iGEM 2017 — Team NJU-China

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# Team:NJU-China/Notebook

NOTEBOOK

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## Calendar

1. [10 Dec](#0)
2. [Jan](#0)
3. [26 Feb](#0)
4. [Mar,Apr](#0)
5. [Mar](#0)
6. [19 Mar](#0)
7. [2 Jun](#0)
8. [17 Jun](#0)
9. [18 Jun](#0)
10. [26 Jun](#0)
11. [5 Jul](#0)
12. [16 Jul](#0)
13. [20 Jul](#0)
14. [6 Aug](#0)
15. [15 Aug](#0)
16. [8 Seq](#0)
17. [10 Seq](#0)
18. [25 Seq](#0)
19. [24 Oct](#0)

* [Prev](#0)
* [Next](#0)

1. *December 10th, 2017*

   NJU China briefing session
2. *January, 2017*

   Team member recruitment
3. *Februry 26th,2017*

   First meet up, laboratory safety regulation learning and task assignment.
4. *March, April, 2017*

   Learn experimental techniques like western blot, RT-PCR, cell culture techniques, etc.

   Data collection and project discussion

   Collaboration with SYSU-Software
5. *March, 2017*

   Human practice：Interview with doctors and patients from Nanjing General Hospital and project design.
6. *March 19th-Jun 8th, 2017*

   Model built
7. *June 2nd-Jun 9th, 2017*

   Anti-Bcl2 plasmid, tpep plasmid design and construction
8. *June 17th-June 22th, 2017*

   Targeting verification in vitro

   HEK293 cell was stained with fluorescence and Tpep plasmid was transfected, two days later, the cells was collected and exosome was extracted. Then the exosomes were co-cultured with C2C12 cell and mouse adipose cell 3T3L1 for 12 hours individually, cell nucleus were dyed with DAPI, then the cells were observed under fluorescent microscope.
9. *June 18th-June 20th, 2017*

   Exosome characterization

   TEM and NTA experiments were carried out in the assay of exosomes.

   Endotoxin test for exosomes
10. *June 26th-July 1st, 2017*

    Anti-Bcl2 and Tpep plasmid were co-transfected into HEK 293 cell, then the exosome extracted was co-cultured with adipose cell. Protein and RNA were extracted and stored for further research.
11. *July 5th-June 13th, 2017*

    Run three times western blot to detect whether there is down-regulated expression of BCL2 protein in adipose cell after plasmids transfection, all failed.
12. *July 16th, 2017*

    Bcl2 RNA reverse transcription and Qpcr to exam the expression level of BCL2 in experimental group, Bcl2 RNA expression marginally reduced.
13. *July 20th-August 4th, 2017*

    Bcl2 plasmid redesign and improvement, repeat experiments to value the effect of the new plasmid.

    Interlab

    Plasmids transformation into E.coli.

    Pick single colonies and run colony PCR.

    OD600 measurement , plasmid extraction and DNA electrophoresis.

    Measure GFP fluorescence intensity with microplate reader

    Flow cytometry
14. *August 6th-August 8th, 2017*

    Egfr tracer experiment in vivo

    4 mice was divided into two groups, experimental group was tail intravenously injected with egfr siRNA+tpep exosome, while control group egfr siRNA exosome. 12hours later, the mice was dissected, their heart, liver, spleen, lung, kidney and adipose tissue were separated.

    RNA extraction of those tissue, reverse transcription and Qpcr was executed to compare egfr expression level in different tissue.
15. *August 15th-September 5th, 2017*

    Aug 15 – Sep 5 Valuation in vivo

    Aug 15- Aug20 Plasmid maxi extraction, cell transfection and exosome extraction.

    Aug21 –Sep 5
    20 mice were fat- fed and divided into fore groups, 5 in each group. Group I was intravenously injected with PBS every two days, Group II egfr-tpep exosome , group III and Group IV Bcl2-tepe exosome, Weight data was collected every day, no obvious tendency in weight lose. After two weeks, took blood by removing eyeball, and heart liver spleen lung kidney and adipose tissue were separated, adipose tissue was weighted.
    ITT
    Starvation treatment in group IV for 6 hours, intraperitoneally inject insulin 0.75 unit per kg, Took blood sample form tail end and measure blood glucose at the time of 0, 15, 30, 45, 60, 90, 120 min.

    GTT
    Starvation treatment overnight and intraperitoneally inject glucose, the following step is as same as ITT
16. *September 8th, 2017*

    LDL, HDL,CHO, TG measurement of blood serum, failed

    protein and RNA extraction of liver and adipose, RNA reverse transcription.
17. *September 10th-September 16th, 2017*

    Western blot of liver and adipose protein to exam Bcl2 expression level, the protein was not so pure that it showed obscure strip

    Repeated experiment: Centrifugate the protein before loading, obscure strip disappeared, still ,no tendency.

    Sep 14 Sent fat tissue to be made into sections, stained by means of hematoxylin eosin (HE) method and calculated under the Image Analyzer.

    Sep 9 Bcl2-mRNA Qpcr with the method of SYBR Green
18. *September 25th-October 23th, 2017*

    Repeated exprement: Validation in vivo, exosomes were injected every two days and weight data was collected everyday. Four weeks later, took blood by removing eyeball, and heart liver spleen lung kidney and adipose tissue were separated. ITT and GTT were also carried out. Western blot and Qpcr were used to exam Bcl2 protein and Bcl2 mRNA individually, failed.

    Oct 9
    Part submission to Nanjing Genescript
19. *October 24th-October 30th, 2017*

    Final Data analysis and preparation.

## PROTOCOL

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# Team:NJU-China/Experiments

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# NJU-China

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

##### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

##### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:NJU-China/Results

PROJECT

[![top](https://static.igem.org/mediawiki/2017/4/41/NJU_China_iGEM_2017_PROJECT_top.png)](#mtop)

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# RESULTS

* [Parts](#parts)
* [Validations](#validations)
* [Safety](#safety)
* [Conclusions](#conclusions)

## Parts

### 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 sequence 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).

![](img/results/Result-1.png)
*Figure 1. The sequence of KRAS shRNA*

#### Usage and Biology

We packaged KRAS siRNA into exosomes by transfecting HEK293 cells with a plasmid expressing KRAS siRNA and then collected siRNA-encapsulated exosomes. When modified exosomes being intravenously injected, they will specifically recognize integrin receptors and fuse with lung adenocarcinoma cells under the direction of the iRGD peptide. Once inside cells, KRAS siRNA will bind to KRAS mRNA through base-pairing and digest the mRNA, resulting in sharp decrease of K-ras in lung cancer cells. As a consequence, K-ras protein’s reduction and disturbed function will both result in the inhabitation of the proliferation of cancer cells, which ultimately have some therapeutic effects on lung cancer (non-small cell lung cancer in this case).

#### Characterization

**Interference efficiency of anti-KRAS siRNA plasmid**

To ensure the interference efficiency of anti-KRAS siRNA plasmid, we transfected it into human lung adenocarcinoma cell line A549 and then extracted protein from these cells to perform western blot. Significant down-regulation of K-ras can be observed in A549 cells treated with anti-KRAS siRNA, demonstrating that anti-KRAS siRNA has the gene silencing effect on lung cancer cells.

![](img/results/Result-2.jpg)
*Figure 2. Protein quantitatively analysis of K-ras extracted from cells without any treatment (Nude) and cells transfected with control siRNA (NC, siRNA targeting a random sequence) or anti-KRAS siRNA, which was made for intuitively support that anti-KRAS siRNA can suppress K-ras expression.*

### BBa\_K1942002

A coding sequence of iRGD peptide and position it outside the membrane.

#### Usage 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 our previously designed siRNA, allowing us to target recipient cells in vivo efficiently. 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 using 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, et al. Biomaterials, 2013). He showed that exosomes, endogenous nano-sized membrane vesicles secreted by most cell types, could 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 αν 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 αν 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 suggested that exosomes modified by targeting ligands could be used therapeutically for the delivery of Dox to tumors, thus having great potential value for clinical applications in our project.

## Validations

### Silencing capability validation in vitro

#### 1.1 KRAS siRNA interference efficiency verification in vitro

To ensure the interference efficiency of anti-KRAS siRNA, we transfected the plasmid loaded with this siRNA into human lung adenocarcinoma cell line A549 and extracted protein to perform western blot. Significant down-regulation of K-ras can be observed in A549 cells treating with anti-KRAS siRNA plasmid, compared with the control group, demonstrating that anti-KRAS siRNA has a gene silencing effect on lung cancer cells.

![](img/results/Result-3.jpg)
*Figure 3. Anti-KRAS siRNA transfected into A549 cells successfully reduced KRAS expression. Left panel: western blot analysis of KRAS protein levels in cells without any treatment (Nude) or treated with negative control siRNA (NC, siRNA targeting at a random sequence) and transfected with anti-KRAS siRNA using Lipo2000. Right panel: protein quantitative analysis made for intuitive support that anti-KRAS siRNA can suppress KRAS expression.*

#### 1.2 TEM imaging of exosomes carrying KRAS siRNA and expressing iRGD peptide on their membrane

After co-transfection of the two plasmids mentioned above, we performed a transmission electron microscopy (TEM) to characterize the iRGD-exosomal KRAS siRNA. The TEM image showed that the exosomes presented normal morphological characteristics after outside modification and siRNA loading, with a diameter of approximately 200 nanometer and a double-layer membrane.

![](img/results/Result-4.png)
*Figure 4. TEM image of iRGD-modified exosomes packaging KRAS siRNA*

### Silencing capability validation in vivo

#### 2.1 Establishment of none-small cell lung cancer mouse model with 40 mice to perform validation experiment in vivo

The iRGD-exosomal KRAS siRNA can be released into A549 cells and suppress the expression of KRAS in vitro. To examine the consequence of KRAS knockdown by anti-KRAS siRNA in vivo, we built a non-small cell lung cancer mouse model for in vivo experiment. Forty mice were subcutaneously injected A549-LUC cells to realize tumor implantation. Then tumor volume were measured through bioluminescent imaging several times after injection. The areas that emit fluorescence in the mice bodies represent labeled tumors (tumors developed from the implanted A549-LUC cells), which helps us to monitor the tumor growth, location and metastasis.

![](img/results/Result-8.jpg)
*Figure 8. In vivo imaging 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. First panel: the imaging of tumor-bearing mice injected with PBS. Second panel: the imaging of tumor-bearing mice treated with iRGD-exosomal KRAS siRNA.*

## Safety

### Endotoxin detecting of anti-KRAS siRNA-loaded exosomes

Endotoxin is a type of natural pyrogen that was found in outer cell membrane of Gram-negative bacteria and can make impact on over 30 biological activities. To ensure the safety of our drug system, avoid toxicities, thus prove that our achievement is of great value for clinical application, a detecting assay was carried out using an endotoxin test kit. The result was negative, demonstrating that our drug system satisfies the safety requirement.

![](img/results/Result-13.png)
*Figure 13. Endotoxin detecting of anti-KRAS siRNA-loaded exosomes*

## Conclusions

### An efficient drug delivery system

KRAS mutation was identified in NSCLC more than 20 years ago, but its clinical importance in cancer therapy just began to be appreciated. Our project aimed to develop a drug system that employed modified exosomes with iRGD peptide on its surface to deliver KRAS-siRNA into lung cancer cells specifically, thus target KRAS gene and down-regulate K-ras protein expression to treat lung cancer cases. Our results had demonstrated that iRGD-exosomal KRAS siRNA can be delivered into tumor cells and efficiently down-regulate KRAS expression both in vitro and in vivo. In silencing validation, we transfected KRAS siRNA into A549 cells using Lipo 2000 and performed western blot to test the function of our siRNA. Subsequently, we collected iRGD-modified exosomes loaded with KRAS siRNA, evaluating its effect on lung cancer cells (A549) by examining KRAS expression after transfection. The result confirmed that iRGD-exosomal KRAS siRNA could effectively down-regulate KRAS transcription level and reduce its protein expression. Later, the EDU assay further ascertained the biological role of KRAS siRNA in cell proliferation suppression in vitro.

To perform in vivo validation, none-small cell cancer mouse model was established after implanting tumor in 40 mice by subcutaneous injection with A549-LUC cells. After a short-time treatment, tumors harvested from killed mice were measured, then protein and RNA extracted from these tissues were examined, results supporting that KRAS siRNA can efficiently suppress KRAS expression, inhibit cell proliferation and thus have its potential to be taken as a cancer treatment. Besides, endotoxin detecting validated that our drug system satisfies the safety requirement and won’t impact on biological activities. To be more meaningful, we purified the batch of iRGD-exosomal KRAS siRNA for liquid fill, completing a full process including experiment design, compounds synthesis, effect test and drug production.

![](img/results/Result-14.jpg)
*Figure 14. Exosome final products*

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