Gene Therapy and the Brain: Neuroimmune Interactions

描述（由申请人提供）：基因治疗提供了令人兴奋的新方法来治疗许多无法治愈的神经退行性疾病，如帕金森病，多发性硬化症或脑癌。不幸的是，对治疗载体的免疫应答仍然是基因治疗临床实现的主要障碍。如果启动，免疫系统会消除大脑中治疗性转基因的表达，从而降低基因治疗的功效。我们将在临床相关模型中研究由临床有效的第一代腺病毒载体介导的转基因表达的免疫调节，以及新型的高容量“无胆”腺病毒载体。在该模型中，将对动物进行腺病毒预免疫，以模拟在接受基因治疗之前暴露于腺病毒的大多数人类患者的免疫状态。在这个提议中，我们将测试免疫系统消除治疗性转基因从大脑中表达的假设，主要是通过细胞毒性，其次是通过非细胞毒性机制。为了解决这些问题，我们开发了一种特异性方法来区分免疫系统诱导的脑细胞死亡与载体介导的转基因表达的选择性下调。该方法基于含有floxed β-gal构建体的转基因小鼠，以及在全细胞和细胞类型特异性启动子下表达Cre的病毒载体。因此，感染的细胞从其基因组表达基因标记，其表达将独立于病毒载体基因组的表达而受到调节。使用我们的新方法的初步实验表明，免疫系统利用两种机制，即它可以通过[1]转导细胞的直接细胞毒性消除脑中治疗性转基因的表达;和[2]转基因表达的功能抑制。在本文中，我们将在雄性和雌性以及两种小鼠品系中测试关于消除脑转基因表达的免疫机制的特定假设;一种显示TH 1偏向免疫应答（C57 BI/6），另一种显示TH 2偏向（DBA/2 J）。作为这项工作的结果，我们将提供增强和更安全的基因治疗方法，以及更有效的临床治疗模式。该提案的结果将对实验和临床基因治疗产生直接影响，并为理解免疫系统如何消除或调节病毒感染脑细胞中的基因表达做出重大贡献。

DESCRIPTION (provided by applicant): Gene therapy provides exciting new approaches to treat numerous incurable neurodegenerative disorders such as Parkinson's disease, multiple sclerosis, or brain cancer. Unfortunately the immune response to therapeutic vectors remains a major obstacle to the clinical realization of gene therapy. If primed, the immune system eliminates expression of therapeutic transgenes from the brain, curtailing gene therapy's efficacy. We will investigate immune regulation of transgene expression mediated by clinically effective first generation adenoviral vectors, and novel high capacity 'gutless' adenoviral vectors in a clinically relevant model. In this model animals will be pre-immunized to adenovirus to mimic immune status in the majority of human patients that were exposed to adenovirus before receiving gene therapy. In this proposal we will test the hypothesis that the immune system eliminates expression of therapeutic transgenes from the brain, primarily through cytotoxic, and secondarily, through non-cytotoxic mechanisms. To address these issues, we developed a specific method to differentiate immune system-induced brain cell death from selective down-regulation of vector-mediated transgene expression. This method is based on transgenic mice containing a floxed beta-gal construct, and viral vectors expressing Cre under pancellular and cell type specific promoters. Infected cells thus express a gene marker from their genomes, expression of which will be regulated independently of expression from the viral vector's genome. Preliminary experiments using our new method demonstrate that the immune system utilizes both mechanisms, namely it can eliminate expression of therapeutic transgenes from the brain by [1] direct cytotoxicity of transduced cells; and, [2] functional inhibition of transgene expression. Herein we will test specific hypothesis concerning the immune mechanisms that eliminate brain transgene expression, in both males and females, and in two mouse strains; one that displays TH1 biased immune responses (C57BI/6), and another one that displays a TH2 bias (DBA/2J). As a result of this work we will make available enhanced and safer gene therapy approaches, and more efficient clinical treatment paradigms. The results from this proposal will have a direct impact on experimental and clinical gene therapy, as well as make major contributions to understanding how the immune system eliminates or regulates gene expression in virally infected brain cells.