Team:NJU-China/Proof
Parts
1.BBa_K1942000
anti-KRAS siRNA (siRNA for KRAS gene silencing)INTRODUCTION
This part is an artificially designed RNA strand. It serves as an element of the Team NJU-CHINA RNAi module which can be used for down-regulation of KRAS expression in Lung adenocarcinoma cells. We designed specific KRAS siRNA with algorithm based on a software developed by SYSU-Software team. This tool can find the best siRNA sequences on target gene KRAS to ascertain the maximum gene-specificity and silencing efficacy and also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. Then we synthesize the shRNA sequence from a DNA synthesis company (Genscript).
Figure 1. The sequence of KRAS shRNA
USAGE AND BIOLOGY
We package KRAS siRNA into exosomes by transfecting HEK293 cells with a plasmid expressing KRAS siRNA and then collect siRNA-encapsulated exosomes modified by iRGD peptide. When inject the modified exosomes into the vein, exosome will specifically recognize integrin receptors and fuse with Lung adenocarcinoma cells under the direction of the iRGD peptide. Once inside cells, KRAS siRNA will degrade KRAS mRNA by base-pairing, resulting in sharp decrease of K-ras in Lung cancer cells. As a consequence, K-ras reduction and disturbed function will result in the inhabitation of the proliferation of cancer cells, which ultimately have some therapeutic effects on Lung cnacer.
CHARACTERIZATION
Interference efficiency of anti-KRAS siRNA plasmidTo ensure the interference efficiency of anti-KRAS siRNA plasmid, we transfected it into human Lung adenocarcinoma cell line A549 and then extracted protein to perform a western blotting. Significant downregulation of K-ras can be observed in A549 cells treating with anti-KRAS siRNA, demonstrating that anti-KRAS siRNA has a gene silencing effect on Lung cancer cells.
Figure 2. Protein quantitatively analysis made for intuitively support that anti-KRAS siRNA can suppress KRAS expression.
2.BBa_K1942002
Coding sequence of iRGD proteins and position it outside the membraneUSAGE AND BIOLOGY
iRGD is a tumor-penetrating peptide that can increase vascular and tissue permeability. Importantly, this effect did not require the drugs to be chemically conjugated to the peptide. To enhance the accuracy of drug delivery system and improve targeting index of drugs, iRGD peptide was displayed on the surface of the exosome containing K-rasour previously designed siRNA, allowing us to target recipient cells in vivo efficiently.
The tumor targeting capability of exosomes was conferred by engineering the immature dendritic cells (imDCs) to express lysosome-associated membrane glycoprotein 2b (Lamp2b), a well-characterized exosomal membrane protein, fused with iRGD (CRGDKGPDC) targeting peptide for av integrin. Immature DCs were transfected with the vector expressing iRGD-Lamp2b fusion proteins using Lipofectamine 2000 transfection reagent. Lamp-2b is a protein found specifically abundant in exosomal membranes. So we connect iRGD with Lamp2b by a glycine-linker, and promote the expression usingby cmv promoter. We engineered our chassis, human embryonic kidney 293 (HEK293) cells, to express iRGD-Lamp2b fusion protein. Therefore, the iRGD exosomes (iRGD-Exos) are endowed with site-specific recognition ability and were purified from cell culture supernatants and loaded with Dox by electroporation
CHARACTERIZATION
The iRGD-Lamp2b expressing vector was thoroughly described in Tian’s article .(Yanhua Tian. etc, Biomaterials, 2013). He shows that exosomes, endogenous nano-sized membrane vesicles secreted by most cell types, can deliver chemotherapeutics such as doxorubicin (Dox) to tumor tissue in BALB/c nude mice. To reduce immunogenicity and toxicity, mouse immature dendritic cells (imDCs) were used for exosome production. Tumor targeting was facilitated by engineering the imDCs to express a well-characterized exosomal membrane protein (Lamp2b) fused to av integrin-specific iRGD peptide (CRGDKGPDC). Purified exosomes from imDCs were loaded with Dox via electroporation, with an encapsulation efficiency of up to 20%. iRGD exosomes showed highly efficient targeting and Dox delivery to av integrin-positive breast cancer cells in vitro as demonstrated by confocal imaging and flow cytometry. Intravenously injected targeted exosomes delivered Dox specifically to tumor tissues, leading to inhibition of tumor growth without overt toxicity. The results suggest that exosomes modified by targeting ligands can be used therapeutically for the delivery of Dox to tumors, thus having great potential value for clinical applications in our project.
Results
According to our study design, we transfected a targeting plasmid into HEK293 cells to express a fusion protein composed of the exosomal membrane protein Lamp2b and the peptide iRGD. Simultaneously, we transfected KRAS siRNA plasmid into HEK293 cells to collect exosomes. Then, the modified exosomes carrying KRAS siRNA were continually produced by the transfected HEK293 cells. Subsequently, we ensure the function of KRAS siRNA and determine whether KRAS siRNA encapsulated by iRGD-modified exosomes had a KRAS gene silencing effect and could effectively suppress the K-ras expression. Generally, the experimental procedure can be divided into three steps:
(1) silencing capability validation in vitro
(2) silencing capability validation in vivo
(3) safety validation
1. Silencing capability validation in vitro
1.1 Verification of interference of efficiency of KRAS siRNA Interference efficiency of anti-KRAS siRNA plasmid in vitro
To ensure the interference efficiency of anti-KRAS siRNA plasmid, we transfected it into human Lung adenocarcinoma cell line A549 and then extracted protein to perform a western blotting. Significant downregulation of K-ras can be observed in A549 cells treating with anti-KRAS siRNA, demonstrating that anti-KRAS siRNA has a gene silencing effect on Lung cancer cells.
igure 3. Anti-KRAS siRNA transfected into A549 cells successfully reduce KRAS expression. Left panel: Western blot analysis of KRAS protein levels in cells without any treatment or treated with negative control siRNA and transfected with anti-KRAS siRNA using Lipo2000. Right panel: Protein quantitatively analysis made for intuitive support that anti-KRAS siRNA can suppress KRAS expression.
1.2 TEM images of exosomes carrying KRAS siRNA inside and expressing iRGD peptide on their membranes
Subsequently, we performed a transmission electron microscopy (TEM) to characterize the iRGD-modified exosomes loaded with KRAS siRNA. The TEM images showed that the exosomes presented normal morphological characteristics after outside modification and siRNA loading, with a diameter of approximately 90 nm and a double-layer membrane. These characteristics indicate that the exosome properties were not affected by these modifications.
Figure 4. TEM image of iRGD-modified exosomes packaging KRAS siRNA
1.3 anti-KRAS siRNA encapsulated by iRGD-modified exosomes suppress the KRAS expression in A549 cells in vitro
We next evaluate the effect of anti-KRAS siRNA packaged by exosomes on KRAS expression in vitro. The KRAS expression levels were assayed in A549 cells after transfected with anti-KRAS siRNA-loaded exosomes. Nonloaded iRGD exosomes were used as control to ascertain that any RNAi response observed did not derive from the exosomes per se. The western electrophoresis and knockdown data obtained from qPCR analysis of KRAS gene expression indicate that KRAS protein and mRNA levels both dramatically decreased in the cells incubating with iRGD exosome-delivered siRNA compared with cells treating with nude exosome or without any treatment. This result suggest that iRGD-modified exosomes containing anti-KRAS siRNA can specifically target A549 cells, deliver siRNA into cells and finally reduce the KRAS expression.
Figure 5. Quantitative RT-pcr analysis of KRAS mRNA levels in A549 cells without any treatment and transfected with nonloaded exosomes or anti-KRAS siRNA-loaded exosomes shows that anti-KRAS siRNA can be transfected into exosomes and target KRAS gene to realize silencing effect, down-regulating KRAS mRNA levels.
1.4 anti-KRAS siRNA loaded into exosomes efficiently arrests cell proliferation in vitro
KRAS over-expression has been demonstrated to promote the growth of Lung adenocarcinoma cells and be involved in migration and invasion of Lung cancer. For further support, we had to examine to ascertain the role of anti-KRAS siRNA-loaded iRGD exosomes in cell proliferation by down-regulating the KRAS expression. An EDU assay was carried out after transfection and as a control, nude exosome also transfected into A549 cells, respectively. The assay result indicates that KRAS knockdown had an anti-proliferative effect on the tumor cells while the nude exosomes were not capable of decreasing the tumor cells growth.
Figure 6.
2. Silencing capability validation in vivo
The anti-KRAS siRNA-loaded exosomes can be transfected into A549 cells and suppress the expression of KRAS in vitro. To examine the consequence of KRAS knockdown by anti-KRAS siRNA packaged by exosomes, we built a non-small cell lung cancer mouse model for in vivo experiment. Forty mice were subcutaneously injected with A549-Luc cells to implant tumors and tumors volumes were measured with bioluminescent imaging several times after injection. The parts emitting fluorescence in the mice bodies represent the tumors xenografted with A549-Luc, which helps monitor the tumor growth, location and metastasis.
Figure 7. Heatmap of tumor-bearing mice. The parts in the mice body representing the tumors xenografted with A549-Luc indicates that the tumors are relatively uniform in size. Left panel: the heatmap of tumor-bearing mice injected with PBS. Right panel: the heatmap of tumor-bearing mice treated with KRAS siRNA packaged by exosomes.
Figure 8. Quantitative analysis of fluorescence intensity of tumor implanted in mice after treatment with PBS and KRAS siRNA injections.The intensity of fluorescence in mice treated with PBS is much more than mice treated with iRGD exosomes losded with KRAS siRNA.
Mice were randomly assigned to 2 groups(n=20 per group) and treated differently. One group received PBS injections, the other group is treated with anti-KRAS siRNA encapsulated by iRGD-modified exosomes via tail-vein injections. The administrations were given five times for 2 successive weeks since contamination of HEK293 cells resulted in exosomes shortage and the treatment of mice were delayed.
Subsequently, mice were sacrificed for tumor harvest after in vivo imaging system. All the tumors were measured for length,volumes and weight. The Small piece of each tumor was cut and fixed with paraformaldehyde to make preparation for histopathological examination.
Figure 9. The tumor harvested from the killed mice and measured for volumes and weight. The first row are tumors from mice injected PBS, the second row are tissues from mice administered with KRAS siRNA encapsulated by iRGD exosomes.
Figure 10. The weight measurement of tumor tissues harvested from model mice With analytical balance
Then, total protein and RNA were extracted from the rest tumor tissues to evaluate the expression levels of KRAS in vivo. As a result, both KRAS protein and mRNA levels were reduced in the tumor cells of mice injected with iRGD exosomes-delivered anti-KRAS siRNA compared with mice treated with PBS. Though the down-regulation is not so evident, taking the short time of treatment into account, it clearly demonstrats that anti-KRAS siRNA using exosomes as efficient RNAi delivery agents were capable of delivering siRNA into cancer cells and regulating target gene expression.
Figure 11. Quantitative RT-pcr analysis of KRAS mRNA levels in tumor cells after treatment of PBS and KRAS siRNA packaged by iRGD-modified exosomes.
3.safety validation
Endotoxin detecting of anti-KRAS siRNA-loaded exosomes
Endotoxins is a type of pyrogen which are natural compounds found in the outer cell membrane of Gram-negative bacteria and can impact over 30 biological activities. To ensure the safety and quality of, avoiding toxicities, proving that our achievement is of great value for clinical application, an endotoxin detecting assay was carried out using endotoxins test kit. The result was negative, demonstrating that our drug system satisfy the safety requirement.
Figure 12. Endotoxin detecting of anti-KRAS siRNA-loaded exosomes
Conclusions
KRAS mutations were identified in NSCLC tumors more than 20 years ago, but the clinic importance of KRAS mutant in cancer therapy just began to be appreciated. This project aimed to develop a drug system that employed modified exosomes expressing the iRGD peptide on the exosomal membrane surface to deliver KRAS siRNA into cancer cells, targeting KRAS gene and down-regulating K-ras expression to treat cancers. Our results had demonstrated that exosomes mediated by iRGD peptide can effectively and specifically help KRAS siRNA into Lung cancer cells and silence KRAS gene to realize the expression level reduction both in vitro and in vivo. In silencing validation , we transfect the KRAS siRNA into A549 cells using Lipo 2000 and performed a western blotting to ensure the function of our RNA sequence. Subsequently, we collected iRGD-modified exosomes loaded with KRAS siRNA, evaluating its effect on Lung cancer cells by examining the KRAS expression after transfection. The result confirmed that iRGD exosomes carrying KRAS siRNA can effectively down-regulate the expression level of KRAS mRNA and reduce the K-ras protein. Later, the EDU assay further ascertained the biological role of KRAS siRNA on cell proliferation suppression in vitro.
In vivo experiment, the none-small cell cancer mouse model was established after implanting tumor in 40 mice by subcutaneous injection with A549-LUC cells. After short-time treatment, the tumor tissues harvested from killed mice were measured, then protein and RNA extracted were tested, which supported that KRAS siRNA can efficiently suppress the KRAS expression, inhibit cell proliferation and have a potential to treat cancers. Besides, the endotoxin detecting validated that our drug system satisfy the safety requirement and will not impact biological activities.
