RNA-BASED GENE THERAPY OF DUCHENNE MUSCULAR DYSTROPHY: PRODUCTION AND VALIDATION OF ANTISENS CONSTRUCTS ABLE TO INDUCE EXON SKIPPING IN DIFFERENT DMD MUTATIONS

Among primary myopathies, the Duchenne Muscular Dystrophy (DMD) is certainly the most relevant because of diffusion and severity. The defect 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. 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 (exon skipping) 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 synthesis was obtained in myoblasts derived from a DMD patient when transduced with genes expressing specific antisense sequences (De Angelis et al., 2002). More recent work in the mdx dystrophic mouse has shown that AAV recombinant viruses are very effective in the in vivo delivery of therapeutic sequences to affected muscles, including the heart and diaphragm, and in restoring both dystrophin synthesis and muscular functionality (Denti et al., 2006 and b). Therefore, this approach seems to be very promising in view of a possible gene therapy of DMD. The aim of this project is on one side to produce antisense constructs able to induce exon skipping in a collection of DMD mutations available to us and to show their effectiveness in dystrophic myoblasts in vitro and on the other to study the features of AAV transduction, and in particular of the serotype 9 known to be very efficient in muscle colonization.