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Antisense sequences delivery for therapeutic intervention on messenger RNAs – GENESPLICE

Submission summary

Up to 50% of disease-associated mutations in humans affect the pre-mRNA splicing and maturation processes, by inactivating splicing signals, creating cryptic ones or modifying binding sites for splicing regulators. They result in exon skipping, inclusion of cryptic exons or intron retention, and in the production of non-functional mRNAs or proteins. In addition, neutral exonic mutations that disrupt splicing enhancers and silencers, can have an important impact on the delicate balance of alternative splicing, a mode of regulation observed in up to 75% of transcription units in the human genome. The processing of pre-mRNA can be modulated with high specificity by antisense oligoribonucleotides (AON) able to mask key determinants of splicing. This approach has been successfully used to counteract the deleterious effect of mutations, and more broadly as a means to influence the cell phenotype by selectively manipulating alternative splicing. More generally, the AON technology emerges as a useful complement to RNA interference because it allows selectively removing information in an mRNA rather than destroying it. Recently, we have adapted viral gene transfer vector for the delivery of antisense sequences and demonstrated their efficacy for mRNA based intervention in the context of Duchenne Muscular Dystrophy. Therapeutic exon-skipping was obtained in the skeletal muscle of diseased mice and dogs and in cells from human patients, when the appropriate antisense sequences were linked to a modified U7 small nuclear RNA (snRNA), which was itself expressed from Adeno-associated or lentiviral vectors. Our goal with the present project is to extend these findings and further develop the technology towards applications to other pathologies. These include genetic diseases affecting the retina and the skin, which are two promising and advanced areas for clinical applications of gene transfer. The four principal aims of the project are: 1. To obtain optimal and controlled levels of antisense sequences following AAV or lentivirus-mediated gene transfer. This will involve the design of efficient, tissue specific and regulatable expression cassettes for improved snRNA antisense carriers, derived from U7 or from snoRNAs. We also propose to test novel approaches for compensating mutations at the 3' end of introns that affect lariat formation or acceptor site recognition, involving either bi-functional ('tailed') antisense RNAs or modified U2 snRNA. 2. To use viral vector mediated delivery of antisense or interfering RNA (shRNA) in the skeletal muscle to further define the tolerance of Dystrophin for exon skipping, and to explore the possibilities of recovering muscular mass in DMD and other wasting diseases by targeting myostatin (GDF8) or its receptor (AcvRIIb). 3. To induce therapeutic exon skipping in a frequent form of Leber Congenital Amaurosis (LCA) due to an intronic mutation in the NPHP6 gene. Alone, this mutation account for 14% of all LCA cases. We will deliver antisense sequences in cells of patients carrying this mutation, in order to induce skipping of a pathologic cryptic exon in the NPHP6 gene. A transgenic mouse expressing the mutant human mRNA will be generated and used for preclinical studies in preparation of a clinical trial involving AAV mediated administration of antisense sequences to the retina. 4. To obtain therapeutic exon skipping in two severe genetic skin diseases, Dystrophic Epidermolysis Bullosa (DEB) and Netherton Syndrome (NS). DEB is caused by recessive loss-of-function or dominant negative mutations in the COL7A1 gene encoding type VII collagen. It is a life-threatening skin and mucosal blistering disorder characterized by impaired dermal-epidermal adherence and severe local and systemic complications. NS is a recessive skin barrier disorder with severe atopic manifestations, arising from the deficiency in a serine protease inhibitor, LEKTI, encoded by the SPINK5 gene. We will demonstrate specific, efficient antisense-mediated exon skipping in vitro in the respective mRNAs; using lentiviral vectors mediating exon skipping, we will generate ex vivo genetically corrected human skin equivalents, to be grafted onto mice for the long-term, functional, biochemical and ultrastructural study of genetic correction; and lastly, we will apply the antisense technology in vivo to transgenic mice models expressing the human COL7A1 or SPINK5 genes. Ultimately, this proof-of-principle study will constitute an essential pre-clinical milestone toward therapies for DEB and NS to improve the patients' functional and vital prognosis using systemic or topical delivery methods.

Project coordination

Olivier DANOS (Organisme de recherche)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Help of the ANR 450,000 euros
Beginning and duration of the scientific project: - 48 Months

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