Cell type gene delivery and alcoholic liver disease

描述(申请人提供)：使用重组基因向肝脏传递基因 腺相关病毒由于转导效率低而受到限制。 不到5%的肝细胞。了解为什么rAAV的能力有限 转导肝脏是开发基因传递方法的关键 酒精性肝损伤。最近有研究表明，rAAV 根据抗原的不同，AAV的血清型有不同的亚型。 不同脑区和细胞类型的信号转导能力 肌肉。因此，根据血清型的不同，推测rAAV载体， 可差异转导肝实质细胞和非肝实质细胞 (即Kupffer细胞)。因此，该提案的目标是解决以下几个问题 具体目的：(1)重组腺相关病毒血清型 不同的实质细胞和非实质细胞的趋向性导致增强 转基因和转基因表达？(2)启动子元件提供什么 转基因在肝实质细胞还是非实质肝细胞中的最佳表达？ (3)能否开发更好的载体来增强转基因表达和 细胞特异性基因打靶，基于目标1和2的结果，以 预防早期酒精性肝炎？我们的第一个目标是比较AAV 全肝的血清型转导差异，随后进行了体外研究 评价Kupffer细胞和肝细胞的血清型趋向性差异。 其次，将针对细胞特异性识别和优化启动子元件 Kupffer细胞和肝细胞中的基因表达。最后，使用鼠标 乙醇性肝炎肠内酒精喂养模型的建立 AIMS 1和AIMS 2中开发的试剂，用于解决氧化剂生成的作用 和氧化还原敏感的转录因子NFB的激活 Kupffer细胞或肝细胞。转录因子NFB的激活是 我们的假设的核心是解释早期的酒精诱导的肝炎；因此， 瞄准库普弗细胞中潜在的氧化剂产生来源或 使用基因转移的肝细胞是这一提议的关键。我们期待着这些 Kupffer细胞NADPH氧化酶是一种 氧化剂的主要来源，导致一连串的炎症反应 (即激活NFB、细胞因子产生、诱导型一氧化氮合酶) 最终会导致组织损伤。此外，这些发现将导致 开发临床上有用的基因转移系统，以及允许我们 解决与细胞类型之间的相互作用相关的关键问题 它们参与了早期酒精性肝损伤的发病机制。 此外，通过授课培训，以及与他的导师和 关键教员，申请者将获得新的技能，使他能够 成为酒精研究社区的一名成功成员。

DESCRIPTION (provided by applicant): 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.