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

产品说明：HIV在人类宿主中的繁殖需要细胞进入、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。目前有抗逆转录病毒药物（ART）针对这些病毒步骤中的每一个，除了前病毒转录。虽然现有的ART的组合控制艾滋病毒在大多数患者中的复制，药物毒性和耐药性仍然是关注的问题，主张发现具有新作用机制的其他ART。特别是，HIV转录抑制剂可能既能提高治疗效果，又能抑制耐药菌株，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（CDK 9）是细胞基因和HIV原病毒转录所必需的。在体外用催化抑制剂、RNAi靶向CDK 9的方法和使用显性阴性形式的直接抑制都表明，无毒性地抑制HIV转录是可能的。由于HIV治疗是终身的，因此确定长期CDK 9抑制在慢性HIV感染中的安全性和抗病毒有效性至关重要。我们和其他人先前已经表明，靛玉红3 '-单肟（IM），一种中药成分的衍生物，抑制CDK 9和HIV在原代淋巴细胞和巨噬细胞中的表达，而没有细胞毒性。在初步研究中，我们表明IM在无毒性的情况下抑制移植人淋巴细胞的NSG小鼠中的血浆HIV RNA，这为CDK 9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> 2 log 10单位，HIV减少的幅度与NRTI EFdA在人源化小鼠中实现的相似。我们还表明，IM和ART从NRTI，蛋白酶和整合酶抑制剂类具有良好的抗HIV的体外相互作用，这表明IM可以与目前的ART组合使用。本申请的目的是通过在慢性感染HIV的人源化小鼠中评价抗病毒机制、耐药性和长期毒性来评估CDK 9抑制剂的抗HIV潜力。我们的假设是，使用CDK 9催化抑制剂长期治疗可以安全地抑制体内HIV转录。我们将使用IM或两种新型CDK 9抑制剂来检验这一假设。有两个具体目标。具体目标1：在移植人CD 34+细胞的NSG小鼠（HSC-NSG小鼠）中评价CDK 9抑制的毒性、药代动力学和抗病毒活性。具体目的2：评价CDK 9抑制剂治疗的HSC-NSG小鼠中HIV的抗病毒活性和长期控制机制。该提案将评估通过长期使用CDK 9抑制剂治疗慢性感染小鼠来阻断HIV转录的潜力，类似于患者的HIV终身治疗。我们假设的成功测试可以通过有效靶向病毒转录来改善HIV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.