RNA therapeutics for epilepsy: An emerging modality for drug discovery.

Drug discovery in epilepsy began with the finding of potassium bromide by Sir Charles Locock in 1857. The following century witnessed the introduction of phenotypic screening tests for discovering antiseizure medications (ASMs). Despite the high success rate of developing ASMs, they have so far failed in eliminating drug resistance and in delivering disease-modifying treatments. This emphasizes the need for new drug discovery strategies in epilepsy. RNA-based drugs have recently shown promise as a new modality with the potential of providing disease modification and counteracting drug resistance in epilepsy. RNA therapeutics can be directed either toward noncoding RNAs, such as microRNAs, long noncoding RNAs (ncRNAs), and circular RNAs, or toward messenger RNAs. The former show promise in sporadic, nongenetic epilepsies, as interference with ncRNAs allows for modulation of entire disease pathways, whereas the latter seem more promising in monogenic childhood epilepsies. Here, we describe therapeutic strategies for modulating disease-associated RNA molecules and highlight the potential of RNA therapeutics for the treatment of different patient populations such as sporadic, drug-resistant epilepsy, and childhood monogenic epilepsies.

MicroRNA-22 Is a Key Regulator of Lipid and Metabolic Homeostasis.

Obesity is a growing public health problem associated with increased risk of type 2 diabetes, cardiovascular disease, nonalcoholic fatty liver disease (NAFLD) and cancer. Here, we identify microRNA-22 (miR-22) as an essential rheostat involved in the control of lipid and energy homeostasis as well as the onset and maintenance of obesity. We demonstrate through knockout and transgenic mouse models that miR-22 loss-of-function protects against obesity and hepatic steatosis, while its overexpression promotes both phenotypes even when mice are fed a regular chow diet. Mechanistically, we show that miR-22 controls multiple pathways related to lipid biogenesis and differentiation. Importantly, genetic ablation of miR-22 favors metabolic rewiring towards higher energy expenditure and browning of white adipose tissue, suggesting that modulation of miR-22 could represent a viable therapeutic strategy for treatment of obesity and other metabolic disorders.

Targeting of microRNA-22 Suppresses Tumor Spread in a Mouse Model of Triple-Negative Breast Cancer.

microRNA-22 (miR-22) is an oncogenic miRNA whose up-regulation promotes epithelial-mesenchymal transition (EMT), tumor invasion, and metastasis in hormone-responsive breast cancer. Here we show that miR-22 plays a key role in triple negative breast cancer (TNBC) by promoting EMT and aggressiveness in 2D and 3D cell models and a mouse xenograft model of human TNBC, respectively. Furthermore, we report that miR-22 inhibition using an LNA-modified antimiR-22 compound is effective in reducing EMT both in vitro and in vivo. Importantly, pharmacologic inhibition of miR-22 suppressed metastatic spread and markedly prolonged survival in mouse xenograft models of metastatic TNBC highlighting the potential of miR-22 silencing as a new therapeutic strategy for the treatment of TNBC.

Assessment of immunostimulatory responses to the antimiR-22 oligonucleotide compound RES-010 in human peripheral blood mononuclear cells.

microRNA-22 (miR-22) is a key regulator of lipid and energy homeostasis and represents a promising therapeutic target for NAFLD and obesity. We have previously identified a locked nucleic acid (LNA)-modified antisense oligonucleotide compound complementary to miR-22, designated as RES-010 that mediated robust inhibition of miR-22 function in cultured cells and in vivo. In this study we investigated the immune potential of RES-010 in human peripheral blood mononuclear cells (PBMCs). We treated fresh human peripheral blood mononuclear cells isolated from six healthy volunteers with different concentrations of the RES-010 compound and assessed its proinflammatory effects by quantifying IL-1ß, IL-6, IFN-γ, TNF-α, IFN-α2a, IFN-ß, IL-10, and IL-17A in the supernatants collected 24 h of treatment with RES-010. The T-cell activation markers, CD69, HLA-DR, and CD25 were evaluated by flow cytometry after 24 and 144 h of treatment, respectively, whereas cell viability was assessed after 24 h of treatment with RES-010. Our results show that RES-010 compound does not induce any significant immunostimulatory responses in human PBMCs in vitro compared to controls, implying that the proinflammatory potential of RES-010 is low.

Knockdown of Circular RNAs Using LNA-Modified Antisense Oligonucleotides.

Circular RNAs (circRNAs) constitute an abundant class of covalently closed noncoding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys, as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. In this study, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We found that, while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes. In summary, our results demonstrate that LNA-modified ASOs complementary to backsplice-junction sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.

Antisense-oligonucleotide-mediated perturbation of long non-coding RNA reveals functional features in stem cells and across cell types.

Within the scope of the FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem cells (iPSCs) and systematically characterize their roles in self-renewal and pluripotency. We find 36 lncRNAs (18%) exhibiting cell growth inhibition. From the knockdown of 123 lncRNAs with transcriptome profiling, 36 lncRNAs (29.3%) show molecular phenotypes. Integrating the molecular phenotypes with chromatin-interaction assays further reveals cis- and trans-interacting partners as potential primary targets. Additionally, cell-type enrichment analysis identifies lncRNAs associated with pluripotency, while the knockdown of LINC02595, CATG00000090305.1, and RP11-148B6.2 modulates colony formation of iPSCs. We compare our results with previously published fibroblasts phenotyping data and find that 2.9% of the lncRNAs exhibit a consistent cell growth phenotype, whereas we observe 58.3% agreement in molecular phenotypes. This highlights that molecular phenotyping is more comprehensive in revealing affected pathways.

Knockdown of the long noncoding RNA PURPL induces apoptosis and sensitizes liver cancer cells to doxorubicin.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with increasing incidence in western countries. Most HCC patients have advanced cancer at the time of diagnosis due to the asymptomatic nature of early-stage HCC and do not qualify for potentially curative surgical treatment, thus, highlighting the need for new therapeutic strategies. Long noncoding RNAs (lncRNAs) comprise a large and heterogeneous group of non-protein coding transcripts that play important regulatory roles in numerous biological processes in cancer. In this study, we performed RNA sequencing of liver biopsies from ten HCC, ten hepatitis C virus-associated HCC, and four normal livers to identify dysregulated lncRNAs in HCC. We show that the lncRNA p53-upregulated-regulator-of-p53-levels (PURPL) is upregulated in HCC biopsies and that its expression is p53-dependent in liver cancer cell lines. In addition, antisense oligonucleotide-mediated knockdown of PURPL inhibited cell proliferation, induced apoptosis, and sensitized HepG2 human HCC cells to treatment with the chemotherapeutic agent doxorubicin. In summary, our findings suggest that PURPL could serve as a new therapeutic target for reversing doxorubicin resistance in HCC.

An intranasal ASO therapeutic targeting SARS-CoV-2.

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5' leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 "variants of concern" tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics.

Systematic Analysis of Long Non-Coding RNA Genes in Nonalcoholic Fatty Liver Disease.

The largest solid organ in humans, the liver, performs a variety of functions to sustain life. When damaged, cells in the liver can regenerate themselves to maintain normal liver physiology. However, some damage is beyond repair, which necessitates liver transplantation. Increasing rates of obesity, Western diets (i.e., rich in processed carbohydrates and saturated fats), and cardiometabolic diseases are interlinked to liver diseases, including non-alcoholic fatty liver disease (NAFLD), which is a collective term to describe the excess accumulation of fat in the liver of people who drink little to no alcohol. Alarmingly, the prevalence of NAFLD extends to 25% of the world population, which calls for the urgent need to understand the disease mechanism of NAFLD. Here, we performed secondary analyses of published RNA sequencing (RNA-seq) data of NAFLD patients compared to healthy and obese individuals to identify long non-coding RNAs (lncRNAs) that may underly the disease mechanism of NAFLD. Similar to protein-coding genes, many lncRNAs are dysregulated in NAFLD patients compared to healthy and obese individuals, suggesting that understanding the functions of dysregulated lncRNAs may shed light on the pathology of NAFLD. To demonstrate the functional importance of lncRNAs in the liver, loss-of-function experiments were performed for one NAFLD-related lncRNA, LINC01639, which showed that it is involved in the regulation of genes related to apoptosis, TNF/TGF, cytokine signaling, and growth factors as well as genes upregulated in NAFLD. Since there is no lncRNA database focused on the liver, especially NAFLD, we built a web database, LiverDB, to further facilitate functional and mechanistic studies of hepatic lncRNAs.

Clinical advances of RNA therapeutics for treatment of neurological and neuromuscular diseases.

RNA therapeutics comprise a diverse group of oligonucleotide-based drugs such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs) that can be designed to selectively interact with drug targets currently undruggable with small molecule-based drugs or monoclonal antibodies. Furthermore, RNA-based therapeutics have the potential to modulate entire disease pathways, and thereby represent a new modality with unprecedented potential for generating disease-modifying drugs for a wide variety of human diseases, including central nervous system (CNS) disorders. Here, we describe different strategies for delivering RNA drugs to the CNS and review recent advances in clinical development of ASO drugs and siRNA-based therapeutics for the treatment of neurological diseases and neuromuscular disorders.Abbreviations 2'-MOE: 2'-O-(2-methoxyethyl); 2'-O-Me: 2'-O-methyl; 2'-F: 2'-fluoro; AD: Alzheimer's disease; ALS: Amyotrophic lateral sclerosis; ALSFRS-R: Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; ARC: Antibody siRNA Conjugate; AS: Angelman Syndrome; ASGRP: Asialoglycoprotein receptor; ASO: Antisense oligonucleotide; AxD: Alexander Disease; BBB: Blood brain barrier; Bp: Basepair; CNM: Centronuclear myopathies; CNS: Central Nervous System; CPP: Cell-penetrating Peptide; CSF: Cerebrospinal fluid; DMD: Duchenne muscular dystrophy; DNA: Deoxyribonucleic acid; FAP: Familial amyloid polyneuropathy; FALS: Familial amyotrophic lateral sclerosis; FDA: The United States Food and Drug Administration; GalNAc: N-acetylgalactosamine; GoF: Gain of function; hATTR: Hereditary transthyretin amyloidosis; HD: Huntington's disease; HRQOL: health-related quality of life; ICV: Intracerebroventricular; IT: Intrathecal; LNA: Locked nucleic acid; LoF: Loss of function; mRNA: Messenger RNA; MS: Multiple Sclerosis; MSA: Multiple System Atrophy; NBE: New Biological Entity; NCE: New Chemical Entity; NHP: Nonhuman primate; nt: Nucleotide; PD: Parkinson's disease; PNP: Polyneuropathy; PNS: Peripheral nervous system; PS: Phosphorothioate; RISC: RNA-Induced Silencing Complex; RNA: Ribonucleic acid; RNAi: RNA interference; s.c.: Subcutaneous; siRNA: Small interfering RNA; SMA: Spinal muscular atrophy; SMN: Survival motor neuron; TTR: Transthyretin.

FibroDB: Expression Analysis of Protein-Coding and Long Non-Coding RNA Genes in Fibrosis.

Most long non-coding RNAs (lncRNAs) are expressed at lower levels than protein-coding genes and their expression is often restricted to specific cell types, certain time points during development, and various stress and disease conditions, respectively. To revisit this long-held concept, we focused on fibroblasts, a common cell type in various organs and tissues. Using fibroblasts and changes in their expression profiles during fibrosis as a model system, we show that the overall expression level of lncRNA genes is significantly lower than that of protein-coding genes. Furthermore, we identified lncRNA genes whose expression is upregulated during fibrosis. Using dermal fibroblasts as a model, we performed loss-of-function experiments and show that the knockdown of the lncRNAs LINC00622 and LINC01711 result in gene expression changes associated with cellular and inflammatory responses, respectively. Since there are no lncRNA databases focused on fibroblasts and fibrosis, we built a web application, FibroDB, to further promote functional and mechanistic studies of fibrotic lncRNAs.

PTENP1-AS contributes to BRAF inhibitor resistance and is associated with adverse clinical outcome in stage III melanoma.

BRAF inhibitors (BRAFi) selectively target oncogenic BRAFV600E/K and are effective in 80% of advanced cutaneous malignant melanoma cases carrying the V600 mutation. However, the development of drug resistance limits their clinical efficacy. Better characterization of the underlying molecular processes is needed to further improve treatments. We previously demonstrated that transcription of PTEN is negatively regulated by the PTEN pseudogene antisense RNA, PTENP1-AS, and here we investigated the impact of this transcript on clinical outcome and BRAFi resistance in melanoma. We observed that increased expression levels of PTENP1-AS in BRAFi resistant cells associated with enrichment of EZH2 and H3K27me3 at the PTEN promoter, consequently reducing the expression levels of PTEN. Further, we showed that targeting of the PTENP1-AS transcript sensitized resistant cells to BRAFi treatment and that high expression of PTENP1-AS in stage III melanoma correlated with poor survival. Collectively, the data presented here show that PTENP1-AS is a promising target for re-sensitizing cells to BRAFi and also a possible prognostic marker for clinical outcome in stage III melanoma.

Development of siRNA Therapeutics for the Treatment of Liver Diseases.

Small interfering RNA (siRNA)-based therapeutics holds the promise to treat a wide range of human diseases that are currently incurable using conventional therapies. Most siRNA therapeutic efforts to date have focused on the treatment of liver diseases due to major breakthroughs in the development of efficient strategies for delivering siRNA drugs to the liver. Indeed, the development of lipid nanoparticle-formulated and GalNAc-conjugated siRNA therapeutics has resulted in recent FDA approvals of the first siRNA-based drugs, patisiran for the treatment of hereditary transthyretin amyloidosis and givosiran for the treatment of acute hepatic porphyria, respectively. Here, we describe the current strategies for delivering siRNA drugs to the liver and summarize recent advances in clinical development of siRNA therapeutics for the treatment of liver diseases.

Long Non-Coding RNAs in Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Although significant progress has been made in recent years to treat DLBCL patients, 30%-40% of the patients eventually relapse or are refractory to first line treatment, calling for better therapeutic strategies for DLBCL. Long non-coding RNAs (lncRNAs) have emerged as a highly diverse group of non-protein coding transcripts with intriguing molecular functions in human disease, including cancer. Here, we review the current understanding of lncRNAs in the pathogenesis and progression of DLBCL to provide an overview of the field. As the current knowledge of lncRNAs in DLBCL is still in its infancy, we provide molecular signatures of lncRNAs in DLBCL cell lines to assist further lncRNA research in DLBCL.

A MicroRNA Linking Human Positive Selection and Metabolic Disorders.

Positive selection in Europeans at the 2q21.3 locus harboring the lactase gene has been attributed to selection for the ability of adults to digest milk to survive famine in ancient times. However, the 2q21.3 locus is also associated with obesity and type 2 diabetes in humans, raising the possibility that additional genetic elements in the locus may have contributed to evolutionary adaptation to famine by promoting energy storage, but which now confer susceptibility to metabolic diseases. We show here that the miR-128-1 microRNA, located at the center of the positively selected locus, represents a crucial metabolic regulator in mammals. Antisense targeting and genetic ablation of miR-128-1 in mouse metabolic disease models result in increased energy expenditure and amelioration of high-fat-diet-induced obesity and markedly improved glucose tolerance. A thrifty phenotype connected to miR-128-1-dependent energy storage may link ancient adaptation to famine and modern metabolic maladaptation associated with nutritional overabundance.

Long Non-Coding RNAs in Liver Cancer and Nonalcoholic Steatohepatitis.

This review aims to highlight the recent findings of long non-coding RNAs (lncRNAs) in liver disease. In particular, we focus on the functions of lncRNAs in hepatocellular carcinoma (HCC) and non-alcoholic steatohepatitis (NASH). We summarize the current research trend in lncRNAs and their potential as biomarkers and therapeutic targets for the treatment of HCC and NASH.

Author Correction: The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Clinical and functional significance of circular RNAs in cytogenetically normal AML.

Circular RNAs (circRNAs) are noncoding RNA molecules that display a perturbed arrangement of exons, called backsplicing. To examine the prognostic and biologic significance of circRNA expression in cytogenetically normal acute myeloid leukemia (CN-AML), we conducted whole-transcriptome profiling in 365 younger adults (age 18-60 years) with CN-AML. We applied a novel pipeline, called Massive Scan for circRNA, to identify and quantify circRNA expression. We validated the high sensitivity and specificity of our pipeline by performing RNase R treatment and RNA sequencing in samples of AML patients and cell lines. Unsupervised clustering analyses identified 3 distinct circRNA expression-based clusters with different frequencies of clinical and molecular features. After dividing our cohort into training and validation data sets, we identified 4 circRNAs (circCFLAR, circKLHL8, circSMC1A, and circFCHO2) that were prognostic in both data sets; high expression of each prognostic circRNA was associated with longer disease-free, overall, and event-free survival. In multivariable analyses, high circKLHL8 and high circFCHO2 expression were independently associated with better clinical outcome of CN-AML patients, after adjusting for other covariates. To examine the biologic relevance of circRNA expression, we performed knockdown screening experiments in a subset of prognostic and gene mutation-related candidate circRNAs. We identified circFBXW7, but not its linear messenger RNA, as a regulator of the proliferative capacity of AML blasts. In summary, our findings underscore the molecular associations, prognostic significance, and functional relevance of circRNA expression in CN-AML.

Publisher Correction: The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Intragenic antagonistic roles of protein and circRNA in tumorigenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.