Upgrading U7snRNA to complete efficient rescue of dystrophin by exon-skipping in DMD patients

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Most cases of Duchenne muscular dystrophy (DMD) are caused by mutations that disrupt the dystrophin mRNA reading frame. In many cases, skipping of a single exon could restore the reading frame, giving rise to a shorter but still functional quasi-dystrophin protein. This exon skipping approach using oligonucleotides is currently in clinical trial for DMD, but only applies to a few patients. We wish to develop constructs which will be applicable to more patients using a viral approach. It has previously been proposed to use small nuclear RNAs, especially U7snRNA, to shuttle antisense sequences designed to mask key elements involved in the splicing of targeted exons.
Our proposed project focuses on the upgrading of U7snRNA to complete rescue of dystrophin by exon-skipping in DMD patients. We therefore propose the development of bifunctional U7snRNAs carrying a complementary sequence to the targeted exon and a free tail harbouring canonical binding sites for the heterogeneous nuclear ribonucleoproteins A1/A2 (hnRNP) that are powerful splicing repressors. The presence of this generic strong silencer tail could indeed circumvent the always tricky and time-consuming specific optimization required for each new exon-target. In addition to the optimization of the mono-exon-skipping, we propose to investigate the possibility of inducing multi-skipping. This approach could considerably increase the number of eligible patients and would reduce the number of U7 constructs that have to be made in order to treat the different kind of mutations found in DMD patients.
The new U7 constructs will be evaluated both in vitro in cells from DMD patients and in vivo in a transgenic humanized mouse model, specifically imported from the Netherlands to our facility. The newly established viral vector production platform in the department will provide the perfect environment to produce the lentiviral and AAV vectors required for both the in vitro and in vivo evaluation of these constructs.
The preliminary results obtained on exon 51 are very encouraging and provide evidence that bifunctional U7snRNA can achieve efficient exon-skipping in myoblasts from DMD patients. Confirmation of their efficiency on other exons and development of the multi-skipping constructs would therefore offer very promising tools for clinical treatment of DMD.