Difference between revisions of "Part:BBa K3335004"

Revision as of 18:31, 22 October 2020

We used it to express iRGD-Lamp2b and siRNA targeted at PD-L1

Tumor cells evade immune system surveillance by overexpressing PD-L1.Therefore, we combined the fusion expression of iRGD and Lamp2b to characterize the exosome targeting.At the same time, siRNA targeting PD-L1 is produced to reduce mRNA expression.This allows the immune system to avoid the effects of PD-L1 and attack tumor cells.

Programmed death-ligand 1 (PD-L1) also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) is a protein that in humans is encoded by the CD274 gene. Programmed death-ligand 1 (PD-L1) is a 40kDa type 1 transmembrane protein that has been speculated to play a major role in suppressing the adaptive arm of immune system during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states. Normally the adaptive immune system reacts to antigens that are associated with immune system activation by exogenous or endogenous danger signals. In turn, clonal expansion of antigen-specific CD8+ T cells and/or CD4+ helper cells is propagated. The binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM). This reduces the proliferation of antigen-specific T-cells in lymph nodes, while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells) - further mediated by a lower regulation of the gene Bcl-2. It appears that upregulation of PD-L1 may allow cancers to evade the host immune system. Many PD-L1 inhibitors are in development as immuno-oncology therapies and are showing good results in clinical trials.Clinically available examples include Durvalumab, pembrolizumab, atezolizumab and avelumab.

PD-L1 siRNA overexpressed in HEK293T cell and exosome

First, we detected the correct expression of PD-L1 siRNA through RT-QPCR to ensure that sufficient targeted siRNA could be produced in cell. CMV-iRGD-siR^P is this part. CMV-iRGD-siR^P+C+K is a Composite part.

Figure.1 PD-L1 siRNA is overexpressed is HEK293T cell

Figure2. PD-L1 siRNA is overexpressed in exosome

Figure.3 Absolute quantification of PD-L1 siRNA

At the same time, We detected siRNA in exosomes produced by HEK293T cells, confirmed that siRNA could be properly wrapped into exosomes, and prepared for its role (Figure.2).

According to the absolute quantification of PD-L1 siRNA, we found that the amount of siRNA in HEK293T cells is 1.973E-08 nmol/L, the amount of siRNA in exosome is 4.370E-09 nmol/L. The exosome siRNA is equivalent to 22% of the siRNA concentration in the cell.

Sequence and Features