A systematic review of non-coding RNA therapeutics in early clinical trials: a new perspective against cancer.

Targeting non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), has recently emerged as a promising strategy for treating malignancies and other diseases. In recent years, the development of ncRNA-based therapeutics for targeting protein-coding and non-coding genes has also gained momentum. This review systematically examines ongoing and completed clinical trials to provide a comprehensive overview of the emerging landscape of ncRNA-based therapeutics. Significant efforts have been made to advance ncRNA therapeutics to early clinical studies. The most advanced trials have been conducted with small interfering RNAs (siRNAs), miRNA replacement using nanovector-entrapped miRNA mimics, or miRNA silencing by antisense oligonucleotides. While siRNA-based therapeutics have already received FDA approval, miRNA mimics, inhibitors, and lncRNA-based therapeutics are still under evaluation in preclinical and early clinical studies. We critically discuss the rationale and methodologies of ncRNA targeting strategies to illustrate this rapidly evolving field.

Delivery vehicle and route of administration influences self-amplifying RNA biodistribution, expression kinetics, and reactogenicity.

Self-amplifying RNA (saRNA) is a next-generation RNA platform derived from an alphavirus that enables replication in host cytosol, offering a promising shift from traditional messenger RNA (mRNA) therapies by enabling sustained protein production from minimal dosages. The approval of saRNA-based vaccines, such as the ARCT-154 for COVID-19 in Japan, underscores its potential for diverse therapeutic applications, including vaccine development, cancer immunotherapy, and gene therapy. This study investigates the role of delivery vehicle and administration route on saRNA expression kinetics and reactogenicity. Employing ionizable lipid-based nanoparticles (LNPs) and polymeric nanoparticles, we administered saRNA encoding firefly luciferase to BALB/c mice through six routes (intramuscular (IM), intradermal (ID), intraperitoneal (IP), intranasal (IN), intravenous (IV), and subcutaneous (SC)), and observed persistent saRNA expression over a month. Our findings reveal that while LNPs enable broad route applicability and stability, pABOL (poly (cystamine bisacrylamide-co-4-amino-1-butanol)) formulations significantly amplify protein expression via intramuscular delivery. Notably, the disparity between RNA biodistribution and protein expression highlight the nuanced interplay between administration routes, delivery vehicles, and therapeutic outcomes. Additionally, our research unveiled distinct biodistribution profiles and inflammatory responses contingent upon the chosen delivery formulation and route. This research illuminates the intricate dynamics governing saRNA delivery, biodistribution and reactogenicity, offering essential insights for optimizing therapeutic strategies and advancing the clinical and commercial viability of saRNA technologies.

Extracellular vesicle transfer of lncRNA H19 splice variants to cardiac cells.

The delivery of therapeutic long non-coding RNAs (lncRNA) to the heart by extracellular vesicles (EVs) is promising for heart repair. H19, a lncRNA acting as a major regulator of gene expression within the cardiovascular system, is alternatively spliced, but the loading of its different splice variants into EVs and their subsequent uptake by recipient cardiac cells remain elusive. Here, we dissected the cellular expression of H19 splice variants and their loading into EVs secreted by Wharton-Jelly mesenchymal stromal/stem cells (WJ-MSCs). We demonstrated that overexpression of the mouse H19 gene in WJ-MSCs induces the expression of H19 splice variants at different levels. Interestingly, EVs isolated from the H19-transfected WJ-MSCs (EV-H19) showed similar expression levels for all tested splice variant sets. In vitro, we further demonstrated that EV-H19 was taken up by cardiomyocytes, fibroblasts, and endothelial cells (ECs). Finally, analysis of EV tropism in living rat myocardial slices indicated that EVs were internalized mostly by cardiomyocytes and ECs. Collectively, our results indicated that EVs can be loaded with different lncRNA splice variants and successfully internalized by cardiac cells.

Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by the absence of dystrophin protein. One current DMD therapeutic strategy, exon skipping, produces a truncated dystrophin isoform using phosphorodiamidate morpholino oligomers (PMOs). However, the potential of exon skipping therapeutics has not been fully realized as increases in dystrophin protein have been minimal in clinical trials. Here, we investigate how miR-146a-5p, which is highly elevated in dystrophic muscle, impacts dystrophin protein levels. We find inflammation strongly induces miR-146a in dystrophic, but not wild-type myotubes. Bioinformatics analysis reveals that the dystrophin 3' UTR harbors a miR-146a binding site, and subsequent luciferase assays demonstrate miR-146a binding inhibits dystrophin translation. In dystrophin-null mdx52 mice, co-injection of miR-146a reduces dystrophin restoration by an exon 51 skipping PMO. To directly investigate how miR-146a impacts therapeutic dystrophin rescue, we generated mdx52 with body-wide miR-146a deletion (146aX). Administration of an exon skipping PMO via intramuscular or intravenous injection markedly increases dystrophin protein levels in 146aX vs. mdx52 muscles while skipped dystrophin transcript levels are unchanged supporting a post-transcriptional mechanism of action. Together, these data show that miR-146a expression opposes therapeutic dystrophin restoration, suggesting miR-146a inhibition warrants further research as a potential DMD exon skipping co-therapy.

Post-transcriptional gene regulation: From mechanisms to RNA chemistry and therapeutics.

A better understanding of the RNA biology and chemistry is necessary to then develop new RNA therapeutic strategies. This review is the synthesis of a series of conferences that took place during the 6th international course on post-transcriptional gene regulation at Institut Curie. This year, the course made a special focus on RNA chemistry.

LNA-i-miR-221 activity in colorectal cancer: A reverse translational investigation.

Colorectal cancer (CRC) is one of the most common malignancies and a relevant cause of cancer-related deaths worldwide. Dysregulation of microRNA (miRNA) expression has been associated with the development and progression of various cancers, including CRC. Among them, miR-221 emerged as an oncogenic driver, whose high expression is associated with poor patient prognosis. The present study was conceived to investigate the anti-CRC activity of miR-221 silencing based on early clinical data achieved from a first-in-human study by our group. Going back from bedside to bench, we demonstrated that LNA-i-miR-221 reduces cell viability, induces apoptosis in vitro, and impairs tumor growth in preclinical in vivo models of CRC. Importantly, we disclosed that miR-221 directly targets TP53BP2, which, together with TP53INP1, is known as a positive regulator of the TP53 apoptotic pathway. We found that (1) both these genes are overexpressed following miR-221 inhibition, (2) the strong anti-tumor activity of LNA-i-miR-221 was selectively observed on TP53 wild-type cells, and (3) this activity was reduced in the presence of the TP53-inhibitor Pifitrin-α. Our data pave the way to further investigations on TP53 functionality as a marker predictive of response to miR-221 silencing, which might be relevant for clinical applications.

Ionizable nanoemulsions for RNA delivery into the central nervous system - importance of diffusivity.

Lipid nanoparticles (LNPs) currently dominate the RNA delivery landscape; however their limited diffusivity hampers targeted tissue dissemination, and, hence, their capacity for intracellular drug delivery. This is especially relevant for tissues such as the central nervous system (CNS), where overcoming proactive brain barriers is crucial for the efficacy of genetic therapeutics. This research aimed to create ionizable nanoemulsions (iNEs), a new generation of RNA delivery systems with enhanced diffusivity. The developed iNEs (consisting of the combination of C12-200, DOPE, Vitamin E, and DMG-PEG) with a size below 100 nm, neutral surface charge, and high RNA loading capacity, showed excellent cell viability and transfection efficiency in various cellular models, including neurons, astrocytes, and microglia. Subsequently, iNEs containing mRNA GFP were tested for CNS transfection, highlighting their exceptional diffusivity and selective transfection of neurons following intra-parenchymal administration.

Out of the dark: the emerging roles of lncRNAs in pain.

The dark genome, the nonprotein-coding part of the genome, is replete with long noncoding RNAs (lncRNAs). These functionally versatile transcripts, with specific temporal and spatial expression patterns, are critical gene regulators that play essential roles in health and disease. In recent years, FAAH-OUT was identified as the first lncRNA associated with an inherited human pain insensitivity disorder. Several other lncRNAs have also been studied for their contribution to chronic pain and genome-wide association studies are frequently identifying single nucleotide polymorphisms that map to lncRNAs. For a long time overlooked, lncRNAs are coming out of the dark and into the light as major players in human pain pathways and as potential targets for new RNA-based analgesic medicines.

RNA nanotechnology on the horizon: Self-assembly, chemical modifications, and functional applications.

RNA nanotechnology harnesses the unique chemical and structural properties of RNA to build nanoassemblies and supramolecular structures with dynamic and functional capabilities. This review focuses on design and assembly approaches to building RNA structures, the RNA chemical modifications used to enhance stability and functionality, and modern-day applications in therapeutics, biosensing, and bioimaging.

Targeting PD-L1 in cholangiocarcinoma using nanovesicle-based immunotherapy.

This study demonstrates the potential of using biological nanoparticles to deliver RNA therapeutics targeting programmed death-ligand 1 (PD-L1) as a treatment strategy for cholangiocarcinoma (CCA). RNA therapeutics offer prospects for intracellular immune modulation, but effective clinical translation requires appropriate delivery strategies. Milk-derived nanovesicles were decorated with epithelial cellular adhesion molecule (EpCAM) aptamers and used to deliver PD-L1 small interfering RNA (siRNA) or Cas9 ribonucleoproteins directly to CCA cells. In vitro, nanovesicle treatments reduced PD-L1 expression in CCA cells while increasing degranulation, cytokine release, and tumor cell cytotoxicity when tumor cells were co-cultured with T cells or natural killer cells. Similarly, immunomodulation was observed in multicellular spheroids that mimicked the tumor microenvironment. Combining targeted therapeutic vesicles loaded with siRNA to PD-L1 with gemcitabine effectively reduced tumor burden in an immunocompetent mouse CCA model compared with controls. This proof-of-concept study demonstrates the potential of engineered targeted nanovesicle platforms for delivering therapeutic RNA cargoes to tumors, as well as their use in generating effective targeted immunomodulatory therapies for difficult-to-treat cancers such as CCA.

Suppressor tRNAs at the interface of genetic code expansion and medicine.

Suppressor transfer RNAs (sup-tRNAs) are receiving renewed attention for their promising therapeutic properties in treating genetic diseases caused by nonsense mutations. Traditionally, sup-tRNAs have been created by replacing the anticodon sequence of native tRNAs with a suppressor sequence. However, due to their complex interactome, considering other structural and functional tRNA features for design and engineering can yield more effective sup-tRNA therapies. For over 2 decades, the field of genetic code expansion (GCE) has created a wealth of knowledge, resources, and tools to engineer sup-tRNAs. In this Mini Review, we aim to shed light on how existing knowledge and strategies to develop sup-tRNAs for GCE can be adopted to accelerate the discovery of efficient and specific sup-tRNAs for medical treatment options. We highlight methods and milestones and discuss how these approaches may enlighten the research and development of tRNA medicines.

Discovery platforms for RNA therapeutics.

RNA therapeutics are emerging as a unique opportunity to drug currently "undruggable" molecules and diseases. While their advantages over conventional, small molecule drugs, their therapeutic implications and the tools for their effective in vivo delivery have been extensively reviewed, little attention has been so far paid to the technological platforms exploited for the discovery of RNA therapeutics. Here, we provide an overview of the existing platforms and ex vivo assays for RNA discovery, their advantages and disadvantages, as well as their main fields of application, with specific focus on RNA therapies that have reached either phase 3 or market approval.

Advancements in clinical RNA therapeutics: Present developments and prospective outlooks.

RNA molecules have emerged as promising clinical therapeutics due to their ability to target "undruggable" proteins or molecules with high precision and minimal side effects. Nevertheless, the primary challenge in RNA therapeutics lies in rapid degradation and clearance from systemic circulation, the inability to traverse cell membranes, and the efficient intracellular delivery of bioactive RNA molecules. In this review, we explore the implications of RNAs in diseases and provide a chronological overview of the development of RNA therapeutics. Additionally, we summarize the technological advances in RNA-screening design, encompassing various RNA databases and design platforms. The paper then presents an update on FDA-approved RNA therapeutics and those currently undergoing clinical trials for various diseases, with a specific emphasis on RNA medicine and RNA vaccines.

RNA therapeutics in targeting G protein-coupled receptors: Recent advances and challenges.

G protein-coupled receptors (GPCRs) are the major targets of existing drugs for a plethora of human diseases and dominate the pharmaceutical market. However, over 50% of the GPCRs remain undruggable. To pursue a breakthrough and overcome this situation, there is significant clinical research for developing RNA-based drugs specifically targeting GPCRs, but none has been approved so far. RNA therapeutics represent a unique and promising approach to selectively targeting previously undruggable targets, including undruggable GPCRs. However, the development of RNA therapeutics faces significant challenges in areas of RNA stability and efficient in vivo delivery. This review presents an overview of the advances in RNA therapeutics and the diverse types of nanoparticle RNA delivery systems. It also describes the potential applications of GPCR-targeted RNA drugs for various human diseases.

Assessment of pDNA isoforms using microfluidic electrophoresis.

The recent rise in nucleic acid-based vaccines and therapies has resulted in an increased demand for plasmid DNA (pDNA). As a result, there is added pressure to streamline the manufacturing of these vectors, particularly their design and construction, which is currently considered a bottleneck. A significant challenge in optimizing pDNA production is the lack of high-throughput and rapid analytical methods to support the numerous samples produced during the iterative plasmid construction step and for batch-to-batch purity monitoring. pDNA is generally present as one of three isoforms: supercoiled, linear, or open circular. Depending on the ultimate use, the desired isoform may be supercoiled in the initial stages for cell transfection or linear in the case of mRNA synthesis. Here, we present a high-throughput microfluidic electrophoresis method capable of detecting the three pDNA isoforms and determining the size and concentration of the predominant supercoiled and linear isoforms from 2 to 7 kb. The limit of detection of the method is 0.1 ng/µL for the supercoiled and linear isoforms and 0.5 ng/µL for the open circular isoform, with a maximum loading capacity of 10-15 ng/µL. The turnaround time is 1 min/sample, and the volume requirement is 10 µL, making the method suitable for process optimization and batch-to-batch analysis. The results presented in this study will enhance the understanding of electrophoretic transport in microscale systems dependent on molecular conformations and potentially aid technological advances in diverse areas relevant to microfluidic devices.

Assembling the RNA therapeutics toolbox.

From the approval of COVID-19 mRNA vaccines to the 2023 Nobel Prize awarded for nucleoside base modifications, RNA therapeutics have entered the spotlight and are transforming drug development. While the term "RNA therapeutics" has been used in various contexts, this review focuses on treatments that utilize RNA as a component or target RNA for therapeutic effects. We summarize the latest advances in RNA-targeting tools and RNA-based technologies, including but not limited to mRNA, antisense oligos, siRNAs, small molecules and RNA editors. We focus on the mechanisms of current FDA-approved therapeutics but also provide a discussion on the upcoming workforces. The clinical utility of RNA-based therapeutics is enabled not only by the advances in RNA technologies but in conjunction with the significant improvements in chemical modifications and delivery platforms, which are also briefly discussed in the review. We summarize the latest RNA therapeutics based on their mechanisms and therapeutic effects, which include expressing proteins for vaccination and protein replacement therapies, degrading deleterious RNA, modulating transcription and translation efficiency, targeting noncoding RNAs, binding and modulating protein activity and editing RNA sequences and modifications. This review emphasizes the concept of an RNA therapeutic toolbox, pinpointing the readers to all the tools available for their desired research and clinical goals. As the field advances, the catalog of RNA therapeutic tools continues to grow, further allowing researchers to combine appropriate RNA technologies with suitable chemical modifications and delivery platforms to develop therapeutics tailored to their specific clinical challenges.

The advent of RNA-based therapeutics for metabolic syndrome and associated conditions: a comprehensive review of the literature.

Metabolic syndrome (MetS) is a prevalent and intricate health condition affecting a significant global population, characterized by a cluster of metabolic and hormonal disorders disrupting lipid and glucose metabolism pathways. Clinical manifestations encompass obesity, dyslipidemia, insulin resistance, and hypertension, contributing to heightened risks of diabetes and cardiovascular diseases. Existing medications often fall short in addressing the syndrome's multifaceted nature, leading to suboptimal treatment outcomes and potential long-term health risks. This scenario underscores the pressing need for innovative therapeutic approaches in MetS management. RNA-based treatments, employing small interfering RNAs (siRNAs), microRNAs (miRNAs), and antisense oligonucleotides (ASOs), emerge as promising strategies to target underlying biological abnormalities. However, a summary of research available on the role of RNA-based therapeutics in MetS and related co-morbidities is limited. Murine models and human studies have been separately interrogated to determine whether there have been recent advancements in RNA-based therapeutics to offer a comprehensive understanding of treatment available for MetS. In a narrative fashion, we searched for relevant articles pertaining to MetS co-morbidities such as cardiovascular disease, fatty liver disease, dementia, colorectal cancer, and endocrine abnormalities. We emphasize the urgency of exploring novel therapeutic avenues to address the intricate pathophysiology of MetS and underscore the potential of RNA-based treatments, coupled with advanced delivery systems, as a transformative approach for achieving more comprehensive and efficacious outcomes in MetS patients.

RNA base editors: The emerging approach of RNA therapeutics.

RNA-based therapeutics offer a flexible and reversible approach for treating genetic disorders, such as antisense oligonucleotides, RNA interference, aptamers, mRNA vaccines, and RNA editing. In recent years, significant advancements have been made in RNA base editing to correct disease-relevant point mutations. These achievements have significantly influenced the fields of biotechnology, biomedical research and therapeutics development. In this article, we provide a comprehensive overview of the design and performance of contemporary RNA base editors, including A-to-I, C-to-U, A-to-m6A, and U-to-Ψ. We compare recent innovative developments and highlight their applications in disease-relevant contexts. Lastly, we discuss the limitations and future prospects of utilizing RNA base editing for therapeutic purposes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Development.

RNA therapeutics for infectious diseases.

Ribonucleic acids (RNAs), including the messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), play important roles in living organisms and viruses. In recent years, the RNA-based technologies including the RNAs inhibiting other RNA activities, the RNAs targeting proteins, the RNAs reprograming genetic information, and the RNAs encoding therapeutical proteins, are useful methods to apply in prophylactic and therapeutic vaccines. In this review, we summarize and highlight the current application of the RNA therapeutics, especially on mRNA vaccines which have potential for prevention and treatment against human and animal infectious diseases.