Improve

Positive feedback

The miRNA hsa-let-7 not only could restore the bactericidal efficacy when delivered to macrophages, but also has the potential to increase the intracellular level of NF-κB in our designed immune-like cells. It is noteworthy that our system is responsively activated by NF-κB depending on the inducible NF-κB promoter, and this activation of hsa-let-7f expression will further produce more NF-κB in turn. As the result, the bactericidal output would be strengthened.

Integration of diagnosis and treatment

Cells can be designed to meet more demands in cell therapy, of which the first comes to our mind is integration of diagnosis and treatment. Things happen that patients die from delayed treatment due to the negative false results in examination. If we can incorporate responsively detectable signals like GFP into this system, both designed cells and remaining bacteria could be monitored.

Prospect of extensive application

Cell therapy, like CAR-T, is known for its therapeutic efficiency. But T cells are difficult to be transfected and lack of derivation, which restrict speed and increase costs of research and development. Eventually the high price would limit the public benefiting from this therapy. If we design immune-like cells that is widely available and easily transfected, it will undoubtedly promote the process and reduce the cost of cell therapy research. With enhanced innate immunity and ingenious delivery strategies, immune-like cells have a more promising perspective, benefiting patients with even more diseases.

Part Improvement

Some viruses, such as the porcine teschovirus-1 (PTV-1) and Thosea asigna virus (TaV), use 2A peptides, or 2A–like sequences, to mediate protein cleavage ^[1]. Through a ribosomal ‘skip’ mechanism, the 2A consensus motif appears to impair normal peptide bond formation between the glycine and the proline ^[2]. We thought this system would be suited for the expression for toll-like receptors and CD14 clusters. In addition, inclusion of a Gly-Ser-Gly (GSG) spacer in front of 2A peptide ensures complete ‘cleavage’, most importantly, no reports claim the improved 2A peptide has deleterious effects on the function of any proteins thus far, so we take this approach to optimize cleavage effectiveness of T2A. In 2019 CPU_CHINA project, we utilize P2A and improved T2A to co-express TLR1:TLR2:CD14 cluster on our designed cell membrane.

Characterization

In order to confirm the cleavage efficiency of the GSG-added T2A was improved, T2A (BBa_K1993019 http://parts.igem.org/Part:BBa_K1993019, Figure1) and our improved T2A (BBa_K2976010 http://parts.igem.org/Part:BBa_K2976010, Figure2) were separately constructed into expression vector for co-expression of TLR1 and CD14. And we performed flow cytometry on cells transfected with plasmid (TLR1-T2A-CD14) and plasmid (TLR1-improved T2A-CD14).

The results show that the amount of CD14 in the cells that were transfected with plasmid (TLR1-improved T2A-CD14) was higher than that of the plasmid (TLR1-T2A-CD14), which proved that the cleavage efficiency of the part is enhanced, and verified our improved part achieved the expected functionality.

Reference

[1] Szymczak, A. L., Workman, C. J., Wang, Y., Vignali, K. M., Dilioglou, S., Vanin, E. F., & Vignali, D. A. (2004). Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide–based retroviral vector. Nature Biotechnology, 22(5), 589-594.

[2] Holst, J., Vignali, K. M., Burton, A. R., & Vignali, D. A. (2006). Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice.. Nature Methods, 3(3), 191-197.

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