Engineering Marker-Free Lettuce Chloroplast Genome to Express Functional Glucagon-Like Peptide-1 Receptor Agonists Exenatide and Lixisenatide.

Diabetes Mellitus is an epidemic affecting > 500 million, claiming 6-7 million lives annually. Chemically synthesised Glucagon-like peptide-1 receptor agonists (GLP-1RAs) containing artificial amino acids reduce haemoglobin A1c and obesity but are not yet affordable and require invasive injections. High dosage requirement and gastrointestinal complications are among the current limitations of oral GLP-1RAs. Therefore, we expressed codon optimised Exenatide and Lixisenatide fused with Cholera-toxin B-subunit (CTB) in lettuce chloroplasts to facilitate their oral delivery, increase affordability, and patient compliance. Site-specific integration of transgene expression cassettes into the chloroplast genome and removal of the selectable marker gene from marker-free lettuce transplastomic lines were confirmed using three sets of PCR primers. Homoplasmy in transplastomic lines was confirmed in Southern blots by the absence of untransformed genomes. CTB-Exenatide and CTB-Lixisenatide expression levels were 1.94 and 3.64 mg/g plant powder in T0 generation and increased ~31 and ~48%, respectively in marker-removed T1 lines. Maternal inheritance of transgenes was confirmed by lack of segregation when seedlings were germinated in the selection medium before removal of the antibiotic resistance gene (aadA). Monosialotetrahexosylganglioside (GM1) ELISA confirmed pentameric assembly efficiency of both CTB-fusion proteins similar to commercial CTB standards. GLP-1 receptor binding confirmed functionality of CTB-Exenatide/CTB-Lixisenatide with statistical significance (***p < 0.001 by t-test) and post-translational amidation in chloroplasts. Expression of functional CTB-Exenatide and CTB-Lixisenatide in an edible marker-free system for the first time and much lower dosage requirement for functionality than recently developed synthetic GLP-1RAs paves the way for clinical studies to advance oral delivery of these affordable biologics.