CRISPR-Cas13-based rapid HIV-1 test

基于 CRISPR-Cas13 的快速 HIV-1 检测

基本信息

批准号：
10593813
负责人：
Peter B Lillehoj
金额：
$ 46.85万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-24 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10593813
关键词：
Acute Antibodies Architecture Bathing Biological Assay Blood Blood specimen Chronic Clinical Clinical Management Clustered Regularly Interspaced Short Palindromic Repeats Communicable Diseases Consumption Data Detection Development Devices Diagnosis Diagnostic Diagnostic tests Early Diagnosis Effectiveness Engineering Enzymes Evaluation FDA approved Freeze Drying General Population Generations HIV HIV Antigens HIV Infections HIV-1 Hand Health Home Human Human immunodeficiency virus test Individual Laboratories Lateral Life Measurement Methods Mission Morbidity - disease rate National Institute of Allergy and Infectious Disease Nucleic Acid Amplification Tests Nucleic Acids Outcome Patient Monitoring Patients Performance Persons Phase Population Heterogeneity Preparation Procedures Public Health RNA RNA amplification RRM2 gene Reaction Refrigeration Research SARS-CoV-2 antigen Saliva Sampling Sensitivity and Specificity Specimen Speed Techniques Technology Telephone Temperature Test Result Testing Time Viral Proteins Water acute infection antiretroviral therapy comparative cost diagnostic tool diagnostic value effectiveness evaluation high reward high risk improved innovation instrument instrumentation isothermal amplification lateral flow assay lateral line mortality novel diagnostics nucleic acid detection operation pre-exposure prophylaxis prototype rapid test salivary assay self testing smartphone application test strip transmission process usability user-friendly viral RNA viral detection viral rebound viral transmission

项目摘要

HIV is one of the most deadly infectious diseases in the world. HIV transmission is largely driven by acutely infected individuals who are unaware of their status or those receiving antiretroviral therapy but not virally suppressed. Currently available rapid HIV tests, including fourth-generation HIV antigen/antibody combo tests, lack the sensitivity needed for detecting HIV during early stages of acute infection and viral rebound. Nucleic acid amplification tests (NAATs) that detect HIV-1 RNA, which can be detected in as early as 5 to 10 days after viral transmission, offer superior sensitivity and are currently the gold standard for detecting HIV during acute infection and viral rebound. However, NAATs are laborious, time-consuming and rely on expensive instrumentation and refrigerated components, hindering their use for self-testing. Therefore, the objective of this project is to develop and validate a shelf-stable, instrument-free CRISPR- Cas13-based rapid test for highly sensitive HIV-1 detection in blood or saliva. This high-risk, high-reward approach is fundamentally different from existing NAATs that involve multiple steps of sample preparation (RNA extraction and amplification) or require laboratory instrumentation or refrigerated components. Our rapid HIV-1 test will employ an engineered Cas13a enzyme with enhanced collateral activity and stability, and lyophilized assay components for enhanced shelf life while offering a simplified workflow that can be performed in home settings (similar to COVID-19 antigen rapid self-tests). A phone app will automatically analyze the readout from the lateral flow device and present the test results in a clear, simple-to-interpret manner. The rationale for this research is supported by the applicants' preliminary data demonstrating highly sensitive detection of viral RNA using a rapid, lateral flow CRISPR-Cas13-based assay and the development of an RRM2-L Cas13a enzyme with enhanced collateral activity and stability. In the R61 phase, we will pursue the following specific aims: 1) Characterize HIV-1 RNA levels in saliva and blood from a diverse population of people with HIV; 2) Investigate strategies for user-friendly device operation; 3) Develop a shelf-stable, instrument-free CRISPR-Cas13 rapid test for highly sensitive HIV-1 detection. If the R61 phase milestones are achieved, we will further develop and characterize this platform and validate its functionality using clinical HIV specimens during the R33 phase by pursuing the following specific aims: 4) Further develop and characterize the lateral flow CRISPR-Cas13-based rapid HIV-1 test; 5) Validate the functionality of the CRISPR-Cas13-based rapid HIV-1 test using clinical specimens. This approach is innovative because it combines the speed, simplicity and affordability of lateral flow-based diagnostic tests with the high sensitivity of CRISPR-Cas-based nucleic acid detection, and it is significant because it will improve the effectiveness of HIV self-testing for early diagnosis and patient monitoring during ART therapy, thus minimizing unintentional HIV transmission and reducing HIV-associated morbidity and mortality.

艾滋病毒是世界上最致命的传染病之一。 HIV传播主要由急性驱动受感染的人不知道自己的地位或接受抗逆转录病毒疗法但没有病毒的人被压制。目前可用的快速HIV测试，包括第四代HIV抗原/抗体组合测试，缺乏在急性感染和病毒反弹的早期阶段检测HIV所需的灵敏度。核检测HIV-1 RNA的酸扩增测试（NAAT），可以在5至10天内检测到HIV-1 RNA 病毒传播，具有较高的灵敏度，目前是急性期间检测HIV的金标准感染和病毒反弹。但是，NAATS是费力的，耗时的，并且依靠昂贵仪器和冷藏组件，阻碍了它们用于自我测试的用途。因此，该项目的目的是开发和验证货架稳定的，无仪器的CRISPR- 基于CAS13的快速测试，用于血液或唾液中高度敏感的HIV-1检测。这个高风险，高回报方法与涉及样品制备多个步骤的现有NAAT根本不同（RNA 提取和放大）或需要实验室仪器或冷藏组件。我们的快速HIV-1 测试将采用具有增强的抵押活动和稳定性的工程CAS13A酶，并冻干分析组件可增强保质期，同时提供可以在家中执行的简化工作流程设置（类似于COVID-19抗原快速自我测试）。电话应用程序将自动分析读数横向流动设备并以清晰，简单的解释方式呈现测试。理由研究得到了申请人的初步数据的支持，证明了病毒RNA的高度敏感性检测使用基于CRISPR-CAS13的快速，横向流量的测定和RRM2-L CAS13A酶的开发具有增强的抵押活动和稳定性。在R61阶段，我们将追求以下特定目标： 1）表征唾液中的HIV-1 RNA水平和来自艾滋病毒众多人群的血液； 2）调查用户友好设备操作的策略； 3）为高度敏感的HIV-1检测开发货架稳定的，无仪器的CRISPR-CAS13快速测试。如果实现了R61阶段里程碑，我们将进一步开发和表征该平台并验证其通过追求以下特定目的，在R33阶段使用临床HIV标本的功能： 4）进一步发展并表征基于CRISPR-CAS13的横向流量HIV-1测试； 5）使用临床标本验证基于CRISPR-CAS13的快速HIV-1测试的功能。这种方法具有创新性，因为它结合了基于横向流的速度，简单性和可负担性诊断测试具有基于CRISPR-CAS的核酸检测的高灵敏度，并且很重要因为它将提高艾滋病毒自我测试在艺术期间早期诊断和患者监测的有效性治疗，从而最大程度地减少了无意的HIV传播，并降低了与HIV相关的发病率和死亡率。