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MicroRNAs (miRNAs or miRs) are small noncoding RNAs derived from genome to control target gene expression. Recently we have developed a novel platform permitting high-yield production of bioengineered miRNA agents (BERA). This study is to produce and utilize novel fully-humanized BERA/miR-328-3p molecule (hBERA/miR-328) to delineate the role of miR-328-3p in controlling nutrient uptake essential for cell metabolism. We first demonstrated successful high-level expression of hBERA/miR-328 in bacteria and purification to high degree of homogeneity (>98%). Biologic miR-328-3p prodrug was selectively processed to miR-328-3p to suppress the growth of highly-proliferative human osteosarcoma (OS) cells. Besides glucose transporter protein type 1, gene symbol solute carrier family 2 member 1 (GLUT1/SLC2A1), we identified and verified large neutral amino acid transporter 1, gene symbol solute carrier family 7 member 5 (LAT1/SLC7A5) as a direct target for miR-328-3p. While reduction of LAT1 protein levels by miR-328-3p did not alter homeostasis of amino acids within OS cells, suppression of GLUT1 led to a significantly lower glucose uptake and decline in intracellular levels of glucose and glycolytic metabolite lactate. Moreover, combination treatment with hBERA/miR-328 and cisplatin or doxorubicin exerted a strong synergism in the inhibition of OS cell proliferation. These findings support the utility of novel bioengineered RNA molecules and establish an important role of miR-328-3p in the control of nutrient transport and homeostasis behind cancer metabolism., Graphical abstract Fully-humanized, bioengineered miR-328-3p agent reduces glycolysis through the suppression of protein levels of targeted transporters GLUT1 and LAT1 in human osteosarcoma cells, leading to synergistic antiproliferative effects when combined with chemotherapeutic drugs.Image 1

Lung cancer remains the leading cause of cancer deaths worldwide and accounts for more than 22% of all cancer-related deaths in the US. Developing new therapies is essential to combat against deadly lung cancer, especially the most common type, non-small cell lung cancer (NSCLC). With the discovery of genome-derived functional small noncoding RNA (ncRNA), namely microRNAs (miRNA or miR), restoration of oncolytic miRNAs lost or downregulated in NSCLC cells represents a new therapeutic strategy. Very recently, we have developed a novel technology that achieves in vivo fermentation production of bioengineered miRNA agents (BERA) for research and development. In this study, we aimed at simultaneously introducing two miRNAs into NSCLC cells by using single recombinant "combinatorial BERA" (CO-BERA) molecule. Our studies show that single CO-BERA molecule (e.g., let-7c/miR-124) was successfully processed to two miRNAs (e.g., let-7c-5p and miR-124-3p) to combinatorially regulate the expression of multiple targets (e.g., RAS, VAMP3 and CDK6) in human NSCLC cells, exhibiting greater efficacy than respective BERA miRNAs in the inhibition of cell viability and colony formation. Furthermore, we demonstrate that CO-BERA let-7c/miR-124-loaded lipopolyplex nanomedicine was the most effective among tested RNAs in the control of tumor growth in NSCLC patient-derived xenograft mouse models. The anti-tumor activity of CO-BERA let-7c/miR-124 was associated with the suppression of RAS and CDK6 expression, and enhancement of apoptosis. These results support the concept to use single ncRNA agent for dual-targeting and offer insight into developing new RNA therapeutics for the treatment of lethal NSCLC.

Rationale: Noncoding RNAs (ncRNAs) such as microRNAs (miRs or miRNAs) play important roles in the control of cellular processes through posttranscriptional gene regulation. However, ncRNA research is limited to utilizing RNA agents synthesized in vitro. Recombinant RNAs produced and folded in living cells shall better recapitulate biologic RNAs. Methods: Herein, we developed a novel platform for in vivo fermentation production of humanized recombinant ncRNA molecules, namely hBERAs, carrying payload miRNAs or siRNAs. Target hBERAs were purified by anion exchange FPLC method. Functions of hBERA/miRNAs were investigated in human carcinoma cells and antitumor activities were determined in orthotopic osteosarcoma xenograft spontaneous lung metastasis mouse models. Results: Proper human tRNAs were identified to couple with optimal hsa-pre-miR-34a as new fully-humanized ncRNA carriers to accommodate warhead miRNAs or siRNAs. A group of 30 target hBERAs were all heterogeneously overexpressed (each accounting for >40% of total bacterial RNA), which facilitated large-scale production (8-31 mg of individual hBERAs from 1L bacterial culture). Model hBERA/miR-34a-5p and miR-124-3p were selectively processed to warhead miRNAs in human carcinoma cells to modulate target gene expression, enhance apoptosis and inhibit invasiveness. In addition, bioengineered miR-34a-5p and miR-124-3p agents both reduced orthotopic osteosarcoma xenograft tumor growth and spontaneous pulmonary metastases significantly. Conclusion: This novel ncRNA bioengineering technology and resulting recombinant ncRNAs are unique additions to conventional technologies and tools for basic research and drug development.

Research on RNA function and therapeutic potential is dominated by the use of chemoengineered RNA mimics. Recent efforts have led to the establishment of novel technologies for the production of recombinant or bioengineered RNA molecules, which should better recapitulate the structures, functions and safety profiles of natural RNAs because both are produced and folded in living cells. Herein, we describe a robust approach for reproducible fermentation production of bioengineered RNA agents (BERAs) carrying warhead miRNAs, siRNAs, aptamers, or other forms of small RNAs, based upon an optimal hybrid tRNA/pre-miRNA carrier. Target BERA/sRNAs are readily purified by fast protein liquid chromatography (FPLC) to a high degree of homogeneity (>97%). This approach offers a consistent high-level expression (>30% of total bacterial RNAs) and large-scale production of ready-to-use BERAs (multiple to tens milligrams from 1 L bacterial culture).

Our group and others have previously shown that genistein combined polysaccharide (GCP), an aglycone isoflavone-rich extract with high bioavailability and low toxicity, can inhibit prostate cancer (CaP) cell growth and survival as well as androgen receptor (AR) activity. We now elucidate the mechanism by which this may occur using LNCaP and PC-346C CaP cell lines, GCP can inhibit intracrine androgen synthesis in CaP cells. UPLC-MS/MS and qPCR analyses demonstrated that GCP can mediate a ~3-fold decrease in testosterone levels (p &lt, 0.001) and cause decreased expression of intracrine androgen synthesis pathway enzymes (~2.5-fold decrease of 3&beta, HSD (p &lt, 0.001), 17&beta, 0.001), CYP17A (p &lt, 0.01), SRB1 (p &lt, 0.0001), and StAR (p &lt, 0.01)), respectively. Reverse-phase HPLC fractionation and bioassay identified three active GCP fractions. Subsequent NMR and LC-MS analysis of the fraction with the highest level of activity, fraction 40, identified genistein as the primary active component of GCP responsible for its anti-proliferative, pro-apoptotic, and anti-AR activity. GCP, fraction 40, and genistein all mediated at least a ~2-fold change in these biological activities relative to vehicle control (p &lt, 0.001). Genistein caused similar decreases in the expression of 17&beta, HSD and CYP17A (2.5-fold (p &lt, 0.001) and 1.5-fold decrease (p &lt, 0.01), respectively) compared to GCP, however it did not cause altered expression of the other intracrine androgen synthesis pathway enzymes, 3&beta, HSD, SRB1, and StAR. Our combined data indicate that GCP and/or genistein may have clinical utility and that further pre-clinical studies are warranted.

The coronavirus disease of 2019 (COVID-19) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global pandemic with increasing incidence and mortality rates. Recent evidence based on the cytokine profiles of severe COVID-19 cases suggests an overstimulation of macrophages and monocytes associated with reduced T-cell abundance (lymphopenia) in patients infected with SARS-CoV-2. The SARS-CoV-2 open reading frame 3 a (ORF3a) protein was found to bind to the human HMOX1 protein at a high confidence through high-throughput screening experiments. The HMOX1 pathway can inhibit platelet aggregation, and can have anti-thrombotic and anti-inflammatory properties, amongst others, all of which are critical medical conditions observed in COVID-19 patients. Here, we review the potential of modulating the HMOX1-ORF3a nexus to regulate the innate immune response for therapeutic benefits in COVID-19 patients. We also review other potential treatment strategies and suggest novel synthetic and natural compounds that may have the potential for future development in clinic.

miR-127 is downregulated in breast cancer, where it has been shown to suppress the proliferation, migration, and invasion of breast cancer cells. In triple-negative breast cancer (TNBC), miR-127 downregulation correlates with decreased disease-free and overall patient survival. Tumor suppressor miRNAs may hold therapeutic promise but progress has been limited by several factors, including the lability and high cost of miRNA mimics. Here, we take a novel approach to produce a miR-127 prodrug (miR-127PD), which we demonstrate is processed to mature, functional miR-127-3p in TNBC tumor cells. miR-127PD decreased the viability and motility of TNBC cells, sensitized TNBC cells to chemotherapy, and restricted the TNBC stem cell population. Furthermore, systemic delivery of miR-127PD suppressed tumor growth of MDA-MB-231 and MDA-MB-468 TNBC cells and spontaneous metastasis of MDA-MB-231 cells. In addition, CERK, NANOS1, FOXO6, SOX11, SOX12, FASN, and SUSD2 were identified as novel, functionally important targets of miR-127. In conclusion, our study demonstrates that miR-127 functions as a tumor and metastasis suppressor in TNBC and that delivery of miR-127 may hold promise as a novel therapy. Significance: Exogenous administration of miR-127, which is functionally activated in target cells, inhibits growth and spontaneous metastasis of triple-negative breast cancer.

Drug-metabolizing enzymes, transporters, and nuclear receptors are essential for the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. MicroRNAs participate in the regulation of ADME gene expression via imperfect complementary WatsonCrick base pairings with target transcripts. We have previously reported that Cytochrome P450 3A4 (CYP3A4) and ATP-binding cassette sub-family G member 2 (ABCG2) are regulated by miR-27b-3p and miR-328-3p, respectively. Here we employed our newly established RNA bioengineering technology to produce bioengineered RNA agents (BERA), namely BERA/miR-27b-3p and BERA/miR-328-3p, via fermentation. When introduced into human cells, BERA/miR-27b-3p and BERA/miR-328-3p were selectively processed to target miRNAs and thus knock down CYP3A4 and ABCG2 mRNA and their protein levels, respectively, as compared to cells treated with vehicle or control RNA. Consequently, BERA/miR-27b-3p led to a lower midazolam 1′-hydroxylase activity, indicating the reduction of CYP3A4 activity. Likewise, BERA/miR-328-3p treatment elevated the intracellular accumulation of anticancer drug mitoxantrone, a classic substrate of ABCG2, hence sensitized the cells to chemotherapy. The results indicate that biologic miRNA agents made by RNA biotechnology may be applied to research on miRNA functions in the regulation of drug metabolism and disposition that could provide insights into the development of more effective therapies. KEY WORDS: Bioengineered RNA, miR-27b, miR-328, CYP3A4, ABCG2, Drug disposition