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Disruption of latent HIV-1 proviruses using CRISPR/Cas endonucleases

使用 CRISPR/Cas 核酸内切酶破坏潜伏的 HIV-1 原病毒

基本信息

批准号：
9427958
负责人：
BRYAN R. CULLEN
金额：
$ 74.17万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2020-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9427958
关键词：
Address Animal Model Anti-Retroviral Agents Antigens Antiviral Agents Biological Assay Biological Models CD34 gene CD4 Positive T Lymphocytes CRISPR/Cas technology Cell Compartmentation Cell Culture Techniques Cell Death Cell Line Cell Nucleus Cells Cessation of life Chimeric Proteins Chronic Clustered Regularly Interspaced Short Palindromic Repeats Color Complex Computer Retrieval of Information on Scientific Projects Database DNA DNA Sequence Deoxyribonuclease I Evaluation Family Gene Targeting Genes Genetic Transcription Genome Goals Grant Guide RNA HIV HIV-1 Highly Active Antiretroviral Therapy Histone Deacetylase Inhibitor Human Infection Lead Lentivirus Vector Methods Modality Modeling Patients Pharmaceutical Preparations Population Proteins Proviruses Reagent Reporter Residual state Rest Site Specificity System T-Lymphocyte Telefacsimile Testing Viral Genome Viral Load result Virion Virus Virus Diseases base cellular transduction design efficacy testing endonuclease experimental study expression vector humanized mouse immunogenicity in vivo in vivo Model in vivo evaluation mouse model novel particle prevent public health relevance reconstitution vector viral DNA viral rebound

项目摘要

DESCRIPTION (provided by applicant): While the infection of activated CD4+ cells by HIV-1 in vivo normally results in a productive viral infection leading to cell death, a small number of infected T cells re-enter the resting state and this can lead to a latent HIV-1 infection. Latent HIV-1 infections result from integrated HIV-1 proviruses that are entirely transcriptionally silent yet can be reactivated from latency by stimulation by an appropriate antigen or reagent. While highly active antiretroviral therapy (HAART) can prevent HIV-1 spread and dramatically reduce viral loads, due to the progressive death of productively HIV-1-infected cells, a small population of latently HIV-1-infected T cells remains intact. As a result, when HAART is discontinued, HIV-1 can rapidly reappear and reconstitute a high level infection. Because the latently infected T-cell pool is extremely long lived, it has proven impossible to "cure" HIV-1 infected patients using HAART alone in the sense that these patients become entirely virus free and are able to discontinue HAART without virus rebound. Because latently HIV-1 infected T cells are, almost by definition, impossible to distinguish from other resting T cells, efforts to deplete the latent pool have previously focused on attempts to activate the proviruses present in latently infected cells using, for example, HDAC inhibitors, in the presence of HAART. However, it has become increasingly clear that this approach is unable to effectively activate the majority of latent proviruses. An alternative strategy, proposed here, is to target and destroy latent HIV-1 proviruses using vector-delivered RNA-guided endonucleases belonging to the CRISPR/Cas family of bacterial antiviral RNA- guided DNA endonucleases. In this application, we propose several approaches to effectively and specifically destroy the latent HIV-1 proviral reservoir using lentiviral vector delivery of Ca9 proteins and small guide RNAs that target one or more highly conserved regions of the HIV-1 genome. We will first optimize proviral targeting and expression of Cas9 and single guide RNAs (sgRNAs) and maximize lentiviral vector titer. Secondly, we will test various transient expression modalities for Cas9/sgRNA combinations and analyze their ability to destroy latent HIV-1 proviruses in culture, and finally, we will determine the ability of lentiviral vector- delivered Cas9/sgRNA complexes to ablate the latent HVI-1 proviral reservoir in vivo, using a humanized mouse model of HIV-1 infection.