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.

测定开发和验证精确抗逆转录病毒疗法以对抗耐药性 项目摘要 该应用是响应于非AI-21-056（HIV耐药性测定和可起作的数据） 传播策略）专注于测定开发和验证以改善患者的护理 耐药性（DR）。大多数HIV阳性个体在抗逆转录病毒疗法（CART）下 可以成功抑制病毒，尽管亚群无法控制病毒载荷。艾滋病病毒 预计博士对购物车的整体有效性产生越来越大的影响，其根本原因是 复杂而多方面。当前的基因型和表型分析在提供一致的方面存在局限性 并准确预测治疗结果，并非所有接受第二或第三线救助治疗的患者 方案可实现病毒学抑制。因此，需要精确和个性化的药物来帮助 照顾这些不幸的人。我们最近建立了采用准备结构的感染评估 携带由NEF启动子驱动的H2B-MRFP报告基因，该记者用红色核突出了感染的细胞 允许在单个细胞分辨率下进行感染定量。通过比较WT和蛋白酶双重 突变体（V77I/V82T，在具有indinavir抗性的患者中鉴定出来），我们的表型分析成功 概括了每个点突变对DR发育的临床表现和定义的贡献 表明突变82T主要赋予indinavir抗性，并增加了达鲁纳韦的敏感性 同时。我们的数据支持共同的共识，即DR可能由多个途径引起 对特定的购物车方案显示出明显的敏感性，这将提供治疗机会 通过根据患者衍生的序列选择抗逆转录病毒药物来最大化效率 - 个性化 医疗处于护理点。该提案的主要目的是确定技术优点和可行性 我们评估准确且一致的DR评估，以指导精确的推车选择。研究 在AIM 1中将提高当前测定的吞吐量能力，并建立表型分析平台 模型淋巴细胞 - HIV感染的自然靶标。 AIM 2的研究将集中于表征 蛋白酶抑制剂抗性相关突变（RAM），以定义单个突变在DR中的作用 开发并验证我们的评估绩效并建立基因型 - 表型相关基线。 AIM 3中的研究将建立和验证评估平台，以评估不同的购物车方案 在 亚型特异性骨架。这些提出的研究的结果将为我们产生关键的概念概念 表型评估提供了可靠且一致的DR评估平台。总体目标 项目是验证我们的表型分析的使用，并以其大量改进的分辨率和准确性来预测 来自HIV感染患者的序列信息的推车效率，从而有助于CART方案 专门针对个人量身定制的选择。