Characterization of Cellular Proteins Involved in HIV Infection

描述（由申请人提供）：在过去二十年中，特别是随着治疗艾滋病毒感染的药物的广泛可用，在治疗病毒感染方面取得了重要进展，但需要开发新的药物，特别是考虑到艾滋病毒疫苗开发面临的挑战和产生耐药性的倾向。随着2007年FDA批准首个靶向细胞基因（HIV共受体CCR5）的抗逆转录病毒药物，现在有证据表明，细胞靶点可以用于开发高效、安全、无毒且耐受性良好的抗逆转录病毒药物。虽然在某种程度上是传染病的范式转变，但事实上，细胞基因通常是针对非传染性疾病的。无论如何，这证明了靶向细胞基因进行抗病毒治疗的潜力。该提案旨在扩展过去几年完成的工作，使用专有技术基因陷阱，这是一种快速有效的方法，用于识别病毒感染，复制或发病所必需的宿主基因。基因诱捕器插入突变已经鉴定出不表达基因的细胞；这些基因的缺失使细胞能够在致命病毒感染期间存活，但显然不是细胞存活所必需的。迄今为止，我们已经确定了许多病毒共同使用的几种途径，这表明可能存在发病所必需的关键细胞机制，并且在体内阻断这种机制可以授予广谱治疗干预。我们的假设是，通过筛选被基因陷阱识别并被流感病毒感染确认的基因的sirna，并在HIV感染的指示细胞系和原代巨噬细胞中进行测试，我们将发现新的宿主基因靶点，可用于开发抗逆转录病毒药物。在第一阶段，我们将根据明确的标准来选择药物发现标准的先导靶点，测试下调特定基因指示细胞系对HIV的影响。最重要的标准是为目标提供高分辨率的三维晶体结构。晶体结构的重要性在于促进将在第一阶段开始的药物发现工作，这将是项目第二阶段的主要重点。使用易于用siRNA转染的指示细胞系U373 MAGI，我们将通过β - gal活性来测试对HIV复制的影响，β - gal活性将确定基因表达之前的复制抑制，包括进入逆转录和整合，我们还将通过HIV p24的产生和HIV RNA的实时PCR来测量细胞外病毒，以捕获病毒生命周期后半段发生的抑制事件。通过并行进行这两种分析，我们确定该基因在早期病毒生命周期事件或后期事件中是否重要。为了确认这些基因与HIV复制的相关性，我们将评估基因下调对原代人巨噬细胞的影响，巨噬细胞是感染的天然靶点。在进行基因诱捕和开发流感病毒感染药物的独立研究中，将评估对艾滋病毒复制的影响的基因将在小鼠研究中同时得到评估。因此，我们将有这些基因的动物安全性数据，在动物中保持一个月的下调。由Dennis Liotta博士领导的埃默里大学的合作者将用小分子文库筛选被确定为对HIV感染重要的基因，并符合我们的药物开发标准。这一建议可能会导致大量新的抗逆转录病毒药物影响新的治疗靶点。公共卫生相关性：在过去十年中，在治疗艾滋病毒感染方面取得了巨大进展，在过去一到两年推出了三种安全、耐受性良好且通常对耐旧药物的病毒株有效的新药。然而，艾滋病毒的治疗渠道并不稳健，在接下来的几年里，整合酶和CCR5等几种针对相同目标的“我也是”药物问世后，临床开发的其他抗逆转录病毒疗法并不多。因此，由于许多患者出现了对旧药物的耐药性，因此迫切需要开发针对新的HIV靶点的新药。我们的建议将确定人类蛋白质作为HIV感染的重要靶标，可用于开发新型抗逆转录病毒药物。PHS 398/2590 （Rev. 11/07）第1页延续格式页

DESCRIPTION (provided by applicant): Important advances have been made in the treatment of viral infections over the last two decades, especially with the extensive availability of drugs to treat HIV infection, but there is a need to develop new drugs particularly with the challenges facing HIV vaccine development and the propensity to develop drug resistance. With FDA approval in 2007 of the first antiretroviral drug targeting a cellular gene, the HIV coreceptor CCR5, there is now proof of concept that a cellular target can be used to develop a highly effective, safe, nontoxic and well-tolerated antiretroviral drug. Although somewhat of a paradigm shift for infectious diseases, in fact, cellular genes are targeted routinely for non-infectious diseases. In any case this demonstrates the potential for targeting cellular genes for antiviral therapy. This proposal aims to extend the work accomplished over the last several years using a proprietary technology, gene-trap, which is a rapid and efficient method for the identification of host genes that are necessary for infection, replication or pathogenesis by viruses. Gene-trap insertional mutagenesis has identified cells with genes that are not expressed; loss of these genes allows the cell to survive during lethal virus infection, but apparently is not required for cell survival. To date, we have identified several pathways used in common by a number of viruses, suggesting that there may be key cellular mechanisms necessary for pathogenesis and that blocking such mechanisms in vivo could confer broad-spectrum therapeutic intervention. Our hypothesis is that by screening siRNAs to genes identified by gene trap and confirmed by influenza virus infection and testing them in indicator cell lines and primary marrophages for HIV infection, we will discover new host gene targets that can be used to develop antiretroviral drugs. During Phase I, we will test the effects of down regulation of specific genes indicator cell lines for effect on HIV based on well-defined criteria to select lead targets for drug discovery criteria. The most important criteria is a high resolution 3D crystal structure available for the target. The importance of crystal structure is to facilitate drug discovery efforts that will begin in phase I and that will be the primary focus in phase II of the project. Using the indicator cell line U373 MAGI, which are readily transfectable with siRNA, we will test the effect on HIV replication both by beta-GAL activity, which will identify inhibition of replication at steps prior to gene expression, including entry reverse transcription and integration, and we will also measure extracellular virus by HIV p24 production and Real-time PCR for HIV RNA, to capture inhibition events that occur in the second half of the virus life cycle. By performing these two assays in parallel we determine whether the gene is important in early viral life cycle events or later ones. To confirm the relevance of these genes for HIV replication we will assess effects of gene down regulation on primary human macrophages, which are natural targets for infection. In independent studies to perform gene-trap and develop drugs for influenza virus infection, the genes that will be assessed for effects on HIV replication will have been assessed in parallel in mouse studies. Therefore, we will have animal safety data for these genes, with down regulation maintained in the animals for a period of one month. Genes identified as important for HIV infection, and meeting our criteria for drug development, will be screened with small molecule libraries with collaborators at Emory, lead by Dennis Liotta, PhD. This proposal will be likely to lead to a large number of new antiretroviral drugs that affect novel targets for therapy. PHS 398/2590 (Rev. 11/07) Page 1 Continuation Format Page PUBLIC HEALTH RELEVANCE: Tremendous advances have been made in treatment of HIV infection over the last ten years, with introduction of three new drugs in the last one to two years that are safe, well-tolerated and typically effective against virus strains resistant to older drugs. The HIV pipeline is not robust, however, and after several "me too" drugs directed to the same targets over the next several years, integrase and CCR5, there are not many other antiretroviral therapies that will be developed clinically. Thus, there is an urgent need to develop new drugs against novel targets for HIV since resistance to older drugs emerges in many patients. Our proposal will identify human proteins as targets important for HIV infection that can be used to develop new classes of antiretroviral drugs. PHS 398/2590 (Rev. 11/07) Page 1 Continuation Format Page