Cell type gene delivery and alcoholic liver disease

描述（由申请人提供）：使用重组质粒将基因递送至肝脏 腺相关病毒由于其低转导效率而受到限制， 少于5%的肝细胞。了解为什么rAAV的能力有限， 肝脏是开发基因递送方法的关键， 酒精性肝损伤最近证实rAAV 血清型，即基于抗原不同性的AAV亚型，具有多样性， 不同区域和细胞类型的大脑中的转导能力， 肌肉.因此，假设rAAV载体，取决于血清型， 能区分肝实质细胞和非实质细胞 (i.e.,枯否细胞）。因此，本提案的目标是解决几个问题， 具体目标：（1）重组腺相关病毒血清型与 不同的实质和非实质细胞向性导致增强的 转导和转基因表达？(2)哪些启动子元件提供 在实质或非实质肝细胞中的最佳转基因表达？ (3)能否开发出更好的载体来增强转基因表达， 基于目标1和2的结果，进行细胞特异性基因靶向， 预防早期酒精性肝炎？我们的第一个目标是比较AAV 全肝中的血清型转导差异，随后进行体外研究 评价枯否细胞和肝细胞中的血清型嗜性差异。 第二，将鉴定启动子元件并优化用于细胞特异性表达。 枯否细胞和肝细胞中的基因表达。最后，使用鼠标 肠内乙醇喂养模型乙醇诱导的肝炎，我们将使用 在目标1和2中开发的试剂，以解决氧化剂生成的作用 和氧化还原敏感性转录因子NFB的激活， 枯否细胞或肝细胞。转录因子NFB的激活是 这是我们解释早期乙醇诱导肝炎的假设的核心;因此， 靶向枯否细胞中氧化剂产生的潜在来源，或 使用基因转移的肝细胞是该提议的关键。我们预计这些 实验，以解决假设，枯否细胞NADPH氧化酶是一个 导致炎症反应级联的氧化剂的主要来源 (ie., NFB的激活、细胞因子的产生、iNOS的诱导）， 最终导致组织损伤。此外，这些发现将导致 临床上有用的基因转移系统的发展，以及允许我们 解决与细胞类型之间的相互作用有关的关键问题， 它们参与早期乙醇诱导的肝损伤的发病机制。 此外，通过教学培训，以及与导师的互动， 关键教师，申请人将获得新的技能，使他能够 成为酒精研究界的成功成员。

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.