P5396CRISPR/Cas9 mediated miR-29b editing restores muscle atrophy and exercise capacity in mice

Abstract

Abstract Background Muscle atrophy is the loss of skeletal muscle mass and strength in response to diversity catabolic stimuli, such as heart failure. At present, no effective treatment except exercise is validated on reducing multiple muscle atrophy clinically. We have recently reported that microRNA-29b (miR-29b) promotes multiple types of muscle atrophy. Purpose The goal of this study was to assess whether genome editing using a clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system can efficiently introduce loss-of-function mutations into the endogenous miR-29b in vivo and as a potential therapy by treating muscle atrophy. Methods We used lentivirus to express CRISPR-associated 9 and a CRISPR guide RNA targeting miR-29b. Mutagenesis rate of miR-29b and off-target mutagenesis were detected by T7 Endonuclease I (T7EI) Assay. The expression level of miR-29b were measured in vitro and vivo after administration of the virus by using qRT-PCR. After intramuscular administration of the virus, the angiotensin II (AngII), immobilization and denervation-induced muscle atrophy were performed. Then muscle function was assessed in exercise capacity, the appearance and weight of muscle, the size of the muscle fibers, molecular and cellular detection. Results Here, we report that CRISPR/Cas9 mediated genome editing through intramuscular administration efficiently targeting the biogenesis processing sites in pre-miR-29b. No off-target mutagenesis was detected in 10 selected sites. This CRISPR-based treatment resulted in decreased miR-29b levels specifically. In vivo, this CRISPR-based treatment could ameliorate the muscle atrophy induced by angiotensin II (AngII), immobilization and denervation via activation of PI3K-AKT-mTOR signaling pathway and protect against AngII-induced apoptosis in mice. Moreover, the exercise capacity is also significantly enhanced. Conclusion Our work establishes CRISPR/Cas9 based gene targeting on miRNA as a potential durable therapy for treatment of muscle atrophy and expands the strategies available interrogating miRNA function in vivo. Acknowledgement/Funding The grants from National Natural Science Foundation of China (81722008, 91639101 and 81570362 to JJ Xiao)