Ricerc@Sapienza

Permanent neonatal diabetes mellitus (PNDM) is a genetic form of diabetes with onset within six months of birth. PNDM can be caused by mutations in the gene encoding insulin (INS/PNDM), the hormone circulating in blood that controls glucose levels. These patients need insulin shots for life. There are two types of INS/PNDM mutations: recessive and dominant. Recent studies diagnosed 14 patients with dominant INS/PNDM characterized by abnormal misfolded insulin that remains trapped inside the endoplasmic reticulum (ER) of beta cells. The insulin misfolding causes a phenomenon called ER-stress that triggers the unfolded protein response (UPR) aimed to restore protein folding homeostasis and leads to beta cell death by apoptosis and consequently to diabetes.
The goal of this project is to establish the proof-of-principle that lentiviral shRNA interfering therapy can rescue diabetic phenotype caused by dominant insulin mutation LB15YB16delinsH (in which of Leu and Tyr are replaced by His at residues 15-16 of the B-chain).
HEK-293T human cells transfected to transiently express this mutation display ER-stress and apoptosis as demonstrated by the increased splicing of X-box binding protein 1 (XBP1, marker of UPR) respect to wt insulin and increased surface expression of GRP78/BiP (ER-stress marker) and Annexin V (early apoptosis marker).
Based on preliminary results showing that HEK-293T human cells transfected with plasmids carrying shRNA sequences directed against this mutation specifically reduce (70%) the expression of the mutated insulin, I will construct interfering lentivirus (LVs) and test them on HEK-293T cells. I will evaluate the reduction of mutated insulin by qPCR and test if LVs are effective in rescuing ER-stress and apoptosis. If successful, this approach could be used on Induced Pluripotent Stem Cells (IPSC) differentiated into pancreatic beta-like-cells, to rescue diabetic phenotype, paving the way for future cell-based therapy of INS/PNDM.