This year we developed an alternative strategy to silence the Immune checkpoint therapy by employing exosome-mediated siRNA delivery. Silencing of immune checkpoints will allow tumor cells to be attacked by the immune system.At the same time, we used iRGD-Lamp2b to achieve exosome targeting.
Particularly, we reprogrammed cells to simultaneously express siRNA and Lamp2b. Lamp2b is an exosomal membrane protein, in fusion with a tumor-penetrating internalizing RGD (iRGD) peptide (CRGDKGPDC), and then produce the tumor-targeting exosomes as siRNA delivery system.
With this system, we can target tumor cells and deliver siRNA.
Figure 1. Basic parts and Composite parts
PD-L1 siRNA overexpressed in HEK293T cell and exosome
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.
Lamp2b is an exosomal membrane protein, in fusion with a tumor-penetrating internalizing RGD (iRGD) peptide (CRGDKGPDC), and then produce the tumor-targeting exosomes as siRNA delivery system.
We detected the high expression of iRGD-Lamp2b after transfection by RT-qPCR, which proved that our parts are effective and provided assistance for the production of targeted exosomes (Figure1.).
Figure 2. iRGD-Lamp2b is overexpressed in HEK293T cell
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-siRP is this part. CMV-iRGD-siRP+C+K is a Composite part.
Figure 3. PD-L1 siRNA is overexpressed is HEK293T cell
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).
Figure 4. PD-L1 siRNA is overexpressed in exosome
Figure 5. Absolute quantification of PD-L1 siRNA
According to the absolute quantification of PD-L1 siRNA, we found that the amount of siRNA in HEK293T cells is 3.946E-4pM, the amount of siRNA in exosome is 0.4370E-4pM. The exosome siRNA is equivalent to 11.07% of the siRNA concentration in the cell.
CD47 siRNA overexpressed in HEK293T cell and exosome
Tumor cells escape macrophage phagocytosis by overexpressing CD47. Therefore, we combined the fusion expression of iRGD and Lamp2b to characterize the exosome targeting.At the same time, siRNA targeting CD47 was produced, and mRNA expression was targeted to be reduced.This allows macrophages to avoid the effects of CD47 and attack tumor cells.
Then, we detected the correct expression of CD47 siRNA. CMV-iRGD-siRC is this part. CMV-iRGD-siRP+C+K is a Composite part.
Figure 6. CD47 siRNA is overexpressed is HEK293T cell
Figure 7. CD47 siRNA is overexpressed in exosome
Figure 8. Absolute quantification of CD47 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 CD47 siRNA, we found that the amount of siRNA in HEK293T cells is 5.710E-4 pM, the amount of siRNA in exosome is 0.2544E-09 pM. The exosome siRNA is equivalent to 4.45% of the siRNA concentration in the cell.
Composite part
After the normal expression of siRNA of KRAS,PD-L1 and CD47 in HEK293T cells and exosomes was verified, the composite Part of three siRNA fusion was designed and each siRNA was separately detected.
Figure 9. siRNA of Composite Part was overexpressed in HEK293T cells
Figure 10. siRNA of Composite Part was overexpressed in exosome
The above experimental results showed that the three types of composite siRNA were strongly expressed in HEK293T cells and exosomes. This opens up the possibility of combining the three siRNA as a treatment.
Figure 11. Absolute quantitative of siRNA
After passing the absolute quantification, siRNA concentration in the Composite Part was measured as shown in the table below.
| Intracellular siRNA Amount (10-4 pM) | Exosome siRNA Amount (10-4 pM) | |
|---|---|---|
| PD-L1 siRNA | 3.818 | 0.1627 |
| KRAS siRNA | 272.9 | 16.14 |
| CD47 siRNA | 49.55 | 2.544 |
As can be seen from the table, the amount of siRNA in exosomes was about 5% of the amount in cells, which laid a foundation and prepared for siRNA to play its role through exosomes.
KIBRA promotes exosomes release
At the same time, in order to promote exosome release and thus enhance the efficacy of exosome therapy, we chose KIBRA as an exosome biological generator. By avoiding ubiquitination of Rab27A, KIBRA stabilizes Rab27A and promotes exosome release.
The expression of each mRNA was confirmed by RT-qPCR after transfected to HEK293T cells.
Figure 12. KIBRA mRNA relative expression in HEK293T cell (vs GADPH)
To further verify the expression of KIBRA at protein level, we used Western Blotting experiment to prove that KIBRA was indeed overexpressed in HEK293T cells.
Figure 13. Western Blotting result shows that KIBRA is overexpressed in HEK293T cell.
In order to further characterize the ability of KIBRA to promote exosome release, NTA analysis was selected to characterize the diameter and number of exosomes produced by cells after transfection with KIBRA Part. The results showed that KIBRA significantly promoted exosome release compared with NC, while the diameter of exosomes did not change significantly. All this shows that KIBRA can play a good role as an exosome biological generator.
Figure 14.(A) Averaged FTLA Concentration / Size for Exosome
(B) Intensity / Size graph for Exosome