Incretin-based muscle-targeted gene therapy for type 2 diabetes

Abstract

According to WHO (August 2011), 346 million people worldwide have diabetes and, of those, 90% have type 2 diabetes (T2DM). This equates to approximately $465 billion (USD) in healthcare costs. Current treatments, which include various different drug types, both oral and injectable, are somewhat effective (i.e. do not prevent associated complications) but also still heavily rely on patient and/or care-giver regimen adherence for the therapies to be effective. One of the most recent classes of drugs is known as Glucagon-like peptide 1 receptor (GLP-1-R) agonists. Current treatment interval extends to only once weekly injections and, whilst this is far superior to daily or even twice daily injections, any possible extension would potentially provide an increased safety profile (by removing nonphysiological peaks in hormone pharmacokinetics) and would generally be better received by the patient. Gene therapy is one area of investigation for T2DM, which aims to provide longer-term therapeutic peptide delivery and therefore reduce the number injections required for satisfactory glucose homeostasis. GLP-1 is a hormone within the group known as incretins. This group comprises hormones which enhance glucose-dependent insulin synthesis and secretion (from pancreatic β-cells) in response to glucose (and other nutrients) and subsequently suppresses post-prandial hyperglycaemia. One of the major pitfalls of GLP-1 is its incredibly short half-life (2-3 minutes) due to degradation by a ubiquitous dipeptidyl peptidase (DPP4). Current therapeutics are small compounds activating incretin functions, for example the GLP-1 analogue mimetics exenatide or liraglutide, or are DPP4 inhibitors, for example stigaliptin. However, due to the progressive nature of the disease and pathophysiological differences between individuals, these treatments may not be suitable for everyone and may not provide a long-term sustainable solution. This thesis aimed to compare the efficacy and efficiency of utilising both plasmid and viral based (AAV) vectors for transgene delivery via skeletal muscle. Skeletal muscle is used mainly for its ease of access and its highly vascularised system, which is extremely capable of uptake and expression of foreign DNA. iv The transgenes chosen for this study encoded unaltered human GLP-1 and the longeracting GLP-1-R agonist, Exendin-4. Exendin-4 shares 53% homology with GLP-1 and exerts insulinotropic activity while lacking the amino acid cleavage site via which DPP4 degrades GLP-1. Enhanced green fluorescent protein (eGFP) was also used, as a control vector and an easy to visualise reporter that would aid in the determination of expression both during the study (for use in live animals) and in post-mortem analysis. In vitro studies for proof of concept were performed in the C2C12 murine muscle cell line, and in a minor part, HEK293 fibroblasts, before being progressed into both normal (CD1) and diabetic (db/db) mouse models. Transgene expression was attained in C2C12 cells, as determined via immunofluorescence staining and ELISA/EIA analysis of both the cells and the cell media. Standard transfection/infection protocols were utilised. The longest study concluded at 5 days post transfection/infection. In vivo studies also attained expression of the three transgenes, eGFP, GLP-1 and Exendin-4 via direct hind limb skeletal muscle injection. In situ plasmid uptake was augmented by pre-injection of the muscle with hyaluronidase and external calliper electroporation. Circulating peptide levels were confirmed via blood serum analysis using ELISA/EIA. Muscle peptide concentration was determined from muscle homogenates and by immunofluorescence staining together with circulating hormone concentration by ELISA/EIA. Pancreas samples were evaluated for β-cell mass. IPGTT data were obtained throughout the study. In addition, each mouse was subject to noninvasive imaging (IVIS), which allowed visualisation of the eGFP reporter gene. Gene expression was followed to a suitable end point, which was determined on a study-bystudy basis. Results showed the delivery system to be effective yielding promising circulating peptide concentrations in all animal models. The data obtained from IPGTT analyses in the diseased model showed the improved capability of glucose clearance when transfected with Exendin-4 transgenes. Expectedly, no differences were noted in the healthy model. The plasmid vectors appeared to give a greater efficacy profile than the AAV vectors, based on both eGFP expression analysed via IVIS and circulating peptide concentrations. In light of this, only the plasmid vector studies were performed in the diseased animal model. v In conclusion, a skeletal muscle based gene delivery system for GLP-1 and longer acting GLP-1-R agonists has proven to be tolerated by both normal and diseased animal models, Furthermore, the administration of the Exendin-4 to diseased animals suggests positive glucose lowering effects. The current work suggests that further studies are worthwhile, including those conducted in a range of animals with overt and perhaps spontaneous diabetes mellitus.