Correction of Diabetic Hyperglycemia and Amelioration of Metabolic Anomalies by Minicircle DNA Mediated Glucose-Dependent Hepatic Insulin Production

Abstract

Type 1 diabetes mellitus (T1DM) is caused by immune destruction of insulin-producing pancreatic β-cells. Commonly used insulin injection therapy does not provide a dynamic blood glucose control to prevent long-term systemic T1DM-associated damages. Donor shortage and the limited long-term success of islet transplants have stimulated the development of novel therapies for T1DM. Gene therapy-based glucose-regulated hepatic insulin production is a promising strategy to treat T1DM. We have developed gene constructs which cause glucose-concentration–dependent human insulin production in liver cells. A novel set of human insulin expression constructs containing a combination of elements to improve gene transcription, mRNA processing, and translation efficiency were generated as minicircle DNA preparations that lack bacterial and viral DNA. Hepatocytes transduced with the new constructs, ex vivo, produced large amounts of glucose-inducible human insulin. In vivo, insulin minicircle DNA (TA1m) treated streptozotocin (STZ)-diabetic rats demonstrated euglycemia when fasted or fed, ad libitum. Weight loss due to uncontrolled hyperglycemia was reversed in insulin gene treated diabetic rats to normal rate of weight gain, lasting ∼1 month. Intraperitoneal glucose tolerance test (IPGT) demonstrated in vivo glucose-responsive changes in insulin levels to correct hyperglycemia within 45 minutes. A single TA1m treatment raised serum albumin levels in diabetic rats to normal and significantly reduced hypertriglyceridemia and hypercholesterolemia. Elevated serum levels of aspartate transaminase, alanine aminotransferase, and alkaline phosphatase were restored to normal or greatly reduced in treated rats, indicating normalization of liver function. Non-viral insulin minicircle DNA-based TA1m mediated glucose-dependent insulin production in liver may represent a safe and promising approach to treat T1DM.

Preparation of Preproinsulin Gene Construct Containing the Metallothionein2A (pBINDMTChIns) and Its Expression in NIH3T3 Cell Line and Muscle Tissue of Alloxan Diabetic Rabbits

“…So far, various therapeutic strategies have been used for improving type 1 diabetes, including functional pancreatic tissue or β cells transplantation in type 1 diabetes patients and conversion of mesenchymal stem cells into pancreatic islet cells (24). Islet β cells transplantation as a promising therapy for type 1 diabetes patients can control blood glucose level and accomplish insulin; however, insulin therapy is still a major approach, because the pancreas donors are limited, and long-term immune system suppression is needed too (22,25,26). Nevertheless, there are many major problems about islet transplantation such as adverse immune responses induced by the islet transplantation process, host autoimmunity recurrence, allorejection and shortage of organ donors (27).…”

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“…Thus, we selected a DNA construct that contained the α-fetoprotein transcriptional enhancer, vascular endothelial growth factor translational enhancer, and albumin 3'-untranslated region in addition to the 3 copies of glucose-inducible responsive elements, albumin promoter and human insulin gene (Figure 1). 34 When testing this insulin construct for its ability to improve the overall phenotype of streptozotocintreated diabetic rats, we were not only able to induce normoglycemia in fasting rats but also rats fed ad libitum (Figure 2). In treated diabetic rats, we were also able to correct weight loss due to uncontrolled hyperglycemia such that the rate of weight gain matched that of healthy control rats for at least 1 month (Figure 3).…”

“…Both viral and nonviral vector-based gene delivery methods have been used for insulin gene therapy applications, with each showing successful amelioration of diabetesassociated hyperglycemia in small animal models. [33][34][35] However, nonviral gene delivery methods are limited by their inefficient delivery to target cells and lack of chromosomal integration, which restricts the longevity of gene expression. As such, viral vectors have been used more prevalently in insulin gene therapy applications and are ultimately the future of the therapy.…”

“…As such, viral vectors have been used more prevalently in insulin gene therapy applications and are ultimately the future of the therapy. Adenoviruses, 34,36,37 adeno-associated viruses, 25,38,39 oncoretroviruses, 40,41 and lentiviruses [42][43][44] all have been used to deliver insulin to hepatocytes, but they vary in their ability to meet the essential criteria described previously. A summary of each viral vector's ability to meet the demands required for insulin gene therapy applications is included in Table 1.…”

“…To do so, many groups have used tissue-specific promoters, and for hepatic insulin gene therapy, a variety of liver-specific promoters have been used. Examples of liver-specific promoters include the insulin-like growth factor binding protein-1 (IGFBP-1), 46 glucose-6-phosphatase (G6Pase), liver-type pyruvate kinase (L-PK), 47 phosphoenolpyruvate carboxykinase (PEPCK), 28 albumin, 34,37 and S-14. While liver-specific transcriptional regulation of insulin gene transcription has been achieved using these promoters, significant limitations have been observed.…”