Selection of RNA aptamers against Ebola virus GP2

描述（由申请人提供）：丝状病毒埃博拉病毒（EBOV）和马尔堡病毒（MARV）在撒哈拉以南非洲引起周期性出血热暴发。由于与这些感染相关的高死亡率以及这些病毒在人群中的高传播性，这类病毒已被列入疾病控制和预防中心建立的A类精选病原体名单。目前还没有针对这些病毒的抗病毒疗法。虽然丝状病毒疫苗的开发看起来很有希望，但对任何疫苗的免疫都不是立即产生的。最近的研究表明，感染期间病毒载量的减少对有害后果有重大影响。因此，病毒载量的短暂减少在降低与丝状病毒感染相关的死亡率方面可能相当有效。在这里，我们建议开发一种抗病毒疗法，可用于减少病毒载量。我们建议选择针对EBOV糖蛋白GP2外域卷曲卷曲区域的RNA适体。适体是一种小的寡核苷酸（通常为20- 50bp），与蛋白质或小靶分子具有高亲和力，不具有免疫原性，可以通过化学修饰稳定较长的体内半衰期。与EBOV GP2羧基末端螺旋区结合的适体可以阻止病毒与细胞膜融合，从而抑制病毒进入允许细胞。HIV-1 TM蛋白类似区域的小抑制剂已被证明在抑制HIV复制方面非常成功，这些抑制剂现在是临床中用于对抗HIV-1的抗病毒武器库的一部分。EBOV糖蛋白GP2将作为适体体的极好靶标，因为该蛋白的这一区域位于细胞外的病毒粒子和受感染细胞上，允许适体体随时进入该蛋白的目标区域。此外，GP2羧基末端线圈区（也称为七tad重复区）在四株EBOV中高度保守，被认为是非结构化的，在融合前的GP结构中可接近。在Aim 1中，我们将选择与GP2羧基末端螺旋区衍生的肽结合的适体，以及与病毒样颗粒表面的EBOV GP结合的适体。在目标2中，我们将优化和表征我们选择的抗EBOV GP2的适体。然后，我们将测试适体阻断EBOV和MARV GP依赖性转导和EBOV感染的功效。开发靶向EBOV GP2并阻断病毒粒子进入的适体将补充Maury实验室正在进行的针对EBOV GP1受体结合域积极选择RNA适体的研究。EBOV GP2适配体的开发在暂时减少感染个体的病毒血症方面应该是非常成功的。即使疫苗开发取得成功，使用针对丝状病毒的抗病毒药物作为应对零星暴发的权宜之计也将非常有益，因为相信在不久的将来会在非洲人口中广泛接种这些病毒疫苗是不现实的。公共卫生相关性：埃博拉和马尔堡丝状病毒引起毁灭性的出血热暴发，目前没有针对这些可怕病毒的抗病毒疗法或疫苗。我们建议选择与埃博拉糖蛋白2结合并阻止病毒与细胞膜融合的小RNA适体。通过这些研究选择的适体可以作为重要的抗病毒药物来降低与这些感染相关的死亡率。

DESCRIPTION (provided by applicant): The filoviruses, Ebola (EBOV) and Marburg (MARV), cause periodic hemorrhagic fever outbreaks in sub- Saharan Africa. Because of the high rates of mortality associated with these infections and high transmissibility of these viruses in the human population, this family of viruses has been placed on the Category A select agent list that has been established by the Centers for Disease Control and Prevention. No anti-viral therapies are currently available against these viruses. While the development of a filoviral vaccine looks promising, immunity to any vaccine is not immediate. Recent studies suggest that reduction in virus load during the infection has a significant impact on deleterious outcomes. Thus, a transient reduction of virus load may be quite effective at decreasing mortality associated with filovirus infection. Here, we propose to develop an antiviral therapy that could be used to reduce virus load. We propose to select RNA aptamers against the coiled coil region of EBOV glycoprotein GP2 ectodomain. Aptamers are small oligonucleotides (generally 20- 50 bp) that specifically bind with high affinity to proteins or small target molecules, are not immunogenic and can be stabilized by chemical modifications for a longer in vivo half life. Aptamer binding to the carboxy terminal coiled coil region of EBOV GP2 would be predicted to prevent virus fusion with cellular membranes thereby inhibiting virus entry into permissive cells. Small inhibitors to the analogous region of the HIV-1 TM protein have proved highly successful at inhibiting HIV replication and these inhibitors are now part of the antiviral arsenal used against HIV-1 in the clinic. The EBOV glycoprotein GP2 will serve as an excellent target for aptamers because this region of the protein resides extracellularly on both virions and infected cells allowing aptamers ready access to the targeted region of the protein. Furthermore, the GP2 carboxy terminal coiled coil region (also called the heptad repeat region) is highly conserved across the four strains of EBOV and is believed to be unstructured and accessible in the pre-fusion GP structure. In Aim 1, we will select aptamers that bind to both a peptide derived from the GP2 carboxy terminal coiled coil region and to EBOV GP on the surface of virus like particles. In Aim 2, we will optimize and characterize the aptamer(s) that we select against EBOV GP2. We will then test the efficacy of the aptamers in blocking EBOV and MARV GP dependent transduction and EBOV infection. Development of aptamers that target EBOV GP2 and block virion entry will complement ongoing studies within the Maury laboratory that are actively selecting RNA aptamers against the EBOV GP1 receptor binding domain. The development of EBOV GP2 aptamers should prove highly successful in transiently reducing viremia in the infected individual. The use of antivirals against filoviruses as a stop-gap measure against sporadic outbreaks will be highly beneficial even if vaccine development is successful as it is unrealistic to believe that wide spread vaccination of African populations against these viruses will occur in the near future. PUBLIC HEALTH RELEVANCE: The filoviruses Ebola and Marburg cause devastating outbreaks of hemorrhagic fever and currently there are no antiviral therapies or vaccines available for these dreadful viruses. We propose to select small RNA aptamers that bind to Ebola glycoprotein 2 and prevent fusion of the virus with cellular membranes. The aptamers selected through these studies may serve as important antivirals to reduce mortality associated with these infections.