Effective Pseudo-Exon Skipping of a COL6A1 Intronic Mutation in Cultured Muscle Interstitial Fibroblasts from a Novel Humanized Mouse Model

Collagen VI-related dystrophies (COL6-RDs) are a group of frequently severe, congenital-onset muscular dystrophies for which there currently is no effective treatment. Our group has recently identified a new recurrent unexpectedly common de novo deep-intronic mutation (c.930+189C>T) associated with a severe COL6-RD phenotype, in the collagen 6 alpha 1 (COL6A1) gene. The intronic mutation creates a new donor splice site and in conjunction with a dormant splice acceptor drives the insertion in about 50% of transcripts of an in-frame pseudo-exon that exerts a dominant-negative effect on the collagen VI extracellular matrix assembly. Using morpholino antisense oligonucleotides (PMOs) targeted at potential splice enhancer sites within the pseudo-exon, we have previously shown that the pseudo-exon was skippable, in patient cultured fibroblasts (Bolduc et al., JCI Insight 2019). We have now created a humanized mouse model by knocking in the relevant COL6A1 genomic region that contains the appropriate intronic context including the mutation, with the goal of testing the pseudoexon skipping approach in preclinical studies. We found that, comparable to human fibroblasts, cultured skeletal muscle interstitial fibroblasts isolated from the KI mouse express the mutant pseudoexon and respond efficiently to the PMOs that were designed for human cells (>90% skipping with 5μM treatment). We synthesized our lead antisense sequence on various chemical scaffolds (phosphorothioate 2’methoxyethyl, and vivo-PMO) to compare their in vivo activity. Data collection for systemic injections is ongoing. The Col6a1/COL6A1 human KI mouse represents an invaluable tool to test human-ready antisense oligonucleotides and assess their potential to target the muscle interstitial fibroblasts, producers of collagen VI.