Recombinant microRNAs in xenobiotic metabolism and disposition

项目摘要 了解可变药物代谢和处置（DMD）背后的分子机制至关重要 来改善药物治疗和药物开发。我研究的最终目标是破译小说 转录后调节机制由非编码microRNAs（miRNAs）控制，并建立 在DMD中的重要性我的实验室在DMD领域开创了miRNA研究， 填补了理解新的转录后调控机制的关键空白。然而，目前 关于DMD中miRNA功能的研究，以及在普通医学科学的广泛领域中的研究，仅限于 使用化学工程的RNA“模拟物”在体外制造，其中，包括广泛的和各种类型的 化学修饰，从根本上不同于天然RNA分子， 在细胞中折叠并且不携带或仅具有最小程度的转录后修饰。这也是 与蛋白质研究形成鲜明对比的是， 在活细胞中产生和折叠的生物工程蛋白质，而不是合成多肽或蛋白质 在试管中制造的。最近，我们已经开发了一种新的重组RNA技术，基于杂交 tRNA/pre-miRNA载体，提供重组miRNA试剂的高产量和大规模生产 （&小干扰RNA或siRNA）通过具有成本效益的细菌发酵。我们的研究也 证明了生物工程miRNA试剂（BERA）作为一种“前药”， 靶向人类细胞中的miRNA分子，以选择性地调节靶基因的表达。此外，Access 到大量（来自1 L细菌培养物的数十毫克）高纯度（>98%）重组miRNA 分子使我们能够容易地研究miRNA对药代动力学的影响， 在体内动物模型中的药效学（PK/PD）。这些高度创新的方法和 独一无二的重组miRNA工具将为miRNA研究和开发开辟新的途径， 以及广泛的普通医学科学。在这个MIRA应用中，我们建议生产和利用 一种重组miRNAs，通过解决DMD中的 以下关键问题：1）基于tRNA/pre-miRNA的技术能否再创新用于生产 全人源化重组miRNAs（hBERA/miRNAs）？2)hBERA/miRNAs是否会被加工成靶向 人肝细胞和肝细胞中选择性调节药物代谢酶或 转运蛋白的表达，并改变细胞DMD的能力？3)miRNA将在多大程度上改变PK和PD 全身系统？

PROJECT SUMMARY Understanding the molecular mechanisms behind variable drug metabolism and disposition (DMD) is critical to improve pharmacotherapy and drug development. The ultimate goal of my research is to decipher novel posttranscriptional regulation mechanisms controlled by noncoding microRNAs (miRNAs) and establish their importance in DMD. My laboratory pioneered miRNA research in the fields of DMD that is expected to fill the critical gaps in understanding new posttranscriptional regulation mechanisms. Nevertheless, current studies on miRNA functions in DMD, as well as in the broad areas of general medical sciences, are limited to the use of chemo-engineered RNA “mimics” made in vitro, which, comprised of extensive and various types of chemical modifications, are fundamentally different from natural RNA molecules that are produced and folded in cells and do not carry or just have minimal degree of posttranscriptional modifications. This is also in sharp contrast to protein research that has found the ultimate success by using recombinant or bioengineered proteins produced and folded in living cells, instead of synthetic polypeptides or proteins made in vitro. Very recently, we have developed a novel recombinant RNA technology, based upon hybrid tRNA/pre-miRNA carriers, which offers high-yield and large-scale production of recombinant miRNA agents (& small interfering RNAs or siRNAs) through cost-effective bacterial fermentation. Our studies have also demonstrated that bioengineered miRNA agent (BERA) acts as a “prodrug”, which is specifically processed to target miRNA molecule in human cells to selectively regulate target gene expression. Further, the access to large quantity (tens milligrams from 1 L bacterial culture) of high-purity (>98%) recombinant miRNA molecules allows us to readily investigate the impact of miRNAs on pharmacokinetics and pharmacodynamics (PK/PD) in animal models in vivo. These highly innovative approaches and one-of-its-kind recombinant miRNA tools shall open up new avenues for miRNA research and development, as well as broadly general medical sciences. In this MIRA application, we propose to produce and utilize one-of-a-kind recombinant miRNAs to dissect new regulatory mechanisms in DMD by addressing the following key questions: 1) Can the tRNA/pre-miRNA-based technology be re-innovated for the production of fully-humanized recombinant miRNAs (hBERA/miRNAs)? 2) Will hBERA/miRNAs be processed to target miRNAs in human hepatocytes and liver cells to selectively modulate drug-metabolizing enzyme or transporter expression, and alter cellular DMD capacity? 3) To what degrees will miRNA alter PK and PD in the whole-body system?