2
3
Splicing messenger RNA 4
5
6
7
RNA copy (pre mRNA) Annemieke Aartsma-Rus
1 2 3 4 5 6 7 8 1 - - - - - - - - - 79
dystrophin protein
Duchenne: reading frame disrupted
Annemieke Aartsma-Rus
Duchenne: reading frame disrupted Exon 46
Exon 47
?
Exon 51
Exon 52
Protein translation stops prematurely
Dystrophin not functional Annemieke Aartsma-Rus
Becker: reading frame maintained
Annemieke Aartsma-Rus
Becker: reading frame maintained Exon 46
Exon 47
Exon 52
Exon 53
Protein translation continues
Dystrophin partly functional Less damage Annemieke Aartsma-Rus
Exon skipping: restore reading frame
Annemieke Aartsma-Rus
Exon skipping • Exon skipping and dystrophin restoration confirmed in patient‐derived cells (deletions, duplications, small mutations) • Exon skipping, dystrophin restoration and muscle function improvement confirmed in mdx mouse model and GRMD dogs (local and systemic!)
Annemieke Aartsma-Rus
Alter et al. Nat Med 2006, 12: 175‐7
Applicability
hotspot
Annemieke Aartsma-Rus
Aartsma‐Rus Hum Mutat 2009, 30:293‐9
AON chemistry • Two different chemistries developed further • GSK/Prosensa: 2’‐O‐methyl phosphorothioate • AVI‐Biopharma: phosphorodiamidate morpholino oligomers • Targeting exon 51
Annemieke Aartsma-Rus
Intramuscular trial (Prosensa) 0.8 mg in TA
Annemieke Aartsma-Rus
Van Deutekom NEJM 2007; 357: 2677‐86
Intramuscular trial (AVI) • Two doses tested (0.09 and 0.9 mg) • EDB muscle • Exon skip in all doses • Dystrophin restoration only for high dose
Annemieke Aartsma-Rus
Kinali Lancet Neurol 2009, 8: 918‐28
Intramuscular trials • Exon skipping observed in all patients • No toxic effects observed! • Dystrophin levels very comparable 17‐35% vs 22‐32% • Number of dystrophin positive cells comparable 64‐97% vs 44‐79% • Systemic treatment needed Annemieke Aartsma-Rus
Systemic trial 2OMePS (GSK/Prosensa) GSK2402968 (0.5, 2, 4 & 6 mg/kg) • Subcutaneous injections (abdomen) • Weekly for 5 weeks • 3 patients per group • Biopsy taken 1 month after last injection • No severe side effects reported Open label extension study initiated • All patients receive 6 mg/kg/week subcutaneous Annemieke Aartsma-Rus
Systemic trial 2OMePS (GSK/Prosensa)
Dose
Pre‐Treatment *
*Revertant Fiber
Dystrophin (ManDys 106)
Annemieke Aartsma-Rus
Goemans et al, NEJM 2011, 364: 1513‐22
Systemic trial 2OMePS (GSK/Prosensa)
Annemieke Aartsma-Rus
Goemans et al, NEJM 2011, 364: 1513‐22
Systemic trial 2OMePS (GSK/Prosensa) • Dystrophin restoration observed in 10/12 patients • 60‐100% muscle fibers dystrophin positive • Dystrophin levels up to 15.6% of control • Dose dependent increase of dystrophin levels • Open label extension study initiated • All patients enrolled • Weekly treatment for 90+ weeks Annemieke Aartsma-Rus
Goemans et al, NEJM 2011, 364: 1513‐22
Systemic trial MDEX/AVI AVI‐4658 (0.5, 1, 2, 4, 10 & 20 mg/kg) • Intravenous infusions • Weekly for 12 weeks • 2‐4 patients per group (total 19) • Biopsies • Before treatment • 2 weeks after last injection • No severe side effects reported Annemieke Aartsma-Rus
Cirak et al, Lancet 2011, 378: 595‐605
Systemic trials MDEX/AVI • Clear dystrophin in 7/19 patients • 7‐10% of control pre‐treatment • 11‐22% of control post‐treatment • 3 good responders (2, 10 & 20 mg/kg group) • Up to 55% dystrophin positive fibers • Up to 18% dystrophin (Western blot) • Inflammatroy infiltrate muscle decreased • No effect on CK or function • Dosing needs to be optimized further Annemieke Aartsma-Rus
Cirak et al, Lancet 2011, 378: 595‐605
Systemic trials MDEX/AVI
Annemieke Aartsma-Rus
Cirak et al, Lancet 2011, 378: 595‐605
Planned/ongoing trials • Exon 51 skipping • Non ambulant trial ongoing USA (GSK/Prosensa) • Dose frequency trial ongoing (GSK/Prosensa) • Phase 3 trial ongoing (GSK/Prosensa) • AVI‐4658 higher dose started (AVI/NWH USA) • Exon 44 skipping Phase I/II ongoing (Prosensa) • Plans for exon 45 and 53 trials (Prosensa) • Optimization other exons ongoing (52 & 55) • Dialogue with regulatory agencies initiated Annemieke Aartsma-Rus
Utrophin upregulation Nerve
Muscle
Dystrophin
Annemieke Aartsma-Rus
Utrophin
Utrophin upregulation • Utrophin upregulation ameliorates disease in mdx mouse model • Need 3‐5 fold increased expression in mouse • Summit/Kay Davies high throughput screening • Drug identified that upregulates utrophin in cells and mdx mouse model • Drug tested in healthy volunteers by Biomarine • Stopped (plasma levels too low) • Working on new formulation (Summit Plc) Annemieke Aartsma-Rus
Myostatin inhibition Muscle growth factors
Muscle growth
Annemieke Aartsma-Rus
Muscle growth inhibitors
Muscle
Myostatin inhibition • Myostatin inhibits muscle growth • Animals/humans without myostatin: big muscles • Inhibit myostatin muscle growth • Compensate for muscle loss in patients?
Annemieke Aartsma-Rus
Myostatin inhibition Normal
Muscle
Annemieke Aartsma-Rus
Myostatin inhibition
Muscle growth
Myostatin inhibition
Muscle strength
Acceloron (Shire): soluble myostatin receptor Tested in healthy mouse • More muscle, less fat Mdx mouse • More muscle, less fat • Stronger muscle
Annemieke Aartsma-Rus
N
mdx
Mdx +
Myostatin inhibition Tested in healthy volunteers • Well tolerated, also repeated treatment • More muscle mass, less fat
Annemieke Aartsma-Rus
Myostatin inhibition Clinical trial in DMD patients (Canada) • 12 week treatment, 3 doses • Placebo arm (1:3 placebo) • Test safety/tolerability • Optimal dose finding Trial terminated • Some patients had unexplained gum/nose bleeds • Shire/Acceleron looking into underlying cause Annemieke Aartsma-Rus
Improve muscle quality No dystrophin/DGC
Muscle damage
Increase calcium levels Activation proteases
Mitochondria damaged
More muscle damage
Oxidatieve stress
Inflammation Fibrosis
Less regeneration
Loss of muscle tissue Loss of muscle function
Annemieke Aartsma-Rus
Improve muscle quality Antioxidants • Idebenone • Improves heart function mdx mouse model • Trials ongoing (Santhera) • Green tea extract • Effects on muscle quality in mdx mouse model • Clinical trail ongoing in Germany in DMD patients Annemieke Aartsma-Rus
Sildenafil (Viagra) • NO: vascular dilation • Dystrophin links nNOS to muscle/heart membrane • Heart problems DMD: lack NO • Sildenafil: more NO • Sildenafil prevents heart problems in mdx mice • Not yet tested in DMD patients! • Side effects? Annemieke Aartsma-Rus
Summary • Many different approaches close to or in trials • Exciting times • Future treatment likely combination • Coordinated effort needed between all parties • Scientists • Clinicians • Industry • Regulatory agencies • Patients!! Annemieke Aartsma-Rus