Selection of RNA aptamers against Ebola virus GP2

描述(申请人提供)：埃博拉丝状病毒(EBOV)和马尔堡丝状病毒(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和这些病毒在人类群体中的高传播性，该病毒家族已被列入疾病控制和预防中心建立的A类选择制剂名单。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力都不是立竿见影的。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，暂时减少病毒载量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，我们建议开发一种可以用来降低病毒载量的抗病毒疗法。我们建议针对EBOV糖蛋白GP2胞外区的卷曲卷曲区域选择RNA适配子。适配子是一种小分子寡核苷酸(一般为20-50bp)，与蛋白质或小靶分子具有高亲和力，不具免疫原性，可通过化学修饰稳定较长的体内半衰期。可以预测，与EBOV GP2的羧基末端卷曲卷曲区域结合的适体可以阻止病毒与细胞膜的融合，从而阻止病毒进入允许细胞。HIV-1 TM蛋白类似区域的小分子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的抗病毒药物的一部分。EBOV糖蛋白GP2将成为适配子的极佳靶点，因为该蛋白的这一区域位于病毒粒子和受感染细胞的细胞外，使适配子能够随时进入蛋白质的靶区域。此外，GP2羧基末端卷曲螺旋区域(也称为七肽重复区域)在EBOV的四个毒株中高度保守，被认为是非结构化的，在融合前的GP结构中是可访问的。在目标1中，我们将选择既能与GP2羧基末端卷曲螺旋区域衍生的肽结合又能与病毒样颗粒表面的EBOV GP结合的适体。在目标2中，我们将优化和鉴定我们选择的针对EBOV GP2的适体(S)。然后，我们将测试适体在阻断EBOV和MARV GP依赖的转导和EBOV感染方面的有效性。针对EBOV GP2并阻止病毒粒子进入的适配子的开发将补充Maury实验室正在进行的研究，这些研究正在积极地选择针对EBOV GP1受体结合域的RNA适配子。EBOV GP2适配子的开发应该被证明在瞬时减少感染者的病毒血症方面非常成功。即使疫苗开发成功，使用抗丝病毒药物作为应对零星暴发的权宜之计也是非常有益的，因为认为在不久的将来将在非洲人口中广泛接种针对这些病毒的疫苗是不切实际的。公共卫生相关性：埃博拉和马尔堡丝状病毒导致毁灭性的出血热暴发，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结合的小RNA适配子，防止病毒与细胞膜融合。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.