Engineered Proteins Program Mammalian Cells to Target Tnf? Sources

In certain diseases (e.g. atherosclerosis and cancer), there is a local formation of cell masses (e.g. plaques and tumours), a low pH extracellular microenvironment and the secretion of various pro-inflammatory cytokines such as TNFα. The ability to engineer a cell to seek these TNFα sources allows for targeted and local delivery of therapeutic intervention to disease sites. To impart this ability into cells that do not naturally target TNFα sources, here we introduced a system of proteins: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca2+-activated RhoA (named CaRQ), VSVG and thymidine kinase. Upon binding of TNFα, TNFR1chi generates a Ca2+ signal. This Ca2+ signal in turn activates CaRQ-mediated non-apoptotic blebs that allow migration towards the TNFα source. Next, with the addition of VSVG, upon low pH induction, these engineered cells fuse with the TNFα source cells. Finally, post-ganciclovir treatment, the cells undergo death via a suicide mechanism assisted by thymidine kinase. Hence, we assembled a system of proteins that forms the basis of engineering a cell to target inflammatory disease sites characterized by the secretion of TNFα and a microenvironment with low pH.