Gene therapy for diabetes : lentiviral expression of insulin in liver cells

University of Technology, Sydney. Faculty of Science.Type 1 diabetes mellitus (T1D) is caused by the autoimmune destruction of the insulin producing pancreatic beta (β)-cells. Current treatment is by multiple daily insulin injections, which cannot mimic the minute-to-minute responsiveness of β-cells in vivo, leading to the development of chronic complications, which increase morbidity and mortality. This study investigated the use of lentiviral vectors to deliver the furin-cleavable proinsulin gene (INS-FUR) to liver cells with the goal of generating glucose-responsive, insulin secreting surrogate β-cells. A methodology for viral titre determination using flow cytometry was developed, optimised and validated. This protocol was compared to the use of fluorescence microscopy for titre determination. The latter underestimated the viral titre as accurate quantification of transduced cells (enhanced green fluorescent protein (EGFP)-positive) was not possible due to the inability to count individual cells, which were in confluent monolayer clusters. Furthermore, cells transduced using a low multiplicity of infection (MOI; a ratio of viral infectious particles per cell) were indistinguishable from background fluorescence, while high MOI significantly underestimated the true functional viral titre. Flow cyton1etry enabled the determination of single or low MOI events and thus increased the accuracy of the viral titre determination. The second aim of the study was to examine liver tissue from spontaneous diabetic non-obese diabetic (NOD) mice, which had normalised their blood glucose following the delivery of furin-cleavable insulin in the HIV/murine stem cell virus promoter/enhancer hybrid (HMD) lentiviral vector (HMD/INS-FUR), for evidence of pancreatic transcription factors and hormones, indicative of liver to pancreas transdifferentiation. The HMD/INS-FUR-treated NOD mice normalised blood glucose levels 24 h after viral vector delivery and normoglycaemia was maintained for 150 days (experimental end point). The transduced liver tissue showed the presence of insulin storage granules and the expression of several pancreatic transcription factors and hormones, including Pdx-1, Neurad1, Ngn3, Pax 4, Nkx2.2, glucagon and somatostatin. Furthermore, the induction of mouse insulin 1 expression was detected and this phenomenon may be attributable to the expression of MafA and MafB transcription factors, which are known to play crucial roles in insulin expression late in the hierarchy of β-cell maturation. These results indicated that the delivery HMD/INS-FUR lentiviral vector to liver tissue induces hepatocyte to β-cell transdifferentiation and therefore holds therapeutic potential for the reversal of autoimmune T1D. The final aim of this study investigated lentiviral vector construct design, and compared the HMD/INS-FUR vector construct with the newly acquired WPT/INS-FUR lentiviral construct, which possessed the elongation factor 1-alpha promoter with the woodchuck hepatitis virus post-transcriptional regulatory element enhancer (WPT). The ability of each lentiviral vector to transduce the Huh7 liver cell line using various MOI, insulin secretion, storage and glucose-regulated secretion were compared. Huh7 cells were readilty transduced with the HMD/INS-FUR vector at an MOI of 75, however attempts at transducing the cells with the WPT/INS-FUR vector at an MOI >50, resulted in inhibition of cell growth. This was not the case with the empty-WPT vector, which implies the INS-FUR gene expressed in this vector putatively inhibited metabolic cell function at the higher MOI of 75. Insulin secretion was the same irrespective of the MOI used to transduce the Huh7 cells. By comparison, the WPT/INS-FUR viral vector-transduced cells stored a significantly higher concentration of insulin when compared to cells transduced with the 50 and 75 MOI of the HMD/INS-FUR viral vector (p < 0.0001 and p = 0.001, respectively). Therefore, fewer viral particles of the WPT/INS-FUR virus were required to achieve similar insulin secretion concentrations as the HMD/INS-FUR virus, while also achieving a higher concentration of insulin storage. However, as the total level of insulin stored after transfection with the WPT/INS-FUR viral vector was low (0.386 ± 0.041 pmoles/10⁶ cells), these results must be viewed with caution. Overall, this study has optimised an appropriate methodology for the accurate determination of viral titre for downstream applications. Furthermore, the study demonstrated the NOD mice transduced with the HMD/INS-FUR viral vector have the ability to store and secrete insulin in a glucose-regulated manner, through the induction of pancreatic β-cell transcription factors and hormones. Moreover, the WPT/INS-FUR viral vector was found to be comparable to HMD/INS-FUR viral vector with respect to insulin secretion. However, as the former stored significantly more insulin in vitro, this suggests that the WPT /INS-FUR viral vector may be a useful construct for future in vivo studies investigating the reversal of T1D in NOD mice via hepatocyte transduction

Long-term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy

Background: Type 1 diabetes (T1D) results from an autoimmune attack against the insulin-producing β-cells of the pancreas. The present study aimed to reverse T1D by gene therapy. Methods: We used a novel surgical technique, which involves isolating the liver from the circulation before the delivery of a lentiviral vector carrying furin-cleavable human insulin (INS-FUR) or empty vector to the livers of diabetic non-obese diabetic mice (NOD). This was compared with the direct injection of the vector into the portal circulation. Mice were monitored for body weight and blood glucose. Intravenous glucose tolerance tests were performed. Expression of insulin and pancreatic transcription factors was determined by the reverse transcriptase-polymerase chain reaction and immunohistochemistry and immunoelectron microscopy was used to localise insulin. Results: Using the novel surgical technique, we achieved long-term transduction (42% efficiency) of hepatocytes, restored normoglycaemia for 150 days (experimental endpoint) and re-established normal glucose tolerance. We showed the expression of β-cell transcription factors, murine insulin, glucagon and somatostatin, and hepatic storage of insulin in granules. The expression of hepatic markers, C/EBP-β, G6PC, AAT and GLUI was down-regulated in INS-FUR-treated livers. Liver function tests remained normal, with no evidence of intrahepatic inflammation or autoimmune destruction of the insulin-secreting liver tissue. By comparison, direct injection of INS-FUR reduced blood glucose levels, and no pancreatic transdifferentiation or normal glucose tolerance was observed. Conclusions: This gene therapy protocol has, for the first time, permanently reversed T1D with normal glucose tolerance in NOD mice and, as such, represents a novel therapeutic strategy for the treatment of T1D. © 2013 John Wiley & Sons, Ltd

Data characterizing the ZMIZ1 molecular phenotype of multiple sclerosis.

The data presented in this article are related to the research article entitled "The autoimmune risk gene ZMIZ1 is a vitamin D responsived marker of a molecular phenotype of multiple sclerosis" Fewings et al. (2017) [1]. Here we identify the set of genes correlated with ZMIZ1 in multiple cohorts, provide phenotypic details on those cohorts, and identify the genes negatively correlated with ZMIZ1 and the cells predominantly expressing those genes. We identify the metabolic pathways in which the molecular phenotype genes are over-represented. Finally, we present the flow cytometry gating strategy we have used to identify the immune cells from blood which are producing ZMIZ1 and RPS6