Phosphorothioate Oligonucleotides as Microbicides against HIV Transmission

描述(由申请人提供)：发展抑制艾滋病毒感染传播的干预措施对遏制艾滋病毒流行至关重要。通常被称为杀微生物剂的局部预防战略已被提议作为阻止或减缓艾滋病毒流行的一种战略。我们已经确定了在体外有效抑制HIV和SIV感染/复制的新型铅杀菌剂。在我们之前提交的报告中，我们报道了一种带有硫代磷酸骨架(OPB)的寡核苷酸，它可以分别抑制HIVBaL或SIVmac251在人或猿外周血单个核细胞中的感染和/或复制。OPB还可抑制HIVBaL和HIVIIIB对P4-R5 MAGI细胞的感染/复制。OPB连续作用24小时，对PBMC或P4-R5 MAGI细胞无明显毒性。初步数据显示，OPB还可能抑制其他病毒，因为它对A型流感病毒也有效。因此，我们的第一代OPB可能是一种有效的抗HIV微生物剂，当局部使用时，可以防止粘膜部位的感染。我们的初步研究是用OPB的13聚体Poly T或Poly A寡核苷酸进行的，这表明这种作用是独立于序列的，甚至可能是由硫代脱氧核糖骨架介导的。事实上，在我们目前的重新提交中，我们提供了关于我们的下一代化合物的数据，这是一种无碱硫代2‘脱氧核糖主链(PDB)，它比OPB具有更强的艾滋病毒抑制活性。我们在这里展示的14聚PDB没有毒性，是HIV的有效抑制剂，并且具有作为TLR7/9拮抗剂的优势，可以抑制HIV诱导的干扰素的产生。后一种特性很重要，因为HIV感染的建立可能取决于TLR引发的HIV诱导的粘膜炎症。重要的是，我们发现，当PDB在pH为4.4的羟乙基纤维素(HEC)凝胶中配制时是活性的，在pH到中性pH的转变中存活下来，并在HEC中长期保持其活性。我们推测，PDB通过作为“化学凝集素”与包膜病毒结合并抑制其感染性。我们进一步假设，PDB可以作为一种抗HIV的杀微生物剂，并可以预防SIV对猕猴的阴道感染。计划在R21阶段进行的研究将进一步优化和表征PDB在体外的安全性和有效性，以及它在瑞士韦氏小鼠阴道/宫颈刺激模型中的安全性。他们将确定对艾滋病毒有效且无毒性的最佳大小和成分。最后，对PDB的作用机理进行了研究，考察了其对羟乙基纤维素凝胶的包结作用，并测定了PDB对共生乳酸菌生长的影响。从R21阶段到R33阶段的进展已经设定了五个具体的里程碑。R33阶段将测试PDB预防阴道SIV感染的有效性，研究精浆和pH变化对OPB疗效的影响，确定其与人类生殖器上皮组织的安全性，并研究其抗HSV-2的有效性。目前的应用将允许对PDB作为可能的新型杀微生物剂候选进行广泛的评估。这里提出的研究涉及开发抑制艾滋病毒感染传播的治疗方法这一重要的公共卫生问题。目前的申请研究了一种可能用于抑制艾滋病毒感染的新化学物质。

DESCRIPTION (provided by applicant): Developing interventions that inhibit the transmission of HIV infection are critical for halting the HIV epidemic. Topical prevention strategies usually termed microbicides have been proposed as one strategy to halt or slow down the HIV epidemic. We have identified novel lead microbicides that potently inhibit HIV and SIV infection/replication in vitro. During our previous submission we reported an oligonucleotide with a phosphorothioate backbone (OPB) that could inhibit HIVBaL or SIVmac251 infection and/or replication in human or simian PBMC, respectively. OPB also inhibited infection/replication in cell-free infections of P4-R5 MAGI cells by HIVBaL and HIVIIIB. OPB exhibited no toxicity against PBMC or P4-R5 MAGI cells after 24h continuous exposure. Preliminary data suggested that OPB may also inhibit other viruses as it was also effective against influenza type A virus. Thus, our first generation OPB may be a potent microbicide against HIV that prevents infection at mucosal sites when topically applied. Our preliminary studies were carried out with a 13mer Poly T or Poly A oligonucleotide of OPB and this suggested that the effect was sequence independent and may even be mediated by the phosphorothioate deoxyribose sugar backbone. Indeed in our current re-submission we present data on our next generation compound, a baseless phosphorothioate 2' deoxyribose backbone (PDB) that has more potent HIV inhibitory activity than OPB. A 14mer PDB we show here has no toxicity, is a potent inhibitor of HIV and has the advantage of being a TLR7/9 antagonist that inhibits HIV-induced IFN¿ production. This later property is important as the establishment of HIV infection may depend on HIV-induced mucosal inflammation triggered by TLR. Importantly, we show that PDB is active when formulated in hydroxyethylcellulose (HEC) gel at pH 4.4, survives pH transition to a neutral pH, and in retains its activity in HEC for long periods. We hypothesize that PDB binds enveloped viruses and inhibits their infectivity by acting as a "chemical lectin". We further hypothesize that PDB can act as a microbicide against HIV and can prevent SIV vaginal infection of rhesus macaques. The studies planned in the R21 phase will further optimize and characterize the safety and effectiveness of PDB in vitro and its safety in the Swiss Webster mouse vaginal/cervical model of irritation. They will determine the optimal size and composition that remains effective against HIV and exhibits no toxicity. Finally, the mechanism of action of PDB will be investigated, the effect of inclusion into hydroxyethylcellulose gel will be tested and PDB's effect on the growth of commensal lactobacilli will be determined. Five specific milestones have been set for the progression from the R21 Phase to the R33 Phase. The R33 phase will test the effectiveness of PDB in preventing vaginal SIV infection, investigate the effect of seminal plasma and pH transition on the efficacy of OPB, determine its safety with human genital epithelial tissue, and investigate its effectiveness against HSV-2. The current application will allow for an extensive evaluation of PDB as possible novel microbicide candidates. The studies proposed here address the important public health problem of developing treatments that inhibit the transmission of HIV infection. The current application investigates a novel chemical that may be used to inhibit infection with HIV.