Research activities of The Gene and Cell Therapy Center

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Insulin Gene Therapy


Recent studies of insulin gene therapy have mainly focused on the transfer of functional insulin genes to non-pancreatic cells to avoid the autoimmune attack, but insulin secreted by non-beta cells were ineffective to properly manage postprandial hyperglycemia. Compared to other cell types, pancreatic beta cells possess exquisite features in terms of controlled transcription and translation of proinsulin, the presence of prohormone convertase expression, and glucose-sensing machinery, in addition to a regulated secretory pathway. To take advantage of the unique properties of pancreatic beta cells, we constructed 3rd generation lentiviral vectors carrying insulin promoter connected to the insulin gene (LentiINS) targeting pancreatic beta cells.

Although intraperitoneal injection of LentiINS to streptozotocin (STZ)-induced diabetic Wistar rats decreased fasting plasma glucose,
improved glucose tolerance, and prevented weight loss, it was insufficient in lowering postprandial blood glucose levels. Only, the
combination of LentiINS and the anti-inflammatory vasoactive intestinal peptide (LentiVIP) gene deliveries successfully lowered post-prandial blood glucose and suppressed diabetes-related inflammation in diabetic rats. Our results suggested that insulin gene therapy
combined with anti-inflammatory/insulinotropic/regenerative gene delivery was more effective than their individual applications.

Combined Efficacy of Insulin and Anti-Inflammatory Gene Therapies for Type 1 Diabetes Mellitus. Fulya Erendor, Yunus Emre Eksi, Elif Ozgecan Sahin, Mustafa Kemal Balci, Thomas S. Griffith and Salih Sanlioglu


Functional consequences of pancreatic beta cell-specific insulin gene expression (LentiINS) complemented with anti-inflammatory gene delivery (LentiVIP). The unique properties of pancreatic beta cells are outlined in the figure. First, pancreatic beta cells possess a regulated secretory pathway allowing insulin storage. Glucose transporter 2 (GLUT2) and glucokinase (GK) function as cellular glucose radars. Prohormone convertase (PC1/3 and PC2) changes proinsulin to insulin. LentiINS gene delivery causes pancreatic beta-cell restricted glucose-regulated insulin gene expression which is augmented by LentiVIP. Concurrent use of LentiINS and LentiVIP stimulates pancreatic beta cell regeneration and proliferation in STZ induced diabetic rats. The anti-inflammatory properties of LentiVIP are not depicted for clarity.


The most of the gene therapy trials dealing with recovering insulin gene expression in Type 1 diabetes patients targeted non-pancreatic tissues to avoid T cell mediated autoimmune response. However, the absence of proinsulin processing enzymes and glucose-dependent insulin secretion in these tissues restricted the success of gene therapy studies. To solve this problem, we aimed to produce a new lentiviral vector (LentiINS) with insulin promoter hooked up to proinsulin gene sequence to provide pancreatic beta cell-specific insulin gene expression and secretion. For this purpose, Multisite Gateway
Technology involving two different entry vector one with minimal insulin promotor and the other with proinsulin gene sequence and a destination vector carrying lentiviral backbone was employed to generate the transfer vector. Following confirmation of the transfer plasmid by restriction enzyme digestion and DNA sequence analysis, high-titer LentiINS vectors at a concentration of 10e9 TU/ml were successfully produced by CaPO4 cotransfection of 293T cells with packaging and transfer plasmids. While transduction of NIT1 mouse pancreatic beta cell lines produced 3 fold increase in insulin gene expression compared to controls, no insulin expression was detected in 293T kidney cell lines as expected. These results indicate that we successfully constructed an HIV-based lentiviral gene therapy vector being capable of beta cell-specific insulin gene expression and secretion.
Generation of a beta cell specific insulin gene therapy vector for diabetes. Sahin EO, Erendor F, Balci MK and Sanlioglu S. ESGCT XXV Anniversary Congress. Human Gene Therapy. Volume:28 Issue:12 Pages:A110 Meeting Abstract: P341. Berlin, Germany OCT 17-20, 2017

Incretin Gene Therapy


New therapy strategies for Type 2 diabetes(T2DM) should be considered to stimulate glucose induced insulin secretion(GSIS); beta cell replication and islet neogenesis and as well as protect islet cells from apoptosis. Previous studies suggested that 50% decrease in incretin (GLP-1) gene expression was detected in T2DM patients[1][2]. Therefore; we aimed to overcome incretin deficiency via lentiviral GLP-1 gene transfer by providing a prolonged GLP-1 expression in a rat model of T2DM. High titered lentiGLP-1 were produced in roller bottles, by CaPO4 transfection to 293T cells. In vitro functionality of LentiGLP-1 vector was proved by an increase in cAMP production and GSIS in transduced MIN6 cells. Previously hyperglycemic diabetic rats exhibit lowered blood glucose levels following in vivo administration of LentiGLP-1. Besides; improved insulin sensitivity and glucose tolerance were observed in treated rats; while triglyceride levels were normalised. Islet cell mass and insulin positive cell assessments revealed 45% increase in islet mass after GLP-1 gene delivery. Additionally, detection of insulin positive cell clusters in aciner regions of pancreatic sections should be considered as an indication of transdifferantiation potential of GLP-1 peptide. TUBITAK-112S114 .
Glucagon like peptide-1 as an antidiabetic gene therapy agent inducing transdifferantiation of pancreatic endocrine cells. Tasyurek H, Altunbas HA and Sanlioglu S. ESGCT XXV Anniversary Congress. Human Gene Therapy. Volume:28 Issue:12 Pages:A93 Meeting Abstract: P277. Berlin, Germany OCT 17-20, 2017

VIP Gene Therapy


For patients with type-1 diabetes (T1DM), immune systemmediated  destruction of the pancreatic beta cells removes the  body of the only cells capable of making insulin. Pancreatic islet  cell death, but not the defect in new islet formation or beta-cell replication, has been blamed for the decrease in beta-cell mass observed in T1DM patients. Thus, therapeutic approaches designed to protect islet cells from apoptosis could significantly improve the management of T1DM. Therefore, an ideal betacell- preserving agent is expected to protect beta cells from apoptosis and stimulate postprandial insulin secretion along with increasing beta-cell replication and/or islet neogenesis. One such potential agent, vasoactive intestinal peptide (VIP) strongly stimulates postprandial insulin secretion. Because of its broad spectrum of actions such as acting as a potent anti-inflammatory factor through suppression of Th1 immune response, and generation
of immune tolerance by way of regulatory T cells, VIP has surfaced as a promising agent for the treatment of T1DM. Since VIP is a neuropeptide with insulinotropic and antiinflammatory functions, 3rd generation lentiviral vectors encoding human VIP (LentihVIP) were constructed using Gateway cloning strategy. Restriction enzyme analysis and DNA sequencing were utilized to confirm the cloning. The lentiviral particle titers were quantified using QuickTiter HIV Lentivirus Quantitation kit (Cell Biolabs). The expression was confirmed by ELISA while functional status was revealed by glucose induced insulin secretion test. Financial support: (TUBITAK- 215S820).
Generation of a lentiviral vector encoding vasoactive intestinal peptide for type 1 diabetes. Erendor F, Sahin EO, Balci MK and Sanlioglu S. ESGCT XXV Anniversary Congress. Human Gene Therapy. Volume:28 Issue:12 Pages:A59-A60 Meeting Abstract: P152. Berlin, Germany OCT 17-20, 2017


CRISPR/Cas9 Mediated Gene Editing


Maturity-onset diabetes of the young 10 (MODY10) is a subtype of MODY characterized by deficiency in insulin synthesis observed due to mutations in the insulin gene. Since
MODY10 carriers eventually need insulin injections, insulin gene transfer through lentiviral vectors can help to eliminate the need for exogenous insulin in such patients. Therefore, to determine the feasibility of insulin gene replacement therapy, we aimed to generate pancreatic beta cell insulin knockout mice (MODY10) using CRISPR / Cas9. Subsequently, beta cells carrying insulin gene defect will be transplanted into the kidney capsule of diabetic subjects to develop MODY10 experimental animal model of diabetes. For this purpose, two different insulin gene therapy vectors with CMV and insulin promoters were constructed. The gene expression profiles of these vectors were first tested in 293T cells and then in NIT1 mouse pancreatic beta cell lines. For the generation of the diabetic experimental animal model, 8 weeks old male C57BL / 6J mice were intraperitoneally
injected with a single dose of 150 mg / kg Streptozotocin. After a month of follow-up, continuous hyperglycemia was observed in the subjects as a sign of destruction of pancreatic beta cells that normally would express endogenous insulin. In the next step, pancreatic beta cells with insulin gene deficiency will be generated using CRISPR / Cas9 technique. TUBITAK(215S820) 
Development of MODY10 diabetic experimental animal model using CRISPR/Cas9 technology for insulin gene transfer studies. Eksi Y, Altunbas HA and Sanlioglu S. ESGCT XXV Anniversary Congress. Human Gene Therapy. Volume:28 Issue:12 Pages: A48-A49 Meeting Abstract: P112. Berlin, Germany OCT 17-20, 2017


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