Exon skipping for DMD Annemieke Aartsma‐Rus February 29 2012
Duchenne Muscular Dystrophy (DMD)
Department of Human Genetics
2
Annemieke Aartsma-Rus
Becker Muscular Dystrophy (BMD)
Clinical symptoms BMD
Age
Department of Human Genetics
3
Annemieke Aartsma-Rus
Genes and proteins • Proteins building blocks of our body • Genes contain blueprint for proteins • Mistake in gene mistake in protein • Genes have a volume switch Only on in necessary tissue • Dystrophin protein has function in muscle • Mistake in dystrophin muscle problems Annemieke Aartsma-Rus
Muscles • 30‐40% of our body consists of muscle • Muscles can grow bigger and smaller • Muscles use lot of energy • Only maintained when needed • Muscles damaged after excessive exercise • Muscles very efficient at repairing damage Bigger when needed Annemieke Aartsma-Rus
Muscles
Annemieke Aartsma-Rus
Muscle fibers Skeleton
Connective tissue
Annemieke Aartsma-Rus
Dystrophin
Dystrophin
Annemieke Aartsma-Rus
Genes and proteins • Proteins building blocks of our body • Genes contain blueprint for proteins • Genes consist of DNA • Cell can translate DNA code into protein
Annemieke Aartsma-Rus
Gene Protein Cell nucleus DNA
Temporary gene copy (RNA) Cell body (cytoplasm)
Protein factory
Annemieke Aartsma‐Rus
Dystrophin gene
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 51 skipping (del exon 48‐50) M NT 51 ‐RT 47
51
52
47
52
0h 4h 8h 16h 24h 48h HC
48 post transfection
NT
MANDYS1
Annemieke Aartsma‐Rus
MANDYS1
DYS2
Aartsma‐Rus HumMolGen 2003 12: 907‐14
Deletion exon 45‐55 ‐AON
+AON
Annemieke Aartsma-Rus
+AON
Exon 44 skipping ‐AON
M NT
44 ‐RT
NT
2d
4d
7d
HC(1:10)
43 44 55 43 55
NT
+AON
48 hours post transfection
MANDYS1
Annemieke Aartsma‐Rus
+AON
MANDYS1
DYS2
Double exon skipping
Annemieke Aartsma-Rus
‐RT
NT 45 & 51
M
Double Exon Skipping NT
2d
3d
HC
44 45 51 52 44 45 52 44 52 Not Treated
48 uur post transfection
Annemieke Aartsma-Rus
Aartsma‐Rus AmJHumGen 2004, 74: 83‐91
Mdx mouse • Natural mouse model • Disrupting mutation exon 23 • Dystrophin deficiency • Muscle function impaired • Less severe disease than humans • Exon 23 skipping can restore genetic code, dystrophin and leads to functional improvement after intramuscular injection of AONs 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 • Prosensa: 2’‐O‐methyl phosphorothioate • AVI‐Biopharma: phosphorodiamidate morpholino oligomers • Targeting exon 51
Annemieke Aartsma-Rus
First clinical trial – set up Set up • Single dose, single center study • 4 pre‐screened DMD patients aged 8‐16 • Single IM injection PRO051 in shin muscle • Biopsy 4 weeks after injection • Exon skip? Dystrophin? Safe? Day ‐60 to ‐7 Pre‐screening
Day 0 Baseline Assessment
Annemieke Aartsma-Rus
Day 0 Treatment
Day 1 to 28 Follow‐up
Day 28 to 35 Safety & Efficacy Assessment
First clinical trial – pre‐screening
Annemieke Aartsma-Rus
First clinical trial – analysis RNA
Annemieke Aartsma-Rus
Annemieke AartsmaRus
Department of Human Genetics
30
725 fibers
Annemieke AartsmaRus
Pt.2
120 fibers
Department of Human Genetics
31 Pt.3
Dystrophin quantification
Annemieke Aartsma-Rus
Second 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 treatment AON levels in muscle and Liver
Exon 23 Skipping
Gastrocn.
WT Mice Mdx Mice
Annemieke Aartsma-Rus
AON Tissue Levels (ug/g)
Systemic treatment
Annemieke Aartsma-Rus
Systemic treatment Tibialis Anterior Tissue levels
RNA
20 15 10 5 0 25 mg/kg
50 mg/kg
16
16 14 12 10 8 6 4 2 0
100 mg/kg
Dose
12 10 8 6 4 2 0
25 mg/kg
50 mg/kg
100 mg/kg
Dose
Similar results for all skeletal muscles Heart lower exon skip and protein levels Annemieke Aartsma-Rus
8 weeks 4 weeks
14
Dystrophin %
% e x o n 2 3 s k ip p in g
T iss ue le vels (ug /g )
25
Protein
25 mg/kg
50 mg/kg
Dose
100 mg/kg
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) • Comparative dose trial planned (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) Annemieke Aartsma-Rus