Gene delivery to liver using recombinant adeno-associated virus has been limited due to low transduction efficiency of less than 5% of hepatocytes. Understanding why rAAV has limited ability to transduce liver is critical for developing gene delivery approaches for ethanol-induced liver injury. It was recently demonstrated that rAAV serotypes, subtypes of AAV based on antigenic dissimilarities, have diverse transduction capabilities in different regions and cell types of brain and muscle. Thus, it is hypothesized that rAAV vectors, depending on the serotype, can differentially transduce hepatic parenchymal and non-parechymal cells (i.e., Kupffer cells). Thus, the goal of this proposal is to address several specific aims: (1) Do recombinant adeno-associated virus serotypes with different parenchymal and non-parenchymal cell tropism lead to enhanced transduction and transgene expression? (2) What promoter elements provide optimal transgene expression in parenchymal or non-parenchymal liver cells? (3) Can better vectors be developed for enhanced transgene expression and cell-specific gene targeting, based on the results from Aims 1 and 2, to prevent early ethanol-induced hepatitis? Our first goal is to compare AAV serotype transduction differences in whole liver, followed by in vitro studies to evaluate serotype tropism differences in Kupffer cells and hepatocytes. Second, promoter elements will be identified and optimized for cell-specific gene expression in Kupffer cells and hepatocytes. Lastly, using the mouse enteral ethanol-feeding model for ethanol-induced hepatitis, we will use reagents developed in Aims 1 and 2 to address the roles of oxidant generation and the redox-sensitive transcription factor NFB activation specifically in Kupffer cells or hepatocytes. Activation of the transcription factor NFB is central to our hypothesis to explain early ethanol-induced hepatitis; thus, targeting potential sources of oxidant production in Kupffer cells or hepatocytes using gene transfer is key to this proposal. We expect these experiments to address the hypothesis that Kupffer cell NADPH oxidase is a primary source of oxidants leading to a cascade of inflammatory responses (ie., activation of NFB, cytokine production, induction of iNOS) which ultimately lead to tissue damage. Moreover, these findings will result in the development of clinically useful gene transfer systems as well as allow us to address critical questions related to interactions between cell types and their involvement in the pathogenesis of early ethanol-induced liver injury. In addition, through didactic training, and interactions with his mentor and key faculty, the applicant will acquire new skills that will allow him to become a successful member of the alcohol research community.
| Isayama, Fuyumi; Moore, Sherri; Hines, Ian N et al. (2016) Fas Regulates Macrophage Polarization and Fibrogenic Phenotype in a Model of Chronic Ethanol-Induced Hepatocellular Injury. Am J Pathol 186:1524-36 |
