Evaluation of Self-delivering FANA Antisense Oligonucleotide Lead Compounds for HIV Therapy

项目摘要/摘要： 人类免疫缺陷病毒1型(HIV-1)是获得性免疫缺陷综合征(AIDS)的主要原因 仅在美国就有100多万人受到影响。治疗方面的进展明显延长了 HIV-1感染者的生命，目前的标准是联合抗逆转录病毒疗法(CART 在治疗护理方面。然而，仅靠CART不能实现病毒的完全根除。此外，缺点是 例如耐药性的产生和严重的副作用(例如早衰、癌症和心血管疾病 疾病)仍然是CART治疗中的关键问题。因此，需要一种高效输送的治疗方法 以及良好的安全性，可以将HIV-1病毒载量降低到无法检测的水平。一种有希望的方法 是通过靶向二聚化起始点(DIS)，一种复制，利用RNA沉默来治疗HIV-1感染 信号位于5‘非翻译区(5’非编码区)。当5‘-非编码区经历一种 构象变化，允许两个RNA基因组的DIS环碱基配对。这形成了一个接吻环(KL) 二聚体，然后导致病毒RNA的后续包装。已知的是突变或抑制 DIS严重限制了病毒的传染性。在这里，我们建议测试2‘-脱氧-2’-氟-D-阿拉伯核酸 针对DIS的反义寡核苷酸(Fana ASO)将阻止病毒复制。法娜·阿索斯将成为 旨在切割(依赖核糖核酸酶H)或阻断病毒RNA，从而抑制暴露后的病毒复制。在一个 利用HIV-1感染的人外周血单个核细胞(PBMC)进行DIS靶向的初步研究 FANAS在单剂400 NM和3微米剂量(IC50)后，抑制HIV-1复制长达两周 =200 NM)。Fana Asos在体外也被测试为避孕药，浓度明显较低，以防止 病毒感染和扩增长达2周。Fana ASO也被设计成与DIS RNA结合，而不需要 切割它，这将防止复制所必需的接吻环结构的形成。奥姆- 在我们的RNaseH依赖的体外研究中，DIS-G9作为先导化合物出现，而AUM-DIS-G0将 作为我们的RNaseH非独立试验的先导化合物。在这项研究中，我们将采用系统的方法来 设计和评估这两个以DIS为目标的Fana Lead Aso。我们的首要目标是评估病毒的减少 根据我们的初步数据，用我们的两种先导化合物治疗后，在人源化的NSG小鼠中进行了复制 易受HIV-1感染的模型。第二个目标是评估Fana Asos作为购物车的潜力 考虑到它们在体外预防方面的成功，在潜伏感染的人源化小鼠中进行体内替代 模特。第三个目标是进行标准的药代动力学和ADME研究 目标1和2活体评估。拟议研究的目标是发展下一代 艾滋病毒的反义治疗。

Project Summary/Abstract: Human immunodeficiency virus 1 (HIV-1) is the primary cause of acquired immune deficiency syndrome (AIDS) that affects over a million people in the United States alone. Advances in treatment have significantly prolonged the lives of those infected with HIV-1, with combinatorial antiretroviral therapy (cART) being the current standard in therapeutic care. However, cART alone cannot achieve complete eradication of the virus. Besides, drawbacks such as drug resistance development and severe side effects (e.g. premature aging, cancer, and cardiovascular disease) remain critical issues in cART therapy. Therefore, there is a need for a treatment, with efficient delivery and a favorable safety profile, that can reduce the HIV-1 viral load to undetectable levels. A promising approach is to utilize RNA silencing to treat HIV-1 infection by targeting the dimerization initiation site (DIS), a replication signal in the 5’ untranslated region (5’-UTR). Dimerization is initiated when the 5'-UTR undergoes a conformational change, allowing the DIS loops of two RNA genomes to base pair. This forms a kissing-loop (KL) dimer, which then leads to the subsequent packaging of the viral RNA. It is known that mutation or inhibition of the DIS severely restricts viral infectivity. Here we propose to test if 2’-deoxy-2’-fluoro-D-arabinonucleic acid antisense oligonucleotides (FANA ASOs), targeting DIS, will prevent viral replication. FANA ASOs will be designed to cleave (RNase H-dependent) or block viral RNA, thus inhibiting viral replication post-exposure. In a preliminary study using HIV-1 infected human peripheral blood mononuclear cells (PBMCs), DIS-targeting FANAs inhibited HIV-1 replication for as long as two weeks, after single doses of 400 nM and 3 µM doses (IC50 = 200 nM). FANA ASOs were also tested as prophylactics in vitro, at significantly lower concentrations, to prevent viral infection and amplification for up to 2 weeks. FANA ASOs were also designed to bind to DIS RNA without cleaving it, which would prevent the formation of the kissing loop structure that is necessary for replication. AUM- DIS-G9 emerged as the lead compound from our RNase H-dependent in vitro studies, while AUM-DIS-G0 will be the lead compound for our RNase H-independent trials. In this study, we will adopt a systematic approach to design and assess these two FANA lead ASOs targeting DIS. Our first aim is to evaluate the reduction in viral replication after treating with our two lead compounds, from our preliminary data, in a humanized NSG mouse model susceptible to HIV-1 infection. The second aim is to assess the potential of FANA ASOs as cART replacement in vivo, considering their prophylactic success in vitro, in a latently infected humanized mouse model. The third Aim is to perform standard pharmacokinetic and ADME studies on the lead compound from Aims 1 and 2 in vivo assessments. The goal of the proposed study is the development of a next generation antisense therapy for HIV.