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.

RNA therapeutics in the clinic.

Ribonucleic acid (RNA) therapeutics are being actively researched as a therapeutic modality in preclinical and clinical studies. They have become one of the most ubiquitously known and discussed therapeutics in recent years in part due to the ongoing coronavirus pandemic. Since the first approval in 1998, research on RNA therapeutics has progressed to discovering new therapeutic targets and delivery strategies to enhance their safety and efficacy. Here, we provide an overview of the current clinically relevant RNA therapeutics, mechanistic basis of their function, and strategies to improve their clinical use. We discuss the 17 approved RNA therapeutics and perform an in-depth analysis of the 222 ongoing clinical trials, with an emphasis on their respective mechanisms and disease areas. We also provide perspectives on the challenges for clinical translation of RNA therapeutics and suggest potential strategies to address these challenges.

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.

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.

The Role of Nanotechnology in Antiviral Regime: An Overview

Nanomedicine or nanotechnology exhibits outstanding features to challenge severe health issues including pathogenic viral infections, the most culpable invaders in the present situation. The perpetual mutational pattern in viruses topped with raising resistance to drug epitomizes the current situation as a trigger to explore nanotech platforms in antiviral therapies. Referring to novel physicochemical features of nanomaterials associated with effective drug delivery, it is viewed as an ideal strategy for treatment of viral infections. The coronavirus induced pathogenesis, including MERS, SARS and SARS-CoV-2 infections, has triggered alarming and highly dangerous precedents against existence of humans. Applications of nanotechnology can serve a new direction for disinfection or treatment of viruses. Presently, various types of nanomaterials, such as nanogels, nanospheres, nanocapsules, liposomes, nanoparticles and many others, that have been investigated in vivo and in vitro for successful drug delivery, vaccination, diagnostic assay and device development with anticipation to be translated in advanced clinical practices, need a collective relook. This paper intents to contribute insightful critique of current studies on the efficacy of nanoplatforms as drug transporter, diagnostic tool and vaccine candidate against pathogenic viruses counting the highly pathogenic and incurable "coronaviruses".

RNA Therapeutics: the Next Generation of Drugs for Cardiovascular Diseases.

PURPOSE OF REVIEW: RNA therapeutics are a new and rapidly expanding class of drugs to prevent or treat a wide spectrum of diseases. We discuss the defining characteristics of the diverse family of molecules under the RNA therapeutics umbrella. RECENT FINDINGS: RNA therapeutics are designed to regulate gene expression in a transient manner. For example, depending upon the strategy employed, RNA therapies offer the versatility to replace, supplement, correct, suppress, or eliminate the expression of a targeted gene. RNA therapies include antisense nucleotides, microRNAs and small interfering RNAs, RNA aptamers, and messenger RNAs. Further, we discuss the mechanism(s) by which different RNA therapies either reduce or increase the expression of their targets. We review the RNA therapeutics approved (and those in trials) to treat cardiovascular indications. RNA-based therapeutics are a new, rapidly growing class of drugs that will offer new alternatives for an increasing array of cardiovascular conditions.

Probing the mutation independent interaction of DNA probes with SARS-CoV-2 variants through a combination of surface-enhanced Raman scattering and machine learning.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution has been characterized by the emergence of sets of mutations impacting the virus characteristics, such as transmissibility and antigenicity, presumably in response to the changing immune profile of the human population. The presence of mutations in the SARS-CoV-2 virus can potentially impact therapeutic and diagnostic test performances. We design and develop here a unique set of DNA probes i.e., antisense oligonucleotides (ASOs) which can interact with genetic sequences of the virus irrespective of its ongoing mutations. The probes, developed herein, target a specific segment of the nucleocapsid phosphoprotein (N) gene of SARS-CoV-2 with high binding efficiency which do not mutate among the known variants. Further probing into the interaction profile of the ASOs reveals that the ASO-RNA hybridization remains unaltered even for a hypothetical single point mutation at the target RNA site and diminished only in case of the hypothetical double or triple point mutations. The mechanism of interaction among the ASOs and SARS-CoV-2 RNA is then explored with a combination of surface-enhanced Raman scattering (SERS) and machine learning techniques. It has been observed that the technique, described herein, could efficiently discriminate between clinically positive and negative samples with ∼100% sensitivity and ∼90% specificity up to 63 copies/mL of SARS-CoV-2 RNA concentration. Thus, this study establishes N gene targeted ASOs as the fundamental machinery to efficiently detect all the current SARS-CoV-2 variants regardless of their mutations.

Inhibition of SARS-CoV-2 by Targeting Conserved Viral RNA Structures and Sequences.

The ongoing COVID-19/Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2) pandemic has become a significant threat to public health and has hugely impacted societies globally. Targeting conserved SARS-CoV-2 RNA structures and sequences essential for viral genome translation is a novel approach to inhibit viral infection and progression. This new pharmacological modality compasses two classes of RNA-targeting molecules: 1) synthetic small molecules that recognize secondary or tertiary RNA structures and 2) antisense oligonucleotides (ASOs) that recognize the RNA primary sequence. These molecules can also serve as a "bait" fragment in RNA degrading chimeras to eliminate the viral RNA genome. This new type of chimeric RNA degrader is recently named ribonuclease targeting chimera or RIBOTAC. This review paper summarizes the sequence conservation in SARS-CoV-2 and the current development of RNA-targeting molecules to combat this virus. These RNA-binding molecules will also serve as an emerging class of antiviral drug candidates that might pivot to address future viral outbreaks.

Antisense Oligonucleotide-Based Therapy of Viral Infections.

Nucleic acid-based therapeutics have demonstrated their efficacy in the treatment of various diseases and vaccine development. Antisense oligonucleotide (ASO) technology exploits a single-strand short oligonucleotide to either cause target RNA degradation or sterically block the binding of cellular factors or machineries to the target RNA. Chemical modification or bioconjugation of ASOs can enhance both its pharmacokinetic and pharmacodynamic performance, and it enables customization for a specific clinical purpose. ASO-based therapies have been used for treatment of genetic disorders, cancer and viral infections. In particular, ASOs can be rapidly developed for newly emerging virus and their reemerging variants. This review discusses ASO modifications and delivery options as well as the design of antiviral ASOs. A better understanding of the viral life cycle and virus-host interactions as well as advances in oligonucleotide technology will benefit the development of ASO-based antiviral therapies.

A rapid RNA extraction-free lateral flow assay for molecular point-of-care detection of SARS-CoV-2 augmented by chemical probes.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the major shortcoming of healthcare systems globally in their inability to diagnose the disease rapidly and accurately. At present, the molecular approaches for diagnosing COVID-19 primarily use reverse transcriptase polymerase chain reaction (RT-PCR) to create and amplify cDNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Although molecular tests are reported to be specific, false negatives are quite common. Furthermore, literally all these tests require a step involving RNA isolation which does not make them point-of-care (POC) in the true sense. Here, we report a lateral flow strip-based RNA extraction and amplification-free nucleic acid test (NAT) for rapid diagnosis of positive COVID-19 cases at POC. The assay uses highly specific 6-carboxyfluorescein (6-FAM) and biotin labeled antisense oligonucleotides (ASOs) as probes those are designed to target N-gene sequence of SARS-CoV-2. Additionally, we utilized cysteamine capped gold-nanoparticles (Cyst-AuNPs) to augment the signal further for enhanced sensitivity. Without any large-stationary equipment and highly trained staffers, the entire sample-to-answer approach in our case would take less than 30 min from a patient swab sample collection to final diagnostic result. Moreover, when evaluated with 60 clinical samples and verified with an FDA-approved TaqPath RT-PCR kit for COVID-19 diagnosis, the assay obtained almost 99.99% accuracy and specificity. We anticipate that the newly established low-cost amplification-free detection of SARS-CoV-2 RNA will aid in the development of a platform technology for rapid and POC diagnosis of COVID-19 and other pathogens.

Nucleic Acid Drugs-Current Status, Issues, and Expectations for Exosomes.

Nucleic acid drugs are being developed as novel therapeutic modalities. They have great potential to treat human diseases such as cancers, viral infections, and genetic disorders due to unique characteristics that make it possible to approach undruggable targets using classical small molecule or protein/antibody-based biologics. In this review, I describe the advantages, classification, and clinical status of nucleic acid therapeutics. To date, more than 10 products have been launched, and many products have been tested in clinics. To promote the use of nucleic acid therapeutics such as antibodies, several hurdles need to be surmounted. The most important issue is the delivery of nucleic acids and several other challenges have been reported. Recent advanced delivery platforms are lipid nanoparticles and ligand conjugation approaches. With the progress of exosome biology, exosomes are expected to contribute to the solution of various problems associated with nucleic acid drugs.

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.

Rapid, Ultrasensitive, and Quantitative Detection of SARS-CoV-2 Using Antisense Oligonucleotides Directed Electrochemical Biosensor Chip.

A large-scale diagnosis of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is essential to downregulate its spread within as well as across communities and mitigate the current outbreak of the pandemic novel coronavirus disease 2019 (COVID-19). Herein, we report the development of a rapid (less than 5 min), low-cost, easy-to-implement, and quantitative paper-based electrochemical sensor chip to enable the digital detection of SARS-CoV-2 genetic material. The biosensor uses gold nanoparticles (AuNPs), capped with highly specific antisense oligonucleotides (ssDNA) targeting viral nucleocapsid phosphoprotein (N-gene). The sensing probes are immobilized on a paper-based electrochemical platform to yield a nucleic-acid-testing device with a readout that can be recorded with a simple hand-held reader. The biosensor chip has been tested using samples collected from Vero cells infected with SARS-CoV-2 virus and clinical samples. The sensor provides a significant improvement in output signal only in the presence of its target-SARS-CoV-2 RNA-within less than 5 min of incubation time, with a sensitivity of 231 (copies µL-1)-1 and limit of detection of 6.9 copies/µL without the need for any further amplification. The sensor chip performance has been tested using clinical samples from 22 COVID-19 positive patients and 26 healthy asymptomatic subjects confirmed using the FDA-approved RT-PCR COVID-19 diagnostic kit. The sensor successfully distinguishes the positive COVID-19 samples from the negative ones with almost 100% accuracy, sensitivity, and specificity and exhibits an insignificant change in output signal for the samples lacking a SARS-CoV-2 viral target segment (e.g., SARS-CoV, MERS-CoV, or negative COVID-19 samples collected from healthy subjects). The feasibility of the sensor even during the genomic mutation of the virus is also ensured from the design of the ssDNA-conjugated AuNPs that simultaneously target two separate regions of the same SARS-CoV-2 N-gene.

Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles.

The current outbreak of the pandemic coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) demands its rapid, convenient, and large-scale diagnosis to downregulate its spread within as well as across the communities. But the reliability, reproducibility, and selectivity of majority of such diagnostic tests fail when they are tested either to a viral load at its early representation or to a viral gene mutated during its current spread. In this regard, a selective "naked-eye" detection of SARS-CoV-2 is highly desirable, which can be tested without accessing any advanced instrumental techniques. We herein report the development of a colorimetric assay based on gold nanoparticles (AuNPs), when capped with suitably designed thiol-modified antisense oligonucleotides (ASOs) specific for N-gene (nucleocapsid phosphoprotein) of SARS-CoV-2, could be used for diagnosing positive COVID-19 cases within 10 min from the isolated RNA samples. The thiol-modified ASO-capped AuNPs agglomerate selectively in the presence of its target RNA sequence of SARS-CoV-2 and demonstrate a change in its surface plasmon resonance. Further, the addition of RNaseH cleaves the RNA strand from the RNA-DNA hybrid leading to a visually detectable precipitate from the solution mediated by the additional agglomeration among the AuNPs. The selectivity of the assay has been monitored in the presence of MERS-CoV viral RNA with a limit of detection of 0.18 ng/µL of RNA having SARS-CoV-2 viral load. Thus, the current study reports a selective and visual "naked-eye" detection of COVID-19 causative virus, SARS-CoV-2, without the requirement of any sophisticated instrumental techniques.