Type 1 Diabetes is characterized by an absolute insulin deficiency due

Type 1 Diabetes is characterized by an absolute insulin deficiency due to the autoimmune destruction of insulin producing -cells in the pancreatic islets. at least in part, for the protective effect of the infectivity-enhanced CA-Akt1 gene delivery vector. Taken together, 161796-78-7 our data suggest CA-Akt1 is usually effective in promoting -cell survival and proliferation in vitro, but direct in vivo use is usually compromised by the efficacy of transgene delivery into -cells. Nonetheless, the vector evoked the manifestation and activation of endogenous Akt in the islets, thus offering beneficial bystander effect against STZ-induced diabetes. myristoylation site. GFP was fused to the C-terminal end of Akt1 to facilitate the detection of transgene manifestation. To achieve -cell specific gene delivery, the rat insulin promoter (Tear), was used to drive CA-Akt1 manifestation. The manifestation cassette RIP-CA-Akt1-GFP was incorporated into the deleted At the1 (At the1) region of Ad5RGDpK7 genome. Since At the1 region is usually essential for the initiation of Ad5 replication, the viral vector was replication deficient. 161796-78-7 The viral vector was subsequently rescued in 293 cells that stably express Ad-E1 genes, and the resultant vector was named Ad5RGDpK7.RIP-CA-Akt1 (Fig.?1A). As unfavorable targeting control, the CA-Akt1 manifestation cassette was incorporated into an At the1-deleted unmodified Ad5 vector, producing in the formation of Ad5.RIP-CA-Akt1 vector (Fig.?1A). Other control vectors encoding RIP-driven reporters such as Ad5.RIP-Luc (for firefly luciferase) and Ad5.RIP-GFP were also constructed in a comparable way (Fig.?1A). Physique?1. Generation and verification of CA-Akt1 gene delivery vectors. (A) Diagram of the vectors used in this study. Luc, firefly luciferase; Tear, Rat Insulin Promoter. GFP was fused to CA-Akt1 at its C-terminal end. The infectivity-enhanced … Next we examined the gene delivery efficiency mediated by the vectors using freshly isolated human islets. The islets were infected with the viruses at an MOI of 250 VPs/cell. Two days later, the islets were either lysed for western blotting assay (Fig.?1B) or processed for immunofluorescence staining (Fig.?1C) to detect Akt1 gene expression. As shown in Physique?1B, CA-Akt1 was successfully delivered into human islets by both Ad5 and Ad5RGDpK7, while the latter showed higher gene delivery efficiency. Staining with antibodies recognizing the phosphorylated 161796-78-7 Akt1 at either Ser473 or Thr308 showed that the transgene was active. Of note, endogenous Akt appeared to be induced by Ad5 vector contamination alone, which was phosphorylated at site Ser473, and to a less degree at Thr308, consistent with our previous observation.6 Of note, it has been shown phosphorylation of Ser473 precedes and facilitates that of Thr308.16 The observation that P-Ser473 staining showed stronger signal than P-Thr308 staining indicates endogenous Akt might not be fully activated by Ad5 infection. Immunofluorescence staining of the human islets with S5mt GFP confirmed CA-Akt1 manifestation and higher gene delivery efficacy that was mediated by the infectivity-enhanced vector (Fig.?1C). Nonetheless, both vectors showed more gene delivery in the peripheral area of human islets, suggesting their penetration into the islet core was limited when applied in culture. Of note, in human islets, -cells and non–cells are intermingled. Therefore, -cell specific manifestation of GFP could be detected in the periphery of the islets. CA-Akt1 manifestation improved the survival of islet cells in vitro Previous studies have shown CA-Akt1 has strong protective effect on islet cells.5,7,12 To examine whether CA-Akt1 delivered by the vectors was functional in islet cells, we performed a WST-1 based viability assay.