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

描述（由申请人提供）：根据CDC的数据，美国有超过110万人，全世界有超过3500万人感染HIV-1。虽然联合抗逆转录病毒疗法（cART）的引入极大地提高了AIDS患者的存活率，但相当一部分HIV-1感染个体由于潜伏感染细胞的再活化而仍然处于发展为全面AIDS的风险中，部分原因是不依从药物治疗和出现耐药病毒。此外，HIV-1阳性的长期存活者继续发展合并症，包括加速老化过程、神经认知障碍、心力衰竭等。从病毒学的角度来看， 目前的治疗方法抑制病毒基因转录，怀疑低，但持续， 具有调节和致病活性的病毒早期蛋白的水平可能有助于这些威胁生活质量的疾病的发展。可悲的是，所有针对HIV-1疫苗的研发工作都没有取得令人鼓舞的成果。因此，通过消除感染受试者中的HIV-1基因组来治愈艾滋病需要一种特异、高效、持续和不可逆的新策略。最近，我们采用了一种遗传方法，使用成簇的调控间隔短回文重复序列辅助系统（Cas）和一种短互补单链RNA（称为向导RNA或gRNA），其特异性靶向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，包括在潜伏感染的小胶质细胞中根除整个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，我们提出开发一种RNA引导的Cas9，其充当分子剪刀，并且通过破坏LTR的各个区域和/或去除整个病毒基因组，消除病毒在巨噬细胞、小胶质细胞和星形胶质细胞中的再活化，这些细胞作为大脑中的病毒储存库。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。这一分子遗传学和病毒学方法的结果将为开发治疗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。

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.