key: cord-0707006-2zyla3k1
authors: Ramezankhani, Roya; Solhi, Roya; Memarnejadian, Arash; Nami, Fatemeharefeh; Hashemian, Seyed MohammadReza; Tricot, Tine; Vosough, Massoud; Verfaillie, Catherine
title: Therapeutic Modalities and Novel Approaches in Regenerative Medicine for COVID-19
date: 2020-10-23
journal: Int J Antimicrob Agents
DOI: 10.1016/j.ijantimicag.2020.106208
sha: 36d687cffc036185a2366f3df60da14dc808a82f
doc_id: 707006
cord_uid: 2zyla3k1

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.

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, (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) . 

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.

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).

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 Tcells (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-angiotensinaldosterone 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.

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).

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).

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) . Interaction of APCs with lymphocytes and cytokine activation (Created by Biorender.com).

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 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 antimalarial 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).

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).

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.

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. 

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 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) . 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). 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) .

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) . (Table   1 ) (131).

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).

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 virusbased 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.

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

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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.

Competing Interests: There is nothing to declare Ethical Approval: Not required