Short-term and long-term effects of methamphetamine exposure on residual viral transcription during treated HIV disease

李淑吉 项目总结/摘要 艾滋病毒感染者最常用的非法兴奋剂是甲基苯丙胺（MA）， 研究表明，强有力的证据表明，MA促进增加艾滋病毒的转录以及免疫 失调艾滋病毒治愈已成为一个重要的临床和研究优先考虑的证据， 尽管有有效的抗逆转录病毒治疗（ART），HIV感染者的免疫功能障碍。一个挑战 在全球范围内根除艾滋病毒的目标是针对最有可能受益的特定弱势群体， 但由于残留的病毒复制， 次优ART依从性和/或来自非法物质使用的直接免疫功能障碍。既往非人类 研究表明，MA直接诱导HIV产生并促进免疫激活， 炎症这些临床前研究结果表明，使用MA的HIV+个体可能会经历更大的 免疫功能障碍，并面临未来根除艾滋病毒的额外挑战。这个实验 R61/R33提案将研究长期和短期MA暴露对HIV+ ART的影响- 有MA使用史的受抑制个体。在目标1中，HIV+ ART抑制的当前MA参与者 将在一项纵向队列研究中招募使用者，以确定长期MA暴露对 残留病毒产生、基因表达、炎症和微量胺相关1（TAAR 1，一种有前途的 精神兴奋剂成瘾的药物靶标）信号传导。在目标2中，HIV+感染的ART抑制个体， 既往有非依赖性MA使用史的患者将入组干预性研究， 口服甲基苯丙胺，以确定短期MA暴露对残留病毒的影响 生产、基因表达、炎症和TAAR 1信号传导。MA暴露将在目标1中量化， 头发样品，以及在目标2中，在24小时监测期内收集多个血浆样品。措施 然后，MA暴露量的增加将与残留病毒的产生（HIV-1细胞相关的未剪接和 多重剪接的RNA，以及作为最近生产的标记物-附加型2-LTR DNA），基因表达 （通过流式细胞术验证蛋白表达）、TAAR 1信号传导（尿β-PEA水平）和炎症 （血浆白介素-6、白介素-10、肿瘤坏死因子-α和转化生长因子-β）。使用此 方法，然后我们将使用一个强大的基因功能验证目标1和2中确定的前60个基因， 使用原代HIV+ CD 4 + T细胞的CRISPR-Cas9编辑技术。这项研究将是第一个 人类遗传学研究，以直接评估MA暴露对残留病毒转录的影响， 这项研究的总体目标是将严格的临床研究设计与高水平的 通过“组学数据”来确定逆转HIV潜伏期、减少炎症的新靶点， 针对使用MA的HIV+ ART抑制个体的个性化未来治疗策略。

Sulggi Lee PROJECT SUMMARY/ ABSTRACT The most commonly used illicit stimulant in HIV-infected individuals is methamphetamine (MA), and prior studies demonstrate strong evidence that MA promotes increased HIV transcription as well as immune dysregulation. HIV cure has emerged as an important clinical and research priority given evidence of ongoing immune dysfunction in HIV-infected individuals despite effective antiretroviral therapy (ART). A challenge in achieving worldwide HIV eradication is targeting specific vulnerable populations who are most likely to benefit from an HIV cure but possess poorer immune responses as a result of residual viral replication due to suboptimal ART adherence and/or direct immune dysfunction from illicit substance use. Prior non-human studies demonstrate that MA directly induces HIV production and promotes immune activation and inflammation. These preclinical findings suggest that HIV+ individuals who use MA may experience greater immune dysfunction and face additional challenges for future HIV eradication. The experiments of this R61/R33 proposal will investigate the effects of long-term and short-term MA exposure in HIV+ ART- suppressed individuals with a history of MA use. In Aim 1, HIV+ ART-suppressed participants with current MA use will be enrolled in a longitudinal cohort study to determine the effects of long-term MA exposure on residual virus production, gene expression, inflammation, and trace amine-associated 1 (TAAR1, a promising drug target for psychostimulant addiction) signaling. In Aim 2, HIV+ infected ART-suppressed individuals with a prior history of non-dependent MA use will be enrolled in an interventional study where they will be administered oral methamphetamine to determine the effects of short-term MA exposure on residual virus production, gene expression, inflammation, and TAAR1 signaling. MA exposure will be quantified in Aim 1 with hair samples, and in Aim 2 with multiple plasma samples collected over a 24-hour monitoring period. Measures of MA exposure will then be associated with residual virus production (HIV-1 cell-associated unspliced and multiply spliced RNA, and, as a marker of recent production – episomal 2-LTR DNA), gene expression (validated by flow cytometry for protein expression), TAAR1 signaling (urinary β-PEA levels), and inflammation (plasma interleukin-6, interleukin-10, tumor necrosis factor-α, and transforming growth factor-β). Using this approach, we will then functionally validate the top 60 genes identified in Aims 1 and 2 using a powerful gene editing technique (CRISPR-Cas9) using primary HIV+ CD4+ T cells. The proposed study will be the first human genetic study to directly evaluate the effect of MA exposure on residual viral transcription during effective ART. The overall goals of the study are to integrate a rigorous clinical study designs with high throughput `omics data to identify novel targets for reversing HIV latency, reducing inflammation, and personalizing future therapeutic strategies specific to HIV+ ART-suppressed individuals who use MA.