Targeted delivery of Cas9/gRNA directed to HIV latent/persistent cells

Cas9/gRNA 靶向递送至 HIV 潜伏/持续细胞

• 批准号: 9112854
• 负责人: MONICA J ROTH
• 金额: $ 19.88万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9112854
• 关键词: Acquired Immunodeficiency Syndrome; Address; Antitoxins; Antiviral Therapy; Area; Bacterial Toxins; Binding; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell model; Cells; Chimeric Proteins; Cleaved cell; Development; Disease; Excision; Genes; Genome; Guide RNA; HIV; HIV Genome; Human; Immune system; Individual; Knowledge; Laboratories; Lead; Life; Life Cycle Stages; Link; Longevity; MS4A1 gene; Mediating; Modification; Molecular; Murine leukemia virus; Normal Cell; Nuclear; Peptide Hydrolases; Proteins; Reagent; Research; Rest; Scheme; Sindbis Virus; Site; Surface; System; Testing; Time; Vertebral column; Viral; Viral Genome; Viral Load result; Virus; Virus-like particle; Visual; antibody conjugate; base; env Gene Products; genome editing; human DNA; interest; knowledge base; memory CD4 T lymphocyte; neutralizing antibody; novel; particle; public health relevance; reactivation from latency; targeted delivery; transcription factor; viral DNA; viral nanoparticle

 DESCRIPTION (provided by applicant): This application develops a targeted delivery system for the Cas9/guide RNA directed against HIV into both latent and persistently infected cells. Although highly active antiviral therapy (HAART) has greatly increased the life-span of HIV infected individual, it requires life-long treatment due to the presence of integrated copies of the viral genome in long-lived reservoirs, including resting CD4+ memory T cells. Reactivation from latency results in releasing the replication-competent virus, replenishing the viral load within the individual. Excision of the integrated virus is not part of the viral life-cycle. In recent months, the possibility of excisingor inactivating the integrated HIV genome through the use of the CRISPR/Cas9 prokaryotic immune system has become a feasible approach, with the first demonstration of effective excision across LTR sequences with limited off-target effects. The key bottleneck now is the development of means to deliver this system specifically to the HIV infected cells. The approach proposed applies two emerging areas of research of our laboratory. The first develops Murine Leukemia Virus (MLV) virus-like particles (VLP) for the delivery of proteins, rather than genes into cells. We have successfully delivered functional nuclear transcription factors and toxic proteins into cells. This system will be modified to incorporate the Cas9/guide RNAs into MLV VLPs. The second specific aim develops a modified Env system that allows entry through scFv moieties. This approach displays the scFv on the outer viral surface and is stoichiometrically linked with the Env proteins. Selective scFv have been identified which will target both latent as well as persistently infected cells. If successful, the study could lead to the elimination of the viral genome from targeted infected cells. Key to this approach is the inherent versatility of the system, allowing for replacement of both the guide RNA targets as well as the scFv used to direct entry. This system could be generalized and expanded to address a wide range of applications and diseases.

 描述（由申请人提供）： 该申请开发了一种靶向递送系统，用于将针对HIV的Cas9/指导RNA递送到潜伏和持续感染的细胞中。尽管高效抗病毒治疗（HAART）已经极大地延长了HIV感染个体的寿命，但是由于在长寿储库（包括静息的CD 4+记忆T细胞）中存在病毒基因组的整合拷贝，因此其需要终身治疗。从潜伏期重新激活导致释放有复制能力的病毒，补充个体内的病毒载量。整合病毒的切除不是病毒生命周期的一部分。近几个月来，通过使用CRISPR/Cas9原核免疫系统切除或灭活整合的HIV基因组的可能性已成为一种可行的方法，首次证明了在有限的脱靶效应下有效切除LTR序列。现在的关键瓶颈是开发将该系统特异性地递送到HIV感染细胞的方法。所提出的方法适用于我们实验室的两个新兴研究领域。第一个是开发小鼠白血病病毒（MLV）病毒样颗粒（VLP），用于将蛋白质而不是基因递送到细胞中。我们已经成功地将功能性核转录因子和毒性蛋白质递送到细胞中。该系统将被修改以将Cas9/指导RNA掺入MLV VLP中。第二个具体目标是开发允许通过scFv部分进入的修饰的Env系统。这种方法将scFv展示在病毒外表面上，并与Env蛋白化学计量连接。已经鉴定了选择性scFv，其将靶向潜伏以及持续感染的细胞。如果成功，该研究可能导致从目标感染细胞中消除病毒基因组。这种方法的关键是系统固有的多功能性，允许替换指导RNA靶标以及用于引导进入的scFv。该系统可以推广和扩展，以解决广泛的应用和疾病。