Discover the 'Decholesterizer' - the IITChicago iGEM team project!

As part of our partnership with iGEM, the biggest synthetic biology competition in the world, we are showcasing some of the exciting projects from teams that share our vision of synbio impacting positively on human health. This week, we've let the IITChicago take over our blog to share what they are working on. Their team is unique in the way it combines a student organisation (IIT iGEM club) with an interdisciplinary, course-linked interprofessional project (IPRO.) IPRO classes bring students from diverse disciplines together to collaboratively address a common problem. This specific IPRO, known as "Engineering Life," attempts to take a multidisciplinary approach to solve pressing problems in health, environment, or energy using synthetic biology.

The problem we want to solve

High cholesterol is a serious health problem affecting 94 million adults in the U.S alone. Cholesterol is a steroid and an important structural element of the cell membrane. In moderate amounts, cholesterol is vital for the production of hormones and vitamin D in the body. However, high cholesterol levels in the blood result in the formation of arterial plaques that can eventually lead to heart attack and stroke, which are the 1st and 3rd leading causes of death in developed societies. The most commonly used treatment for high cholesterol are drugs such as statins which are used by over 92 million Americans. Unfortunately, the prolonged intake of cholesterol-lowering medication might cause mild side effects such as headache or nausea, as well as serious side effects such as muscle damage or risk for diabetes; therefore, there is a need for a more permanent and less adverse solution. Another important aspect is that statins target just one of the enzymes in the cholesterol biosynthesis pathway. Since many of the enzymes in that pathway have been difficult to target using small-molecule drugs, synthetic biology offers an opportunity to regulate their activity. 

Our solution 

Using synthetic biology, we aim to harness the power of a patient's own genetic system to regulate cholesterol production. To validate, our team is developing a synthetic cholesterol regulatory circuit to regulate cholesterol synthesis in HepG2 liver cells or hepatocytes. We chose hepatocytes because they are the primary site of cholesterol synthesis in the body. The synthetic circuit is composed of two major components: the detection mechanism and the regulatory mechanism. The detection mechanism detects high levels of cholesterol in the liver cells and induces the response of the regulatory mechanism. In response to the induction, the regulatory mechanism inhibits the production of cholesterol in the liver cells.

The liver cells modified by this circuit would be able to detect changes in cholesterol levels and down-regulate cholesterol synthesis when levels are high. Our approach allows us to target multiple steps in the cholesterol biosynthetic pathway in an effort to provide an alternative, patient-tailored treatment for hyperlipidemia.

Given the nature of our project, we have elected to name it aptly: The Decholesterizer.

The approach and the concept 

Our team decided the best way to tackle this project was to separate into 4 subteams: The cell Maintenance team, the Vector Design team, the Regulatory Response team, and the Public Relations team - each responsible for different aspects of the project.

Cell Maintenance

Our work depends on healthy cell lines of HepG2 cells. In order to keep the cells happy and healthy, the Cell Maintenance team performs media changes and cell splitting on a near-daily basis. The Cell team is also working towards developing stable HepG2 cell lines in 3 different levels of cholesterol: low, regular, and high, as we need a basis for future testing of the detection and regulatory mechanisms. The cell team is also responsible for testing the activity of the promoter designed by the Vector Design team using a Dual-Luciferase Assay. The Cell team also contributes to the work of the Regulatory Response Team by performing small interfering RNA (siRNA) transfections of HepG2 cells for validation purposes. Most recently, however, the Cell team conducted a transfection optimisation experiment using GFP DNA in our HepG2 cells to determine the most effective ratio of transfection agent Lipofectamine to DNA for consistent, successful transfections.

Detection Mechanism 

The detection mechanism of our genetic circuit relies on the sterol regulatory element binding proteins (SREBP) system which contains sterol regulatory elements (SRE). These SRE elements were transferred into a cytomegalovirus (CMV) promoter creating an engineered pSRE promoter that acts as a cholesterol biosensor. Cholesterol-induced activation of the promoter initiates the transcription of the vector, including the short hairpin RNA (shRNA) which acts as a regulatory mechanism for cholesterol synthesis. Based on a dual-luciferase assay, performed to test the activity of the promoter, the current pSRE promoter responds to low levels of cholesterol. The Vector Design team is actively working on improving the promoter system by inverting the detection mechanism so that it can be activated only by high cholesterol concentration. Once the Vector Design team identifies the desired promoter mechanism, it will be incorporated into a cholesterol regulatory circuit along with the shRNA, the regulatory portion of our circuit, designed and tested by the Regulatory Response team.

Our initial circuit design was composed of a cholesterol sensor, the SRE promoter, and a response element, siRNA which inhibits some gene in cholesterol biosynthesis. There are many genes that we are investigating; our team has so far validated an effective siRNA against SQLE, squalene epoxidase. In this fashion, the cell can sense cholesterol level and adjust cholesterol biosynthesis.

However, when we began to validate the sensor, we realized that our circuit worked in the wrong direction, inhibiting cholesterol synthesis when cholesterol is low and allowing it to continue when cholesterol is high. Which is why we created circuit design V3.

In our most current circuit design, we added an inverter element between the SRE promoter and the siRNA response element to invert the regulation of siRNA.

In a low cholesterol condition, the SRE promoter will be activated causing repressor X to be transcribed. Which will then bind to promoter X, the promoter for the siRNA sequence, and prevent the siRNA from being transcribed. Thus, allowing cholesterol synthesis to proceed.

The X protein is engineered to contain a degron, which are tunable elements providing various degradation rates. This allows us to tune the degradation rate to eliminate X rapidly enough when the SRE promoter is off, but allow it to accumulate when the promoter is on. We are currently investigating vaious X repression systems and degron elements.

In a high cholesterol condition, the SRE promoter will not be active, stopping the expression of X which then allows promoter X to turn on, producing the siRNA. Therefore inhibiting cholesterol
Production.

Regulatory Mechanism

The Regulatory Response team has, through RNA extraction and quantification of gene expression using qPCR, identified 5 viable gene targets for inhibitory response. All target genes code for enzymes catalysing the reactions in the cholesterol synthesis pathway. Ideally, inhibition of those genes would stop the production of cholesterol and lower the levels of cholesterol in the body. The Regulatory Response team designed the siRNAs that would block the transcription of the target genes. Currently, the team is working towards validating which of the siRNAs can successfully inhibit the transcription of target genes through the comparison of the quantified gene expression between the regular cells and cells transfected with the siRNA. Once the siRNAs are validated, one or multiple designed siRNAs will be incorporated in the circuit in the form of shRNAs, along with the promoter designed by the Vector Design Team to serve as a regulatory mechanism.  

Currently, we are working with 5 enzymes: HMGCR, MVD, FDFT1, SQLE, and DHCR24. Each catalyses a unique step in the cholesterol biosynthesis pathway.

Public Relations (PR)

The PR team is responsible for broadcasting our project to a wider audience, as well as coordinating communication with iGEM and sponsors supporting our project. The PR team is responsible for all of IITChicago’s promotional materials including our 2023 iGEM Project Wiki Page and our 15-minute presentation video for our project: the Decholesterizer. Currently, they are working on the development of a new confluency tool that will aid our team in its lab efforts.