EV-Based Therapeutics against SARS-CoV-2: History
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Tamanna Mustajab
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Moriasi Sheba Kwamboka
,
Da Ae Choi
,
Dae Wook Kang
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Junho Kim
,
Kyu Ri Han
,
Yujin Han
,
Sorim Lee
,
Dajung Song
,
Yong-Joon Chwae

Extracellular vesicles (EVs) , comprising a variety of nano-scale vesicles ranging from 50 to 1000 nm in size, are released from all types of cells carrying a variety of lipids, proteins, and nucleic acids in a more protective manner than un-enveloped circulating biomolecules such as antibodies and cytokines from cellular DNases, RNases, proteases, and other degrading materials, due to the presence of the lipid bilayer membrane. EVs contribute to the dissemination and persistence of genetic material and proteins of SARS-CoV-2 due to the similarity in the entrance, budding, and mechanisms of biogenesis during infection. 

  • extracellular vesicles
  • COVID-19
  • SARS-CoV-2

1. Extracellular Vesicles Tagged with Receptor Binding Domain (RBD)

The very first target of the SARS-CoV-2 virus is type 2 alveolar epithelial cells of the lungs, where it binds to the highly expressing ACE2 receptors [1]. Although EVs have a great capacity as a natural carrier to deliver cargos to the recipient cells, a majority of externally injected EVs are absorbed by the liver, spleen, and pancreas instead of being taken up by the specific target. Therefore, for the satisfactory localization of specifically targeted therapeutic particles, it is suitable to tag EVs with tissue-specific peptides or antibodies targeting specific antigens [2].
In previous studies, vesicular stomatitis virus-G protein (VSVG) has been used as a fusion backbone with a variety of reporter proteins that include luciferase, green fluorescent protein (GFP), and red fluorescent protein (RFP) for the tracking and detection of EVs [3]. Recently, VSVG has been engineered to be fused with RBD of SARS-CoV-2 virus protein, the key domain in virus attachment and entry, by replacing the ectodomain of VSVG with RBD, resulting in the production of pseudoviral particles expressing RBD–VSVG fusion protein, without changing the physical properties of the modified EVs [4]. This is because it has been found that EVs and VSVs have similar lipid envelope compositions resulting from shared intracellular trafficking [5].
The association between the RBD of SARS-CoV-2 and the ACE2 of the host has been thought to be the most crucial to be targeted for vaccine development [6], antibody neutralization, and small-molecule inhibitors [7], which block the entry of virus into the cell. The host cell receptor ACE2 is present in most cell types, including the heart, intestine, kidneys, and lungs, among which the lungs show the highest expression of ACE2s and are proven to have a stronger affinity for SARS-CoV-2 than other organs [1][8][9][10]. Similarly, it has been found that the cellular presence of ACE2 receptors was needed for the entry of RBD-tagged EVs, which is why they were successfully targeted to the lungs and other tissues expressing ACE2 and reduced the infection of SARS-CoV-2 by delivering siRNA incorporated into RBD-tagged EVs, which was conducted in a transgenic hACE2 mouse model. This study suggested that SARS-CoV-2 infection could be efficiently treated by the delivery of antiviral drugs and that RBD-tagged EVs could be a potential therapeutic approach for other diseases [4].

2. EVs Expressing Tetraspanins Fusion

Tetraspanins, CD9, CD63, CD81, CD82, and CD151, are transmembrane proteins concentrated in the plasma membrane lipid microdomain and largely expressed in the exosomes, which makes them exosomal markers. They have also been known to contribute to the biogenesis and cargo sorting of exosomes [11]. In the field of COVID-19 therapeutics, engineered EVs expressing a novel fusion of a tetraspanin, i.e., CD63 with anti-SARS-CoV-2 nanobody, inhibited the binding of SARS-CoV-2 to ACE2, thereby neutralizing the SARS-CoV-2 infection [12]. Similarly, EVs expressing soluble ACE2 on their surface by the fusion of truncated scaffold of CD9 serve as decoy receptors for SARS-CoV-2 and block infection by SARS-CoV-2 variants including D614G, δ, and β, as well as the wild type [13]. Consistent with the current data, EVs expressing ACE2 inhibited the infection of pseudotyped lentiviruses from variants of concern of SARS-CoV-2, which was even more enhanced along with TMPRSS2 [14].
In addition to engineered EVs expressing specific proteins for hindering the cellular entrance of SARS-CoV-2, EVs produced from cell lines such as Vero CCL-81 and Vero E6 infected with SARS-CoV-2 can display surface proteins of SARS-CoV-2 that can recognize alveolar macrophages. Therefore, these EVs can be used as vehicles encapsulating drug delivery platforms [15][16].

3. ACE2 Loading onto EVs to Block Virus Entry

Because of the quickly evolving variants of SARS-CoV-2, COVID-19 remains a matter of concern and a major challenge despite tremendous advances in vaccine development and novel therapeutics [17][18][19]. Therefore, it is crucial to develop a strategy to cope with newly emerging variants of SARS-CoV-2. In this critical situation, EVs expressing ACE2 (evACE2) could be an alternative strategy. Supporting these data, small EVs, as well as non-membranous extracellular nanoparticles, exomeres, are reported to express ACE2 on their surface which subsequently inhibit SARS-CoV-2 infection, acting as a decoy by binding to the virus [20].
Moreover, the loading of ACE2 onto EVs is highly dependent on ACE2 palmitoylation at two major sites, i.e., Cys141 and Cys498, called S-palmitoylation. EVs secretion of ACE2 from the plasma membrane is increased by palmitoylation through the zinc finger DHHC-Type palmitoyltransferase 3 (ZDHHC3) or decreased by de-palmitoylation through acyl protein thioesterase 1 (LYPLA1). During viral infection, ACE2 present on the membrane is secreted along with EVs, and it blocks infection with SARS-CoV-2 by binding to the RBD domain, preventing the binding of SARS-CoV-2 with cellular ACE2. Therefore, engineered EVs enriched with ACE2 on their surface by palmitoylation were bound efficiently to RBDs and subsequently neutralized both pseudo-typed and wild-type SARS-CoV-2 in human ACE2 transgenic mice [21].

4. CD24-Loaded EVs

CD24, expressed in a variety of cells, is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein and is localized in lipid microdomain [22][23]; its interaction with Siglecs, which are mostly expressed by cells of the immune system, has been shown to transduce inhibitory signals on Siglec-expressing inflammatory cells [24][25][26]. Therefore, CD24 fused with Fc fragment of Ig (CD24-Fc) was tested to evaluate its effect in reducing over-activated inflammation in COVID-19, which is similar to human immunodeficiency virus type-1/simian immunodeficiency virus infection, where it provides protection to Chinese rhesus macaques (ChRMs) against disease progression [27][28]. Intriguingly, CD24 is also highly expressed in EVs, possibly due to its localization in lipid microdomain and GPI anchorage, prompting the development of CD24-expressing EVs as a novel therapeutic. Consequently, exosomes derived from CD24-overexpressing 293T cells, T-Rex™ (EXO-CD24), are developed for the treatment of COVID-19 cytokine storms with the format of inhalation. The rationale of this new drug is that exosomes expressing CD24 can attenuate the cytokine storm via activating anti-inflammatory immune cells through CD24 signaling by over-activating innate immune responses of SARS-CoV-2 patients [29].

5. EVs from Convalescents

Exosomes from the plasma of the convalescent phase of COVID-19 patients were recently reported to be equipped with all the components, including viral proteins, peptides, and RNAs for successful adaptive immune responses to SARS-CoV-2 [30].
The release of EVs expressing ACE2 (evACE2) was markedly increased in the convalescent serums of severely infected COVID-19 patients. They inhibited infections of SARS-CoV-2 including α, β, and δ strains with 135 times higher efficiency than recombinant human ACE2 (rhACE2), by competing with cellular ACE2 in the binding with RBD of SARS-CoV-2. In vivo, the ACE2-transgenic mouse model evACE2 was 60 to 80 times more potent than rhACE in inhibiting infection from both original and pseudotyped viruses and also protected mice from SARS-CoV-2-induced lung injury [31].
Following on this finding, virus-specific T cells should have similar capabilities with the plasma exosomes, and they are currently in clinical trials. COVID-19-specific T-cell-derived exosomes (CSTC-Exo) were purified from T cells of convalescent COVID-19 patients and expanded in vitro by stimulation with SARS-CoV-2-specific peptides and cytokines. An inhalable format of CSTC-Exo is now in clinical trials in COVID-19 patients with early pneumonia. Action mechanisms of CSTC-Exo have suggested that cytokines, including IFN-γ within the exosomes prepared from activated SARS-CoV-2-specific T cells, have anti-viral effects on COVID-19 patients [32].
Convalescent plasma infusion has been successfully tried in severe COVID-19 patients for treatment [33][34] and is now considered a possible treatment choice in severe COVID-19 patients not responding to existing therapies [35]. Among the components of convalescent plasma, it has been proposed that EVs from platelets are the major constituents responsible for the regeneration of damaged tissues [36]. Therefore, platelet-derived EVs could be used as a COVID-19 therapeutic. Supporting this notion, platelet-derived exosomes loaded with an anti-inflammatory drug, [5-(p-fluorophenyl)-2-ureido] thiophene-3-carboxamide (TPCA-1), have been shown to alleviate cytokine storm in pneumonia in a mice model [37].

This entry is adapted from the peer-reviewed paper 10.3390/ijms231911247

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