Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signaling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIPR or GLP-1R were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic β-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts. cAMP production in response to GIPR or GLP-1R activation was abolished and GIP or GLP-1 induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate knockout cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9 engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant β-cell system. This demonstrates the broad utility of CRISPR/Cas9 applied to natively expressing systems in developing drugs targeting multiple receptors where the balance of receptor activity is critical.
- glucagon-like peptide-1
- glucose-dependent insulinotropic polypeptide
- pancreatic beta cell
- ©2016 The Author(s)
This is an Accepted Manuscript; not the final Version of Record. Archiving permitted only in line with the archiving policy of Portland Press Limited. All other rights reserved.