Assay Development and Validation for Precision Antiretroviral Therapy to Combat Drug Resistance

对抗耐药性的精准抗逆转录病毒疗法的测定开发和验证

• 批准号: 10882256
• 负责人: CHAOPING CHEN
• 金额: $ 34.04万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-23 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10882256
• 关键词: Acquired Immunodeficiency Syndrome; Anti-Retroviral Agents; Biological Assay; Caring; Cells; Clinical; Clinical Treatment; Coin; Complex; Consensus; Data; Drug resistance; Effectiveness; Evolution; FDA approved; Family; Genotype; Goals; HIV; HIV Infections; HIV Seropositivity; HIV drug resistance; Human; Indinavir; Individual; Integrase Inhibitors; Laboratories; Left; Lentivirus; Life; Lymphocyte; Modeling; Mutation; Outcome; Pathway interactions; Patient Care; Patient Selection; Patients; Peptide Hydrolases; Performance; Pharmaceutical Preparations; Phenotype; Plasma; Point Mutation; Population; Prediction of Response to Therapy; Predisposition; Prognosis; Protease Inhibitor; RNA-Directed DNA Polymerase; Red nucleus structure; Regimen; Reporter; Reporting; Resistance; Resistance development; Resistance profile; Resolution; Role; Salvage Therapy; Side; Testing; Therapeutic; Time; Transfection; Treatment Efficacy; Treatment outcome; Validation; Vertebral column; Viral; Viral Load result; Virus; Virus Replication; antiretroviral therapy; assay development; clinical application; clinical phenotype; clinical prognosis; combat; data dissemination; defined contribution; detection sensitivity; dissemination strategy; drug resistance development; experience; experimental study; improved; individualized medicine; inhibitor; insight; member; mutant; outcome prediction; particle; personalized medicine; point of care; promoter; resistance mechanism; response; success; vaccine access

Assay Development and Validation for Precision Antiretroviral Therapy to Combat Drug Resistance Project Summary This application is in response to NOT-AI-21-056 (HIV Drug Resistance Assays and Actionable Data Dissemination Strategies) focusing on assay development and validation to improve care for patients experiencing drug resistance (DR). Most HIV-positive individuals under combination antiretroviral therapy (cART) can have successful viral suppression, although, a subpopulation is unable to keep viral load under control. HIV DR is expected to have a growing impact on the overall effectiveness of cART with the underlying causes being complex and multifaceted. Current genotype and phenotype analyses have limitations in providing consistent and accurate prediction of treatment outcome, and not all patients receiving second- or third-line salvage therapy regimen achieve virological suppression. Therefore, precise and personalized medicines are needed to help care for these misfortunate individuals. We recently established an infectivity assay employing proviral constructs carrying an H2B-mRFP reporter driven by the nef promoter, which highlights the infected cells with a red nucleus allowing for infectivity quantification at a single cell resolution. By comparing the WT and a protease double mutant (V77I/V82T, identified in a patient experiencing indinavir resistance), our phenotype analysis successfully recapitulated the clinical manifestation and defined contributions of each point mutation to DR development showing that mutation 82T predominantly confers indinavir resistance and increases darunavir susceptibility at the same time. Our data support the common consensus that DR can be caused by multiple pathways with each displaying distinct susceptibility towards specific cART regimens, which would provide a therapeutic opportunity to maximize efficacy by selecting antiretroviral drugs based on patient-derived sequences – personalized medicine at point of care. The main objective of this proposal is to determine the technical merit and feasibility of our assay for accurate and consistent DR assessment in guiding precision cART regimen selection. Studies in Aim 1 will improve throughput capacity of the current assay and establish a platform for phenotype analysis of model lymphocytes – the natural targets of HIV infection. Studies in Aim 2 will focus on characterization of protease inhibitor resistance-associated mutations (RAMs) to define the role of individual mutations in DR development and to validate our assay performance and to establish genotype-phenotype correlation baselines. Studies in Aim 3 will establish and validate assay platforms for DR assessment of different cART regimens containing various combinations of protease, reverse transcriptase, and integrase inhibitors in the context of subtype-specific backbones. Results of these proposed studies will generate critical proof-of-concept for our phenotype assay in providing a reliable and consistent platform for DR assessment. The overall goal of this project is to validate the use of our phenotype assay with its much-improved resolution and accuracy to predict cART efficacy from the sequence information found in HIV-infected patients and thereby assist in cART regimen selection specifically tailored to the individual.

用于精确抗逆转录病毒治疗以对抗耐药性的检测方法开发和验证 项目摘要 本申请是对NOT-AI-21-056（HIV耐药性测定和可操作数据）的响应 传播策略），侧重于检测开发和验证，以改善患者护理 耐药性（DR）。大多数接受抗逆转录病毒联合治疗的艾滋病毒阳性者 可以成功地抑制病毒，尽管亚群不能控制病毒载量。艾滋病毒 DR预计将对cART的整体有效性产生越来越大的影响，其根本原因是 复杂而多面的。目前的基因型和表型分析在提供一致的 并准确预测治疗结局，并非所有接受二线或三线挽救治疗的患者 方案实现病毒学抑制。因此，需要精确和个性化的药物来帮助 照顾这些不幸的人。我们最近建立了一个感染性试验采用前病毒的结构 携带由nef启动子驱动的H2 B-mRFP报告基因，该基因突出显示了具有红色核的感染细胞 允许以单细胞分辨率进行感染性定量。通过比较WT和蛋白酶双 突变体（V77 I/V82 T，在经历茚地那韦耐药的患者中鉴定），我们的表型分析成功 概括了临床表现和每个点突变对DR发展的明确贡献 表明突变82 T主要赋予茚地那韦耐药性，并增加地瑞那韦敏感性， 同时还研究与讨论我们的数据支持这样一个共识，即DR可以由多种途径引起， 显示出对特定cART方案的明显敏感性，这将提供治疗机会， 通过基于患者衍生序列选择抗逆转录病毒药物来最大限度地提高疗效-个性化 在护理点的药物。本提案的主要目的是确定技术优点和可行性 我们的检测试剂盒用于准确和一致的DR评估，以指导精确的cART方案选择。研究 目的1中的方法将提高当前检测的通量能力，并建立表型分析平台 模型淋巴细胞-艾滋病毒感染的天然目标。目标2中的研究将侧重于 蛋白酶抑制剂耐药相关突变（RAM），以确定单个突变在DR中的作用 开发和验证我们的检测性能，并建立基因型-表型相关基线。 目标3中的研究将建立和验证用于不同cART方案的DR评估的测定平台 含有蛋白酶、逆转录酶和整合酶抑制剂的各种组合， 亚型特异性骨架。这些拟议研究的结果将为我们的 表型分析为DR评估提供了可靠和一致的平台。总的目标是 该项目是验证我们的表型测定的使用，其分辨率和准确性大大提高，以预测 从HIV感染患者中发现的序列信息中获得cART疗效，从而辅助cART方案 为个人量身定制的选择。