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Therapeutic Modalities and Novel Approaches in Regenerative Medicine for COVID-19

MacKenzieOct 27, 2020, 5:40:19 AM

https://www.sciencedirect.com/science/article/pii/S0924857920304192

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https://doi.org/10.1016/j.ijantimicag.2020.106208 Get rights and content

Highlights

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Direct cytopathic effect and immunopathological pathogenesis are as two main underlying mechanisms for severe pulmonary injury in COVID-19 infections.

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Repurposed antiviral agents are among the most promising therapeutics for overcoming the COVID-19 pandemic.

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Immunomodulatory effects of mesenchymal stem/stromal cells (MSCs) hold the potential for preventing the immune mediated consequences of SARS-CoV-2.

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Nucleic acid- based approaches have gained much attention for treating COVID-19 infections.

Abstract

The recent coronavirus disease 2019 (COVID-19) outbreak around the world has an enormous impact on the global health burden, threatening the lives of many individuals, and has severe socio-economic consequences. Many pharmaceutical and biotechnology companies have begun intensive research on different therapeutic strategies, from repurposed antiviral drugs to vaccines and monoclonal antibodies to prevent the spread of the disease and treat infected patients. Among the various strategies, advanced therapeutic approaches including cell- and gene editing-based therapeutics are also being investigated and initial results in in vitro and early phase I studies were promising, however, still further assessments are required. In this paper, we first review the underlying mechanisms for severe acute respiratory syndrome coronavirus (SARS-CoV-2) pathogenesis and further discuss available therapeutic candidates and advanced modalities that are being currently evaluated in in vitro/in vivo models and of note in clinical trials.

Keywords

SARS-CoV-2

COVID-19

Advanced therapeutic approaches

ATMP

1. SARS-CoV-2 background

The recent outbreak of COVID-19, caused by a novel beta-coronavirus (CoV), is now a major worldwide medical (and economical) challenge. Therefore, specifying the therapeutic approaches and the mechanisms which lead to these strategies are of outmost importance. Reviewing the published papers in regards with the mechanisms and the state of art medications, we have tried to draw an overall picture of the involved mechanism and the related therapeutic approaches. The type of documents used to obtain the data were original articles, review articles, and HTML documents from the official websites (e.g. WHO). Search terms included MeSH (Medical Subject Headings) terms, “coronavirus, severe acute respiratory syndrome coronavirus 2, 2019-nCoV, along with focusing on novel therapeutic approaches”. The registered and active clinical trials were found on ClinicalTrials.gov and the index of studies of novel coronavirus pneumonia in the Chinese Clinical Trial Registry. The cut-off date for the data search was “September 2020.

CoVs are enveloped viruses, wherein the 27-32kb genomic RNA is capped and polyadenylated (1). They are subdivided into four distinct groups; alpha, beta, delta and gamma (2). The CoV species HKU1, NL63, OC43 and 229E cause common cold symptoms. Others, like the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Coronavirus (MERS-CoV) result in fatal diseases. SARS-CoV-2 is the third virus in the CoV family with the potential to cause life-threatening disease in a wide range of individuals (3).

An Approximately 30 kb single‐stranded positive‐sense RNA (+ssRNA) forms the genomic content of SARS-CoV-2, and makes it the largest known RNA virus (4). The genome interacts with the nucleoprotein, N, which is bound to the membrane (M) protein. The M protein plays a crucial role in viral assembly and budding. The envelop (E) protein functions in viral morphogenesis, release and pathogenesis (5). Finally, the spike (S) protein is a trimeric glycoprotein including two subunits of S1 and S2 (Figure 1). Using the S1 and S2 subunits of S glycoprotein, coronaviruses have acquired the ability to attach and fuse to the target cell membrane, respectively (Figure 2b) (6). M, E, and S make up the virus envelop (5). The genome and sub-genome of a typical CoV contain at least six ORFs (4). Two-thirds of that include ORF1a, and 1b, which are translated into the polyproteins 1a (pp1a) and pp1ab, respectively (Figure 3a). The remaining ORFs on the one-third of the genome encode for the main structural [S, E, M, and N] and accessory proteins. After translation, the viral genome initiates replication. Most of the 16 nonstructural proteins (nsps), which are produced from pp1a and pp1ab form a very large protein complex, responsible for viral genome replication and subgenomic mRNA (sgmRNA) synthesis (7). The viral life cycle is completed by fusion of virus particles with the plasma membrane and release into the extracellular space. While MERS-CoV S protein binds to the dipeptidyl peptidase 4 (DPP4) receptor, to gain entry into the cells, the receptor for both SARS-CoV and SARS-CoV-2 is hACE2 (8).

Figure 1. An overview of SARS-CoV-2 structure.

Figure 2. Pathogenesis of SARS-CoV-2. A. fever and coughing along with headache are the common symptoms of COVID-19. ARDS along with multi organ failure account for the critical stage of the disease (Left). Type II alveolar cells highly express ACE2, the known receptor for SARS-CoV-2 (Right). B. Virus entry mechanism along with the involved enzymes in this process is shown.

Figure 3. A. Viral genome replication and subgenomic mRNA (sgmRNA) synthesis represented. B. Interaction of APCs with lymphocytes and cytokine activation (Created by Biorender.com).

2. Pathogenesis

Aside from the oral mucosa, especially the tongue, high expression levels of the SARS-CoV-2 receptor, hACE2, have been reported on lung type II alveolar cells (AT2), upper and stratified esophageal epithelial cells, absorptive enterocytes from ileum and colon, cholangiocytes, myocardial cells, kidney proximal tubule cells, podocytes, bladder urothelial cells, male reproductive cells, placental trophoblasts, eye, and vascular endothelial cells (9, 10, 11). Two different pathophysiological patterns may be responsible for severe pulmonary injury in COVID-19 infections, direct cytopathic effect (CPE) and immunopathological pathogenesis. The cytopathic effect may be related to a high level of viral load, while the role of immune mediated pulmonary effects are more prevalent in late respiratory failure where the viral load has already been reduced (12). Both of these events are discussed in the following paragraphs.

2.1. Direct cytopathic effect

The exact mechanism(s) underlying the CPE of SARS-CoV-2 are not yet completely understood. However, as SARS-CoV-2 has high similarities with SARS-CoV (structure and entry-receptor specificity) as well as MERS-CoV (structure, but not entry-receptor), insights from how SARS-CoV and MERS-CoV cause pulmonary cell will undoubtedly speed the discovery of SARS-CoV-2 mediated pulmonary cell death (13). SARS-CoV causes cell death via both apoptosis and necrosis. Also, MERS-CoV has been shown to induce apoptosis in both immune and non-immune cells such as lung and kidney cells (14, 15). These findings can form the basis for possible underlying mechanisms through which SARS-CoV-2 may prompt its CPE, which has been demonstrated in human airway epithelial cells following the virus inoculation along with the cessation in cilia movements (3). Regarding the kidney cells there is evidence for a direct CPE of SARS-CoV-2 on various renal cells (16). For other cell types with ACE2 receptors, further studies need to be done to determine the probable direct CPE of SARS-CoV-2, as, for instance, in one study no apparent histological alterations in heart tissue was reported in the postmortem histopathological study on a COVID-19 infected patient other studies have suggested cardiac injury due to the direct effect of virus entry into myocardial tissue (17, 18, 19). Also, very little is known regarding the CPE of SARS-Cov-2 in gastrointestinal (GI) cells (20). Nevertheless, the increased rate of CPE after inoculation of human intestinal organoids and liver organoids with SARS-Cov-2 has been seen (21, 22). ACE2 is not expressed on hematopoietic cells; hence, direct infection of immune cells by SARS-CoV-2 may not be likely. However, if SARS-CoV-2 can infect the immune cells directly still needs to be studied (23).

2.2. Immunological effects

The precise mechanisms through which SARS-CoV-2 affects the immune system are not yet fully known. Similarities in the pathogenesis of coronaviruses, especially between SARS-CoV and SARS-CoV-2, have, however, provided valuable insights in how SARS-CoV-2 might affect the immune system (24).

Both innate and adaptive immune responses are involved in COVID-19 pathogenesis. Several components of the innate immune system have been reported to be over activated or increased in number. In fact, macrophage activation syndrome (MAS) is suggested as one of the possible reasons of COVID-19-related hyperinflammation (25). It is because SARS-CoV-2 has been shown to cause the activation of NLRP3 inflammasome in macrophages, which leads to an increased level of proinflammatory cytokines production (26). Moreover, neutrophils have also been predominantly found in lung infiltration of COVID-19 patients. The elevated amount of neutrophils and neutrophil-to-lymphocyte ratio (NLR) usually predict poor clinical outcome (27). In addition, it is shown that necroinflammation is one of the results of neutrophils infiltration and neutrophil extracellular traps (NETs) formation in COVID-19 patients (28). On the other hand, recognition of the pathogen-associated molecular pattern (PAMP) of SARS-CoV (genomic RNA) by TLR3 and TLR7 and the cytosolic RNA sensor, RIG-I/MDA5 subsequently lead to pro-inflammatory cytokine induction, particularly, type I IFN. However, both structural and non-structural proteins from coronaviruses interfere with the type I IFN-related signaling pathways (9, 29). The delayed type I IFN response along with the confirmed increase in neutrophils and monocytes/macrophages influx (30), followed by an excessive production of type I IFN in the later phases may explain part of the symptoms of the virus.

In addition to innate immunity, both humoral and cellular immune responses also play significant roles in coronavirus clinical complications. Many attempts to identify T and B cells epitopes for the virus structural proteins have been undertaken (31). Cytotoxic CD8 T-cells (cytotoxic T cells) and helper CD4 T-cells (helper T cells), as key parts of antiviral immunity, require the presentation of viral antigens through HLA I and II molecules on the surface of antigen presenting cells (APCs). Genetic polymorphisms in components of the antigen presentation system appear to account at least in part for the risk of SARS-CoV infections (29). In this regards, one in silico study showed that the expression of HLA-B*46:01 may make the individuals more susceptible to COVID-19 as the binding peptides for SARS-CoV-2 is predicted to be the least, while HLA-B*15:03 expression may enable cross-protective T-cell-based immunity (32). However, the correlation between different allele frequencies and susceptibility to SARS-CoV-2 infection needs more investigation. CD8+ T cells can destroy virally infected cells. CD4+ cells have a key role in promoting the activation of T‐dependent B cells and the production of proinflammatory cytokines leading to recruitment of monocytes and macrophages and over production of cytokine and chemokines (33). Reduction of T helper cells therefore, might lead to a strong immune‐mediated interstitial pneumonitis and delayed clearance of SARS‐CoV from lungs (34). It is also noteworthy that all the SARS‐CoV-related memory T cells found in SARS‐CoV convalescent patients, mediated an anti-SARS‐CoV structural protein response, whereby the S protein was mostly involved in these T cell responses (35, 36). This implies a role of the structural proteins as candidate for designing efficient SARS vaccines. Furthermore, clinical observations in COVID-19 patients confirm the reduction of excessively activated CD4+ and CD8+ T cells (18). Production of early stage-related IgM, long lasting specific IgG, and IgA forms the main B cell immune response to SARS-CoV (29). In this regards, the isolation of specific B-cell clones that produce neutralizing monoclonal antibodies has been already shown in a SARS-CoV convalescent patient (37). Although neutralizing antibodies may have the potential to block the viruses entry into human cells (38), anti-S protein neutralizing IgGs might also hold the risk for fatal acute lung injury (39).

On the other hand, it is interesting to note that ACE2 through which SARS-CoV-2 enters into cells has been reported to be downregulated in one mouse model of SARS-CoV infection and pulmonary disease, and that this downregulation may lead to more severe lung injuries (40). In the renin–angiotensin–aldosterone system (RAAS), ACE converts angiotensin I to angiotensin II, while ACE2 has a role in angiotensin II inactivation (40). Therefore, it is possible that SARS-CoV-2 can cause an increase in blood flow not only by the acute inflammation response, but also via an increase in angiotensin II due to ACE2 downregulation. In addition, the circulatory fraction of immunosenescent T cells (i.e. CD8+CD28−CD57+ cells that accumulate with aging and in chronic inflammatory conditions (41)) have been found in larger numbers of patients with high blood pressure, which may raise the risk for severe forms of COVID-19. This together with elevated levels of C-X-C chemokine receptor type 3 (CXCR3) chemokines and serum granzyme B, also hypothesized to be involved in T-cell–driven inflammation in human hypertension (42), may explain why aging and / or hypertension worsen the incidence and prognosis of COVID-19.

2.3. Cytokine storm-based effects

Acute respiratory distress syndrome (ARDS), a common immunopathological process, might be the leading cause of death in COVID-19 patients (Figure 3b) (43). In addition, failure of several other organs has been reported in severe cases of COVID-19 patients (18). The uncontrolled anti-COVID-19 proinflammatory cytokine and chemokine production, also termed cytokine storm, causes ARDS (44).

However, it is important to note that there are doubts regarding the relevancy of this event to COVID-19. In this regards, Sinha et al. have questioned the precise function of imbalanced cytokine responses in COVID-19 patients. They suggest that lung injury in COVID-19 patients is not solely attributed to “cytokine storm”. Of note, they mention that although the level of IL-6, a key cytokine in acute inflammation, in patients with COVID-19 is higher than the median value, it is less than what is measured in individuals affected by ARDS (10-200 fold higher in ARDS). Based on some available evidences, they note that, alveolar micro thrombi might be the possible culprits for lung injury in COVID-19 (45). Moreover, there are evidences indicating that the defect in both innate and adaptive immune responses is of more importance in comparison with the hypercytokinemia-induced organ injury, regarding the pathophysiological abnormalities in COVID -19 patients (46).

2.4. Coagulation dysfunctions

High blood pressure, pulmonary embolism and thrombosis are among the symptoms seen in COVID-19 patients, proposing the hypothesis that COVID-19 is an endothelial dysfunction disease (47, 48). In fact, significant higher levels of D-dimer and fibrin degradation products (FDP) together with longer prothrombin time (PT) has been confirmed in survivors compared to non-survivors upon admission to hospital (49). Coagulation dysfunction is related to the imbalanced immune response and massive inflammatory reactions, leading to microvascular system damage and activation of coagulation processes (50). This in turn leads to extensive microthrombosis (51). Due to the widespread inflammation, negative control mechanisms by which the thrombin production is monitored can be inhibited (51). The inflammatory reactions caused by the overproduction of proinflammatory cytokines also promote vascular permeability (51). On the other hand, reduced activity of ACE2 in lungs of animal models with coronavirus-induced severe ARDS may increase the risk of vascular hyperpermeability and pulmonary edema, as ACE2 is a negative regulatory factor for severity of lung edema (52). Pulmonary embolus is shown to be frequent in COVID-19 patients (53).

2.5. Circulating ACE2

ADAM17 and TMPRSS2 proteases are responsible for the cleavage of ACE2, which is mostly anchored at the apical surface of the cell (Figure 2b). However, while the first may have protective roles against SARS-CoV-2, the latter facilitates the virus entry (54, 55). In fact, metalloprotease ADAM17 cleaves the N-terminal catalytic domain of ACE2, which is also the coronavirus-binding site, and releases it into circulation. The exact role of cleaved ACE2 in circulation of COVID-19 patients still needs to become clear, however it was previously showed that serum ACE2 activity is elevated during hypertension and progression of cardiovascular disease (56, 57).

3. Therapeutics

Currently, the main strategy for managing the disease, focuses on supportive treatments such as oxygen therapy, fluid management, and ventilator support. However, it is of note to mention that despite the controversy on using the noninvasive ventilation (NIV) for managing ARDS in COVID-19 patients, still there might be a selected subpopulation of patients who may benefit from NIV (58).

Disease specific therapies might include available anti-viral medications as well as advanced strategies, such as cellular and gene-based protocols. In the following sections, we classified some of the currently suggested and available therapeutics based on the stated order for the virus pathogenesis mechanism (i.e. 1- Direct CPE, 2- Immunological effects and 3- Coagulation dysfunction) for management of COVID-19 patients (Figure 4). The defined therapeutics involves repurposed antiviral agents, cell/gene-based strategies, anti-cytokine modalities, and the anti-coagulation therapies.

Figure 4. Therapeutics. A. The current therapeutics used for treating COVID-19 patients. B. Advanced therapeutic medicinal products (ATMP). Cell- and gene-based therapies that might be candidate therapies (Created by Biorender.com).

4. Prevention of direct cell death due to virus

4.1. Repurposed antiviral agents

Several existing antiviral drugs have been repurposed and used to treat COVID-19 infected patients. Remdesivir (RDV), an adenosine analogue, was originally developed for the treatment of Ebola virus disease. RDV may have antiviral activity against a number of other RNA viruses, including SARS–CoV and MERS–CoV (59, 60). The triphosphate form of RDV competes with adenosine triphosphate (ATP) for incorporation into the genome and inhibits the RNA-dependent RNA polymerase (59). Following promising results of the drug in vitro and in vivo studies against MERS-CoV and SARS-CoV (61), RDV is a FDA- approved emergency use authorized anti-COVID-19 drug which has been tested in multi-site clinical trials. Initial studies suggest that treating COVID-19 patients with intravenous RDV improves the clinical condition of patients, even if side effects such as temporary gastrointestinal upset have been noted (62). Recently, Beigel et al. evaluated beneficial effects of RDV in hospitalized COVID-19 patients. Based on their findings, RDV treatment can shorten the recovery time in patients (63).

Other nucleotide analogues such as favipiravir (nucleoside analog), ribavirin (guanosine analog), galidesivir (adenosine analog), sofosbuvir (pyrimidine nucleotide analogue), alovudine (thymidine dideoxynucleoside analogue), Zidovudine (ZDV) (thymidine analogue), etc. are also under investigation for treatment of COVID-19 (29, 64). Favipiravir is an RNA polymerase inhibitor for a number of RNA viruses, and is approved for treatment of influenza in China and Japan. Several clinical trials are investigating the therapeutic effect of favipiravir for COVID-19 (65). Favipiravir may have a more potent antiviral action than some protease inhibitors such as kaletra (lopinavir/ritonavir) (65). However, compared to arbidol (an inhibitor of membrane fusion between the virus and the plasma membrane and also endocytic vesicle membranes), no significant improvement was reported in clinical recovery rate of patients after 7 days of favipiravir therapy (66). It is of note that recently a small phase 3 trial in India with 150 patients showed faster viral clearance in mild to moderate COVID-19 cases who had received Favipiravir (67). Accordingly, favipiravir is approved for restricted emergency use in moderate COVID-19 cases by Drugs Controller General of India (DCGI) (68).

Other antiviral drugs such as oral oseltamivir (a neuraminidase inhibitor), intravenous ganciclovir, and chloroquine phosphate tablets are candidate drugs that may reduce the infection symptoms (69). At the beginning of the pandemic, there were some reports indicating that chloroquine phosphate (an anti-malarial agent), that has both anti-viral and anti-inflammatory activities, might prevent worsening of pneumonia. However, due to the serious cardiac adverse events and other potential serious side effects of chloroquine phosphate and hydroxychloroquine sulfate and the low efficacy against COVID-19, the U.S. Food and Drug Administration (FDA) has recently cancelled its emergency use authorization (EUA) (69, 70). Also, the combination of hydroxychloroquine and azithromycin has recently been shown to have no significant effect on the rate of virologic clearance in patients with COVID-19 (71). Consistently, Cavalcanti et al. observed that using hydroxychloroquine, alone or with azithromycin, has no effects in the clinical status of patients with COVID-19 (72).

Chymotrypsin-like proteases (3CLpro), such as cinanserin, and flavonoids along with papain-like proteases (PLP) like diarylheptanoids may prevent coronavirus replication and are also considered as candidates to battle the virus (29). Finally, low-dose systemic administration of corticosteroids in addition to inhalation of interferon are other anti-COVID-19 strategies (69). Recently, it has been shown that dexamethasone appears promising in reducing the mortality rate in critically ill COVID-19 patients, which is appreciated by WHO (73). In fact, it is shown that the use of dexamethasone (at a dose of 6 mg once daily) in patients hospitalized with COVID-19 leads to lower mortality rate (74).

As mentioned in the pathogenesis section, ACE2 is a receptor responsible for entry of the virus. On the other hand, S protein and TMPRSS2 are among the molecules that are essential for viral entry. By using neutralizing antibodies against S protein and also TMPRSS2 inhibitors (camostate mesylate), viral entry was blocked (75). Moreover, recombinant ACE2 (APN01) could reduce both angiotensin ІІ and IL-6 levels in ARDS patients. This agent is currently under investigation for COVID-19 patients in China (76). However, there is a challenge regarding the use of ACE inhibitors in the context of cardiovascular diseases. For example, high levels of urinary ACE2 has been detected in patients who received the angiotensin-receptor blocker Olmesartan, but this was not seen in patients receiving the ACE inhibitor, enalapril or other angiotensin-receptor blocker, losarthan (77). Thus, use of angiotensin-receptor blockers or ACE inhibitors in the treatment of COVID-19 needs further investigation (78).

4.2. mAbs

Researchers tested to neutralize SARS-CoV-2 with mAbs previously shown to bind to SARS-CoV receptor binding domain (RBD)-directed mAbs. However, no significant binding to SARS-CoV-2 was seen (79). On the other hand, SARS-specific human monoclonal antibody CR3022 may have some cross-reactive binding between SARS-CoV-2 and SARS-CoV (80). Therefore, investigating the cross reactivity of other mAbs against SARS-CoV, including m396 and CR3014, may have the potential for the treatment of COVID-19 patients (29).

Moreover, neutralizing antibodies in the plasma of patients recovering from SARS, MERS, or the 2009 H1N1 pandemic, could modify the disease progression of other patients with these infections. Therefore, applying convalescent plasma (CP) from COVID-19 patients might hold promise to attenuate the clinical symptoms and mitigating the pulmonary damage, and eliminate SARS-CoV-2 RNA clearance (81, 82). In this regards, FDA has issued an EUA for COVID-19 convalescent plasma, although the significant efficacy of this approach for the treatment of COVID-19 patients is yet to be demonstrated in placebo-controlled randomized controlled trials (RCTs) (83).

4.3. Gene therapy-based strategies

In the context of development of novel treatments for viral infections including SARS-CoV-2, nucleic acid- based strategies as well as clustered regularly interspaced short palindromic repeats (CRISPR) associated protein nuclease (Cas)-based approaches could be used.

4.3.1. siRNA

The potential of siRNA technology for treating viral infections have been previously shown (84). Therefore, siRNAs may be considered as potential candidates to be used against SARS-CoV-2. A number of studies in this regard will be discussed.

In several studies, different structural and functional proteins of coronavirus were targeted using siRNA. Zhang et al. provide evidence that RNAi tool can be a therapeutic approach for SARS-CoV infection. Using spike-specific siRNAs they showed that the siRNAs effectively inhibited the expression of spike protein in cultured cells (85). He et al. evaluated the efficacy of six distinct siRNAs designed for different regions of SARS-CoV replicase gene. Although they did not report any synergistic effects of these siRNA, inhibitory effects of each of the siRNAs were demonstrated in vitro (86). However, later they reported synergistic antiviral activities of siRNAs targeting structural and replicase genes of SARS-associated coronavirus (87). There is also a report suggesting that targeting different regions of SARS-CoV viral genome using siRNA may inhibit viral infection and replication (88). Li et al. demonstrated the potential prophylactic as well as therapeutic ability of SARS coronavirus specific siRNA therapy in Rhesus macaque, with decreased body temperature recorded, lower levels of viral RNA and some decrease in lung damage (89).Overall, there are already several numbers of siRNA related patents regarding the SARS-CoV (90, 91). However, to the best of our knowledge, there are not any active clinical trials using the potential siRNAs for COVID-19.

4.3.2. CRISPR-Cas system

The CRISPR/ Cas system is an adaptive immune system in archaea and bacteria to protect against foreign DNA or in some cases RNA coming from viruses or mobile genetic elements. This immune response consists of three steps: 1. Acquisition of the foreign DNA; during which foreign DNA segments (spacers) are inserted to the genome of the host in between the repeats in CRISPR locus and therefore storing the memory of the previous invader 2. Transcription of the CRISPR locus long transcript and subsequent processing into short CRISPR RNAs (crRNAs) which guides Cas effector proteins to the complementary DNA or RNA sequence in the invading organism 3. Interference happens when the target region is cleaved by a single-protein, Cas effector protein (class 2) or large multisubunit protein complexes (class 1) (92). The CRISPR system comprises two classes and different types and subtypes. Class1 consists of Type I, III and IV and class2 consists of type II (Cas9 effector), V and VI; each having different subtypes. The CRISPR system has been mostly used to perform genome editing and transcription modification. However, this system has been naturally evolved in bacteria to defend against invading phages. This suggests that this system can be repurposed in mammalian cells to defend against RNA and DNA viruses. Type III and Type VI CRISPR systems have RNA targeting activities while the rest target DNA, and therefore they can target RNA and DNA viruses, respectively (93). Successful examples of targeting DNA viruses by CRISPR system type II (Cas9) have been provided in cell culture and animal models for HIV, HBV, herpesvirus, HPV, and many other viruses (94). Recent studies of type IV CRISPR-Cas (effector Cas13) have suggested that they may be able to efficiently target and degrade RNA (95, 96, 97, 98). Therefore, this system provides a potential therapeutic approach for elimination of RNA viruses. Additionally, Cas13 can process the long transcript crRNA and therefore can be used for multiplex targeting. Another advantage of Cas13 is the minimal off target activity on the host transcriptome, as shown in some recent studies (96, 99). Freije et al. showed 94.6% of the 396 single strand RNA (ssRNA) human associated viruses have >10 putative Cas13a target sites (93). Potent Cas13 activity against three different ssRNA viruses (lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV)) was described by Freije et al. The first proof of Cas13 for targeting SARS-CoV-2 has been suggested in the recent study by Abbott et al. In this study, CRISPR-Cas13 was tested as a prophylactic antiviral CRISPR in human cells (PAC-MAN), and showed efficient degradation of SARS-CoV-2 sequences as well as influenza A genome in vitro in human lung epithelial cells (100). This approach could constitute a pan-coronavirus inhibitory strategy, as a group of six cr-RNA might target more than 90% of coronaviruses. Although this study has provided a first proof for the potential usefulness of CRISPR-Cas to target SARS-CoV-2, further studies using replication-incompetent viruses and validation in animal models are required.

5. Immune-based approaches

5.1. Modulating immune responses

5.1.1. Cell based therapy approaches

Mesenchymal stem/stromal cells (MSCs), that can be relatively easily harvested either from fat tissue, bone marrow or placenta, among others, hold the potential for preventing the immune mediated consequences of SARS-CoV-2 and for restoring damaged tissues, by production of both trophic factors and anti-inflammatory molecules (101, 102). These include prostaglandin 2, indoleamine 2,3-dioxygenase (IDO), transforming growth factor-β, HLA-G5, interleukin (IL)-10, nitric oxide, and tumor necrosis factor (TNF)-α-induced gene/ protein 6 (TSG-6) (103). The anti-inflammatory feature of MSCs is exploited for treatment of steroid-resistant, severe, acute graft-versus-host disease (104, 105), and is being tested in the setting of a number of lung inflammatory disorders (106, 107). Moreover, positive effects of SCs in the setting of other viral diseases of the lung have been reported (108). In addition, MSCs have been suggested to potentially ameliorate ARDS, such as what is seen in severely affected COVID-19 patients (109). The immune modulatory role of MSCs in the context of COVID-19 is dual, including antiviral protection mediated by increased interferon stimulated genes (ISGs) expression and a secondary response to IFN, resulting in ISG stimulation and widespread viral resistance (110). Leng et al. showed that infusion of MSCs may improve some laboratory and clinical parameters of patients with COVID-19, and specifically might affect the dysregulated inflammatory responses. For instance, MSC therapy increased peripheral lymphocyte and also regulatory DC cells, while reducing levels of TNF-α and the over-activated cytokine-secreting immune cells, CXCR3+CD4+ T cells, CXCR3+CD8+ T cells and CXCR3+ NK cells (111). Liang et al. reported that the clinical manifestations of a 65- year old woman infected with COVID-19, treated by human umbilical cord MSCs, improved (112).

An alternative approach that might be considered as therapy for COVID-19 related lung disease, are stem cell educator (SCE) therapies. SCE cells are created by culturing patient lymphocytes with cord-blood derived stem cell (CB-SC), after which the autologous lymphocytes can be reinfused into patients. SCE has been demonstrated to potentially restore the balance of immune responses in a variety of autoimmune disease (113). Considering the immunomodulatory properties of SCE cells, researchers are now addressing their efficacy in SARS-related pneumonia (Table 2).

Natural killer (NK) cells are a subset of lymphocytes that exert cytotoxic effects against tumor cells and virus infected cells. In fact, cytokine production (mainly IFNs), by dendritic cells (DCs) stimulates activation of NK cells and these cells perform their cytotoxic activity by secretion of perforin and limiting viral replication (114). Of particular interest is CYNK-001, a NK therapy being developed from placenta derived hematopoietic stem cells (HSC) that is introduced as a potential therapeutic option for a variety of malignancies from hematological to solid tumors (NCT04310592). Celularity Inc., developing allogeneic cellular therapies from human placenta, recently announced the U.S. Food and Drug Administration (FDA) has cleared the company's Investigational New Drug (IND) application for the use of its proprietary CYNK-001 in individuals infected with COVID-19. They will commence a Phase I/II clinical study including up to 86 patients with COVID-19 (Not mentioned in Table 2) (115).

5.2. Anti-cytokine storm therapies

Cytokine release syndrome (CRS), first described in patients received CAR-T cell therapy, characterized by the release of a large body of cytokines including IL-6 and IL-1ß, as well as IL-2, IL-8, IL-17, G- CSF, GM- CSF, IP10, MCP1, MIP1a (also known as CCL3) and TNF (27). TH17 responses, which lead to vascular permeability and leakage, are enhanced by both IL-1β and TNF-α. Moreover, Th-17 shows a wide inflammatory response through producing G-CSF, IL-1β, TNF-α. TH-17 cells are among the cells involved in the COVID-19 cytokine storm. There is evidence that circulating levels of TH-17 cells are increased in COVID-19, suggesting the use of TH-17 blockers as a potential treatment for the present threat, COVID-19. STAT-3 is a transcription factor that mediates inflammatory response of TH-17 cells, while IL-6 activates STAT3 through JAK2 pathway. In the context of COVID-19, Wu et al. proposed that the FDA approved drug, Fedratinib (a JAK2 pathway inhibitor) might be a promising candidate treatment (116). Baricitinb, another member of JAK inhibitors, can improve the clinical status of COVID-19 patients. The related mechanism is by exerting inhibitory effects on numb-associated kinase (NAK) (117).

Type-I interferon (IFN-I) is involved in intracellular pathogen defense in the context of both innate and adaptive immunity. The DNA sensor cyclic GMP–AMP synthase (cGAS) and its downstream effector STING (stimulator of interferon genes) regulate transcription of many inflammatory molecules, including type I and type III interferons. However, dysregulation of IFNs production may lead to inflammatory diseases. Delayed IFN-I production in SARS infected animals, causes the accumulation of pathogenic monocyte-macrophages, resulting in lung immunopathology, vascular leakage, and suboptimal T cell responses. Therefore, targeting cGAS-STING pathway may be a suitable strategy for treatment of severe lung diseases caused by SARS-CoV and SARS-CoV-2. In line with this, Deng et al. evaluated the efficacy of some of the FDA approved drugs for targeting the STING pathway and found that approved drugs, such as suramin and ALK inhibitors, might be efficient and therefore worth testing in clinical trials. Adalimumab (TNF-α) and CMAB806 (IL-6) are among the cytokine-directed antagonists that are in clinical trials for COVID-19 (118). Indeed, anti-IL-6 receptor antibody (anti-IL-6RAb), (chemical name: Tocilizumab), a humanized monoclonal antibody, was developed and showed prophylactic efficacy against a broad spectrum of autoimmune diseases. However, as it was previously mentioned, there is a possibility that as a result of inaccurate attribution of “cytokine storm” with COVID-19, the related therapies would also need reconsideration (45).

Moreover, considering the profound lymphopenia in COVID-19 patients which can lead to severe pathological symptoms, treatments regarding the restoration of lymphocyte numbers have gain attentions. IL-7 has a key role in survival and expansion of lymphocytes. In this regards, Laterre et al. have showed the safety of IL-7 administration in patient with COVID -19 (119).

Interestingly, restoration of Th17/Treg imbalance seems to be the mechanism through which Tocilizumab confers protection over a range of diseases (120, 121). The therapeutic potential of Tocilizumab for treatment of COVID-19 patients is therefore being evaluated. These initial studies suggest that Tocilizumab might revert lymphopenia, lower oxygen need, and improve lung lesion, and therefore suggest that Tocilizumab might be a promising therapeutic strategy for individuals infected with COVID-19 (122). Results of a retrospective cohort study suggest that administered Tocilizumab might mitigate the risk of mechanical ventilation or death in COVID-19 patients with severe pnemonia (123).

5.3. Preventive strategies

5.3.1. Vaccines

Researchers in biotech companies and academic laboratories are making many efforts to develop efficient vaccines against COVID-19 with an unprecedented speed. The number of vaccines that are currently under preclinical investigations confirms this endeavor. A number of approaches have shown sufficiently promising in vitro and in vivo animal results such that they are already moving to early the clinical phase studies (124). In the following, examples of such vaccines discussed. The mRNA-1273 is a lipid nanoparticle encapsulated platform, which encodes the S protein of SARS-CoV-2, and therefore has the potential to elicit a robust immune response (NCT04283461) (125).

Recently, Zhang et al. has developed a novel thermostable mRNA- based vaccine, named ARCoV, which is encoding receptor binding domain of SARS-CoV-2 and elicited a protective response in animals challenged with SARS-CoV-2 (ChiCTR2000034112) (126).

Ad5-nCoV (NCT04313127) and INO-4800 (NCT04336410) vaccine candidates employ the same strategy but using different delivery platforms of adenovirus type 5 and DNA plasmid, respectively. LV-SMENP-DC (NCT04276896) and pathogen-specific aAPC (NCT04299724) are dendritic cells (DCs) and artificial antigen-presenting cells (aAPCs), which are manipulated with lentiviral vector (LV) to express mini-genes based on conserved and critical structural and protease protein domains of virus, respectively. However, developing new vaccination strategies may take months to years in terms of getting approval for both safety and efficacy.

5.3.2. Recombinant nucleic acids

5.3.2.1. DNA vaccines

Vaccination is among the most efficient measures against a vast variety of communicable diseases, a permanent global problem. In DNA vaccine technology, the gene encoding an immunogen, is inserted into a suitable vector, which may after administration lead to humoral and / or cellular immunity against this immunogen. Compared with traditional vaccination with an inactivated virus, DNA vaccination holds less risk for infection, is inexpensive, safe and more stable (127). Yang et al. reported that a DNA vaccine encoding for the S glycoprotein of SARS-CoV, exerts T-cell mediated immune response that is accompanied by increased neutralizing antibodies, and protects mice from being infected with SARS-CoV (128). Wang et al. demonstrated that a DNA vaccine encoding for the combination of 2 immunogens, namely the S1 subunit and full-length protein, confers protection against several strains of MERS in non- human primates and mice. Thus, in the context of MERS-CoV, this study is of interest, as the spike protein and S1 subunit DNA vaccine provides potent protection in animal models. Of note, there are some advantages regarding employing spike DNA prime-S1 protein boost over a single protein as the immunogen and for boosting immunization, which involves the production of Th1 immune response along with yielding various neutralizing antibodies (129).

5.3.2.2. mRNA-based strategies

An alternative to DNA vaccines, are RNA-based approaches. The problems with instability of RNA, have been in part overcome over the last decade (130). As compared with DNA, RNA-based therapeutics do not suffer from the risk of insertional mutations, and are only short-term present due to the faster RNA degradation. Nowadays, in vitro transcribed (IVT) mRNA has received increasing attention. The mRNA-based drugs are being developed as protein-replacement therapies, immunotherapeutics and in regenerative medicine. Preclinical and clinical applications of IVT-mRNA have been evaluated in a wide range of diseases including viral infections. However, there are some disadvantages regarding these molecules such as their stimulatory effect on immune responses through inducing interferons production. mRNA-1273 is one example of an IVT-mRNA as a novel lipid nanoparticle (LNP)-encapsulated mRNA-based vaccine that encodes for a full-length, spike (S) protein of SARS-CoV-2 (Table 1) (131).

Table 1. An overview of Phase 1, 2 clinical trials regarding gene-based therapy of COVID-19.

	Status	Interventions	Population	Phase	Location	NCT number
1	Recruiting	mRNA-1273	45	1	•Emory Vaccine Center – The Hope Clinic, Decatur, Georgia, United States •National Institutes of Health Clinical Center – Vaccine Research Center Clinical Trials Program, Bethesda, Maryland, United States •Kaiser Permanente Washington Health Research Institute Vaccines and Infectious Diseases, Seattle, Washington, United States	NCT04283461
2	Recruiting	Injection and infusion of LV-SMENP-DC vaccine and antigen-specific CTLs	100	1,2	•Shenzhen Geno-immune Medical Institute, Shenzhen, Guangdong, China •Shenzhen Second People's Hospital, Shenzhen, Guangdong, China •Shenzhen Third People's Hospital, Shenzhen, Guangdong, China	NCT04276896
3	Recruiting	Pathogen specific aAPC	100	1	•Shenzhen Geno-immune Medical Institute, Shenzhen, Guangdong, China	NCT04299724
4	Active, not recruiting	Recombinant Novel Coronavirus Vaccine (Adenovirus Type 5 Vector)	108	1	•Hubei Provincial Center for Disease Control and Prevention, Wuhan, Hubei, China	NCT04313127
5	Recruiting	INO-4800	40	1	•Center for Pharmaceutical Research, Kansas City, Missouri, United States •University of Pennsylvania, Philadelphia, Pennsylvania, United States	NCT04336410

NCT number: The National Clinical Trial number

Table 2. An overview of cell-based clinical trials for COVID-19 infections.

1	Status	Interventions	Population	Phase	Location	NCT number
2	Recruiting	Umbilical cord Wharton's jelly-derived human	40	1,2	Hôpital Pitié-Salpêtrière - APHP, Paris, France Hôpital Européen Georges Pompidou - APHP, Paris, France	NCT04333368
3	Recruiting	MSC Treatment	30	1,2	Istinye University, Istanbul, Turkey	NCT04392778
4	Recruiting	MSC therapy	60	2,3	Royan Institute Tehran, Iran, Islamic Republic of	NCT04366063
5	Enrolling byinvitation	HB-adMSCs	56	2	Hope Biosciences Stem Cell Research Foundation, Sugar Land, Texas, United States	NCT04349631
6	Active, not recruiting	UCMSCs	100	2	General Hospital of Central Theater Command, Wuhan, Hubei, China Maternal and Child Hospital of Hubei Province, Wuhan, Hubei, China Wuhan Huoshenshan Hospital, Wuhan, Hubei, China	NCT04288102
7	Recruiting	MSC	20	2	Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Punjab, Pakistan	NCT04444271
8	Recruiting	Human cord tissue mesenchymal stromal cells		1,2	Duke Hospital, Durham, North Carolina, United States	NCT04399889
9	Not yet recruiting	Wharton's jelly-MSC	40	1,2	Clinical Somer, Rionegro, Antioquia, Colombia	NCT04390152
10	Enrolling by invitation	Allogenic pooled olfactory mucosa-MSC	40	1,2	Institute of Biophysics and Cell Engineering of National Academy of Sciences of Belarus, Minsk, Belarus	NCT04382547
11	Not yet recruiting	MSC	40	2	University Hospital Tuebingen, Tuebingen, Germany	NCT04377334
12	Not yet recruiting	HB-adMSC	100	2	River Oaks Hospital and Clinics, Houston, Texas, United States	NCT04362189
13	Recruiting	Umbilical Cord Mesenchymal Stem Cells	24	1,2	Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, Florida, United States	NCT04355728
15	Enrolling by invitation	HB-adMSCs	100	2	Hope Biosciences Stem Cell Research Foundation, Sugar Land, Texas, United States	NCT04348435
16	Not yet recruiting	BMMSCs	20	1,2	Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China	NCT04346368
17	Not yet recruiting	Dental pulp mesenchymal stem cells	24	Early phase 1	Not defined	NCT04302519
18	Not yet recruiting	UC-MSCs	48	Not applicable	Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China	NCT04273646
19	Recruiting	UC-MSCs	10	2	Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China	NCT04269525
20	Recruiting	Wharton's Jelly-MSCs	5	1	Stem Cells Arabia Amman, Jordan	NCT04313322
21	Recruiting	MSCs	20	1	Beijing 302 Military Hospital of China Beijing, China	NCT04252118
22	Recruiting	UC-MSCs	30	1,2	Puren Hospital Affiliated to Wuhan University of Science and Technology Wuhan, Hubei, China	NCT04339660
22	Recruiting	Wharton's Jelly-MSCs	30	1,2	Hospital del Mar Recruiting Barcelona, Spain, 08003	NCT04390139
23	Recruiting	MSCs	300	3	United States	NCT04371393
24	Recruiting	MSCs	20	1,2	CHU de Liège Liège, Belgium	NCT04445454
25	Recruiting	Human umbilical cord derived CD362 enriched MSCs	75	1,2	Belfast Health and Social Care Trust, Royal Hospitals Belfast, Northern Ireland, United Kingdom	NCT03042143
26	Recruiting	MSC	10	2	Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán Mexico City, Mexico	NCT04416139
27	Recruiting	MSC	24	2	Hospital Universitario Rio Hortega Valladolid, Spain	NCT04361942
28	Not yet recruiting	Autologous adipose-derived stem cells	200	2	Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Punjab, Pakistan	NCT04428801
29	Recruiting	Intravenous Infusions of Stem Cells	20	2	Jinnah Hospital, Lahore, Punjab, Pakistan	NCT04437823
30	Active, not recruiting	COVID-19 Specific T Cell derived exosomes (CSTC Exo)	60	1	GENKOK, Kayseri, Melikgazi, Turkey	NCT04389385
31	Recruiting	CYNK-001	86	1,2	Hackensack University Medical Center, Hackensack, New Jersey, United States Atlantic Health, Morristown, New Jersey, United States Atlantic Health, Summit, New Jersey, United States Multicare Health System, Tacoma, Washington, United States	NCT04365101
32	Recruiting	NK cell	30	1	The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, China	NCT04280224
33	Not yet recruiting	SCE-Treated Mononuclear Cells Apheresis	20	2	Not defined	NCT04299152
34	Not yet recruiting	MSCs-derived exosomes	30	1	Not defined	NCT04276987

MSC: Mesenchymal Stem Cell, SCE: Stem Cell Educator

5.4. Anticoagulation therapies

Coagulopathy, characterized by high D-dimer levels, increased fibrinogen along with low anti thrombin, is one of the hallmarks associated with patient death due to COVID-19. “Fibrinolytic therapy”, using tissue plasminogen activator (t-PA) could improve survival in animal. models and patients suffering from ARDS. A study by Wand et al. demonstrated that administration of Alteplase (t-PA) was able to improve P/F ratio (the arterial pO2 divided by the fraction of inspired oxygen, expressed as a decimal that the patient is receiving) in COVID-19 patients. However, since the improvement was transient they suggested that re-dosing the anti-fibrotic drug might result in more durable response (132). Disseminated intravascular coagulation (DIC) caused by endothelial dysfunction due to excessive production of thrombin as well as decreased fibrinolysis, is also responsible for COVID-19 lethality. This suggests that anticoagulant agents (e.g. heparin) may be considered as potential anti-COVID-19 candidates. Supporting this idea, Tang et al. showed that anticoagulation therapy using heparin, is presumably associated with a better prognosis in patients infected with COVID-19 (133). In addition, due to some critical features of unfractionated heparin (UFH), also called nebulized heparin, including anti-coagulant, anti-inflammatory and mucolytic effects, UFH may be a potentially effective treatment for COVID-19 (134).

6. Conclusion

The global challenge of the COVID-19 pandemic that is associated with an enormous morbidity and mortality highlights the urgent need for developing efficient therapeutic strategies. Tremendous advances in understanding the molecular basis of the disease pathogenesis in various corona virus-based diseases, and the very fast insights gained in COVID-19 pathogenesis offer opportunities to take a leap in introducing novel and efficient therapeutics as well as preventive measures against COVID-19. Assessing the efficiency of previously approved antiviral agents for inhibiting SARS-CoV-2 is one of the main areas that is being pursued clinically, in the short term. Despite the encouraging results with some of these agents, more research should be done regarding the safety and efficiency, as exemplified by the initial adoption and later withdrawal of the use of chloroquine as a therapy for COVID-19. Different novel therapeutic strategies including cell and gene-based therapies against SARS-CoV-2 are now being developed, of which some are already being tested in early phase clinical trials worldwide. Dysregulation of inflammatory responses, a manifestation of individuals infected with SARS-CoV-2, can be modulated using MSCs. Likewise, SCE cells are another cell based strategy, which will be assessed for its efficiency in relieving SARS- related inflammation. Yet an alternative cell-based strategy is NK cell therapy to inhibit the viral replication. Another approach is based on nucleic acid-based therapies and includes CRISPR methods along with DNA vaccines and mRNA molecules, which are also being intensively studied and some have shown encouraging effects in preclinical trails. Overall, all aforementioned studies inspire further investigation of advanced therapeutic strategies as novel treatment options for COVID-19.

Declaration of Competing Interest

There is nothing to declare

Acknowledgment

The authors would like to express their gratitude to Royan institute and the Katholieke Universiteit Leuven for their support throughout the course of this work.

Funding

The authors received no financial support for authorship, and/or publication of this article.

Ethical Approval

Not required

References

L van der Hoek, K Pyrc, MF Jebbink, W Vermeulen-Oost, RJM Berkhout, KC Wolthers, et al.Identification of a new human coronavirus

Nature Medicine, 10 (4) (2004), pp. 368-373

View Record in Scopus Google Scholar

CI Paules, HD Marston, AS FauciCoronavirus Infections—More Than Just the Common Cold

JAMA, 323 (8) (2020), pp. 707-708

CrossRef View Record in Scopus Google Scholar

N Zhu, D Zhang, W Wang, X Li, B Yang, J Song, et al.A Novel Coronavirus from Patients with Pneumonia in China, 2019

New England Journal of Medicine., 382 (8) (2020), pp. 727-733

CrossRef View Record in Scopus Google Scholar

Y Chen, Q Liu, D GuoEmerging coronaviruses: Genome structure, replication, and pathogenesis

Journal of Medical Virology, 92 (4) (2020), pp. 418-423

CrossRef View Record in Scopus Google Scholar

S Kang, W Peng, Y Zhu, S Lu, M Zhou, W Lin, et al.Recent progress in understanding 2019 novel coronavirus (SARS-CoV-2) associated with human respiratory disease: detection, mechanisms and treatment

Int J Antimicrob Agents (2020), Article 105950

Article Download PDF Google Scholar

X Ou, Y Liu, X Lei, P Li, D Mi, L Ren, et al.Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Nature Communications, 11 (1) (2020), p. 1620