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Gene replacement therapies in genetic medicine primarily rely on adeno-associated viral (AAV) vectors for transgene expression. However, episomal expression can decline over time due to epigenetic silencing. CRISPR-based integration methods offer promise for long-term transgene insertion. While the development of transgene integration methods has made substantial progress, identifying optimal insertion loci remains challenging. Skeletal muscle is a promising tissue for gene replacement owing to the ease of access, relative proportion of body mass, the multinucleated nature of muscle, and the potential for reduced adverse effects. Leveraging endogenous promoters in skeletal muscle, we evaluated two high-expressing loci using homology-independent targeted integration (HITI) to integrate reporter or therapeutic genes in mouse myoblasts. We hijacked the muscle creatine kinase (Ckm) and myoglobin (Mb) promoters by co-delivering CRISPR-Cas9 and a donor plasmid with promoterless constructs encoding green fluorescent protein (GFP) or human Factor IX (hFIX). Additionally, we deeply profiled our genome and transcriptome outcomes from targeted integration and evaluated the safety of the proposed sites. This study introduces a proof-of-concept technology for achieving high-level therapeutic gene expression in skeletal muscle, with potential applications in targeted integration-based medicine and synthetic biology.
Padmaswari et al. (Tue,) studied this question.