key: cord-0919922-hrj17lxv
authors: Gasparello, Jessica; Finotti, Alessia; Gambari, Roberto
title: Tackling the COVID-19 “cytokine storm” with microRNA mimics directly targeting the 3’UTR of pro-inflammatory mRNAs
date: 2020-11-25
journal: Med Hypotheses
DOI: 10.1016/j.mehy.2020.110415
sha: fb1e84376a1373d61ecedb9e6bb27757e045711e
doc_id: 919922
cord_uid: hrj17lxv

COVID-19 is characterized by two major clinical phases, the SARS-CoV-2 infection of target cells and tissues, and a deep inflammatory state, known as “cytokine storm”, caused by activation of pro-inflammatory genes, such as NF-kB, STAT-3, IL-6, IL-8, IL-1ß. Among possible anti-inflammatory agents, the “microRNA targeting” should be carefully considered, since it is well known that microRNAs are deeply involved in the expression of cytokines, chemokines and growth factors. The working general hypothesis is that targeting of the microRNA network might be important for the development of therapeutic approaches to counteract the COVID-19 induction of inflammatory response. This hypothesis is based on several publications demonstrating the use of miRNA mimics for inhibitory functions on the production of proteins characterizing the COVID-19 “cytokine storm”.

COVID-19 is characterized by two major clinical phases [1] . The first is the SARS-CoV-2 infection of target cells and tissues, leading to important clinical manifestations and complication, such as pulmonary failure [1] . The second phase is a deep inflammatory state, known as "cytokine storm", caused by activation of pro-inflammatory genes, such as NF-kB, STAT-3, IL-6, IL-8, G-CSF [2] [3] [4] [5] [6] [7] [8] [9] . In COVID-19 cytokine storm, this perturbation is initiated via attachment of the SARS-CoV-2 spike protein to its receptor, ACE2, followed by the ACE/Ang II/AT1R axis activation leading to hyperactivation of NF-κB by IL-6 STATs axis [3] .

Although SARS-CoV-2 itself activates NF-κB through pattern recognition receptors, it is the simultaneous activation of NF-κB and STAT3 that enhances NF-κB activation machinery (the IL-6 amplifier) [4] . This hyper-activation of NF-κB via the IL-6 Amp in the lungs induces a cytokine storm with subsequent ARDS (Acute Respiratory Distress Syndrome) that has been observed in severe COVID-19 patients [6, 7] . In fact, several studies found a clear relationship between the hyper-inflammatory state and the severity of the disease [6] [7] [8] [9] . The pharmacological approach for treating ARDS needs of novel anti-inflammatory reagents as different COVID-19 patients might respond differently to these treatments [10] [11] [12] [13] [14] [15] [16] [17] . Table 1 shows a partial list of studies outlining the induction of cytokines, chemokines and growth factors in COVID-19 [8, [18] [19] [20] [21] [22] [23] [24] [25] . As clearly evident, IL-1β, IL-6 and IL-8 are found up-regulated in most studies. Interestingly, upregulation of these proteins following SARS-CoV-2 infection is associated with poor outcome of the COVID-19 patients [7, 8] . Finally, the therapeutic importance of these molecules is supported by the ongoing clinical trials based on their targeting, such as NCT04381052 (based on the IL-6 inhibitor Clazakizumab), NCT04247226 (based on the IL-8 neutralizing agent BMS-986253) and NCT04603742 (based on Anakinra, a recombinant IL-1 receptor antagonist, inhibiting both IL-1-alpha and IL-1-beta).

Among possible anti-inflammatory agents, the "microRNA targeting" should be carefully considered [26] , since it is well known that microRNAs are deeply involved in the expression of cytokines, chemokines and growth factors [27] .

MicroRNAs are from 19 to 25 nucleotides noncoding RNAs that regulate gene expression by targeting mRNAs, leading to a translational repression or mRNA degradation [28, 29] . Since their discovery the number of microRNA sequences deposited in the miRBase databases is significantly growing [30] . The complex networks constituted by miRNAs and RNAs lead to the control of highly regulated biological functions, such as differentiation, cell cycle and apoptosis [31] . Some miRNA-binding sites (which are boxed) are in common might participate to a network responsible to a co-regulation of these genes in COVID-19. The list of the miRNA binding sites that are present in the 3'UTR of IL-1β, IL-6 and IL-8 mRNA is enlisted in Table 2 . The data are derived from Chou et al. [32] and Huang et al. [33] .

The working general hypothesis is that targeting of the microRNA network might be reported that polyethylenimine (PEI) was successfully employed to deliver plasmid DNA containing miR-200c into target cells, and that this procedure caused an increased expression of miR-200c associated with effective inhibition of IL-6, IL-8, and CCL-5 [36] .

They reported that miR-200c directly targets the 3'UTR of IL-6, IL-8 and CCL-5. 

This is a key step in designing a meaningful approach to negatively control the COVID-19 associated "cytokine storms". If fact, several microRNA target sites are present in the 3'UTR of cytokine/chemokine mRNAs. An in silico analysis based on the analysis of molecular interactions between microRNAs and target sites present in the 3'UTR of proinflammatory mRNAs might be of great help for understanding the theoretical stability and possible importance of these interaction and for designing potential bioactive sequences. This is shown in Fig. 3 (boxed area) . The sequence of the agomiR might be designed to display an even increased affinity to the target mRNA in respect to the original miRNA sequence. The miRNA mimicking approach (outlined in Fig. 2 ) might help in verifying which miRNA should be mimicked in order to decrease mRNA/protein expression.

In order to validate the hypothesis cytokines/chemokines/growth factors should be analyses using well known biochemical approaches (for instance those based on ELISA and

Bio-plex assays). One example has been reported in several studies and it is based on the use of a 27-plex analyzing, among others, most of the COVID-19 associated cyto/chemokines, such as IL-6, IL-8, IP-10, G-CSF and TNF-alpha. The experimental approach that might be employed is described in Fig. 3 as well as the use of novel delivery reagents, such argininocalix [4] arene macrocycles as reported by our research group for miRNA delivery [41] .

The expected outcomes of the approach described in this paper are the following.

 Development of a protocol based on the exposure of target cells to the SARS-CoV-2 spike protein, in order to induce high expression levels of genes involved in the COVID-19 "cytokine storm".

 Protocols for the alteration of the inflammosome using miRNA targeting and/or mimicking.

 Protocols for combined treatments using pre-miRNA molecules in combination with anti-inflammatory drugs already used in COVID-19 therapeutic protocols.

The proposed approaches might lead to the development of protocols for the reduction of the expression of key components of the COVID-19 "cytokine storm" [1] [2] [3] [4] [5] . This is a major issue in the management of COVID-19 patients [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . As far other DNA-and RNA-based therapeutic interventions [42] suitable delivery systems should be considered for optimizing the proposed treatment.

The authors declare that they have no competing interests. The level of IL-6 in peripheral blood is an early indicator of cytokine release syndrome in COVID-19-associated pneumonia. IL-2R, IL-6, IL-10, TNF-α were markedly higher in severe cases than in moderate cases. Of note, IL-6 levels were increased in both moderate and severe cases.

McElvaney et al., 2020 [24] The listed ILs levels were detected in healthy volunteers, hospitalized but stable patients with COVID-19 (COVID stable patients), patients with COVID-19 requiring ICU admission (COVIDICU patients). IL-1b, IL-6, IL-8, and sTNFR1 were all increased in patients with COVID-19. COVIDICU patients could be clearly differentiated from COVID stable patients, and demonstrated higher levels of IL-1b, IL-6, and sTNFR1 but lower IL-10.

TNF-α, IL-6, IL-10 Serum Diao et al., 2020 [25] Levels of TNF-α, IL-6, and IL-10 were significantly increased in infected patients, and their levels in ICU patients were significantly higher than in non-ICU patients 

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G-CSF and IL-8 but not GM-CSF correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome

An inflammatory cytokine signature helps predict COVID-19 severity and death

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miR-20a-5p, let-7b-5p, miR-16-5p, miR-376a-5p, miR-335-5p, miR-98-5p, miR-124-3p, miR-1-3p, miR-34a-5p, miR-98-5p, miR-99a-5p, miR-191-5p, miR-128-3p, miR-138-5p, miR-182-5p, miR-195-5p, miR-203a-3p, miR-205-5p, miR-21-3p, miR-21-5p, miR-221-3p, miR-27a-3p, miR-27a-5p

miR-23a-3p, miR-23b-3p, miR-296-3p, miR-302c-5p, miR-302d-5p, miR-450a-5p, miR-493-5p, miR-499a-3p, miR-519d-3p, miR-520a-3p, miR-526b-3p, miR-5582-3p, miR-587, miR-664a-3p, miR-1-3p, miR-429, miR-34a-5p, miR-155-5p, let-7b-5p, miR-124-3p, miR-126-3p, miR-16-5p, miR-27a-3p, miR-335-5p, miR-1291, miR-138-5p, miR-101-3p, miR-107, miR-129-2-3p, miR-130a-3p, miR-146a-5p, miR-147a, miR-194-5p, miR-203a-3p, miR-21-3p, miR-21-5p, miR-210-3p, miR-212-3p, miR-214-3p, miR-221-3p, miR-29a-5p, miR-29a-3p, miR-30d-5p, miR-376a-5p