COL6 related congenital muscular dystrophy (CMD) is caused by mutations in either COL6A1, COL6A2 or COL6A3 genes that disrupt the normal assembly of the COL6A1, COL6A2, and COL6A3 chains into heterotrimer fibrils essential for integration into the extracellular matrix. Autosomal recessive Ullrich CMD patients carrying mutations that lead to premature termination of translation present with severe phenotypes due to extremely low levels or lack of COL6. This is a proof-of-concept study to ascertain if an exon skipping therapy we have developed for the treatment of Duchenne muscular dystrophy could be applied to bypass mutated exons and rescue defective COL6 production in Ullrich CMD patients. We explored whether: (i) small exons in COL6 transcripts can be efficiently and specifically excised; (ii) the equivalent exons in COL6A1, COL6A2, and COL6A3 gene transcripts can be simultaneously excised; and (iii) these modified COL6A isoforms can assemble into functional trimer fibrils.
We analyzed COL6 protein assembly and designed antisense oligonucleotides to induce skipping of the corresponding exons of COL6A1, COL6A2, and COL6A3 transcripts, which encode part of the triple helix structure of COL6. The initial screen for the best exon skipping sequences was performed using oligomers synthesized as 2’-O-methyl modified bases on a phosphorothioate backbone, and the top performing sequences are then synthesized as clinically relevant, phosphorodiamidate morpholinos oligomer. Efficient exon 23 skipping of the COL6A2 transcript in healthy fibroblasts disrupted COL6 assembly as expected, indicating that the exon skipping we observed at the RNA level was translated into protein. We are currently screening for antisense oligomers which induce exon skipping in similar size exons in COL6A1 and COL6A3 transcripts to rescue a proper assembly of COL6 in the cells treated with COL6A2 exon 23 skipping oligomer.