Aminoglycoside microbicides restore natural expression of anti-HIV-1 retrocyclins

描述（由申请人提供）：通过粘膜表面的性传播是HIV-1在世界范围内最常见的传播途径。尽管对艾滋病毒-1疫苗的开发给予了很大的关注，但进展缓慢，迫切需要找到替代方法，例如局部杀微生物剂，以防止艾滋病毒-1的传播。人体防御素和防御素有助于对微生物和病毒感染的先天免疫防御；然而，这些内源性因素在预防艾滋病毒传播方面收效甚微。某些非人类灵长类动物也会产生？-防御素- 18残基环肽，作为HIV-1进入抑制剂。多个人？-防御蛋白基因是存在的，但它们含有一个阻止翻译的过早终止密码子。因此，？在人类基因组中编码的防御素（在人类中称为逆转录细胞周期蛋白）不以肽的形式表达。基于完整的反转录细胞周期蛋白基因所包含的遗传密码，我们合成了反转录细胞周期蛋白，并发现它们对来自多种HIV-1分支的R5和X4分离株具有显著活性。而在体内产生？恒河猴的防御素涉及两个9残基肽的翻译后连接，直到现在，这一独特过程的机制和它在人类细胞中的存在都不为人所知。我们提出了一种模式的转变，即杀菌剂如何通过使用廉价和广泛可用的氨基糖苷类抗生素来增强阴道对HIV-1的先天免疫，从而预防HIV-1的传播。在真核生物中，氨基糖苷可以通过在其位置上结合氨基酸来抑制过早终止密码子。在初步研究中，我们利用氨基糖苷抑制逆转录细胞周期蛋白的过早终止密码子，恢复了人早幼粒细胞和HeLa细胞中逆转录细胞周期蛋白肽的表达，并发现这些天然的逆转录细胞周期蛋白对HIV-1有活性。根据我们的研究，我们假设氨基糖苷可以恢复宫颈阴道粘膜中逆转录细胞周期素的自然产生，并且可以制成快速释放膜，以增强对性传播HIV-1的保护。在一个多学科团队的帮助下，我们将在R21期通过以下方法来验证这些假设：1)评估氨基糖苷类药物在人阴道上皮细胞、器官型宫颈阴道组织和宫颈器官培养中诱导逆转录细胞周期蛋白持续表达的能力；2)评估局部应用氨基糖苷类药物在阴道粘膜中诱导体外和离体毒性和促炎细胞因子的能力。一种表现出最大活性且对阴道粘膜不良影响最小的氨基糖苷将进入R33阶段，我们将：3)将氨基糖苷制成快速释放膜，诱导天然抗hiv -1逆转录细胞周期蛋白的表达；4)评估膜制氨基糖苷在尾尾猕猴中的阴道安全性。氨基糖苷介导的反转录细胞周期蛋白翻译可能导致一类新的杀微生物剂来预防HIV-1的传播。

DESCRIPTION (provided by applicant): Sexual transmission through mucosal surfaces has been the most common route of HIV-1 spread throughout the world. Although much attention has been focused on vaccine development for HIV-1, progress has been slow and there is an urgent need to find alternative approaches, such as topical microbicides, to target the spread of HIV-1. Human a- and ¿-defensins contribute to innate immune defenses against microbial and viral infections; however, these endogenous factors are minimally effective in preventing the spread of HIV. Certain nonhuman primates also produce ?-defensins - 18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human ?-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the ?-defensins encoded within the human genome (called retrocyclins in humans) are not expressed as peptides. Based on the genetic code contained within the otherwise intact retrocyclin genes, we recreated retrocyclins synthetically, and revealed them to be remarkably active against R5 and X4 isolates from a wide variety of HIV-1 clades. While in vivo production of ?-defensins in rhesus macaques involves the post-translational ligation of two nine-residue peptides, neither the mechanism of this unique process nor its existence in human cells was known - until now. We are proposing a paradigm shift in how a microbicide could act to prevent HIV-1 transmission, through the use of inexpensive and widely available aminoglycoside antibiotics to enhance vaginal innate immunity against HIV-1. In eukaryotes, aminoglycosides can suppress premature termination codons through the incorporation of an amino acid in its place. In preliminary studies, we restored the expression of retrocyclin peptides by human promyelocytic and HeLa cells using aminoglycosides that suppressed retrocyclin's premature termination codon, and revealed that these natural retrocyclins were active against HIV-1. Based on our studies, we hypothesize that aminoglycosides can restore the natural production of retrocyclins in the cervicovaginal mucosa, and can be formulated as quick-release films to confer increased protection against sexually transmitted HIV-1. With assistance from a multidisciplinary team, we will test these hypotheses in the R21 phase by: 1) assessing the ability of aminoglycosides to induce sustained expression of retrocyclins in human vaginal epithelial cells, organotypic cervicovaginal tissues and cervical organ cultures, and 2) evaluating the ability of topically applied aminoglycosides to induce in vitro and ex vivo toxicity and proinflammatory cytokines in the vaginal mucosa. A lead aminoglycoside, exhibiting the greatest activity while exhibiting minimal adverse effects to the vaginal mucosa, will proceed to the R33 phase in which we will: 3) formulate the aminoglycoside as a quick-release film to induce natural anti-HIV-1 retrocyclin expression, and 4) evaluate the vaginal safety of the film-formulated aminoglycoside in pigtailed macaques. Aminoglycoside-mediated translation of retrocyclins could result in a new class of microbicide to prevent HIV-1 transmission.