Preproinsulin mRNA engineering and its application to the regulation of insulin secretion from human hepatomas.
Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include non-beta cells genetically engineered to secrete insulin in response to physiologic cues; responsiveness can be introduced at the transcriptional level to regulate preproinsulin (PPI) mRNA biosynthesis. However, these cells exhibit sluggish secretion dynamics, which is not appropriate for achieving euglycemia in higher animals and, eventually, humans. In this work, we have engineered the PPI mRNA so as to destabilize it through nonsense-mediated mRNA decay (NMD). When expressed under transcriptional regulation in HepG2 hepatomas, the engineered PPI mRNA level and of the insulin secretion rate declined faster upon switching off transcription, compared to the one-copy non-engineered control. Our work provides a simple and straightforward method to improve the dynamics of transcriptionally regulated insulin secretion, which can be a useful tool in developing cell-based therapies for IDD.
Tang
Shiue-Cheng
SC
School of Chemical Engineering, Georgia Tech-Emory Center for the Engineering of Living Tissues, Atlanta 30332, USA.
Sambanis
Athanassios
A
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D017931:DNA Primers; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin / Q000235:genetics* / Q000378:metabolism*; D000078790:Insulin Secretion; D008113:Liver Neoplasms; D008156:Luciferases / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011384:Proinsulin / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007328:Insulin; D011498:Protein Precursors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C013737:preproinsulin; D011384:Proinsulin; D008156:Luciferases