COLVI-related dystrophies are one of the two most common forms of congenital muscular dystrophy (CMD) and are associated with a clinical spectrum with Ullrich CMD at the severe end. They are caused by pathogenic variants in the genes encoding COLVI: COL6A1, COL6A2, and COL6A3. One recurrent variant is the dominantly acting deep intronic c.930+189C>T in COL6A1 intron 11, which creates a donor splice site that allows for the insertion of a translated 72-nucleotide pseudo-exon sequence that disrupts the proteins’ structure and function.
Native U7snRNPs can be repurposed into a splice modulation tool, as has been successfully demonstrated for targets such as exon 2 of the dystrophin gene. Here, we aim to develop an AAV-delivered U7snRNA-based ‘exon-skipping’ therapy to precisely skip the pseudo-exon insertion to result in the wild-type mRNA isoform. Ten candidate antisense U7snRNA sequences that spanned the pseudo-exon region were selected and cloned for individual AAV2 packaging. The AAVs were applied in vitro to patient fibroblast cultures. Four promising constructs emerged that showed a significant reduction of pseudo-exon expression. Currently, different combinations of these AAVs are being tested in vitro with the possibility of generating a bi- or tri-specific cassette to be delivered in a single AAV. The most successful sequence will also be tested in a humanized mouse model of the COL6A1 c.930+189C>T variant. These findings are promising given the potential for long-term splicing correction with this novel targeted antisense approach.