Examination of Pancreatic Transdifferentiation in the Livers of Animals

La, Que Tran

Examination of Pancreatic Transdifferentiation in the Livers of Animals

La, Que Tran

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Publication Type:: Thesis
Issue Date:: 2021

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Field	Value	Language
dc.contributor.author	La, Que Tran
dc.date.accessioned	2022-06-03T05:02:51Z
dc.date.available	2022-06-03T05:02:51Z
dc.date.issued	2021
dc.identifier.uri	http://hdl.handle.net/10453/157904
dc.description	University of Technology Sydney. Faculty of Science.	en_US.UTF-8
dc.description.abstract	Type I diabetes mellitus (T1D) is a chronic metabolic disorder resulting from the autoimmune destruction of the pancreatic β-cells, leading to insulin deficiency and hyperglycaemia. T1D is currently managed by insulin therapy, which delays but does not fully prevent long-term complications. The only cure for the disease is pancreas or islet transplantation; however, due to the lack of available organs and the complications from immunosuppression, transplantation is not widely applied. Gene therapy is one treatment being considered to treat or cure T1D. Our group previously reversed diabetes in several animal models by the delivery of a lentiviral vector (HMD) carrying the furin-cleavable human insulin gene (INS-FUR) using intervallic infusion of the vectors in full flow occlusion (FFO), a surgical technique that was thought to improve the transduction efficiency by isolating the liver from the circulation. The main aim of the current study was to explore the possibility of reproducing the results of the HMD/INS-FUR transduction using less invasive delivery techniques. In order to achieve a high level of transduction and to limit the expression of the transgenes to the livers, the adeno-associated-viral vectors serotype 8 (AAV8) that have a high tropism toward the mouse hepatocytes and the liver specific promoter (LSP) were studied. Two different vector systems: the traditional AAV8-LSP vectors that predominantly allowed episomal transgene expression and the hybrid AAV8/piggyBac-LSP system that could facilitate the integration of the transgene into the genome were used to deliver the INS-FUR gene and the β-cell transcription factors Pdx1 and NeuroD1. The AAV8-LSP-Pdx1 or AAV8-LSP-NeuroD1 transduction could not correct hyperglycaemia in streptozotocin (STZ)-induced diabetic NOD/scid mice. The delivery of the AAV8-LSP-INS-FUR led to hypoglycaemia in both the STZ-induced diabetic NOD/scid mice and the autoimmune non-obese-diabetic (NOD) mice. Using the same vector system to deliver various combinations of INS-FUR, Pdx1 and NeuroD1 caused hypoglycaemia in the diabetic NOD/scid mice. The combination of AAV8-LSP-INS-FUR and AAV8-LSP-Pdx1 transduction or the combination of AAV8-LSP-INS-FUR transduction and FFO surgery was ineffective in treating diabetes in NOD mice. The AAV8/piggyBac-INS-FUR transduction could not normalise the random blood glucose levels in the NOD mice, but normal glucose tolerance was achieved. Interestingly, NOD mice that received FFO surgery after the AAV8/piggyBac-LSP-INS-FUR transduction had normal blood glucose levels, normal glucose tolerance and possibly glucose-responsive insulin secretion without the expression of any endogenous β-cell transcription factors. The study suggested that AAV8/piggyBac system may be further developed to become an alternative therapy for T1D.	en_US.UTF-8
dc.format	Thesis (PhD)
dc.language.iso	en_US	en_US.UTF-8
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/157904/2/02whole.pdf
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	au.edu.uts.lib/ppc
dc.rights	info:eu-repo/semantics/openAccess
dc.title	Examination of Pancreatic Transdifferentiation in the Livers of Animals	en_US.UTF-8
dc.type	Thesis
utslib.copyright.status	open_access	*

Abstract:

Type I diabetes mellitus (T1D) is a chronic metabolic disorder resulting from the autoimmune destruction of the pancreatic β-cells, leading to insulin deficiency and hyperglycaemia. T1D is currently managed by insulin therapy, which delays but does not fully prevent long-term complications. The only cure for the disease is pancreas or islet transplantation; however, due to the lack of available organs and the complications from immunosuppression, transplantation is not widely applied. Gene therapy is one treatment being considered to treat or cure T1D. Our group previously reversed diabetes in several animal models by the delivery of a lentiviral vector (HMD) carrying the furin-cleavable human insulin gene (INS-FUR) using intervallic infusion of the vectors in full flow occlusion (FFO), a surgical technique that was thought to improve the transduction efficiency by isolating the liver from the circulation. The main aim of the current study was to explore the possibility of reproducing the results of the HMD/INS-FUR transduction using less invasive delivery techniques. In order to achieve a high level of transduction and to limit the expression of the transgenes to the livers, the adeno-associated-viral vectors serotype 8 (AAV8) that have a high tropism toward the mouse hepatocytes and the liver specific promoter (LSP) were studied. Two different vector systems: the traditional AAV8-LSP vectors that predominantly allowed episomal transgene expression and the hybrid AAV8/piggyBac-LSP system that could facilitate the integration of the transgene into the genome were used to deliver the INS-FUR gene and the β-cell transcription factors Pdx1 and NeuroD1. The AAV8-LSP-Pdx1 or AAV8-LSP-NeuroD1 transduction could not correct hyperglycaemia in streptozotocin (STZ)-induced diabetic NOD/scid mice. The delivery of the AAV8-LSP-INS-FUR led to hypoglycaemia in both the STZ-induced diabetic NOD/scid mice and the autoimmune non-obese-diabetic (NOD) mice. Using the same vector system to deliver various combinations of INS-FUR, Pdx1 and NeuroD1 caused hypoglycaemia in the diabetic NOD/scid mice. The combination of AAV8-LSP-INS-FUR and AAV8-LSP-Pdx1 transduction or the combination of AAV8-LSP-INS-FUR transduction and FFO surgery was ineffective in treating diabetes in NOD mice. The AAV8/piggyBac-INS-FUR transduction could not normalise the random blood glucose levels in the NOD mice, but normal glucose tolerance was achieved. Interestingly, NOD mice that received FFO surgery after the AAV8/piggyBac-LSP-INS-FUR transduction had normal blood glucose levels, normal glucose tolerance and possibly glucose-responsive insulin secretion without the expression of any endogenous β-cell transcription factors. The study suggested that AAV8/piggyBac system may be further developed to become an alternative therapy for T1D.

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http://hdl.handle.net/10453/157904