RNA-BASED GENE THERAPY OF PRIMARY MYOPATHIES

Among primary myopathies, the Duchenne Muscular Dystrophy (DMD) is certainly the most relevant because of diffusion and severity. The molecular basis of this degenerative muscular disease resides in mutations in the X-linked dystrophin gene. In the absence of such protein the muscles gradually begin to deteriorate. Since this disease is a monogenic disorder, it was considered from the very beginning for a gene therapy approach, although technically very challenging due to the large size of the protein whose coding sequences are difficult to accomodate in most viral vectors. Currently, several different approaches have been conceived for gene therapy of primary myopathies: i) development of viral vectors capable of delivering a dystrophin mini-gene to muscle fibers in vivo; ii) functional rescue of the tissue by the delivery of genetically corrected autologous myogenic cells. In the last years we have pioneered a strategy, different from gene replacement, consisting in the modification at the post-transcriptional level of the coding capacity of the dystrophin mRNA. Most of the mutations in the dystrophin gene produce non-functional mRNAs; by preventing the inclusion of specific mutant exons in the mature mRNA, it is possible to restore a partially corrected phenotype. In a previous work we showed that this strategy is very effective: specific exon skipping and rescue of dystrophin sysnthesis was obtained in myoblasts derived from a DMD patient (2). In the present project we want to produce molecules able to induce such post-transcriptional modifications in the mRNA of several other different dystrophin mutations; in addition, and more important, we want to extend and test the validity of our approach to the mdx animal model. Delivery of the “therapeutic molecules” to the different muscular districts of the mutant animal will be accomplished through transplantation of engineered stem cells or delivery by AAV vectors.