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

描述(申请人提供)：根据美国疾病控制与预防中心的数据，美国有超过110万人感染艾滋病毒-1，全球有超过3500万人感染。虽然联合抗逆转录病毒疗法CART的引入极大地提高了艾滋病患者的存活率，但由于潜伏感染细胞的重新激活，相当一部分HIV-1感染者仍然面临着发展为全面艾滋病的风险，部分原因是不坚持药物治疗和出现抗药性病毒。此外，HIV-1阳性的长期幸存者继续发展为共病，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，因为没有 目前的治疗方法抑制病毒基因转录，据怀疑，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疾病的生活质量的发展。可悲的是，所有针对HIV-1疫苗的开发努力都没有显示出有希望的结果。因此，通过根除感染者中的HIV-1基因组来治愈艾滋病需要一种特异、高效、持续和不可逆转的新策略。最近，我们采用了一种遗传方法，使用了簇调控间隔短回文重复序列辅助系统(Cas)和一个短的互补单链RNA，称为GUIDE RNA或gRNA，它专门针对HIV-1 LTR启动子的U3区域，并精确地切除其表达所需的一段病毒调控序列。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，从而取消病毒基因的表达和转录。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的不同区域和/或移除整个病毒基因组，在巨噬细胞、小胶质细胞和星形胶质细胞中消除病毒的重新激活，这些细胞充当大脑中的病毒库。此外，我们将探索我们的单一和多重Cas9系统作为预防化合物用于体外HIV-1感染培养模型的可行性。这种分子遗传学和病毒学方法的结果将为开展治疗艾滋病及其相关的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.