Gene Therapy of Duchenne Muscular Dystrophy using U7 snRNA ...

Gene Therapy of Duchenne MuscularDystrophy using U7 snRNA-mediated ExonSkippingLuis Garcia & Olivier DanosCNRS UMR8115, Généthon, Evry-FRFrench-American Innovation DayHarvard Medical School,BostonOctober 17, 2005

Duchenne Muscular Dystrophy• 1 / 3500 boys• Clinical signs appear after 3 year ofage.• Walking is lost at 10 year• Scoliosis• Severe respiratory problems• Heart disease in 1/3 of patients• Occasional cognitive deficit

Dystrophin and associatedproteinsLaminin-2extracellular matrix(basal lamina)sarcolemmaDystrophinDystroglycansGrb2NOsF-ActinSarcoglycansSarcospanFilamin 2SyntrophinsDystrobrevinCalmodulinSyncoilinDesmincytoskeleton

Duchenne Muscular DystrophyΔdystrophinfiber weaknessregenerationnecrosisatrophiafibrosisadipocytosisN/R

Gene Therapy for MuscularDystrophies• Treat before irreversible destruction ofthe muscle tissue• Restore muscle function permanently inmost muscles including heart anddiaphragm• Avoid immune reactions and acutetoxicity

MolecularInterventions inDMD• Gene replacement– Dystrophin & Microdystrophin– Utrophin• Gene compensation– Intégrin α7– NO synthase– GalNAc transferase– ADAM12 metalloprotease• Muscle mass enhancement– IGF-1– Anti Myostatin• Gene repair– Exon skipping– Supressor tRNA– Chimeraplasts

Adeno-Associated Viral Vectors•Non pathogenic humanparvovirus (5.0 kb ssDNA)•Helper-free production ofhigh titer vectors•Stable (up to 6 year) genetransfer into the musclewithout vector genomeintegration•Serotypes 1,6 and 8 highlyefficient in muscle

High Pressure Intra-Arterial Injection ofrAAV into the Rat Hind-LimbGonin et al. 2004

Hierarchy of Gene Therapy ClinicalTrials in Muscular Dystrophies• Local– No expected benefit– Toxicity and safety– Gene transfer efficiency– Immune response– AAV dose : 10e11 – 10e12 gc• Regional– Functional improvement of muscle function– AAV dose : 10e14 – 10e15 gc• Multiple Regional– Immunosuppression• Systemic ?

Modular Organization ofDystrophinNH 2R1H3R20R21R22R23R24H4CRCOOHNH 2ABDH1ActinR1R2R3H2R4R5R6R7R8R9R10R11R12R R13 14R15R16R17hingecryptic internal actinbinding sitespectrin-like repeatsR18R19H3R20R21R22R23R24H4CRβ-DystroglycanCTCOOHα1,β1-Syntrophins(nNOS)α-DystrobrevinFabb et al (2002)

Deletions in the Dystrophin Gene ofDuchenne and Becker Patients277 DMD deletions203 BMD deletions0 0,5 1 1,5 2 2,5Million base pairsDMD-UMD database, JC Kaplan

Exon Chaining in the Dystrophin Gene

Target Sequences for ExonSkippingSDBPSA ESEU1U2/U6

Restoring the dystrophin orf by exonskippingin the mdx mouse21 22 23 2425 Pre-mRNAC > T3185cgtgttagtgtaaatgaacttctatttaattttgagGCTC… …TCAGgtaagccgaggtttggcctttaaaBP22SD23

AAV-2/1-Mediated Transfer ofU7-SD23/BP22Goyenvalle et al. Science (2004)

Detection of Exon 23-SkippedDystrophin mRNA and Proteinmonths0 0.5 1 2 3 6***901bp688bp***RT-PCRC57/BlWestern blot

Dystrophin rescue following administration ofAAV-U7SD23/BP2212 months

Functional recovery in treated musclesC57/BlmdxTreated (45 days)contralateraltreated

Animal Models of DuchenneMuscular DystrophyExon #1Dp 42730Dp 260Dp 140Dp 116Dp 7144 56 633 ’I 6 E 10 E 23 E 42 E 53E 58E 79mousemdx Cv5 mdx mdx Cv2 mdx Cv4mdx Cv3dogGRMDRotweilerGSHP

Multiple exon-skipping in GRMDAAV(U7 C6ESE2) & AAV(U7 C8ESE1), 2 months

Rescue of the Dystrophin-Associated Complexdystrophinβ-dystroglycanγ-sarcoglycanα-sarcoglycanGRMD Treated dog Normal dog

Deletions around exon 51 inDuchenne and Becker Patientsn=12n=4n=17n=9n=4n=0 +1n=0n=1n=3 +1n=1n=1n=11UMD-DMD patient databaseJC Kaplan

AAV-U7 mediated skipping ofhuman exon 51 in the hDMD mouse*ITRU7-Ex51ITRINTRON.50…ctcaaattgttactcttcaattaaatttgacttattgttattgaaattggctctttagcttgtgtttctaatttttctttttcttcttttttccttctttgcaaaaacccaaaatattttagCTCCTACTCAGACTGTTACTCTGGTGACACAACCTGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGGTACCTGCTCTGGCAGATTTCAACCGGGCTTGGACAGAACTTACCGACTGGCTTTCTCTGCTTGATCAAGTTATAAAATCACAGAGGGTGATGGTGGGTGACCTTGAGGATATCAACGAGATGATCATCAAGCAGAAGgtatgagaaaaaatgataaaagttggcagaagtttttctttaaaatgaagattttccaccaatcactttactctcctagaccatttcccaccagttctta…INTRON.511 month*Bremer-Bout et al. (2004)

Lentiviral vector (U7-ex51)ASDINTRON.50…ctcaaattgttactcttcaattaaatttgacttattgttattgaaattggctctttagcttgtgtttctaatttttctttttcttcttttttccttctttgcaaaaacccaaaatattttagCTCCSTACTCAGACTGTTACTCTGGTGACACAACCTGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGGTACCTGCTCTGGCAGATTTCAACCGGGCTTGGACAGAACTTACCGACTGGCTTTCTCTGCTTGATCAAGTTATAAAATCACAGAGGGTGATGGTGGGTGACCTTGAGGATATCAACGAGATGATCATCAAGCAGAAGgtatgagaaaaaatgataaaagttggcagaagtttttctttaaaatgaagattttccaccaatcactttactctcctagaccatttcccaccagttctta…INTRON.51H51a(+68+88)H51b(+161+183)ctrlLent(U7-H51ab)Lent(U7-ASDS)∆51 RT-PCR (human myoblasts)

Conclusions• Therapeutic levels of dystrophin can be restoredusing exon skipping• The approach solves the problem of tissue specifictransgene expression and is immunologically safer• Up to 80% DMD patients are eligible for monoskippingtherapy. Multiple skipping increases thisproportion• Targeting the spliceosome with modified snRNAcould be applied to other diseases (exoninclusion/exclusion, splice site mutations).• Exon skipping can be used for gene knock-down, asan alternative to siRNA.

CNRS UMR 8115 /GénéthonLuis GarciaOlivier DanosAurélie GoyenvalleAdeline VulinRachid BenchaouirFrançoise FougerousseCécile PeccateGénéthon Core FacilitiesENVAStéphane BlotCHU MontpellierChristophe BéroudSylvie TufferyMireille ClaustreLeiden University (NL)Judith Van DeutekomInstitut CochinJean-Claude KaplanFrance LeturcqJamel Chelly