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

BACKGROUND: Current 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 RESULTS: In 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 > or = 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 > or = 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. CONCLUSIONS: We 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

Susceptibility of insulin-secreting hepatocytes to the toxicity of pro-inflammatory cytokines

The liver has been suggested as a suitable target organ for reversing type I diabetes by gene therapy. Whilst gene delivery systems to the hepatocyte have yet to be optimized in vivo, whether insulin-secreting hepatocytes are resistant to the autoimmune process that kills pancreatic β-cells has never been addressed. One of the mechanisms by which β-cells are killed in type I diabetes is by the release of the cytokines interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) by immune cells. To test the effect of the cytokines on insulin-secreting hepatocytes in vitro we exposed the betacyte, also called the HEP G2ins/g cell which possesses cytokine receptors and can synthesize, store and secrete insulin in a regulated fashion to a glucose stimulus, to the above mentioned cytokines for 14 days. Viability of the HEP G2ins/g cells was similar to that of other liver cell lines/primary cells which were more resistant to the cytokines than the β-cell line NIT-1. The cytokines had no adverse effect for the first six days on insulin secretion, content and mRNA levels of the HEP G2ins/g cells and insulin secretion in response to 1-h exposure to 20 mM glucose was enhanced 14-fold. Our results indicate that genetically engineered hepatocytes and primary liver cells are more resistant than pancreatic β-cells to the adverse effects of cytokines offering hope that insulin secreting hepatocytes in vivo made by gene therapy are less likely to be destroyed by cytokines released during autoimmune destruction. © 2001 Academic Press

Comparison of size, viability, and function of fetal pig islet-like cell clusters after digestion using collagenase or liberase

Liberase is a highly purified blend of collagenases that has been specifically developed to eliminate the numerous problems associated with the conventional use of crude collagenase when isolating islet-like cell clusters (ICCs) from pancreases of different species. The influence of Liberase on yield, size, viability, and function of ICCs has been documented when this enzyme was used to digest adult but not fetal pancreases. In this study, we compared the effects of collagenase and Liberase on fetal pig ICCs. A total of eight fetal pig pancreas digestions were analyzed. Fetuses were obtained from Large White Landrace pigs of gestational age 80 +/- 2.1 days. The pancreases were digested with either 3 mg/ml collagenase P or 1.2 mg/ml Liberase HI. The time taken to digest the pancreas was shorter for collagenase when compared with Liberase (22 +/- 2 vs. 31 +/- 2 min). The size of ICCs was similar for both collagenase (83 +/- 0.5 microm) and Liberase (79 +/- 0.4 microm) as was the number of ICCs produced per pancreas (7,653 +/- 1,297 vs. 8,101 +/- 1,177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 +/- 1% vs. 76 +/- 1%,

Lowering of blood glucose to nondiabetic levels in a hyperglycemic pig by allografting of fetal pig isletlike cell clusters

Background. Fetal pig isletlike cell clusters (ICCs) will differentiate when grafted into the thymus gland of outbred immunosuppressed nondiabetic pigs for up to 3 months. Whether these cells will survive for a similar period in a diabetic recipient and will mature with secretion of insulin to ameliorate the hyperglycemia is unknown. Methods. Between 40,000 and 125,000 ICCs (7,000 to 11,400 ICCs/kg) were injected into the thymus gland of five juvenile pigs immunosuppressed with cyclosporine and deoxyspergualin, and the animals were subsequently made diabetic by the injection of streptozotocin. Insulin was administered subcutaneously, with one pig dying from hypoglycemia. The animal with the least number of ICCs transplanted was killed 81 days later, and the graft was analyzed histologically. Blood glucose levels and porcine C-peptide in the remaining animals were monitored for a median of 101 days. Results. Histological analysis of the graft showed numerous epithelial cell clusters; the percentage of cells that contained insulin, glucagon, somatostatin, and pancreatic polypeptide were 61%, 64%, 25%, and 18%, respectively. Some cells contained more than one hormone. Porcine C-peptide was detected from 21 days after induction of diabetes but not before. In the pig receiving the most ICCs, blood glucose levels were lowered to nondiabetic levels 109 days after transplantation. Plasma C-peptide levels in response to glucagon in this pig steadily increased after grafting; peak levels were 0, 0.21, 0.45, and 0.52 ng/ml at 4, 21, 49, and 80 days after induction of diabetes compared to 0.09 ng/ml in control diabetic pigs. The secretion of C-peptide in response to oral and intravenous glucose and arginine also was greater than in untransplanted diabetic pigs, the pattern of secretion being consistent with developing fetal β cells as the source of the C-peptide. Pancreatic insulin content was 0.1 mU/mg, 4% of that in nondiabetic pigs, and the number of β cells per islet was 3 to 6 compared to 90 in nondiabetic controls. Conclusions. ICCs will differentiate and function for up to 111 days when transplanted into outbred immunosuppressed pigs rendered diabetic. Blood glucose levels can be lowered to nondiabetic levels when sufficient numbers of ICCs are grafted

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic β cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose-response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for β cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic β cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic β cells and become a therapy for the treatment of type I diabetes

Viral-Mediated Gene Therapy for the Generation of Artificial Insulin-Producing Cells as a Therapeutic Treatment for Type 1 Diabetes Mellitus

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