Gene therapy and genome editing technologies for the study and potential treatment of :

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1 WORKSHOP ON GENOME EDITING 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

2 Duchenne Muscular Dystrophy Duchenne muscular dystrophy (DMD) is the most common severe form of childhood muscular dystrophy affecting 1: male births (Emery AEH. The muscular dystrophies. The Lancet.2002 Feb ;359(9307): ) DMD : A dystrophinopathy with a X linked inheritance Alteration(s) in the DMD gene coding Dystrophin Life expectancy with ventilatory assistance is superior to 36 years (Kieny P1, Evolution of life expectancy of patients with Duchenne muscular dystrophy at AFM Yolaine de Kepper centre between 1981 and Ann Phys Rehabil Med Sep;56(6): doi: /j.rehab Epub 2013 Jun 24.) No actual cure 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 Dystrophin is formed by 3,685 residues Skeletal muscle isoform : 427 kda, (260 kda, 140 kda, 116 kda and 71 kda)

4 Dystrophin: a sub-sarcolemmal protein

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

7 Overview of therapeutic approaches : RNA repair : Exon skipping EXONDYS 51 (eteplirsen) Sarepta : Treatment by skipping exon 51 of the DMD gene using phosphorodiamidate morpholino oligomer (PMO) chemistry (~ 13% of DMD patients) Principle of exon skipping Deletion of exons 45 to 50 in DMD gene : Exon skipping of Exon 51 No Dystrophin DMD Truncated Dystrophin

8 Overview of therapeutic approaches : RNA repair : Exon skipping EXONDYS 51 (eteplirsen) Sarepta : Treatment by skipping exon 51 of the DMD gene using phosphorodiamidate morpholino oligomer (PMO) chemistry (~ 13% of DMD patients) Studies over 4 years prolonged ambulation and 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: ]. FDA grants accelerated approval to first drug for Duchenne muscular dystrophy Gene replacement therapy but the DMD gene is too large to be packaged in the vector : adeno-associated virus (AAV). Therefore, a mini-/microdystrophin (4-5 kb) with reduced size must replace the full-length cdna ( ~14 kb). Clinical trial phase I (safety) R. Mendell, Nationwide Children's Hospital Others : Translarna (ataluren) PTC Phase III ACT DMD Clinical Trial in DMD Patients: To bypass nonsense point mutation Totality of clinical data demonstrate Translarna's ability to slow disease progression for patients with nonsense mutation Duchenne muscular dystrophy - Summit Drug SMT C1100 Upregulation of utrophin using a drug its efficacy is being tested in a Phase II clinical trial

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 gene DMD mouse model STOP Premature stop codon in exon 23 of DMD gene

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

11 Long C, Amoasil L, Mireault AA, et al. Science 2016; 351: Principle Delivery tools : vector Associated adenovirus : AAV In vivo

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

13 Hum Genet (2016) 135: Local injection systemic injection

14 Statement of accounts Each of the three studies demonstrated efficacy using a two-vector system of AAV-CRISPR rather than single vectors for both the guide RNA and the Cas9 nuclease. The cdnas from Staphylococcus aureus 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 Functional recovery was demonstrated in CRISPR/Cas9 treated mice: increased grip strength, improved force generation, resistance against eccentric contraction, reduced serum creatine kinase (CK) Improved cardiac function

15 The dark side 1/Genome editing has repeatedly been charged with the concerns of off-target effects. All three studies explored deep sequencing of grna target sites with specific attention to 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 phenotypic evidence of toxicity. 2/Host response online 5 september 2016; doi: /nmeth.3993 Cas9 evokes cellular immune responses while AAV9 does not Cas9 evokes humoral immune responses AAV9 evokes humoral immune responses AAV CRISPR Cas9 does not evoke extensive cellular damage

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

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