A humanized knock-inCol6a1mouse recapitulates a deep-intronic splice-activating variant

Abstract

AbstractAntisense therapeutics such as splice-modulating antisense oligonucleotides (ASOs) are promising tools to treat diseases caused by splice-altering intronic variants. However, their testing in animal models is hampered by the generally poor sequence conservation of the intervening sequences between human and other species. Here we aimed to model in the mouse a recurrent, deep-intronic, splice-activating,COL6A1variant, associated with a severe form of Collagen VI-related muscular dystrophies (COL6-RDs), for the purpose of testing human-ready antisense therapeuticsin vivo. The variant, c.930+189C>T, creates a donor splice site and inserts a 72-nt-long pseudoexon, which, when translated, acts in a dominant-negative manner, but which can be skipped with ASOs. We created a unique humanized mouse allele (designated as “h”), in which a 1.9 kb of the mouse genomic region encoding the amino-terminus (N-) of the triple helical (TH) domain of collagen α1(VI) was swapped for the human orthologous sequence. In addition, we also created an allele that carries the c.930+189C>T variant on the same humanized knock-in sequence (designated as “h+189T”). We show that in both models, the human exons are spliced seamlessly with the mouse exons to generate a chimeric mouse-human collagen α1(VI) protein. In homozygousCol6a1h+189T/h+189Tmice, the pseudoexon is expressed at levels comparable to those observed in heterozygous patients’ muscle biopsies. WhileCol6a1h/hmice do not show any phenotype compared to wild-type animals,Col6a1h/h+189TandCol6a1h+189T/h+189Tmice have smaller muscle masses and display grip strength deficits detectable as early as 4 weeks of age. The pathogenic h+189T humanized knock-in mouse allele thus recapitulates the pathogenic splicing defects seen in patients’ biopsies and allows testing of human-ready precision antisense therapeutics aimed at skipping the pseudoexon. Given that theCOL6A1N-TH region is a hot-spot for COL6-RD variants, the humanized knock-in mouse model can be utilized as a template to introduce otherCOL6A1pathogenic variants. This unique humanized mouse model thus represents a valuable tool for the development of antisense therapeutics for COL6-RDs.