Long-Term Inhibition of HIV transcription by targeting cellular CDK9 in vivo.

描述：HIV在人类宿主中的传播需要细胞进入、病毒RNA的逆转录、整合到人类基因组中、整合的前病毒的转录以及新病毒颗粒的组装/释放。目前，除了前病毒转录外，有针对这些病毒步骤的抗逆转录病毒药物(ART)。尽管现有ART的组合控制了大多数患者的艾滋病毒复制，但药物毒性和耐药性仍然令人担忧，因此需要发现更多具有新作用机制的ART。特别是，艾滋病毒转录抑制物既可能增加治疗效力，又可能抑制耐药菌株，从而改善患者的生活。我们的长期目标是通过靶向病毒生命周期中必不可少的细胞因子来改善艾滋病毒患者的治疗。蜂窝 细胞周期蛋白依赖性激酶9(CDK9)是细胞基因和HIV前病毒转录所必需的。在体外用催化抑制剂、RNAi和使用显性阴性形式直接抑制CDK9的方法都表明，在没有毒性的情况下抑制HIV转录是可能的。由于HIV治疗是终生的，因此确定CDK9长期抑制在慢性HIV感染中的安全性和抗病毒效果至关重要。我们和其他人之前已经证明，蓝宝石3‘-单肟(IM)是一种中药成分的衍生物，在没有细胞毒性的情况下抑制原代淋巴细胞和巨噬细胞中CDK9和HIV的表达。在初步研究中，我们发现IM在无毒性的情况下抑制与人淋巴细胞移植的NSG小鼠的血浆HIV RNA，为CDK9催化抑制剂在体内抑制HIV提供了第一个证据。IM单独抑制HIV RNA的作用单位为2log10，与NRTI EFdA在人源化小鼠中实现的HIV减少幅度相似。我们还表明，来自NRTI、蛋白酶和整合酶抑制剂类别的IM和ARTS在体外具有良好的抗HIV作用，提示IM可以与现有的ARTS结合使用。这项应用的目的是通过评估抗病毒机制、耐药性和对慢性感染HIV的人源化小鼠的长期毒性来评估CDK9抑制剂的抗HIV潜力。我们的假设是，使用CDK9催化抑制剂进行慢性治疗可以安全地抑制HIV在体内的转录。我们将使用IM或两种新的CDK9抑制剂来验证这一假设。有两个具体目标。具体目的1：评价抑制CDK9对人CD34+细胞移植NSG小鼠(HSC-NSG小鼠)的毒性、药代动力学和抗病毒活性。特定目的2：评价CDK9抑制剂对HSC-NSG小鼠HIV的抗病毒活性和长期控制作用的机制。这项建议将评估通过延长CDK9抑制剂在慢性感染小鼠中的治疗来阻断艾滋病毒转录的可能性，类似于患者对艾滋病毒的终身治疗。我们假设的成功测试可以通过有效地靶向病毒转录来改善艾滋病毒的治疗。

DESCRIPTION: Propagation of HIV in the human host requires cell entry, reverse transcription of viral RNA, integration into the human genome, transcription of the integrated provirus, and assembly/release of new virus particles. Currently there are antiretrovirals (ARTs) against each of these viral steps, except for provirus transcription. Although combinations of existing ARTs control HIV replication in most patients, drug toxicity and drug resistance are remaining concerns, arguing for the discovery of additional ARTs with novel mechanisms of action. In particular, an inhibitor of HIV transcription might both increase potency of treatment and suppress drug-resistant strains, improving the lives of patients. Our long-term goal is to improve treatment in HIV patients by targeting cellular factors essential in the virus life cycle. Cellular cyclin-dependent kinase 9 (CDK9) is required for transcription of both cellular genes and the HIV provirus. Approaches targeting CDK9 in vitro with catalytic inhibitors, RNAi, and direct inhibition using a dominant negative form, have all suggested that inhibition of HIV transcription without toxicity might be possible. Because HIV therapy is life-long, it is critical to determine safety and antiviral effectiveness of prolonged CDK9 inhibition in chronic HIV infection. We, and others, have previously shown that Indirubin 3'-monoxime (IM), a derivative of an ingredient in Chinese traditional medicine, inhibits CDK9 and HIV expression in primary lymphocytes and macrophages without cytotoxicity. In Preliminary Studies we show that IM suppresses plasma HIV RNA in NSG mice transplanted with human lymphocytes in the absence of toxicity, providing the first evidence for HIV inhibition by a CDK9 catalytic inhibitor in vivo. IM alone suppressed HIV RNA by > 2 log10 units, a magnitude of HIV reduction similar to that achieved with the NRTI EFdA in humanized mice. We also show that IM and ARTs from the NRTI, protease and integrase inhibitor classes have favorable anti-HIV interactions in vitro, suggesting IM could be used in combination with current ARTs. The goal of this application is to assess the anti-HIV potential of CDK9 inhibitors by evaluating antiviral mechanism, drug resistance and long- term toxicity in humanized mice chronically infected with HIV. Our hypothesis is that chronic treatment with CDK9 catalytic inhibitors can safely inhibit HIV transcription in vivo. We will test this hypothesis using IM or, alternatively, two novel CDK9 inhibitors. There are two Specific Aims. Specific Aim 1: To evaluate toxicity, pharmacokinetics, and antiviral activity of CDK9 inhibition in NSG mice transplanted with human CD34+ cells (HSC-NSG mice). Specific Aim 2: To evaluate mechanism of antiviral activity and long-term control of HIV in HSC-NSG mice treated with a CDK9 inhibitor. This proposal will assess the potential of blocking HIV transcription by prolonged treatment with a CDK9 inhibitor in chronically infected mice, resembling life-long therapy of HIV in patients. Successful testing of our hypothesis could improve HIV therapy by effectively targeting virus transcription.