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

描述(申请人提供)：通过粘膜表面的性传播一直是HIV-1在世界各地传播的最常见途径。尽管人们对艾滋病毒-1疫苗的开发给予了很大关注，但进展缓慢，迫切需要找到替代方法，如局部使用杀微生物剂，以控制艾滋病毒-1的传播。人类防御素有助于对微生物和病毒感染的先天免疫防御；然而，这些内源性因素在防止艾滋病毒传播方面的效果微乎其微。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进入的抑制剂。存在多种人类防御素基因，但它们含有阻止翻译的提前终止密码子。因此，人类基因组中编码的？-防御素(在人类中称为逆周期蛋白)不以肽的形式表达。基于包含在其他完整的逆转录周期蛋白基因中的遗传密码，我们合成了逆转录周期蛋白，并发现它们对来自各种HIV-1分支的R5和X4分离株具有显著的活性。虽然猕猴体内产生的？-防御素涉及两个九个残基的翻译后连接，但这一独特过程的机制及其在人类细胞中的存在至今尚不清楚。我们正在提出一种范式转变，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艾滋病毒-1的天然免疫力，从而改变杀微生物剂如何作用于预防艾滋病毒-1传播。在真核生物中，氨基糖苷类化合物可以通过在其位置掺入一种氨基酸来抑制提前终止密码子。在初步研究中，我们使用抑制逆转录周期蛋白提前终止密码子的氨基糖苷类化合物恢复了人早幼粒细胞和HeLa细胞逆转录周期素肽的表达，并揭示了这些天然逆转录周期蛋白对HIV-1具有活性。根据我们的研究，我们假设氨基糖苷类可以恢复宫颈阴道粘膜中逆转录周期蛋白的自然产生，并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阶段测试这些假设：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周期蛋白持续表达的能力，以及2)评估局部应用氨基糖苷类药物在阴道黏膜诱导体外和体外毒性和促炎细胞因子的能力。具有最大活性而对阴道黏膜的不良反应最小的氨基糖苷铅将进入R33阶段，在此阶段我们将：3)将氨基糖苷制成快速释放膜以诱导天然抗HIV-1逆转录周期蛋白的表达，以及4)评估该膜配方的氨基糖苷对小尾猴的阴道安全性。氨基糖苷类药物介导的逆转录周期蛋白的翻译可能导致一类新的杀微生物剂，以防止艾滋病毒-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.