Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci.

Autor:	Padmaswari MH; Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA.; Cellular and Molecular Biology, University of Arkansas, Fayetteville, AR, USA., Bulliard G; Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA., Agrawal S; Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA., Jia MS; Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA., Khadgi S; Exercise Science Research Center, Molecular Muscle Mass Regulation Laboratory, Department of Health, Human Performance, and Recreation, University of Arkansas, Fayetteville, AR, USA., Murach KA; Cellular and Molecular Biology, University of Arkansas, Fayetteville, AR, USA.; Exercise Science Research Center, Molecular Muscle Mass Regulation Laboratory, Department of Health, Human Performance, and Recreation, University of Arkansas, Fayetteville, AR, USA., Nelson CE; Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA.; Cellular and Molecular Biology, University of Arkansas, Fayetteville, AR, USA.
Jazyk:	angličtina
Zdroj:	Molecular therapy. Nucleic acids [Mol Ther Nucleic Acids] 2024 Sep 02; Vol. 35 (4), pp. 102320. Date of Electronic Publication: 2024 Sep 02 (Print Publication: 2024).
DOI:	10.1016/j.omtn.2024.102320
Abstrakt:	Gene replacement therapies primarily rely on adeno-associated virus (AAV) vectors for transgene expression. However, episomal expression can decline over time due to vector loss or 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 low invasiveness of intramuscular injections, 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 highly expressing loci using homology-independent targeted integration (HITI) to integrate reporter or therapeutic genes in mouse myoblasts and skeletal muscle tissue. 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. Competing Interests: M.H.P. and C.E.N. are named inventors on patents and patent applications related to genome editing. (© 2024 The Author(s).)
Databáze:	MEDLINE
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