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

项目摘要 该项目的目标是开发小的非编码RNA指导的转录基因沉默作为治疗HIV-1感染的治疗方式。我们已经了解到，靶向HIV-1或CCR 5启动子中特定位点的小的非编码RNA可以导致HIV-1或CCR 5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治的。我们 最近还开发并人源化了来自嗜热四膜虫的Pddlp DNA切除机制，并发现该系统可用于切除被小的非编码RNA靶向转录沉默的那些基因座。本文提出的工作将从机制上验证几种不同的互补方法，这些方法可能会产生一种能够长期调节HIV-1或CCR 5转录或切除的新型治疗方法。这些方法围绕着3种有针对性的 交货期：（1）条件复制型HIV-2载体，（2）CCR 5或gp 120结合适体和（3）CXCR 4或CCR 5结合纳米颗粒，以便在HIV-1感染的和相关的细胞类型中引入能够转录沉默和切除HIV-1或CCR 5的非编码RNA和切除复合物。这些方法将在体外和体内进行开发和机械验证，并对非预期的继发脱靶效应进行严格评估。该提案将是验证几种靶向递送方法的第一阶段，这些方法将用作非编码RNA指导的转录基因沉默和RNA指导的基因切除的细胞特异性递送策略，这是一种有可能在不存在病毒抗性的情况下导致感染个体中病毒表达长期稳定沉默的机制。

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.