Team:CPU CHINA/design
DESIGN
Description and Overall Design
Rheumatoid arthritis (RA) is a serious chronic, inflammatory and systemic autoimmune disease. It occurs virtually in all races of the world. RA presents great pain and financial burden for patients, but for the time being, there is no radical cure for RA. Therefore, it is necessary to develop a kind of novel targeted cell therapy for RA.
To solve the problems existing in the current treatment of RA, we design and build a brand new immunotherapy. FOXP3+ regulatory T cells, which can suppress and regulate immune reaction, are engineered by inserting a chimeric antigen receptor (CAR) using lentiviral vectors to recognize RA related inflammatory cells. At the same time, we insert another receptor to activate the functional stability pathway of regulatory T cells in the inflammatory environment, and thus making it possible for them to be more immunosuppressive and more stable in lesions in hope for a better anti-RA effect. These two redirections of the two different systems on the native regulatory T cell response ensure both the efficacy and efficiency of our novel immunotherapy for RA.
Figure 1
SynNotch System
SynNotch is a system that is capable of specifically activating the expression of the USP7 gene in an inflammatory environment, and thereby maintaining the activity of Treg cells by stabilizing the FOXP3 proteins.
Figure 2
In our SynNotch system, we retained the functional sequence of the transmembrane domain and the cleavage site of the Notch 1 protein. At the N-terminus, we fused the extracellular domain of IL17RA with Notch 1 to specifically recognize IL-17A secreted by Th17 cells, so that our regulatory T cells to obtain the ability to response to IL-17A. We also connected the gene of Gal4-vp64 (a fusion protein) in the downstream of Notch 1. In the presence of inflammatory cytokines IL17A, SynNotch protein is cleaved, and thus Gal4-VP64 fusion protein is detached from the cell membrane.
The released Gal4-VP64 will recognize UAS sequence in the upstream of promoter USP7 (in our another part BBa_K ) and then these two proteins combine together, which enable USP7 gene express with high efficiency. USP7 proteins will deubiquitinate the ubiquitinated FOXP3, so that enhance the stability of FOXP3 protein in the inflammation environment by protecting FOXP3 from degradation via ubiquitination. In this way, Treg cells can survive and play a role of immunosuppression.
CAR System
In our design, our CAR system was fused from the following protein constructs, first of all the extracellular fraction, and we chose to use the variable regions of the CD20 monoclonal antibody to form a scFv fragment that accurately recognizes and binds to B lymphocytes Surface antigen CD20, then we connected a CD3Z sequence as a stimulus signal, two 4-1-BB sequence as a co-stimulatory signal, so that a high level of signal transmission to the cells, thus activating the effective response of Treg cells. In order to facilitate the detection, at the end of the CAR fusion protein, we linked a red fluorescent protein mCherry (BBa_K106005) as a reporter signal.
Therefore, we designed such a CAR system can be used as a sharp spear, so that engineering Treg cells effectively reduce rheumatoid arthritis in patients with inflammation levels, and thus play a role in the treatment of rheumatoid arthritis.
Figure 3
