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。鉴于艾滋病毒对已批准的PR、RT和进入抑制剂迅速产生耐药性的能力，以及目前抗逆转录病毒治疗方案的毒性问题，需要开发更多种类的抗病毒药物。作为HIV-1复制的必需酶，整合酶（IN）是一个有吸引力的抗病毒靶点。几种IN抑制剂（INIs）的临床评估（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。该项目的最终目标是验证临床应用快速、敏感的表型和基因型分析，以评估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（INIs）。试验开发将涉及评估两种替代载体系统（pol或RHIN），它们产生含有从患者分离物中提取的HIV-1 pol蛋白的病毒颗粒。这些检测将基于现有的基于细胞的抗病毒药物敏感性检测技术PhenoSense HIVTM，用于评估蛋白酶（PR）和逆转录酶（RT）抑制剂。该项目的具体目标是：目标1：选择用于临床验证的检测格式（第一年）A.完善pol检测的RT-PCR扩增步骤，目标是尽可能地复制现有的CLIA验证的用于pi和rti的PhenoSense和GeneSeq检测，以及用于ini的RUO验证的RHIN检测的性能特征。B.将单pol测定的表型和基因型结果与当前验证的PhenoSense测定（对pi和RTIs的敏感性，复制能力）和I期开发的RHIN测定（对INIs的敏感性）进行比较。选择pol或RHIN测定进行预验证和最终的CLIA验证。目标2：对RHIN或pol表型和基因型分析进行预验证研究（如果选择的话，包括pol分析的RUO验证）（YEAR 2）。目标3:在商业临床实验室验证RHIN或pol检测格式的性能（YEAR 3） a .临床验证RHIN或pol表型检测的性能B.临床验证RHIN或pol基因型检测的性能C.获得已验证基因型和表型检测的CLIA认证HIV-1对已批准药物快速进化耐药性的能力，以及当前抗逆转录病毒治疗方案的毒性问题。需要开发更多种类的抗病毒药物。作为HIV-1复制的必需酶，整合酶（IN）是一个有吸引力的抗病毒靶点。几种IN抑制剂的临床评估正在进行中。该项目的最终目标是验证临床应用快速、敏感的表型和基因型分析，以评估HIV-1分离株对所有HIV-1病毒酶抑制剂的敏感性，特别是对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.