Exosomes booster module
Considering that TIL cells don’t secret enough exosomes under normal condition, we fuse three genes to increase the number of exosomes (Alenquer, 2015), thus increasing the transfer efficiency of exosomes.They are STEAP3(boost exosomes biogenesis), hSDC4 (supports budding of endosomal membranes to form multivesicular bodies) and nadB (boost cellular metabolism by tuning up with the citric acid cycle).
They combine with each other to boost exosomal transfer efficiency：
In previous article, it use IRES (internal ribosome entry site,IRES) to connect three genes, we change it into linkers T2A and P2A to ensure proper expression of three genes.
Then we use use reporter gene copGFP to manifest the expression of exosome booster with the promoter of 5-HRE-PminCMV.
To find out which miRNA or combination of miRNA can best assist Ms.T to arrest cancer cells, we give Mr.T different weapons under different inducer.
Induced by tetracycline,we give Mr.T a gun (miR-135b-3p).
Induced by biotin,we give Mr.T a bloodhound (miR-769-5p).
Induced by galactose, we give Mr.T a spontoon (miR-942-5p).
Exosomes booster module and miRNA module assembly
Because different miRNA can show variable anti-tumor activity.What’s their comprehensive effect on tumor cells? Will their comprehensive effect higher than separate impact? To explore this question, we fuse exosome booster module and three miRNA module into one plasmid.
By adding different inducers into the cell culture, we can prompt engineered 293T cells to express different concentrations of miRNA.There are six combination:
Then we can find the optimum combination of miRNA to achieve our goal——kill tumor cells.
When TIL cell sense the condition of hypoxia in tumor microenvironment, HIF-1(hypoxia inducible factor 1) is produced, and it can bind to HRE(hypoxia response elements), then activating the minimized CMV promoter. Through this mechanism, we can switch the interest gene specifically in tumor microenvironment.
BS-Biotin-On system module
BirA, a bifunctional protein, can activate biotin and connect it with specific protein receptor in the presence of ATP, and can also bind to activated biotin to produce conformational changes, and then bind to the specific operon sequence (OBirA) located upstream of biotin biosynthesis operon to inhibit the transcription of this operon. The system utilizes the above characteristics of BirA to construct an artificial transcription activator, BirA-VP16, with the transcription activation domain of herpes simplex virus transcription activator VP16. The obtained BirA-VP16 can bind and activate the minimum CMV promoter containing three copies of OBirA in the presence of biotin, thereby activating the downstream genes.