Difference between revisions of "Part:BBa K3335003"

Latest revision as of 09:35, 27 October 2020

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

Lung cancer is the major cause of cancer-related deaths globally. Mutant KRAS is a feature of 15%–50% of lung cancer cases[1]. Unfortunately, although much effort has been spent on searching for small molecule inhibitors of KRAS, KRAS gene has proven extraordinarily difficult to target by current pharmacological agents.

This year we developed an alternative strategy to silence the so-called untargetable and undruggable KRAS gene by employing exosome-mediated siRNA delivery.At the same time, we used iRGDLamp2b to achieve exosome targeting. Particularly, we reprogrammed cells to simultaneously express KRAS 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 KRAS siRNA delivery system.[1]

With this system, we can target tumor cells and deliver siRNA.

[1]Yu Zhou et al.,Tumor-specific delivery of KRAS siRNA with iRGD-exosomes efficiently inhibits tumor growth. ExRNA,2019.

Usage and Biology

Functional Parameters

KRAS siRNA overexpressed in HEK293T cell and exosome

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

Figure.1 KRAS siRNA is overexpressed is HEK293T cell

Figure2. KRAS siRNA is overexpressed in exosome

Figure.3 Absolute quantification of KRAS 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 KRAS siRNA, we found that the amount of siRNA in HEK293T cells is 9.138E-02 pM, the amount of siRNA in exosome is 2.848E-03 pM. The exosome siRNA is equivalent to 3.11% of the siRNA concentration in the cell.

Figure. 4 iRGD-siRK part inhibits mRNA expression in vitro

Since the iRGD-siR^K part is improved on the basis of the iRGD guiding part, we further validated whether the iRGD-siR^K part can inhibit KRAS mRNA in vitro<i>. After transfecting HEK293T cells with the iRGD-siR^K part, we detected the KRAS mRNA expression level with RT-qPCR and GAPDH was used as the internal control. As shown in Figure 4, the relative expression level of KRAS gene was downregulated after transfection, which indicates the improved part was functional <i>in vitro<i>.</p> Sequence and Features