Novel RNA molecular bioengineering technology efficiently produces functional miRNA agents.

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNAGly and BioRNALeu, respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.

RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges.

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.

Poor survival and prediction of prolonged isolated thrombocytopenia post umbilical cord blood transplantation in patients with hematological malignancies.

Prolonged isolated thrombocytopenia (PIT) is a common complication after umbilical cord blood transplantation (UCBT). However, data on PIT prediction and impacts on transplantation outcomes for UCBT patients are rare. We retrospectively analyzed 244 patients with hematological malignancies who received single-unit UCBT at the First Affiliated Hospital of USTC between August 2018 and December 2019. Among them, PIT occurred in 49 recipients, with a crude incidence of 20.1%. In the PIT patients, the 2-year cumulative incidence of transplant-related mortality (TRM) was significantly higher, and the probabilities of 2-year overall survival, leukemia-free survival and graft-versus-host disease (GVHD)-free relapse-free survival were significantly poorer (57.1% vs. 88.6%; 53.1% vs. 81.9%; 22.4% vs. 59.8%; p < 0.001), without remarkable increases in the cumulative incidence of relapse or chronic GVHD. Importantly, the multivariate analysis revealed that lower high-resolution HLA compatibility (≤6/10), lower infused CD34+ cell count (≤1.78 × 105 /kg), grade II-IV acute GVHD preplatelet engraftment, a lower pretransplantation platelet count (≤100 × 109 /L), and a longer neutrophil engraftment time (≥17 days) were independent risk factors for PIT after UCBT. These results demonstrate that PIT is common after UCBT, predicting inferior survival and the need for more monitoring during the early phase.

A Novel Integrated Pharmacokinetic-Pharmacodynamic Model to Evaluate Combination Therapy and Determine In Vivo Synergism.

Understanding pharmacokinetic (PK)-pharmacodynamic (PD) relationships is essential in translational research. Existing PK-PD models for combination therapy lack consideration of quantitative contributions from individual drugs, whereas interaction factor is always assigned arbitrarily to one drug and overstretched for the determination of in vivo pharmacologic synergism. Herein, we report a novel generic PK-PD model for combination therapy by considering apparent contributions from individual drugs coadministered. Doxorubicin (Dox) and sorafenib (Sor) were used as model drugs whose PK data were obtained in mice and fit to two-compartment model. Xenograft tumor growth was biphasic in mice, and PD responses were described by three-compartment transit models. This PK-PD model revealed that Sor (contribution factor = 1.62) had much greater influence on overall tumor-growth inhibition than coadministered Dox (contribution factor = 0.644), which explains the mysterious clinical findings on remarkable benefits for patients with cancer when adding Sor to Dox treatment, whereas there were none when adding Dox to Sor therapy. Furthermore, the combination index method was integrated into this predictive PK-PD model for critical determination of in vivo pharmacologic synergism that cannot be correctly defined by the interaction factor in conventional models. In addition, this new PK-PD model was able to identify optimal dosage combination (e.g., doubling experimental Sor dose and reducing Dox dose by 50%) toward much greater degree of tumor-growth inhibition (>90%), which was consistent with stronger synergy (combination index = 0.298). These findings demonstrated the utilities of this new PK-PD model and reiterated the use of valid method for the assessment of in vivo synergism. SIGNIFICANCE STATEMENT: A novel pharmacokinetic (PK)-pharmacodynamic (PD) model was developed for the assessment of combination treatment by considering contributions from individual drugs, and combination index method was incorporated to critically define in vivo synergism. A greater contribution from sorafenib to tumor-growth inhibition than that of coadministered doxorubicin was identified, offering explanation for previously inexplicable clinical observations. This PK-PD model and strategy shall have broad applications to translational research on identifying optimal dosage combinations with stronger synergy toward improved therapeutic outcomes.

MicroRNA-1291-5p Sensitizes Pancreatic Carcinoma Cells to Arginine Deprivation and Chemotherapy through the Regulation of Arginolysis and Glycolysis.

Cancer cells are dysregulated and addicted to continuous supply and metabolism of nutritional glucose and amino acids (e.g., arginine) to drive the synthesis of critical macromolecules for uncontrolled growth. Recent studies have revealed that genome-derived microRNA (miRNA or miR)-1291-5p (miR-1291-5p or miR-1291) may modulate the expression of argininosuccinate synthase (ASS1) and glucose transporter protein type 1 (GLUT1). We also developed a novel approach to produce recombinant miR-1291 agents for research, which are distinguished from conventional chemo-engineered miRNA mimics. Herein, we firstly demonstrated that bioengineered miR-1291 agent was selectively processed to high levels of target miR-1291-5p in human pancreatic cancer (PC) cells. After the suppression of ASS1 protein levels, miR-1291 perturbed arginine homeostasis and preferably sensitized ASS1-abundant L3.3 cells to arginine deprivation therapy. In addition, miR-1291 treatment reduced the protein levels of GLUT1 in both AsPC-1 and PANC-1 cells, leading to a lower glucose uptake (deceased > 40%) and glycolysis capacity (reduced approximately 50%). As a result, miR-1291 largely improved cisplatin efficacy in the inhibition of PC cell viability. Our results demonstrated that miR-1291 was effective to sensitize PC cells to arginine deprivation treatment and chemotherapy through targeting ASS1- and GLUT1-mediated arginolysis and glycolysis, respectively, which may provide insights into understanding miRNA signaling underlying cancer cell metabolism and development of new strategies for the treatment of lethal PC. SIGNIFICANCE STATEMENT: Many anticancer drugs in clinical use and under investigation exert pharmacological effects or improve efficacy of coadministered medications by targeting cancer cell metabolism. Using new recombinant miR-1291 agent, we revealed that miR-1291 acts as a metabolism modulator in pancreatic carcinoma cells through the regulation of argininosuccinate synthase- and glucose transporter protein type 1-mediated arginolysis and glycolysis. Consequently, miR-1291 effectively enhanced the efficacy of arginine deprivation (pegylated arginine deiminase) and chemotherapy (cisplatin), offering new insights into development of rational combination therapies.

Pharmacokinetic and Pharmacodynamic Factors Contribute to Synergism between Let-7c-5p and 5-Fluorouracil in Inhibiting Hepatocellular Carcinoma Cell Viability.

Pharmacological interventions for hepatocellular carcinoma (HCC) are hindered by complex factors, and rational combination therapy may be developed to improve therapeutic outcomes. Very recently, we have identified a bioengineered microRNA let-7c-5p (or let-7c) agent as an effective inhibitor against HCC in vitro and in vivo. In this study, we sought to identify small-molecule drugs that may synergistically act with let-7c against HCC. Interestingly, we found that let-7c exhibited a strong synergism with 5-fluorouracil (5-FU) in the inhibition of HCC cell viability as manifested by average combination indices of 0.3 and 0.5 in Hep3B and Huh7 cells, respectively. By contrast, coadministration of let-7c with doxorubicin or sorafenib inhibited HCC cell viability with, rather surprisingly, no or minimal synergy. Further studies showed that protein levels of multidrug resistance-associated protein (MRP) ATP-binding cassette subfamily C member 5 (MRP5/ABCC5), a 5-FU efflux transporter, were reduced around 50% by let-7c in HCC cells. This led to a greater degree of intracellular accumulation of 5-FU in Huh7 cells as well as the second messenger cyclic adenosine monophosphate, an endogenous substrate of MRP5. Since 5-FU is an irreversible inhibitor of thymidylate synthetase (TS), we investigated the interactions of let-7c with 5-FU at pharmacodynamic level. Interestingly, our data revealed that let-7c significantly reduced TS protein levels in Huh7 cells, which was associated with the suppression of upstream transcriptional factors as well as other regulatory factors. Collectively, these results indicate that let-7c interacts with 5-FU at both pharmacokinetic and pharmacodynamic levels, and these findings shall offer insight into molecular mechanisms of synergistic drug combinations. SIGNIFICANCE STATEMENT: Combination therapy is a common strategy that generally involves pharmacodynamic interactions. After identifying a strong synergism between let-7c-5p and 5-fluorouracil (5-FU) against hepatocellular carcinoma cell viability, we reveal the involvement of both pharmacokinetic and pharmacodynamic mechanisms. In particular, let-7c enhances 5-FU exposure (via suppressing ABCC5/MRP5 expression) and cotargets thymidylate synthase with 5-FU (let-7c reduces protein expression, whereas 5-FU irreversibly inactivates enzyme). These findings provide insight into developing rational combination therapies based on pharmacological mechanisms.

Novel approaches for efficient in vivo fermentation production of noncoding RNAs.

Genome-derived noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), play an essential role in the control of target gene expression underlying various cellular processes, and dysregulation of ncRNAs is involved in the pathogenesis and progression of various diseases in virtually all species including humans. Understanding ncRNA biology has opened new avenues to develop novel RNA-based therapeutics. Presently, ncRNA research and drug development is dominated by the use of ncRNA mimics that are synthesized chemically in vitro and supplemented with extensive and various types of artificial modifications and thus may not necessarily recapitulate the properties of natural RNAs generated and folded in living cells in vivo. Therefore, there are growing interests in developing novel technologies for in vivo production of RNA molecules. The two most recent major breakthroughs in achieving an efficient, large-scale, and cost-effective fermentation production of recombinant or bioengineered RNAs (e.g., tens of milligrams from 1 L of bacterial culture) are (1) using stable RNA carriers and (2) direct overexpression in RNase III-deficient bacteria, while other approaches offer a low yield (e.g., nano- to microgram scales per liter). In this article, we highlight these novel microbial fermentation-based technologies that have shifted the paradigm to the production of true biological ncRNA molecules for research and development.

Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs.

Noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), regulate target gene expression and can be used as tools for understanding biological processes and identifying new therapeutic targets. Currently, ncRNA molecules for research and therapeutic use are limited to ncRNA mimics made by chemical synthesis. We have recently established a high-yield and cost-effective method of producing bioengineered or biologic ncRNA agents (BERAs) through bacterial fermentation, which is based on a stable tRNA/pre-miR-34a carrier (~ 180 nt) that accommodates target small RNAs. Nevertheless, it remains a challenge to heterogeneously express longer ncRNAs (e.g., > 260 nt), and it is unknown if single BERA may carry multiple small RNAs. To address this issue, we hypothesized that an additional human pre-miR-34a could be attached to the tRNA/pre-miR-34a scaffold to offer a new tRNA/pre-miR-34a/pre-miR-34a carrier (~ 296 nt) for the accommodation of multiple small RNAs. We thus designed ten different combinatorial BERAs (CO-BERAs) that include different combinations of miRNAs, siRNAs, and antagomirs. Our data showed that all target CO-BERAs were successfully expressed in Escherichia coli at high levels, greater than 40% in total bacterial RNAs. Furthermore, recombinant CO-BERAs were purified to a high degree of homogeneity by fast protein liquid chromatography methods. In addition, CO-BERAs exhibited strong anti-proliferative activities against a variety of human non-small cell lung cancer cell lines. These results support the production of long ncRNA molecules carrying different warhead small RNAs for multi-targeting which may open avenues for developing new biologic RNAs as experimental, diagnostic, and therapeutic tools.

Bioengineered Noncoding RNAs Selectively Change Cellular miRNome Profiles for Cancer Therapy.

Noncoding RNAs (ncRNAs) produced in live cells may better reflect intracellular ncRNAs for research and therapy. Attempts were made to produce biologic ncRNAs, but at low yield or success rate. Here we first report a new ncRNA bioengineering technology using more stable ncRNA carrier (nCAR) containing a pre-miR-34a derivative identified by rational design and experimental validation. This approach offered a remarkable higher level expression (40%-80% of total RNAs) of recombinant ncRNAs in bacteria and gave an 80% success rate (33 of 42 ncRNAs). New FPLC and spin-column based methods were also developed for large- and small-scale purification of milligrams and micrograms of recombinant ncRNAs from half liter and milliliters of bacterial culture, respectively. We then used two bioengineered nCAR/miRNAs to demonstrate the selective release of target miRNAs into human cells, which were revealed to be Dicer dependent (miR-34a-5p) or independent (miR-124a-3p), and subsequent changes of miRNome and transcriptome profiles. miRNA enrichment analyses of altered transcriptome confirmed the specificity of nCAR/miRNAs in target gene regulation. Furthermore, nCAR assembled miR-34a-5p and miR-124-3p were active in suppressing human lung carcinoma cell proliferation through modulation of target gene expression (e.g., cMET and CDK6 for miR-34a-5p; STAT3 and ABCC4 for miR-124-3p). In addition, bioengineered miRNA molecules were effective in controlling metastatic lung xenograft progression, as demonstrated by live animal and ex vivo lung tissue bioluminescent imaging as well as histopathological examination. This novel ncRNA bioengineering platform can be easily adapted to produce various ncRNA molecules, and biologic ncRNAs hold the promise as new cancer therapeutics.

Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells.

The nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor in the regulation of many oxidative enzymes and efflux transporters critical for oxidative stress and cellular defense against xenobiotics. NRF2 is dysregulated in patient osteosarcoma (OS) tissues and correlates with therapeutic outcomes. Nevertheless, research on the NRF2 regulatory pathways and its potential as a therapeutic target is limited to the use of synthetic small interfering RNA (siRNA) carrying extensive artificial modifications. Herein, we report successful high-level expression of recombinant siRNA against NRF2 in Escherichia coli using our newly established noncoding RNA bioengineering technology, which was purified to >99% homogeneity using an anion-exchange fast protein liquid chromatography method. Bioengineered NRF2-siRNA was able to significantly knock down NRF2 mRNA and protein levels in human OS 143B and MG63 cells, and subsequently suppressed the expression of NRF2-regulated oxidative enzymes [heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1] and elevated intracellular levels of reactive oxygen species. In addition, recombinant NRF2-siRNA was effective to sensitize both 143B and MG63 cells to doxorubicin, cisplatin, and sorafenib, which was associated with significant downregulation of NRF2-targeted ATP-binding cassette (ABC) efflux transporters (ABCC3, ABCC4, and ABCG2). These findings support that targeting NRF2 signaling pathways may improve the sensitivity of cancer cells to chemotherapy, and bioengineered siRNA molecules should be added to current tools for related research.

Co-administration of nuciferine reduces the concentration of metformin in liver via differential inhibition of hepatic drug transporter OCT1 and MATE1.

Nuciferine (NF), one of the main and effective components in Nelumbo nucifera Gaertn. leaf extracts, is a promising drug candidate for the treatment of obesity-related diseases, while metformin is a first line therapeutic drug for type 2 diabetes mellitus. Since nuciferine and metformin are likely to be co-administered, the aim of the present study was to evaluate whether co-administration of nuciferine would influence the liver (target tissue) distribution and the anti-diabetic effect of metformin by inhibiting hepatic organic cation transporter 1 (OCT1) and multidrug and toxin extrusion 1 (MATE1). The data demonstrated that nuciferine significantly reduced metformin accumulation in MDCK cells stably expressing human OCT1 (MDCK-hOCT1) or hMATE1 (MDCK-hMATE1), and primary cultured mouse hepatocytes. Furthermore, the presence of nuciferine in the basal compartment caused a concentration-dependent reduction of intracellular metformin accumulation in MDCK-hOCT1/hMATE1 cell monolayers. Compared with the metformin treatment-alone group, co-administration of nuciferine (40 mg/kg) markedly reduced the metformin concentration in mouse livers at 30 and 60 min after a single oral dose of metformin (200 mg/kg), and subsequently impaired the glucose-lowering effect of metformin (200 mg/kg), but the glucose-lowering effect became no different at 90 and 120 min. Therefore, nuciferine influenced the liver concentration and glucose-lowering effect of metformin only for a period of time after dose, administration of nuciferine and metformin with an interval might prevent the drug-drug interaction mediated by OCT1 and MATE1.

MicroRNA Pharmacoepigenetics: Posttranscriptional Regulation Mechanisms behind Variable Drug Disposition and Strategy to Develop More Effective Therapy.

Knowledge of drug absorption, distribution, metabolism, and excretion (ADME) or pharmacokinetics properties is essential for drug development and safe use of medicine. Varied or altered ADME may lead to a loss of efficacy or adverse drug effects. Understanding the causes of variations in drug disposition and response has proven critical for the practice of personalized or precision medicine. The rise of noncoding microRNA (miRNA) pharmacoepigenetics and pharmacoepigenomics has come with accumulating evidence supporting the role of miRNAs in the modulation of ADME gene expression and then drug disposition and response. In this article, we review the advances in miRNA pharmacoepigenetics including the mechanistic actions of miRNAs in the modulation of Phase I and II drug-metabolizing enzymes, efflux and uptake transporters, and xenobiotic receptors or transcription factors after briefly introducing the characteristics of miRNA-mediated posttranscriptional gene regulation. Consequently, miRNAs may have significant influence on drug disposition and response. Therefore, research on miRNA pharmacoepigenetics shall not only improve mechanistic understanding of variations in pharmacotherapy but also provide novel insights into developing more effective therapeutic strategies.

Chimeric MicroRNA-1291 Biosynthesized Efficiently in Escherichia coli Is Effective to Reduce Target Gene Expression in Human Carcinoma Cells and Improve Chemosensitivity.

In contrast to the growing interests in studying noncoding RNAs (ncRNAs) such as microRNA (miRNA or miR) pharmacoepigenetics, there is a lack of efficient means to cost effectively produce large quantities of natural miRNA agents. Our recent efforts led to a successful production of chimeric pre-miR-27b in bacteria using a transfer RNA (tRNA)-based recombinant RNA technology, but at very low expression levels. Herein, we present a high-yield expression of chimeric pre-miR-1291 in common Escherichia coli strains using the same tRNA scaffold. The tRNA fusion pre-miR-1291 (tRNA/mir-1291) was then purified to high homogeneity using affinity chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-1291 was readily processed to mature miR-1291 in human carcinoma MCF-7 and PANC-1 cells. Consequently, recombinant tRNA/mir-1291 reduced the protein levels of miR-1291 target genes, including ABCC1, FOXA2, and MeCP2, as compared with cells transfected with the same doses of control methionyl-tRNA scaffold with a sephadex aptamer (tRNA/MSA). In addition, tRNA-carried pre-miR-1291 suppressed the growth of MCF-7 and PANC-1 cells in a dose-dependent manner, and significantly enhanced the sensitivity of ABCC1-overexpressing PANC-1 cells to doxorubicin. These results indicate that recombinant miR-1291 agent is effective in the modulation of target gene expression and chemosensitivity, which may provide insights into high-yield bioengineering of new ncRNA agents for pharmacoepigenetics research.

[Establishment of MDCK cell models expressing human MATE1 or co-expressing with human OCT1 or OCT2].

To establish single- and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindIII and Kpn I restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)- hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 µg · mL(-1), all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 µmol · L(-1) cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.

The contribution of human OCT1, OCT3, and CYP3A4 to nitidine chloride-induced hepatocellular toxicity.

Nitidine chloride (NC), a quaternary ammonium alkaloid, has numerous pharmacological effects, such as anticancer activity. However, it was found that NC also has hepatocellular toxicity. Because organic cation transporters 1 and 3 (OCT1 and OCT3) might mediate the influx of NC into hepatocytes, multidrug and toxin extrusion 1 (MATE1) probably mediates the efflux of NC from hepatocytes, while cytochrome P450 (P450) enzymes might contribute to NC metabolism, the present study was to evaluate the contribution of OCT1, OCT3, MATE1, and P450 enzymes to NC-induced hepatocellular toxicity. Our results showed that the uptake of NC in Madin-Darby canine kidney (MDCK) cells expressing human (h) OCT1 and OCT3 (MDCK-hOCT1 and MDCK-hOCT3) was significantly higher than that in mock cells; the hOCT1- and hOCT3-mediated uptake followed typical Michaelis-Menten kinetics. Meanwhile, NC was also a substrate of hMATE1, although its transport capacity was much lower than that of OCT1 NC-induced cytotoxicity in MDCK-hOCT1 or MDCK-hOCT3 cells was obviously higher than that in mock cells. Quinidine and (+)-tetrahydropalmatine [(+)-THP], OCT1 and OCT3 inhibitors, significantly reduced the uptake of NC in MDCK-hOCT1 cells, MDCK-hOCT3 cells, and rat primary hepatocytes, but only (+)-THP markedly attenuated the NC-induced toxicity. In addition, P450 enzymes, such as CYP3A4, mediated the metabolism of NC, and NC-induced toxicity in MDCK-hOCT1/hCYP3A4 cells was lower than that in MDCK-hOCT1 cells. Our results indicated that NC is a substrate of hOCT1, hOCT3, and CYP3A4; that OCT1 and OCT3 mediate the uptake of NC in hepatocytes and subsequently cause hepatotoxicity; and that NC-induced toxicity could be attenuated by CYP3A4-mediated metabolism.

[In vitro interaction of deferiprone with cellular membrane transporters of hOCTs and hOAT1].

OBJECTIVE: To develop a LC-MS/MS method for determination of deferiprone in cell lysate and to study the potential interaction between deferiprone and hOCTs or hOAT1 transporters in vitro. METHODS: The determination was performed on an Agilent Eclipse Plus C18 column(3.5 µm, 2.1 mm×50 mm).The gradient mobile phase was composed of solvent A:0.1% formic acid in water, and B:0.1% formic acid in acetonitrile. The mass spectrometer with an electrospray interface was operated in positive ion mode with multiple reaction monitoring (MRM) scan mode monitored the ion pair of deferiprone at m/z 140→96, or phenacetin at m/z 180→110. The effects of deferiprone on the accumulation of typical substrates of hOCTs and hOAT1 were evaluated by MDCK-hOCTs and MDCK-hOAT1 cells respectively. The accumulation of deferiprone was also investigated in MDCK-hOCTs cells and mock cells with or without typical inhibitors. RESULTS: The standard curve was linear over the range of 5-300 nmol/L. The assay recovery of deferiprone was above 94%, and the intra-day precision (RSD) was less than 2.0%. The accumulation of MPP(+) in MDCK-hOCTs cells with 300 µmol/L deferiprone were 73.5%, 87.1% and 70.4%, respectively. The uptake of deferiprone in MDCK-hOCTs and mock cells did not show significant difference. Deferiprone of 100 µmol/L did not significantly affect the accumulation of 6-CF in MDCK-hOAT1 cell. CONCLUSION: The method is sensitivity and suitable for the determination of deferiprone in cell lysate. Deferiprone can significantly inhibit hOCT1 and hOCT3, but has no effects on hOCT2 and hOAT1. hOCTs may not play a major role in the transport of deferiprone.

Molecular Engineering of Functional SiRNA Agents.

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (<5 EU/µg RNA). Furthermore, we demonstrate that three representative BioRNA/siRNAs against GFP, BCL2, and PD-L1 are biologically active and can specifically and efficiently silence their respective targets with the potential to effectively produce downstream antiproliferation effects by PD-L1-siRNA. With these promising results, we aim to advance the field of synthetic biology by offering a novel platform to bioengineer functional siRNA agents for research and drug development.

Efflux ABC transporters in drug disposition and their posttranscriptional gene regulation by microRNAs.

ATP-binding cassette (ABC) transporters are transmembrane proteins expressed commonly in metabolic and excretory organs to control xenobiotic or endobiotic disposition and maintain their homeostasis. Changes in ABC transporter expression may directly affect the pharmacokinetics of relevant drugs involving absorption, distribution, metabolism, and excretion (ADME) processes. Indeed, overexpression of efflux ABC transporters in cancer cells or bacteria limits drug exposure and causes therapeutic failure that is known as multidrug resistance (MDR). With the discovery of functional noncoding microRNAs (miRNAs) produced from the genome, many miRNAs have been revealed to govern posttranscriptional gene regulation of ABC transporters, which shall improve our understanding of complex mechanism behind the overexpression of ABC transporters linked to MDR. In this article, we first overview the expression and localization of important ABC transporters in human tissues and their clinical importance regarding ADME as well as MDR. Further, we summarize miRNA-controlled posttranscriptional gene regulation of ABC transporters and effects on ADME and MDR. Additionally, we discuss the development and utilization of novel bioengineered miRNA agents to modulate ABC transporter gene expression and subsequent influence on cellular drug accumulation and chemosensitivity. Findings on posttranscriptional gene regulation of ABC transporters shall not only improve our understanding of mechanisms behind variable ADME but also provide insight into developing new means towards rational and more effective pharmacotherapies.