Permanent neonatal diabetes mellitus (PNDM) is a type of diabetes characterized by the onset of hyperglycemia within the first six months of life. PNDM could be caused by mutations in INS1 gene both recessive and dominant. The dominant mutations affect proinsulin folding and trafficking; misfolded (pro)insulin is retained inside ER and triggers the unfolded protein response (UPR), an adaptive cellular mechanism aimed at restoring protein-folding homeostasis in specialized secretory cells and apoptosis which leads in turn to the swift decline of insulin secretion. Still, how sustained ER-stress can induce pancreatic beta cell apoptosis is not fully elucidated. INS1L39Y40delinsH mutation is one of the dominant mutations associated to PNDM. Actually INS/PNDM patients depend on insulin for survival, which calls for investment into the design of innovative therapeutic strategies.
A proposed new therapeutic approach was based on lentiviral mediated expression of shRNAs designed to allow specific reduction of mutated INS1 expression. For INS1L39Y40delinsH mutation a shRNA capable of reducing mutated expression up to 70% leaving almost unaltered INS1 expression was already selected. Preliminary results indicate that this shRNA could be efficient in promoting the rescue of CHOP expression induced by mutated INS1 to promote apoptosis triggered by ER stress.
The aim of this project is to complete the characterization of the rescue capability of the selected shRNA expressing lentivector analyzing other PNDM-associated intracellular biomarkers, including: XBP1 splicing products and apoptosis markers (by annexin V and PI). Moreover I will assay corrected or not, INS1 mutated cells by UPR-specific PCR microarrays comparing them with wild type cells. If successful, this approach can be applied to obtain mutant allele-specific silencing in IPSC deriving from INS/PNDM patients that could be differentiated into pancreatic beta-like cells in order to rescue the diabetic phenotype.
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 (19) only partially impacting because of the dominant property of the mutation, which calls for investment into the design of innovative therapeutic strategies.
In the optic of identification of new therapeutic approaches for INS/PNDM we proposed the use 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. An indication of the potential success of this kind of approach came from the consideration 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.
Moreover the lab in which I'm working already identified the more efficient and selective shRNA sequence among a panel of sequences designed on the genomic region surrounding the mutation and the lentivectors encoding this shRNA is already available in the lab.
Based on these robust preliminary results, I'll propose in this project to continue the characterization of the therapeutic potential of this construct going on with the analysis of its capability of rescuing the ER-stress and apoptosis induced by INS1 mutation.
I'll propose moreover to analyze a panel of gene involved in UPR both in lentivector rescued HEK-293T and in affected cells expressing mutated INS1 using UPR-specific PCR microarrays. This, beyond giving information on the efficiency of the rescue of ER-stress induced by interfering lentivector will also allow to acquire new information about UPR genes activated by insulin misfolding that lead to apoptosis and consequently to diabetes. 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.
Finally the success of this kind of approach in rescuing the phenotypic effect caused by INS1 mutation could become a proof of principle for the therapeutic treatment of other form of dominant INS1/PNDM caused by other mutations.
References of the entire project:
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2 Russo L et al, 2011, Diabetologia
3 Støy J et al, 2007, PNAS
4 Colombo C et al, 2008, J Clin Invest
5 Garin I et al, 2010, PNAS
6 Liu M et al, 2015, Mol Aspects Med
7 Scheuner D, Kaufman RJ, 2008, Endocr Rev
8 Bonfanti R et al, 2009, Diabetes Care
9 Wang J et al, 1999, J Clin Invest
10 Hodish I et al, 2011, Diabetes
11 Araki E et al, 2003, Intern Med
12 Lamkamfi M et al, 2007, Cell Death Differ
13 Oyadomari S et al, 2002, J Clin Invest
14 Nobrega C et al, 2013, PLoS One
15 Jiang J et al, 2013, Science
16 Piersanti S et al, 2010, J Bone Miner Res
17 Lahav R et al,2004, Antonie Van Leeuwenhoek
18 Dragosits M et al, 2010, BMC Genomics
19 Reynolds A et al, 2004, Nature biotechnology
20 Piersanti S et al, 2006, Calcif Tissue Int
21 Yoshida H et al, 2001, Cell