Safety-by-design

Our application, NanoBlade, is a targeted, light-inducible bacteria-based tumor therapy designed to be as safe and efficient as possible.

Biosafety is “containment principles, technologies, and practices that are implemented to prevent unintentional exposure to biological agents or their inadvertent release,” as defined by the WHO. [1] In other words, genetically modified organisms should not leave their isolated system for the safety of our environment.

In our view, this interpretation is not a holistic approach to biosafety; thus, it neglects the safety issues within the system, which is an essential aspect when it comes to therapeutic GMOs. These organisms should not only be retained but their interaction with their host must be limited and highly controlled to prevent adverse side effects.

During our design, we identified safety issues that we need to address: off-target toxicity and targeting efficiency, producing a sufficient amount of drugs, a safe way of inducing drug production, genetic instability, and lateral genetic transfer. [2]

Reducing potential off-target toxicity can be done by restricting therapeutic expression to sites of disease. Spatial control of such systems could be carried out using quorum sensing, as facultative anaerobic bacteria strains (like Salmonella or Escheria sp.) show intrinsic high density in solid tumors. The same rationale applies to other methods harnessing tumor microenvironment conditions, such as acidic pH, hypoxia, or increased body temperature-induced protein expression. [2, 3] Although these approaches serve as safety measures, they alone are insufficient for achieving effective colonization and therapeutic effect. Therefore, targeting strategies should be implemented to tackle these obstacles. [4]

Because of this, we decided to use a targeting molecule, a Nanobody displayed on the bacterial outer membrane that can bind to tumor cells selectively. For Surface Display, we used a synthetic adhesion molecule (described by Pinero-Lambeau et al.) composed of a beta-barrel domain derived from intimin that can embed the Nanobody in the outer membrane. Combining this with a fluorescent reporter, GFP makes our Nanobody Display System, which serves diagnostic purposes.

We explored safe ways to produce drug molecules locally. Although TME condition-driven protein expression is a popular choice among bacterial therapies, it suffers from limitations such as leakage or lack of control. Instead of them, we choose an external trigger, a light-inducible expression system called BLADE, described by Romano et al., since light-related techniques provide excellent spatiotemporal control of biological processes. [6]

One of the main issues associated with bacterial therapies is their genetic instability, which can occur as mutations causing unforeseen side effects or, more commonly, loss of function. Producing proteins puts a heavy burden on bacteria which they do not preferentially carry. Therefore, one must keep the introduced system as simple as possible, using the least number of Parts. For this aim, we used the BLADE system because it is one of the few published one-component light-inducible protein expression systems. [7] (We must mention, though, that this decision decreased the therapeutic applicability of our system; thus, blue light has limited tissue penetration features. [8]) Following the same line of thought, we improved our Nanobody Display System to be one component as well. We call this MiniNano, which combines tumor-specific attachment with deep-tissue visualization built upon the work of Team UPENN from 2012. [9]

For the same reason, we did not plan to introduce Kill switches into our project because it would have been too much pressure on our bacteria. Team Oxford from 2019 had a similar approach to this problem. They had worked on a probiotic therapy for C. difficile infection to replace antibiotic-based methods. As they mentioned, many switches go against one of their core value because they are antibiotic-inducible. [10]

Beyond the scope of our project

Genomic integration or creating chromosome-free cells could resolve genetic instability and lateral gene transfer. The latter would be the direction we would choose for our project. For example, Fan and her coworkers described an I-CeuI endonuclease-induced method for producing chromosome-free SimCells, which can process designed DNA and express target genes for an extended period without the ability to proliferate. [11]

Safe work

Throughout our work, we have always placed great emphasis on safety. At the very beginning of our journey, our Advisor, Boglárka Schilling-Tóth gave all of us a Safety training.

[1] World Health Organization. Laboratory Biosafety Manual. Fourth edition. Geneva: World Health Organization; 2020. https://www.who.int/publications/i/item/9789240011311 Accessed October 2, 2022.

[2] Riglar, D. T.; Silver, P. A. Engineering Bacteria for Diagnostic and Therapeutic Applications. Nat. Rev. Microbiol. 2018 , 16 , 214–225. https://doi.org/10.1038/nrmicro.2017.172

[3] Forbes, N. S. Engineering the Perfect (Bacterial) Cancer Therapy. Nat. Rev. Cancer 2010, 10, 785–794. https://doi.org/10.1038/nrc2934

[4] Toso, J. F.; Gill, V. J.; Hwu, P.; Marincola, F. M.; Restifo, N. P.; Schwartzentruber, D. J.; Sherry, R. M.; Topalian, S. L.; Yang, J. C.; Stock, F.; Freezer, L. J.; Morton, K. E.; Seipp, C.; Haworth, L.; Mavroukakis, S.; White, D.; MacDonald, S.; Mao, J.; Sznol, M.; Rosenberg, S. A. Phase I Study of the Intravenous Administration of Attenuated Salmonella Typhimurium to Patients with Metastatic Melanoma. J. Clin. Oncol. 2002, 20, 142–152. https://doi.org/10.1200/jco.2002.20.1.142

[5] Piñero-Lambea, C.; Bodelón, G.; Fernández-Periáñez, R.; Cuesta, A. M.; Álvarez-Vallina, L.; Fernández, L. Á. Programming Controlled Adhesion of E. Coli to Target Surfaces, Cells, and Tumors with Synthetic Adhesins. ACS Synth. Biol. 2014, 4, 463–473. https://doi.org/10.1021/sb500252a

[6] Romano, E.; Baumschlager, A.; Akmeriç, E. B.; Palanisamy, N.; Houmani, M.; Schmidt, G.; Öztürk, M. A.; Ernst, L.; Khammash, M.; Ventura, B. D. Engineering AraC to Make It Responsive to Light Instead of Arabinose. Nat. Chem. Biol. 2021, 17, 817–827. https://doi.org/10.1038/s41589-021-00787-6

[7] Lindner, F.; Diepold, A. Optogenetics in Bacteria – Applications and Opportunities. FEMS Microbiol. Rev. 2021, 46. https://doi.org/10.1093/femsre/fuab055

[8] Ash, C.; Dubec, M.; Donne, K.; Bashford, T. Effect of Wavelength and Beam Width on Penetration in Light-Tissue Interaction Using Computational Methods. Laser Med Sci 2017, 32, 1909–1918. https://doi.org/10.1007/s10103-017-2317-4

[9] https://2012.igem.org/Team:Penn/SurfaceDisplay Accessed October 2, 2022.

[10] https://2019.igem.org/Team:Oxford/Safety Accessed October 2, 2022.

[11] Saltepe, B.; Wang, L.; Wang, B. Synthetic Biology Enables Field-Deployable Biosensors for Water Contaminants. Trends Anal. Chem. 2022, 146, 116507. https://doi.org/10.1016/j.trac.2021.116507