Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice

Eriya Kenjo,Akitsu Hotta,Naoto Inukai,Masataka Ifuku,Youichi Naoe,Naoko Fujimoto,Yuichiro Amano,Kumiko A Iwabuchi,Hiroyuki Hozumi,Yukimasa Makita,Satoru Matsumoto,Maya Kimura

doi:10.1038/s41467-021-26714-w

Eriya Kenjo, Akitsu Hotta + Show 10 more

Open Access

https://doi.org/10.1038/s41467-021-26714-w

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Journal: Nature Communications	Publication Date: Dec 1, 2021
Citations: 113	License type: open-access

Affiliation: Takeda (Japan), Kyoto University

Abstract

Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, however, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. In general, AAV vectors are used for in vivo delivery, but AAV injections cannot be repeated because of neutralization antibodies. Here we report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas9 mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. Although the expressions of Cas9 protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. Furthermore, administration of our LNP via limb perfusion method enables to target multiple muscle groups. The repeated administration and low immunogenicity of our LNP system are promising features for a delivery vehicle of CRISPR-Cas9 to treat skeletal muscle disorders.

Highlights

Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues
To target broader muscle tissues, we propose the injection of lipid nanoparticle (LNP)-CRISPR via intravenous limb perfusion to induce genomic exon skipping and restoration of dystrophin protein in various muscle groups
We first screened for LNP formulations that could efficiently deliver mRNA into skeletal muscle

Summary

Introduction

Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. We report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas[9] mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. The expressions of Cas[9] protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. To induce genomic exon skipping in DMD patients’ muscle tissue, a proper delivery vehicle is needed to introduce CRISPR-Cas[9] and sgRNA targeting the human dystrophin sequence. To this end, many groups have utilized AAV (adeno-associated virus) vectors to deliver and express Cas[9] and sgRNA3–14. LNPs were developed as a vaccine platform by intramuscular injection, and two LNP-based vaccines against SARS-CoV-2 spike protein (mRNA-127323 and BNT162b124) have been authorized for emergency use in the United States and other countries

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