Resumen de Aav-mediated genetic engineering of the pancreas and adipose tissue for the study and treatment of diabetes and obesity
Pancreatic ß-cells play a primary role in the maintenance of glucose homeostasis by means of insulin secretion in response to nutrients, hormones and neuronal stimuli. Likewise, adipose tissue is pivotal in the modulation of energy homeostasis both by balancing energy intake and expenditure and as a secretory organ of FFA and glycerol, hormones and cytokines. An inadequate mass of functional ß-cells and alterations in the metabolic and endocrine functions of adipose tissue are the main features of insulin resistance, diabetes and obesity. In vivo genetic engineering of the pancreas, especially of ß-cells, and of adipose tissue may prove useful to studies designed to fully understand the metabolic and molecular mechanism(s) underlying such events. Moreover, the unravelling of such pathogenic process/es is the cornerstone for the development of new, more effective, and potentially curative therapies for diabetes, obesity and their associated diseases. Nevertheless, despite the progress in in vivo tissue engineering, effective gene transfer to the pancreas and the adipose tissue remains elusive.
In this study, we conducted a detailed comparative evaluation of the ability of single-stranded adeno-associated viral vectors (AAV) to in vivo genetically engineer the pancreas, with special emphasis on gene transfer to ß-cells, and white and brown adipose tissue. AAV are attractive vehicles for gene transfer because of their lack of pathogenicity, low immunogenicity, stability and ability to mediate long-term expression of the gene of interest.
The intraductal delivery of ssAAV vectors of serotypes 6, 8 and 9 led to highly efficient and long-term transduction of ß-cells and exocrine pancreas. In addition, the use of the rat insulin promoters I and II or the rat elastase I (-205/+8) promoter restricted AAV-mediated transgene expression to ß-cells and acinar cells, respectively. Moreover, as a proof of concept, we showed that the AAV-mediated pancreatic overexpression of hepatocyte growth factor prevented autoimmune diabetes in a mouse model of type 1 diabetes.
Local administration of ssAAV8 and ssAAV9 vectors into epididymal white adipose tissue (eWAT) and brown adipose tissue led to highly efficient gene transfer to white and brown adipocytes. When the expression of genes was under the control of the basal promoters combined with the enhancer elements that confer adipose specificity of the murine aP2 or rat UCP1 genes, highly specific, long-term transgene expression in white and/or brown adipocytes was achieved. As proof of concept of this technology, the genetic engineering of eWAT by AAV9 vectors coding for the enzyme hexokinase II led to increased in vitro and in vivo glucose uptake specifically by adipocytes. Similarly, AAV-transduced white adipocytes were able to secrete stable high levels of the murine secreted alkaline phosphatase marker protein into the bloodstream.
Therefore, the results obtained in this thesis demonstrate that ssAAV vectors, in particular serotypes 8 and 9, may prove useful as a new tool for genetic engineering of the pancreas and adipose tissue for the study and treatment of diabetes and obesity.
