Recombinant microRNAs in xenobiotic disposition

 描述(由申请人提供)了解microRNAs(miRNAs或miRs)在调节异物处置和行为中的功能和意义对于开发新的合理治疗方法非常重要。然而，目前还缺乏有效的手段来生产大量天然的、具有生物活性的miRNA试剂，用于体内预期的机制和治疗研究。为了填补这一空白，PI的实验室研究了生产重组前miRNA(Mir)试剂的可行性，这种试剂可能会在人类细胞中加工成功能成熟的miRNA。这些研究使PI开发了一种新的基于前miRNA的优化非编码RNA支架(OnRS)，以实现在普通大肠杆菌中一致、高产和大规模表达重组OnRS/miRNAs和OnRS/siRNAs。经过纯化和结构鉴定，重组的ncRNAs被发现在不同的人类细胞系中被选择性地加工成靶向miRNAs(或siRNAs)。因此，重组的miRNAs降低了靶基因的表达，改变了细胞的异种代谢能力，并使细胞对小分子药物敏感。此外，在体内-jetPEI配方的重组ncRNAs在小鼠模型中具有良好的耐受性，并在体内抑制了靶基因的表达。鉴于这些令人兴奋的初步发现，我们假设具有生物活性的miRNA制剂(例如miR-27b、miR-34a和miR-124)可以使用这种基于OnRS的新型重组RNA技术进行大规模工程，OnRS/miRNAs可能作为独特的工具来定义miRNAs在调节药物处置和疗效中的重要性。为了验证这一假说，我们将改进和标准化OnRS技术，生产一套OnRS/miRNA和OnRS/siRNA试剂，并定义它们的主要结构特征，包括转录后修饰(目标1)，描述OnRS/miRNAs在控制细胞外异物代谢和体外处置中的机制功能(目标2)，并建立OnRS/miRNAs在体内临床相关动物模型中调节药代动力学和治疗结果的有效性(目标3)。本研究将建立一种新的基于OnRS的重组RNA技术，并为miRNA药物表观遗传学开辟新的途径。

 DESCRIPTION (provided by applicant) Understanding the function and significance of microRNAs (miRNAs or miRs) in the regulation of xenobiotic disposition and actions is important for developing new rational therapies. However, there is a lack of efficient means to produce large quantities of natural, biologically active miRNA agents for prospective mechanistic and therapeutic studies in vivo. To fill this gap, the PI's lab has investigated the feasibility of producing recombinant pre-miRNA (mir) agents, which may be processed to functional mature miRNAs in human cells. These studies have led the PI to develop a novel pre-miRNA-based optimal noncoding RNA scaffold (OnRS) for achieving a consistent, high-yield, and large-scale expression of recombinant OnRS/miRNAs and OnRS/siRNAs in a common strain of E. coli. After purification and structural characterization, recombinant ncRNAs were found to be selectively processed to target miRNAs (or siRNAs) within various human cell line systems. Consequently, recombinant miRNAs reduced target gene expression, altered cellular xenobiotic metabolism capacity, and sensitized the cells to small-molecule drugs. Moreover, in vivo-jetPEI-formulated recombinant ncRNAs were well tolerated in mouse models and suppressed target gene expression in vivo. Given these exciting preliminary findings, we hypothesize that biologically active miRNA agents (e.g., miR-27b, miR-34a, and miR-124) can be engineered on a large scale using this novel OnRS-based recombinant RNA technique, and the OnRS/miRNAs may serve as unique tools to define the importance of miRNAs in the modulation of drug disposition and efficacy. To test this hypothesis, we will refine and standardize OnRS technique, produce a set of OnRS/miRNA and OnRS/siRNA agents, and define their primary structural characteristics including posttranscriptional modifications (Aim 1), delineate the mechanistic functions of OnRS/miRNAs in the control of cellular xenobiotic metabolism and disposition in vitro (Aim 2), and establish the efficacy of OnRS/miRNAs in the modulation of pharmacokinetics and therapeutic outcomes in clinically relevant animal models in vivo (Aim 3). This research shall establish a novel OnRS-based recombinant RNA technique and open new avenues to miRNA pharmacoepigenetics.