abcd Our project used modified attenuated Salmonella to carry our unique co-silencing plasmid for targeted killing. At the same time, the special delayed lysis modification also raised our safety to a new level. Some texts. At the same time, we have also preliminary established the efficacy scoring model of the entire research design, hoping to provide a dynamic efficacy evaluation model for the iGEM team participating in the treatment track in the future.
In addition, we completed a reference manual on microbial-mediated tumor therapy in synthetic biology with BNUZH-China, Guangxi-U-China, PekingHSC, Peking, and Tsinghua. Our goal is to provide practical and meaningful solutions to common challenges faced by iGEM teams working on future projects for microbiome-mediated therapeutics in tumors, including safety, ethics, and engineering design. Our work is expected to provide a new understanding of bacterial treatment of tumors and propose a new strategy for co-silencing in iGEM.
Arabinose-delayed lysis of bacterial systems for biosafety
Our long-term goal is to develop capsule drugs for oral administration that protect humans against various intestinal tumors. Treatment with live Salmonella vaccines may release bacteria into the environment, possibly resulting in unintended biological releases. After reviewing the literature, JLU-NBBMS learned about the Regulated programmed lysis of recombinant Salmonella [1] as a strain for immune vaccination. Based on this, we constructed and evaluated a biocontainment system that meets our current team's needs for delivery to the colorectum. The requirements of the functional capsule carrier, together with the above-mentioned knockout of asnB and insertion of flaB, are met to colonize deep in the tumor tissue for a long enough time to ensure sufficient time to activate the immune system.
After seeing the current difficulties RASV faces and the solutions provided by the scientific field, we hope to apply such a containment system to engineered bacteria to treat tumors. We used the tightly regulated araC PBAD activator-promoter system to construct a strain/plasmid system that directs regulated arabinose-dependent, programmed lysis. An arabinose-regulated cell lysis system should not be undermined by release into the environment where arabinose stream and groundwater levels are in the submicromolar range.[5]
Data show that our designed system results in cell lysis in the absence of arabinose and clearance of strains from host tissues (see Project for details).
The system for antigen delivery can be customized to fit various requirements. By introducing mutations that target genes associated with Salmonella flagella and motility, we can enhance the colonization deeper into the tumor tissue. For instance, we have eliminated additional genes from the arabinose operon to block arabinose metabolism and maintain effective arabinose concentrations in the cytoplasm for longer periods.
The regulated lysis system also has the potential as a delivery system for DNA vaccine vectors. AsdA deletion mutants of Shigella flexneri have been used to deliver animal DNA [6]. Still, immune responses were weak, possibly because the cells did not persist long enough to invade host tissue effectively. The Δ asdA mutant of E. coli has also been used to deliver DNA in tissue culture [7].
However, our system, whether used for Shigella, E. coli, or Salmonella [8], provides sufficient time for the vaccine to establish itself in the host tissue before lysis occurs, thereby increasing the likelihood of differential delivery. Finally, the system can be modified to provide effective biocontainment of genetically engineered bacteria for multiple purposes besides therapy.
Through our model, we have made efforts to make it easier to work with Salmonella-based Colorectal, which simulates the effects of this bacteria to a certain extent and simulates the changes in the bacteria in tumor cells, and the IGEM teams in the future can apply it to other bacteria by modifying infection rates or adding rates. In the model's design, tumor markers' content was proposed as the evaluation index, which provided a compelling idea for the dynamic efficacy evaluation model. See Model for details.
In education, we have planned different activities based on people with varying levels of knowledge. From the perspective of acceptance, we understand that other age groups and different abilities to accept knowledge will lead to differences in interest in other activities. In this context, we have chosen to use various means, such as sitcoms, clay, popular science manuals, knowledge competitions, etc., to attract the attention and attention of people from all walks of life to synthetic biology and biosafety (more details: Education). We are still exploring more educational and entertaining ways to spread scientific knowledge to more people in a dynamic and life-friendly way.
Through discussions and exchanges with BNUZH-China and Peking University collaboration teams this year (more details: Collaborations), we found that there are inevitably many problems in designing and conceptualizing microbial-mediated tumor treatments. For example, how do we deal with the ethical issues engineered bacteria raise? How do we solve the safety problem of tumor treatment? How we ensure that engineered bacteria can specifically colonize tumor areas and minimize damage to normal tissue cells?
These questions are common ones encountered by teams working on microbiome-mediated cancer therapies. We are very happy and excited to have BNUZH-China, a team working with us on Salmonella as a chassis organism, on this year's team. In order to discuss these issues together, we established a microbial cancer treatment discussion group in China on May 8 this year in cooperation with BNUZH-China and Peking University.
After the China iGEMers Community Conference on the 10th, our seminar group attracted more iGEM teams to participate, including Guangxi-U-China, PekingHSC, Tsinghua, USTC, CPU-China, Jilin-China, etc. On July 27 this year (more details: Collaboration), under the proposal of BNUZH-China and us, a reference manual of "Microbiome-Mediated Tumor Treatment in Synthetic Biology" was compiled. We aim to provide iGEM teams and other scientific groups aiming at future microbial therapeutic oncology projects with practical and meaningful solutions to the typical difficulties faced by safe, ethical, engineering design.
Through several meetings, we clarified the framework and content of the manual as well as the division of labor among the participating teams. Subsequently, we began a long writing process, constantly optimizing the details of the manual, until the final draft was completed at the end of September this year.
In developing this manual, we sought input from numerous former iGEM team members and consultants. Our intention was to create a playbook that could be used by any future team in need and easily integrated into their unique team design. The content of our manual covers a variety of aspects, including background information on biotherapeutics in cancer treatment, engineering chassis design, ethical considerations of biotherapeutics, and human practices. The six teams worked together throughout the writing process to build incredibly close relationships. We extensively reviewed previous iGEM projects and research papers on tumor microbiome therapeutics.
From determining the corresponding framework to collecting drafts from each team and finally sorting out the layout design, we are grateful to BNUZH-China for their contribution. They spent much time and energy designing the entire brochure's style. Sun Jicheng also worked hard to communicate with Chao Pan, the leader of BNUZH-China, to achieve the best display effect.
Finally, at the end of September, representatives of each team completed their signatures on the back of the manual. On the one hand, we hope this manual will provide solutions from multiple perspectives to the many common questions that future iGEM teams encounter on related topics and consider issues they may have overlooked. We also hope this manual can provide a reference for other researchers and be further improved. On the other hand, the instructions are illustrated and vivid in content. We hope to apply the manual to popular science on university campuses so that more students interested in biology and medicine or eager to learn about cancer can benefit from it.
[1] Kong W, Wanda SY, Zhang X, Bollen W, Tinge SA, Roland KL, Curtiss R 3rd. Regulated programmed lysis of recombinant Salmonella in host tissues to release protective antigens and confer biological containment. Proc Natl Acad Sci U S A. 2008 Jul 8;105(27):9361-6. doi: 10.1073/pnas.0803801105. Epub 2008 Jul 7. PMID: 18607005; PMCID: PMC2453710.
[2] Kang HY, Curtiss R., III Immune responses dependent on antigen location in recombinant attenuated Salmonella typhimurium vaccines following oral immunization. FEMS Immunol Med Microbiol Lett. 2003;37:99–104.
[3] Hess J, et al. Superior efficacy of secreted over somatic antigen display in recombinant Salmonella vaccine induced protection against listeriosis. Proc Natl Acad Sci USA. 1996;93:1458–1463.
[4] Curtiss R, III, et al. Recombinant Salmonella vectors in vaccine development. Dev Biol Stand. 1994;82:23–33.
[5] Cheng X, Kaplan LA. Simultaneous analyses of neutral carbohydrates and amino sugars in freshwaters with HPLC-PAD. J Chromatogr Sci. 2003;41:434–438.
[6] Sizemore DR, Branstrom AA, Sadoff JC. Attenuated bacteria as a DNA delivery vehicle for DNA-mediated immunization. Vaccine. 1997;15:804–807.
[7] Grillot-Courvalin C, Goussard S, Huetz F, Ojcius DM, Courvalin P. Functional gene transfer from intracellular bacteria to mammalian cells. Nat Biotechnol. 1998;16:862–866.
[8] Loessner H, Weiss S. Bacteria-mediated DNA transfer in gene therapy and vaccination. Expert Opin Biol Ther. 2004;4:157–168.