A humanized knock-in COL6A1 mouse recapitulates a splicing defect correctable with exon skipping

Authors

• HG Tran
• MA Kwarteng
• AJH Sedlack
• AZ Hawa
• KA Link
• R Penjweini
• JR Knutson
• M Garmendia-Cedillos
• TJ Pohida
• RW Robey
• MM Gottesman
• NY Morgan

Abstract

Splice-modulating antisense oligonucleotides have recently entered the clinical space as a viable therapeutic strategy for personalized medicine. Our goal is to develop a splice-modulating therapy to skip a pathogenic pseudoexon in the collagen 6 alpha 1 (COL6A1) gene, associated with a severe form of collagen VI-related muscular dystrophy. The pseudoexon insertion is driven by an unexpectedly common deep-intronic variant (c.930+189C>T) in intron 11 of COL6A1, that our group recently identified. We previously showed in cultured primary fibroblasts that phosphorodiamidate morpholino (PMO) antisense oligonucleotides efficiently skip the pseudoexon and restore COL6A1 expression. Here, we aim at creating a preclinical mouse model to test our exon-skipping therapy in vivo. Modeling splicing defects caused by deep-intronic variants, however, is complicated by the poor degree of conservation of intronic sequences between human and mouse. To address this challenge, we knocked in 1.9 kb of the human COL6A1 genomic sequence into the corresponding mouse locus, encompassing human exons 9-14 and their corresponding human introns. We generated one humanized knock-in (KI) mouse line that carries the reference C nucleotide (Col6a1HumC/HumC) at the c.930+189 position in intron 11, and one line that carries the pathogenic variant T nucleotide (Col6a1HumC/HumT, and Col6a1HumT/HumT). Using long-read sequencing, we validated that the human exons are properly spliced into the Col6a1 transcripts, including the 72-nt pseudoexon in Col6a1HumC/HumT, and Col6a1HumT/HumT. Males and females show grip strength deficit as early as one month of age. The Col6a1 humanized KI mouse represents an invaluable tool to assess the potential of human-ready exon-skipping antisense oligonucleotides.

Scientific Focus Area: RNA Biology

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