(338e) Targeted siRNA Delivery to ACE2-Expressing Lung Cells Using MSC-Derived Extracellular Vesicles Functionalized with Receptor-Binding Domain of Sars-Cov-2 Spike Protein

Introduction: Extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) have been studied extensively for their potential therapeutic applications in lung injury. MSC-derived EVs could reduce lung inflammation by suppressing the production of pro-inflammatory cytokines. As a proof-of-concept study, MSC-derived EVs were tethered with the receptor-binding domain (RBD) of SARS-CoV-2 spike protein and carried small interfering RNA green fluorescent protein (siRNA GFP). The functionalized EVs are expected tospecifically target ACE2 receptors on A549 lung adenocarcinoma cells, thereby silencing GFP expression.

Methods: The gene encoding RBD of SARS-CoV-2 spike protein was amplified by PCR using pcDNA3-SARS-CoV-2-S-RBD-8his vector (Addgene), forward primer (5’- TAAGATATCGCCACCATGAAGCCATC-3’, and reverse primer 5’-ACTAGAATTCAC-CGGATCCCTTTTTG G-3’ (Integrated DNA Technologies). The purified PCR products treated with EcoRV and EcoRI restricted enzymes were inserted and ligated into pET30a (+)-streptavidin (SA) plasmid, and then expressed in BL21 (DE3) E. coli. The RBD-SA fusion protein purified by immobilized metal affinity chromatography was characterized by Western blot and bound to MSC-derived EVs biotinylated with Biotin-Cap-PE (Avanti Polar Lipids). The functionalized EVs, loaded with siRNA GFP usingCaCl-mediated precipitation and heat shock, were added to GFP-expressing A549 cells to evaluate the silencing efficacy at various times (8, 12, 24, and 48 hr).

Results: The purified RBD-SA fusion protein revealed MW of 37 kDa by Western blot analysis against His-tag and streptavidin monoclonal antibodies. The ACE2 receptors expressed on A549 cells were confirmed by detecting red fluorescent dye-conjugated anti-mouse IgG against anti-ACE2 primary antibody. For the ACE2-and GFP-expressing A549 lung cells targeted by RBD-tethered MSC-derived EVs encapsulated with siRNA GFP, the level of green fluorescence detected by plate reader and flow cytometry was notably dropped ove rtwo days. In fact, there was nominal no GFP expression 24 hr post-treatment. As a negative control, the EVs loaded with scramble siRNA illustrated high GFP expression over 48-hr cultivation.

Conclusions: The purified MSC-derived EVs tethered with RBD of SARS-CoV-2 spike protein and encapsulated with siRNA GFP could specifically target ACE2 receptors on GFP-expressing A549 lung cells and dramatically diminish the level of GFP expressed within the cells.