Pluronic® L64-mediated stable HIF-1α expression in muscle for therapeutic angiogenesis in mouse hindlimb ischemia

Abstract

Hongmei Song,1 Sijia Liu,1 Caixia Li,1,2 Yanyan Geng,1,3 Gang Wang,1 Zhongwei Gu1 1National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, People’s Republic of China; 2Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, People’s Republic of China; 3Hebei University of Engineering, Handan, Hebei, People’s Republic of China Abstract: Intramuscular injection of plasmid DNA (pDNA) to express a therapeutic protein is a promising method for the treatment of many diseases. However, the therapeutic applications are usually hindered by gene delivery efficiency and expression level. In this study, critical factors in a pDNA-based gene therapy system, such as gene delivery materials, a therapeutic gene, and its regulatory elements, were optimized to establish an integrated system for the treatment of mouse hindlimb ischemia. The results showed that Pluronic® L64 (L64) was an efficient and safe material for gene delivery into mouse skeletal muscle. It also showed intrinsic ability to promote in vivo angiogenesis in a concentration-dependent manner, which might be through the activation of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB)-regulated angiogenic factors. The combination of 0.1% L64 with a hybrid gene promoter (pSC) increased the gene expression level, elongated the gene expression duration, and enhanced the number of transfected muscle fibers. In mice ischemic limbs, a gene medicine (pSC-HIF1αtri/L64) composed of L64 and pSC-based expression plasmid encoding hypoxia-inducible factor 1-alpha triple mutant (HIF-1αtri), improved the expression of stable HIF-1α, and in turn, the expression of multiple angiogenic factors. As a result, the ischemic limbs showed accelerated function recovery, reduced foot necrosis, faster blood reperfusion, and higher capillary density. These results indicated that the pSC-HIF1αtri/L64 combination presented a potential and convenient venue for the treatment of peripheral vascular diseases, especially critical limb ischemia. Keywords: gene therapy, Pluronic L64, angiogenesis, SV40 enhancer, HIF-1α