1. Gene therapy and genome editing technologies for the study and potential treatment of :
 Duchenne Muscular Dystrophy by Dr France Piétri-Rouxel, Institut de Myologie
Centre de Recherche en Myologie UMRS 974 UPMC - INSERM - FRE 3617 CNRS - AIM
WORKSHOP ON GENOME EDITING

2. Duchenne Muscular Dystrophy
Duchenne muscular dystrophy(DMD) is the most common severe form of childhoodmusculardystrophy
affecting 1: 3 500 male births (Emery AEH. The muscular dystrophies. The Lancet.2002 Feb 23 2002;359(9307):687-695)
 DMD : A dystrophinopathywith a X linked inheritance
 Alteration(s) in the DMD gene coding Dystrophin
 Life expectancy with ventilatoryassistance is superior to 36 years (Kieny P1,Evolutionof life expectancyof patientswithDuchenne muscular
dystrophyatAFM Yolaine de Keppercentre between1981 and2011. AnnPhys Rehabil Med.2013 Sep;56(6):443-54. doi:10.1016/j.rehab.2013.06.002. Epub2013 Jun24.)
 No actualcure is available

3. Duchenne Muscular Dystrophy : A school case
Genetic feature: The DMD gene is one of the largest known gene :
 2.5 mega bases
 Transcript length:14,040 bps/Exons: 79
 Dystrophinis formed by 3,685 residues
 Skeletal muscle isoform : 427 kDa, (260 kDa, 140 kDa, 116 kDa and 71 kDa)

4.  Dystrophin:a sub-sarcolemmalprotein

6.  Alteration(s) in the DMD gene coding Dystrophin leads to Dystrophinopathies

7.  RNA repair : Exon skipping
EXONDYS 51 (eteplirsen) Sarepta : Treatment by skipping exon 51 of the DMD gene using phosphorodiamidate
morpholinooligomer (PMO) chemistry (~ 13% of DMD patients)
Overview of therapeutic approaches:
Deletion of exons 45 to 50 in DMD gene : ∆45-50
42 4443 535251
Exon skipping of Exon 51
42 4443 535251
No Dystrophin  DMD
Truncated Dystrophin
Principle of exon skipping

8.  RNA repair : Exon skipping
EXONDYS 51 (eteplirsen) Sarepta : Treatment by skipping exon 51 of the DMD gene using phosphorodiamidate
morpholinooligomer (PMO) chemistry (~ 13% of DMD patients)
Studies over 4 years  prolonged ambulationand change in the rate of decline compared to age matched
controls measured by the 6 minute walk test [Mendell JR, Goemans N, Lowes LP, et al. Ann Neurol 2016; 79:257-271. ].
FDA grants accelerated approval to first drug for Duchenne muscular dystrophy
Overview of therapeutic approaches:
 Gene replacementtherapy  but the DMD gene is too large to be packaged in the vector : adeno-associatedvirus
(AAV). Therefore, a mini-/microdystrophin(4-5 kb) with reduced size must replace the full-length cDNA ( ~14 kb).
Clinicaltrial phase I (safety) R. Mendell,NationwideChildren'sHospital
 Others :
Translarna™(ataluren)PTC  Phase III ACT DMD ClinicalTrialin DMD Patients: To bypass nonsense point mutation
Totalityof clinicaldata demonstrate Translarna'sabilityto slow disease progression for patientswith nonsense mutation
Duchenne muscular dystrophy -
Summit Drug SMT C1100  Upregulationof utrophinusing a drug its efficacy is being tested in a Phase II clinicaltrial
………

9. A novel approach for DMD : gene correction by genome editing
Analogousto exon-skipping therapies (RNA repear),
CRISPR-Cas9 mediated removal of one or more exons from the genomic DNA
DMD mouse model
Premature stop codon in exon 23 of DMD gene
DMD gene
STOP

10. Three separate laboratoriespublished in Science in 2016 in vivo genome editing with CRISPR/Cas9
In all three studies, the CRISPR/Cas9 system targeted the point mutationin exon 23 of the mdx mouse
 Long C, Amoasil L, Mireault AA, et al. Science 2016; 351:400-403.
 Nelson CE, Hakim CH, Ousterout DG, et al. Science 2016; 351:403-407.
 TabebordbarM, Zhu K, Cheng JK, et al. Science 2016; 351:407-411.
A novel approach for DMD : gene correction by genome editing

11. Long C, Amoasil L, Mireault AA, et al. Science 2016; 351:400-403
Principle
In vivo
Delivery tools :vector  Associatedadenovirus :AAV

12. EXON SKIPPING
Dystrophin positive fibers
Force
Long C, Amoasil L, Mireault AA, et al. Science 2016; 351:400-403

13. Hum Genet (2016)
135:1029–1040
Local injection
systemic injection

14. Each of the three studies demonstratedefficacy using a two-vector system of AAV-CRISPR rather than single
vectors for both the guide RNA and the Cas9 nuclease.
The cDNAs from Staphylococcusaureus Cas9 and Streptococcus pyogenes Cas9 were both effective in these in
vivo pre-clinical
Studies and delivery of vectors using either AAV serotypes, AAV9 or AAV8, performed well.
Dystrophin expression: skeletal muscle, vascular smooth muscle, cardiomyocytes
Functionalrecovery was demonstratedin CRISPR/Cas9 treated mice:
 increased grip strength,
 improved force generation,
 resistance against eccentric contraction,
 reduced serum creatine kinase (CK)
 Improved cardiac function
Statement of accounts

15. 1/Genome editing has repeatedlybeen charged with the concerns of off-target effects.
 All three studies explored deep sequencing of gRNA target sites with specific attentionto the top 10
predicted possibilities.
 None of the studies showed evidence of off-target gene editing that would be of concern clinically and
CRISPR-treated mice showed no phenotypicevidence of toxicity.
2/Host response
The dark side
Cas9 evokes cellularimmune responses while AAV9 does not
Cas9 evokes humoral immune responses
AAV9 evokes humoral immune responses
AAV–CRISPR–Cas9 does not evoke extensive cellulardamage
online 5 septeMbeR 2016; doi:10.1038/nMeth.3993

16. CRISPR-Cas9 : the tool to develop animal models to test therapeutic strategies
 Establishmentof dystrophic models
The DMD-modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and
decliningthickness of smooth muscle in the stomach and intestine.
 Mimic rare DMD cases : duplication of exons, 5’ or 3’ mutation
 Mimic BMD cases