Genetic analysis of unspliced HIV RNA produced during HDAC inhibitor therapy

项目摘要 目前的抗逆转录病毒疗法有效地抑制但不能根除HIV-1感染。化疗期间联合 抗逆转录病毒疗法（cART），通常可将HIV-1 RNA水平降至50拷贝/ml以下; 然而，使用超灵敏测定法在血浆中检测到持续的低水平病毒血症。源极和 目前正在研究这种持续病毒血症的动态。HIV-1的一个明确的储存库是记忆 CD 4 + T细胞，其中当活化的CD 4 + T细胞被HIV-1感染时，HIV-1潜伏期建立，但 转化为终末分化的记忆T细胞，而不是经历裂解性感染。上调细胞 转录诱导HIV基因表达已被提出作为减少潜伏- 感染的静息记忆CD 4 + T细胞和可能携带病毒基因组整合形式的骨髓细胞。 这包括抑制细胞组蛋白脱乙酰酶（HDAC），因为这些HDAC通过以下方式促进潜伏期： 调节基因组结构和转录活性。 最近用单剂量组蛋白治疗8名接受抑制性cART的HIV感染患者 脱乙酰酶抑制剂（HDACi）伏立诺他导致细胞相关未剪接（CA-US） 静息记忆CD 4 + T细胞中的HIV RNA。在澳大利亚的一项临床试验中，CA- 在接受14天治疗的HIV感染患者血液中的CD 4 + T细胞中观察到US HIV RNA。 HDACi伏立诺他。来自丹麦的HDACi帕比司他的多剂量研究的初步数据也 显示CA-US HIV RNA增加。 鉴于这些发现，我们假设：1）HDACi治疗后CA-US HIV RNA的序列将 是克隆性的，这与来自响应于免疫应答的受感染的静息记忆T细胞亚群的转录一致。 血液和肠道中的HDAC抑制剂; 2）特定细胞类型正在产生CA-US HIV RNA; 3）这种CA-US HIV RNA将在遗传上类似于治疗前（pre-cART和pre-HDACi）血浆衍生的HIV-1序列， 表明该储库在cART启动之前建立; 4）如果病毒在分析治疗期间反弹 在帕比司他治疗后中断，它将在遗传上类似于HDACi期间重新激活的CA-US HIV RNA 疗法这些假设将在以下具体目的的实验中得到解决：（1）确定 在多次免疫治疗前后，来自患者CD 4 + T细胞的CA-US HIV RNA的遗传组成和多样性， vorinostat或panobinostat剂量治疗;（2）确定未剪接的基因组成和多样性， 骨髓细胞和特异性记忆性CD 4 + T细胞亚群中的HIV-1 RNA;（3）比较 CA-US HIV RNA与来自治疗前样本和治疗后采集的样本的血浆来源HIV-1序列的比对 帕比司他治疗后分析治疗中断。 如果这项探索性研究揭示了CA-US HIV RNA在骨髓细胞中的克隆扩增和特异性静息记忆， 从HIV-1感染患者的HDAC抑制剂的T细胞，这将提供直接的证据，这些化合物 正在破坏这些细胞内的前病毒潜伏期，并且HIV潜伏期可以在人类中进行治疗。

PROJECT SUMMARY Current antiretroviral therapy effectively suppresses but does not eradicate HIV-1 infection. During combination antiretroviral therapy (cART), reduction of HIV-1 RNA levels to less than 50 copies/ml is frequently achieved; however, persistent low-level viremia has been detected in plasma using ultrasensitive assays. The source and dynamics of this persistent viremia is currently under investigation. One well-defined reservoir of HIV-1 is memory CD4+ T cells, where HIV-1 latency is established when an activated CD4+ T cell becomes infected by HIV-1 but transitions to a terminally differentiated memory T cell instead of undergoing lytic infection. Upregulating cellular transcription to induce HIV gene expression has been proposed as a strategy for reducing the pool of latently- infected resting memory CD4+ T cells and possibly myeloid cells carrying an integrated form of the viral genome. This includes inhibiting cellular histone deacetylases (HDACs) because these HDACs promote latency by regulating genome structure and transcriptional activity. A recent treatment of eight HIV-infected patients on suppressive cART with a single dose of the histone deacetylase inhibitor (HDACi), vorinostat, resulted in a significant increase in cell associated unspliced (CA-US) HIV RNA in resting memory CD4+ T cells. During a clinical trial in Australia, a similar significant increase in CA- US HIV RNA was observed in CD4+ T-cells in blood from HIV-infected patients treated with 14 days of the HDACi vorinostat. Preliminary data from a multidose study of the HDACi panobinostat in Denmark also demonstrates an increase in CA-US HIV RNA. Given these findings, we hypothesize that: 1) the sequence of CA-US HIV RNA following HDACi treatment will be clonal which is consistent with transcription from a subset of infected resting memory T cells responsive to HDAC inhibitors in the blood and gut; 2) specific cell types are producing CA-US HIV RNA; 3) this CA-US HIV RNA will be genetically similar to pre-therapy (pre-cART and pre-HDACi) plasma-derived HIV-1 sequences, indicating this reservoir was established prior to cART initiation; 4) if virus rebounds during an analytical treatment interruption post-panobinostat therapy, it will be genetically similar to CA-US HIV RNA reactivated during HDACi therapy. These hypotheses will be addressed in the experiments of the following Specific Aims: (1) to determine the genetic makeup and diversity of CA-US HIV RNA from CD4+ T cells of patients prior to and following multi- dose treatment with vorinostat or panobinostat; (2) to determine the genetic makeup and diversity of unspliced HIV-1 RNA in myeloid cells and specific memory CD4+ T cell subsets; and (3) to compare the genetic makeup of CA-US HIV RNA to plasma-derived HIV-1 sequences from pre-therapy samples and samples taken after an analytical treatment interruption following panobinostat therapy. If this exploratory study reveals clonal expansion of CA-US HIV RNA in myeloid cells and specific resting memory T cells from HIV-1 infected patients on HDAC inhibitors, this would provide direct evidence that these compounds are disrupting proviral latency within these cells and that HIV latency can be therapeutically targeted in humans.