In vivo fermentation production of humanized noncoding RNAs carrying payload miRNAs for targeted anticancer therapy

Abstract

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.

MicroRNAs in the Regulation of Solute Carrier Proteins Behind Xenobiotic and Nutrient Transport in Cells

“…An alternative approach to miRNA mimics has been developed to produce bioengineered RNA molecules ( Chen Q.-X. et al, 2015 ; Ho et al, 2018 ; Li P.-C. et al, 2018 ; Deng et al, 2021 ; Li P.-C. et al, 2021 ; Tu et al, 2021 ). The challenges of developing miRNA therapeutics have been discussed in recent articles ( Segal and Slack, 2020 ; Kara et al, 2022 ; Yu and Tu, 2022 ), which include minimizing the degradation from nucleases, improving target cell uptake, and avoiding off-target or unwanted side effects.…”

Bioengineered miR-34a modulates mitochondrial inner membrane protein 17 like 2 (MPV17L2) expression toward the control of cancer cell mitochondrial functions

“…Recent studies have also demonstrated that miR-34a is involved in the modulation of cancer metabolism and tumor immune responses through the regulation of several metabolic enzymes [ 20 , 21 ] and programmed death-ligand 1 (PD-L1) [ 22 , 23 ], respectively. As a result, restoration of miR-34a function has been shown to be effective in controlling many types of malignancies including lung cancer [ 24–26 ], osteosarcoma (OS) [ 14 , 27 , 28 ], prostate cancer [ 19 , 29 ], liver cancer [ 15 ], and colorectal cancer [ 30 ] in animal models.…”

“…At present, miRNA research and experimental therapy depend predominantly upon the use of synthetic miRNA “mimics.” Noticing the fundamental differences between miRNA ”mimics” synthesized chemically or biochemically in vitro and natural RNAs produced and folded in vivo [ 31–33 ], we have established a novel technology, based on the stable tRNA-fused precursor miRNA (pre-miRNA) carriers identified in our laboratory, to achieve in vivo fermentation production of bioengineered miRNA agents (BERAs) [ 26 , 34 , 35 ] that should better recapitulate the physicochemical and biological properties of natural RNAs because both are produced and folded in live cells. Further identification of proper human tRNAs and optimal hsa-pre-miRNA coupled carriers leads to high-yield and large-scale production of “humanized“ BERAs (hBERAs) that are more compatible to human cells [ 28 ]. Omics-based studies and targeted analyses have demonstrated that oncolytic miRNAs or small interfering RNAs (siRNAs) are precisely released from the BERAs or hBERAs in human cells to selectively control target gene expression and regulate various cancer cellular processes in vitro and subsequently suppress tumor growth and metastasis and improve chemotherapy in animal models in vivo [ 25 , 26 , 28 , 34 , 36–44 ].…”

“…Further identification of proper human tRNAs and optimal hsa-pre-miRNA coupled carriers leads to high-yield and large-scale production of “humanized“ BERAs (hBERAs) that are more compatible to human cells [ 28 ]. Omics-based studies and targeted analyses have demonstrated that oncolytic miRNAs or small interfering RNAs (siRNAs) are precisely released from the BERAs or hBERAs in human cells to selectively control target gene expression and regulate various cancer cellular processes in vitro and subsequently suppress tumor growth and metastasis and improve chemotherapy in animal models in vivo [ 25 , 26 , 28 , 34 , 36–44 ].…”

MicroRNA-22-3p targeted regulating transcription factor 7-like 2 (TCF7L2) constrains the Wnt/β-catenin pathway and malignant behavior in osteosarcoma

“…Recently, exploring the character of miRNAs in cancer development has become a research hotspot. Recent studies have manifested that both bioengineered miR-34a-5p and −124-3p can suppress tumor growth and spontaneous lung metastasis of raw OS [ 9 ]. Wang Y et al .…”