RNA editing: Expanding the potential of RNA therapeutics.

RNA therapeutics have had a tremendous impact on medicine, recently exemplified by the rapid development and deployment of mRNA vaccines to combat the COVID-19 pandemic. In addition, RNA-targeting drugs have been developed for diseases with significant unmet medical needs through selective mRNA knockdown or modulation of pre-mRNA splicing. Recently, RNA editing, particularly antisense RNA-guided adenosine deaminase acting on RNA (ADAR)-based programmable A-to-I editing, has emerged as a powerful tool to manipulate RNA to enable correction of disease-causing mutations and modulate gene expression and protein function. Beyond correcting pathogenic mutations, the technology is particularly well suited for therapeutic applications that require a transient pharmacodynamic effect, such as the treatment of acute pain, obesity, viral infection, and inflammation, where it would be undesirable to introduce permanent alterations to the genome. Furthermore, transient modulation of protein function, such as altering the active sites of enzymes or the interface of protein-protein interactions, opens the door to therapeutic avenues ranging from regenerative medicine to oncology. These emerging RNA-editing-based toolsets are poised to broadly impact biotechnology and therapeutic applications. Here, we review the emerging field of therapeutic RNA editing, highlight recent laboratory advancements, and discuss the key challenges on the path to clinical development.

Antisense oligonucleotide: A promising therapeutic option to beat COVID-19.

The COVID-19 crisis and the development of the first approved mRNA vaccine have highlighted the power of RNA-based therapeutic strategies for the development of new medicines. Aside from RNA-vaccines, antisense oligonucleotides (ASOs) represent a new and very promising class of RNA-targeted therapy. Few drugs have already received approval from the Food and Drug Administration. Here, we underscored why and how ASOs hold the potential to change the therapeutic landscape to beat SARS-CoV-2 viral infections. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions.

Efficient Targeted Delivery of Bifunctional Circular Aptamer-ASO Chimera to Suppress the SARS-CoV-2 Proliferation and Inflammation.

Inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and excessive inflammation is the current task in the prevention and treatment of corona vireus disease 2019 (COVID-19). Here, a dual-function circular aptamer-ASO chimera (circSApt-NASO) is designed to suppress SARS-CoV-2 replication and inflammation. The chemically unmodified circSApt-NASO exhibits high serum stability by artificial cyclization. It is also demonstrated that the SApt binding to spike protein enables the chimera to be efficiently delivered into the host cells expressing ACE2 along with the infection of SARS-CoV-2. Among them, the SApt potently inhibits spike-induced inflammation. The NASO targeting to silence N genes not only display robust anti-N-induced inflammatory activity, but also achieve efficient inhibition of SARS-CoV-2 replication. Overall, benefiting from the high stability of the cyclization, antispike aptamer-dependent, and viral infection-mediate targeted delivery, the circSApt-NASO displays robust potential against authentic SARS-CoV-2 and Omicron, providing a promising specific anti-inflammatory and antiproliferative reagent for therapeutic COVID-19.

Antisense oligonucleotides targeting ORF1b block replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The ongoing COVID-19 pandemic continues to pose a need for new and efficient therapeutic strategies. We explored antisense therapy using oligonucleotides targeting the severe acute respiratory syndrome coronavirus (SARS-CoV-2) genome. We predicted in silico four antisense oligonucleotides (ASO gapmers with 100% PTO linkages and LNA modifications at their 5' and 3'ends) targeting viral regions ORF1a, ORF1b, N and the 5'UTR of the SARS-CoV-2 genome. Efficiency of ASOs was tested by transfection in human ACE2-expressing HEK-293T cells and monkey VeroE6/TMPRSS2 cells infected with SARS-CoV-2. The ORF1b-targeting ASO was the most efficient, with a 71% reduction in the number of viral genome copies. N- and 5'UTR-targeting ASOs also significantly reduced viral replication by 55 and 63%, respectively, compared to non-related control ASO (ASO-C). Viral titration revealed a significant decrease in SARS-CoV-2 multiplication both in culture media and in cells. These results show that anti-ORF1b ASO can specifically reduce SARS-CoV-2 genome replication in vitro in two different cell infection models. The present study presents proof-of concept of antisense oligonucleotide technology as a promising therapeutic strategy for COVID-19.

Transcript-Targeted Therapy Based on RNA Interference and Antisense Oligonucleotides: Current Applications and Novel Molecular Targets.

The development of novel target therapies based on the use of RNA interference (RNAi) and antisense oligonucleotides (ASOs) is growing in an exponential way, challenging the chance for the treatment of the genetic diseases and cancer by hitting selectively targeted RNA in a sequence-dependent manner. Multiple opportunities are taking shape, able to remove defective protein by silencing RNA (e.g., Inclisiran targets mRNA of protein PCSK9, permitting a longer half-life of LDL receptors in heterozygous familial hypercholesteremia), by arresting mRNA translation (i.e., Fomivirsen that binds to UL123-RNA and blocks the translation into IE2 protein in CMV-retinitis), or by reactivating modified functional protein (e.g., Eteplirsen able to restore a functional shorter dystrophin by skipping the exon 51 in Duchenne muscular dystrophy) or a not very functional protein. In this last case, the use of ASOs permits modifying the expression of specific proteins by modulating splicing of specific pre-RNAs (e.g., Nusinersen acts on the splicing of exon 7 in SMN2 mRNA normally not expressed; it is used for spinal muscular atrophy) or by downregulation of transcript levels (e.g., Inotersen acts on the transthryretin mRNA to reduce its expression; it is prescribed for the treatment of hereditary transthyretin amyloidosis) in order to restore the biochemical/physiological condition and ameliorate quality of life. In the era of precision medicine, recently, an experimental splice-modulating antisense oligonucleotide, Milasen, was designed and used to treat an 8-year-old girl affected by a rare, fatal, progressive form of neurodegenerative disease leading to death during adolescence. In this review, we summarize the main transcriptional therapeutic drugs approved to date for the treatment of genetic diseases by principal regulatory government agencies and recent clinical trials aimed at the treatment of cancer. Their mechanism of action, chemical structure, administration, and biomedical performance are predominantly discussed.

Delivery of RNA Therapeutics: The Great Endosomal Escape!

RNA therapeutics, including siRNAs, antisense oligonucleotides, and other oligonucleotides, have great potential to selectively treat a multitude of human diseases, from cancer to COVID to Parkinson's disease. RNA therapeutic activity is mechanistically driven by Watson-Crick base pairing to the target gene RNA without the requirement of prior knowledge of the protein structure, function, or cellular location. However, before widespread use of RNA therapeutics becomes a reality, we must overcome a billion years of evolutionary defenses designed to keep invading RNAs from entering cells. Unlike small-molecule therapeutics that are designed to passively diffuse across the cell membrane, macromolecular RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and are instead taken up into cells by endocytosis. However, similar to the cell membrane, endosomes comprise a lipid bilayer that entraps 99% or more of RNA therapeutics, even in semipermissive tissues such as the liver, central nervous system, and muscle. Consequently, before RNA therapeutics can achieve their ultimate clinical potential to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a clinically acceptable manner.

RNA Therapeutic Options to Manage Aberrant Signaling Pathways in Hepatocellular Carcinoma: Dream or Reality?

Despite the early promise of RNA therapeutics as a magic bullet to modulate aberrant signaling in cancer, this field remains a work-in-progress. Nevertheless, RNA therapeutics is now a reality for the treatment of viral diseases (COVID-19) and offers great promise for cancer. This review paper specifically investigates RNAi as a therapeutic option for HCC and discusses a range of RNAi technology including anti-sense oligonucleotides (ASOs), Aptamers, small interfering RNA (siRNA), ribozymes, riboswitches and CRISPR/Cas9 technology. The use of these RNAi based interventions is specifically outlined in three primary strategies, namely, repressing angiogenesis, the suppression of cell proliferation and the promotion of apoptosis. We also discuss some of the inherent chemical and delivery problems, as well as targeting issues and immunogenic reaction to RNAi interventions.

Deliver the promise: RNAs as a new class of molecular entities for therapy and vaccination.

The concepts of developing RNAs as new molecular entities for therapies have arisen again and again since the discoveries of antisense RNAs, direct RNA-protein interactions, functional noncoding RNAs, and RNA-directed gene editing. The feasibility was demonstrated with the development and utilization of synthetic RNA agents to selectively control target gene expression, modulate protein functions or alter the genome to manage diseases. Rather, RNAs are labile to degradation and cannot cross cell membrane barriers, making it hard to develop RNA medications. With the development of viable RNA technologies, such as chemistry and pharmaceutics, eight antisense oligonucleotides (ASOs) (fomivirsen, mipomersen, eteplirsen, nusinersen, inotersen, golodirsen, viltolarsen and casimersen), one aptamer (pegaptanib), and three small interfering RNAs (siRNAs) (patisiran, givosiran and lumasiran) have been approved by the United States Food and Drug Administration (FDA) for therapies, and two mRNA vaccines (BNT162b2 and mRNA-1273) under Emergency Use Authorization for the prevention of COVID-19. Therefore, RNAs have become a great addition to small molecules, proteins/antibodies, and cell-based modalities to improve the public health. In this article, we first summarize the general characteristics of therapeutic RNA agents, including chemistry, common delivery strategies, mechanisms of actions, and safety. By overviewing individual RNA medications and vaccines approved by the FDA and some agents under development, we illustrate the unique compositions and pharmacological actions of RNA products. A new era of RNA research and development will likely lead to commercialization of more RNA agents for medical use, expanding the range of therapeutic targets and increasing the diversity of molecular modalities.

Current Advances in RNA Therapeutics for Human Diseases.

Following the discovery of nucleic acids by Friedrich Miescher in 1868, DNA and RNA were recognized as the genetic code containing the necessary information for proper cell functioning. In the years following these discoveries, vast knowledge of the seemingly endless roles of RNA have become better understood. Additionally, many new types of RNAs were discovered that seemed to have no coding properties (non-coding RNAs), such as microRNAs (miRNAs). The discovery of these new RNAs created a new avenue for treating various human diseases. However, RNA is relatively unstable and is degraded fairly rapidly once administered; this has led to the development of novel delivery mechanisms, such as nanoparticles to increase stability as well as to prevent off-target effects of these molecules. Current advances in RNA-based therapies have substantial promise in treating and preventing many human diseases and disorders through fixing the pathology instead of merely treating the symptomology similarly to traditional therapeutics. Although many RNA therapeutics have made it to clinical trials, only a few have been FDA approved thus far. Additionally, the results of clinical trials for RNA therapeutics have been ambivalent to date, with some studies demonstrating potent efficacy, whereas others have limited effectiveness and/or toxicity. Momentum is building in the clinic for RNA therapeutics; future clinical care of human diseases will likely comprise promising RNA therapeutics. This review focuses on the current advances of RNA therapeutics and addresses current challenges with their development.

Small Drugs, Huge Impact: The Extraordinary Impact of Antisense Oligonucleotides in Research and Drug Development.

Antisense oligonucleotides (ASOs) are an increasingly represented class of drugs. These small sequences of nucleotides are designed to precisely target other oligonucleotides, usually RNA species, and are modified to protect them from degradation by nucleases. Their specificity is due to their sequence, so it is possible to target any RNA sequence that is already known. These molecules are very versatile and adaptable given that their sequence and chemistry can be custom manufactured. Based on the chemistry being used, their activity may significantly change and their effects on cell function and phenotypes can differ dramatically. While some will cause the target RNA to decay, others will only bind to the target and act as a steric blocker. Their incredible versatility is the key to manipulating several aspects of nucleic acid function as well as their process, and alter the transcriptome profile of a specific cell type or tissue. For example, they can be used to modify splicing or mask specific sites on a target. The entire design rather than just the sequence is essential to ensuring the specificity of the ASO to its target. Thus, it is vitally important to ensure that the complete process of drug design and testing is taken into account. ASOs' adaptability is a considerable advantage, and over the past decades has allowed multiple new drugs to be approved. This, in turn, has had a significant and positive impact on patient lives. Given current challenges presented by the COVID-19 pandemic, it is necessary to find new therapeutic strategies that would complement the vaccination efforts being used across the globe. ASOs may be a very powerful tool that can be used to target the virus RNA and provide a therapeutic paradigm. The proof of the efficacy of ASOs as an anti-viral agent is long-standing, yet no molecule currently has FDA approval. The emergence and widespread use of RNA vaccines during this health crisis might provide an ideal opportunity to develop the first anti-viral ASOs on the market. In this review, we describe the story of ASOs, the different characteristics of their chemistry, and how their characteristics translate into research and as a clinical tool.

COVID-19 associated Multisystem Inflammatory Syndrome in Children (MIS-C) guidelines; revisiting the Western New York approach as the pandemic evolves.

Multisystem inflammatory syndrome of children (MIS-C) continues to be a highly concerning diagnosis in those recently infected with SARS-CoV-2. The diagnosis of MIS-C cases will likely become even more challenging as vaccine uptake and natural immunity in previously infected persons leads to lower circulating rates of SARS-CoV-2 infection and will make cases sporadic. Febrile children presenting with cardiac dysfunction, symptoms overlapping Kawasaki disease or significant gastrointestinal complaints warrant a thorough screen in emergency departments, urgent care centers, and outpatient pediatric or family medicine practices. An increased index of suspicion and discussion regarding higher level of care (transferring to pediatric tertiary care centers or to intensive care) continues to be recommended. Herein we outline a broad approach with a multidisciplinary team for those meeting the case definition and believe such an approach is crucial for successful outcomes.

Nucleic Acid-Based Technologies Targeting Coronaviruses.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently creating a global health emergency. This crisis is driving a worldwide effort to develop effective vaccines, prophylactics, and therapeutics. Nucleic acid (NA)-based treatments hold great potential to combat outbreaks of coronaviruses (CoVs) due to their rapid development, high target specificity, and the capacity to increase druggability. Here, we review key anti-CoV NA-based technologies, including antisense oligonucleotides (ASOs), siRNAs, RNA-targeting clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas), and mRNA vaccines, and discuss improved delivery methods and combination therapies with other antiviral drugs.