RNA directed silencing and excision of HIV-1 and CCR5

描述（由申请人提供）：该项目的目标是开发小非编码RNA定向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，针对HIV-1或CCR5启动子中特定位点的小非编码rna可以导致HIV-1或CCR5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嗜热四膜菌的Pddip DNA切除机制，并发现该系统可用于切除那些被小非编码RNA转录沉默的靶向位点。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3种靶向递送方法：(1)有条件地复制HIV-2载体，(2)CCR5或gp120结合适体和(3)CXCR4， 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。这些方法将在体外和体内进行开发和机械验证，并对意外的继发性脱靶效应进行严格评估。该建议将是验证几种靶向递送方法的第一阶段，这些方法将被用作非编码RNA定向转录基因沉默和基因切除的细胞特异性递送策略，这种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。这样的结果可以被认为是功能性治愈。

DESCRIPTION (provided by applicant): 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 Pddip 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, CCR5 binding nanoparticles, in order to introduce non-coding RNAs 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 severa targeted delivery approaches to be used as a cell specific delivery strategy for non-coding RNA directed transcriptional gene silencing and 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. Such an outcome could be considered a functional cure.