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

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

Why focusing on exon duplication?

1 2 32

• 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

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 2 2

Center for Gene Therapy Muscle and Skin Cell Line

• 73 primary lines established

• 38 dystrophinopathy (DMD/IMD/BMD)

Cellline

Exon(s)duplicated

1 22 23 124 185 216 217 438 449 2-1910 3-411 3-712 3-1613 8-914 8-1115 10-1716 42-4317 44-5118 46-4719 55-63

In vitro testing of antisense sequence using fibroblasts derived myoblasts

Non

-ind

uce

d FM

D7

Ind

uce

d FM

Muscle specific

alpha actinin

MF20 hMyosin

Heavy Chain

Desmin

In vitro testing of antisense sequence using fibroblasts derived myoblasts

Exon skipping restores a normal dystrophin reading frame

Normal DMD RNA

• 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

31 2 2

Bl6du

p2

The new Dup2 mouse:

Bl6du

p2

Vulin, Wein et al., 2015

- First dmd exonic duplication - Platform to test exon skippingBut:What if you skip too much?

• 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.

31 22

Nonsense mutations in exon 1 cause very mild BMD

Gurvich OL et al., Human Mutation 2009Flanigan 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)

Mechanism in case of nonsense

N-truncated dystrophin

IRES

Wein N. et al., Nat Med. 2014

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

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

AAV

Intramuscular injection at 2months collected at 3months

5e11vg/TA

Intramuscular injection

Dup2+U7

N-truncated isoform protects against Evans Blue dye uptake

Muscle protection

Intact fiber Fiber w/o dystrophin

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 linesAccording to the skipping efficiency

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

Too efficient exon skipping

untre

ated

H44

A+H

45A

150m

M

H45

A - 1

50m

M

H44

A - 1

50m

M

Becker Muscular dystrophy

Muscle histology at 12 months

In vitro evaluation of exon 2 duplication

Duplication of exon 2 blocks IRES activity/access

?

Most frequent duplicated exon (10%) -> DMD

?

Can we use exon-skipping to generate premature stop codon and forcing IRES activation as a therapeutic strategy ?

Antisense

Out-of-frame exon skipping