Frameworks for transformational breakthroughs in RNA-based medicines.

RNA has sparked a revolution in modern medicine, with the potential to transform the way we treat diseases. Recent regulatory approvals, hundreds of new clinical trials, the emergence of CRISPR gene editing, and the effectiveness of mRNA vaccines in dramatic response to the COVID-19 pandemic have converged to create tremendous momentum and expectation. However, challenges with this relatively new class of drugs persist and require specialized knowledge and expertise to overcome. This Review explores shared strategies for developing RNA drug platforms, including layering technologies, addressing common biases and identifying gaps in understanding. It discusses the potential of RNA-based therapeutics to transform medicine, as well as the challenges associated with improving applicability, efficacy and safety profiles. Insights gained from RNA modalities such as antisense oligonucleotides (ASOs) and small interfering RNAs are used to identify important next steps for mRNA and gene editing technologies.

Breaking the mold with RNA-a "RNAissance" of life science.

In the past decade, RNA therapeutics have gone from being a promising concept to one of the most exciting frontiers in healthcare and pharmaceuticals. The field is now entering what many call a renaissance or "RNAissance" which is being fueled by advances in genetic engineering and delivery systems to take on more ambitious development efforts. However, this renaissance is occurring at an unprecedented pace, which will require a different way of thinking if the field is to live up to its full potential. Recognizing this need, this article will provide a forward-looking perspective on the field of RNA medical products and the potential long-term innovations and policy shifts enabled by this revolutionary and game-changing technological platform.

Discovery and preclinical evaluation of anti-miR-17 oligonucleotide RGLS4326 for the treatment of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in either PKD1 or PKD2 genes, is one of the most common human monogenetic disorders and the leading genetic cause of end-stage renal disease. Unfortunately, treatment options for ADPKD are limited. Here we report the discovery and characterization of RGLS4326, a first-in-class, short oligonucleotide inhibitor of microRNA-17 (miR-17), as a potential treatment for ADPKD. RGLS4326 is discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney and collecting duct-derived cysts, displaces miR-17 from translationally active polysomes, and de-represses multiple miR-17 mRNA targets including Pkd1 and Pkd2. Importantly, RGLS4326 demonstrates a favorable preclinical safety profile and attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models after subcutaneous administration. The preclinical characteristics of RGLS4326 support its clinical development as a disease-modifying treatment for ADPKD.

microRNA-17 family promotes polycystic kidney disease progression through modulation of mitochondrial metabolism.

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent genetic cause of renal failure. Here we identify miR-17 as a target for the treatment of ADPKD. We report that miR-17 is induced in kidney cysts of mouse and human ADPKD. Genetic deletion of the miR-17∼92 cluster inhibits cyst proliferation and PKD progression in four orthologous, including two long-lived, mouse models of ADPKD. Anti-miR-17 treatment attenuates cyst growth in short-term and long-term PKD mouse models. miR-17 inhibition also suppresses proliferation and cyst growth of primary ADPKD cysts cultures derived from multiple human donors. Mechanistically, c-Myc upregulates miR-17∼92 in cystic kidneys, which in turn aggravates cyst growth by inhibiting oxidative phosphorylation and stimulating proliferation through direct repression of Pparα. Thus, miR-17 family is a promising drug target for ADPKD, and miR-17-mediated inhibition of mitochondrial metabolism represents a potential new mechanism for ADPKD progression.

Assessing Anti-miR Pharmacology with miRNA Polysome Shift Assay.

Target engagement measurements are critical for evaluating developmental drug candidates and their pharmacological activity. microRNA (miRNA) Polysome Shift Assay enables measurement of anti-miR drug target engagement (i.e. extent of miRNA inhibition) without the need to pre-identify or pre-validate downstream miRNA-regulated genes. This makes it useful for assessing anti-miR activity in target tissues or cells where biology of the inhibited miRNAs may not be well understood. In addition, miRNA Polysome Shift Assay can be multiplexed to assess inhibition of multiple miRNAs by a single anti-miR, thus guiding drug optimization for enhancing or avoiding these activities as desired. This chapter outlines the miRNA Polysome Shift Assay technique, describes sample preparation and quality control, and how to calculate and interpret results.

Competitive Argonaute-Based RNA Immunoprecipitation for Investigation of Transcriptomic Response to Anti-miR.

Identification and validation of microRNA (miRNA) target genes is essential for gaining a better understanding of the many different functions miRNAs have in healthy and diseased cells. From a practical standpoint, validated target genes are also useful for monitoring pharmacological activity of developmental therapeutics that modulate miRNAs, such as anti-miRNA oligonucleotides (anti-miR). Here, we describe a method that uses changes in Argonaute 2-RNA immunoprecipitation in response to competition by anti-miR, titrated ex vivo, as physical evidence for target validation.

Polysome shift assay for direct measurement of miRNA inhibition by anti-miRNA drugs.

Anti-miRNA (anti-miR) oligonucleotide drugs are being developed to inhibit overactive miRNAs linked to disease. To help facilitate the transition from concept to clinic, new research tools are required. Here we report a novel method--miRNA Polysome Shift Assay (miPSA)--for direct measurement of miRNA engagement by anti-miR, which is more robust than conventional pharmacodynamics using downstream target gene derepression. The method takes advantage of size differences between active and inhibited miRNA complexes. Active miRNAs bind target mRNAs in high molecular weight polysome complexes, while inhibited miRNAs are sterically blocked by anti-miRs from forming this interaction. These two states can be assessed by fractionating tissue or cell lysates using differential ultracentrifugation through sucrose gradients. Accordingly, anti-miR treatment causes a specific shift of cognate miRNA from heavy to light density fractions. The magnitude of this shift is dose-responsive and maintains a linear relationship with downstream target gene derepression while providing a substantially higher dynamic window for aiding drug discovery. In contrast, we found that the commonly used 'RT-interference' approach, which assumes that inhibited miRNA is undetectable by RT-qPCR, can yield unreliable results that poorly reflect the binding stoichiometry of anti-miR to miRNA. We also demonstrate that the miPSA has additional utility in assessing anti-miR cross-reactivity with miRNAs sharing similar seed sequences.

Non-inhibited miRNAs shape the cellular response to anti-miR.

Identification of primary microRNA (miRNA) gene targets is critical for developing miRNA-based therapeutics and understanding their mechanisms of action. However, disentangling primary target derepression induced by miRNA inhibition from secondary effects on the transcriptome remains a technical challenge. Here, we utilized RNA immunoprecipitation (RIP) combined with competitive binding assays to identify novel primary targets of miR-122. These transcripts physically dissociate from AGO2-miRNA complexes when anti-miR is spiked into liver lysates. mRNA target displacement strongly correlated with expression changes in these genes following in vivo anti-miR dosing, suggesting that derepression of these targets directly reflects changes in AGO2 target occupancy. Importantly, using a metric based on weighted miRNA expression, we found that the most responsive mRNA target candidates in both RIP competition assays and expression profiling experiments were those with fewer alternative seed sites for highly expressed non-inhibited miRNAs. These data strongly suggest that miRNA co-regulation modulates the transcriptomic response to anti-miR. We demonstrate the practical utility of this 'miR-target impact' model, and encourage its incorporation, together with the RIP competition assay, into existing target prediction and validation pipelines.

High-resolution three-dimensional mapping of mRNA export through the nuclear pore.

The flow of genetic information is regulated by selective nucleocytoplasmic transport of messenger RNA:protein complexes (mRNPs) through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the three-dimensional (3D) pathway taken by mRNPs as they transit through the NPC, and the kinetics and selectivity of transport, remain obscure. Here we employ single-molecule fluorescence microscopy with an unprecedented spatiotemporal accuracy of 8 nm and 2 ms to provide new insights into the mechanism of nuclear mRNP export in live human cells. We find that mRNPs exiting the nucleus are decelerated and selected at the centre of the NPC, and adopt a fast-slow-fast diffusion pattern during their brief, ~12 ms, interaction with the NPC. A 3D reconstruction of the export route indicates that mRNPs primarily interact with the periphery on the nucleoplasmic side and in the centre of the NPC, without entering the central axial conduit utilized for passive diffusion of small molecules, and eventually dissociate on the cytoplasmic side.

Disease-linked microRNA-21 exhibits drastically reduced mRNA binding and silencing activity in healthy mouse liver.

MicroRNAs (miRNAs) bind to mRNAs and fine-tune protein output by affecting mRNA stability and/or translation. miR-21 is a ubiquitous, highly abundant, and stress-responsive miRNA linked to several diseases, including cancer, fibrosis, and inflammation. Although the RNA silencing activity of miR-21 in diseased cells has been well documented, the roles of miR-21 under healthy cellular conditions are not well understood. Here, we show that pharmacological inhibition or genetic deletion of miR-21 in healthy mouse liver has little impact on regulation of canonical seed-matched mRNAs and only a limited number of genes enriched in stress response pathways. These surprisingly weak and selective regulatory effects on known and predicted target mRNAs contrast with those of other abundant liver miRNAs such as miR-122 and let-7. Moreover, miR-21 shows greatly reduced binding to polysome-associated target mRNAs compared to miR-122 and let-7. Bioinformatic analysis suggests that reduced thermodynamic stability of seed pairing and target binding may contribute to this deficiency of miR-21. Significantly, these trends are reversed in human cervical carcinoma (HeLa) cells, where miRNAs including miR-21 show enhanced target binding within polysomes and where miR-21 triggers strong degradative activity toward target mRNAs. Taken together, our results suggest that, under normal cellular conditions in liver, miR-21 activity is maintained below a threshold required for binding and silencing most of its targets. Consequently, enhanced association with polysome-associated mRNA is likely to explain in part the gain of miR-21 function often found in diseased or stressed cells.

Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly.

MicroRNAs (miRNAs) associate with components of the RNA-induced silencing complex (RISC) to assemble on mRNA targets and regulate protein expression in higher eukaryotes. Here we describe a method for the intracellular single-molecule, high-resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore-labelled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules. Mobility and mRNA-dependent assembly changes suggest the existence of two kinetically distinct pathways for miRNA assembly, revealing the dynamic nature of this important gene regulatory pathway. iSHiRLOC achieves an unprecedented resolution in the visualization of functional miRNAs, paving the way to understanding RNA silencing through single-molecule systems biology.

Chemical modulators of heat shock protein 70 (Hsp70) by sequential, microwave-accelerated reactions on solid phase.

Molecular chaperones, such as Hsp70 and Hsp90, are responsible for a variety of protective, anti-apoptotic functions. While inhibitors of Hsp90, such as geldanamycin and its derivative 17-AAG, are well known and important anti-cancer leads, Hsp70 has received less attention. Interesting lead candidates for Hsp70 share a dihydropyrimidine core; however, the preferred display of pendant functionality is still not clear. Here, we take advantage of the versatility of peptides to explore the requirements for activity. An exploratory compound collection was assembled by performing a Biginelli cyclocondensation at the terminus of a resin-bound beta-peptide. Liberation from solid support yielded peptide-modified dihydropyrimidines and, within this series, we uncovered compounds that alter the ATPase activity of Hsp70 and its bacterial ortholog, DnaK. Moreover, we identified important contributions made by aromatic, hydrophobic groups. These chemical probes could be used to study the roles of this molecular chaperone in disease.