Our long-term goal is to develop safe and effective gene therapy methods for permanent correction of inherited metabolic diseases, using Crigler-Najjar syndrome-1 (CN-1) as a model target. CN-1, which is caused by the lack of UGT1A1 activity that is critical for glucuronidation and subsequent biliary excretion of bilirubin. Currently, liver transplantation is the only definitive cure for this potentially lethal disease. The Gunn rat, an animal model of CN-1, provides a convenient model for evaluating the efficiency of liver-directed gene therapy in vivo. Based on considerable progress during the current funding cycle, we will now focus specifically on safe and repeatable nucleic acid delivery methods that permit permanent phenotypic correction of mutant hepatocytes in vivo. The first specific aim is to optimize recombinant SV40 (rSV40) vectors. We hypothesize that rSV40 may need lipid rafts to enter cells, but unlike the wildtype virus, does not require caveolae. We will test this using sterol binding agents, protein kinase C activators and by studies on caveolin-1 knockout mice. We will improve the vector by progressively deleting the viral genes to accommodate potent internal promoters. """"""""Insulator"""""""" sequences will be inserted as """"""""boundaries"""""""" against heterochromatin effect that silences transgene expression in vivo, and as """"""""barriers"""""""" to prevent the enhancers used in the vector from affecting the expression of neighboring host genes. The second specific aim will utilize receptor mediated nucleic acid delivery into hepatocytes in vivo. Novel plasmid constructs utilizing the """"""""Sleeping Beauty"""""""" transposon-transposase system will be targeted to the liver to promote transgene integration into the host genome. In another approach, we will use RNA-DNA chimera or novel single strand all-DNA oligonucleotides to trigger cellular DNA repair mechanisms for correction of the genetic lesion in the Gunn rat in vivo. Successful completion of this project will provide effective methods for correction of genetic liver diseases, which will avert the untoward side effects that have plagued the clinical application of gene therapy in recent years.
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