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

基本信息

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
项目类别：
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

项目摘要

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.

对抗耐药性的精准抗逆转录病毒疗法的测定开发和验证项目概要此应用程序是为了响应 NOT-AI-21-056（HIV 耐药性测定和可操作数据）传播策略）专注于检测开发和验证，以改善对患者的护理大多数 HIV 阳性个体正在接受联合抗逆转录病毒治疗 (cART)。可以成功抑制病毒，但有一个亚群无法控制艾滋病病毒载量。预计 DR 将对 cART 的整体有效性产生越来越大的影响，其根本原因是目前的基因型和表型分析在提供一致性方面存在局限性。并准确预测治疗结果，并非所有患者都接受二线或三线挽救治疗因此，需要精准和个性化的药物来帮助实现病毒学抑制。我们最近建立了一种采用前病毒构建体检测的感染性。携带由 nef 启动子驱动的 H2B-mRFP 报告基因，突出显示带有红核的感染细胞通过比较 WT 和蛋白酶双倍，可以在单细胞分辨率下进行感染性定量。突变体（V77I/V82T，在茚地那韦耐药患者中发现），我们的表型分析成功概括了临床表现以及每个点突变对 DR 发展的明确贡献显示突变 82T 主要赋予茚地那韦耐药性并增加达芦那韦敏感性同时，我们的数据支持了这样的共识：DR 可能由多种途径引起。对特定 cART 方案表现出明显的敏感性，这将提供治疗机会根据患者来源的序列选择抗逆转录病毒药物，以最大限度地提高疗效——个性化该提案的主要目的是确定技术优点和可行性。我们的准确一致的 DR 评估方法可指导精准的 cART 方案选择。目标 1 将提高当前检测的通量能力并建立表型分析平台目标 2 中的研究将集中于模型淋巴细胞的特征——HIV 感染的自然目标。蛋白酶抑制剂耐药相关突变 (RAM) 来定义单个突变在 DR 中的作用开发并验证我们的检测性能并建立基因型-表型相关基线。目标 3 的研究将建立并验证用于不同 cART 方案的 DR 评估的检测平台含有蛋白酶、逆转录酶和整合酶抑制剂的各种组合这些拟议研究的结果将为我们提供关键的概念验证。表型测定为 DR 评估提供可靠且一致的平台。该项目的目的是验证我们的表型测定的使用，其分辨率和预测准确性大大提高从 HIV 感染者中发现的序列信息得出 cART 疗效，从而协助 cART 治疗方案专门针对个人的选择。