Team:KEYSTONE A/Poster

Poster: KEYSTONE_A

It's not ALRIGHT It's ARTHRITIS

Symonne Liu, Lavender Li, Alex Wu, Wendy Wu, Annie Sun, Sarina Liang, Anna Ren, Benedict Lu, Kevin Deng

Presented by KEYSTONE_A 2020

Abstract

Inspired by “Gaoyao” (an active patch with medical properties) in traditional Chinese medicine, we aim to tackle arthritis by designing a living Gaoyao. Its matrix is bacterial cellulose, a biomaterial with excellent biological compatibility and physical properties. It is embedded with engineered mammalian therapeutic cells harboring a modular genetic circuit, which contains: (1) a sensor with (2) an amplifier to detect trace amounts of inflammation signals at the early stage; (3) an effector that can secret antagonists of pro-inflammatory cytokines to suppress inflammation, BMP2 to activate skeletal stem-cells, and glucosamine to provide building blocks for cartilage; (4) a safety switch, enabling people to externally control the system. It can provide long-term treatment for arthritis, which not only suppresses inflammation before damages arise, but also promotes cartilage regeneration.

Introduction

Arthritis is a common disease caused by infection, inflammation, degeneration, and trauma. People encountering this disease usually have extreme pain and swell, which possibly could evolve as malformation of the joint for some extreme cases. The usual symptoms of arthritis includes extreme pain when lifting their arms, legs, and other joints in our body, which is typically unavoidable in modern treatments. In this aging society, arthritis hunts 355 million people around the world, yet there is no radical cure for it. Driven by the desire to help these patients, and inspired by traditional Chinese medicine concept, ”Gaoyao” (an active patch with medical properties), we aim to tackle arthritis by designing a living “Gaoyao”. Its matrix is bacterial cellulose, a biomaterial with excellent biological compatibility and physical properties. It is embedded with engineered mammalian therapeutic cells harboring a modular genetic circuit. Overall, our living “Gaoyao” is aimed to provide a long-term treatment for arthritis, which not only suppresses inflammation before damages arise, but also promotes cartilage regeneration.

Our Goal

Although none of the members of KEYSTONE_A are patients of arthritis, we have witnessed our parents and grandparents suffering from it. On march 2020, nine of the group members came to the common realization: arthritis is not alright. Through the power of synthetic biology, we want to enlighten and inspire the modern treatment, allowing the numerous patients to step into a life without constraints. To achieve this goal, we want to design a long-term cure to reduce arthritis, which can: 1) Reduce inflammation in the synovial capsule. 2) Encourage cartilage regeneration.

Inspiration

One special thing about our design is that we are deeply inspired by traditional Chinese medicine (TCM). In fact, “Gaoyao” is a common external TCM treatment for arthritis. TCM practitioner immerses a layer of skin-like material in the effective Chinese herbal medicine, creating a “Gaoyao”. Then, the practitioner applies this “Gaoyao” to the affected area of the arthritis patient. As the medical substances of the herbs diffuse under the skin, the patient feels less pain. Although the clinical value of “Gaoyao” is not fully recognized by the western scientific community, members of KEYSTONE_A find the concept of “Gaoyao” conceptually intriguing: applying for herbal medicine on a soft material, and then apply this material on the affected part. How smart is that! By drawing inspiration from TCM in this synthetic biology competition, KEYSTONE_A wants to show the world the scientific potential in traditional Chinese culture.

Problem

In this aging society, arthritis hunts 355 million people around the world, yet there is no radical cure for it. It is a common disease caused by infection, inflammation， degeneration, and trauma. People encountering this disease usually have extreme pain and swell, which possibly could evolve as malformation of the joint for some extreme cases. The usual symptoms of arthritis includes extreme pain when lifting their arms, legs, and other joints in our body, which is typically unavoidable in modern treatments.

Statistics

“Arthritis affects more than one in four adults.”

“9.6% men and 18.0% women Over age 60 suffer from symptomatic osteoarthritis.”

“Of the 54.4 million adults diagnosed with arthritis by doctors, 43.5% are restricted in daily activities”

Although none of the members of KEYSTONE_A are patients of arthritis, we have witnessed our parents and grandparents suffering from it. On march 2020, nine of the group members came to the common realization: arthritis is not alright. Through the power of synthetic biology, we want to enlighten and inspire the modern treatment, allowing the numerous patients to step into a life without constraints.

Our Idea

Our Gaoyao aims at providing long-term treatment for arthritis, which not only suppresses inflammation before damages and pains, but also promotes cartilage regeneration. It is composed of two components: the bacterial cellulose matrix, and engineered mammalian cells.

The bacterial cellulose, a biomaterial with excellent biological compatibility and physical properties, is used to provide a foundation to harbor engineered mammalian cells. Meanwhile, it is embedded with engineered mammalian therapeutic cells harboring a modular genetic circuit, which contains:

1.A sensor.

2.An amplifier to amplify system’s output.

3.An effector that can secret antagonists of pro-inflammatory cytokines to suppress inflammation, BMP2 to activate skeletal stem-cells, and glucosamine to provide building blocks for cartilage.

4.A thresholder to prevent the over-activation of the system.

5.A safety switch enabling people to externally control the system.

Engineering

We designed to have engineered mammalian therapeutic cells to harbor a modular genetic circuit, which contains a sensor with an amplifier to detect trace amounts of inflammation signals at the early stage, an effector that can secret antagonists of pro-inflammatory cytokines (IL4) to suppress inflammation, BMP2 to activate skeletal stem-cells, glucosamine to provide building blocks for cartilage, a thresholder to avoid the over-activation, and a safety switch, enabling people to externally control the system.

Sensor

NF-kB acts like sensor to sense the common immune factor released from both innate and adaptive immune reaction. The NF-kB factor binds to the sensor to initiate the translation of transcription factor, GVP16. GVP16 as a transcription factor will bind to the amplifier UAS regulatory region to to initiate more expression of GVP16. The transcription factor will also bind to the UAS regulatory region on the effector to produce more target modified mRNA. The RNA will translate through multi copy expression. The SD sequence and translation codon is prepared before each desired protein, same as the stop codon. This helps the expressed mRNA to translate three desired protein at the same time without messing up its amino acid sequence and structure. However, the effector gene needs only one promoter to guide the transcription.

Thresholder

The thresholder circuit is made possible because of Gal4 protein, a transcriptional factor without VP16 activation domain. The idea of Gal4 acts like a threshold is based on the competition between Gal4 and Gv16 acting on the UAS site. This transcriptional factor will bind to the UAS operator in the amplifier and effector module, which acts as a threshold for the system activation. According to the model simulations, the thresholder decreased the unwanted background activity while retaining high amplification.

Repressor and inhibition circuit

To enable the external control over the activity of the device, an inducible OFF-switch should be constructed to shutdown the system. A repressor named rtTR-KRAB is introduced through another independent module (PCMV-rtTR-KRAB). The switch can reset the expression of effectors, anti-inflammatory factors the product aims to produce. The binding site of rtTR-KRAB, TRE, is set into other three main expression-related module. The rtTR-KRAB is designed to locate on the upper stream of other binding sites and promoter to avoid further disruption of pre-existed sections, which could potentially impair the function (TRE-PNF-kB2-PMIN-GV16-myc; TRE-UAS-PMIN-GV16- myc). The repressor only works efficiently when the addition of a simple molecule, Dox. Efficient and dose-dependent repression was achieved through the requirement of combining simple molecule DOX and repressor rtTR-KRAB to activate the repression.

Bacterial Cellulose

Bacterial cellulose (BC), also known as microbial cellulose, is a biodegradable, natural cellulose that is synthesized by bacteria. The diameter of BC fibers is 20–100 nm. BC has a special physical and mechanical characteristic. We designed a method to produce composites with homogeneous size distribution of silver nanoparticles. This structure provides a robust, highly-porous and self-sustaining structure with large surface area, which is essential to facilitate incorporation of the silver ions in the metallization process to give a high silver loading content. The combination of the antibacterial efficacy of the silver nanoparticles and the biodegradability of the BC fibers in the composite fibers can make them practical for use as antimicrobial membranes in medical applications.

We designed this procedure because The implementing of our ”Gaoyao” must be done through a medical surgery, where an incision is needed to operate on the patient’s knee joint. When an incision takes place, possibility of postoperative infection (POI) becomes inevitable. In order to prevent POI, we designed this approach to attach silver microparticles on the bacterial cellulose film to give the matrix an anti-microbial ability.

The antibacterial principle of silver ion interferes with the synthesis of the cell wall. Peptidoglycan is an important component of the bacterial cell wall. The interference effect of silver ion antibacterial agent on cell wall mainly inhibits the connection between polysaccharide chain and polypeptide and makes the whole peptidoglycan wall deform. That makes the cell wall loses its integrity, protective effect on osmotic pressure, resulting in the death of bacteria.

modelling

A simple deterministic model with ordinary differential equations(ODEs) was constructed using CellDesigner 4.4 software. The model guided us to design and optimize our system step by step. It also demonstrate the feasibilityof our project, since all expected performance of our system can be observed in the simulation result, although no experiment was done.

State transition diagram

First, we constructed a state transition diagram representing the genetic circuit. The usage of symbols obeys certain rules described in reference and. The background color of DNA-protein complex indicates its state (red-blocked, green-transcriptional activated). The identification number, meaning, parameter of each element and reaction are described in detail, respectively, which are at the end of model section.

Engineering Success

The simulations were performed using built-in ODE Solver. We were particularly interested in a behavior of the device in the two states: where there was no input signal (non-stimulated), i.e. the NF-κB protein complex particle number was set to zero, and where the input signal had maximal value (stimulated).

Potential

Social Value

In this aging society, there is a huge population of arthritis patients. If our product can get successfully popularized, the easier method, simple implementation, and high suitability to the physiology of the aging population would significantly reduce the number and statistics of the arthritis patient. Hence, not only would the aging population gain better life, enhancing their treatment and status in the current Chinese society, the elimination of the constraints to the elderly’s life would also contribute to reducing the pressure on child-raising affairs in the younger population. Especially considering that the wide-spread diagnosis of arthritis have already been reaching the younger generation, in the working teenagers and young adults due to their long use of arm and leg joints, this product also provides a solution to their problems, helping enhance the working efficiency of firms. On the other hand, the invention of a simpler treatment would also alleviate working pressure of the doctors at the hospital, reducing the needs for them to repeatedly and frequently provide treatment to the same patient, providing better working space and healthy time schedule for doctors—a rare resource in China.

Business Value

Tour “Gaoyao” faces two end users: patients who have relevantly mild osteoarthritis- symptoms. Our project would raise sufficient societal value if implemented. Already did the Chinese society crowded with the aged population who suffers from arthritis. If our product can get successfully popularized, we can significantly reduce the suffering of arthritis patients. Besides, our project demonstrates business value. Since there is a huge arthritis patient population around the world, our product has an enormous potential customer base. If executed, this can be lucrative.

References and Acknowledgements

Acknowledgements

We thank Songyuan Zhang, the senior iGEMer from Peking University with 3 years‘ experience in iGEM. He has provided us insightful comments about synthetic biology. We thank Ms. Mahendran, the secondary school teacher at Keystone Academy, Beijing. She helped to coordinate and manage our members.

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