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Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity

https://doi.org/10.1016/j.jbc.2021.101266Get rights and content
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Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.

Keywords

COVID19
spike
nucleocapsid
exosome
mRNA
cationic lipid
lipofection
antibody
T-cell
extracellular vesicles

Abbreviations

ACE2
angiotensin-converting enzyme II
BCA
bicinchoninic acid
BLI
bioluminescent imaging
CDM
chemically defined media
CTCS
clarified tissue culture supernatant
CVS
concentrated vesicle suspension
DOPE
dioleoyl phosphatidylethanolamine
DOTAP
dioleoyl-3-trimethylammonium propane
DTZ
diphenylterazine
ER
endoplasmic reticulum
EV
extracellular vesicle
HUVEC
human umbilical vein endothelial cell
LNP
lipid nanoparticle
NTA
nanoparticle tracking analysis
ORF
open reading frame
pfPBS
particle-free PBS
SARS-CoV-2
severe acute respiratory syndrome coronavirus 2
SEC
size-exclusion chromatography

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Shang Jui Tsai is a graduate student working in the Department of Biological Chemistry at Johns Hopkins University. His research has two broad goals, one of which is to develop exosomes as a vehicle for delivering nucleic acid therapeutics, while the other is to develop genetically encoded modulators of neuronal signaling. His long-term goal is to develop novel therapeutics for the treatment of chronic pain and other neurological disorders.

These authors contributed equally to this work.