Team:NJU-China/Poster

Poster: NJU-China



ExosomeBomb SiRNA
Presented by Team：NJU-China

Tianyu Ma1, Clare Li1, Mingrun Shi1, Mingkai Wu2, Yingfan Liu2, Huilian Luo2, Hao Sun2, Disheng Qiao2, Chenyu Tao2, Kai Mei2, Xiaohan Song2, Yanyan Guo2, Shurui Mao2, Zeping Qu2, Xiyao Li2, Nannan Jiang3, Yuanyuan Su4, Xu Guo4, Hongyuan Guo4, Yu Zhou5, Zheng Fu5, Chao Yan6, Xi Chen7.

1Student Leaders.
2Student Members.
3Mentor.
4Advisors.
5Instructors.
6Secondary PI.
7Primary PI.


Project Abstract


siRNAs offer an opportunity to specifically target mRNAs and modulate the expression of therapeutic targets which can facilitate cancer treatment. Here, we designed modular and programmable genetic circuits that use the liver as a tissue chassis to direct the self-assembly of exogenous siRNAs into secretory exosomes. The circuits include core parts to express siRNAs that not only inhibit the cancer cells, but also immune evasion.  Enhancer parts facilitate the production of more anti-cancer exosomses, while supporting parts direct the exosome to target cancer cells. Taken together, these genetic parts assemble into anti-tumor exosomes, are secreted into circulation, delivered to cancer cells, and eliminate them. This strategy provides a controllable, efficient, and convenient cancer treatment which may even provide a personalized treatment strategy that can address a broad range of problems in biomedicine.
Cancer Worldwide



An estimated 14.1 million new cancer cases and 8.2 million cancer deaths occurred in 2012 worldwide. Lung and breast cancer are the most frequently diagnosed cancers and the leading causes of cancer death in men and women, respectively, both overall and in less developed countries[1].



The International Agency for Research on Cancer (IARC) estimates that there were 4285,000 new cancer cases in China in 2018, accounting for 23.7% of the world's new cancer cases.

It is estimated that about 2814,000 Chinese will die from cancer in 2015, corresponding to over 7500 cancer deaths on average per day. The 5 leading causes of cancer death among both men and women are cancers of the lung and bronchus, stomach, liver, esophagus, and colorectum, accounting for about three‐quarters of all cancer deaths. Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death in men aged 75 years or older. Most new cancer cases and cancer deaths in men occur in the age range from 60 to 74 years[2].

COVID-19



Globally, as of 2:46pm CET, 9 November 2020, there have been 50,266,033 confirmed cases of COVID-19, including 1,254,567 deaths, reported to WHO.

A report released by the World Health Organization on 1 June shows that noncommunicable disease prevention and treatment services have been severely disrupted since the start of the COVID-19 pandemic. Of the 155 countries surveyed, 42 percent had partial or complete disruption of cancer treatment. Because malignancies and anticancer treatments such as chemotherapy or surgery can lead to systemic immunosuppression, cancer patients are more likely than non-cancer patients to be infected by epidemic diseases[3]. Therefore, cancer patients may be more susceptible to COVID-19 and have a poor prognosis. The treatment status of cancer patients with COVID-19 in China shows that compared with patients without cancer, cancer patients are at higher risk of serious events and their disease deteriorates faster[4].

Under these conditions, it is difficult for cancer patients to go to the hospital for treatment. So we thought about whether there could be a way of giving drugs in the body for a long time so that cancer patients could be treated without having to go to the hospital. And it would improve on existing therapies.

So we asked the question, can we design a treatment for cancer that can be done without having to go to the hospital or reduce the number of trips to the hospital? This will be extremely useful during the COVID-19 pandemic or other infectious diseases and will be a great relief to patients.

Our Solution

As we mentioned before, cancer is a complex group of diseases with many possible causes, and discovered many genes as targets for cancer treatment. After communication with Dr. Fu Zheng, we learned that the drawback of current cancer therapy is targeting, siRNAs offer an opportunity to specifically target mRNAs and modulate the expression of therapeutic targets before their biogenesis.

Meanwhile, the delivery of siRNA is a big hassle, so we design to use the host's liver cells as a chassis to produce siRNA containing therapeutic effects so as to achieve the purpose of treatment. In the subsequent design, we continuously improved and completed the design.


KRAS

RAS mutations are genetic drivers in numerous cancer types including CRC, pancreatic ductal adenocarcinoma (PDAC), lung adenocarcinoma (LUAD; a subtype of non-small-cell lung cancer (NSCLC))[5-6]. Large percentage of LUAD (32%), PDAC (86%) and CRC (41%) are driven by KRAS mutations, which predominantly occur at codon 12 in these tumour types.

The mutation of KRAS may cause abnormal cell proliferation and even carcinogenesis. Therefore, we regard KRAS as one of the targets.

PD-L1 and CD47

CD47 and PD-L1 may contribute to the formation of tumor microenvironment by affecting macrophages, preventing phagocytosis and T cell activation. PD-L1 is overexpressed in tumor cells and sends out Don't Find Me signal, while CD47 is overexpressed and sends out Don't Eat Me signal[7].

Therefore, knockdown of these two protein expressions can promote the recognition and clearance of tumor by the immune system, which is why we use this as the targets.

Final Design
After determining that we will use siRNA to knock down the expression of KRAS, CD47 and PD-L1, we completed the general design.Meanwhile, in the interviews with professors and doctors, our project has been continuously improved. Tissue specific albumin promoter, Tet system, iRGD-Lamp2b targeting system and Cre-LoxP system were added.


iRGD-Lamp2b


iRGD peptide was reported to specifically bind to the highly expressed αv integrin in tumor cells. We chimerize the iRGD peptide segment to the N-terminus of Lamp2b, a protein expressed on exosome membrane. The fused protein will be incorporated to the exosome memberane and display the iRGD guiding tag on the outside. This design will enhance our targeting ability and increase the delivery efficiency and therapeutic efficacy[8].


KIBRA

To further enhance the efficiency of exosome delivery, we designed an exosome booster system. KIBRA has been reported to stabilize Rab27 by inhibiting the degradation of Rab27a by proteasomes, which has been shown to promote the release of exosomes in vivo and in vitro. By overexpressing KIBRA in siRNA producing cells, we can speed up the production of therapeutic exosomes and boost the whole therapeutic systems[9].

Tet-On System

The Tet-on system consists of a regulatory expression module and a response expression module. The regulatory expression module contains a human cytomegalovirus early promoter (PhCMV) and a reverse tetracycline transcriptional activator (rtTA). Response expression module consists of Tet-responsive element (TRE) and minimal CMV promoter (PminCMV) In the absence of Dox, rtTA cannot bind to TRE, resulting in suppressed gene expression; in the presence of Dox, rtTA can bind to TRE, which in turn activates PminCMV to enable gene expression[10].

Cre-loxP

The Cre recombinase recognizes reverse repeats at both ends of the loxP site The DNA sequence between the two loxP sites was cut off by Cre recombinase and quickly link by DNA ligase. The result of recombination depends on the location and direction of loxP sites. In our design, two loxP sites are located on the same DNA strand in the opposite direction, the Cre recombinase will inverse the sequence between loxP[10].
Experimental Design
In order to realize our idea, we rationally designed hierarchical genetic circuits that allows the verification of each parts and the free combination of different functional modules. Then we confirm the expression and function of each part in vitro and in vivo, followed by the confirmation of each system. After that, we will package the whole system with into a lentivirus delivery system, of which the pathogenic genes have been deleted. The lentivirus can effectively infect human cells and be reprogrammed into a exosome production unit by continuous expression of the circuits. Eventually, we will test the therapeutic effect of our strategy in KRAS mutation driven lung cancer mice model.

pCMV-nSMase2 and pCMV-SDC4-STEAP3-NadB plasmids used by the previous iGEM team to increase the efficiency of cell exosomes were compared with our KIBRA part.

Expression of siRNA
First, we detected the correct expression of KRAS, PD-L1 and CD47 siRNA through RT-QPCR to ensure that sufficient targeted siRNA could be produced in cell. CMV-iRGD-siRP/C/K is the part which expressed siRNA targeted PD-L1/CD47/KRAS. CMV-iRGD-siRP+C+K is a Composite part which expressed all three siRNA. At the same time, we detected siRNA in exosomes produced by HEK293T cells to verify the expression of siRNA in exosomes, confirmed that siRNA could be properly wrapped into exosomes, and prepared for its role (Figure.2).

Figure1. siRNA is overexpressed in HEK293T cell.



Figure2. siRNA is overexpressed in exosome.



In order to ensure the content of siRNA in exosomes, siRNA standard was used as the absolute quantification.

Figure.3 Absolute quantification of siRNA


Table1. siRNA Concentration in exosome and cell




According to the absolute quantification of siRNA, we found that the concentrations of all three kinds of siRNA are appropriate. Meanwhile, the concentration of siRNA in exosomes is about 5% of the siRNA in cells which can complete the treatment.
Exosome Booster part
As previously reported, pCMV-hSDC4-STEAP3-NadB can strongly promote exosome release. To develop the more efficient booster part for our strategy, we designed another two parts (pCMV-KIBRA and pCMV-nSMase2) to find out the most efficient design for our project. We tested all the three designs in vitro. The expression of each mRNA was confirmed by RT-qPCR after transfected to HEK293T cells.

To further verify the expression of KIBRA and nSMase2 at protein level, we used Western Blotting experiment to prove that KIBRA and nSMase2 was indeed overexpressed in HEK293T cells.

Figure 1. KIBRA, NadB, nSMase2 mRNA relative expression in HEK293T cell (vs GADPH)


Figure 2. (A)Western Blotting result shows that nSMase2 is overexpressed in HEK293T cell.
(B) Western Blotting result shows that KIBRA is overexpressed in HEK293T cell.


Figure 3. Total amounts of exosomes (shown as total protein) secreted by HEK293 cells with the introduction of nSMase2, KIBRA and hSDC4-STEAP3-NadB.


After confirming the correct expression of these three genes, we further compared their ability to promote exosome secretion. To simplify the process, we first detected the total concentration of the exosomes produced by cells transfected each part. The result indicated that overexpression of KIBRA generates the highest amount of exosomes among these three groups. So we chose KIBRA as the candidate for further characterization.


KIBRA promotes exosome release

To further evaluate the effeciency of KIBRA in promoting exosome release, HEK293T cells were transfected with the KIBRA part, and the exosomes in cell culture medium were characterized. Nanoparticle tracking analysis (NTA) revealed that higher amount of exosomes was secreted in KIBRA-overexpressing group than the control group, while similar size distribution peaking at 124-127 nm was observed in both groups. The result verified that the exosome booster part can significantly promote exosome release without changing the natural size of exosomes produced.

Figure 4.(A) Averaged FTLA Concentration / Size for Exosome
(B) Intensity / Size graph for Exosome

Expression of iRGD-Lamp2b

We transfected the relevant plasmids into HEK293T cells, and iRGD-Lamp2b overexpression was detected by RT-QPCR.The targeting of iRGD-Lamp2b to lung cancer cells has been fully studied, so we have sufficient reasons to believe that it can achieve a good target.

Figure 1. iRGD-Lamp2b is expressed in HEK293T cells.

Conclusions and future work

Conclusion

We verified siRNA overexpression at the cellular level and measured the relative content of siRNA in cells and exosomes. The modeling results shows that packaging efficiency and transfer efficiency of siRNA.

We also demonstrated that KIBRA could effectively promote exosome release, which would facilitate siRNA to function as a drug.

The modeling result shows the pharmacokinetic characteristics of doxycycline in mice and the initiation of different concentrations of doxycycline in the Tet-on system, which is very helpful for our subsequent experiments.

Future work

We will verify the pharmacokinetic parameters of doxycycline and fit the relevant data in the experiment.

We will characterize the effect of siRNA on the knockdown of related mRNA in lung cancer cells and verify the efficacy in mice.

We will choose the two-phase project and complete the overall experiment of the project next year.
Human Practice
Our project is closely integrated with society. We started with a thesis about cancer patients during the epidemic. Through the paper and the information we have collected, we understood the difficult situation of cancer patients during the epidemic. We decided to do something to help them. Then we interviewed some cancer doctors and professors who research cancer therapy. According to their suggestions, we added KIBRA protein and Dox switch, which made our project safer and reliable. Our goal is to create a safe and effective treatment. However, scientific perfection does not mean social acceptance. Therefore, we launched investigations into the acceptance and ethical issues of modified organs. Through interview and questionnaire, we proved that if our project is safe enough, it can be recognized by the society. We also interviewed the company, which promoted the completion of industrialization of our project.
References and Acknowledgements

Acknowledgement

It has been a long way for NJU-China from organizing the team to writing this. Because of COVID-19, this team should not have existed, but it was Professor Chen Xi and Gao Zhanyu who helped make it happen. It's been 239 days since we started forming our team at the end of February. In the process, we thought about giving up and failing many times, but it was PI, Instructor and Advisor who helped us along the way. We really appreciate your help.


At the same time, I would also like to thank every student for their contributions and efforts, and it is your efforts that have finally turned our project into this.From a vision to a reality, thank you all for your efforts.


Thanks for the funding support from M3 lab, Department of Life Science and Office of Academic Affairs of Nanjing University, who provided the laboratory and required material involved in the experiments of our project. Thank NJU Advanced Institution for Life Science and Jiangsu Engineering Center for MicroRNA Biology and Technology for the technical support. Thank Nanjing Drum-tower Hospital and Tianjin Medical University Cancer Hospital, General Hospital of Eastern Theater Command, for providing the opportunity of human practice. Thank GenScript and GeneChem for the opportunity to participate in the exchange and discussion, for the industrialization of the project to provide the possibility. Thank Snapgene for your sponsorship.



References

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[2]Chen, W., Zheng, R., Baade, P.D., Zhang, S., Zeng, H., Bray, F., Jemal, A., Yu, X.Q. and He, J. (2016), Cancer statistics in China, 2015. CA: A Cancer Journal for Clinicians, 66: 115-132.
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[7]Kristina Iribarren, Aitziber Buque, Laura Mondragon, Wei Xie, Sarah Lévesque, Jonathan Pol, Laurence Zitvogel, Oliver Kepp & Guido Kroemer (2019) Anticancer effects of anti-CD47 immunotherapy in vivo, OncoImmunology, 8:3.
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[9]Song L, Tang S, Han X, et al. KIBRA controls exosome secretion via inhibiting the proteasomal degradation of Rab27a. Nat Commun. 2019;10(1):1639. Published 2019 Apr 9.
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