New Phenotypic HIV-1 Drug Resistance Assay Validation

描述(由申请人提供)：改进的表型敏感性 艾滋病患者HIV-1耐药性监测方法的研究进展 对耐药的次要物种的早期检测是必要的。我们是 一种利用分子载体系统产生指示剂的新分析方法 过度表达CD4、CXCR4和CCR5基因的细胞，使它们 允许T嗜性和M嗜性HIV-1感染，使药物 耐药性分析可以使用直接感染的方法进行， 病人血清。这些指示细胞，包含TAT激活的分子 控制GFP报告基因开关，提供了一种敏感的检测机制 并在药物存在和不存在的情况下量化病毒复制。系统 允许根据感染病毒的实际水平进行病毒载量分析 现在时。它应该可以很容易地检测到低水平的抗药性病毒颗粒 在与主要是野生型病毒的混合物中，因为只有抗药性病毒 会在药物存在的情况下在指示细胞中复制并触发 报告基因的表达。由于不涉及聚合酶链式反应或克隆步骤， 这种化验方法直接测定病人体内的内在耐药性。 原始病毒种群。新的化验方法有望成为一种简单、灵敏和 用于HIV-1耐药性监测的高容量表型试验将是 适用于所有类别的抗艾滋病毒药物。此第一阶段SBIR-AT-NIAID赠款 应用程序请求资金以优化、格式化和验证新的化验方法， 根据HIV-1耐药合作小组发布的指南 (RCG)，在商业化之前。这将需要对准确性进行研究， 该方法的灵敏度、精密度、特异度和稳定性。它将涉及到 对大量具有良好特征的HIV-1进行广泛的回顾性研究 临床研究中的临床分离株(野生型和耐药株) 合作者和来自制药公司的存储库，诊断 公司和美国国家卫生研究院及其相对敏感性的确定 到目前批准的抗逆转录病毒药物。 建议的商业应用： 这项研究项目是为了验证一个显著改进的新表型 具有很大商业市场潜力的抗HIV耐药性监测方法 在艾滋病患者身上。它的应用包括通过临床参考进行分析的性能 实验室支持患者管理和临床试验，开发诊断 用于全球分发的试剂盒，基于传染性病毒水平的病毒负载分析，以及高 抗HIV药物发现的吞吐量分析。

DESCRIPTION (Provided by the applicant): Improved sensitivity of phenotypic assays for monitoring HIV-1 drug-resistance in AIDS patients, with an emphasis on early detection of drug-resistant minor species, is needed. We are developing a new assay using a molecular vector system to produce indicator cells that overexpress the genes for CD4, CXCR4, and CCR5, making them permissive to both T-tropic and M-tropic HIV-1 infection so that drug resistance assays can be performed using a direct infection approach with patient sera. These indicator cells, containing a tat-activated molecular switch controlling a GFP reporter gene, provide a sensitive mechanism to detect and quantify virus replication in the presence and absence of drugs. The system allows for viral load assays based on the actual levels of infectious virus present. It should readily detect low levels of drug-resistant virus particles in a mixture with predominantly wild-type virus, since only the resistant virus will replicate in the indicator cells in the presence of drug and trigger the expression of the reporter gene. Since PCR or cloning steps are not involved, this assay measures directly the intrinsic drug resistance of the patient's original virus population. The new assay promises to be a simple, sensitive and high-capacity phenotypic test for HIV-1 drug resistance monitoring and will be applicable to all classes of anti-HIV drugs. This Phase I SBIR-AT-NIAID grant application requests funding to optimize, format, and validate the new assay, according to guidelines issued by the HIV-1 Drug Resistance Collaborative Group (RCG), prior to commercialization. This will require studies of the accuracy, sensitivity, precision, specificity and stability of the assay. It will involve extensive retrospective studies with large numbers of well-characterized HIV-1 clinical isolates (both wild-type and resistant strains) from clinical research collaborators and from repositories at pharmaceutical companies, diagnostic companies, and the NIH and the determination of their relative susceptibilities to currently approved antiretroviral drugs. PROPOSED COMMERCIAL APPLICATION: This research project is for the validation of a significantly improved new phenotypic assay with a very large commercial market potential for anti-HIV drug resistance monitoring in AIDS patients. Its applications include performance of the assay by clinical reference laboratories in support of patient management and clinical trials, development of a diagnostic kit for worldwide distribution, viral load assays based on levels of infectious virus, and high- throughput assays for anti-HIV drug discovery.