Engineering multiple levels of specificity in an RNA viral vector
Synthetic molecular circuits could provide powerful therapeutic capabilities, but delivering them to specific cell types and controlling them remains challenging. An ideal "smart" viral delivery system would enable controlled release of viral vectors from "sender" cells, conditional entry into target cells based on cell-surface proteins, conditional replication specifically in target cells based on their intracellular protein content, and an evolutionarily robust system that allows viral elimination with drugs. Here, combining diverse technologies and components, including pseudotyping, engineered bridge proteins, degrons, and proteases, we demonstrate each of these control modes in a model system based on the rabies virus. This work shows how viral and protein engineering can enable delivery systems with multiple levels of control to maximize therapeutic specificity.
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Posted May 27, 2020. We thank J. Ruan and A. Erskine for technical assistance; R. Zhu, Z. Singer, H. McBride, and L. Luo for critical feedback. The research was funded by DARPA (HR0011-17-2-0008, M.B.E.), the Gordon and Betty Moore Foundation (GMBF2809, M.B.E.), NIH (T32 GM07616, L.S.C., 1K99EB027723-01, X.J.G.), National Science Foundation Graduate Research Fellowship Program (DGE-1745301 ,L.S.C) and the Helen Hay Whitney Foundation (F1047, X.J.G.). M.B.E is a Howard Hughes Medical Institute investigator. Author contributions: X.J.G. and L.S.C. conceived of the project. X.J.G., L.S.C., M.S.K., and M.B.E. designed experiments. X.J.G., L.S.C., M.H.I. and M.S.K. performed experiments. X.J.G., L.S.C. and M.B.E. analyzed data. X.J.G., L.S.C., and M.B.E. wrote the manuscript, with input from all authors. Competing interests: X.J.G, L.S.C., M.S.K., and M.B.E. are inventors on a U.S. patent provisional application related to this work. Data and materials availability: All DNA constructs will be available from Addgene, and cell lines are available from M.B.E. under a material transfer agreement with Caltech. The datasets generated and analyzed and the computer code used during the current study are available at data.caltech.edu, DOI 10.22002/D1.1438 . Flow cytometry data analysis software used for this study is available at https://antebilab.github.io/easyflow/.
Submitted - 2020.05.27.119909v1.full.pdf
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