(641c) Tuning the Tropism and Infectivity of Sars-Cov-2 Virus-like-Particles (VLP) for mRNA Delivery

SARS-CoV-2 virus-like-particles (VLP) are ~100 nm minimalist units composed of the four structural proteins of the authentic virus. We undertook extensive molecular engineering to optimize these VLPs, to determine if they can be used for mRNA delivery. Cloning the N protein upstream of M-IRES-E resulted in a 3 plasmid (3P) VLP system that displayed ~7-fold higher viral entry efficiency compared to VLP formed by co-transfection with 4 plasmids. >90% of ACE2 expressing cells could be transduced using these 3P VLPs. Viral tropism could be readily modified by trans-complementing with spike protein from other coronavirus, and also the Vesicular stomatitis virus G protein (VSV-G). VLP synthesis could be further simplified into a 2 plasmid (2P) system where one vector carried the viral surface glycoprotein and the second carried the remaining SARS-CoV-2 structural proteins and reporter gene. This 2P system could greatly streamlined wet-lab experimental workflows. The SARS-CoV-2 VLP could be engineered to carry up to four genes, including functional Cas9 mRNA for genome editing. Gene editing is specific target cell types was possible by switching viral surface glycoprotein to tune VLP tropism. Successful mRNA delivery to mouse lungs, suggests that the SARS2 VLPs could be used for pulmonary gene delivery. Overall, the study describes the systematic advancement of SARS-CoV-2 VLP platform for robust mRNA delivery both in vitro and in vivo.