Synthetic antisense oligoribonucleotides can be used to modulate gene splicing by masking key motifs on the pre-mRNA required for spliceosome assembly. Yet, intracellular expression of oligoribonucleotides generates only a transitory effect whereas stable delivery of antisense sequences can be achieved by linking them to chimeric small RNAs delivered and expressed by viral vectors. In the murine model of Duchenne Muscular Dystrophy a chimeric U7 snRNA (U7Dtex23) induces skipping of the mutated exon 23 and restores the Dystrophin mRNA reading frame. The main limitation of this approach remains the large amount of snRNA vector needed to be produced and administered to patients. To optimize this system we used self-complementary AAV vectors (scAAV) to express the U7snRNA shuttles. ScAAV vectors were tested in mouse myoblast cultures and we observed an increase in U7Dtex23 expression and in dystrophin exon 23 skipping compared to single-stranded AAV, highlighting the potential for this strategy to reduce the vector dose. Alternatively, we have used a muscle and heart-specific enhancer (MHCK) to drive the expression of U7Dtex23 cassettes delivered with AAV vectors and our results showed that MHCK improves chimeric U7snRNA expression and increases dystrophin exon 23 skipping in vitro and in vivo. However, additional U7snRNA species were produced following gene transfer, pointing at a possible limitation of the cellular processing machinery capability with saturating levels of U7 shuttles. We have also explored the possibility of using small nucleolar (sno) RNAs as novel molecular platforms for antisense delivery. We replaced the original antisense of MBII-52 snoRNA with the Dtex23 sequence and observed low levels of exon 23 skipping in AAV-transduced myotubes. While our observation validates the approach, the efficiency of skipping is still considerably lower than with the U7snRNA cassette. As a last approach, we engineered the human C/D box U24snoRNA to specifically target the methylation of an adenosine branch point in a luciferase reporter pre-mRNA in order to induce the skipping of the downstream exon. We were not able to observe any modulation of splicing using this strategy
