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

 描述(由申请人提供)： 这项应用为针对HIV的Cas9/GUIDE RNA开发了一种靶向递送系统，使其同时进入潜伏和持续感染的细胞。尽管高效抗病毒疗法(HAART)极大地延长了HIV感染者的寿命，但由于病毒基因组的完整拷贝存在于长期存活库中，包括静止的CD4+记忆T细胞，因此需要终生治疗。从潜伏期重新激活会导致释放具有复制能力的病毒，补充个体内的病毒负荷。切除整合病毒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艾滋病毒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非靶点效应的情况下对LTR序列进行有效的切除。目前的关键瓶颈是开发专门将该系统传递给艾滋病毒感染细胞的方法。提出的方法适用于我们实验室的两个新兴研究领域。第一种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输送到细胞中。我们已经成功地将功能性核转录因子和有毒蛋白输送到细胞中。将对该系统进行修改，将Cas9/GUIDE RNA纳入MLV VLP。第二个具体目标是开发一种改进的Env系统，允许通过scFv部分进入。这种方法将scFv显示在病毒的外表面，并与Env蛋白化学计量相联系。已经确定了选择性单链抗体，它既可以针对潜伏感染细胞，也可以针对持续感染的细胞。如果成功，这项研究可能会导致从目标感染细胞中消除病毒基因组。这一方法的关键是该系统固有的多功能性，允许替换引导RNA靶标以及用于直接进入的单链抗体。该系统可以推广和扩展，以解决广泛的应用和疾病。

 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.