No abstract

Muscular dystrophies are a group of genetically distinct diseases for which no treatment exists. While gene transfer approach is being tested for several of these diseases, such strategies can be hampered when the size of the corresponding complementary DNA (cDNA) exceeds the packaging capacity of adeno-associated virus vectors. This issue concerns, in particular, dysferlinopathies and titinopathies that are due to mutations in the dysferlin (DYSF) and titin (TTN) genes. We investigated the efficacy of RNA trans-splicing as a mode of RNA therapy for these two types of diseases. Results obtained with RNA trans-splicing molecules designed to target the 3' end of mouse titin and human dysferlin pre-mRNA transcripts indicated that trans-splicing of pre-mRNA generated from minigene constructs or from the endogenous genes was achieved. Collectively, these results provide the first demonstration of DYSF and TTN trans-splicing reprogramming in vitro and in vivo. However, in addition to these positive results, we uncovered a possible issue of the technique in the form of undesirable translation of RNA pre-trans-splicing molecules, directly from open reading frames present on the molecule or associated with internal alternative cis-splicing. These events may hamper the efficiency of the trans-splicing process and/or lead to toxicity.

Mutations in dysferlin and anoctamin 5 are the cause of muscular disorders, with the main presentations as limb-girdle muscular dystrophy or Miyoshi type of distal myopathy. Both these proteins have been implicated in sarcolemmal resealing. On the basis of similarities in associated phenotypes and protein functions, we tested the hypothesis that ANO5 protein could compensate for dysferlin absence. We first defined that the main transcript of ANO5 expressed in skeletal muscle is the 22-exon full-length isoform, and we demonstrated that dysferlin-deficient (Dysf (prmd)) mice have lower Ano5 expression levels, an observation that further enhanced the rational of the tested hypothesis. We then showed that AAV-mediated transfer of human ANO5 (hANO5) did not lead to apparent toxicity in wild-type mice. Finally, we demonstrated that AAV-hANO5 injection was not able to compensate for dysferlin deficiency in the Dysf (prmd) mouse model or improve the membrane repair defect seen in the absence of dysferlin. Consequently, overexpressing hANO5 does not seem to provide a valuable therapeutic strategy for dysferlin deficiency.