Team:UCAS-China/Description
Background
“Microbial therapy allows for the creation of engineered systems that intelligently sense and respond to diverse environments, ultimately adding specificity and efficacy that extends beyond the capabilities of molecular-based therapeutics.”[1]
Bacteria-based microbial therapies have the potential for solving many clinical problems, which remain unmet by traditional therapies, in treating oncological, metabolic, and gastrointestinal diseases[2].
Some strains of bacteria show preferential accumulation at tumour sites[3], and these characters can be utilized for tumour targeting, avoiding the influence of mutations in cancer cells and immunosuppressive microenvironment, which was inaccessible to traditional molecular drugs[4]. The diversity and plasticity of bacteria allow treatment for multiple diseases[4], and through genetic manipulations sensitive switches with high dynamic ranges can be incorporated into microbial candidates, enabling efficient and safe cancer therapies.
The ability for commensal bacteria to colonize the host also makes it possible for integration of recombinant microbes into host microbiome, providing long-term solutions for metabolic and gastrointestinal diseases. Recombinant bacteria are able to achieve continuous and on-demand drug release, improving the bioavailability of drug molecules and restoring host homeostasis[5]. Degradation and metabolism of poisonous metabolites can also be achieved, which promotes the emergency of more direct and flexible therapies to treat metabolic disorders[6].
However, safety concerns still exist. The proliferation of bacteria at tumor sites brings challenges to dose determination and control[4]. And the off-target effects on healthy tissues need to be considered and reduced. But few safety circuits are incorporated into therapeutic microbes[2], which may lose their function due to mutations, and have potential influence on hosts and environment. Therefore, there is a strong need for better combination of safety and therapeutic genetic circuits in bacteria.
Overview
As there is a strong need for better combination of safety and therapeutic genetic circuits in bacteria, we designed a novel,flexibility and versatility platform, ARK. micro, to address the safety issues in bacteria-based microbial therapies, and to translate more laboratory researches into clinical applications.
ARK. micro, our platform, consists of thermo-sensitive switch module, using temperature to switch on or off the expression of targeted gene, and biosafety module, concerning avoiding unintended proliferation into the environment and healthy parts of human body.
To show and prove the advantage of microbial therapeutics, we constructed two therapeutic genetic circuits for Parkinson’s disease, a serious disease which needs long-term treatment and take drugs on-demand, and cancer, whose off-target effect needs to be reduced urgently, respectively. What’s more, we combined the platform with therapeutic genetic circuits in both two diseases to show flexibility and versatility of our platform.
In the treatment of cancer, we also built a double-status switch, which can regulate the expression of lysis gene to lowering off-target effect based on ordinary thermo-sensitive switches.
Finally, because we need a heat device to achieve the function of avoiding unintended proliferation into healthy parts of human body and lowering off-target effect, which both use the heat-inducible ON-switch to control the expression of target genes, our hardware developed an innovative electronic capsule, cPlus, which can perfectly assist diagnosis and treatment of intestinal diseases.
By developing a novel, flexible and versatile platform, ARK. micro, and a innovative electronic capsule, cPlus, we promote the development, clinical and commercial application of microbial therapeutics, a emerging therapeutics with great potential for basic research, as well as industrial and biomedical applications.
Components of ARK. micro
Switch module of ARK. micro
In the switch module, we chose temperature as the trigging factors of the switch because of its unique advantages in microbial therapy: non-invasive nature, good penetrability and reversibility.
We developed a series of thermo-sensitive parts, including transcription factors and proteases, and built different cold-inducible ON-switches and heat-inducible ON-switches with narrow transition ranges and high-fold induction.
Figure 1. The cold-inducible ON-switch is encoded on two plasmids. The circuit is shown with the genetic parts and relationships among them.
Figure 2. The heat-inducible ON-switch is encoded on one plasmid. The circuit is shown with the genetic parts and relationships among them.
What’s more, by combining the heat-inducible ON-switch with the cold-inducible ON-switch, we can get a double-status switch to response more different temperatures and realize more functions in one creature.
Figure 3. The double-status switch is encoded on three plasmids. The circuit is shown with the genetic parts and relationships among them.
With high performance and versatility, our thermo-sensitive switches truly make engineered bacteria precisely controlled and have huge potential for basic research, as well as industrial and biomedical applications.
Biosafety module of ARK. micro
Figure 4. The mind map of biosafety design
After constructing switch module, to satisfy the need of biocontainment and lowering off-target effect, we also constructed a biosafety module to enhance ARK. micro.
Current strategies mainly include integrating toxin/antitoxin ‘kill switches’ and establishing auxotrophies for essential compounds. However, either of them has its own obstacles. [7]
This year, we provided our own solutions and improvements.
By combining our high-performance cold-inducible on-switch with the toxin system, we optimized the ‘kill switch’ from its foundation, improving the response speed also the efficiency.
By developing a ‘synthetic auxotrophy’ for non-canonical amino acids, our therapeutic bacteria are robust against environmental supplementation. [7]
Additionally, considering the dependence on exogenous source, we innovatively combined our heat-inducible on-switch with ncAA system, exploring greater potential for our ark to sail to the real world.
Journey of ARK. micro
Parkinson’s disease
Parkinson’s disease
To prove the potential of microbial therapeutics and our platform’s universality and unique advantage on microbial therapeutics, we chose Parkinson's disease as an example of long-term medication diseases, which needs frequent and precise doses of L-dopa.
We firstly transferred the tyrosine hydroxylase(TyrOH) gene, which can catalyze tyrosine to L-dopa, into our engineered E.coli to provide a stable and sustainable concentration of DOPA in the patient’s blood.
Figure 5. TyrOH catalyzes tyrosin to levodopa
Figure 6. Gene of TyrOH under a constitutive promoter
Compared with the traditional therapy by taking pills regularly for a long time, microbial therapeutics can achieve a constant and stable supply of DOPA.
Finally, combining biocontainment module of our platform with the circuit for releasing TyrOH can make this microbial therapeutic for Parkinson's disease safer and meet the requirements of clinical and commercial applications.
Figure 7. The combination of biocontainment module and therapeutic module
Cancer
Considering the unique advantages of microbiological therapy in treating cancer, we selected cancer as one of our application and built a double-status switch in our platform to solve the problem of biocontainment and biosafety, which is used to lower the off-target effect simultaneously.
In our project, we used CD47 nanobody as a drug molecule to block CD47 on the surface of cancer cells, so that they could be killed by microphages.[1]
Figure 8. CD47 nanobody interacts with CD47 on cancer cells
Take advantage of the natural targeting property of certain microorganisms to approach the microoxygen area near the tumor tissue, we put CD47 nanobody gene under an anaerobic induced promoter so that we can primarily lower the off-target effect.
Figure 9. CD47nanobody under the anaerobic induced promoter, PfnrS
To further make sure the release of CD47 nanobody could not hurt healthy tissues, we used our thermo-sensitive switches to construct a double-status switches, in which the cold-inducible ON-switch for the toxin gene and the heat-inducible ON-switch for the drug releasing gene avoiding unintended proliferation into the environment and healthy parts of human body respectively.
Finally, combining this double-status switch with the circuit of microbial therapeutics, we can lower the off-target effect significantly and make full use of unique advantage of microbial therapeutics.
Figure 10. The combination of double-status switch and the circuit of microbial therapeutics
Hardware
Figure 11. Overview of our cPLUS
We developed an innovative electronic capsule, cPlus, which can perfectly assist diagnosis and treatment of intestinal diseases. cPlus consists of a heating system, a sampling device, a clamping device, a positioning device and a remote-control system. With the accurate positioning(<1cm) and heating (<1000s to reach the target temperature within 1cm range) control, cPlus can be precisely located and heat the specific lesion to assist the targeted drug release from the engineered therapeutic bacteria. Not only for treatment, but cPlus contains a sampling device and can be combined with current imaging system for diagnosis. In the present research of intestinal flora, original technology of sampling from the feces is difficult to meet the requirements of sampling intestinal flora at specific location (including long-term fixed flora), cPlus fills a vacancy of intestinal flora accurate sampling. We believe cPlus will bring an evolution in the industry of medical microdevice.
Model
Our model consists of three parts.
In the first part, we establish OED-based models to verify the superiority of our design of cold-induced gene circuit, and to describe and predict the function of our gene circuit.
In the second part, we employ an OED-based model to compare the distribution of drug by two different ways of drug delivery--pills and bacteria, and illustrates that delivering drug by our bacteria is better than traditional method.
The third part is about our hardware. We simulate the temperature distribution in intestinal tract when the capsule heats. We also implement the location algorithm of magnetic capsule based on Levenberg-Marquardt Method.
Parts
This year, we developed a collection of thermosensitive parts with high-performance, versatility and robustness.
Based on TCI transcription factor family and TlpA family, we collected some heat-inducible ON-switches, which can open the gene expression under high temperature. More importantly, we developed a series of cold-inducible ON-switches which are active under low temperature and inactive when temperature rises.
We fully characterized the performance of them in both Top10 strain and Nissle 1917, a probiotic with more than 100 years of medical application and most of them show robustness and high performance, some can even reach more than 100 fold-change under different temperatures.
We also constructed a double-status switch with two kinds of switches, which can turn on different genes‘ expression at different temperatures.
Our parts collection shows high performance and versatility, which ensure the potential for basic research, as well as industrial and biomedical applications, and truly makes engineered bacteria precisely controlled.
Human practice
After many brainstormings and researches, we identified the theme of our project this year – constructing an efficient temperature-sensitive switch system as a safe platform for microbial therapy.
In order to have a more comprehensive understanding of the current state of microbial therapy use, we have conducted a large number of data searches and have a preliminary understanding of microbial therapy related drugs. After obtaining these data, we try to make them more fresh. Live, through dialogue with the public, we know the public's understanding of microbial therapy, and what microbial therapy looks like in their eyes; through the field visits of microbial therapy companies, we understand their prospects for the development of microbial therapy. Expected and some obstacles encountered at present; through talks with researchers, we have learned about microbial therapy from a technical level; through communication with doctors, we understand some conditions that microbial therapy related drugs are accepted by doctors and patients.Safety and effectiveness occur frequently in these conversations.
After interacting with them, we realized what we might be able to do for the development of microbial therapies. Our project tried to solve some of the problems they mentioned and made microbial therapies better.At the same time, we know that the protection of ethical issues and laws and regulations is the necessary factor for every technology to be touched and benefited from the laboratory to the public. Therefore, we interviewed some ethics scholars and legal experts.We also hope that we can make some efforts to make up for the gaps in the laws and regulations and ethics on microbial therapy, so we put forward some suggestions.
Volkswagen, companies, researchers, doctors and patients, ethics scholars, legal persons, and the six-in-one make our projects connect with the world, and it is the exchange with them that makes our projects perfect.Of course, we are also very grateful to other teams for their support, and their exchanges have made us better.Finally, we hope that more microbial therapies will benefit from our security platform, so we are planning to write a business plan and hope to promote our security platform application in the near future.
References
[1]Chowdhury, S., Castro, S., Coker, C., Hinchliffe, T. E., Arpaia, N., & Danino, T. (2019). Programmable bacteria induce durable tumor regression and systemic antitumor immunity. Nature Medicine, 25(7), 1057-1063.
[2]Higashikuni, Y.; Chen, W. C.; Lu, T. K., Advancing therapeutic applications of synthetic gene circuits. Curr Opin Biotechnol 2017, 47, 133-141.
[3]Wu, M. R.; Jusiak, B.; Lu, T. K., Engineering advanced cancer therapies with synthetic biology. Nat Rev Cancer 2019, 19 (4), 187-195.
[4]Forbes, N. S.; Coffin, R. S.; Deng, L.; Evgin, L.; Fiering, S.; Giacalone, M.; Gravekamp, C.; Gulley, J. L.; Gunn, H.; Hoffman, R. M.; Kaur, B.; Liu, K.; Lyerly, H. K.; Marciscano, A. E.; Moradian, E.; Ruppel, S.; Saltzman, D. A.; Tattersall, P. J.; Thorne, S.; Vile, R. G.; Zhang, H. H.; Zhou, S.; McFadden, G., White paper on microbial anti-cancer therapy and prevention. J Immunother Cancer 2018, 6 (1), 78.
[5]Mimee, M.; Citorik, R. J.; Lu, T. K., Microbiome therapeutics - Advances and challenges. Adv Drug Deliv Rev 2016, 105 (Pt A), 44-54.
[6]Isabella, V. M.; Ha, B. N.; Castillo, M. J.; Lubkowicz, D. J.; Rowe, S. E.; Millet, Y. A.; Anderson, C. L.; Li, N.; Fisher, A. B.; West, K. A.; Reeder, P. J.; Momin, M. M.; Bergeron, C. G.; Guilmain, S. E.; Miller, P. F.; Kurtz, C. B.; Falb, D., Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat Biotechnol 2018, 36 (9), 857-864.
[7] Mandell, D. J. et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55-60, (2015).
