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. Such cellular models would play a key role in the elucidation of the function of the two genes of interest- ABCC8 and HADH. In addition, this cell model would be used to develop and screen for novel therapeutic drugs.
Methods: Several CRISPR sgRNAs were designed to target each gene and tested to identify the best sgRNAs to generate the KO cellular models. 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 Sanger sequencing and T7 Endo assay. As a pilot, optimisation of ELISA using wild type (WT) TC6 cells to demonstrate glucose-stimulated insulin secretion (GSIS) has been undertaken.
Results: Progress so far has addressed the optimisation of transfection conditions to deliver CRISPR/Cas9. Several sgRNAs have been designed for the disruption of the Abcc8 and Hadh genes. The molecular validation of Abcc8 KO model had been demonstrated by heteroduplexes in the T7 Endo assay. In addition, the optimisation of the ELISA insulin assay in wild type TC6 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 gene; create a KO allele of hadh gene in the TC6 cell line 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.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology