Engineering Endosymbionts Capable of Controlling Mammalian Cell Gene Expression

Directed cellular reprogramming has the potential to impact human health through guiding tissue regeneration, advancing cell-based therapeutics and modulating immune responses. However, controlling cellular fates and function in complex tissue environments has remained a significant challenge. To address this challenge, we have developed an engineered endosymbiont (EES) designed to escape destruction in mammalian cell vacuoles and reside in the cytoplasm where it can control gene expression and guide host cell fate and function. EES can be engineered with regulatable operons that can be induced to express and secrete reporter genes for imaging, or transcription factors that direct cellular function. We evaluated EES for modulating immune cells both as a demonstration of EES-driven cellular programming, and as a means of controlling the host immune response for therapeutic applications. After transfer to host cells, the EES was shown to escape endosomes and replicate within the cytoplasm via confocal and live cell microscopy. We used the lacZ gene encoding β-galactosidase with a secretion sequence and nuclear localization sequence to demonstrate delivery of proteins made in the EES to the nuclei of host cells as visualized by fluorescence microcopy. Operons encoding transcription factors, Stat-1 and Klf6 or Klf4 and Gata-3, capable of polarizing macrophages into M1 or M2 phenotypes, respectively, were developed and cloned into the EES. Intracellular EES may be able to polarize resting state J774 macrophage/monocyte cells into M1 or M2 phenotypes after induction of transcription factors. Accordingly, the EES could be used as a therapeutic for treating cancer or arthritis by polarizing macrophages to M1 or M2 phenotype, respectively. EES are versatile cellular control modules that could direct cellular functions for the treatment of diseases, or for guided regeneration and replacement of lost or damaged tissues. The regulation of immune function would demonstrate the utility of EES to control regenerative processes.