1. Exon Duplication and 5’ mutations in the DMD gene Nicolas Wein, PhD Flanigan Lab Center for Gene Therapy Nationwide Children’s Hospital Columbus, OH, USA

2. Dystrophin Mutations Dystrophin gene (Xp21.1) is huge: –2.4 million nucleotides –79 exons and 8 promoters ~5% have duplications Large deletions (≥ 1 exon) account for ~65% of DMD/BMD patients ~15% of boys have nonsense mutations Remainder are frameshifting insertions/deletions, splice site mutations, missense mutations

3. Why focusing on exon duplication? 1 2 3 2 Skipping of exons adjacent to large deletions turns a DMD gene message into a BMD gene message However, skipping a duplicated exon has the potential to turn a DMD gene message into an entirely normal message

4. 3 Why focusing on exon duplication? Skipping of exons adjacent to large deletions turns a DMD gene message into a BMD gene message However, skipping a duplicated exon has the potential to turn a DMD gene message into an entirely normal message 1 22

5. Center for Gene Therapy Muscle and Skin Cell Line 73 primary lines established 38 dystrophinopathy (DMD/IMD/BMD) Cell line Exon(s) duplicated 12 22 312 418 521 6 743 844 92-19 103-4 113-7 123-16 138-9 148-11 1510-17 1642-43 1744-51 1846-47 1955-63

6. In vitro testing of antisense sequence using fibroblasts derived myoblasts

7. Non-induced FM D7 Induced FM Muscle specific alpha actinin MF20 hMyosin Heavy Chain Desmin In vitro testing of antisense sequence using fibroblasts derived myoblasts

8. Exon skipping restores a normal dystrophin reading frame  Normal DMD RNA

9. No animal model for exon duplication mutations Exon 2 duplication is the most frequent The new Dup2 mouse: Why a make a new mouse model? Will allow us to test whether skipping of only one copy of a duplicated single exon can restore an entirely normal DMD mRNA and expression of a wild-type dystrophin protein 3 1 22

10. Bl6 dup2 The new Dup2 mouse: Bl6 dup2 Vulin, Wein et al., 2015 -First dmd exonic duplication -Platform to test exon skipping But:  What if you skip too much?

11. Skipping of both copies of a duplicated exon 2 would be expected to result in an out-of-frame DMD mRNA and no dystrophin expression. 3 122

12. Nonsense mutations in exon 1 cause very mild BMD Gurvich OL et al., Human Mutation 2009 Flanigan et al, Neuromuscular Disorders 2009  Due to the presence of an N-truncated protein produced by alternative initiation that begins in exon 6 C-Term N-Term (exon 1)

13. Mechanism in case of nonsense N-truncated dystrophin IRES Wein N. et al., Nat Med. 2014

14. Why is this important?  The duplication of exon 2 is a particular case as :  We can skip partially to make normal protein  We can “overskip” to make a very highly functional N-truncated protein

15. Exon skipping using an scAAV-U7 system  Delivery in a virus into muscle  AAV are non-integrative small viruses  Good tropism for muscle (AAV1, 6,8 and 9)  Mediation of exon skipping AAV_U7 delivery  U7 :  deliver antisense sequence:  Longer and continuous expression of the antisense sequence

16. Treatment of FibroMyoD from exon 2 duplicated patient using out of frame exon skipping  Out-of-frame exon-skipping induces expression of a N-truncated dystrophin in patient derived fibroblasts

17. Intramuscular injection at 2months collected at 3months 5e11vg/TA

18. Intramuscular injection Dup2+U7

19.  N-truncated isoform protects against Evans Blue dye uptake Muscle protection Intact fiber Fiber w/o dystrophin

20. Future directions  Preclinical dose-response studies of our AAV-U7 vector are underway  Planning the toxicity studies that will allow us to proceed to human trials (preIND meeting with FDA in Q4 2015)  In parallel, testing skipping of single, double and multiple exon duplications in patient cell lines  According to the skipping efficiency

21. Acknowledgments Department of Human Genetics (Salt Lake City, UT, USA): Dunn DM, Weiss RB Howard M Center for Gene Therapy (NCH, Columbus, OH, USA): Simmons T, Vulin A, Findlay A, Gummiesy F. Huang N. Yurkosi J. Flanigan KM Queen square centre for neuromuscular disease (UCL, London, UK) Francesco Muntoni Department of Medical Science (Ferrara, Italy): Falzarano MS, Bovolenta M, Gualandi F, Ferlini A Royal Institute of Technology (Stockholm, Sweden): Al-Khalili Szigyarto C, Uhlen M Centre for Neuromuscular and Neurological Disorders, (Perth, Australia): Fletcher S, Wilton SD Department of Molecular and Cellular Biochemistry (Columbus, OH, USA): Bakthavachalu B, Schoenberg D Center for Gene Therapy (NCH, Columbus, OH, USA): Heller KN, Rodino-Kaplac L

22. Too efficient exon skipping untreated H44A+H45A 150mM H45A - 150mMH44A - 150mM  Becker Muscular dystrophy

23. Muscle histology at 12 months

24. In vitro evaluation of exon 2 duplication  Duplication of exon 2 blocks IRES activity/access ?  Most frequent duplicated exon (10%) -> DMD

25. ? Can we use exon-skipping to generate premature stop codon and forcing IRES activation as a therapeutic strategy ? Antisense Out-of-frame exon skipping