key: cord-0800836-28kqxyxz
authors: Scott, Tristan A.; Supramaniam, Aroon; Idris, Adi; Cardoso, Angelo A.; Shrivastava, Surya; Kelly, Gabrielle; Grepo, Nicole A.; Soemardy, Citradewi; Ray, Roslyn M.; McMillan, Nigel A.J.; Morris, Kevin V.
title: Engineered extracellular vesicles directed to the spike protein inhibit SARS-CoV-2
date: 2022-02-02
journal: Mol Ther Methods Clin Dev
DOI: 10.1016/j.omtm.2022.01.015
sha: b6fd1344d87121bdae5a69661f744d6b221b5701
doc_id: 800836
cord_uid: 28kqxyxz

SARS-CoV-2 (CoV-2) viral infection results in COVID-19 disease, which has caused significant morbidity and mortality worldwide. A vaccine is crucial to curtail the spread of SARS-CoV-2, while therapeutics will be required to treat ongoing and reemerging infections of SARS-CoV-2 and COVID-19 disease. There are currently no commercially available effective anti-viral therapies for COVID-19 urging the development of novel modalities. Here, we describe a molecular therapy specifically targeted to neutralize SARS-CoV-2, which consists of extracellular vesicles (EVs) containing a novel fusion tetraspanin protein, CD63, embedded with an anti-CoV-2 nanobody. These anti-CoV-2 enriched EVs binds SARS-CoV-2 spike protein at the receptor-binding domain (RBD) site and can functionally neutralize SARS-CoV-2. This work demonstrates an innovative EV targeting platform that can be employed to target and inhibit the early stages of SARS-CoV-2 infection.

Introduction 30 31 SARS-CoV-2 (CoV-2) has emerged on the world stage as a highly infectious agent that can spread 32 rapidly geographically, causing significant mortality globally. 1 While current vaccines have 33 proven useful at preventing severe infection; vaccine hesitancy, the rapid emergence of variants of 34 concerns (VOC) that could escape pre-exiting immunity, and the possibility of the endemic 35 establishment of SARS-CoV-2 suggests COVID-19 cases will continue to be observed into the 36 foreseeable future, which will require the development of novel approaches that directly target and 37 inhibit SARS-CoV-2 during infection. EUA. SARS-CoV-2 intracellular entry is mediated through the spike protein consisting of S1 and 44 infection. 3 This binding results in the reorganization of the S2 subunit into a fusogenic state, which 47 is further matured by transmembrane serine protease 2 (TMPRSS2). 3 Although other target sites 48 in the spike protein have been found to be important, the RBD is considered the major target for 49 antibody-mediated neutralization, 4 which blocks the interaction of this target site with ACE2 50 preventing infection, representing a site for targeted virus inactivation. 51 52 Extracellular vesicle (EV) is an umbrella term for a wide range of nano-size particles secreted from 53 cells comprising of, but not limited to, the broadly defined multivesicular body-derived exosomes 54 (30-150 nm), membrane-derived microvesicles (100-1000 nm), and apoptotic bodies. 5 These 55 particles have lipid bilayer membranes, a feature that allows these systems to be engineered to 56 present artificial targeting ligands on their surface or the incorporation of various payloads like 57 RNA, DNA and protein in the luminal compartment. 6 They are biodegradable, potentially 58 allogenic, biocompatible and safe. 7 Importantly, EVs are emerging as a therapeutic platform with 59 applications as anti-inflammatory, 8 anti-viral 9 , and anti-cancer agents. 10 60 61 Artificial proteins have been incorporated onto EVs to inhibit SARS-CoV-2 intracellular entry, 62 whereby a decoy hACE2 protein presented on the EV surface can inhibit the entry of spike 63 pseudotyped virions. 11 However, the impact of administrating EVs expressing ACE2, which is 64 known to function in the regulation of the cardiovascular system, is currently unknown, and 65 targeting the virus directly may prove more specific and therapeutically relevant. Furthermore, the 66 EV surface can be engineered with various overexpressed receptors. Not all receptors are 67 efficiently incorporated into EVs (e.g. the spike protein of SARS-CoV-2), 12 and therefore using 68 enriched scaffolds could be exploited for this purpose. EVs have a wide range of specifically 69 J o u r n a l P r e -p r o o f

To determine if the VHH72 on the surface of EVs was able to bind its cognate target, we performed 141 an enrichment assay where beads were coated with an anti-spike antibody to the S1 ectodomain 142 and then bound to the SARS-CoV-2 trimeric spike, which were subsequently incubated with 143 VHH72-CD63 EVs. An Nluc signal was observed with the VHH72-CD63 EVs with increasing 144 amounts of recombinant trimeric spike protein (Figure 2A , Supp. Figure 2C and 2D) . Notably, 145 an enrichment was not observed with the control EVs. To further verify that the modified EVs 146 were binding to the RBD, VHH72-CD63 EVs were bound to beads and then incubated with SARS-147

CoV-2 RBD before determining the levels of bound RBD by flow cytometry. A higher signal was 148 observed with VHH72-CD63 EVs demonstrating the EVs were binding the RBD ( Figure 2B) . 149

Furthermore, TEM imaging revealed that the surface of VHH72-CD63 EVs bound SARS-CoV-2 150 RBD when detected using gold-nanoparticle labeling, which often appeared as a cluster on the EV 151 surface (Figure 2C and Supp. Figure 2E) . We also observed an increased uptake of VHH72-152 CD63 EVs in HEK293 cells transfected with a vector expressing the spike protein ( Figure 2D and 153 Supp. Figure 2F) . Lastly, to determine mechanistically if the VHH72 containing EVs could block 154 an RBD interaction with the ACE2 protein, a ACE2 blocking assay was performed. The VHH72-155

EVs were incubated at increasing doses with the SARS-CoV-2 RBD and then added to a plate 156 coated with recombinant ACE2, and the amount of ACE2 bound to RBD was determined. A 157 significant reduction in signal was observed with increasing amounts of VHH72-EVs compared 158 to the control CD63 EVs ( Figure 2E ). Collectively, these data verify that VHH72-CD63 EVs were 159 able to bind the SARS-CoV-2 spike protein on the membrane surface and block its interaction with To assess if the VHH72-CD63 EVs were able to neutralize SARS-CoV-2 spike, we utilized a 165 pseudotyped lentiviral vector assay. We first optimized cellular and viral conditions to facilitate 166 spike infection of the target cells. Lentiviral particles 20 were packaged with a GFP-firefly 167 luciferase reporter (GFP-Fluc) and pseudotyped with a WT spike protein. These pseudotyped 168 lentiviral particles were able to transduce stable hACE2-HEK293 cells (Supp. Figure 3A and 169 4A), which was further increased in hACE2-hTMPRSS2-HEK293 cells in a dose-responsive 170 manner (Supp. Figure 3B and 4B). The virus was unable to transduce WT HEK293 cells. VERO-171 E6 have been described to be SARS-CoV-2 permissive, but we proceeded with the HEK293 cells 172 as the VERO-E6 cells showed lower transduction even with stable hACE2-hTMPRSS2 173 expression, possibly because of restriction factors in these cells (Supp. Figure 4C) . Furthermore, 174

we introduced the dominant D614G mutation present in circulating strains, which improves ACE2 175 binding, virus transmissibility and infection, 21-23 alone or in combination with a mutation in the 176 furin cleavage site, R682Q. 24 The highest infection levels observed was with the D614G-R682Q 177 combination (Supp. Figure 4D ). Finally, we tested a C-terminal truncated spike protein, which 178 has been shown to improve spike incorporation into lentiviral particles and virus transduction, but 179 only showed modest improvements in transduction and was not explored further (Supp. Figure  180 4E). Furthermore, the truncated spike had increased non-specific entry into HEK293 cells, which 181 may result from the truncation increasing the spikes fusogenic properties. 25 A combination assay 182 of the relevant individually optimized conditions is presented in Supp. Figure 4F . R682Q virus was dose dependent, which increased with a higher ratio of VHH72-CD63 EVs to 191 viral particles ( Figure 3B ). COVID-19 convalescent plasma (CPP) from patients recovered from 192 SARS-CoV-2 infection was able to reduce transduction by ~50% (Supp. Figure 5A) , which was 193 comparable to VHH72-CD63 EVs suggesting effective inactivation of the D614G-R682Q spike. 194

Of interest, the RBD-targeting S35 mAb was ineffective at neutralizing the D614G-R682Q 195 pseudotyped virion. 196 197 To assess if the VHH72-CD63 can inactivate spike proteins derived from SARS-CoV-2 VOC, the 198 EVs were incubated with lentivirus pseudotyped with the spike from Alpha (B.1.1.7), Beta 199 (B.1.351), Gamma (P.1), and Epsilon (B.1.429) variants. These preparations were then transduced 200 onto the hACE2-hTMPRSS2-HEK293 cells, and high levels of neutralization were observed with 201 the VHH72-CD63 EVs for all variants tested, compared to the CD63 control ( Figure 3C) . 202 Furthermore, the neutralization was comparable to a recombinant bivalent VHH72. A ten-fold 203 lower number of EVs reduced the neutralization of the pseudotyped lentiviral variants by the 204 VHH72-CD63, demonstrating a dosing effect (Supp. Figure 5B) . Importantly, the anti-RBD S35 205 mAb and CPP (P9K) were unable to effectively neutralize the spikes from the Beta and Gamma Effective neutralization with the ancestral strains was observed at >1E8 total particles, and so a 219 starting dose of 5E8 EV particles was used and effective neutralization of the VOC was observed. 220

However, higher doses of VHH72-CD63 EVs did not significantly improve inhibition. 221

Collectively, these data show that the VHH72-CD63 EVs can inhibit live SARS-CoV-2 VOC, 222 mediated through the inhibition of the spike protein. Here we describe a novel EV fusion receptor that can be used to redirect EVs to viral targets. 227

Specifically, we characterized its binding to SARS-CoV-2 spike protein and the inhibition of both 228 pseudotyped and authentic virus. There are a number of methods to attach ligands pre-and post-229 production to EVs, 26 but a feature of the lipidic bilayer membrane allows for transmembrane 230 protein incorporation, which can be leveraged to express artificial receptors during production, a 231 powerful tool for EV retargeting. The EV CD63 scaffold has been used functionally in various 232 ways: embedding inhibitory peptides to treat muscular dystrophy, 27 fusing an Apolipoprotein A-I 233 (Apo-A1) ligand to the C-terminus to direct EVs to liver cancer cells, 28 presenting antigens for 234 vaccine augmentation 29 , or real-time EV imaging through embedded reporters. 16 This work adds 235 to the growing versatility of tetraspanins by showing that antibody-derived fragments can be 236 embedded into CD63 for functional receptor targeting. Furthermore, the EC1 of CD63 has been 237 used for protein presentation, 16,27 which was further corroborated in this study ( Figure 1B enriched scaffolds. Furthermore, Lamp2b has been shown be proteolytically unstable requiring 255 additional optimization to produce stable versions, 13,31 which is not the case for the ubiquitous, 256 stable tetraspanins that are known to be enriched in EVs from various cell types, making 257 tetraspanins as an attractive scaffold for further development. 258

The TEM images presented here showed that not all EVs bound gold-labelled RBD and often the 260 fusion proteins were found in clusters on the EV surface ( Figure 2C and Supp. Figure 2D ). This 261 observation may reflect binding to VHH72-CD63 in tetraspanin enriched microdomains, with a 262 similar phenomenon observed with GPI scaffolds associated with lipid rafts. 32 Supporting this 263 notion are observations that the pHluorin-CD63 reporter was found in heterogeneous clusters on 264 EV particles, and mainly enriched in EVs from the smaller range, preferentially in exosomes. 16 265

Selective isolation methods will need to be explored to improve product purity of EVs modified 266 with artificial scaffolds to ensure optimal products for therapeutic applications. 267

Furthermore, the N6-CD63 scaffold, although functional, had significantly reduced levels of 268 expression (Supp. Figure 1C) . To a lesser extent, the VHH72 had reduced expression compared 269 to the CD63 control ( Figure 1F) , which highlights the need to empirically identify stable ligands, 270 a common process in developing targeted vesicles. As a result of the simplicity of ligands that have 271 single effector modules, such as nanobodies and Darpins 33 , these modalities may be attractive 272 ligands for further development into tetraspanin scaffolds. Although effective neutralization of the D614G-R682Q spike lentivirus was observed with the 278 VHH72-CD63 EVs, suggesting conservation of the target epitope ( Figure 3B) , neutralization 279 clearly affected the S35 mAb and CPP (Supp. Figure 5A ) that readily inhibited several 280 pseudotyped VOC ( Figure 3D) . These data suggest possible structural changes in the spike 281 because of the furin mutation which impacts antibody binding, advising caution when using furin-282 mutated spikes for neutralization studies. 283

The emergence of VOC has caused great concern as these can escape from vaccinated and COVID-285 19 patient serum 37 as well as from therapeutic monoclonal antibodies, 38 resulting in the need for 286 antibody cocktails to prevent escape. 39 Alternatively, targeting conserved regions in 287 betacoronavirus spike could counter this issue. The VHH72 targets a region adjacent to the ACE2 288 binding site, a conserved site, that can cross neutralize SARS-COV-1 and 2, and bind spike protein 289 from a bat coronavirus, WIV1-CoV. 40 The data presented here demonstrates that the VHH72-290 CD63 EVs can neutralize a range of VOC ( Figure 3C and 3E, Figure 4) . Interestingly, anti-viral 291 activity with recombinant VHH72 has been shown to improve against the N501K mutation 41 as 292 this mutation reestablished a positively charged amino acid present at the equivalent position in 293 SARS-CoV-1, 40 suggesting that VHH72-CD63 EVs may even have improved activity against 294 some variants. 295

EVs represent a multi-functional system that could be adapted to inhibit the virus through 297 mechanisms other than neutralization. EVs are able to transfer incorporated therapeutic payloads 298 and loaded with chemotherapeutic agents have been shown to improve drug potency to cancer 299 cells, 10 deliver RNA interference effectors, 42 or gene-regulatory proteins 43 . Spike-targeted EVs 300 could be adapted to deliver anti-CoV-2 payloads to infected tissues in combination with 301 neutralizing effects. Furthermore, inflammation is a crucial factor in severe COVID-19 and stem- with the mutations were used to amplify the sequence by PCR. For the D614G vector, two 321 fragments were amplified with one containing the D614G mutation (primers: Spike-F + D614G-322 R) and a second WT sequence fragment (primers: D614G-F and Spike-R). To generate the double 323 mutation D614G-R682Q, an amplicon was generated with the R682Q mutation (primers: R682Q-324 F + Spike-R) and a second amplicon (primers: R614G-F + R682Q-R), which were both diluted down 325 1:100 in sterile water and mixed at a 1:1 ratio then amplified in a fusion PCR (primers R614G-F + 326 spike-R) to generate a single fragment with the R682Q mutation. The WT spike vector was 327 digested with Bsu36I and BsrGI and the D614G fragment was mixed either with the WT fragment 328 or the R682Q fragment and cloned into the digested vector. The truncated spike vector was 329 generated by PCR amplification of the spike sequence (Spike-F2 and spike-R2) and cloned into a 330

EcoRI and NotI digested pCAGGS vector. were washed in PBS, spun at 3000 x g for 10 min and then incubated with an anti-hCD63 antibody 415 in PBS for 1 hr at 4°C and then centrifuged at 3000 x g for 10 min and blocked in 1xPBS with 0.2% 416 BSA for 30 min at 4°C. The sample was then centrifuged at 3000 x g for 10 min and the VHH72-417 CD63 or control CD63 EVs were incubated with the sample in PBS for 1 hr at 4°C. The beads were 418 centrifuged again at 3000 x g for 10 min, and then incubated with 5 µg/ml SARS-CoV-2 RBD (Cat. No. 78205-1), and Delta variants (B.1.617.2, Cat. No. 78215-1). The described amount of EV 506 particles were incubated with 1 µl of pseudotyped lentiviral vector (Alpha, Beta, Gamma=1.2 E5 507 TU/ml; Epsilon=1.5E5 TU/ml; Kappa=7E5 TU/ml; Delta 3E5 TU/ml), and incubated at 37°C for 30 508 min, and then added to HEK293-hACE2-hTMPRSS2 cells. The recombinant VHH72 bivalent 509 nanobody was included as a positive control. Virus only controls were incubated in PBS. The S35 510 (Cat. No. SAD-S35, Acrobiosystems, DE USA) was added to the pseudotyped virus at the described 511 final concentrations. The Covid-19 convalescent plasma (CPP) was obtained from individuals 512 previously exposed to SARS-CoV-2 (P9K and LE4) and incubated with the pseudotyped virus at 513 the described dilutions. Plasma from a COVID-19 convalescent patient (P9KMAW8T) was used as 514 reference. This plasma was collected under IRB-20204 (PI: Dr. John A. Zaia), with appropriate 515 informed consent, upon confirmation of COVID-19 diagnosis. This plasma was collected at 94 516 days post-diagnosis and showed high titer of anti-CoV-2 IgG in a serological assay and potent 517 RBD-neutralizing activity (>97% inhibition) in the sVNT assay as described above (data not 518 shown). The cell final volume of media on the cells was 50 µl when the virus:EV mixture was 519 added. At 48 hrs, the media was removed and the Bright-Glo™ Luciferase Assay System was used 520 to assess luciferase activity (Promega, WI, USA). The luciferase activity was measured on a 521

GloMax® Explorer Multimode Microplate Reader (Promega, WI, USA). 522

To perform the SARS-CoV-2 neutralization assay, the ancestral (VIC1) strain 49 , Beta variant 523 (VIC18383), Kappa variant (VIC18447) and Delta variant (VIC18440) of SARS-CoV-2 were cultured 524 in Vero E6 cells and originally obtained from the Peter Doherty Institute for Infection and 525

Immunity and Melbourne Health, Victoria, Australia. Viral titer was determined by the viral 526 immunoplaque assay as previously described 50 . EVs or the mAb anti-SARS-CoV-2 RBD antibodies 527 (Clone# CB6 and 5309) were incubated with 250 PFUs of SARS-CoV-2 at the described 528 concentrations for 30 min at room temperature before infecting Vero E6 cells for 1 hr at 37°C. 529

The virus was then removed, and the wells layered with MC agar. The numbers of plaques were 530 assessed 4 days after infection. 

Exosomal vaccines containing the 600 S protein of the SARS coronavirus induce high levels of neutralizing antibodies

A versatile platform for 604 generating engineered extracellular vesicles with defined therapeutic properties

Analysis of ESCRT functions in exosome 608 biogenesis, composition and secretion highlights the heterogeneity of extracellular 609 vesicles

Identification of a CD4-Binding-Site Antibody 612 to HIV that Evolved Near-Pan Neutralization Breadth

A live cell reporter of exosome secretion and 616 uptake reveals pathfinding behavior of migrating cells

Structural 620 Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid 621

Quantitative proteomics identifies the core proteome of exosomes 624 with syntenin-1 as the highest abundant protein and a putative universal biomarker

Engineered 627 extracellular vesicles as versatile ribonucleoprotein delivery vehicles for efficient and safe 628 CRISPR genome editing

Comprehensive toxicity and immunogenicity 632 studies reveal minimal effects in mice following sustained dosing of extracellular vesicles 633 derived from HEK293T cells

SARS-CoV-2 D614G variant exhibits 637 efficient replication ex vivo and transmission in vivo

SARS-CoV-2 spike D614G change 641 enhances replication and transmission

The Spike D614G mutation increases SARS-CoV-2 infection of multiple human 645 cell types

Optimized Pseudotyping Conditions for the SARS-648 COV-2 Spike Glycoprotein

Genetic analysis of the SARS-coronavirus spike glycoprotein 651 functional domains involved in cell-surface expression and cell-to-cell fusion

Engineering exosomes for targeted drug 654 delivery

Effects of exosome-656 mediated delivery of myostatin propeptide on functional recovery of mdx mice

Engineered exosome-659 mediated delivery of functionally active miR-26a and its enhanced suppression effect in 660 HepG2 cells

Extracellular Vesicles via DNA Vaccination Results in Robust CD8(+) T Cell Responses

Sequential deletion of CD63 identifies topologically distinct scaffolds for surface 667 engineering of exosomes in living human cells

Stabilization of Exosome-targeting Peptides via 670 Engineered Glycosylation*

Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles 674 promotes tumour cell targeting

Surface-Engineered Lentiviral Vectors for Selective 676

Optimized Pseudotyping Conditions for 680 the SARS-COV-2 Spike Glycoprotein

SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are 683 generated in vitro during propagation in TMPRSS2-deficient cells

Loss of furin cleavage site 687 attenuates SARS-CoV-2 pathogenesis

Evidence of 691 escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera

CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization

Antibody cocktail to SARS-CoV-2 spike protein 699 prevents rapid mutational escape seen with individual antibodies

Structural Basis 703 for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies

Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-708 mediated immunity

Delivery 711 of siRNA to the mouse brain by systemic injection of targeted exosomes

Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis 715 transmembrane conductance regulator

Migration of human neural 718 stem cells toward an intracranial glioma

Designer exosomes produced by implanted cells 722 intracerebrally deliver therapeutic cargo for Parkinson's disease treatment

HIV-1 ENVELOPE. Effect of the cytoplasmic domain on antigenic 726 characteristics of HIV-1 envelope glycoprotein

Conditionally Replicating Vectors Mobilize Chimeric Antigen 730

Receptors against HIV

Antibody-mediated 734 immunotherapy of macaques chronically infected with SHIV suppresses viraemia

Isolation and rapid sharing of the 738 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with

An 742 Optimized High-Throughput Immuno-Plaque Assay for SARS-CoV-2

EC1 and EC2 denote the 750 two main loops of CD63 and cystine disulphide bonds are highlighted. An Nluc was fused in-751 frame to the C-terminus. (B) The N6-CD63 EVs were bound to beads and then incubated with 752 GP120 and binding was assessed by flow cytometry

Errors bars represent standard deviation generated from samples treated in triplicate. The p-values 754 were generated using a one-way ANOVA

EVs and the levels of Nluc 756 were assessed at 18 hrs post-addition. The Nluc levels were normalized to HEK293 WT cells and 757 made relative to the CD63 control set at 100%. Errors bars represent standard deviation generated 758 from samples treated in triplicate. The p-values were generated using an unpaired student's t

EVs and cell lysates were assessed by western blot for known EV markers 761 (TSG101, ALIX, CD81) and the components of the CD63 fusion protein (Nluc and CD63)

GAPDH was included as a loading control. Ladder molecular weights are indicated

Figure 2: VHH72-CD63 EVs can bind SARS-CoV-2 spike. (A) Beads were coated with an anti-765

SARS-CoV-2 spike antibody and bound to increasing concentrations of recombinant trimeric spike 766 (0.1-10 µg/ml). The spike bound beads were incubated with VHH72-CD63 EVs and the levels of 767

Nluc were assessed, which were made relative to the beads without spike, set at 100%. Errors bars 768 represent standard deviation generated from samples treated in duplicate

EVs were bound to beads, incubated with SARS-CoV-2 RBD, and then flow cytometry was used 770 to assess binding. Errors bars represent standard deviation generated from samples

The p-values were generated using an unpaired student's t-test compared to RBD 772 negative samples (*p<0.05). (C) TEM analysis of the VHH72-CD63 and CD63 containing EVs 773 bound to gold-nanoparticle labelled SARS-CoV-2 RBD. Red arrows highlight bound gold 774 particles. Scale bar represents 200 nm. (D) HEK293 cells were transfected with a spike expressing 775 vector

The Nluc levels were normalized to untransfected HEK293 cells and made relative to the CD63 777 control. Errors bars represent standard deviation generated from samples treated in triplicate

p-values were generated using an unpaired student's t-test compared to the control (***p<0.005)

RBD fused to an HRP was pre-incubated with 2E9, 1E9, 5E8, and 1E8 total 780 particles of the CD63 or VHH72-CD63 EVs, and the amount of RBD bound to ACE2 was assessed 781 through a colorimetric assay. The % inhibition was calculated from samples treated in duplicate

A recombinant VHH72 was included as a positive control

VHH72-CD63 EVs broadly neutralize SARS-CoV-2 pseudovirus. (A) The

and then 786 transduced on HEK293-hACE2-hTMPRSS2 cells and the levels of Fluc and Nluc were assessed 787 at 72 hrs post-transduction. Errors bars represent standard deviation generated from samples 788 treated in triplicate. The p-values were generated using an unpaired student's t-test compared to 789 the CD63 control treated samples (**p<0.01). (B) The EVs were incubated at increasing 790 concentrations with a set number