RNA-mediated excision of the HIV-1 genome from latently infected cells in CNS

RNA介导的中枢神经系统潜伏感染细胞中HIV-1基因组的切除

• 批准号: 8736299
• 负责人: Wenhui Hu
• 金额: $ 38.92万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-15 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8736299
• 关键词: Ablation; Acquired Immunodeficiency Syndrome; Adenoviruses; Aging; Aging-Related Process; Anti-Retroviral Agents; Astrocytes; Bioinformatics; Biological Assay; Biological Models; Biology; Bone Diseases; Brain; Cell Culture Techniques; Cell model; Cells; Centers for Disease Control and Prevention (U.S.); Central Nervous System Diseases; Chromosomes; Clinical Research; Comorbidity; Complementary RNA; DNA; DNA Sequence; Detection; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Drug resistance; Employment; Enzyme-Linked Immunosorbent Assay; Epidemic; Excision; Exhibits; Gene Expression; Gene Mutation; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Genotype; Guide RNA; HIV-1; Heart failure; Hela Cells; Human; In Vitro; Individual; Infection; Knowledge; Laboratories; Lentivirus Vector; Long-Term Survivors; Luciferases; Mediating; Microglia; Modeling; Molecular; Molecular Genetics; Neurocognitive; Neuropathogenesis; Outcome; Outcome Study; Patients; Pharmaceutical Preparations; Phenotype; Population; Production; Property; Proteins; Quality of life; RNA; Reporter; Reporter Genes; Reporting; Rest; Restriction fragment length polymorphism; Risk; Solid; Southern Blotting; Specificity; Survival Rate; System; Technology; Therapeutic; Uncertainty; United States; Viral; Viral Genes; Viral Genome; Viral Proteins; Virus; Virus Diseases; antiretroviral therapy; arm; base; drug resistant virus; experience; genetic analysis; improved; interest; macrophage; nervous system disorder; neurobehavioral disorder; novel; novel strategies; nuclease; preclinical study; promoter; prophylactic; public health relevance; resistant strain; screening; tool; vaccine development; viral DNA

DESCRIPTION (provided by applicant): According to the CDC, greater than 1.1 million people in the United States and more than 35 million people worldwide are infected with HIV-1. While the introduction of combined antiretroviral therapy, cART, has greatly improved survival rates among AIDS patients, a substantial portion of HIV-1 infected individuals remain at risk for the development of full blown AIDS as a result of reactivation of latently infected cells, partly due t nonadherence to medication and emergence of drug resistant viruses. Moreover, HIV-1 positive long term survivors continue to develop comorbidities including an accelerated aging process, neurocognitive disorders, heart failure, and others. From the virological point of view, as none of the current treatments suppress viral gene transcription, it is suspected that low, yet continuous, levels of viral early proteins with regulatory and pathogenic activities may contribute to the development of these quality of life threating illnesses. Sadly, none of the efforts toward the development of vaccines against HIV-1 have shown promising outcomes. Thus, curing of AIDS by eradicating the HIV-1 genome in infected subjects requires a novel strategy that is specific, highly effective, sustained, and irreversible. Recently, we have adapted a genetic approach using the clustered regulatory interspaced short palindromic repeat-assisted system (Cas) and a short complementary single-stranded RNA, called guide RNA or gRNA, which specifically targets the U3 region of the HIV-1 LTR promoter and precisely excises a segment of the viral regulatory sequence required for its expression. In addition, the employment of single and multiplex gRNA in our Cas system show promising results that include eradication of the entire HIV-1 genome in latently infected microglial cells, thus abrogating viral gene expression and transcription. Based on this preliminary observation, we propose to develop an RNA-guided Cas9 that acts as molecular scissors and, by disrupting various regions of the LTR and/or removing the entire viral genome, abrogates reactivation of the virus in macrophages, microglia and astrocytes which serve as the viral reservoir in the brain. Furthermore, we will explore the feasibility of our single and multiplex Cas9 system for use as a prophylactic compound in in vitro HIV-1 infection culture models. The outcome of this molecular genetic and virological approach will provide a solid platform for developing preclinical and clinical studies toward the treatment f AIDS and its associated neurological and neurobehavioral disorders.

描述（由申请人提供）：根据 CDC 的数据，美国有超过 110 万人，全球有超过 3500 万人感染 HIV-1。虽然联合抗逆转录病毒疗法（cART）的引入大大提高了艾滋病患者的生存率，但由于潜伏感染细胞的重新激活，相当一部分HIV-1感染者仍然面临发展为全面艾滋病的风险，部分原因是不坚持用药和耐药病毒的出现。此外，HIV-1 呈阳性的长期幸存者会继续出现合并症，包括加速衰老过程、神经认知障碍、心力衰竭等。从病毒学的角度来看，没有 目前的治疗方法抑制病毒基因转录，怀疑其水平较低但持续， 具有调节和致病活性的病毒早期蛋白水平可能有助于这些威胁生活质量的疾病的发展。遗憾的是，开发 HIV-1 疫苗的努力均未显示出有希望的结果。因此，通过根除感染者体内的HIV-1基因组来治愈艾滋病需要一种特异性、高效、持续和不可逆转的新策略。最近，我们采用了一种遗传方法，使用簇状调控间隔短回文重复辅助系统（Cas）和短互补单链RNA（称为向导RNA或gRNA），它特异性靶向HIV-1 LTR启动子的U3区域，并精确切除其表达所需的病毒调控序列片段。此外，在我们的Cas系统中使用单一和多重gRNA显示出有希望的结果，包括根除潜伏感染的小胶质细胞中的整个HIV-1基因组，从而消除病毒基因表达和转录。基于这一初步观察，我们建议开发一种RNA引导的Cas9，它充当分子剪刀，通过破坏LTR的各个区域和/或去除整个病毒基因组，消除病毒在作为大脑病毒储存库的巨噬细胞、小胶质细胞和星形胶质细胞中的重新激活。此外，我们将探索我们的单一和多重 Cas9 系统在体外 HIV-1 感染培养模型中用作预防性化合物的可行性。这种分子遗传学和病毒学方法的成果将为开发艾滋病及其相关神经和神经行为障碍的临床前和临床研究提供坚实的平台。