Fundamentals of RNA based gene silencing and excision of HIV-1 and CCR5

项目总结 该项目的目标是开发小的非编码RNA定向转录基因沉默，作为治疗HIV-1感染的一种方式。我们已经了解到，针对HIV-1或CCR5启动子中特定位点的小的非编码RNA可以导致HIV-1或CCR5的长期稳定的表观遗传沉默。值得注意的是，在HIV-1的背景下，这种形式的沉默对病毒突变是难以实现的。我们 最近，他们还开发了嗜热四膜虫的Pddlp DNA切除机制并将其人源化，发现该系统可用于切除那些被小的非编码RNA定位为转录沉默的基因座。这里提出的工作将从机械上验证几种不同的互补方法，这些方法可能导致一种新的治疗方法，能够长期调节HIV-1或CCR5的转录或切除。这些方法主要围绕以下三个方面： 传递：(1)有条件地复制HIV-2载体，(2)CCR5或gp120结合适体和(3)CXCR4或CCR5结合纳米颗粒，以便在HIV-1感染和相关细胞类型中引入能够转录沉默和切除HIV-1或CCR5的非编码RNA和切除复合体。这些方法将被开发出来，并在体外和体内进行机械验证，以及对意外的次要脱靶效应进行严格的评估。这项建议将是验证几种靶向传递方法的第一阶段，这些方法将被用作细胞特异性传递策略，用于非编码RNA定向转录基因沉默和RNA定向基因切除，这一机制有可能在没有病毒耐药性的情况下导致感染个体中病毒表达的长期稳定沉默。

PROJECT SUMMARY The goal of this project is to develop small non-coding RNA directed transcriptional gene silencing as a therapeutic modality for the treatment of HIV-1 infection. We have learned that small non-coding RNAs targeted to specific loci in the HIV-1 or CCR5 promoters can result in long-term stable epigenetic silencing of HIV-1 or CCR5. Notably, this form of silencing in the context of HIV-1 is refractory to viral mutation. We have also recently developed and humanized the Pddlp DNA excision machinery from Tetrahymina thermophila and found that this system can be used to excise those loci targeted for transcriptional silencing by the small non-coding RNA. The work proposed here will mechanistically validate several different complimentary approaches that may result in a novel therapeutic capable of regulating transcription or excision of HIV-1 or CCR5 in a long-term manner. These approaches center around 3 methods of targeted delivery: (1) conditionally replicating HIV-2 vectors, (2) CCR5 or gp120 binding aptamers and (3) CXCR4 or CCR5 binding nanoparticles, in order to introduce non-coding RNAs and excision complex capable of transcriptionally silencing and excising HIV-1 or CCR5 in HIV-1 infected and relevant cell types. These approaches will be developed and mechanistically validated in vitro and in vivo as well as critically assessed for unintended secondary off-target effects. This proposal will be the first stage of validating several targeted delivery approaches to be used as a cell specific delivery strategy for non-coding RNA directed transcriptional gene silencing and RNA directed gene excision, a mechanism that has the potential to result in long-term stable silencing of viral expression in infected individuals in the absence of viral resistance.