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Review
. 2023;24(3):175-189.
doi: 10.2174/1389200224666230425232433.

Recent Advances in Novel Recombinant RNAs for Studying Post-transcriptional Gene Regulation in Drug Metabolism and Disposition

Affiliations
Review

Recent Advances in Novel Recombinant RNAs for Studying Post-transcriptional Gene Regulation in Drug Metabolism and Disposition

Mei-Juan Tu et al. Curr Drug Metab. 2023.

Abstract

Drug-metabolizing enzymes and transporters are major determinants of the absorption, disposition, metabolism, and excretion (ADME) of drugs, and changes in ADME gene expression or function may alter the pharmacokinetics/ pharmacodynamics (PK/PD) and further influence drug safety and therapeutic outcomes. ADME gene functions are controlled by diverse factors, such as genetic polymorphism, transcriptional regulation, and coadministered medications. MicroRNAs (miRNAs) are a superfamily of regulatory small noncoding RNAs that are transcribed from the genome to regulate _target gene expression at the post-transcriptional level. The roles of miRNAs in controlling ADME gene expression have been demonstrated, and such miRNAs may consequently influence cellular drug metabolism and disposition capacity. Several types of miRNA mimics and small interfering RNA (siRNA) reagents have been developed and widely used for ADME research. In this review article, we first provide a brief introduction to the mechanistic actions of miRNAs in post-transcriptional gene regulation of drug-metabolizing enzymes, transporters, and transcription factors. After summarizing conventional small RNA production methods, we highlight the latest advances in novel recombinant RNA technologies and applications of the resultant bioengineered RNA (BioRNA) agents to ADME studies. BioRNAs produced in living cells are not only powerful tools for general biological and biomedical research but also potential therapeutic agents amenable to clinical investigations.

Keywords: Drug-metabolizing enzyme; microRNA; recombinant RNA; regulation; siRNA; transporter.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors are named inventors of issued and pending patents related to RNA bioengineering technology and use that are owned by the University of California, Davis; and Dr. Yu is a founder of AimRNA, Inc. that intends to license the intellectual property.

Figures

Fig. (1).
Fig. (1).
Role of miRNAs in ADME/PK involving post-transcriptional gene regulation of metabolic enzymes, transporters, and their regulators. Genes encoding drug-metabolizing enzymes and transporters that are directly involved in the absorption, distribution, metabolism, and excretion of drugs are commonly referred to as ADME genes. MiRNAs have shown to regulate ADME gene expression at the post-transcriptional level via direct _targeting of ADME gene transcripts and/or acting on their regulators, such as nuclear receptors (NRs). Nuclear receptors typically bind to the DNA promoter regions of _targeted ADME genes to achieve transcriptional gene regulation. Alteration of drug-metabolizing enzyme and transporter levels could result in changes in the ADME/PK properties of related drugs. Vice versa, the processing of miRNAs could be controlled by multiple regulators that may be affected by drugs or xenobiotics.
Fig. (2).
Fig. (2).
Mechanisms of miRNA- and siRNA-mediated ADME gene regulation. The functional strand of the miRNA or siRNA duplex is loaded into an Argonaute protein (AGO) to form the RNA-induced silencing complex (RISC) and subsequently binds to _target mRNA through complementary base paring to exert RNA degradation and/or translation inhibition for the control of drug-metabolizing enzyme, transporter, and NR protein levels. MiRNAs usually bind to the 3’-untranslated region (3’UTR) of _target transcript via imperfect complementarity, while siRNAs are often designed to _target the coding region, and now increasingly 3’UTR, of _target mRNA through perfect base pairing.
Fig. (3).
Fig. (3).
MicroRNA biogenesis and common RNAi agents used in ADME research. Production of endogenous miRNAs is initiated by transcription of miRNA-encoding genes to produce primary miRNAs (pri-miRNA) that are subsequently sliced by nucleic RNase III enzyme Drosha to release precursor miRNAs (pre-miRNAs). Pre-miRNAs are then exported to the cytoplasm by Exportin-5 in a Ran guanosine triphosphate (Ran-GTP) dependent way. The cytoplasmic pre-miRNAs are further cleaved by another RNase III enzyme Dicer to produce miRNA duplexes. (A) MiRNA mimics or shRNA expressing plasmids or virus are the conventional RNAi agents designed by mimicking the natural miRNA biogenesis pathways. After transfection or infection, desired small RNA-encoding genes are integrated into the genome of the host cells, and then the miRNAs or siRNAs are derived by following the natural miRNA biogenesis processes, except that some designs of short, stem-loop shRNAs bypass the pri-miRNA production step. (B) Small RNA analogs produced by chemical synthesis or in vitro transcription are the most widely used RNA reagents in basic and clinical studies. In addition to double-stranded miRNA mimics and siRNAs, another kind of synthetic RNAs, single-stranded antisense oligos, namely antagomirs, are also used for the inhibition of miRNAs. (C) Recombinant or bioengineered RNA agents made in live cells using the tRNA fused pre-miRNA as a carrier are a novel class of RNAi agents, which, upon introduced into cells, are processed to _target miRNA or siRNA duplex in a Dicer-dependent or independent manner.
Fig. (4).
Fig. (4).
Schematic illustration of the novel tRNA/pre-miRNA carrier-based RNA bioengineering platform technology. (A) _target recombinant RNA or bioengineered RNA (BioRNA) is designed using a tRNA/pre-miRNA carrier with payload miRNA or siRNA or other small RNA. (B) The coding sequence of the BioRNA is cloned into a _target vector to offer the BioRNA-expressing plasmid. (C) The plasmids are transformed into E. coli to express the BioRNAs through overnight fermentation. (D) Overexpression of the _target RNA is readily verified by urea polyacrylamide gel electrophoresis (PAGE) analysis. The strong extra band shown at the expected size in the BioRNA expressing sample, as compared to the wild-type E. coli sample, indicates the successful high-level expression of _target BioRNA. (E) _target BioRNA is separated from the total bacterial RNA by an anion-exchange fast protein liquid chromatography (FPLC) method. (F) The purity of the final BioRNA product is further quantified by high-performance liquid chromatography (HPLC) analysis. BioRNAs with high homogeneity (> 97%) can be used for further studies.

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