Abstract
Introduction: Type 1 diabetes is an autoimmune disorder characterized by the destruction of pancreatic islet beta cells responsible for insulin production, leading to insulin deficiency. The PDX1 and NKX6-1 genes are essential to beta cell development and maturation in the pancreas. Based on previous studies, Pdx1 is downregulated in diabetics, and higher Nkx6.1 expression has been shown to induce high cell turnover. This proposal aims to investigate the potential therapeutic effects of gene therapy on streptozotocin-induced non-obese diabetic/severe-combined immunodeficient mice in treating Type 1 Diabetes. It is hypothesized that the use of gene therapy to overexpress PDX1 and NKX6-1 into the pancreas of experimental diabetic mice will result in increased beta cell production and improved glucose metabolism. Methods: Both male and female streptozotocin-induced non-obese diabetic/severe-combined immunodeficient experimental mice will receive human islets from individuals 32-55 years old. The PDX1 and NKX6-1 genes will be overexpressed in a shuttle vector and incorporated into the gutless adenovirus vector backbone through co-transfection using the Cre293/lox P cell line. Verification of successful vectors containing the gutless adenovirus coupled with a helper plasmid will be done using gel electrophoresis. Incubation with beta cells will stimulate the transduction process after which beta cells will be delivered into mice pancreas’ through endoscopic retrograde cholangiopancreatography. An intraperitoneal glucose tolerance test, pancreatic biopsy with immunohistochemical staining, and islet calculations will be done. Results: An intraperitoneal glucose tolerance test is expected to show improved blood glucose levels in experimental mice post-treatment, while a pancreatic biopsy with immunohistochemical staining and subsequent islet diameter and volume calculations are expected to depict an increase in the number of beta cells. Discussion: The experimental mice are anticipated to show an improvement in blood glucose levels and increased production of beta cells following treatment due to the overexpression of PDX1 and NKX6-1. The normal control mice and diabetic untreated mice are expected to show normal and high glucose levels, as well as normal and deficient beta cells respectively. Conclusion: This proposal may provide a better understanding of the pathophysiology in Type 1 Diabetes and pave a new path for its treatment.
Highlights
Type 1 diabetes is an autoimmune disorder characterized by the destruction of pancreatic islet beta cells responsible for insulin production, leading to insulin deficiency
It is hypothesized that the use of gene therapy to overexpress PDX1 and NKX6-1 into the pancreas of experimental diabetic mice will result in increased beta cell production and improved glucose metabolism
Independent human islet batches will be harvested for the genes of interest, PDX1 and NKX6-1 which will be isolated, overexpressed in the gutless adenovirus vector coupled to the helper plasmid and introduced into mice
Summary
Type 1 diabetes is an autoimmune disorder characterized by the destruction of pancreatic islet beta cells responsible for insulin production, leading to insulin deficiency. It is hypothesized that the use of gene therapy to overexpress PDX1 and NKX6-1 into the pancreas of experimental diabetic mice will result in increased beta cell production and improved glucose metabolism. Results: An intraperitoneal glucose tolerance test is expected to show improved blood glucose levels in experimental mice post-treatment, while a pancreatic biopsy with immunohistochemical staining and subsequent islet diameter and volume calculations are expected to depict an increase in the number of beta cells. In 2018, a study done by Xiao et al found that the use of an adeno-associated virus (AAV) to deliver and express MAFA and PDX1 genes encoding MAF BZIP Transcription Factor A (MafA) and Pancreas/Duodenum Homeobox Protein 1 (Pdx1) respectively into mice results in the conversion of alpha cells (α-cells) to β-cells in vivo [2]. Both genes code for transcription factors, MafA binds to promoter/ enhancer regions of the insulin
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