Exploring the future of SARS-CoV-2 treatment after the first two years of the pandemic: A comparative study of alternative therapeutics


 One of the most pressing challenges associated with SARS-CoV-2 treatment is the emergence of new variants that may be more transmissible, cause more severe disease, or be resistant to current treatments and vaccines. The emergence of SARS-CoV-2 has led to a global pandemic, resulting in millions of deaths worldwide. Various strategies have been employed to combat the virus, including neutralizing monoclonal antibodies (mAbs), CRISPR/Cas13, and antisense oligonucleotides (ASOs). While vaccines and small molecules have proven to be an effective means of preventing severe COVID-19 and reducing transmission rates, the emergence of new virus variants poses a challenge to their effectiveness. Monoclonal antibodies have shown promise in treating early-stage COVID-19, but their effectiveness is limited in severe cases and the emergence of new variants may reduce their binding affinity. CRISPR/Cas13 has shown potential in targeting essential viral genes, but its efficiency, specificity, and delivery to the site of infection are major limitations. ASOs have also been shown to be effective in targeting viral RNA, but they face similar challenges to CRISPR/Cas13 in terms of delivery and potential off-target effects. In conclusion, a combination of these strategies may provide a more effective means of combating SARS-CoV-2, and future research should focus on improving their efficiency, specificity, and delivery to the site of infection. It is evident that the continued research and development of these alternative therapies will be essential in the ongoing fight against SARS-CoV-2 and its potential future variants.



Introduction
Throughout the history of the 21st century, there has been no such pandemic as formidable as the recent Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic.At the end of February 2003, the World Health Organization identified the outbreak that emerged in China and spread to more than 24 countries in North and South America, Europe, and Asia before it was controlled [1,2].SARS claimed the lives of 774 with an incidence of 8098 cases globally [2].A similar event played out in late December 2019, identified to be airborne (Fig. 1).An outbreak that claimed led to 6945,714 deaths from 768,187,096 reported confirmed cases were reported to have originated from Wuhan in China, and was soon worldwide (WHO, 2023).This led to policies that applied to every location of the world in curbing the spread of the virus.
Multiple therapeutic approaches have been employed to combat COVID-19, with a significant focus on antiviral drugs.Notably, regulatory agencies have granted emergency use authorization to certain antiviral drugs such as Remdesivir for the treatment of COVID-19, primarily by inhibiting viral replication [3].In the pursuit of effective treatments, virtual screening and in vitro studies have explored the potential of antiretroviral and antiviral drugs, considering their applicability to COVID-19 treatment [3].This strategy is justified by the presence of shared protein targets among various viruses, leading to the investigation of drugs with established efficacy against related viral diseases [4][5][6].For instance, Lopinavir, a protease inhibitor primarily used for HIV-1, demonstrated a significant reduction in β-coronavirus viral loads when combined with Ritonavir [3].However, a study by Cao [7] found no additional benefits in terms of viral RNA detection or overall patient outcomes when treating adults with lopinavir-ritonavir, but observed gastrointestinal adverse effects.As of October 22nd, 2020, the Food and Drug Administration (FDA) had only approved Remdesivir for the treatment of SARS-CoV-2 [8].Originally developed for Ebola and Marburg infections, Remdesivir targets the RNA-dependent RNA polymerase (RdRp), a key enzyme responsible for viral RNA synthesis and replication [9].Inhibiting RdRp, which has been identified as a primary drug target for SARS-CoV-2, impedes viral growth and replication [4,6].Promising outcomes from the administration of Remdesivir in COVID-19 patients have been reported by Wang et al. [10], Holshue et al. ( 2019), and others.However, Diaz et al. [11] observed that patients treated with Remdesivir still experienced mortality, albeit at a lower rate compared to those treated with chloroquine.Furthermore, the effectiveness of Remdesivir against emerging variants such as Omicron remains inconclusive, and severe side effects have been documented [9,12].These side effects encompass respiratory failure, organ dysfunction, coagulopathy, decreased red blood cell count, elevated liver enzyme levels indicating inflammation or liver damage, hypotension, nausea, vomiting, and other adverse events [13,14].It is worth noting that treating viral infections, including COVID-19, is challenging due to the ability of viruses to evade the immune system once they enter host cells.This characteristic poses a significant threat to the body's defense mechanisms.
In addition to their efficacy and side effects, the cost of current antiviral drugs targeting SARS-CoV-2 is a significant concern.Margaret (2022) highlighted that the commercialization of Remdesivir by Gilead Sciences has resulted in a cost of $390 per vial.A week-long treatment course would amount to $2730, while a 14-day course would cost $5460.This high cost poses a financial burden for individuals with low to middle incomes and may also impact the finances of high-income earners.The affordability of such treatment becomes even more critical in regions with limited resources, where the expense may render it unattainable during similar outbreaks, potentially leading to complications and fatalities.Nature's report draws a parallel between the future of viral diseases and the current prevalence of malaria, a preventable and treatable disease that still claims a significant number of lives annually (Schudellari, 2019).While the emergence of RNA vaccines has been advantageous, the effectiveness of these vaccines relies on the durability of immunity they provide.
Despite considerable research efforts focused on the development of anti-SARS-CoV-2 drugs, the number of novel drugs in the pipeline remains limited.Furthermore, there is a noticeable scarcity of pharmaceutical companies involved in the production of these drugs [15][16][17].This can be attributed to the global landscape of the disease, which may not offer substantial financial incentives for pharmaceutical ventures.
In the scientific literature, there is an increasing focus on the exploration and discovery of novel compounds targeting SARS-CoV-2.There is a growing body of research dedicated to identifying alternative therapeutics that offer targeted efficacy and potential costeffectiveness compared to current standard treatments.These alternative options aim to provide more durable solutions for the treatment of SARS-CoV-2 infections.The emergence of new variants of the virus presents a significant challenge in combating the disease, as these variants may exhibit increased transmissibility, more severe symptoms, or resistance to existing treatments and vaccines.Consequently, continuous research and development of new and alternative therapies are imperative to address the evolving nature of the virus.This narrative Fig. 1.An illustration of SARS-CoV-2 infection through the atmosphere.
B.A. Babalola et al. review aims to present a fresh perspective on alternative therapeutic approaches against SARS-CoV-2.Specifically, three promising options are explored: antisense oligonucleotides, neutralizing monoclonal antibodies, and the CRISPR/Cas system.This study comprehensively examines the mechanisms of action, efficacy, and clinical and economic considerations of these alternative therapies, providing insights into their potential as viable treatment options for SARS-CoV-2.

Method
A systematic search of scientific databases including PubMed, Scopus, and Web of Science was conducted to identify relevant articles on alternative therapeutics for SARS-CoV-2.The search strategy included a combination of keywords and subject headings related to "COVID-19," "SARS-CoV-2," "Monoclonal antibody," "Antisense Oligonucleotides," CRISPR-Cas", and "post-pandemic."Two authors independently screened titles and abstracts of the articles obtained from the search for relevance to the study objectives.Inclusion criteria for the study were articles reporting alternative therapeutics for SARS-CoV-2, published in English, and available in full-text.Any discrepancies between the two authors were resolved through discussion with a third author.Full-text articles that met the inclusion criteria were assessed for their suitability for inclusion in the study.Articles that reported on alternative therapies that were not supported by scientific evidence or studies conducted in vitro were excluded from the paper.To ensure the completeness of the search strategy, reference lists of the selected articles were reviewed for additional relevant studies.

Mechanism of action of ASO against CoVs
In the context of COVID-19, ASOs could be used to target specific viral RNA sequences and inhibit the replication of the virus.One potential target for ASOs in COVID-19 is the RNA genome of the Sars-CoV2 virus.The genome of SARS-CoV-2 is a single-stranded RNA molecule that serves as the template for the production of new viral particles.By designing ASOs that target specific regions of the viral transcriptome, researchers could potentially inhibit the virus from replicating (Fig. 2).SARS-CoV-2 produces several non-structural proteins that are essential for viral replication [18].By targeting the RNA transcripts that encode these proteins, ASOs could potentially inhibit the production of the proteins, thus inhibiting viral replication.Once ASOs are bound to these target RNAs of SARS-CoV-2, they can regulate their function by either binding to the RNA to hinder their function without facilitating the RNA degradation-this could be by translation inhibition or modulation of RNA processing.ASOs also promote the degradation of RNAs via endogenous enzymes such as RNaseH or argonaute-2 (RNA interference (RNAi)) [11,19].In addition to targeting the virus itself, ASOs could also be used to modulate the host's immune response to the virus.One of the key features of severe COVID-19 is an overactive immune response that can cause inflammation and damage to the lungs and other organs.ASOs could be designed to target specific immune signaling molecules or receptors, thus modulating the immune response and potentially reducing Fig. 2. Proposed mechanism of ASO against SARS-CoV-2.In this illustration, the conjugated ASO binds to the receptor which is internalized and taken up by the endosome.The conjugate then dissociates from the ASO.From the endosome, there is an endosomal escape of the ASO.There are three mechanisms ASO functions: (1) ASO is associated with splicing modification which causes the inhibition of target protein production of SARS-CoV-2.(2) Also, the pre-mRNA of relevant target proteins can be spliced and undergo post-translational modification in the nucleus.Outside of the nucleus, the ASO can inhibit the translation of the target protein by causing the steric hindrance of the ribosome function.(3) The ASO recruits RNaseH which degrades the target mRNA from SARS-CoV-2, thereby reducing or inhibiting the target protein production.the severity of the disease.

Pre-pandemic knowledge of antisense oligonucleotides (ASO)
Over a few decades ago, ASOs were identified to control RNA processing and regulate protein expression [20].ASOs are short, chemically modified nucleotides usually 12-24 bases in length, that can be designed to target specific RNA sequences [21].Antisense oligonucleotides bind to their target nucleic acid by Watson-Crick base pairing, inhibiting or altering gene expression through steric hindrance, splicing modifications, target degradation initiation, and other mechanisms [22].
Stephenson and Zamecnik developed the first ASO therapy with the aim of blocking the replication of the Rous sarcoma virus and preventing the oncogenic transformation of chicken fibroblast infected with the Rous virus [23].And the first ASO to receive FDA approval was designed to treat CMV retinitis, which is a remarkable fact [23].Influenza A genes, which are highly conserved, have been successfully inhibited with ASOs in vitro and in vivo.While there are several ASO therapies for neurodegenerative diseases in various stages of clinical development, only two of them, nusinersen and eteplirsen, have been approved by the FDA for use in treating neurodegenerative diseases [24].According to Fusco et al. [21], the use of antisense-based therapies for inflammatory bowel diseases like Crohn's disease and ulcerative colitis is supported by positive results from preclinical models and initial clinical studies, as well as the safety profiles of the compounds.However, large clinical trials have not yet confirmed the promising results seen in preclinical models.ASOs have been studied over the years for use against viral infection, but only a few numbers have reported therapeutic applications; most have been developed to treat genetic diseases [25].RNAs frequently fold into structures that obstruct ASO hybridization, and this limits the efficiency of ASOs [25].As a result, therapeutic ASO techniques frequently target RNA sections that are known to not fold or rely on three-dimensional structures to keep the strands in a stable conformation [25].
Short-interfering RNAs (siRNAs) are double-stranded RNA oligonucleotides that catalyze the degradation of complementary mRNAs.Because RNA virus infection relies on the transport, replication, and translation of viral RNA, these pathogens are an obvious target for sequence-specific treatments, which are fast improving.SARS-CoV-2 contains several highly conserved and functionally important regions that could be used as therapeutic targets (Manfredonia et al., 2020).The replicase gene, which has two open reading frames, ORF1a and 1b, which overlap at the frameshifting stimulation element (FSE), essential for ORF1b translation, dominates the 5' end of the genome [26].Therefore, ASO may be an effective and alternative therapeutic approach to combating SARS-CoV-2 through the inhibition of the production of important targets for are relevant for its survival.

Pandemic correlations/data of antisense oligonucleotides (ASOs)
The potential use of antisense oligonucleotides (ASOs) as a therapeutic strategy for COVID-19 is still being investigated.However, preclinical studies have shown promising results in targeting specific viral RNA sequences and modulating the host immune response.For example, Lulla et al. [27] demonstrated that an ASO-modified stem-loop motif of SARS-CoV-2 leads to RNA cleavage in vitro.Similarly, Pfafenrot et al. [28] reported that a circular ASO targeting a non-structural protein of SARS-CoV-2 reduced viral replication in cell cultures by 90 % for over 48 h.Su et al. [29] developed a chimeric ASO that activated the host RNase L pathway, leading to viral RNA cleavage and a significant reduction in viral replication and infectivity in vitro.In another study, Zhu et al. [30] designed and tested a pool of ASOs targeting the conserved regions of the SARS-CoV-2 RNA genome in human respiratory cells and mice.The ASOs efficiently reduced viral replication, transcription, and the expression of viral genes in human cells, and resulted in a significant reduction in viral load and inflammation in the lungs of mice.Furthermore, Stincarelli et al. [31] tested a panel of ASOs targeting the conserved stem-loop structures of the viral RNA, which are essential for viral replication.The ASOs efficiently reduced viral replication and transcription in SARS-CoV-2 infected cells.Another study reported a 94 % reduction in SARS-COV-2 RNA-dependent RNA polymerase RNA levels in infected lung cell lines treated with ASOs, and a 98 % reduction in virus RNA levels in COVID-19 hospitalized patients.Overall, while the use of ASOs as a therapeutic strategy for COVID-19 is still in its early stages, preclinical studies have shown promising results in reducing viral replication and infectivity and modulating the host immune response to the virus.Further studies are needed to evaluate the safety and efficacy of this approach in clinical settings.

Limitations and benefits of ASOs as therapy
Delivery is the most difficult obstacle for oligo-based therapeutic techniques [32].Systemic administration by intravenous or subcutaneous injection is used in the majority of oligotherapeutic applications [32].In this case, an inhalation delivery system would be excellent to get the oligos straight into the airways and lungs (Rossi and Rossi, 2020).Several scientists have been working on this mode of delivery for respiratory disorders over the last decade, and its successful deployment in patients with COVID-19 could make many of the aforementioned techniques truly useful [32].Oligo therapies are simple to design, manufacture, and have numerous optimizing chemical alterations already recognized for usage in various diseases, facilitating design, production, and medicinal chemistry optimization during a crisis when speedy medication development is critical [32,33].
Only a few complementary oligonucleotides can successfully hybridize to a specific mRNA in practice [34].This is thought to be due to difficulties with target accessibility, which could be caused by the secondary or tertiary mRNA structure and/or proteins linked to the RNA [34].Several predictive algorithms have recently been developed to determine the optimal mRNA hybridization sites [34].[35] suggested a method for determining RNA structures based on algorithms and the RNA's thermodynamic and structural properties.They identified advantageous local target sequences using a systemic alignment of computer-predicted secondary structures of local sequences of the targeted RNA and then designed more efficient antisense oligonucleotides [34].Another way of selection is based on determining melting temperatures or the free energies of oligonucleotide/RNA duplex formation [34,36].Combinatorial oligonucleotides, which are employed to identify the hybridization sites directly inside the RNA, have also been utilized in recent approaches [34].RNase H cleavage, microarrays, and MALDI-TOF mass spectrometry can all reveal these locations [34].Despite the fact that these approaches are time-consuming, they may eventually lead to the identification of great target areas [34].
Limited target engagement, low biological activity, and off-target toxic effects have impeded progress in translating ASO therapeutic options into the clinic [20].However, chemical modifications of ASOs can be employed to address these issues [20,22].These changes, together with a better understanding of the mechanism of action of ASOs and better clinical trial design will help to accelerate the translation of ASO-based tactics into medicines [20].Higher affinity due to the development of chemical modifications that boost affinity, selectivity while reducing toxicity due to off-target effects are all advantages of ASOs [22].Because oligonucleotides of 15-20 nucleotides long can uniquely attach to a target RNA, the length of ASOs helps to improve their specificity [37].
Antisense treatments can be developed and tested far more swiftly and rationally than standard pharmaceuticals [38], with antisense libraries regularly outperforming traditional libraries by several orders of magnitude [39].The success rate for traditional libraries, that is, the possibility of finding a chemical that would bind to a target protein by large-scale screening, has been reported as high as 0.02 % [38].When compared to antisense libraries, which have a reported success rate of around 10 %, this is a significant difference [38].From both an economic and a medical standpoint, the potential benefits in terms of reduced development durations and a faster rate of getting medications B.A. Babalola et al. to patients who need them are intriguing [38].

Clinical considerations for COVID-19
ASOs have shown great efficacy in blocking viral RNA regions, such as AUG start sites and critical regulatory motifs, through steric blockage.Three patents were simultaneously published describing the development of ASOs targeting different regions of the SARS-CoV-2 genome.Stein et al. [40] developed ASOs that target the translational initiation start site (AUG) of the first open reading frame (ORF1) of the viral genome, while Ionis Pharmaceutical [41] developed ASOs to disrupt the pseudoknot in the frameshift site, which is essential for viral replication.AVI Biopharma, now known as Sarepta Therapeutics, developed ASOs to target the stem-loop secondary structure at the 3' terminal end of the negative strand of viral RNA to inhibit replication [42].This targeting strategy was also proposed for the treatment of infections caused by other ssRNA viruses such as flavivirus, picornavirus, tagovirus, and calicivirus due to the highly conserved secondary structure of the stem-loop motif.
For the past 40 years, scientists have been pursuing the idea of utilizing synthetic oligonucleotides to control the expression of diseasecausing genes [43].Clinical success had been elusive for a long time and with good cause.Antisense therapy encompasses a variety of sequence types, such as siRNA, ASOs, ribozyme, DNAzyme, and aptamers, each with distinct features and mechanisms of action.RNAi has identified many viral genes, both structural and non-structural, that can be targeted, making it a promising therapeutic tool against infections.Oligo therapeutics are cost-effective to produce, and chemical modifications make them easier to optimize during drug development emergencies.While many potential targets for SARS-CoV-2 have been identified, the most effective sequence targets remain unclear.Although the recent approval of siRNA-based therapies is promising for antiviral agents, delivering oligonucleotides directly to the lungs is still challenging, and research is needed to find safe and effective delivery vehicles.

Mechanism of action
The pathophysiology of COVID-19 involves the entry of the causative virus, SARS-CoV-2, into the host's cells.This is achieved through the binding of the SARS-CoV-2 spike or S protein (S1) to angiotensinconverting enzyme 2 (ACE2) receptors that are highly expressed on the respiratory epithelium, specifically on type II alveolar epithelial cells.The spike protein's receptor-binding domain (RBD) regulates this process, which is followed by the priming of the spike protein (S2) by the host transmembrane serine protease 2 (TMPRSS2) (Babalola et al., 2021).This facilitates cell entry and subsequent viral replication.The mAbs may act through several direct and indirect mechanisms and may confer multiple mechanisms of action on SARS-CoV-2.They can have direct actions on the Spro target of SARS-CoV-2 by receptors, blocking cell-cell interactions, activating signaling pathways, or inducing cell death (Fig. 3).Neutralizing mAbs can work by targeting the Spro of the SARS-CoV-2 virus.Neutralizing mAbs can bind to specific regions on the spike protein, preventing it from binding to its ACE2 receptor in human cells.This inhibits the ability of the virus to cause disease and allows the immune system to clear the virus from the body.Another mechanism of action of neutralizing mAbs is to enhance the immune response.It recruits immune cells to attack and eliminate the virus, which can help to speed up the clearance of the virus from the body (Taylor et al., 2021).Additionally, neutralizing mAbs can activate the complement system, which is part of the immune system that helps to clear pathogens from the body [44].To explain this better, the constant regions (Fc) of Immunoglobulin (IgG) antibodies have conserved differences that distinguish them into four subclasses: IgG1, IgG2, IgG3, and IgG4.These Fc regions play a role in binding to Fc receptors (FcγR), complement factor component 1q (C1q), and the neonatal receptor (FcRn).The IgG subclasses' effector functions, such as phagocytosis, antibody-dependent cell-mediated cytotoxicity, complement activation, and their half-life and capacity for transplacental transport and transport through mucosal surfaces, are determined by these interactions.Thus, IgG1 mAbs can activate natural killer (NK) cells through CD16A, promote antibody-dependent phagocytosis (ADPh) by binding to macrophage CD16A, CD32A, and CD64, and activate the complement leading to  complement-dependent cytotoxicity (CDC) towards the destruction of SARS-CoV-2.Neutralizing mAbs can also neutralize viral particles in circulation.By binding to viral particles, neutralizing mAbs prevents them from infecting human cells and promotes their clearance by the immune system.

Pre-pandemic correlations/data of neutralizing monoclonal antibodies
Monoclonal antibodies have been a game-changing tool in medical treatments for several decades, particularly in cancer and autoimmune disease therapies.These antibodies can specifically recognize and target proteins on cancer cells or modulate immune system activity in autoimmune disorders, providing effective, targeted treatments.Wofsy and Seaman [45] carried out an experiment in which monoclonal antibodies to the L3T4 antigen were used to successfully treat autoimmune disease in NZB/NZW F1 mice.The mice were treated with monoclonal antibodies for a period of time, and the results were compared to a control group of untreated mice.The researchers found that the monoclonal antibody treatment significantly reduced the severity of the autoimmune disease in the mice [45], Ciazza et al. [46], demonstrated that the treatment of breast cancer cells with monoclonal antibodies resulted in a decrease in cancer cell growth, indicating the that-tumor potential of monoclonal antibodies.Furthermore, N-cadherin antibody reduced cancer cell proliferation, migration, and invasion in vitro, and inhibited tumor growth and metastasis in vivo [47].Interestingly, this antibody treatment delayed the onset of castration resistance in a mouse model of prostate cancers.Another study reported that monoclonal antibodies body targeting the Notch1 ligand-binding domain, effectively deplete a subpopulation of putative breast cancer stem-like cells, both in vitro and in vivo [48].Before the COVID-19 pandemic, monoclonal antibodies had already been approved, for multiple sclerosis [49], Alzheimer's disease [50], and HIV/AIDS [51], showcasing their versatility as a therapeutic tool.Overall, monoclonal antibodies have proven to be a valuable therapeutic tool in various medical fields showcasing their versatility as a therapeutic tool.

Pandemic data on neutralizing monoclonal antibodies (mAbs)
Mayer et al. (2019), described that administration of monoclonal antibodies including casirivimab and imdevimab reduced the risk of developing COVID during pregnancy.When mAbs are introduced into host cells, they target the RBD of SARS CoV2 and bind with their Spro, thereby disrupting the interaction between the ACE2 and Spro of the virus and preventing fusion and subsequent entry of the virus.However, some mAbs that do not target the receptor-binding-domain (RBD) of Spro recognize specific epitopes different from the ACE2-binding motif or the non-RBD regions.These antibodies are also classified as virusneutralizing.In other cases, mAbs (such as S309) may bind cells containing the Fcγ receptor (FcγR), leading to antibody-dependent cell cytotoxicity (mediated by natural killer cells) or to antibody-dependent cellular phagocytosis [52].
Bamlanivimab is a recombinant human IgG1 neutralizing mAb isolated from convalescent plasma obtained from patients with COVID-19, and it was developed by AbCellera biologics and Eli Lilly.This mAb binds to the receptor-binding domain of the spike protein of SARS-CoV-2 and thereby preventing the attachment of the spike protein to ACE2 [53,54].Etesevimab is also a neutralizing IgG1 mAb, isolated from convalescent plasma of COVID-19 patients, binding to the receptor-binding domain of spike protein of SARS-CoV-2 to different but overlapping epitopes [55].
Bamlanivimab and Etesevimab are neutralizing IgG1 mAbs that bind to overlapping epitopes of the receptor-binding domain (RBD) of Spro [55].While bamlanivimab binds to specific epitopes, some of these epitopes might have undergone mutation and become resistant to the bamlanivimab.These variants are being neutralized by the etesevimab which binds to a different epitope.Combining these two neutralizing antibodies may enhance viral load reduction and reduce resistant variants [56].To add, REGEN-COV or antibody cocktail is a combination of casirivimab (REGN10933) and Imdevimab (REGN10987).This type of human IgG1 mAbs targets the receptor-binding domain of the spike protein of SARS-CoV-2 [57,58].

Efficacy
Vaccines usually take several weeks to induce the production of antibodies in immunized individuals, but the neutralizing mAbs may produce an immediate response and therefore be a suitable option for populations with high-risk and immunocompromised individuals.It can also be considered efficient for individuals of all ages [59].The dosage needed to achieve a certain level of antiviral activity efficiency and the administration regimen determines the number of antibodies required for the treatment of a specific viral disease.Also, this dosage is influenced by the half-life and potency of the mAbs with an extended half-life.Better neutralizing activity is still significant, which can be aided through genetic engineering [60].
The reduction of SAR-CoV-2 viral load observed in convalescent plasma (CP) pilot trials suggests that SARS-CoV-2 infection can be controlled using passive immunization in clinical settings (Jaworski, 2021).The efficacy of nMAbs over CPs has been observed in recent studies in patients with Ebola [61].Ansuvimab (MAb114) was found to be safe and effective in reducing the mortality rate of the Ebola virus from 67 % to 34 %.This underscores the application of nMAbs for infectious and deadly diseases [61,62].Compared to HIV-1, SARS-CoV-2 accounts for a lower mutation rate.Currently, the combination of HIV-1-nMAbs is being tested to avoid virus resistance emergence, proof of concept phase 2 trials showed a partial efficacy of single nMAbs due to the selection of resistant viral mutants [60].
In November 2020, the US Food and Drug Administration (FDA) authorized the medication for emergency use (EUA).However, the EUA was withdrawn by the FDA in April 2021.Bamlanivimab, derived from the convalescent plasma who had COVID-19 [63,64], was used as a potent neutralizing monoclonal antibody (IgG1 with an unmodified Fc region) to the Spro.Bamlanivimab like other mAbs binds the Spro RBD, engaging its cognate epitope in both up and down conformations, making it potentially useful as a monotherapy.There have been historical precedents for the effectiveness of neutralizing mAbs as a monotherapy such as MAb114.Bamlanivimab at concentrations down to 100-fold below the effective concentration for a half-maximum response was studied in primary human macrophages and immune cell lines exposed to SARS-CoV-2 and it did not demonstrate productive viral infection [65].Prophylactic efficacy was tested in rhesus macaques given bamlanivimab 24 h before a virus challenge [64].The symptoms in this model were mild overall, but the treatment significantly decreased viral load and replication in the respiratory tract following inoculation, supporting its antiviral efficacy [66].
Depending on the infectious agent and the target epitope, combinations of mAbs may be required to maintain efficacy and prevent treatment failure.From past observations from HIV-1 which also has a high mutation rate, suggestions were made that two or more mAbs should be combined in order to improve the efficacy of this therapy [67,68] although this is not required always as in the case of Ansumivab monotherapy which targets a broadly conserved epitope on Ebola's RBD and found to be more effective than ZMapp triple cocktail and equally as effective as REGN-EB3 [60].The advantage of using this combination (REGEN-COV or antibody cocktail) is to prevent mutational escape (Weinreich et al., 2019).Bamlanivimab and etesevimab significantly reduced viral load and this led to a 70 % reduction in COVID-19 hospitalization and deaths; this data led to the issuance of EUA of bamlanivimab together with etesevimab [66].

Clinical consideration
Some neutralizing antibodies REGEN COV constituting casirivimab and imdevimab are already being used to treat mild to moderate COVID-19 in adults and pediatric patients.It was developed by the American biotechnology company Regeneron Pharmaceuticals and was clinically tried on non-hospitalized adults.The patients were given different doses of this combination, and some were treated with a placebo; results showed a decrease in the viral load of patients that were administered with the combination therapy compared to those treated with a placebo.Possible side effects of casirivimab and imdevimab include anaphylaxis, fever, chills, hives, itching, reactions due to infusion, and flushing [69].The rate of hospitalization of patients and admission into the emergency room also decreased.This shows that combination therapy will play a huge role in the treatment and prevention of COVID-19.
Monoclonal antibodies, such as bamlanivimab (LY-CoV555), may be associated with worse clinical outcomes when hospitalized patients with COVID-19 requiring high-flow oxygen or mechanical ventilation are being administered to.Clinical studies [70] have shown that this antibody actively reduced viral load and that patients should be given treatment in a facility that is equipped to manage anaphylaxis, and during the administration of the drug, patients should be monitored for hypersensitivity to the drug for not less than an hour infusion has been completed [70].Bamlanivimab is not authorized for use in patients who are hospitalized or require oxygen therapy because of COVID-19.These neutralizing IgG1 mAb was however granted an FDA EUA for treatment in patients diagnosed with mild to moderate COVID-19 and were at high risk of being hospitalized or becoming severe [70].
Neutralizing monoclonal antibodies, most of which are specific to the RBD of the SARS-CoV-2 Spro is still being developed using single B cells from individuals already infected with COVID-19 [71][72][73][74].
Some SARS-CoV targeting mAbs can also be considered as some of them neutralize SARS-CoV-2.It is noteworthy however that the mAbs of SARS-CoV although bind the RBD of SARS-CoV-2, they do not with the receptor binding motif, thereby failing to cross-neutralize SARS-CoV-2 infections [52] The mAbs of SARS-CoV can however still be purposed for neutralizing SARS-CoV-2 infections but should be genetically engineered to improve the neutralizing activity of the mAbs.The combination of nAbs of SARS-CoV with low neutralizing activity with nMAbs of SARS-CoV-2 can also be considered for better cross-neutralizing activity [59].
An important restriction of the use of mAbs in clinical settings is related to the large-scale production of mAbs, as well as the high costs associated with it.Currently, the platforms for the production of mAbs are based on the cloning of Ig-encoding genes from isolated specific-B cells.PCR amplification of Ig-encoding genes from B cells, and cloning them into an expression vector are carried out in this technique (Jaworski, 2021).

Limitations and benefits of monoclonal antibodies
Monoclonal antibodies have shown promise as a treatment option for SARS-CoV-2, but there are some limitations to their use.Firstly, monoclonal antibodies are not effective in patients with severe diseases who require hospitalization or mechanical ventilation [75].They are most effective when given early in the course of the disease, ideally within the first few days of symptoms.Secondly, the emergence of new variants of the virus may affect the effectiveness of monoclonal antibodies [76].Some variants may have mutations in the spike protein that could reduce the binding affinity of monoclonal antibodies.Thirdly, the production and distribution of monoclonal antibodies can be a challenge, as they are complex molecules that require specialized manufacturing and storage conditions.
Monoclonal antibodies are often big and the majority of them must be made in mammalian cell expression to maintain their functionality and shape.Such mammalian cell expressions have a high cost of production due to poor expression yield [59]. Production of large quantities of neutralizing mAbs with a low cost of production that is affordable will be of great advantage.Monoclonal Abs that neutralize a wide spectrum of SARS CoV2 variants need to also be produced to cover a larger population of COVID-19-infected patients [77].Due to the high cost of production, the availability of this therapy is limited to developed/high-income countries and as such would have a very limited impact globally.It's therefore suggested that means of producing these antibodies in mass should be discovered for accessibility by larger populations of other low-income countries.Public research institutions and even private-sector industries should deploy innovative technologies necessary for cutting of the production cost of mAbs.More methods for product and process improvement should be discovered and employed.The establishment of procurement and strategies for delivery may also improve the accessibility of a larger population to these antibodies.For example, the government of different countries can merge to purchase vaccines and medicines in bulk to reduce prices; this is known as pooled procurement (IAVA).This method has made vaccines and medicines available and accessible even in poor countries and, therefore, might work for them mAbs therapy.

CRISPR/Cas system 3.3.1. Mechanism of action
The CRISPR/Cas system represents a powerful tool in the fight against viral infections, including SARS-CoV-2 [78].The mechanism of action of this system is rooted in its ability to target and degrade viral RNA, thereby hindering the virus's ability to replicate and transcribe its proteins.The CRISPR/Cas system is modeled after the defense mechanisms utilized by bacteria to protect themselves from viruses.It consists of the Cas9 nuclease, which acts as a molecular pair of scissors, and a guide RNA that directs the Cas9 nuclease to the specific target within the genome.In the case of SARS-CoV-2, the CRISPR-CAS system could work through two main steps.The virus targets the ACE2 receptor to release its RNA into host cells.After the viral RNA is reverse-transcribed into double-stranded DNA, the pre-designed sgRNA-Cas13 nuclease specifically cleaves the viral RNA.(Fig. 4).

Pre-pandemic correlations/data of CRISPR/Cas system
The CRISPR-CAS system is a revolutionary gene-editing tool that has gained widespread attention since its discovery in 2012.Prior to the pandemic, the CRISPR-CAS9 system has been employed to treat various DNA viral infections.For example, in a study carried out by Ramanan et al. [79], the CRISPR/cas9 system was employed to specifically knockdown HBV in vitro and in vivo respectively in hepatocyte cell line and a mouse model of HBV respectively with no off-target cleavage reported even after some weeks of constant expression of the CRISPR/cas9 system.Cas9 protein has also been used to knock down the Hepatitis B virus (HBV) in-vitro [80].However, in this case, a special Cas9 protein was used which alters the nucleotide leading to a nonsense modification, producing a nonsense codon and ultimately stopping the translation of viral protein.Despite the fact HIV presents itself as a recalcitrant organism over time, researchers have also developed a CRISPR/Cas9 system to target HIV-1 genes present inside the host both invitro [81], in vivo [82] and ex vivo [83] respectively preventing the replication in this hosts.Furthermore, the CRISPR CAS system has also been used to target and knock down Epstein Barr Virus [84], and Human papillomavirus [85].

Pandemic data on CRISPR/Cas system
In response to the covid 19 pandemic, the CRISPR/Cas13 with its unique ability to break down RNA was developed, as a promising solution for the treatment of RNA virus infections.Researchers have proposed the use of CRISPR/Cas13 to tackle the positive-strand RNA viruses (ssRNA) genome of SARS-CoV-2, as this system has been shown to effectively knock down bacteriophage genes using sequence-specific trans-activating CRISPR-RNA (crRNAs), thereby offering immunity to bacterial hosts.The advantage of this Cas13d protein is that it can be used to knock down any new unfolding variant of this virus by the quick development of crRNAs.Nguyen et al. [86] designed a CRISPR/Cas13d system and used crRNAs to knock down the replicase transcriptase (ORF1ab) and spikes genes of SARS-CoV-2.Additionally, the CRISPR/Cas13 system can also target the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2, which is responsible for the replication of the viral RNA.This gene has a highly conserved amino acid sequence and molecular structure, making it an optimal target for CRISPR/Cas13-mediated RNA degradation [87].Prophylactic Antiviral CRISPR in human Cells (PAC-MAN) has also been successfully used by Abott et al. (2020) to knockdown genomic sequence of SARS-CoV-2 and Influenza virus, preventing the replication of these viruses in airway epithelial cells.In addition, Blanchard et al. [88] employed CRISPR/-Cas13 to treat SARS-CoV-2 infection in rodents.Interestingly, the researcher discovered that cas13 impeded SARS-CoV-2 replication and alleviated covid-19 symptoms in the rodent [88].

Limitations and benefits of CRISPR/Cas as therapy
The CRISPR/Cas13 system has effectively knocked down viral genes, including the replicase transcriptase (ORF1ab), spikes genes, and RNAdependent RNA polymerase (RdRp) gene of SARS-CoV-2, which are essential for viral replication.Although CRISPR/Cas13 is perceived to be promising, it has some limitations, which have impeded it from being used clinically.One big concern is the potential for off-target effects.This means that the CRISPR/Cas13 system could potentially target and degrade the wrong RNA sequences, leading to unintended consequences such as changes to the host's genetic makeup.Another limitation of CRISPR/Cas13 is its efficiency and specificity.Although CRISPR/Cas13 is capable of targeting RNA sequences with high precision, it may not always be effective in degrading the target RNA in all cases.This could be due to several factors, including the length of the target RNA, the presence of other RNA molecules in the host, and the overall efficiency of the CRISPR/Cas13 system (Burmistrz et al., 200).The delivery of CRISPR/Cas13 to the site of infection is also a major limitation.In order to be effective, the CRISPR/Cas13 system needs to reach the site of infection and target the virus's RNA.This can be a challenging task, especially in cases where the virus has already spread throughout the body.In addition, the successful transfer of the CRISPR/Cas13 system to virus-infected cells is also a challenge, but as the technology improves and becomes more efficient and sensitive, these limitations may become less of a concern.

Clinical consideration
So far there are no reports on the clinical considerations of the use of the CRISPR/Cas system in human clinical practice for SRAS-CoV-2 treatment.This is due to some major setbacks which include safety, delivery, and effectiveness.However, it could be used as an immediate therapeutic option that could be deployed to treat the infections caused by the SARS-cov2 virus because it targets highly conserved regions in this virus.

Discussion
With the emergence of new variants of SARS-CoV-2, it is expedient that various therapeutics are researched and developed for the containment of the COVID-19 disease.Various therapies are being considered to have the potential in tackling this disease based on the stage of infection as opposed to vaccination only.
The three strategies discussed in the previous prompts are ASOs, monoclonal antibodies, and CRISPR/Cas13.Each of these strategies has shown promise in the treatment of SARS-CoV-2, but they also have limitations that impede their use.Antisense oligonucleotides (ASOs) have shown potential as a treatment option for COVID-19.ASOs are short DNA or RNA molecules that can bind to a complementary RNA sequence and prevent it from being translated into protein.By targeting specific RNA sequences in SARS-CoV-2, ASOs potentially inhibit viral replication and reduce the severity of COVID-19 symptoms.One advantage of ASOs is that they can be designed to target highly conserved regions of the viral genome, making it less likely for the virus to develop resistance to the treatment.ASOs can also be delivered directly to the lungs, where the virus primarily replicates, making them an attractive option for treating respiratory infections like COVID-19.However, like the other strategies discussed, ASOs have some limitations.One challenge is delivering them to the site of infection in sufficient quantities.ASOs are large molecules and may have difficulty crossing the cell membrane to reach the target RNA.In addition, ASOs can have off-target effects, binding to unintended RNA sequences and potentially causing unintended consequences.
Monoclonal antibodies have been shown to be effective in treating COVID-19 patients, especially when administered early in the course of the disease.However, the emergence of new variants and the high cost of production and distribution limit their availability to low-income countries.To increase accessibility to a larger population, innovative technologies are necessary to cut production costs and improve the manufacturing process.The establishment of procurement and delivery strategies may also improve accessibility.Therefore, future recommendations should focus on discovering more product and process improvement methods while exploring strategies to reduce the cost of production and distribution.
CRISPR/Cas13 has effectively knocked down viral genes of SARS-CoV-2.However, its potential for off-target effects and challenges in delivery and specificity limits its clinical use.Future research should focus on developing more efficient and sensitive delivery systems for CRISPR/Cas13 while improving its efficiency and specificity to overcome these limitations.This will allow for better targeting and degradation of the viral RNA, making CRISPR/Cas13 a more viable treatment option.

Conclusion
In conclusion, each of the three strategies discussed -ASOs, monoclonal antibodies, and CRISPR/Cas13 -has its own advantages and limitations for the treatment of COVID-19.While monoclonal antibodies have shown promise in clinical trials, they are not effective in all patients and may be limited by the emergence of new viral variants.CRISPR/Cas13 and ASOs are still in the early stages of development and face challenges in delivery and off-target effects.In the future, it may be beneficial to explore combination therapies that utilize multiple strategies to target different aspects of the virus's life cycle.This could potentially improve treatment efficacy and reduce the likelihood of viral resistance.Additionally, continued research into the development of safe and effective delivery methods for these therapies will be critical for their success.

Fig. 3 .
Fig. 3. Proposed mechanism of action of neutralizing monoclonal antibodies as a therapeutic option against SARS-CoV-2.MAbs may act through direct (a) or indirect (b) mechanisms (a) One direct mechanism of monoclonal antibodies involves binding to a cell-bound ligand or receptor to block ligand-receptor interactions, which inhibits downstream signaling events.(b) The majority of monoclonal antibodies have a human IgG1 Fc region that can activate effector cells such as natural killer (NK) cells, leading to antibody-dependent immune cell cytotoxicity (ADCC), or macrophages, leading to antibody-dependent phagocytosis (ADPH), by interacting with their FCγ receptors.Additionally, the Fc region of monoclonal antibodies can activate the complement, resulting in complement-dependent cytotoxicity (CDC) of SARS-CoV-2.