Optimization of miR-22 expression cassette for rAAV delivery on diabetes

Molecular Biomedicine

Birong Dong¹  Qiukui Hao¹  Biao Dong¹  Li Yang¹  Jun Li¹  Jiao Zhou¹  C. Alexander Valencia¹  Hoi Yee Chow¹  Jintao Du²  Lin Xiao³  Qingzhe Yang³  Wenya Du³  Zhaoyue Zheng³  Li Wang³  Xianghui Fu³ 

[1] Department of Geriatrics and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University;Department of Otorhinolaryngology Head & Neck Surgery, West China Hospital, Sichuan University;State Key Laboratory of Biotherapy, West China Hospital, Sichuan University;

关键词: Recombinant adeno-associated virus;    MicroRNA;    Expression cassettes;    MiR-22;    Insulin;   

DOI  :  10.1186/s43556-021-00063-y

来源: DOAJ

【 摘 要 】

Abstract MicroRNA-22 (miR-22) was suggested to be important for type 2 diabetes but its functions for this disease remained unclear. Recombinant adeno-associated virus (rAAV)-mediated miR delivery is a powerful approach to study miR functions in vivo, however, the overexpression of miR-22 by rAAV remains challenging because it is one of the most abundant miRs in the liver. In this study, a series of expression cassettes were designed and compared. It was shown that different lengths of primary miR-22 were overexpressed in HEK293 and HeLa cells but the longer ones were more efficiently expressed. miR-22 may be placed in either introns or the 3′ UTR of a transgene for efficient overexpression. RNA polymerase III or II promoters were successfully utilized for miR expression but the latter showed higher expression levels in cell lines. Specifically, miR-22 was expressed efficiently together with an EGFP gene. After screening, a liver-specific TTR promoter was chosen to overexpress miR-22 in diabetic mice fed a high-fat diet. It was shown that miR-22 was overexpressed 2-3 folds which improved the insulin sensitivity significantly. The approach utilized in this study to optimize miR overexpression is a powerful tool for the creation of efficient rAAV vectors for the other miRs.

【 授权许可】

Unknown