Gerace, D, Martiniello-Wilks, R, Habib, R & Simpson, AM 2019, 'Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells.', PloS one, vol. 14, no. 7.View/Download from: UTS OPUS or Publisher's site
Due to their ease of isolation, gene modification and tumor-homing properties, mesenchymal stem cells (MSCs) are an attractive cellular vehicle for the delivery of toxic suicide genes to a variety of cancers in pre-clinical models. In addition, the incorporation of suicide genes in stem cell-derived cell replacement therapies improves their safety profile by permitting graft destruction in the event of unexpected tumorigeneses or unwanted differentiation. Due to the functional requirement of ATP for the Firefly luciferase gene Luc2 to produce light, luciferase-based reporting of cytotoxicity can be engineered into potential cell therapies. Consequently, we nucleofected mammalian expression plasmids containing both the Luc2 and the yeast fusion cytosine deaminase uracil phosphoribosyltransferase (CDUPRT) genes for expression in murine MSCs to assess luciferase as a reporter of suicide gene cytotoxicity, and MSC as vehicles of suicide gene therapy. In vitro bioluminescence imaging (BLI) showed that following the addition of the non-toxic prodrug fluorocytosine (5-FC), CDUPRT-expressing MSCs displayed enhanced cytotoxicity in comparison to Luc2 reporter MSC controls. This study demonstrates the utility of luciferase as a reporter of CDUPRT-mediated cytotoxicity in murine MSC using BLI.
Gerace, D, Martiniello-Wilks, R, Habib, R, Ren, B, Nassif, NT, O'Brien, BA & Simpson, AM 2019, 'Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells.', Stem Cells International, vol. 2019.View/Download from: UTS OPUS or Publisher's site
Combinatorial gene and cell therapy as a means of generating surrogate β-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 ex vivo, 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 ex vivo expanded bone marrow-derived murine MSCs for their persistence in immune-competent and immune-deficient animal models and their ability to differentiate into surrogate β-cells. CD45-/Ly6+ murine MSCs were isolated from the bone marrow of nonobese diabetic (NOD) mice and nucleofected to express the bioluminescent protein, Firefly luciferase (Luc2). The persistence of a subcutaneous (s.c.) transplant of Luc2-expressing MSCs was assessed in immune-competent (NOD) (n = 4) and immune-deficient (NOD/Scid) (n = 4) animal models of diabetes. Luc2-expressing MSCs persisted for 2 and 12 weeks, respectively, in NOD and NOD/Scid mice. Ex vivo expanded MSCs were transduced with the HMD lentiviral vector (MOI = 10) to express furin-cleavable human insulin (INS-FUR) and murine NeuroD1 and Pdx1. 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). INS-FUR-expressing MSCs were assessed for their ability to reverse diabetes after transplantation into streptozotocin- (STZ-) diabetic NOD/Scid mice (n = 5). Transduced MSCs did not undergo pancreatic transdifferentiation, as determined by RT-PCR analyses, lacked glucose responsiveness, and upon transplantation did no...
Ren, B, La, QT, O'Brien, BA, Nassif, NT, Tan, Y, Gerace, D, Martiniello-Wilks, R, Torpy, F, Dane, AP, Alexander, IE & Simpson, AM 2018, 'Partial pancreatic transdifferentiation of primary human hepatocytes in the livers of a humanised mouse model.', The journal of gene medicine, vol. 20, no. 5.View/Download from: UTS OPUS or Publisher's site
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.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.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.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.
Gerace, D, Martiniello-Wilks, R, Nassif, NT, Lal, S, Steptoe, R & Simpson, AM 2017, 'CRISPR-targeted genome editing of mesenchymal stem cell-derived therapies for type 1 diabetes: a path to clinical success?', STEM CELL RESEARCH & THERAPY, vol. 8.View/Download from: UTS OPUS or Publisher's site
Gerace, D, Martiniello-Wilks, R & Simpson, AM 2015, 'Diabetes reversal via gene transfer: building on successes in animal models', Research and Reports in Endocrine Disorders, vol. 5, pp. 15-29.View/Download from: UTS OPUS or Publisher's site
Type 1 diabetes (T1D) is caused by the autoimmune destruction of the insulin-producing pancreatic β-cells. People with T1D manage their hyperglycemia using daily insulin injections; however, this does not prevent the development of long-term diabetic complications such as retinopathy, nephropathy, neuropathy, and various macrovascular disorders. Currently, the only "cure" for T1D is pancreas transplantation or islet-cell transplantation; however, this is hampered by the limited number of donors and the requirement for life-long immunosuppression. As a result, the need for alternative therapies is vital. One of the strategies employed to correct T1D is the use of gene transfer to generate the production of an “artificial” β-cell that is capable of secreting insulin in response to fluctuating glucose concentrations that normally occurs in people without T1D. The treatment of many diseases using cell and gene therapy is generating significant attention in the T1D research community; however, for a cell therapy to enter clinical trials, success and safety must first be shown in an appropriate animal model. Animal models have been used in diabetes research for over a century, have improved our understanding of the pathophysiology of diabetes, and have led to the discovery of useful drugs for the treatment of the disease. Currently, the nonobese diabetic mouse is the animal model of choice for the study of T1D as it most closely reflects disease development in humans. The aim of this review is to evaluate the success of cell and gene therapy to reverse T1D in animal models for future clinical application.
Gerace, D, Martiniello-Wilks, R, O'Brien, BA & Simpson, AM 2015, 'The use of beta-cell transcription factors in engineering artificial beta cells from non-pancreatic tissue', GENE THERAPY, vol. 22, no. 1, pp. 1-8.View/Download from: UTS OPUS or Publisher's site
Gerace, D, Ren, B, Hawthorne, W, Byrne, M, Phillips, P, O'Brien, B, Nassif, N, Alexander, I & Simpson, AM 2013, 'Pancreatic transdifferentiation in porcine liver following lentiviral delivery of human furin-cleavable insulin', Transplantation Proceedings, vol. 45, no. 5, pp. 1869-1874.View/Download from: UTS OPUS or Publisher's site
Type I diabetes mellitus (TID) results from the autoimmune destruction of the insulin-producing pancreatic ß-cells. Gene therapy is one strategy being actively explored to cure TID by affording non-ß-cells the ability to secrete insulin in response to physiologic stimuli. In previous studies, we used a novel surgical technique to express furin-cleavable human insulin (INS-FUR) in the livers of streptozotocin (STZ)-diabetic Wistar rats and nonobese diabetic (NOD) mice with the use of the HMD lentiviral vector. Normoglycemia was observed for 500 and 150 days, respectively (experimental end points). Additionally, some endocrine transdifferentiation of the liver, with storage of insulin in granules, and expression of some ß-cell transcription factors (eg, Pdx1, Neurod1, Neurog3, Nkx2-2, Pax4) and pancreatic hormones in both studies. The aim of this study was to determine if this novel approach could induce liver to pancreatic transdifferentiation to reverse diabetes in pancreatectomized Westran pigs. Nine pigs were used in the study, however only one pig maintained normal fasting blood glucose levels for the period from 10 to 44 days (experimental end point). This animal was given 2.8 × 10(9) transducing units/kg of the lentiviral vector expressing INS-FUR. A normal intravenous glucose tolerance test was achieved at 30 days. Reverse-transcription polymerase chain reaction analysis of the liver tissue revealed expression of several ß-cell transcription factors, including the key factors, Pdx-1 and Neurod1, pancreatic hormones, glucagon, and somatostatin; however, endogenous pig insulin was not expressed.
Gerace, D, Ren, B, Hawthorne, WJ, Byrne, MR, Phillips, P, O'Brien, BA, Nassif, N, Alexander, IE & Simpson, AM 2012, 'Pancreatic Transdifferentiation in Porcine Liver Following Lentiviral Delivery of Human Furin-Cleavable Insulin', TRANSPLANTATION, vol. 94, no. 10, pp. 172-172.View/Download from: Publisher's site
Gerace, D, Ren, B, Martiniello-Wilks, R & Simpson, AM 2019, 'High-efficiency lentiviral gene modification of primary murine bone-marrow mesenchymal stem cells' in Methods in Molecular Biology, pp. 197-214.View/Download from: Publisher's site
© Springer Science+Business Media, LLC, part of Springer Nature 2019. 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.
Gerace, D, Martiniello-Wilks, R & Simpson, AM 2016, 'Viral-Mediated Gene Therapy for the Generation of Artificial Insulin-Producing Cells as a Therapeutic Treatment for Type 1 Diabetes Mellitus' in Hardikar, AA (ed), Pancreatic Islet Biology, Springer, Germany, pp. 241-257.View/Download from: UTS OPUS or Publisher's site
Over the past decade, several approaches have been employed to develop cell and gene therapy strategies that generate artificial insulin-producing cells (IPCs) for potential therapeutic applications in the treatment of type 1 diabetes mellitus (T1D) . The genetic engineering of functional IPCs necessitates a broad understanding of the pancreatic developmental process and the β cell transcription factors that govern mature β cell differentiation and function. To successfully obtain functional IPCs, the type of vectors utilised for gene transfer and the selection of a suitable target cell for subsequent differentiation into IPCs is of fundamental importance. Techniques for manufacturing IPCs include the dedifferentiation and directed transdifferentiation of autologous or allogeneic cells ex vivo followed by transplantation and the in vivo transdifferentiation of target tissue via viral gene transfer. Ultimately, the goal is to construct IPCs that have the capacity to process, store and secrete insulin in response to fluctuating blood glucose levels, whilst avoiding the administration of immunosuppressants and recurrent autoimmune destruction, thereby indefinitely restoring normoglycaemia.
Ren, B, La, QT, O'Brien, BA, Nassif, NT, Tan, Y, Gerace, D, Martiniello-Wilks, R, Torpy, F, Dane, AP, Alexander, IE & Simpson, AM 2017, 'EXPRESSION OF HUMAN PANCREATIC TRANSCRIPTION FACTORS IN THE LIVERS OF FRG MICE', JOURNAL OF GENE MEDICINE, Joint 10th Annual Scientific Meeting of the Australian-Gene-and-Cell-Therapy-Society (AGCTS) and Australasian-Society-for-Stem-Cell-Research (ASSCR), WILEY, Univ Technol Sydney, Sydney, AUSTRALIA.View/Download from: UTS OPUS
Gerace, D, Martiniello-Wilks, R, Nassif, NT, Ren, B & Simpson, AM 2017, 'Ex Vivo Expanded Murine Mesenchymal Stem Cells as Targets for the Generation of a Cell Replacement Therapy for Type 1 Diabetes', DIABETES, 77th Scientific Sessions of the American-Diabetes-Association, AMER DIABETES ASSOC, San Diego, CA, pp. A83-A83.View/Download from: UTS OPUS
Gerace, D, Martiniello-Wilks, R & Simpson, AM 2015, 'BIOLUMINESCENT IMAGING OF MESENCHYMAL STEM CELL ENGRAFTMENT IN IMMUNE COMPETENT AND IMMUNE DEFICIENT ANIMAL MODELS OF TYPE 1 DIABETES', JOURNAL OF GENE MEDICINE, 9th Biennial Meeting of the Australasian-Gene-and-Cell-Therapy-Society (AGCTS), WILEY-BLACKWELL, Univ Melbourne, Univ Coll, Parkville, AUSTRALIA, pp. 201-201.
Gerace, D, Martiniello-Wilks, R & Simpson, AM 2015, 'Persistence of Luciferase Expressing Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs) in Non-Obese Diabetic (NOD) and NOD/Scid Mice', MOLECULAR THERAPY, 18th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT), NATURE PUBLISHING GROUP, New Orleans, LA, pp. S101-S101.View/Download from: Publisher's site
Gerace, D, Ren, B, Hawthorne, WJ, Byrne, MR, Phillips, P, O'Brien, BA, Nassif, N, Alexander, IE & Simpson, AM 2013, 'REVERSAL OF DIABETES IN A PORCINE MODEL FOLLOWING LIVER-DIRECTED GENE THERAPY', JOURNAL OF GENE MEDICINE, 8th Meeting of the Australasian-Gene-Therapy-Society, WILEY-BLACKWELL, Univ Technol, Sydney, AUSTRALIA, pp. 326-326.View/Download from: UTS OPUS
Gerace, D, Ren, B, Hawthorne, WJ, Byrne, MR, Phillips, P, O'Brien, BA & Simpson, AM 2012, 'Reversal of Diabetes in a Pig Model Following Lentiviral Delivery of Human Furin-Cleavable Insulin (INS-FUR)', The Annual Scientific Meeting of the Australian Diabetes Society and the Australian Diabetes Educators Association, Gold Coast, Australia.
Gerace, D, Ren, B, Byrne, MR, O'Brien, BA & Swan, A 2011, 'Pancreatic Transdifferentiation in the Livers of Non-Obese Diabetic (NOD) Mice Following Lentiviral Delivery of Furin-Cleavable Insulin', RNSH/UTS/USYD/KIMR Annual Scientific Meeting, Kolling Institute of Medical Research.