Recombinant microRNAs in xenobiotic disposition

 说明（申请人提供）理解microRNA（miRNAs或miRs）在调节异生物质处置和作用中的功能和意义对于开发新的合理疗法是重要的。然而，缺乏有效的手段来生产大量的天然的、生物活性的miRNA试剂用于体内的前瞻性机制和治疗研究。为了填补这一空白，PI的实验室已经研究了生产重组pre-miRNA（mir）试剂的可行性，这些试剂可以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pre-miRNA的最佳非编码RNA支架（OnRS），用于在常见的E.杆菌在纯化和结构表征后，发现重组ncRNA在各种人类细胞系系统中被选择性地加工成靶向miRNA（或siRNA）。因此，重组miRNAs降低了靶基因的表达，改变了细胞的异生物质代谢能力，并使细胞对小分子药物敏感。此外，体内jetPEI配制的重组ncRNA在小鼠模型中耐受良好，并抑制体内靶基因表达。鉴于这些令人兴奋的初步发现，我们假设生物活性miRNA试剂（例如，miR-27 b、miR-34 a和miR-124）可以使用这种新的基于OnRS的重组RNA技术进行大规模工程化，并且OnRS/miRNAs可以作为独特的工具来定义miRNAs在调节药物处置和功效中的重要性。为了验证这一假设，我们将完善和标准化OnRS技术，产生一组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（Aim 1），描述OnRS/miRNAs在体外控制细胞异源生物质代谢和处置中的机制功能（Aim 2），并确定OnRS/miRNA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功效（目的3）。该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.