Publications by authors named "Binhai Ren"

8 Publications

  • Page 1 of 1

Use of a Hybrid Adeno-Associated Viral Vector Transposon System to Deliver the Insulin Gene to Diabetic NOD Mice.

Cells 2020 10 2;9(10). Epub 2020 Oct 2.

School of Life Sciences, University of Technology Sydney, 15 Broadway, Ultimo, NSW 2007, Australia.

Previously, we used a lentiviral vector to deliver furin-cleavable human insulin () to the livers in several animal models of diabetes using intervallic infusion in full flow occlusion (FFO), with resultant reversal of diabetes, restoration of glucose tolerance and pancreatic transdifferentiation (PT), due to the expression of beta (β)-cell transcription factors (β-TFs). The present study aimed to determine whether we could similarly reverse diabetes in the non-obese diabetic (NOD) mouse using an adeno-associated viral vector (AAV) to deliver - ± the β-TF to the livers of diabetic mice. The traditional AAV8, which provides episomal expression, and the hybrid AAV8/ that results in transgene integration were used. Diabetic mice that received AAV8- became hypoglycaemic with abnormal intraperitoneal glucose tolerance tests (IPGTTs). Expression of β-TFs was not detected in the livers. Reversal of diabetes was not achieved in mice that received AAV8--FUR and AAV8- and IPGTTs were abnormal. Normoglycaemia and glucose tolerance were achieved in mice that received AAV8/-/FFO. Definitive evidence of PT was not observed. This is the first in vivo study using the hybrid AAV8/ system to treat Type 1 diabetes (T1D). However, further development is required before the system can be used for gene therapy of T1D.
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http://dx.doi.org/10.3390/cells9102227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600325PMC
October 2020

High-Efficiency Lentiviral Gene Modification of Primary Murine Bone-Marrow Mesenchymal Stem Cells.

Methods Mol Biol 2019 ;2029:197-214

The School of Life Sciences and Centre for Health Technologies, University of Technology Sydney, Sydney, NSW, Australia.

Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However, the efficiency with which they transduce target cells varies significantly, in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning, production, and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gene modification of MSCs into insulin-producing cells via overexpression of the furin-cleavable human insulin (INS-FUR) gene.
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http://dx.doi.org/10.1007/978-1-4939-9631-5_16DOI Listing
April 2020

Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic -Cells.

Stem Cells Int 2019 10;2019:1395301. Epub 2019 Mar 10.

The School of Life Sciences and Centre for Health Technologies, Faculty of Science, University of Technology Sydney, Sydney, Australia.

Combinatorial gene and cell therapy as a means of generating surrogate -cells has been investigated for the treatment of type 1 diabetes (T1D) for a number of years with varying success. One of the limitations of current cell therapies for T1D is the inability to generate sufficient quantities of functional transplantable insulin-producing cells. Due to their impressive immunomodulatory properties, in addition to their ease of expansion and genetic modification , mesenchymal stem cells (MSCs) are an attractive alternative source of adult stem cells for regenerative medicine. To overcome the aforementioned limitation of current therapies, we assessed the utility of expanded bone marrow-derived murine MSCs for their persistence in immune-competent and immune-deficient animal models and their ability to differentiate into surrogate -cells. CD45/Ly6 murine MSCs were isolated from the bone marrow of nonobese diabetic (NOD) mice and nucleofected to express the bioluminescent protein, . The persistence of a subcutaneous (s.c.) transplant of -expressing MSCs was assessed in immune-competent (NOD) ( = 4) and immune-deficient (NOD/) ( = 4) animal models of diabetes. -expressing MSCs persisted for 2 and 12 weeks, respectively, in NOD and NOD/ mice. expanded MSCs were transduced with the HMD lentiviral vector (MOI = 10) to express furin-cleavable human insulin () and murine and . This was followed by the characterization of pancreatic transdifferentiation via reverse transcriptase polymerase chain reaction (RT-PCR) and static and glucose-stimulated insulin secretion (GSIS). -expressing MSCs were assessed for their ability to reverse diabetes after transplantation into streptozotocin- (STZ-) diabetic NOD/ mice ( = 5). Transduced MSCs did not undergo pancreatic transdifferentiation, as determined by RT-PCR analyses, lacked glucose responsiveness, and upon transplantation did not reverse diabetes. The data suggest that expanded MSCs lose their multipotent differentiation potential and may be more useful as gene therapy targets prior to expansion.
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http://dx.doi.org/10.1155/2019/1395301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431458PMC
March 2019

Partial pancreatic transdifferentiation of primary human hepatocytes in the livers of a humanised mouse model.

J Gene Med 2018 05 16;20(5):e3017. Epub 2018 Apr 16.

School of Life Sciences, University of Technology Sydney, Sydney, Australia.

Background: Gene therapy is one treatment that may ultimately cure type 1 diabetes. We have previously shown that the introduction of furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes resulted in the reversal of diabetes and partial pancreatic transdifferentiation of liver cells. The present study investigated whether streptozotocin-diabetes could be reversed in FRG mice in which chimeric mouse-human livers can readily be established and, in addition, whether pancreatic transdifferentiation occurred in the engrafted human hepatocytes.

Methods: Engraftment of human hepatocytes was confirmed by measuring human albumin levels. Following delivery of the empty vector or the INS-FUR vector to diabetic FRG mice, mice were monitored for weight and blood glucose levels. Intraperitoneal glucose tolerance tests (IPGTTs) were performed. Expression levels of pancreatic hormones and transcription factors were determined by a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry.

Results: Diabetes was reversed for a period of 60 days (experimental endpoint) after transduction with INS-FUR. IPGTTs of the insulin-transduced animals were not significantly different from nondiabetic animals. Immunofluorescence microscopy revealed the expression of human albumin and insulin in transduced liver samples. Quantitative RT-PCR showed expression of human and mouse endocrine hormones and β-cell transcription factors, indicating partial pancreatic transdifferentiation of mouse and human hepatocytes. Nonfasting human C-peptide levels were significantly higher than mouse levels, suggesting that transdifferentiated human hepatocytes made a significant contribution to the reversal of diabetes.

Conclusions: These data show that human hepatocytes can be induced to undergo partial pancreatic transdifferentiation in vivo, indicating that the technology holds promise for the treatment of type 1 diabetes.
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http://dx.doi.org/10.1002/jgm.3017DOI Listing
May 2018

Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line.

Int J Mol Sci 2016 Apr 8;17(4):534. Epub 2016 Apr 8.

School of Life Sciences and Centre for Health Technologies, University of Technology Sydney, P.O. Box 123, Broadway, 2007 Sydney, NSW, Australia.

Due to the limitations of current treatment regimes, gene therapy is a promising strategy being explored to correct blood glucose concentrations in diabetic patients. In the current study, we used a retroviral vector to deliver either the human insulin gene alone, the rat NeuroD1 gene alone, or the human insulin gene and rat NeuroD1 genes together, to the rat liver cell line, H4IIE, to determine if storage of insulin and pancreatic transdifferentiation occurred. Stable clones were selected and expanded into cell lines: H4IIEins (insulin gene alone), H4IIE/ND (NeuroD1 gene alone), and H4IIEins/ND (insulin and NeuroD1 genes). The H4IIEins cells did not store insulin; however, H4IIE/ND and H4IIEins/ND cells stored 65.5 ± 5.6 and 1475.4 ± 171.8 pmol/insulin/5 × 10⁶ cells, respectively. Additionally, several β cell transcription factors and pancreatic hormones were expressed in both H4IIE/ND and H4IIEins/ND cells. Electron microscopy revealed insulin storage vesicles in the H4IIE/ND and H4IIEins/ND cell lines. Regulated secretion of insulin to glucose (0-20 mmol/L) was seen in the H4IIEins/ND cell line. The H4IIEins/ND cells were transplanted into diabetic immunoincompetent mice, resulting in normalization of blood glucose. This data shows that the expression of NeuroD1 and insulin in liver cells may be a useful strategy for inducing islet neogenesis and reversing diabetes.
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http://dx.doi.org/10.3390/ijms17040534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848990PMC
April 2016

Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells.

Mol Ther Methods Clin Dev 2015 8;2:15011. Epub 2015 Apr 8.

School of Medical and Molecular Biosciences and Centre for Health Technologies, University of Technology Sydney , Sydney, Australia.

As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of β-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known β-cell cytotoxins was associated with the expression of NF-κB and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial β-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D.
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http://dx.doi.org/10.1038/mtm.2015.11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445011PMC
June 2015

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

J Gene Med 2013 Jan;15(1):28-41

School of Medical & Molecular Biosciences, University of Technology Sydney, Sydney, Australia.

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.
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http://dx.doi.org/10.1002/jgm.2692DOI Listing
January 2013
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