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.
INS/PNDM patients (3-6,8), depend on insulin for survival. To date, the most advanced form of treatment for patients with INS/PNDM is sensor-augmented continuous subcutaneous insulin infusion (26). The approach that I propose in this project consists in testing the ability of lentiviral interfering shRNA(s) directed against the insulin dominant mutation LB15YB16delinsH to inhibit the production of the mutant gene product abolishing in turn ER-stress. If successful this approach can be applied to obtain mutant allele-specific silencing in Induced Pluripotent Stem Cells (IPSC) differentiated into pancreatic beta-like cells, deriving from INS/PNDM patients in order to rescue the diabetic phenotype.
One may also hypothesize that even a partial inhibition of the mutant allele may result in a significant reduction of the apoptotic process, as can be inferred by the fact that the Akita mouse, with one mutant allele out of four (Ins1 and Ins2), doesn't develop diabete in the neonatal period, but at 6-8 weeks of age (9).
Thus this strategy could pave the way for the iPSC-based cure of patients with neonatal diabetes due to proteotoxic insulin gene mutations.
In addition the dissection of UPR and ER stress-induced apoptosis mechanism(s) caused by INS/PNDM mutations is instrumental to identify specific therapeutic strategies. To date, knowledge in this area comes prevalently from experiments conducted in mice with naturally occurring Ins2 mutations and in murine cell model of beta-like cell INS1-E or MIN6 (4). Taking into account that mouse/rat pancreatic islets differs from human islets in several ways, studying this phenomena in a human cell line model might reveal some abnormalities specific to the human beta-cell that could be exploited for therapeutic intervention.