Long-term correction of diabetes in rats after lentiviral hepatic insulin gene therapy

Aims/hypothesis: Type 1 diabetes results from the autoimmune destruction of pancreatic beta cells. Exogenous insulin therapy cannot achieve precise physiological control of blood glucose concentrations, and debilitating complications develop. Lentiviral vectors are promising tools for liver-directed gene therapy. However, to date, transduction rates in vivo remain low in hepatocytes, without the induction of cell cycling. We investigated long-term transgene expression in quiescent hepatocytes in vitro and determined whether the lentiviral delivery of furin-cleavable insulin to the liver could reverse diabetes in rats. Materials and methods: To improve transduction efficiency in vitro, we optimised hepatocyte isolation and maintenance protocols and, using an improved surgical delivery method, delivered furin-cleavable insulin alone or empty vector to the livers of streptozotocin-induced diabetic rats by means of a lentiviral vector. Rats were monitored for changes in body weight and blood glucose, and intravenous glucose tolerance tests were performed. Expression of insulin was determined by RT-PCR, immunohistochemistry and electron microscopy. Results: We achieved long-term transgene expression in quiescent hepatocytes in vitro (87 ± 1.2% transduction efficiency), with up to 60 ± 3.2% transduction in vivo. We normalised blood glucose for 500 days-a significantly longer period than previously reported-making this the first successful study using a lentiviral vector. This procedure resulted in the expression of genes encoding several beta cell transcription factors, some pancreatic endocrine transdifferentiation, hepatic insulin storage in granules, and restoration of glucose tolerance. Liver function tests remained normal. Importantly, pancreatic exocrine transdifferentiation did not occur. Conclusions/interpretation: Our data suggest that this regimen may ultimately be employed for the treatment of type 1 diabetes

Therapeutic Potential of HDL in Cardioprotection and Tissue Repair

Epidemiological studies support a strong association between high-density lipoprotein (HDL) cholesterol levels and heart failure incidence. Experimental evidence from different angles supports the view that low HDL is unlikely an innocent bystander in the development of heart failure. HDL exerts direct cardioprotective effects, which are mediated via its interactions with the myocardium and more specifically with cardiomyocytes. HDL may improve cardiac function in several ways. Firstly, HDL may protect the heart against ischaemia/reperfusion injury resulting in a reduction of infarct size and thus in myocardial salvage. Secondly, HDL can improve cardiac function in the absence of ischaemic heart disease as illustrated by beneficial effects conferred by these lipoproteins in diabetic cardiomyopathy. Thirdly, HDL may improve cardiac function by reducing infarct expansion and by attenuating ventricular remodelling post-myocardial infarction. These different mechanisms are substantiated by in vitro, ex vivo, and in vivo intervention studies that applied treatment with native HDL, treatment with reconstituted HDL, or human apo A-I gene transfer. The effect of human apo A-I gene transfer on infarct expansion and ventricular remodelling post-myocardial infarction illustrates the beneficial effects of HDL on tissue repair. The role of HDL in tissue repair is further underpinned by the potent effects of these lipoproteins on endothelial progenitor cell number, function, and incorporation, which may in particular be relevant under conditions of high endothelial cell turnover. Furthermore, topical HDL therapy enhances cutaneous wound healing in different models. In conclusion, the development of HDL-targeted interventions in these strategically chosen therapeutic areas is supported by a strong clinical rationale and significant preclinical data.status: publishe

Verification and synthesis of co-simulation algorithms subject to algebraic loops and adaptive steps

Simulation-based analyses are becoming increasingly vital for the development of cyber-physical systems. Co-simulation is one such technique, enabling the coupling of specialized simulation tools through an orchestration algorithm. The orchestrator describes how to coordinate the simulation of multiple simulation tools. The simulation result depends on the orchestration algorithm that must stabilize algebraic loops, choose the simulation resolution, and adhere to each simulation tool's implementation. This paper describes how to verify that an orchestration algorithm respects all contracts related to the simulation tool's implementation and how to synthesize such tailored orchestration algorithms. The approaches work for complex and adaptive co-simulation scenarios and have been applied to several real-world case studies

Immunosuppression overcomes insulin- and vector-specific immune responses that limit efficacy of AAV2/8-mediated insulin gene therapy in NOD mice

We report the restoration of euglycaemia in chemically induced diabetic C57BL/6 mice and spontaneously diabetic Non Obese Diabetic (NOD) mice by intravenous systemic administration of a single-stranded adeno-associated virus (ssAAV2/8) codon optimised (co) vector encoding furin cleavable human proinsulin under a liver-specific promoter. There were no immunological barriers to efficacy of insulin gene therapy in chemically induced C57BL/6 mice, which enjoyed long-lasting correction of hyperglycaemia after therapy, up to 250 days. Euglycaemia was also restored in spontaneously diabetic NOD mice, although these mice required a 7–10-fold higher dose of vector to achieve similar efficacy as the C57BL/6 mice and the immunodeficient NODscid mice. We detected CD8+ T cell reactivity to insulin and mild inflammatory infiltration in the livers of gene therapy recipient NOD mice, neither of which were observed in the treated C57BL/6 mice. Efficacy of the gene therapy in NOD mice was partially improved by targeting the immune system with anti-CD4 antibody treatment, while transfer of NOD mouse AAV2/8-reactive serum to recipients prevented successful restoration of euglycaemia in AAV2/8-HLP-hINSco-treated NODscid mice. Our data indicate that both immune cells and antibodies form a barrier to successful restoration of euglycaemia in autoimmune diabetic recipient mice with insulin gene therapy, but that this barrier can be overcome by increasing the dose of vector and by suppressing immune responses