Background: Congenital Hyperinsulinism(CHI) is characterized by the unregulated secretion of insulin in the presence of hypoglycaemia. The mutations in ABCC8 and KCNJ11, which encode the sulfonylurea receptor 1 (SUR1) and potassium inward-rectifying 6.2 (Kir6.2) subunits of ATP-sensitive potassium channel (K channel), are the most common identified cause of the condition. Defects in the HADH gene are responsible for SCHAD- HI, a rare form of the disease caused by the disruption of fatty acid oxidation.
Aims: The aim of this project is to use the novel CRISPR/Cas9 gene editing technique to create a KO mouse cell model of Congenital Hyperinsulinism. The two genes of interest are ABCC8 and HADH. This cell model would be used for molecular and functional interrogation and may further aid in development of novel therapeutic drugs for CHI.
Methods: Three CRISPR sgRNAs were designed to target both genes of interest. Optimisation of the delivery of CRISPR/Cas9 system included the evaluation of different formats such as plasmid DNA, mRNA and RNP complex using a reporter gene. At the molecular level, the disruption of the gene was confirmed by T7 Endo assay. Single cell cloning is being attempted prior to Sanger sequencing. As a pilot, optimisation of ELISA using wild type (WT) βTC6 cells to demonstrate glucose-stimulated insulin secretion (GSIS) has been undertaken. Optimisation of Western Blot analysis looking for reduced protein expression is being undertaken concurrently.
Results: Progress so far has addressed the optimisation of transfection conditions to deliver CRISPR/Cas9. LPR nanocomplexes were used successfully for the first time in the transfection of βTC6 cells.
The molecular validation of Abcc8 and Hadh KO models has been demonstrated by heteroduplexes in the T7 Endo assay. In addition, the optimisation of the ELISA insulin assay in wild type βTC6 cells has demonstrated a dose dependent GSIS which can be used as a standard to compare the GSIS from the KO cell model.
Conclusions: The results of our study so far has demonstrated the potential of the use of Cas9/gRNA system as an efficient reverse genetic tool in studying the molecular mechanisms underlying CHI. Our future aims are to: conduct further molecular interrogation to confirm the KO in Abcc8 and Hadh gene; and further, use the newly generated KO mutant cells to analyse the function of these genes and furthermore, to test and develop novel therapeutic drugs for CHI.
19 - 21 Sep 2019
European Society for Paediatric Endocrinology