Team:SCU-WestChina/Poster

Lung Knight

Presented by SCU-WestChina 2020

Wang Mingyao¹,Liu Haoyang¹,Zhang Zhidong¹,Wang Kang¹,Hu Jungen²,Yang Hao³,Xie Huiqi³, and State Key Laboratory of Biotherapy^§

¹iGEM Student Member,²iGEM team mentor,³iGEM team PI,⁴ faculty sponsor,State Key Laboratory of Biotherapy,Sichuan University,Chengdu,Sichuan,China

Abstract

Our project has three aims: to design functional peptide drugs for pulmonary fibrosis patients and test their function, to establish an appropriate liposome delivery system and make convenience for patients’ application, and to propose a screening platform for drugs swift selection and optimization. We applied ITAASER and Z-DOCK to select our peptides, then express them in vitro and in vivo to achieve quick selection and verification. After that, we use SDS-PAGE and HPLC to verify their expression, and use RT-qPCR, IHC to verify their function. Then, Gastroplus was applied to verify our delivery system can improve the function of our peptide drugs. Furthermore, we implement our concept of drug selection and optimization platform so that we can form a virtuous cycle to find better drugs for PF patients. We are honored to provide protocols of expression in vitro and pharmacokinetic study with Gastroplus, and we believe our project can explore the application of synthetic biology to drugs development and bring new ideas for iGEM community.

What is PF？

Pulmonary fibrosis, known as “non-cancerous cancer,” is a chronic, progressive lung disease of unknown etiology that causes pulmonary fibrosis and leads to loss of lung function.

In recent years, the incidence of PF in China has been rising gradually, with a high mortality rate and a short survival time. According to statistics, there are more than 300 million idiopathic pulmonary fibrosis patients in China, costing nearly 2 billion US dollars. There is no clear cause of IPF. Smoking, dust and infection are all risk factors for IPF

PF drug demanding

Both nidanib and pirfenidone, commonly used in IPF in The Chinese market, are faced with such adverse factors as high price, poor curative effect, lack of complete treatment, high drug dependence, and severe toxic and side effects.

Therefore, it is of great significance to create and invent a highly targeted drug to overcome the shortcomings of existing treatment schemes, precisely target pulmonary fibrosis tissue, and reverse the fibrosis process.

Integrate HP

I:Pathway Blocking

II: Digestion Activation

III: Liposome Delivery

IV: Future Work

Design

TGF-β truncated Receptor

TGF-β is a key factor in triggering fibrosis by inducing the activation and differentiation of myofibroblasts, which leads to excessive production of extracellular matrix then forming fibrosis in organs or tissues.

PDGFβ truncated Receptor

Activated monocytes and macrophages in culture have been shown to produce several growth factors including platelet-derived growth factor (PDGF). PDGF is a potent mitogen and chemoattractant for fibroblasts and smooth muscle cells and a stimulator of collagen synthesis. Therefore, PDGFβ truncated receptor also has therapeutic potential for pulmonary fibrosis.

Plasmin has been used in the treatment of thrombosis. As a proteolytic enzyme, plasmin is also promising in applications in terminal pulmonary fibrotic diseases .
In consideration of controlling side effects, we did not choose plasminogen activating peptides with enzymatic activity, but peptides with carboxyl terminal lysine.

A2-Antiplasmin (a2AP) interferes with the binding of plasminogen to fibrin because lysine residues in its carboxy-terminal region compete with those in fibrin.

We encapsulate the drug proteins into liposomes and make aerosols. In this case, liposome agents will be inhaled into the lungs in the form of aerosols to achieve a local high concentration, limiting the scope of drug action from a general level, and improving drug targeting. We inserted a lung-targeted probe into the liposomes to achieve the 3-step system: nidus-targeted, collagen-targeted and disease-treatment.

Seen from above, in our several experimental phases, we have effectively followed and used the practical model of "design-expression-verification-optimized design-optimized configuration" in an innovative way. This model has brought us more inspiration and made our projects better, and we want to further iterate and optimize the protein drug. We also hope to promote the cell-free protein expression technology through the iGEM community and exchange design ideas with researchers in related research fields, so as to jointly promote the research of fibrosis treatment

Result

We successfully synthesized M7824-probes by cell-free expression system and PDGFβR-TRIAL by E.Coli system,and verified its expression with SDS-PAGE.

And we proved that PDGF-truncated-receptor worked well to depress tubular epithelial-myofibroblast trans-differentiation and collagen I deposition by IHC and RT-qpCR.

Modeling

Z-DOCK to obtain the parameters of four truncated receptors.
I-TASSER for molecular docking and stability experiments to select the collagen-binding targeting probes with suitable KD values.We first chose M7824 as our truncated receptor （strong binding ability to TGF-β ） Next, we wanted to choose a suitable probe. As the affinities of the three selected probes differ by 100 times, we are interested in which affinity level is better for fibrosis treatment.

Our next task is to simulate the pharmacokinetics and pharmacodynamic parameters of protein drugs in animal models and human bodies to understand the metabolic process of drugs. We use the PBPK model prediction software GastroPlus^TM to predict concentration-time (Cp-time) curves. AP26 and SAK (two activation peptides) are our research object.
The results show that :
(1)compared with intravenous administration, lung administration can obtain a higher bioavailability, both in humans and rats.
(2)AP26 showed a higher drug deposition rate in lung, higher bioavailability and faster clearance rate, representing a potential smaller side effect.

We predict the effect of liposome delivery system on drug metabolism. We successfully construct the release curve model of sustained-release preparations based on transdermal administration in vitro and predicted the PK curve which shows a significantly increased exposure and bioavailability after the use of sustained-release preparations.

We wanted to provide more options for probes loaded on liposomes. The first step in designing a probe is to choose a suitable target which can localize the liposomes to fibrotic area. We selected three targets after investigation: TGF-β, type Ⅰ collagen and macrophages related to fibrosis. We then screened for proteins that can bind to these targets and use I-TASSER to find the alpha helix in recognition domain of the selected proteins. These sequences composed of alpha helix may become potential probes. We will use wet experiments to verify the affinity and other properties of these probes in the future.

Street Interview

Use the experience of wearing a mask to introduce disease introduction We asked respondents how they feel when they wear multi-layer masks and when they exercise or sleep. To trigger interviewees’ feelings about the symptoms of patients with pulmonary fibrosis

Use sponge balls to simulate lungs to introduce pulmonary fibrosis We compared a sponge ball that was fully immersed in water and expanded and softened with a sponge ball filled with glue to simulate normal lungs and lungs of patients with pulmonary fibrosis.

Introduce the project through ppt Explain our project to interviewees.
Respondents put forward prospects after understanding the project Respondents expressed their expectation of the project and their prospects and development.

Sunny

In order to promote the knowledge of wildlife protection, we cooperated with the creative volunteer organization SK Sunny to jointly plan and carry out a series of public welfare activities.
The theme of our charity event is "There will be no winner after the gunfire". During the event, We launched activities as : interesting painting creation, public science education, video dubbing etc. . At least 2500 people participated in or viewed our online activities.

Our team members also created a series of paintings to have a charity sale with the SK Sunny and finally donated the proceeds to the Chengdu Longqiao Black Bear Rescue Center

Sharing: MoClo Kit

We made a specific business plan according to the market research and questionnaire survey. We have obvious price advantage comparing with other treatment method like Nintedanib and lung transplantation.

The drug research and development is directly carried out by the Lung Knight team.

After OEM mass production, drugs will be sold and promoted in general hospitals and chronic disease rehabilitation centers, and later technological innovation and upgrades will be carried out based on patient feedback.