Cell type gene delivery and alcoholic liver disease

细胞类型基因传递和酒精性肝病

基本信息

批准号：
6751869
负责人：
MICHAEL D WHEELER
金额：
$ 11.34万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-06-01 至 2007-05-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：使用重组体将基因递送至肝脏腺相关病毒由于转导效率低而受到限制肝细胞少于5%。了解为什么 rAAV 的能力有限转导肝脏对于开发基因传递方法至关重要乙醇引起的肝损伤。最近的研究表明，rAAV 基于抗原差异的 AAV 血清型、亚型具有多种特征大脑不同区域和细胞类型的转导能力肌肉。因此，推测 rAAV 载体根据血清型，可以差异转导肝实质和非实质细胞（即库普弗细胞）。因此，本提案的目标是解决几个问题具体目的：（1）重组腺相关病毒血清型不同的实质和非实质细胞趋向性导致增强转导和转基因表达？ (2) 启动子元件提供什么实质或非实质肝细胞中的最佳转基因表达？ (3) 能否开发更好的载体来增强转基因表达和根据目标 1 和 2 的结果，进行细胞特异性基因靶向预防早期乙醇诱发的肝炎？我们的首要目标是比较 AAV 全肝血清型转导差异，随后进行体外研究评估库普弗细胞和肝细胞的血清型向性差异。其次，将针对细胞特异性鉴定和优化启动子元件库普弗细胞和肝细胞中的基因表达。最后，使用鼠标对于乙醇诱发的肝炎，我们将使用肠内乙醇喂养模型目标 1 和 2 中开发的试剂用于解决氧化剂生成的作用以及氧化还原敏感转录因子 NFB 激活，特别是在枯否细胞或肝细胞。转录因子 NFB 的激活是我们的假设的核心是解释早期乙醇诱发的肝炎；因此，针对库普弗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案的关键。我们期待这些实验证明库普弗细胞 NADPH 氧化酶是一种假设导致一系列炎症反应的氧化剂的主要来源（即 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，这些发现将导致开发临床上有用的基因转移系统，并使我们能够解决与细胞类型之间相互作用相关的关键问题它们参与早期乙醇引起的肝损伤的发病机制。此外，通过教学培训以及与导师和其他人的互动关键教师，申请人将获得新技能，使他能够成为酒精研究界的成功成员。