Nonvirally Modified Autologous Primary Hepatocytes Correct Diabetes and Prevent Target Organ Injury in a Large Preclinical Model

Abstract

BackgroundCurrent gene- and cell-based therapies have significant limitations which impede widespread clinical application. Taking diabetes mellitus as a paradigm, we have sought to overcome these limitations by ex vivo electrotransfer of a nonviral insulin expression vector into primary hepatocytes followed by immediate autologous reimplantation in a preclinical model of diabetes.Methods and ResultsIn a single 3-hour procedure, hepatocytes were isolated from a surgically resected liver wedge, electroporated with an insulin expression plasmid ex vivo and reimplanted intraparenchymally under ultrasonic guidance into the liver in each of 10 streptozotocin-induced diabetic Yorkshire pigs. The vector was comprised of a bifunctional, glucose-responsive promoter linked to human insulin cDNA. Ambient glucose concentrations appropriately altered human insulin mRNA expression and C-peptide secretion within minutes in vitro and in vivo. Treated swine showed correction of hyperglycemia, glucose intolerance, dyslipidemia and other metabolic abnormalities for ≥47 weeks. Metabolic correction correlated significantly with the number of hepatocytes implanted. Importantly, we observed no hypoglycemia even under fasting conditions. Direct intrahepatic implantation of hepatocytes did not alter biochemical indices of liver function or induce abnormal hepatic lobular architecture. About 70% of implanted hepatocytes functionally engrafted, appeared histologically normal, retained vector DNA and expressed human insulin for ≥47 weeks. Based on structural tissue analyses and transcriptome data, we showed that early correction of diabetes attenuated and even prevented pathological changes in the eye, kidney, liver and aorta.ConclusionsWe demonstrate that autologous hepatocytes can be efficiently, simply and safely modified by electroporation of a nonviral vector to express, process and secrete insulin durably. This strategy, which achieved significant and sustained therapeutic efficacy in a large preclinical model without adverse effects, warrants consideration for clinical development especially as it could have broader future applications for the treatment of other acquired and inherited diseases for which systemic reconstitution of a specific protein deficiency is critical.

Insulin expressed from endogenously active glucose-responsive EGR1 promoter in bone marrow mesenchymal stromal cells as diabetes therapy

“…4 Similarly, intrahepatic (IH) implantation of autologous primary hepatocytes in diabetic pigs showed durable (X47 weeks) correction of hyperglycemia, glucose intolerance, dyslipidemia and other metabolic abnormalities, which correlated significantly with the number of hepatocytes implanted. 5 Importantly, we observed no hypoglycemia, regardless of feeding behavior, that is, the 'nibbling' behavior of mice and 'humanized' mealtime feeding of pigs, and even under fasting conditions in both animal models.…”

“…13 Transgene The reference construct that served as comparator in these experiments was the human metallothionein IIA promoter in p3MTchINS, which drives insulin expression in hepatocytes. 5,6 pEGFP served as negative control. (d) PBMMSC electroporated with the aforementioned plasmid constructs (5 mg DNA/ 2 Â 10 6 cells) were cultured in DMEM with different glucose concentrations for 30 min, after which the culture medium was aspirated for human insulin and human proinsulin radioimmunoassays (1.5 Â 10 5 cells per well, three wells for each glucose concentration).…”

High efficiency non-viral transfection of retinal and iris pigment epithelial cells with pigment epithelium-derived factor

“…For example, islet cells that express and secrete insulin have been used to deliver insulin in diabetic patients 15 and recently the use of nonvirally transfected autologous liver cells producing insulin has been suggested. 16 The most ideal system for subretinal delivery of bioactive factors would be to transfect RPE cells with the appropriate factors and transplant them into the subretinal space. However, allogeneic RPE cells are rejected and autologous RPE cells are difficult to obtain.…”

Failure to Achieve Normal Metabolic Response in Non-Obese Diabetic Mice and Streptozotocin-Induced Diabetic Mice After Transplantation of Primary Murine Hepatocytes Electroporated With the Human Proinsulin Gene (p3MTChins)

“…This method has known advantages over other cell replacement therapies, prompting efforts to examine this method’s efficacy and stability in vivo [1,2]. Freshly isolated hepatocytes are readily electroporated with a non-viral vector [3,4], efficiently engraft in structurally normal liver, and have been shown to have fewer inflammatory and tumorigenic properties compared with transfection with adenovirus, retroviral vectors [5–7], and pluripotent stem cells [8–11].…”

“…A recent study by Chen et al [1,2] described the therapeutic effectiveness of this strategy in murine and porcine models. By delivering a human proinsulin cDNA plasmid construct (p3MTChins, National Cancer Centre, Singapore) to primary hepatocytes through ex vivo electroporation, the authors were able to achieve durable treatment of streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM).…”