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的非编码RNA定向转录基因沉默作为治疗HIV-1感染的方法。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。我们最近还开发了嗜热四膜虫的PDDIP DNA切割机制并将其人源化，发现该系统可以用来切割那些被小的非编码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。这些方法围绕3种靶向传递方法：(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。这项提议将是验证几种靶向递送方法的第一阶段，该递送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，这一机制有可能在没有病毒耐药性的情况下导致感染个体的病毒表达长期稳定沉默。这样的结果可以被认为是一种有效的治疗方法。

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.