Acute/chronic limitations to transcriptional RNAi therapies for infectious and other liver diseases

描述（由申请人提供） 基于肝脏的RNA干扰（RNAi）疗法的有限早期成功正在实现额外的临床前和临床方法。与任何治疗剂一样，当递送高RNAi剂量时，实现功能效果会受到毒副作用的影响。基于高水平转录RNAi的小鼠临床前研究的毒性甚至致死性首先由我们的小组报道，并由其他人证实。在过去的5年中，我们对microRNA生物学的基本理解做出了重要贡献，包括pri/pre-miRNA加工，Dicer切割机制以及与miRNA-RISC加载相关的特性。这些研究导致了改进的RNAi设计策略，旨在提高疗效和最大限度地减少毒性，特别是那些面向更好地治疗HBV和HCV感染的策略。与RNAi相似但不同的是，反义寡核苷酸临床试验越来越受欢迎，包括靶向高度丰富的肝脏特异性miR- 122（70%的肝脏miRNAs）以防止HCV复制。我们最近发现，新转录的miR-122- 5 p-22 nt miRNA亚型在AAV-U6-shRNA递送后被选择性地从活性RISC复合物中置换，并且miR-122的功能降低是毒性的原因。我们计划研究miR-122的这些变化如何发生并导致毒性的机制。我们提出了一个竞争模型，其中从DNA模板转录的shRNA取代miR 122，然后导致含有miR 122靶基因的上调和miR 122基因位点的转录改变，最终破坏正常肝细胞的稳态并诱导肝损伤。我们还认为，来自miR 122基因座的前体转录物-一组长非编码RNA不仅仅是一组前体中间体，而且在细胞编程中具有单独的功能。我们将使用各种遗传工具和各种小鼠模型来解偶联来自miR 122位点的各种RNA的表达，并确定它们对基因调控、肝细胞稳态和生长状态的影响。我们还将评估miR 122基因对参与肝细胞癌形成的chr 12 qF 1 microRNA位点激活的可能调控作用。总之，我们的提案将进一步阐明RNAi诱导毒性的机制，并确定最高表达和组织特异性miRNA基因座-miR 122在健康和患病肝脏状态中的生物学功能。

DESCRIPTION (provided by applicant) Limited early successes in liver-based RNA interference (RNAi) therapies are enabling additional pre-clinical and clinical approaches. As with any therapeutic, achieving a functional effect is tempered by toxic side effects when a high RNAi dose is delivered. Toxicity and even fatality from high-level transcriptional RNAi based preclinical studies in mice was first reported by our group and confirmed by others. During the last 5 years, we made important contributions to our basic understanding of microRNA biology including pri/pre-miRNA processing, mechanism of Dicer cleavage, and properties related to miRNA-RISC loading. These studies have resulted in improved RNAi design strategies aimed at increasing efficacy and minimizing toxicity, especially those geared towards better treatments for HBV and HCV infection. Similar but distinct from RNAi, antisense oligonucleotide clinical trials are gaining in popularity includig targeting the highly abundant liver-specific miR- 122 (70% of liver miRNAs) to prevent HCV replication. We recently found that the newly transcribed miR-122- 5p-22nt miRNA isoform was selectively displaced from active RISC complexes after AAV-U6-shRNA delivery and that the functional reduction in miR-122 was responsible for the toxicity. We plan to investigate the mechanism(s) involved in how these changes in miR-122 occur and lead to toxicity. We propose a competition model where the shRNAs transcribed from a DNA template displaces miR122, which then causes both an up regulation of genes containing a miR122 target and altered transcription from the miR122 gene locus ultimately disrupting normal hepatocyte homeostasis and inducing liver injury. We also believe that precursor transcripts from the miR122 locus - a set of long-noncoding RNAs are not merely a set of precursor intermediates but have separate functions in cellular programming. We will use a variety of genetic tools and various mouse models to uncouple the expression of the various RNAs derived from the miR122 locus and determine their effect on gene regulation, hepatocyte homeostasis, and growth states. We will also evaluate the possible regulatory role that the miR122 gene has on the activation of the chr12qF1 microRNA locus implicated in the formation of hepatocellular carcinoma. In total, our proposal will further unravel the mechanism(s) of RNAi-induced toxicity and define the biological function of the most highly expressed and tissue specific miRNA gene locus- miR122 in healthy and diseased liver states.