Proposed Implementation
Abstract
The synthetic biological system called “SWIFT", which is a research product of iGEM UTokyo has the ability to detect the ligand and secrete the target protein accordingly. With its feature of rapid response to various ligands, the system is potentially useful in situations where speed and scalability are required.
The system would be very useful as a biosensor or for medical applications. Examples for the former include environmental monitoring, drug screening, toxicity evaluation. For the latter, CRS (cytokine release syndrome) and peptide hormone regulation are expected as examples.
As mentioned above, its novelty lies in its scalability that makes it possible to detect and appropriately respond to various kinds of ligands. Considering this, the proposed system would lead to the mitigation of CRS as side effects of CAR-T cell therapy, hematopoietic stem cell transplantation, and viral infections, which is strongly required in the medical field. We are sure SWIFT will contribute to the further development of synthetic biology.
Implementation for CRS
CRS is a systemic inflammation caused by an overproduction of cytokines that results in an immune response. This potentially serious inflammation can occur as a side effect of CAR-T cell therapy, hematopoietic stem cell transplantation, or as a complication of viral infection.
Human Practices with many clinicians and researchers has provided many suggestions for improving our project. Detailed feedback from stakeholders on our project can be found on the Integrated Human Practices page .
Their input has helped us progress towards a more effective and secure implementation of SWIFT's CRS handling.
Suggested stakeholder input is as follows.
Why SWIFT is Needed
Our project was originally to address CRS by regulating gene expression at the transcriptional level. The specific gene sequence and design is as follows
However, with input from research physicians, we learned that the CRS response is instantaneous and that responding to this instantaneous response is critical in dealing with CRS.
This led us to change our project to the following system of secretion control, which became the original proposal for SWIFT.
In addition, existing therapies suppress CRS by administering antibodies against cytokines and their receptors. However, discussions with experts revealed that there were safety concerns regarding CRS, mainly for infectious diseases, due to the fact that the method of introducing antibodies suppressed immunity too much.
In particular, with respect to CAR-T cell therapy, there is a possibility that too much immunosuppression may reduce the function of CAR-T cells, and with respect to infectious diseases, too much immunosuppression may lead to severe viral infections. In fact, it has been pointed out that the use of existing therapies for CRS as a complication of COVID-19 may increase the risk of severe infection.
It was suggested that the advantages of SWIFT over existing therapies could be demonstrated in this situation.
SWIFT is capable of transmitting protease signals by forming dimers of MESA in a ligand concentration-dependent manner. Researchers suggested that future success in designing MESAs and proteases that adjust their secretion in response to extracellular cytokine concentrations could reduce the risk of severe disease caused by excessive cytokine suppression.
Cell Type Selection
Which cells to incorporate the SWIFT system into for CRS handling was a major point of debate. Initially, we envisioned utilizing immune cells, such as T and B cells, which would be directly harvested from the patient, transfected, and subsequently reintroduced into the body. However, this approach encountered the following issues.
Gene Transfer is Costly and Time-consuming
It is time-consuming and expensive to take cells directly from the patient and then introduce the gene.
A similar cell therapy is CAR-T cell therapy, but in the case of CAR-T cell therapy, it takes one to two months from the time the cells are harvested from the patient to the time the gene is introduced and administered to the patient, and the SWIFT system must be incorporated into each CAR-T cell, which is time consuming and expensive.
In addition, the use of the viral vector method requires the construction of a factory to manufacture the cells, which costs billions of dollars and is another reason for the high cost.
There is No Factory in Japan that Manufactures CAR-T Cells.
As mentioned earlier, CRS can manifest as a side effect of CAR-T cell therapy. Initially, when we considered introducing CRS as a potential side effect of CAR-T cell therapy, we contemplated integrating the SWIFT system directly into the CAR-T cells themselves. However, during discussions with experts, we learned that CAR-T cells are not manufactured in Japan. Furthermore, it was highlighted that in order to incorporate these systems into CAR-T cells, collaboration with a company offering CAR-T cell therapy for their production would be essential, which was not feasible.
Clinicians and medical researchers have proposed the introduction of SWIFT into MSCs (mesenchymal stem cells) as a transduced cell solution to these problems. Advantages of introduction into MSCs include:
- Low expression of HLA and can be derived from others.
- Immunosuppressive effects on immune cells such as T cells and B cells1.
- Clinical trials have already started in Japan, and the safety aspects have been well investigated, so there is little risk of cancer due to genetic modification, as is the case with hematopoietic stem cells.
- MSCs are less likely to cause cell death due to ER stress in introducing the SWIFT system because of their considerable secretory capacity2.
Conversely, the disadvantages are
- Short lifespan of the cells around 2-3 weeks.
- Since MSCs are trapped in the lungs, cytokine inhibition is mainly performed from the lungs.
- The number of divisions is limited to 10~20 times, so it is difficult to select good cells.
The following are some of the reasons why MSCs are used in this way.
After discussion with the experts, it was suggested that the first disadvantage of a short lifespan could actually be an advantage of safety, as CRS treatment would be completed in about two to three weeks.
Next, regarding the selection of good cells, he suggested that it would be good to use a technology that has already been established for introducing iPS cells and then differentiating them into MSCs.
And as for being trapped in the lungs, this could be a safety concern. It was suggested that a solution could be found by introducing a kill-switch for actual clinical application.
For these reasons, it was decided to introduce SWIFT as a CRS coping mechanism in MSCs.
Cytokines to be suppressed and inhibitors to be secreted
Initially our project was aimed at suppressing IL-6 as the main working cytokine.
However, during discussions with experts, we discovered that there are cytokines besides IL-6 that can serve as mediators of CRS. Furthermore, there are variations among individuals and in symptomatology regarding which cytokines act as the primary mediators, and instances in which CRS occurs even when IL-6 is successfully suppressed.
This led us to contemplate the feasibility of targeting multiple cytokines for suppression.
As mentioned earlier, we believed that by detecting various cytokines and addressing them individually, we could effectively manage CRS, regardless of which cytokine plays a prominent role.
Additionally, our initial consideration involved soluble gp130 (sgp130), which comprises solely the extracellular domain of gp130, as a potential inhibitor of IL-6.
We designed our circuit with sgp130 as the inhibitor, and it has a certain effect of suppression. However in the discussion with medical researchers, An option of a more effective inhibitor was proposed. It is the B-Cell Receptor (BCR), a soluble form of the membrane antigen utilized for IL-6 binding.
Future Prospects
As a future perspective, we have drawn a roadmap of how we should implement SWIFT in CRS coping situations in the future.
Things to check before experimenting with MSCs
Prior to experiments with MSCs, matters that should be experimentally confirmed in model cells such as HEK293 include the following.
- Design of cytoplasmic linker length, receptor extracellular domain, and protease of MESA such that the secreted amount of cytokine can be suppressed at an appropriate cytokine concentration to the extent that infection does not become severe.
- Investigation of inhibitors and the specific therapeutic effects of such inhibitors.
- Selection of proteases that will not cleave proteins in vivo.
in vitro experiments with MSCs
Once the above items have been experimentally confirmed in model cell lines such as HEK293, they can be introduced into MSCs for in vitro experiments. At that time, it is necessary to experimentally confirm the following items.
- Confirmation that SWIFT actually works in MSCs at the same level of expression as in HEK293 by introducing it in the same way as in HEK293.
- Confirmation that there is no stress on the cells due to the cleavage of intracellular proteins by the proteases selected in the HEK293 experiment.
- Confirmation of the operation of the kill switch introduced to address concerns that MSCs could be trapped in the lungs and pose a safety risk.
There is also a model in which MSCs are immortalized by expressing telomerase and other enzymes. It was suggested that this model could be used for validation.
Animal Experiments with Mice
After the in vitro experiments have been demonstrated, animal testing is the next step. Animal testing is conducted to see if the product is actually effective and if there are any unknown adverse safety risks.
What should be kept in mind when conducting animal testing is the law. In Japan, the Animal Welfare Law exists, so it is necessary to check and comply with the law.
It is also necessary to check the guidelines of the Ministry of Health, Labor and Welfare, and then go through the university's ethical review process. The university's ethics review committee must include people who are representative of the general public.
Careful consideration must be given to whether human MSCs or mouse MSCs will be used when conducting animal experiments on mice.
Mouse MSCs are much more difficult to genetically manipulate than human MSCs, and even if they are transfected, the cell division cycle is fast and they are no longer usable.
Human MSCs can only be evaluated transiently when they are injected into mice, and cannot be evaluated over a long period of time.
Safety Evaluation by Human Subjects
Once safety and practicality have been demonstrated in animal studies, the next step is to evaluate safety by human subjects at a lower dose level than in animal studies. In this case, it is important to provide sufficient explanations to the trial participants and ensure that there are no conflicts of interest.
When conducting a clinical trial on human subjects, it is desirable to administer the drug to healthy subjects, but since our project cannot administer the drug to healthy subjects, it was suggested that we obtain consent in advance from the patient or obtain consent on behalf of a family member when the actual clinical trial stage is reached.
Implementation beyond CRS
This system of rapid soluble ligand detection and protein secretion is not only clearly useful in dealing with CRS as described above, but also in a wide range of fields like medical applications, bioremediation, material production, agriculture, and many other aspects.
Implementation in medical applications
Non-medical Applications
A non-medical application would be to introduce SWIFT into cells other than mammalian cells. In this case, a problem appears in the MESA section. We have investigated the introduction of SWIFT into bacteria and yeast, and the presence of an outer wall or outer membrane between the cell membrane has been described as a major problem. The next step is to establish a method to get rid of peel off the outer wall and membrane.
Regulation of GMOs in Japan
Implementation in Bioremediation
Implementation in Bioproduction
Implementation in Plant Pathogen Detection
References
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Makoto Murata. (2019). bone marrow-derived mesenchymal cell. Japanese Journal of Internal Medicine, 108, 1369–1374. https://www.jstage.jst.go.jp/article/naika/108/7/108_1369/_pdf ↩
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Wu, J., Kaufman, R. From acute ER stress to physiological roles of the Unfolded Protein Response. Cell Death Differ 13, 374–384 (2006). https://doi.org/10.1038/sj.cdd.4401840 ↩
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Japan bioindustry association. (2022, January). Cartagena Law Guidebook. https://www.jba.or.jp/link_file/publication/2201_cartagena.pdf ↩
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METI. (2021, February 26). Revision of the Commentary on the Guidelines for Bioremediation Applications. https://www.meti.go.jp/policy/mono_info_service/mono/bio/cartagena/new_info/210226_bio-remediation_kaisetsu-amendment.html ↩