Enhancing the Intracellular Functioning of anti-HIV RNAs

增强抗 HIV RNA 的细胞内功能

基本信息

批准号：
7197099
负责人：
John Joseph Rossi
金额：
$ 63.87万
依托单位：
BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-07-03 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7197099
关键词：
Acquired Immunodeficiency Syndrome Antiviral Agents Binding Sites Catalytic Domain Catalytic RNA Cell Differentiation process Cells Chimera organism Cleaved cell Collaborations Colorado Condition Cytoplasm DNA DNA Polymerase III Development Diffusion Effectiveness Escape Mutant Face Gene Expression Genes Genomics Goals HIV HIV Infections HIV-1 Hand Hematopoietic Hematopoietic stem cells Highly Active Antiretroviral Therapy Human In Situ Hybridization In Vitro Inbred NOD Mice Infection Knock-out Libraries Localized MicroRNAs Molecular Conformation Nucleotides Physiological Plants Polymerase Chain Reaction Population Positioning Attribute Procedures Production Publications RNA RNA Interference RNA Sequences RNA primers Relative (related person)Research Small Interfering RNA Standards of Weights and Measures Structure Testing Toxic effect Transcript Transfer RNA Variant Viral Virion Viroids base cellular targeting design fascinate gene therapy genetic regulatory protein hammerhead ribozyme in vivo inhibitor/antagonist mouse model novel particle programs promoter trafficking

项目摘要

HIV-1 infection and AIDS continues to be a worldwide problem, even in the face of new combinations of highly active antiretroviral therapies. This research program continues to focus upon the development of novel RNA based inhibitors of HIV-1 replication for use in anti-HIV-1 gene therapy applications. RNA interference has proven to be a remarkably fascinating compilation of cellular mechanisms that regulate gene expression from the transcriptional through post-transcriptonal levels. With the potency of small interfering RNAs still greatly exceeding that of any single ribozyme, there has been a major emphasis shift in this program from ribozymes to RNA interference as an antiviral agent. Our goals are to enhance the functioning of anti-HIV siRNAs, and to elucidate the mechanism of action of effective siRNAs or micro RNAs with the goals of developing non-toxic anti-HIV siRNAs that minimize viral escape mutants and provide long-term inhibition of viral replication. Despite the growing and realistic excitement over RNAi, ribozymes can still perform useful functions. Recent findings emulate the native catalytic domain of plant viroids and virusoids to achieve more efficient ribozymes under physiological conditions. We have exploited this finding to provide the first demonstration that the native structure of the hairpin domain in plant viroids and virusoids can be applied to trans-cleaving ribozymes. This program will continue to exploit hammerhead ribozymes by incorporating this native conformation into the design of our anti-HIV ribozymes and continue to study the most effective ways of expressing these more highly active ribozyme motifs in hematopoietic cells. We have recently identified a new version of a potent anti-HIV-1 nucleolar localizing ribozyme from a ribozyme library. We are now in a position to design new ribozyme expression constructs that combine the most recent structural constraints with nucleolar trafficking ribozymes. In vivo testing of the most promising anti-HIV constructs will be carried out via collaboration with Dr. Ramesh Akkina at Colorado State Univ. He has established both a standard SCID-hu and the Rag2-/-yc-\- double knockout mouse models to study hematopoietic stem cell differentiation. In accordance with the changes that have taken place in this research program over the past four years, the specific aims have been adjusted accordingly. The revised aims are:Aim 1) Intracellular expression and functional analyses of anti-HIV shRNAs and micro RNAs: Aim 2) Intracellular expression, mechanism of action, and anti-HIV-1 studies of nucleolar localizing ribozymes and new ribozyme catalytic domain motifs: Aim 3) Functional analyses and differentiation potentials of anti-HIV RNA expressing hematopoietic cells in vitro and in vivo. The overall goal of this program remains that of developing potent RNA based anti-HIV inhibitory agents for use in human gene therapy treatment of HIV infection.

即使面对新的组合，HIV-1 感染和艾滋病仍然是一个世界性问题高活性抗逆转录病毒疗法。该研究计划继续关注开发新型基于 RNA 的 HIV-1 复制抑制剂，用于抗 HIV-1 基因治疗应用。核糖核酸干扰已被证明是调节基因的细胞机制的一个非常令人着迷的汇编从转录到转录后水平的表达。干扰小 RNA 仍然大大超过任何单一核酶，这方面的重点发生了重大转变从核酶到RNA干扰作为抗病毒剂的程序。我们的目标是增强功能抗 HIV siRNA 的研究，并阐明有效 siRNA 或 micro RNA 的作用机制开发无毒的抗 HIV siRNA 的目标是最大限度地减少病毒逃逸突变体并提供长期的抑制病毒复制。尽管人们对 RNAi 的兴趣日益浓厚，但核酶仍然可以执行有用的功能。最近的研究结果模拟了植物类病毒和类病毒的天然催化结构域在生理条件下获得更有效的核酶。我们利用这一发现来提供首次证明植物类病毒和类病毒中发夹结构域的天然结构可以应用于反式切割核酶。该计划将继续利用锤头核酶将这种天然构象纳入我们的抗 HIV 核酶的设计中，并继续研究在造血细胞中表达这些活性更高的核酶基序的最有效方法。我们有最近从核酶文库中鉴定出一种新版本的有效抗 HIV-1 核仁定位核酶。我们现在能够设计新的核酶表达构建体，该构建体结合了最新的核仁运输核酶的结构限制。最有希望的抗艾滋病毒的体内测试该构建将通过与科罗拉多州立大学的 Ramesh Akkina 博士合作进行。他有建立标准 SCID-hu 和 Rag2-/-yc-\- 双敲除小鼠模型进行研究造血干细胞分化。根据本次变更的情况四年来研究计划、具体目标都作了相应调整。修订后的目标是：目标 1) 抗 HIV shRNA 和 micro RNA 的细胞内表达和功能分析：目标 2) 核仁定位核酶的细胞内表达、作用机制和抗 HIV-1 研究新的核酶催化结构域基序：目标3）抗HIV的功能分析和分化潜力体外和体内表达造血细胞的 RNA。该计划的总体目标仍然是开发基于 RNA 的有效抗 HIV 抑制剂，用于人类 HIV 基因治疗感染。