key: cord-347221-g98q9cga
authors: Piyush, Ravikant; Rajarshi, Keshav; Chatterjee, Aroni; Khan, Rajni; Ray, Shashikant
title: Nucleic acid-based therapy for coronavirus disease 2019
date: 2020-09-19
journal: Heliyon
DOI: 10.1016/j.heliyon.2020.e05007
sha: 
doc_id: 347221
cord_uid: g98q9cga

The coronavirus disease 2019 (COVID-19), the pandemic that originated in China has already spread into more than 190 countries, resulting in huge loss of human life and many more are at the stake of losing it; if not intervened with the best therapeutics to contain the disease. For that aspect, various scientific groups are continuously involved in the development of an effective line of treatment to control the novel coronavirus from spreading rapidly. Worldwide scientists are evaluating various biomolecules and synthetic inhibitors against COVID-19; where the nucleic acid-based molecules may be considered as potential drug candidates. These molecules have been proved potentially effective against SARS-CoV, which shares high sequence similarity with SARS-CoV-2. Recent advancements in nucleic acid-based therapeutics are helpful in targeted drug delivery, safely and effectively. The use of nucleic acid-based molecules also known to regulate the level of gene expression inside the target cells. This review mainly focuses on various nucleic acid-based biologically active molecules and their therapeutic potentials in developing vaccines for SARS-CoV-2.

The coronavirus disease 2019 , the pandemic that originated in China has already spread into more than 190 countries, resulting in huge loss of human life and many more are at the stake of losing it; if not intervened with the best therapeutics to contain the disease. For that aspect, various scientific groups are continuously involved in the development of an effective line of treatment to control the novel coronavirus from spreading rapidly. Worldwide scientists are evaluating various biomolecules and synthetic inhibitors against COVID-19; where the nucleic acid-based molecules may be considered as potential drug candidates. These molecules have been proved potentially effective against SARS-CoV, which shares high sequence similarity with SARS-CoV-2. Recent advancements in nucleic acid-based therapeutics are helpful in targeted drug delivery, safely and effectively. The use of nucleic acid-based molecules also known to regulate the level of gene expression inside the target cells. This review mainly focuses on various nucleic acid-based biologically active molecules and their therapeutic potentials in developing vaccines for SARS-CoV-2.

The world has already witnessed many viruses causing disease outbreaks in various regions across the globe in the past two decades; such as Severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic in 2002-2003 originated in China [1] , Influenza A pandemic in 2009, first reported in Spain [2] , Middle East respiratory syndrome (MERS) pandemic in 2012, first identified in Saudi Arabia and the current [3] , Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or COVID-19) pandemic in 2019-2020, originating from China. SARS-CoV-2 consists of a positive sense (+) single-strand RNA genome [4, 5] . It belongs to the β-coronavirus sub-family along with SARS-CoV and MERS-CoV [4, 6] . The whole-genome sequence of SARS-CoV-2 consists of 29,903 nucleotides assigned with GenBank accession number MN908947 and the order of gene present was:

replicase ORF1ab, spike (S), envelope (E), membrane (M) and nucleocapsid (N) in 5´ to 3´ direction of the viral genome [7] [8] [9] . WHO officially designated this new disease as coronavirus disease-2019, i.e., COVID-19. To date, the virus has infected more than 17 million people, and more than 680,000 people lost their lives across the globe [10].

This unprecedented global crisis has posed a significant challenge for the human and has brought forward our therapeutic limitations to fight against an unpredictable deadly virus. In recent years significant progress has been made in the understanding of viral gene functioning, viral genomics, and target-based drug designing which have stimulated the development of many therapeutic strategies capable of efficiently blocking viral gene expression [11] . Among them, the nucleic acid-based therapeutic molecules have shown significant potential as therapeutic agents with potential anti-viral properties [12] . These proposed anti-viral drugs, feature a versatile mode of action and has been designed to specifically arrest viral disease progression [13] . The nucleic acid-based drugs have been shown to elicit a broad spectrum of anti-viral immunity in the body along with suppressing viral replication and gene expression [14] . This phenomenon of producing an effective immunity is particularly important in their use against the development of nucleic acid based therapeutic drugs for the treatment of SARS-CoV-2.

By the introduction of nucleic acid-based therapeutic technologies, the gene expression can be regulated either at the transcriptional or post-transcriptional level [15] . Nucleic acid-based therapeutic molecules try to restore the homeostatic balance in two ways: overexpression of protective genes and silencing of damaged genes. Nucleic acid-based therapeutic biomolecules have also shown some promising results in pulmonary diseases [16] . Nucleic acid-based therapies, especially, RNA therapies including RNAi (RNA interference), siRNAs (small interfering RNA) and RNA aptamers, Ribozymes and ASOs (antisense oligonucleotides) target and neutralize the crucial components of the virus-like specific mRNA molecules, viral proteins like E (envelope), M (membrane), or N (nucleocapsid), or SARS helicase, etc. These biomolecules have also been found to be effective during the previous epidemic due to SARS-CoV [17] . The phylogenetic analysis of SARS-CoV-2 with SARS-CoV has shown that they share 89.1% similarities among each other [7] [8] [9] , the use of nucleic acid-based molecules against SARS-CoV-2 may emerge as a potential therapeutic strategy.

Several viruses have been reported to show tropism towards the cells of respiratory tracts which not only help in the entry of virus particles inside respiratory tracts but it also helps to causes the infection in the host cells [13] . The physiological function of the lungs makes it highly exposed to pollutants and viral particles from outside [13] . Due to this reason, lungs get susceptible to several diseases ranging from common viral infection to lung cancer.

Viruses that infect the respiratory tract spread rapidly among the population due to their J o u r n a l P r e -p r o o f simple and easy mode of transmission [13] . The transmission of such viruses mainly takes place either by physical contact with the infected person or by droplets containing the viral particles [13] .

Currently, there is no therapeutic agent or vaccination for the treatment of SARS-CoV-2. In the present scenario, patients are administrated with a combination of drugs such as remdesivir, lopinavir, etc. [18] , hydroxychloroquine, azithromycin, zinc sulfate and corticosteroids. Patients with severe infections are treated by collateral care like ventilation and fluid management. So, discovering a novel therapeutic approach that could help in the control, prevention, and treatment of SARS-CoV-2 infection is an urgent need to save the entire globe from this pandemic situations.

The objective of this review is to put forward the current scenario of the development and efficacy of nucleic acid-based therapeutics as potential anti-viral agents. Here, we have also summarized the potential benefits and challenges in the application of these anti-viral agents in the context of SARS-CoV-2 infection.

The entry of virus particles inside the host cells determines the viral infectivity and pathogenicity [19] . The spike protein present on the surface of SARS-CoV-2 facilitates the entry of virus particles through the human angiotensin-converting enzyme 2 (hACE2) receptors binding domain [20] and is proteolytic activated by host cell proteases [19] . Therefore, several research groups are trying to decipher the interaction of hACE2 and spike protein as a novel drug target site to stop the pathogenicity [5, 21] . The hACE2 receptors present on the lungs, arteries, heart, kidneys and small intestine, colon, thymus, bone marrow, lymph nodes, the brain of the host cells [22] . The S1 subunit of the spike protein binds to the receptor-binding domain and assists the attachment of spike protein to the receptor, resulting in conformational changes into the spike protein [21] . TMPRSS211, a serine protease and lysosomal proteases cathepsins produced by the host cell cleaves the spike protein at the boundary of S1/S2 in such a way that S1 dissociates from the complex [23] and intense structural change in S2 domain was observed, which is necessary for the fusion of the virus into the host cell membrane [24, 25] . Membrane fusion and internalization of the virus are carried out via the S2 domain of the spike protein [5] . Unlike SARS-CoV, the pre-activation of SARS-CoV-2 entry inside the host cell is caused by proprotein convertase furin, which reduces its dependency on target cell proteases for invasion [19] .

J o u r n a l P r e -p r o o f

Progression of the virus and failure of the immune system causes severe damage to the other parts of the body, especially those organs which express the hACE2 receptors highly, such as kidneys, lungs, and intestines [26] . The immune response towards any disease differs due to genetic variations from individual to individual. The SARS-CoV-2 infection can be classified into two categories: initial (non-severe) and latter (severe) [27] . In the initial stages, macrophages and granulocytes mediate the inflammatory responses. SARS-CoV-2 infection in the respiratory system has been reported to activate nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) through pattern recognition receptors (PRRs), leading to the activation of pro-inflammatory cytokines, including interleukin-6 (IL-6), chemokines and tumor necrosis factors (TNFs) [28] . Signal transducer and activator of transcription 3 (STAT3) is required for the hyper-activation of NF-κB via activation of the IL-6 amplifier (IL-6 Amp), leading to autoimmune and multiple inflammatory diseases in the patients [29] .

But in severe cases, a powerful chronic inflammation due to cytokine response from both immune and non-immune cells causes severe damage to the host or even death due to immune-mediated Adverse Drug Reactions [30, 31] (ADRs) [28] .

Although lots of efforts are being put into research and development of DNA or RNA vaccine over the past few years, currently it has not been perfected enough to be used in humans [32] . Though several DNA vaccines have been accepted to be used for trials on animals [32] . Improvements in the DNA and RNA vaccine development strategy might turn out to be very crucial, keeping in view the increased frequency of epidemics, also efficient development of the vaccine may prevent infections caused by highly transmittable pathogens.

Since synthetic DNA and RNA are easier to construct, therefore, DNA and RNA based approach could provide for more quick development of vaccines [33] .

The nucleic acid-based vaccination technologies involve the use of RNA (mRNA) [34] or plasmid DNA, which encodes for antigen. These antigens encoded by the nucleic acid can trigger humoral as well as cell-mediated immune responses upon their expression after cellular uptake [35] . The nucleic acid-based vaccination technology is considered versatile and flexible as it allows easy maneuvering and manipulation of the antigen. The advantage of producing antigens in the target cells is that it imitates the protein synthesis during the infection as the protein remains localized in the plasma membrane, and protein modification J o u r n a l P r e -p r o o f processes like glycosylation can occur with great extents of fidelity [36] . Notably, they assist the delivery of an antigen of choice, irrespective of whether it was originated from a bacteria, parasite, or virus, thus facilitating the development of vaccines against a wide range of pathogens [32] . The different nucleic acid-based vaccine candidates which are in the different phase in clinical trails are described in Table 2 Several nucleic acid-based molecules such as aptamers, siRNA, and miRNA have been used for the treatment of severe viral infections including HIV-1 (human immunodeficiency virus) [37, 38] 

Incorporation of a eukaryotic expression cassette that encodes for single or multiple antigens of interest into a bacterial plasmid leads to the generation of DNA vaccines. The plasmid backbone is comprised of the origin of replication and the sequences for antibiotic resistance genes which is used as a selection marker. Mostly, these selectable markers include antibiotic resistance genes against antibiotics like kanamycin [30] . DoggyboneTM (covalently closed linear DNA construct) [31] and Minicircle DNA [32] , comprised of the gene expressing 

Small interfering RNAs (siRNAs) are 21-23 base pairs long double-stranded molecules, which is used for silencing the target genes in a sequence-specific manner Another mechanism by which this antisense technology works is by restricting the binding of either the ribosome or the polymerase to the 5´ terminus of the target sequence, thus inhibiting the translation by preventing the congregation of the target machinery.

In 

The mRNA-based vaccine has emerged out to a hopeful choice against SARS-CoV-2 for the development of the vaccine [141] . The mRNA based vaccines possess a beneficial edge over other biomolecules as it lacks the ability of integration into the genome, has the ability to induce autoantibodies, lack of persistence over time, their high purity and can be produced in large quantity to meet the need of vaccination [142] . Moderna Inc. The USA, and Pfizer in collaboration with BioNTECH has selected mRNA based vaccine candidates against SARS-CoV-2. Arcturus Therapeutics and Imperial College London are utilizing self-replicating or self-amplifying mRNA [143, 144] , Translate Bio and Curevac are using optimized mRNA sequences which are unmodified [145, 146] , while BioNTech, at present, is yet evaluating its three different RNA formats derived from different antigenic regions of spike glycoprotein [147] . Most of the companies are expected to target the major spike protein (structural J o u r n a l P r e -p r o o f protein) as the gene of choice, but all developers have not clearly stated so [148] . BioNTech offers three platforms for lipid-based delivery systems, i.e., lipoplexes, LNPs (lipid nanoparticles), and polyplexes [149] . The LUNAR system of Synthetic Genomics, which is the delivery platform to be used by Arcturus Therapeutics, seems to be widely applicable for several target tissues through multiple target routes [144] .

One of the first vaccine, mRNA-1273, developed by the National Institute of Allergy and Infectious Diseases in association with Moderna, is under phase1/ phase 2 of the clinical trial.

An mRNA-1273 encoding for S protein of SARS-CoV-2 is encapsulated into lipid nanoparticles and delivered into the cell to generate an immune response against S protein [141] .

Using lipid nanoparticles (LNPs) as a carrier, the mRNA sequence of the recombinant target protein is delivered to the somatic cytoplasm for direct translation and encoding of the target protein [150] (Figure 2 ). The antigen-presenting cells quickly recognize these target proteins when released from the host cell [151] . Processing of these target proteins and their presentation is an important step for subsequent activation of both T and B cells resulting in cytotoxicity and humoral responses [151] .

The therapeutic effect resulting from the antibodies against S protein may include the clearance of viral load from the infected cells, and reduced bioavailability of hACE2 might reduce the proliferation and spread of SARS-CoV-2 [152] .

The inherent ratio of nucleotides to S protein (n/s) and outer surface protein to SARS-CoV-2 virion (s/v), both the ratio will play a crucial role to determine the amount of IgG antibody titer to be present and the number of booster doses to be given, to effectively neutralize the virus in the body [141] . A previous study on HIV has shown that low spike density with large spacing among them is incapable of activating B cells [153, 154] . 

Based on the drugs approved by FDA between 2009 to 2018, the mean research and development investment required to bring a new drug to the market was estimated to be $1336 million (M), and the median was estimated to be $985M [155] . The major components of cost driver include product development, facilities and equipment, direct labor, overhead, and licensing and commercialization [156] . In product development, the estimated cost range >500M USD with the risk-adjusted cost of 135 to 350M USD. In facilities and equipment, the estimated cost range between 50 to 700M USD. Direct labor and overhead cost range has been estimated to be 25% less than of total manufacturing costs, and up to 45% of labor and raw materials cost combined together respectively. For licensing and commercialization, the WHO process 300 (thousand) K USD for a site audit, 25K to 100K USD for evaluation and annual fees of 4.8K to 140K USD [157] [158] [159] [160] . mRNA-1273 being developed by Moderna has received funding of $483M in April and $472M in July with total funding of $955M approximately whereas another leading vaccine, ChAdOx1 being developed by University of Oxford has received funding of £84M approximately [161, 162] . Based on this finding, the major challenge to the world after vaccine development will be of vaccine distribution across the globe.

The high potency of mRNA vaccines with only one or two low dose immunization is capable of generating potent antiviral neutralizing antibodies by activating both CD4 + and CD8 + T cells [164] [165] [166] . The structural modification of mRNA results in higher immunogenicity by improving its stability and translation efficacy [164, 165] . The potential risk of infection and insertion induced mutagenesis are minimized by mRNA based vaccines due to its natural degradation in cells [167] . In order to treat large populations, engineered mRNA facilitates the large-scale production of the required vaccine dose [168, 169] .

The mRNA vaccination may be detrimental due to local and systemic inflammatory responses, possible development of autoreactive antibodies, persistence, and bio-distribution of induced immunogenic responses and toxic effect of delivery system components and nonnative nucleotides [170] [171] [172] . Fatigue, chills, headache, myalgia, and pain at injection site are some of the solicited systemic and local adverse effect that occurred in more than half participants on which vaccination trail of mRNA-1273 was carried out [173] . Mutation in the spike protein increases the possibility that the vaccine will not be very effective in the long term [141] .

Like mRNA vaccines, DNA vaccines generate effective antiviral neutralizing antibodies by activating CD4 + and CD8 + T cells [174] [175] [176] . The DNA drug product is stable for a longer duration and can be deployed in an effective and executable manner [177] . To meet the large demand to treat patients across the globe, the DNA plasmid manufacture process facilitates the scaled manufacture of the drug [176, 177] .

The major cons associated with DNA vaccines were reported more prominent in humans and other large animals rather than small animal models [177] . It have been reported that in human and large animals DNA vaccine causes lower immunogenicity in comparison to inactivated vaccines, autoimmune responses, and DNA integration in the host genome, etc. [176, 178] . J o u r n a l P r e -p r o o f The desired gene is introduced into the target cell by applying an electric pulse to it. The electric pulse generates electric pores into the cell membrane through which the desired gene gets into it. As soon as the electric pulse is removed, the gene remains into the cell as the electric pores are closed and the cell membrane is in normal state. A similar method has been adopted for the delivery of INO-4800 into the host.

J o u r n a l P r e -p r o o f The nucleic acid can be either DNA or mRNA. A) In case of mRNA-1273, mRNA encoding prefusion spike protein is encapsulated in lipid nanoparticle. It is administrated intravenously into the patient. The ligand on lipid nanoparticle helps it to bind to the receptor and gets internalized through endocytosis. It is then, released into the cytosol where the gene enclosed in it gets released. mRNA is then translated into protein and the protein is translocated to the cell surface where it activates the immune cells to produce antibodies against it. B) If the nucleic acid is DNA, then it first goes into the nucleus to get transcribed into mRNA. In the cytoplasm, mRNA gets translated into the protein and the protein helps to generate an immune response in a similar way as stated above.

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