Ribozymes for studies and treatment of cytomegalovirus infections

用于研究和治疗巨细胞病毒感染的核酶

• 批准号: 7916993
• 负责人: Fenyong Liu
• 金额: $ 17.17万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-12 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7916993
• 关键词: Acquired Immunodeficiency Syndrome; Address; Animal Model; Animals; Antiviral Agents; Biochemical; Biological Models; Catalytic RNA; Cells; Cleaved cell; Clinical Research; Code; Cytomegalovirus; Cytomegalovirus Infections; Development; Drug resistance; Engineering; Environment; Foscarnet; Ganciclovir; Gastroenteritis; Gene Expression; Gene Targeting; Genes; Genetic Materials; Genetic Transcription; Growth; HIV; Homebound Persons; Human; Human Virus; Hydrolysis; In Vitro; Infection; Messenger RNA; Methods; Murid herpesvirus 1; Mus; Opportunistic Infections; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Physiological; Pneumonia; Proteins; RNA; RNase P; Reporting; Research; Retinitis; Specificity; Therapeutic Agents; Variant; Viral; Viral Genes; Virus Diseases; Vision; clinically relevant; combat; in vivo; insight; macromolecule; novel; novel strategies; prevent; programs; public health relevance; tool

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) causes one of the most common opportunistic infections in patients with AIDS. Disseminated HCMV infection in these patients is usually associated with gastroenteritis, pneumonia, and sight-threatening retinitis. The emergence of drug-resistant HCMV strains to currently available drugs (e.g. ganciclovir and foscarnet) has posed a need to develop new drugs and novel strategies to combat HCMV infections. This proposal represents our continued effort to develop ribonuclease P (RNase P) ribozyme as gene-targeting agents for therapy of infections by human viruses including HCMV. Recently, we have shown that RNase P ribozyme (M1GS RNA) can cleave the mRNAs coding for the HCMV essential protease (PR) and transcription regulator IE1 and IE2, and block viral gene expression and growth in cells. However, little is known about the mechanism of how RNase P ribozymes achieve efficient cleavage activity under physiological cellular environment. The specificity and antiviral activity of M1GSs in human cells that are clinically relevant to HCMV infections have not been studied. Furthermore, whether M1GS RNA is effective in down-regulating gene expression in animal models in vivo has not been reported. We propose in this research program to address these issues. In the initial part of the study, we will generate ribozyme variants that are highly active under physiological conditions. In vitro biochemical analyses will be carried out to characterize these ribozymes, and their interactions with cellular proteins will be investigated. Then, the engineered ribozymes will be expressed in human cells that are known to be infected by HCMV in vivo, and the antiviral activity of these ribozymes will be determined and their mechanisms in inhibiting viral replication will be investigated. Finally, using murine cytomegalovirus infection of mice as a model system, we will determine whether M1GS RNAs are effective in shutting down viral infection and preventing viral diseases in vivo in animals. These studies will generate novel ribozyme variants that are highly active in cells and can be used for gene-targeting applications. Moreover, these studies will provide insight into the mechanism of how M1GS ribozymes achieve efficient activity in physiological cellular conditions, and will reveal whether RNase P ribozymes are effective in downregulating gene expression in clinically-relevant human cells and in animals. Our proposed research will facilitate the development of M1GS ribozymes as gene-targeting therapeutic agents for treatment of infections by HCMV and other AIDS-associated human viruses. PUBLIC HEALTH RELEVANCE: The objective of the proposed studies is to develop a new method to inhibit specific gene expression, which may be applied in both basic and clinical research, such as treatment of infection of human cytomegalovirus (HCMV), one of the most common opportunistic infections encountered in AIDS patients. This project aims to construct a macromolecule (RNA), termed RNase P ribozyme, which will be introduced into cells and will hydrolyze a specific RNA, e.g. HCMV messenger RNA and the genetic material of HIV which is made of RNA. Our study will facilitate the development of novel macromolecules that can be used as research tools and therapeutic agents for studies and treatment of infections of human viruses including HCMV.

描述（由申请人提供）：人巨细胞病毒（HCMV）是艾滋病患者最常见的机会性感染之一。这些患者中的播散性HCMV感染通常与胃肠炎、肺炎和威胁视力的视网膜炎相关。对目前可用的药物（例如更昔洛韦和膦甲酸）具有耐药性的HCMV毒株的出现已经提出了开发新药物和新策略以对抗HCMV感染的需要。该建议代表了我们继续努力开发核糖核酸酶P（RNase P）核酶作为治疗人类病毒感染（包括HCMV）的基因靶向药物。最近，我们发现RNase P核酶（M1GS RNA）可以切割编码HCMV必需蛋白酶（PR）和转录调节因子IE1和IE2的mRNA，并阻断病毒基因在细胞中的表达和生长。然而，关于RNase P核酶在生理细胞环境下如何实现高效切割活性的机制知之甚少。尚未研究M1GS在临床上与HCMV感染相关的人细胞中的特异性和抗病毒活性。此外，M1GS RNA是否在体内动物模型中有效下调基因表达尚未报道。我们建议在本研究计划中解决这些问题。在研究的最初部分，我们将产生在生理条件下具有高度活性的核酶变体。将进行体外生化分析来表征这些核酶，并研究它们与细胞蛋白质的相互作用。然后，工程化的核酶将在已知体内被HCMV感染的人细胞中表达，并且这些核酶的抗病毒活性将被确定，并且它们抑制病毒复制的机制将被研究。最后，使用鼠巨细胞病毒感染小鼠作为模型系统，我们将确定M1GS RNA是否有效地关闭病毒感染和预防动物体内的病毒性疾病。这些研究将产生新的核酶变体，这些变体在细胞中具有高度活性，可用于基因靶向应用。此外，这些研究将深入了解M1GS核酶如何在生理细胞条件下实现有效活性的机制，并将揭示RNase P核酶是否有效下调临床相关人类细胞和动物中的基因表达。我们提出的研究将促进M1GS核酶作为基因靶向治疗剂的发展，用于治疗HCMV和其他艾滋病相关的人类病毒感染。 公共卫生关系：该研究的目的是开发一种新的方法来抑制特定的基因表达，这可能会应用于基础和临床研究，如治疗感染的人巨细胞病毒（HCMV），最常见的机会性感染的艾滋病患者中遇到的。该项目旨在构建一种称为RNase P核酶的大分子（RNA），将其引入细胞并水解特定RNA，例如HCMV信使RNA和由RNA组成的HIV遗传物质。我们的研究将促进新型大分子的开发，这些大分子可用作研究工具和治疗剂，用于研究和治疗人类病毒（包括HCMV）感染。