title: Use of CRISPR/Cas9-engineered INS-1 pancreatic β cells to define the pharmacology of dual GIPR/GLP-1R agonists.
abstract: 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 signalling 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 GIP receptors (GIPR) or GLP-1 receptors (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 (KOs). 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 KO 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 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical.
authors.affiliation: Cardiovascular and Metabolic Disease, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Naylor
authors.affiliation: MedImmune LLC, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Arthur T
authors.initials: AT
authors.lastname: Suckow
authors.affiliation: Cardiovascular and Metabolic Disease, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: Asha
authors.initials: A
authors.lastname: Seth
authors.affiliation: Cardiovascular and Metabolic Disease, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: David J
authors.initials: DJ
authors.lastname: Baker
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Sermadiras
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: Peter
authors.initials: P
authors.lastname: Ravn
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: Rob
authors.initials: R
authors.lastname: Howes
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: Jianliang
authors.initials: J
authors.lastname: Li
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: Mike R
authors.initials: MR
authors.lastname: Snaith
authors.affiliation: Cardiovascular and Metabolic Disease, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Coghlan
authors.affiliation: Cardiovascular and Metabolic Disease, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
authors.forename: David C
authors.initials: DC
authors.lastname: Hornigold
mesh_terms: D000818:Animals; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D052216:Glucagon-Like Peptide 1 / Q000819:agonists*; D005947:Glucose / Q000494:pharmacology; D005951:Glucose Tolerance Test; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology*; D007621:Karyotyping; D051379:Mice; D018345:Mice, Knockout; D011964:Receptors, Gastrointestinal Hormone / Q000819:agonists* / Q000235:genetics
chemical_list: D007328:Insulin; D011964:Receptors, Gastrointestinal Hormone; D052216:Glucagon-Like Peptide 1; C041086:gastric inhibitory polypeptide receptor; D005947:Glucose
keywords: CRISPR; glucagon-like peptide-1; glucose-dependent insulinotropic polypeptide; pancreatic β cell