UPF_Barcelona

Hormonic

An intein-mediated control system for thyroid homeostatic regulation

Abstract: One-third of the treated hypothyroidism patients do not respond correctly to current therapies. To solve that, a system capable of restoring hormonal feedback was designed, offering an alternative to the 10% of Spaniards and 5% of the global population that suffer from this endocrine disorder. Its functioning is based on our successfully engineered intein-mediated Escherichia coli biosensor, which showed a great response to distinct T3 thyroid hormone levels giving rise to different superfolder Green Fluorescent Protein (GFP) concentrations. This was designed to be coupled to an external controller, which computes the dosage that compensates for hormonal disbalances. As a proof of concept for the validation of this regulatory mechanism, a lactone cell circuit incorporating a turbidostat was developed, showing a high correlation between the experimental and modelling results. Finally, a detailed future implementation plan was defined so that it included the necessary steps towards a socially responsive, safe and inclusive implementation of this innovative treatment.

Clinical need

Need of automatizing the treatment of hormonal imbalances

Hypothyroidism is a condition characterized by the lack of thyroid hormones, usually raised by dysregulations of the thyroid feedback. Despite it affects 5% of the overall population, its treatment is not well optimized [1]. Patients are prescribed levothyroxine tablets, which are not effective in one-third of the cases, and their doses, besides acting in a narrow range without adverse effects, are adjusted by trial-and-error [2]. Moreover, the tracking methodology to adjust the doses has evidenced to not properly adapt to the requirements of the current COVID pandemic situation, interrupting the treatment of the patients [3].

All in all, it is clear that a new therapy approach that considers the whole feedback system and that allows a telematic treatment is needed.

Figure 1. Map of overt hypothyroidism prevalence (selective populations used when representative data not available), 2018 [4].

[1]Chiovato, L., Magri, F. & Carlé, A. (2019) Hypothyroidism in Context: Where We’ve Been and Where We’re Going. Adv Ther, 36, 47–58. DOI:10.1007/s12325-019-01080-8
[2]Fliers, E., Demeneix, B., Bhaseen, A., & Brix, T. H. (2018) European Thyroid Association (ETA) and Thyroid Federation International (TFI) Joint Position Statement on the Interchangeability of Levothyroxine Products in EU Countries. European thyroid journal, 7(5), 238–242
[3] Gorgojo Martínez, J., Zugasti Murillo, A., Rubio Herrera, M. and Bretón Lesmes, I. (2020) TELECONSULTA En Endocrinología Y Nutrición En Tiempos De La Pandemia COVID-19 Y Más Allá. [online] Seen.es. Available at: https://www.seen.es/ModulGEX/workspace/publico/modulos/web/docs/apartados/1433/160620_105727_7128864936.pdf [Accessed 22 October 2020]
[4] Taylor, P. N., Albrecht, D., Scholz, A., Gutierrez-Buey, G., Lazarus, J. H., Dayan, C. M., & Okosieme, O. E. (2018). Global epidemiology of hyperthyroidism and hypothyroidism. Nature Reviews Endocrinology, 14(5), 301–316. doi:10.1038/nrendo.2018.18

T3 biosensor

Sensing T3 hormone levels through an intein-mediated bacteria

We developed an Intein Mediated T3 (IMT3) biosensor in E. coli. Using the protein self-splicing properties of inteins [1] and binding domain of T3, we could create a chimeric fluorescent sensing protein:

T3 Ligand Binding domain (from the Thyroid hormone receptor β1): to recognize T3 present in the media [2].
Splicing intein domains (from the Mycobacterium tuberculosis RecA protein): to join the two superfoldingGFP (sfGFP) halves when activated by the ligand domain [3].
Split superfolder GFP: to report the T3 concentration as fluorescence [4].

Figure 1. Final constructs of the T3 biosensor with sfGFP.

We expressed our IMT3 biosensor in E. coli and exposed them to different T3 concentrations in the media. Proportional fluorescence with T3 concentration was reported.

Figure 2. Transfer function of IMT3_sfGFP_pUC-Amp correlating the steady fluorescence with the amount of T3. Experimental data (red dots) and fitted model (blue line).

FURTHER STEPS

These IMT3 biosensors can be further improved to increase its precision, widening dynamic range or lowering detection limit. Different approaches have been explored such as more ligand sites [5] or directed evolution [6].

[1] Skretas, G. (2005). Regulation of protein activity with small-molecule-controlled inteins. Protein Science, 14(2), 523–532. DOI:10.1110/ps.04996905
[2] Yen, P. M. (2001). Physiological and Molecular Basis of Thyroid Hormone Action. Physiological Reviews, 81(3), 1097–1142. DOI:10.1152/physrev.2001.81.3.1097
[3] Wood, D. W., Wu, W., Belfort, G., Derbyshire, V., & Belfort, M. (1999). A genetic system yields self-cleaving inteins for bioseparations. Nature Biotechnology, 17(9), 889–892. DOI:10.1038/12879
[4] Gangopadhyay, J. P., Jiang, S., & Paulus, H. (2003). An in Vitro Screening System for Protein Splicing Inhibitors Based on Green Fluorescent Protein as an Indicator. Analytical Chemistry, 75(10), 2456–2462. DOI:10.1021/ac020756b
[5] Gonzalez-Flo, E., Alaball, M. E., Macia, J. (2020).Two-Component Biosensors: Unveiling the Mechanisms of Predictable Tunability. ACS Synthetic Biology, 9(6), 1328-1335. DOI: 10.1021/acssynbio.0c00010
[6] Badran, A., Liu, D. (2015). Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Nat Commun, 6, 8425. https://doi.org/10.1038/ncomms9425

Hormonic

Goals and achievements

Hormonic is designed to reestablish the hormonal feedback system thanks to an external PID controller coupled to an Intein-Mediated T3 (IMT3) biosensor. But beyond that, it also aims to bring synthetic biology to the society. For this purpose, in the UPF_Barcelona team we have:

Designed a groundbreaking therapy approach.

Successfully engineered the IMT3 biosensor.

Used a lactone circuit as a proof of concept.

Engaged with the scientific & educational community.

Explored society needs & received experts advice.

Autorregulation system

Controlled administration of levothyroxine

Once sensing the amount of T3 hormone, our computational unit has to interpret and react accordingly. In order to close the hormonal feedback loop, a PID controller was selected due to its great static performance [1]. It continuously computes the difference between the measured hormonal level and the desired one so as to calculate the adequate levothyroxine injection.

Figure 1: General schematic showing the application of the PID controller in Hormonic.

This correction action (levothyroxine injection) will compensate for the error depending on proportional, integrative and derivative constants. They can be tuned with the aim of preventing hyperthyroidism (reduce overshoot), while maintaining the levothyroxine injection rate under its maximum (enlarge rise-time) and within the drug’s effective range (eliminate steady-state error). To do so, a mathematical model of subcutaneous injection of levothyroxine must be developed and characterized [2].

Figure 2: Graph showing the PID response.

[1] Chen, S., Zhao, J., & Qian, J. (2007). A Design Method of Bang-Bang and PID Integrated Controller Based on Rough Set. Fourth International Conference on Fuzzy Systems and Knowledge Discovery (FSKD 2007). doi:10.1109/fskd.2007.17
[2] Li, J., & Johnson, J. D. (2009). Mathemical models of subcutaneous injection of insulin analogues: a mini-review. Discrete and continuous dynamical systems. Series B, 12(2), 401–414. https://doi.org/10.3934/dcdsb.2009.12.401

Proof of concept

Lactone circuit as an analogy to MINIMAL HORMONAL REGULATION MODEL

As a proof of concept for our device, we decided to implement a closed biological circuit, which could not be done using our T3 biosensor. For this reason, we used a bacterial lactone circuit, using lactone-producer cells and lactone-sensing cells[1][2].

Figure 1. Diagram for a general closed circuit.

Figure 2. Comparison between the minimal hormonal regulation model and hypothyroidism condition.

The producer cells were induced by arabinose and inhibited by glucose, and were provided by the lab. In a hypothyroidism model, these cells would mimic the peripheral tissue cells, responsible for metabolizing levothyroxine into T3 hormones[3], lactone in our case.

The sensor cells produced superfolder GFP (sfGFP) when lactone was present in the media. In these cells, LuxR was expressed constitutively and sfGFP was induced by a pLux promoter, dependent on the binding of LuxR and lactone.

All these biological behaviours were characterized and demonstrated using Plate-Reader analysis.

[1] Ballestero, M. C., Duran-Nebreda, S., Monta, R., Solé, R., Macía, J., Rodríguez-Caso, C. A bottom-up characterization of transfer functions for synthetic biology designs: lessons from enzymology. Nucleic Acids Research, 2014, Vol. 42, No. 22
[2] Garcia-Ojalvo, J., Elowitz, M.B., Strogatz, S.H. Modeling a synthetic multicellular clock: repressilators coupled by quorum sensing. Proc. Natl Acad. Sci. U.S.A., 101, 10955–10960
[3] Brent G. A. (2012). Mechanisms of thyroid hormone action. The Journal of clinical investigation, 122(9), 3035–3043. https://doi.org/10.1172/JCI60047

Proof of concept

Turbidostat, our continuous microbiological culture device

In order to perform a long-lasting in vitro demonstration of our feedback system, the turbidostat can maintain a user-fixed optical density for any kind of cell culture (producer + reporter cells in our proof of concept). This ensures constant conditions during the whole experiment avoiding thus interferences in the sensing process.

Figure 1. Diagram for the use of the turbidostat and PID controller in conjunction with the lactone circuit.

[1] Wong BG, Mancuso CP, Kiriakov S, Bashor CJ, Khalil AS. Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER. Nat Biotechnol, 2018 Aug;36(7):614-623. doi: 10.1038/nbt.4151. Epub 2018 Jun 11. PMID: 29889214; PMCID: PMC6035058.

Modelling

Two ordinary differential equation (ODE) models have been developed to characterize and understand the behaviour of the T3 intein-mediated biosensor and the lactone proof of concept system. These models allow us to predict specific hormone concentration, and therefore, to regulate the wanted feedback loops. They were fitted with experimental data and were able to successfully reproduce it. Furthermore, since we are developing biosensors, we computed an analysis on the sensitivity and other parameters that affect the ability of the cell to sense, thus showing a balance between robustness and hypersensitivity. The T3 biosensor model sets a precedent for any kind of future intein-mediated biosensor.

On top of that, we have designed a model to characterize the ASV tag [1] and to mathematically demonstrate its ability to increase the protein degradation rate, thus reducing sensing time.

T3 intein-mediated biosensor

Equation 1. Final ODE system of the T3 sensor cell model.

Lactone circuit

Equation 2. Final ODE system of the lactone sensor cell model.

Equation 3. Final ODE system of the lactone producer cell model.

[1] Andersen, J.,Sternberg, C., Poulsen, L. K., Bjørn, S. P., Givskov, M. New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria. Applied and environmental microbiology, June 1998, p. 2240–2246

Human practices

Integrated Human Practices

Feedback from multiple stakeholders has been gathered throughout all the stages of the project as to pursue a human-centered solution search process.

Figure 1. Diagram of the journey of our UPF_Barcelona team.

Doctor’s physiological knowledge was complimented with the demands of national and international endocrine associations, which arose biosafety and bioethical concerns that were also tackled. The defined scientific approach helped us design the future implementation that was later used for the entrepreneurship planification.

Public engagement

Bringing synthetic biology to the world is a very important task, but how can it be done in a year like 2020?

Figure 2. Scheme of our public engagement tasks.

Future implementation

How can we deliver Hormonic to the world?

Hormonic’s final prototype was envisioned: a microneedle array coupled to a cell-free T3 sensor and to a Levothyroxine reservoir, whose administration is controlled by a PID. Also the biosafety and biosecurity was taken into account in order to make Hormonic safe for everyone.

Figure 1. Design of the Hormonic prototype.

A rigorous study of the potential users was also performed, including patients during the Covid-19 context, the ones who still exhibit symptoms after treatment or pregnant people among others.

According to EU regulation, Hormonic was classified as a III class device.

Business models are key to bring projects to the final customer. To make Hormonic a reality we elaborated a business plan, using a business model canvas, and the costs and revenues were studied in order to design a financial plan.

Figure 2. Costs & Revenues analysis for Hormonic..

Acknowledgements and Sponsors

Acknowledgements

EXPERIMENTAL SUPPORT
Our instructors: Marc Güell, Avencia Sánchez-Mejías, Dimitrije Ivančić & Sira Mogas
Other researchers that have helped us: Javier Macia, Guillermo Nevot, Nuria Rafel, Guillem Lopez-Grado, Nastassia Knödlseder, Jessica Jaraba, Maria Pallarès, Carlos Toscano, Jordi Garcia Ojalvo & Òscar Fornas

HARDWARE DESIGN SUPPORT
Laura Becerra, Aracelys Garcia & Mario Gomez

SPECIAL THANKS TO
Dr. Ricard Torras, Dr. Albert Goday Arno, Dr. Eric Fliers, Professor Vanesa Daza, Carles Taulé Flores & all the entities that have helped us: