| Glucagon-like peptide-1 (GLP-1) is an important incretin hormone which is synthesized and secreted by intestinal L-cells in response to nutrition ingestion. GLP-1 has a variety of attractive biological functions, including glucose-dependent stimulation of insulin secretion from pancreatic beta cells and inhibition of glucagon release from pancreatic alpha cells. GLP-1 also stimulates cell proliferation and neogenesis, while inhibiting cell apoptosis, therefore increasing the cell mass and enhancing cell function. Furthermore, GLP-1 improves the insulin sensitivity in peripheral tissues including liver and muscle. The hormone also slows down gastrointestinal motility, induces satiety and decreases the bodyweight. GLP1 has a very short half-life (less than 2 min), whichlimits its value for clinic use. Great efforts thus have been made in developing effective therapies to enhance GLP-1 action. The GLP-1 analogues which significantly prolong the half-life of.GLP-1, however, still need to be administrated once or twice a day. GLP-1 gene therapy achieved effective glycemic control. While in these studies, the GLP-1 gene existed as a episomal form in the transfected cells.Adeno-associate virus (AAV) is a nonpathogenic and replication defective human parvovirus. Wild AAV can site-specifically integrate its DNA into human chromosome 19q13.4, named AAV binding site 1 (AAVS1) which is believed as safe integration site. Thus, the recombinant vector originated from AAV (rAAV) is considered as promising vector for gene therapy, due to its potential of achieving persistent expression of transgene and minimizing risk of developing insertional mutagenesis and immune response against vector transduced cells.. AAV Rep68/78 protein and RBE element are sufficient to mediate the genome integration. Rep68/78 also participates in the AAV DNA replication, therefore the recombinant AAV viral vectors loss the ability of site-specific genome integration., Thus, we need to find a new gene therapy strategy, which would mediate the efficient transduction of rAAV and site-specific integration of transgene at AAVS1 site.Here, we develop a non-viral gene therapy strategy with two plasmids, one coding for the Rep78, and the other carrying GLP-1/Fc flanked by a RBE element. When two plasmids were co-transferred into target cells, the transiently expressed Rep78 protein and RBE element would mediate the genome integration of GLP-1/Fc gene. PCR-based DNA hybridization was used for detection of genome integration. In vitro assays showed that GLP-1/Fc gene integrated into the AAVS1 site in 293 cells in a Rep protein dependent way. In vivo expression of GLP-1 was made in transgenic mice carrying hAAVSl through intramusclular injection of the plasmid RBE/GLP-1/Fc and Rep78/VRnew followed by an electroporation. We found that GLP-1/Fc gene was integrated into AAVS1 site other than mir-341 locus which site insertion is reported to be related to the high incidence of carcinoma. We detected GLP-1/Fc fusion protein in the DNA injected muscle and circulation by immonohistochemistry and enzyme-liked immunoabsorbent assay (ELISA) techniques and suggesting that the GLP-1/Fc fusion proteins were processed and expressed in the mice after the gene transfection. In high fat diet (HFD) feeding mice, the GLP-1/Fc gene therapy significantly decreased the food intake and reduced the body weight gain. Interestingly we also found that the blood triglyceride was decreased, while the leptin and ghrelin levels were normalized, as well as improved insulin sensitivity in these GLP-1/Fc treated mice on HFD feeding. Our study revealed a novel non-viral targeted genome strategy for GLP-1 therapy with the apparent absence of side effects, which may has clinical relevance. |
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