RNA Interference for the Treatment of Ebola and Marburg Hemorrhagic Fever

RNA 干扰治疗埃博拉和马尔堡出血热

基本信息

批准号：
8146966
负责人：
Thomas William Geisbert
金额：
$ 58.51万
依托单位：
UNIVERSITY OF TEXAS MED BR GALVESTON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-24 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8146966
关键词：
Address Angola Animals Antiviral Agents Biological Response Modifier Therapy Bioterrorism Case Fatality Rates Categories Cavia Cells Centers for Disease Control and Prevention (U.S.)Democratic Republic of the Congo Disease Outbreaks Disorder by Site Ebola virus Encapsulated Filovirus Frankfurt-Marburg Syndrome Virus Gene Expression Gene Targeting Genes Goals Guanosine Guidelines Human Immune Immunohistochemistry In Vitro Infection Injection of therapeutic agent Lead Licensing Lipids Macaca mulatta Messenger RNA Methods Modality Modeling Mus National Institute of Allergy and Infectious Disease Nucleic Acids Organ Pharmacologic Substance Plasmids Polymerase Preventive Process Property Public Health Publishing RNA Interference Reporting Safety Sequence Alignment Small Interfering RNA Sudan System Technology Testing Therapeutic Therapeutic Agents Time Titrations Uridine Vaccines Vero Cells Viral Viral Hemorrhagic Fevers Viral Proteins Viremia Virus Virus Diseases Work base design disorder prevention in vitro activity in vivo laboratory accident nonhuman primate novel particle pathogen protective efficacy response treatment strategy vector

项目摘要

DESCRIPTION (provided by applicant): The CDC and NIAID have classified the filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), as Category A priority pathogens. These viruses cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates with case fatality rates as high as 90% for some species or strains such as Zaire ebolavirus (ZEBOV). There are presently no approved active or passive therapeutic modalities for EBOV or MARV infections resulting from a natural outbreak, laboratory accident, or deliberate misuse. Recently, we demonstrated the first complete postexposure protection of nonhuman primates against a lethal ZEBOV challenge using a combination of small interfering RNA (siRNA) targeting the ZEBOV L polymerase, viral protein (VP) 24, and VP35 formulated in stable nucleic acid-lipid particles (SNALP). The goal of this proposal is to develop siRNA-based therapies for treating EBOV and MARV infections. Specifically, we will: (1) Identify candidate siRNAs targeting Ebola and Marburg virus genes and evaluate antiviral activity in vitro. siRNA will be designed using sequences for EBOV and MARV. When possible, conserved siRNAs between virus species or strains will be chosen based on multiple sequence alignments. Lead candidate siRNAs will be identified by testing their ability to inhibit replication of infectious EBOV and MARV in Vero cells and also by using a nonviral plasmid-based expression system. (2) Evaluate the immune stimulatory activity of modified siRNAs targeting Ebola and Marburg virus genes in mice. Candidate siRNAs that have sufficient antiviral activity against filoviruses will be modified by substituting 2'-O-methyl (2'OMe) guanosine and/or uridines in the sense and antisense strands to eliminate the immune stimulatory capacity of the siRNA. These modified siRNAs will be tested for their ability to inhibit replication of infectious EBOV and MARV in Vero cells and administered to mice to demonstrate that they do not activate a nonspecific immunostimulatory response. (3) Determine the efficacy of non-immunostimulatory siRNAs against Ebola and Marburg viruses in guinea pigs. Promising modified siRNA candidates that demonstrate significant inhibition of filoviral replication in vitro and do not activate an immunostimulatory response in mice will be manufactured at a scale sufficient to support animal studies. siRNAs delivered by SNALP will be administered to guinea pigs i.v. by retroorbital injection shortly after filoviral challenge to determine protective efficacy. (4) Determine the efficacy of non-immunostimulatory siRNAs against Ebola and Marburg viruses in nonhuman primates. Modified siRNA candidates that show complete protection of guinea pigs against either SEBOV, ZEBOV, or against each of four strains of MARV (MARV-Angola, MARV-Ci67, MARV-Musoke, and MARV-Ravn) will be evaluated in rhesus monkeys to determine the protective efficacy against each of these viruses when administered at various times after filovirus challenge.

描述（由申请人提供）：CDC和NIAID已将埃博拉病毒（EBOV）和MARBURG病毒（MARV）（MARV）分类为优先病原体。这些病毒在人类和非人类灵长类动物中引起严重且经常致命的出血热（HF），其中某些物种或菌株（例如Zaire Ebolavirus（Zebov））的病例死亡率高达90％。目前，由于自然爆发，实验室事故或故意滥用而导致的EBOV或MARV感染目前尚无批准的主动或被动治疗方式。最近，我们使用了针对Zebov L聚合酶，病毒蛋白（VP）24和VP35的小型干扰RNA（siRNA）对非人类灵长类动物对致命的Zebov挑战的第一个完整暴露后保护。该提案的目的是开发基于siRNA的治疗EBOV和MARV感染的疗法。具体而言，我们将：（1）确定靶向埃博拉病毒和马堡病毒基因的候选siRNA，并在体外评估抗病毒活性。 siRNA将使用EBOV和MARV的序列设计。在可能的情况下，将根据多个序列比对选择病毒或菌株之间的保守siRNA。铅候选siRNA将通过测试其抑制Vero细胞中传染性EBOV和MARV复制的能力，并使用基于非病毒质粒的表达系统抑制其复制的能力。（2）评估小鼠中靶向埃博拉病毒基因的改性siRNA的免疫刺激活性。在意义上和反义链中取代2'-O-甲基（2'Ome）鸟苷和/或尿液来，可以改变具有足够抗病毒病毒活性的候选siRNA。这些改良的siRNA将被测试，以抑制Vero细胞中传染性EBOV和MARV复制的能力，并向小鼠施用，以证明它们不会激活非特异性的免疫刺激反应。（3）确定豚鼠中针对埃博拉病毒和马堡病毒的非免疫刺激性siRNA的疗效。有望在体外表现出显着抑制丝病毒复制并且不激活小鼠的免疫刺激反应的有希望的修饰的siRNA候选物，将以足以支持动物研究的规模制造。 SNALP交付的siRNA将用于豚鼠i.v.在犯罪病毒挑战后不久，通过恢复注射以确定保护效果。（4）确定非人类灵长类动物中针对埃博拉病毒和马尔堡病毒的非免疫刺激性siRNA的功效。经过改进的siRNA候选者显示，对豚鼠对Sebov，Zebov的完全保护，或对Marv（Marv-Gangola，Marv-Gangola，Marv-CI67，Marv-Ci67，Marv-Musoke和Marv-Ravn）的四种菌株进行评估，将在恒河猴中进行评估，以确定这些病毒效应的各种时代的保护效率。