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

精确抗逆转录病毒治疗对抗耐药性的检测方法的建立和验证 项目摘要 此应用程序是对NOT-AI-21-056(艾滋病毒耐药性分析和可操作数据)的响应 传播策略)专注于分析开发和验证，以改善对患者的护理 出现耐药(DR)。大多数HIV阳性患者正在接受联合抗逆转录病毒治疗(CART) 可以成功地抑制病毒，尽管一个亚群无法控制病毒载量。艾滋病病毒 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 复杂而多方面的。目前的基因型和表型分析在提供一致性方面存在局限性 和对治疗结果的准确预测，并不是所有接受二线或三线抢救治疗的患者 该方案实现了病毒学的抑制。因此，需要精准和个性化的药物来帮助 关心这些不幸的人。我们最近建立了一种使用前病毒构建物的传染性分析方法。 携带由nef启动子驱动的h2B-MRFP报告，该报告突出显示带有红核的受感染细胞 允许在单个细胞分辨率下对传染性进行量化。通过比较WT和一个蛋白水解酶 突变(V77I/V82T，在一名对依地那韦耐药的患者中发现)，我们的表型分析成功 总结了每个点突变的临床表现和明确的对DR发生的贡献 显示突变82T主要导致依地那韦耐药，并增加达鲁那韦的易感性 同一时间。我们的数据支持这样一个普遍共识，即每条路径都可能由多条路径引起 对特定的CART方案表现出明显的敏感性，这将提供治疗机会 通过根据患者衍生序列选择抗逆转录病毒药物来最大化疗效-个性化 医疗点的药物。该提案的主要目标是确定技术优势和可行性。 我们的试验用于准确和一致的DR评估，以指导精密CART方案的选择。研究 目标1将提高当前分析的吞吐能力，并建立一个表型分析平台 模型淋巴细胞--艾滋病毒感染的自然目标。在目标2中的研究将集中在表征 蛋白水解酶抑制物耐药相关突变(RAMS)确定个体突变在DR中的作用 开发和验证我们的检测性能，并建立基因-表型相关性基线。 AIM 3中的研究将建立和验证不同CART方案的DR评估的分析平台 在以下上下文中包含各种组合的蛋白酶、逆转录酶和整合酶抑制剂 特定于子类型的主干。这些建议研究的结果将为我们的 表型分析为DR的评估提供了可靠和一致的平台。这个项目的总体目标是 项目是验证我们的表型分析的使用，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获得CART疗效，从而辅助CART方案 专门为个人量身定做的选择。

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.