The MIT iGEM team sought to create a new mode of engineered intercellular communication for use in synthetic biology by modifying the contents of existing exosomes through the use of naturally occurring miRNA and the protein domain Acyl-TyA. We built on existing research targeting proteins to exosomes to enable intercellular communication by targeting signal proteins into exosomes and into HEK 293 receiver cells.
Over the summer, we accomplished the following:
(1) Exosomal Cell-Cell Communication with miRNA
Jurkat T cells are known to produce a large number of exosomes which naturally contain high levels of miRNA 451. Using this natural system, our initial goal is to create a miRNA 451/Exosome sensor to begin our work with Exosomal communication.
(2) Creating/Testing miRNA Sensor
We constructed an EYFP fluorescent gene with four miRNA 451 target sites which will allow the EYFP reporter to be repressed by the miRNA 451. By expressing our reporter along with synthetic siRNA 451, we saw repression of our reporter.
(3) Exosomes + Sensor
After seeing our sensor work with siRNA 451, we then isolated exosomes from Jurkat T cells and used them to treat HEK 293 expressing our reporter. We observed similar repression of our reporter.
(4) Jurkat T Cells + Sensor
With our reporter sensing isolated exosomes, we proceeded to coculture both Jurkat T cells producing exosomes with HEK 293 cells transfected with our reporter. We observed repression of our reporter indicating that we have achieved CELL-CELL COMMUNICATION!
(5) Exosomal Cell-Cell Communication with Proteins
Proteins have been shown to be targeted into exosomes with the addition of a high-order oligomerizing protein Acyl-TyA. By fusing a protein signal to an Acyl-TyA domain, we could send the protein from cell to cell through exosomes.
(6) Protein Targeting
We began by fusing Acyl-TyA to GFP and observing colocalization of the Acyl-TyA-GFP with the membrane stain Rh-PE, which has been shown to be targeted to the site of exosomal biogenesis. Then, we demonstrated through a western blot that our protein signal exists within the exosome rich media.
(7) Testing our Protein Signal
We demonstrated retained functionality of our protein signal after fusion with Acyl-TyA (Acyl-TyA-rtTA3). In addition, we tested our reporter construct (TRE-tight_mkate), which allowed us to assay for the function of our protein signal.
(8) Application - Endogenous Gene Activation
Most regulated promoters in Synthetic Biology have been engineered to have upstream regulatory sites, but endogenous genes don't have such features. Activating an endogenous gene would require targeting an activator to an arbitrary sequence upstream of an endogenous promoter. Cas9 allows us to target arbitrary sequences using a guide RNA specific to that sequence. By fusing a VP16 domain to Cas9 we're able to create a programmable DNA binding trans-activator.
(9) Testing Cas9-VP16
We tested our Cas9-VP16 fusion protein by transfecting it into HEK 293 cells along with a guide RNA which will target the Cas9-VP16 to upstream regulatory sites on our reporter construct and activate the expression of EYFP. We showed that our Cas9-VP16 fusion is indeed functional.
(10) Future Work
We currently have being constructed an Acyl-TyA-Cas9-VP16 fusion protein to send through exosomes to activate endogenous genes within receiver cells.
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