MOLECULAR THERAPEUTICS FOR HIV COGNITIVE DYSFUNCTION

描述（改编自申请人摘要）：感染的星形胶质细胞和 小胶质细胞已被证明表达早期基因产物达特， 参与导致CNS内发病的多种途径 通过直接和间接的机制。因此，达特是一个有吸引力的目标， 设计用于治疗HIV-1诱导的CNS病症的治疗剂。我们 建议利用两种天然存在的蛋白质作为起始平台， 它们与达特、YB-1和Vpr相互作用。我们最近确定YB-1是一种 与活化反应元件达特相互作用的转录因子 TAR，以及HIV-1 LTR中存在的GC富集区。YB-1激活 HIV-1基因在中枢神经系统细胞中的表达，我们设计了突变形式的 蛋白质，其功能上无活性并且可以抑制Tat介导的 病毒LTR启动子的反式激活。我们提出了一个全面的分析， YB-1突变体战略性地设计治疗蛋白， 与达特，但抑制而不是激活病毒转录。Vpr激活了一个 多种细胞启动子，参与病毒核心的易位 颗粒到受感染细胞的细胞核，也能够失调， 细胞周期我们提出了达特与Vpr相互作用的证据， 一种基于Vpr设计反式显性负突变体的策略， 抑制达特功能。为了开发更有效的达特拮抗剂 功能使用YB-1和Vpr，我们建议分析的抑制特性， 含有转录阻遏元件的YB-1和Vpr融合蛋白， ERF-1是ETS家族蛋白，具有很强的阻遏活性。 我们将测试这些策略在原代CNS细胞培养物中的功效， 纯化的小胶质细胞、星形胶质细胞以及混合的CNS细胞培养物中。为了 为了递送靶向达特蛋白的治疗基因，我们将利用 最近开发的HSV-1基因递送系统允许表达 CNS细胞中的外源/治疗性基因。为此，我们将利用 组织特异性启动子、大唾液酸蛋白和胶质细胞酸性蛋白 （GFAP），为了将基因表达靶向小胶质细胞和星形胶质细胞， 分别

Description (adapted from applicant's abstract): Infected astrocytes and microglia have been shown to express the early gene product, Tat, which has been implicated in a variety of pathways leading to pathogenesis within the CNS via direct as well as indirect mechanisms. Thus Tat is an attractive target for the design of therapeutics for treatment of HIV-1 induced CNS disorders. We propose to utilize as a starting platform two naturally occurring proteins, which interact with Tat, YB-1 and Vpr. We have recently identified YB-1 as a transcription factor which interacts with Tat, the activation response element TAR, as well as the GC rich region present in the HIV-1 LTR. YB-1 activates HIV-1 gene expression in CNS cells and we have designed mutant forms of the protein, which are functionally inactive and can inhibit Tat-mediated transactivation of the viral LTR promoter. We propose a thorough analysis of YB-1 mutants to strategically design therapeutic proteins, which can interact with Tat, but inhibit rather than activate viral transcription. Vpr activates a variety of cellular promoters, is involved in translocation of viral core particles to the nucleus of infected cells, and also is able to dysregulate the cell cycle. We present evidence for the interaction of Tat with Vpr and present a strategy to design transdominant negative mutants based on Vpr which can inhibit Tat function. In order to develop more potent antagonists of Tat function using YB-1 and Vpr, we propose to analyze the inhibitory properties of YB-1 and Vpr fusion proteins containing the transcriptional repressor element, ERF-1, an ETS family protein which exhibits strong repressor activity. We will test the efficacy of these strategies in primary CNS cell cultures of purified microglia, astrocytes, as well as in mixed CNS cell cultures. In order to delivery therapeutic genes targeting Tat protein, we will utilize the recently developed HSV-1 gene delivery system permitting the expression of foreign/therapeutic genes in CNS cells. Toward this end, we will utilize the tissue specific promoters, macrosialin and glial fibrillary acidic protein (GFAP), in order to target gene expression to microglia and astrocytes, respectively.