This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This program will evaluate the suitability of the marmoset as a small primate model for the delivery of antiviral siRNA to the liver by the use of Adeno-assoicated virus gene therapy vectors. This project will be conducted in three phases; 1) development of a tissue culture system for AAV using primary marmoset hepatocytes; 2) evaluation of different AAV vectors for infection of marmoset liver in vivo using vectors that express Beta-galactosidase; 3) and evaluation of antiviral efficacy of AAV vectors encoding siRNA directed at viral sequences of GBV-B, a surrogate model of hepatitis C virus infections. Primary hepatocytes will be isolated from a marmoset using collagenase perfusion and grown in a defined serum free medium. In the initial experiments, hepatocytes will be infected with different dilutions of AAV vectors encoding ?-galactosidase for detection of infected cells with ?-gal histochemical staining. AAV vectors of three serotypes (2, 6 and 8) will be compared for infection of marmoset hepatocytes. In the second phase, marmosets will be inoculated with AAV vectors of different serotypes (2, 6 and 8) via portal vein following laparotomy. The vectors will encode ?-galactosidase for histochemical detection of infected cells. Two marmosets will be inoculated with each AAV serotype and one animal will serve as an uninfected control (7 animals). In the final phase, efficacy of silencing GBV-B replication with AAV-shRNA will be examined in marmosets. Animals will be inoculated IV with a GBV-B/HCV chimeric virus and 2 weeks later will be inoculated with the AAV-siRNA vector. Blood and tissues will be monitored to determine effect of siRNA on GBV-B replication.
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