Novel Assays for measuring Integrase and RNase-H Inhibition

描述（由申请人提供）：目前批准的药物通过干扰蛋白酶（PR）、逆转录酶（RT）的酶活性或进入过程来抑制HIV-1复制。HIV对批准的PR、RT和进入抑制剂快速发展耐药性的能力，以及当前抗逆转录病毒方案的毒性问题，需要开发额外类别的抗病毒药物。整合酶（integrase，IN）作为HIV-1复制的必需酶，是一个很有吸引力的抗病毒靶点。几种IN抑制剂（INI）的临床评价（1期和2期）正在进行中，预计至少有一项3期试验将于2006年开始。本项目的最终目标是验证临床使用快速、灵敏的表型和基因型检测方法，以评价HIV-1分离株对所有HIV-1病毒酶抑制剂的敏感性，特别强调IN抑制剂（INI）。检测试剂盒开发将涉及对两种替代载体系统（pol或RHIN）的评价，这两种载体系统可产生含有来自患者分离株的HIV-1 pol蛋白的病毒颗粒。这些检测将基于PhenoSense HIVTM的技术基础，PhenoSense HIVTM是一种现有的基于细胞的抗病毒药物敏感性检测，用于评估蛋白酶（PR）和逆转录酶（RT）的抑制剂。本项目的具体目标是：目标1：选择临床验证（第1年）A的测定格式。优化pol检测试剂盒的RT-PCR扩增步骤，目的是尽可能接近复制现有CLIA验证的PI和RTI PhenoSense和GeneSeq检测试剂盒以及RUO验证的INI RHIN检测试剂盒的性能特征。B。将单pol检测试剂盒的表型和基因型结果与当前经验证的PhenoSense检测试剂盒（PI和RTI敏感性、复制能力）以及I期C期间开发的RHIN检测试剂盒（INI敏感性）的结果进行比较。选择pol或RHIN检测试剂盒进行预验证和最终CLIA验证。目的2：对RHIN或pol表型和基因型测定（第2年）进行预验证研究（包括pol测定的RUO验证（如选择））。目的3：验证商业临床实验室中RHIN或pol检测试剂盒形式的性能（第3年）A。临床验证RHIN或pol表型测定B的性能。临床验证RHIN或pol基因型检测试剂盒C的性能。获得CLIA认证，以进行经验证的基因型和表型检测HIV-1能够迅速发展出对获批药物的耐药性，加上目前抗逆转录病毒治疗方案的毒性问题，需要开发其他类别的抗病毒药物。整合酶（integrase，IN）作为HIV-1复制的必需酶，是一个很有吸引力的抗病毒靶点。几种IN抑制剂的临床评价正在进行中。本项目的最终目标是验证临床使用快速、灵敏的表型和基因型检测方法，以评价HIV-1分离株对所有HIV-1病毒酶抑制剂（特别是IN抑制剂）的敏感性。

DESCRIPTION (provided by applicant): Currently approved drugs inhibit HIV-1 replication by interfering with the enzymatic activities of either protease (PR), reverse transcriptase (RT), or with the entry process. The ability of HIV to rapidly evolve drug resistance to approved PR, RT and entry inhibitors, together with toxicity problems of current antiretroviral regimens, requires the development of additional classes of antiviral drugs. As an essential enzyme for HIV-1 replication, integrase (IN) is an attractive antiviral target. Clinical evaluation (phase 1 and 2) of several IN inhibitors (INIs) is underway, with at least one phase 3 trial expected to begin in 2006. The ultimate goal of this project is to validate for clinical use rapid, sensitive phenotypic and genotypic assays to evaluate the susceptibility of HIV-1 isolates to inhibitors of all HIV-1 viral enzymes, with particular emphasis on IN inhibitors (INIs). Assay development will involve evaluation of two alternative vector systems (pol or RHIN) that produce virus particles containing HIV-1 pol proteins derived from patient isolates. These assays will be based on the technology underlying PhenoSense HIVTM, an existing cell-based antiviral drug susceptibility assay that evaluates inhibitors of protease (PR) and reverse transcriptase (RT). Specific aims for this project are: Aim 1: Select assay format for Clinical Validation (YEAR 1) A. Refine the RT-PCR amplification steps of the pol assay, with the objective being to replicate as closely as possible the performance characteristics of the existing, CLIA validated PhenoSense and GeneSeq assays for PIs and RTIs, and the RUO validated RHIN assays for INIs. B. Compare phenotypic and genotypic results from the single pol assay to those from the currently validated PhenoSense assay (susceptibility to PIs and RTIs, Replication Capacity), and from the RHIN assay developed during Phase I (susceptibility to INIs) C. Select either the pol or RHIN assays for pre-validation and eventual CLIA validation. Aim 2: Perform pre-validation studies (including RUO validation for pol assay if selected) for RHIN or pol phenotypic and genotypic assay (YEAR 2). Aim 3: Validate the performance of the RHIN or pol assay format in a commercial clinical laboratory (YEAR 3) A. Clinically validate the performance of the RHIN or pol phenotypic assay B. Clinically validate the performance of the RHIN or pol genotypic assay C. Obtain CLIA certification for validated genotypic and phenotypic assays The ability of HIV-1 to rapidly evolve resistance to approved drugs, together with toxicity problems of current antiretroviral regimens, requires the development of additional classes of antiviral drugs. As an essential enzyme for HIV-1 replication, integrase (IN) is an attractive antiviral target. Clinical evaluation of several IN inhibitors is underway. The ultimate goal of this project is to validate for clinical use rapid, sensitive phenotypic and genotypic assays to evaluate the susceptibility of HIV-1 isolates to inhibitors of all HIV-1 viral enzymes, with particular emphasis on IN inhibitors.