Gene and stem cell therapy for type 1 diabetes mellitus

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Type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing pancreatic β-cells. As a result, people with T1D suffer from high blood glucose which requires exogenous insulin therapy to maintain within the normal physiological range. However, exogenous insulin therapy does not mimic the tightly regulated function of the pancreas, and as a result does not prevent the development of diabetic complications. Currently, the only cure is either pancreas or islet transplantation; however these treatments are hampered by a shortage of donors. As a result, the generation of an alternative cell replacement therapy would overcome the aforementioned limitations with current treatments. Gene therapy as a means of generating “artificial” insulin-producing cells (IPCs) is being considered as a potential cure for T1D. Previous research has shown that the viral-mediated transfer of the pancreatic transcription factor NeuroD1 and human furin-cleavable insulin (INS-FUR) genes to hepatocytes resulted in their transdifferentiation into pancreatic-like cells capable of synthesising, storing and secreting insulin in response to fluctuating glucose concentrations. Due to their ease of isolation, ease of genetic modification and immune-modulatory properties, the aim of this study was to evaluate the utility of ex vivo expanded murine bone marrow-derived mesenchymal stem cells (BMSCs) as gene therapy targets for the development of a T1D cell replacement therapy following the lentiviral over-expression of murine NeuroD1 and Pdx1, and INS-FUR. Non-invasive bioluminescence imaging (BLI) technology is an established and sensitive tool for accessing cell replacement therapy efficacy and treatment outcome in living preclinical small animal models. Furthermore, preclinical BLI results often serve as the decision point of a cell replacement therapy’s suitability (efficacy and safety) for clinical trial testing in humans. This study utilised the Firefly luciferase reporter protein Luc2, a Luc2 specific light producing substrate D-luciferin and an IVIS Lumina II imaging system. First, a unique sub-population of BMSCs were isolated from the bone marrow of non-obese diabetic (NOD) mice. These BMSCs displayed potent clonogenicity and tri-lineage differentiation potential, hallmark characteristics of mesenchymal stem cells, when compared to the plastic adherent bone marrow stromal cell starting population. Second, BMSCs were nucleofected to express the yeast fusion cytosine deaminase uracil phosphoribosyltransferase (CDUPRT) and/or Luc2 genes (BMSC-Luc2/CDUPRT; BMSC-Luc2). Luc2 was utilised as a reporter protein for evaluating the in vitro and in vivo persistence of transgene expression in BMSCs and the in vivo persistence of gene-modified BMSCs in immune intact and immune-compromised mice. CDUPRT is a pro-drug converting enzyme, otherwise known as a suicide gene that was utilised as a cell therapy experimental ‘off’ switch. CDUPRT converts the non-toxic pro-drug 5-fluorocytosine (5-FC) into the toxic metabolite 5-fluorouracil (5-FU) that causes BMSC death. In vitro functional characterisation of CDUPRT using a Luc2 based cytotoxicity assay showed that following exposure to 5-FC, BMSC-Luc2/CDUPRT demonstrated increased cell death when compared to BMSC-Luc2 and parental BMSC controls. A subcutaneous transplant of Luc2/CDUPRT-expressing BMSCs in immune-intact (NOD; n=4) and immune-deficient (NOD/Scid; n=4) mice persisted for 2 weeks and 12 weeks respectively. These results show a BMSC transplant survival providing an experimental window of 12 weeks in NOD/Scid mice and the rapid immune-mediated destruction of BMSC carrying non-mammalian genes in NOD mice. Ex vivo culture-expanded BMSCs were subsequently transduced with the HMD lentiviral vector (MOI=10) to express INS-FUR, and murine NeuroD1 and Pdx1 as mediators of β-cell differentiation. Pancreatic transdifferentiation was characterised via reverse transcriptase polymerase chain reaction (RT-PCR), followed by the assessment of insulin storage and secretion. INS-FUR-expressing BMSCs lacked glucose-responsiveness and secreted large amounts of human insulin chronically, whereas NeuroD1 and Pdx1-expressing BMSCs lacked glucose-responsiveness and insulin secretion capabilities. Furthermore, RT-PCR analysis showed that BMSC did not undergo pancreatic transdifferentiation when transduced with pancreatic transcription factors, and did not store insulin within secretory granules as determined by acid/ethanol extraction. A subcutaneous transplant of 1x10⁷ and 5x10⁷ INS-FUR-expressing BMSCs were assessed for their ability to reverse diabetes in STZ-NOD/Scid mice (n=5), which failed to do so upon transplantation. This study showed ex vivo expanded BMSC multipotential differentiation into fat and bone diminishes with increasing passage, and therefore BMSC may be more useful as gene therapy targets prior to expansion. This correlates with other studies where ex vivo expansion of MSCs is associated with a loss of MSC characteristics (phenotype, proliferative capacity, self-renewal, immunomodulation) and negative T1D clinical outcomes.
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