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

基本信息

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
项目类别：
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

项目摘要

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）：该应用开发了一种针对 HIV 的 Cas9/引导 RNA 靶向递送系统，将其导入潜伏和持续感染的细胞中。由于长寿命储存库中存在病毒基因组的整合副本，包括休眠的 CD4+ 记忆 T 细胞，从潜伏期重新激活会释放具有复制能力的病毒，从而补充个体内的病毒载量。整合的病毒不属于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，并首次证明了有效切除。现在的关键瓶颈是开发专门针对 HIV 感染细胞的系统。所提出的方法适用于我们实验室的两个新兴研究领域。 (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/引导 RNA 整合到 MLV 中。第二个具体目标是开发一种改良的 Env 系统，允许通过 scFv 部分进入，这种方法将 scFv 展示在病毒外表面，并与已鉴定的选择性 scFv 进行化学计量连接。如果成功，该研究可能会消除目标感染细胞中的病毒基因组，这种方法的关键是系统固有的多功能性，可以替换引导RNA靶标。以及用于直接进入的 scFv，该系统可以推广和扩展以解决广泛的应用和疾病。