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Description

An In-depth Overview and Objective.

Description

Abstract

Time is of the Essence. Synthetic biology has led to extraordinary progress on extensive issues: environmental, clinical, and industrial. Yet many of them fail to achieve practical application. Biomolecular signals do not travel through the cell at the speed of electricity. To make biological systems available to solve the world's ongoing problems, swiftness of response is often required. In 2022 UTokyo tackled the security concern of synthetic biology with the optogenetic passcode system, Optopass. Although we constructed a light-induced unlocking mechanism, it lacked rapid reacting. As the complexity of this system increases, the longer it takes to function. We strongly recognized the need to fundamentally address this problem. “SWIFT" is a revolutionary proposal for genetic circuit design that achieves the required speed and has the scalability to be optimally developed to meet the challenges of society. In this manner, we intend to bring about a transformation from the basic system of obtaining the protein of interest upon detection of a certain ligand.

Detection Unit

Synthetic biologists and iGEMers commonly require systems capable of detecting specific biomarkers and releasing specified substances when implementing engineering solutions in synthetic biology. Advances in synthetic biology have facilitated the programming of cells to exhibit new biological behaviors by employing synthetic receptors for ligand detection, with CAR-T therapy serving as a prime example. For the broad applicability of such innovative systems utilizing synthetic receptors, they must be "scalable" to detect various ligands. We have chosen the MESA system as the SWIFT detection unit and adapted it for rapidity. In principle, MESA can be utilized with any synthetic receptor that undergoes dimerization and can detect a wide array of ligands, thereby supporting "scalability" in SWIFT detection.

MESA

MESA is a synthetic receptor system capable of independent signaling distinct from endogenous signaling pathways 1. MESA utilizes the binding of ligands to synthetic receptors as input, leading to the release of the Protein Of Interest (POI) into the cytoplasm as the output. Because the signaling is exclusively triggered by the synthetic receptor response, MESA can function autonomously, operating independently of endogenous signaling pathways. The following provides an overview of the MESA system.

Overview_of_MESA Figure 1: Overview of MESA MESA comprises two types of chains: protease chains (PC) and target chains (TC). The protease chain consists of an ectodomain (ECD) that binds to the ligand, a scaffold on the cell membrane (SCF), a transmembrane domain (TM), a linker domain (LD), and a protease (PR). On the other hand, the target chain consists of a Cleavage Sequence (CS) of PR located below LD. The reaction mechanism of MESA is described below.

The binding of the ligand to the receptor induces receptor dimerization. Receptor dimerization brings the protease chain (PC) and target chain (TC) into physical proximity, and the protease downstream of the protease chain specifically recognizes and cleaves the Cleavage Sequence of the target chain. This process releases the Protein Of Interest (POI) into the cytoplasm, which was initially bounded to the cell membrane.

MESA is recognized as an easily tunable response system due to its modular components, including synthetic receptors and proteases. The ectodomain plays a crucial role by providing both specificity and affinity for the ligand. Ectodomain materials may consist of proteins that undergo dimerization upon ligand binding, such as ligand-binding domains derived from natural receptors or short-chain variable fragments (scVf) derived from monoclonal antibodies. Ligand binding can be homotypic, where the ectodomain on each MESA chain recognizes the same ligand for multivalent ligands (e.g., homodimers of many cytokines), or heterotypic, where the ectodomain on each MESA chain binds to different portions of a given ligand.

Furthermore, since MESAs operate through a Protease cleavage mechanism triggered by dimerization via synthetic receptors, MESAs comprising new EctoDomains and Proteases can effectively detect and signal various ligands while maintaining orthogonality with in vivo reactions. In summary, MESA demonstrates the capability to detect and transmit a wide variety of ligands.

In conclusion, MESA exhibits the ability to detect a diverse range of ligands, offers ease of response adjustments, and maintains orthogonality with in vivo responses, thereby establishing SWIFT as a fundamental and versatile system.

Why MESA?

Other response systems utilizing synthetic receptors include RASSL, Tango, SynNotch, CAR, and GEMS 23.

Other_response_system Figure 2: Other Response Systems

In comparison to these, MESA offers several advantages:

  1. MESA can detect a broader range of soluble ligands, facilitated by its ability to be designed in the presence of synthetic receptors.

  2. Design improvements are easily proposed for MESA due to the availability of existing measurement data on Protease and Ectodomain, essential components of MESAs.

  3. The addition of a new MESA allows for the detection of multiple types of ligands within a single cell.

In contrast:

  • RASSL is limited to detecting synthetic ligands and is not suitable for biomarkers like cytokines2.

  • SynNotch is unable to detect soluble ligands4.

  • GEMS and CAR face challenges in connecting to the Secretion system and lacks rapidity3.

  • Tango has limitations in detecting a large number of ligands4.

After considering these factors in comparison with other synthetic receptor-based detection systems, we have chosen to use MESA as our preferred detection unit.

Our Design

Detection_Unit Figure 3:Detection Unit

In the conventional MESA, transcription factors (TFs) such as tTA and Gal4 are typically chosen as the Protein Of Interest (POI), and gene expression is regulated by the release of TFs into the cytoplasm. In our modification, we replaced the TF component with a protease and integrated it with the subsequent Secretion Module, achieving the desired "rapidity" in SWIFT. Further details on the development of this Engineering cycle can be found on the Engineering Page.

Additionally, our research demonstrates that having an ample amount of protease in the early stages allows for a greater secretion compared to TF. Further information is available in the Modeling Page.

To sum up, we opted for MESA as the detection unit and adapted the conventional MESA by substituting the POI from TF to Protease. This design not only imparts "scalability" to SWIFT but also ensures "rapidity," thereby establishing SWIFT as a fundamental and versatile system.

Secretion Unit

By secreting the target protein in response to detection, we do not regulate at the transcriptional level, but at the secretory level.

Why Secretion?

As mentioned earlier, SWIFT is a system that requires "rapidity". In secretory control, secreted proteins are stored in the cell in advance for rapid secretion without transcription and translation by using KKYL, an ER retention signal. This design was provided by the results of the Dry lab.

Protease Inducible Secretion

We designed KKYL to cause rapid secretion from Protease input from MESA without transcription or translation.

KKYL is a Lys-Lys-Tyr-Leu amino acid sequence that is attached to the C-terminus of proteins that penetrate the ER membrane and are exposed on the cytoplasmic side, where it is recognized by specific receptors in the ER and cis-GA. When the recognized protein with KKYL is transported out of the ER, it is recovered back into the ER by retrograde transport of COP I-coated vesicles. By cutting off this KKYL with protease, the protein with KKYL attached can be sent to the downstream transport pathway5.

There are a variety of amino acid sequences for ER retention signals, but we chose KKYL because it has been reported to be a signal with particularly high retention performance6.

Previous Studies by Other iGEM Team

Previous iGEM projects have also used ER retention signals; Slovenia's 2016 project is designed to cut off retention signals by inducing protease activation from external mechanical stimuli7.

UPenn's 2022 project also uses ER retention signals; UPenn cuts off retention signals by light-induced activation of proteases8.

Our Design

Secretion_Unit Figure 4: Secretion Unit

In SWIFT, secreted proteins are retained in the ER and cis-GA in advance by KKYL. In addition, an input protease’s CS is inserted between KKYL and TM, because KKYL is not in the ER but on the cytosolic side. Proteases released into the cytosol by the MESA reaction can cleave this CS, thus separating KKYL from the secretory proteins.The KKYL-detached secretory protein is then transported through the Golgi layer.The secretory protein has a TM when KKYL is detached and cannot be secreted out of the cell if it is transported as is. Therefore, furin, a protease native to trans-GA, is used to separate the TM from the secretory protein. 5 For this purpose, SWIFT is designed with a furin cleavage site inserted between the TM and the secreted protein.

Amplification Unit

As mentioned above, our goals for SWIFT include orthogonality, which allows detection of multiple ligands without interfering with each other, and versatility, which allows the design to be reconfigured to meet the needs of various users. To achieve this, we have designed an Amplification System using Protease.

Our Design

Amplification_System Figure 5: Amplification Unit

First, a suitable quantity of a molecule that connects Protease (P2) is synthesized, intended for subsequent release, with a peptide (inhibitor domain) designed to block its activity within the cell, utilizing a linker. When designing this molecule, make sure that the linker is cleaved by Protease (P1) released by MESA. P1 is released from the dimerized MESA that detects the ligand and encounters the inhibitor domain of P2, it cleaves the linker connecting P2 and the inhibitor domain of P2. Then P2 and the inhibitor domain are separated, and P2 is activated. Once P1, which has cut the linker once, can cut other linkers without becoming inactive, Amplification is possible as a whole9.

Protease Precursor

In the method of using Protease inhibitors as the aforementioned inhibitor domain, the liberated inhibitor domain continues to inhibit P2 10. On the other hand, by utilizing the precursor of Protease2 and cleaving a part of its sequence with P1, P2 can be activated. Since this reaction is irreversible, it prevents inhibition of P2 after cleavage10. Therefore, although there is a disadvantage that P1 is determined from the sequence of P2 precursor, it becomes a superior system in terms of induction ratio10.

Design of Inhibitory Peptides

If the design using Protease precursors is available, there is no problem. However, this is because the sequence of the already known Protease precursor is used as it is, so the Protease that can be used as P1 is limited. This is because the sequence that P1 can cleave must be included in the precursor of P2.

To enhance SWIFT's goal of customization, we worked on designing an inhibitory peptide. Specifically, we first decided on the P1 and P2 we wanted to use, replaced part of the P2 precursor with P1's CleaveSite, confirmed the stereo structure and actual function, and checked for any problems. We were able to develop a perspective on the design of this inhibitory peptide through HP.

Advantages Over Other Systems

Combining the above three systems, the overall picture of SWIFT, is shown in Figure 6 below;

Our_system Figure 6: Our System - SWIFT

This system has the following advantages over existing systems.

Advantages Over Other Systems in Terms of Speed

Several methods can release proteins upon signal reception, apart from the Secretion Unit used in SWIFT. However, none of these methods are as rapid as the Secretion Unit. Let's take a closer look at each of these methods.

Transcriptional Activation System

One method used for programmable secretion control is the transcriptional activation system. In this approach, the release of a sequence known as the transcription factor (TF) initiates the transcription of a target gene, referred to as GOI (Gene Of Interest), as shown in the Figure 1 below.

Transcriptional_System Figure 7: Transcriptional System

TF is composed of two main components: a binding domain, which specifically attaches to the sequence right before the promoter, and an activating domain that stimulates the promoter. When the binding domain recognizes and binds to its target, it facilitates promoter activation11.

An exemplary setup uses TetR as the binding domain and VP16, a protein from the herpes simplex virus, as the activating domain. The introduction of doxycycline has been shown to enhance the GOI expression level by over 1000-fold, as it prompts the binding of TetR to the activating domain12.

However, before the desired protein is secreted using this method, it must undergo the following processes: (1) Transcription and translation of the protein (2) Secretion from the cell via the Golgi apparatus Step (1) consumes a notable amount of time, but our Secretion System has the advantage of pre-completing this phase. By initiating step (1) even before receiving the signal, our method facilitates a more swift secretion process. For specific time comparisons, please refer to the Modeling Page.

Riboswitch

A riboswitch regulates transcription and translation by capitalizing on conformational changes in mRNA, which occur due to the binding of small molecules. This molecular shift activates or deactivates a specific segment of the sequence, subsequently promoting or repressing transcription. Various riboswitch types have been documented in prokaryotes 13.

An illustrative approach to artificially implement a riboswitch involves modifying the activity of the IRES, the initiation site for protein translation, as depicted in Figure 2 below 14.

Riboswitch Figure 8: Riboswitch

In this approach, in the absence of Ligand, IRES is suppressed by anti-IRES, leading to a halt in translation. However, when Ligand is introduced, an mRNA segment termed 'aptamer' binds to the ligand. This binding instigates a conformational shift, activating IRES. It's crucial to understand that only the mRNA's three-dimensional structure undergoes this change; its primary structure remains unaffected. Consequently, the presence of Ligand can induce a spatial reconfiguration in mRNA, governing its translation.

Yet, as one might observe, this strategy, akin to the earlier mentioned transcriptional activation system, necessitates a translation phase prior to protein secretion through the Golgi apparatus. This makes it less expedient compared to our Secretion system.

Other Inducible Secretion Systems

SWIFT stands out for its 'rapidity' and 'extensibility.' While there are several known inducible secretion methods besides our approach in the Secretion Unit, most don't involve transcription, making their speed roughly comparable to ours. However, when we shift our focus to scalability, these alternate methods fall short. Let's dive deeper to elucidate this.

VSVG Method

The VSVG method is known as the classical method of controlling secretion15. This method takes advantage of the property of ER to retain misfolded proteins16.

VSVG_method Figure 9: VSVG Method

In the VSVG method, the lumenal domain of the temperature-sensitive viral glycoprotein, VSVGtsO45, is fused to the protein slated for secretion. At around 32 degrees Celsius, VSVGtsO45 folds correctly, but at 40 degrees, it misfolds. This misfolding at 40 degrees causes it to be retained in the ER, preventing the protein's extracellular secretion.

The primary trigger in this method is temperature, which poses challenges. Not only does it make ligand detection and secretion induction difficult, but it also limits the environmental contexts where this method can be employed. Moreover, if having VSVGtsO45 fused to the secreted protein is undesirable, there's a need to devise an additional mechanism to detach it post-secretion.

RUSH Method

The RUSH method, also recognized as a secretion control technique17 , employs a distinctive mechanism. In this approach, two proteins are expressed on the ER membrane: one is a fusion of the 'hook' and streptavidin, and the other combines a reporter with SBP. Upon the addition of Biotin, a vitamin, it binds competitively to Streptavidin. This binding event releases the reporter-SBP from the hook-Streptavidin complex on the ER, facilitating its transport to the Golgi. By fusing the desired protein with reporter-SBP, it is possible to control the secretion of the protein you want to secrete.

RUSH_method Figure 10: RUSH Method

In the RUSH method, the trigger is confined to molecules that can permeate membranes, such as Biotin. This constraint hinders its compatibility with ligand detection systems like MESA, making it less versatile compared to our Secretion system when integrating into diverse platforms.

Application to CRS

SWIFT is useful in various situations where "scalability" and "speed" are required, such as bioremediation, therapeutics, and biomanufacturing. As an example of where speed can be the deciding factor between life and death, we worked on a social implementation in therapeutics, particularly in dealing with Cytokine Release Syndrome.

Swift_for_CRS Figure 11: SWIFT for CRS

Current CRS Situation

Cytokine release syndrome (CRS) is "a systemic inflammatory response that can be triggered by a variety of factors such as infections and certain drugs."18 It is caused by a rapid increase in inflammatory cytokines such as IL-6 and IL-1. It is a frequent side effect of CAR-T cell therapy treatment and hematopoietic stem cell transplantation, and is also caused by viral infections. It brings various symptoms such as high fever, headache, low blood pressure, and nausea in patients. In severe cases, it is called cytokine storm, and there have been reports of deaths, making it a serious problem that needs to be addressed in medicine.

CRS is a systemic inflammatory reaction that occurs instantaneously and requires a rapid response 19, and currently, tocilizumab, an IL-6 receptor antibody, is mainly administered to patients. However, there are precedents in which CRS occurs even when the function of IL-6 is suppressed 20, suggesting that IL-6 is not the only mediator that causes CRS. In fact, there are several CRS cases that cannot be suppressed with tocilizumab. Given that CRS mediators vary by individual and condition, new CRS coping systems need to be scalable to address a variety of inflammatory cytokines in addition to rapidity.

SWIFT for CRS

SWIFT for CRS is based on an auto-regulation system that detects and immediately suppresses inflammatory cytokines by combining the Detection / Secretion / Amplification unit described above.

There are individual and symptomatic differences in which inflammatory cytokines are the major mediators in CRS. Therefore, by creating a system like the one shown in the figure below, we can create a system that detects various cytokines and returns separate responses accordingly.

MESA_Do_not_Engage_Each_Other Figure 12: Scalability of SWIFT's Detection

The rapidity and scalability of SWIFT's detection targets can thus be optimized and applied to the medical problem of CRS. (For more information, please See Proposed Implementation Page.)*

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