Ternalys’ Technology & MicroRNAs

Articles

Noncoding RNA Therapeutics – Challenges and Potential Solutions

Nature Reviews Drug Discovery, Vol 20, 629-651 (2021)

WINKLE M, EL-DALY SM, FABBI M, ET AL.

Therapeutic targeting of noncoding RNAs (ncRNAs), such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), represents an attractive approach for the treatment of cancers, as well as many other diseases. Over the past decade, substantial effort has been made towards the clinical application of RNA-based therapeutics, employing mostly antisense oligonucleotides and small interfering RNAs, with several gaining FDA approval. However, trial results have so far been ambivalent, with some studies reporting potent effects whereas others demonstrated limited efficacy or toxicity. Alternative entities such as antimiRNAs are undergoing clinical testing, and lncRNA-based therapeutics are gaining interest. In this Perspective, we discuss key challenges facing ncRNA therapeutics — including issues associated with specificity, delivery and tolerability — and focus on promising emerging approaches that aim to boost their success.

The Nuclear DICER–circular RNA Complex Drives the Deregulation of the Glioblastoma Cell microRNAome

Science Advances. Vol. 6, Issue 51 (2020)

BRONISZ A, ROOJ AK, KRAWCZYNSKI K, ET AL.

The assortment of cellular microRNAs (“microRNAome”) is a vital readout of cellular homeostasis, but the mechanisms that regulate the microRNAome are poorly understood. The microRNAome of glioblastoma is substantially down-regulated in comparison to the normal brain. Here, we find malfunction of the posttranscriptional maturation of the glioblastoma microRNAome and link it to aberrant nuclear localization of DICER, the major enzymatic complex responsible for microRNA maturation. Analysis of DICER’s nuclear interactome reveals the presence of an RNA binding protein, RBM3, and of a circular RNA, circ2082, within the complex. Targeting of this complex by knockdown of circ2082 results in the restoration of cytosolic localization of DICER and widespread derepression of the microRNAome, leading to transcriptome-wide rearrangements that mitigate the tumorigenicity of glioblastoma cells in vitro and in vivo with correlation to favorable outcomes in patients with glioblastoma. These findings uncover the mechanistic foundation of microRNAome deregulation in malignant cells.

Engineering, Delivery, and Biological Validation of Artificial microRNA Clusters for Gene Therapy Applications

Nature Protocols. 14, 3538–3553 (2019)

BHASKARAN V, YAO Y, BEI F, ET AL.

The cellular machinery regulating microRNA biogenesis and maturation relies on a small number of simple steps and minimal biological requirements and is broadly conserved in all eukaryotic cells. The same holds true in disease. This allows for a substantial degree of freedom in the engineering of transgenes capable of simultaneously expressing multiple microRNAs of choice, allowing a more comprehensive modulation of microRNA landscapes, the study of their functional interaction, and the possibility of using such synergism for gene therapy applications. We have previously engineered a transgenic cluster of functionally associated microRNAs to express a module of suppressed microRNAs in brain cancer for therapeutic purposes. Here, we provide a detailed protocol for the design, cloning, delivery, and utilization of such artificial microRNA clusters for gene therapy purposes. In comparison with other protocols, our strategy effectively decreases the requirements for molecular cloning, because the nucleic acid sequence encoding the combination of the desired microRNAs is designed and validated in silico and then directly synthesized as DNA that is ready for subcloning into appropriate delivery vectors, for both in vitro and in vivo use. Sequence design and engineering require 4–5 h. Synthesis of the resulting DNA sequence requires 4–6 h. This protocol is quick and flexible and does not require special laboratory equipment or techniques, or multiple cloning steps. It can be easily executed by any graduate student or technician with basic molecular biology knowledge.

The Functional Synergism of MicroRNA Clustering Provides Therapeutically Relevant Epigenetic Interference in Glioblastoma

Nature Communications. 10, 442 (2019)

BHASKARAN V, NOWICKI MO, IDRISS M, ET AL.

MicroRNA deregulation is a consistent feature of glioblastoma, yet the biological effect of each single gene is generally modest, and therapeutically negligible. Here we describe a module of microRNAs, constituted by miR-124, miR-128 and miR-137, which are co-expressed during neuronal differentiation and simultaneously lost in gliomagenesis. Each one of these miRs targets several transcriptional regulators, including the oncogenic chromatin repressors EZH2, BMI1 and LSD1, which are functionally interdependent and involved in glioblastoma recurrence after therapeutic chemoradiation. Synchronizing the expression of these three microRNAs in a gene therapy approach displays significant anticancer synergism, abrogates this epigenetic-mediated, multi-protein tumor survival mechanism and results in a 5-fold increase in survival when combined with chemotherapy in murine glioblastoma models. These transgenic microRNA clusters display intercellular propagation in vivo, via extracellular vesicles, extending their biological effect throughout the whole tumor. Our results support the rationale and feasibility of combinatorial microRNA strategies for anticancer therapies.

MicroRNA Therapeutics: Towards a New Era for the Management of Cancer and Other Diseases

Nature Reviews Drug Discovers, Vol 16, 203-222 (2017)

RUPAIMOOLE R AND SLACK FJ

In just over two decades since the discovery of the first microRNA (miRNA), the field of miRNA biology has expanded considerably. Insights into the roles of miRNAs in development and disease, particularly in cancer, have made miRNAs attractive tools and targets for novel therapeutic approaches. Functional studies have confirmed that miRNA dysregulation is causal in many cases of cancer, with miRNAs acting as tumour suppressors or oncogenes (oncomiRs), and miRNA mimics and molecules targeted at miRNAs (antimiRs) have shown promise in preclinical development. Several miRNA-targeted therapeutics have reached clinical development, including a mimic of the tumour suppressor miRNA miR-34, which reached phase I clinical trials for treating cancer, and antimiRs targeted at miR-122, which reached phase II trials for treating hepatitis. In this article, we describe recent advances in our understanding of miRNAs in cancer and in other diseases and provide an overview of current miRNA therapeutics in the clinic. We also discuss the challenge of identifying the most efficacious therapeutic candidates and provide a perspective on achieving safe and targeted delivery of miRNA therapeutics.

MicroRNA-128 Coordinately Targets Polycomb Repressor Complexes in Glioma Stem Cells

Neuro-Oncology 15, 1212–1224 (2013)

PERUZZI, P, BRONISZ A, NOWICKI MO, ET AL.

We show that microRNA-128 (miR-128) directly targets mRNA of SUZ12, a key component of PRC2, in addition to BMI1, a component of PRC1 that we previously showed as a target as well. This blocks the partially redundant functions of PRC1/PRC2, thereby significantly reducing PRC activity and its associated histone modifications. MiR-128 and SUZ12/BMI1 show opposite expression in human glioblastomas versus normal brain and in glioma stemlike versus neural stem cells. Furthermore, miR-128 renders glioma stemlike cells less radioresistant by preventing the radiation-induced expression of both PRC components. Finally, miR-128 expression is significantly reduced in neural stem cells from the brain of young, presymptomatic mice in our mouse model of glioblastoma. This suggests that loss of miR-128 expression in brain is an early event in gliomagenesis. Moreover, knockdown of miR-128 expression in nonmalignant mouse and human neural stem cells led to elevated expression of PRC components and increased clonogenicity.

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