Selection of RNA aptamers against Ebola virus GP2

描述（由申请方提供）：丝状病毒埃博拉（EBOV）和马尔堡（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和这些病毒在人群中的高传播性，该病毒家族已被置于疾病控制和预防中心建立的A类选择剂列表中。目前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力都不是立即的。最近的研究表明，在感染过程中减少病毒载量对有害结果有显着影响。因此，病毒载量的瞬时减少可能在降低与丝状病毒感染相关的死亡率方面非常有效。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。我们建议针对EBOV糖蛋白GP 2胞外域的卷曲螺旋区域选择RNA适体。适体是小寡核苷酸（通常为20- 50 bp），以高亲和力特异性结合蛋白质或小靶分子，不具有免疫原性，并且可以通过化学修饰稳定化以获得更长的体内半衰期。结合到EBOV GP 2的羧基末端卷曲螺旋区域的适体将被预测为防止病毒与细胞膜融合，从而抑制病毒进入容许细胞。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上用于抗HIV-1的抗病毒武器库的一部分。EBOV糖蛋白GP 2将作为适体的极好靶标，因为蛋白质的该区域存在于病毒体和感染细胞两者的细胞外，允许适体容易地接近蛋白质的靶向区域。此外，GP 2羧基末端卷曲螺旋区（也称为七肽重复区）在EBOV的四种毒株中高度保守，并且被认为是非结构化的并且在融合前GP结构中是可接近的。在目标1中，我们将选择结合至源自GP 2羧基末端卷曲螺旋区的肽和病毒样颗粒表面上的EBOV GP两者的适体。在目标2中，我们将优化和表征我们针对EBOV GP 2选择的适体。然后，我们将测试适体在阻断EBOV和MARV GP依赖性转导和EBOV感染中的功效。靶向EBOV GP 2并阻断病毒体进入的适体的开发将补充Maury实验室内正在进行的研究，这些研究正在积极选择针对EBOV GP 1受体结合结构域的RNA适体。EBOV GP 2适体的开发应证明在暂时减少感染个体的病毒血症方面是非常成功的。即使疫苗开发成功，使用抗丝状病毒的抗病毒药作为针对零星爆发的权宜之计也将是非常有益的，因为认为在不久的将来非洲人群将广泛接种这些病毒是不现实的。公共卫生相关性：丝状病毒埃博拉病毒和马尔堡病毒导致毁灭性的出血热爆发，目前还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结合并防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，以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.