Team:BNUZH-CHINA/Safety
Overview
Our project, FeFighterS, is focused on utilizing the mechanism of ferroptosis and
enhancing its induction in tumor cells. The primary objective is to develop a safer and more efficient
approach for eliminating tumor cells and treating cancer. Our ultimate goal is to apply this system to the
clinical treatment of cancer. Therefore, in the project design, we have considered the potential impacts
of this system on the human body. We have implemented a safe implementation plan and designed a safety
module to prevent plasmid loss and to prevent the escape of engineered bacteria in the late stage of
treatment, reducing the adverse effects that they may have on the external environment and on normal human
cells while killing tumor cells. In addition, we have also taken the safety of the experimental personnel
during the experimental operation process, laboratory safety, and other safety issues in the project into
account. We will demonstrate the safety and reliability of the FeFighterS project from various aspects.
Design safety
Safety of attenuated Salmonella typhimurium
Salmonella is an invasive intracellular parasite that can introduce exogenous proteins into tumor
cells through a type III secretion system. Among a variety of bacteria used in tumor treatment research,
Salmonella is widely used in various solid tumor treatment studies because of its good tumor
targeting and anti-tumor effects.
VNP20009 is an attenuated S. typhimurium strain with a higher safety profile compared to wild-type Salmonella. It has been studied in animal tumor models and phase I clinical trials in cancer patients1. First of all, the VNP20009 strain used in the project is a S. typhimurium strain with msbB and purI gene deletion, which is genetically stable and susceptible to common antibiotics. The protein encoded by the msbB is required for lipid acylation to endotoxin, and its absence prevents the lipid-like A terminal from acylation, thus reducing the virulence of this strain. The purI is essential in purine metabolism, and its deletion leads to the requirement exogenous purines for bacterial reproduction, so that the strain can better target the tumor microenvironment. This indicates that VNP20009 offers the advantages of robust treatment effects and low toxic side effects. In addition, VNP20009 can specifically target hypoxia microenvironment caused by tumor tissues, multiply in anoxic areas of tumor tissues, and show good anti-tumor efficacy, thereby reducing the impact on other normal tissues. It is a typical representative of bacteria-mediated tumor treatment and is currently widely used as an anti-tumor carrier.
In summary, VNP20009 has low pathogenicity, high intratumor targeting and anti-tumor activity, and is a safe and reliable anti-tumor drug carrier.
VNP20009 is an attenuated S. typhimurium strain with a higher safety profile compared to wild-type Salmonella. It has been studied in animal tumor models and phase I clinical trials in cancer patients1. First of all, the VNP20009 strain used in the project is a S. typhimurium strain with msbB and purI gene deletion, which is genetically stable and susceptible to common antibiotics. The protein encoded by the msbB is required for lipid acylation to endotoxin, and its absence prevents the lipid-like A terminal from acylation, thus reducing the virulence of this strain. The purI is essential in purine metabolism, and its deletion leads to the requirement exogenous purines for bacterial reproduction, so that the strain can better target the tumor microenvironment. This indicates that VNP20009 offers the advantages of robust treatment effects and low toxic side effects. In addition, VNP20009 can specifically target hypoxia microenvironment caused by tumor tissues, multiply in anoxic areas of tumor tissues, and show good anti-tumor efficacy, thereby reducing the impact on other normal tissues. It is a typical representative of bacteria-mediated tumor treatment and is currently widely used as an anti-tumor carrier.
In summary, VNP20009 has low pathogenicity, high intratumor targeting and anti-tumor activity, and is a safe and reliable anti-tumor drug carrier.
Safety of the targeting modules
Transgenic attenuated S. typhimurium VNP20009 is an important carrier for cancer treatment and
vaccine development, but only exhibits limited tumor targeting in vivo. Therefore, in order to
enhance the ability of engineered bacteria targeting tumors and to simultaneously reduce the toxicity on
healthy tissue cells, we designed the lipopolysaccharide-outer membrane protein (Lpp-OmpA) display system
for the expression of carcinoembryonic antigen (CEA)-specific single-chain antibody fragments (scFv) on
the surface of engineered bacteria. The Lpp-OmpA system is a technique that uses OmpA to efficiently
anchor the outer membrane and allows displaying its coupled passenger proteins that fuse at the C-terminus
of the OmpA protein. The signal peptide of Lpp fuse with OmpA fractional peptide to form a composite
display system that has successfully fulfilled for many macromolecular proteins.
CEACAM5 belongs to the immunoglobulin family of CEA-associated cell adhesion molecules (CEACAMs), which are closely related to various functions of tumor cells, including the adhesion, proliferation, and migration of the tumor cells. Carcinoembryonic antigen is widely expressed in many human cancer cells such as gastrointestinal cancer, pancreatic cancer, non-small cell lung cancer, and breast cancer. It is a widely studied tumor biomarker with a broad spectrum. Based on the above research, we hope to rely on the Lpp-OmpA expression system to express scFv on the surface of engineered bacteria, so as to improve the effect of tumor targeting.
In summary, our targeting module is safe and reliable, which provide an important guarantee for the design safety of the project.
CEACAM5 belongs to the immunoglobulin family of CEA-associated cell adhesion molecules (CEACAMs), which are closely related to various functions of tumor cells, including the adhesion, proliferation, and migration of the tumor cells. Carcinoembryonic antigen is widely expressed in many human cancer cells such as gastrointestinal cancer, pancreatic cancer, non-small cell lung cancer, and breast cancer. It is a widely studied tumor biomarker with a broad spectrum. Based on the above research, we hope to rely on the Lpp-OmpA expression system to express scFv on the surface of engineered bacteria, so as to improve the effect of tumor targeting.
In summary, our targeting module is safe and reliable, which provide an important guarantee for the design safety of the project.
Safety of the suicide module
In the safety module, we developed a “logical suicide circuit” to maintain two functional plasmids and
induce engineered bacteria to commit suicide through intaking the doxycycline. This ensures the safety and
feasibility of our project.
In the design of the artificial suicide switch, antitoxin (Sok RNA) will be steadily transcribed to protect engineered bacteria from toxin (Hok protein). When the patients’ condition improves after tumor regression, they can take doxycycline (Dox) orally to repress the expression of Sok gene through the “logical suicide circuit”, while the expression of Hok gene remains affected. Ultimately, this leads to the death of engineered bacteria. Dox is a tetracycline analog that is well-tolerated and can be absorbed almost completely by the digestive system. It has a wide range of therapeutic properties2,3, including controlling invasive and metastatic cancer cells, inhibiting tumor growth, and preventing cancer metastasize4. Studies have also shown its potential as an anti-tumor drug for the treatment of cancer patients4. Furthermore, clinical trials have demonstrated the use of oral doxycycline for the treatment of other diseases5. Therefore, we believe that our oral doxycycline program is safe and feasible.
To maintain the presence of both plasmids in the engineered bacteria, we utilized the different half-lives of hok protein and sok RNA in the “logical suicide circuit”. Through the intricate interaction between the gene circuit contained in the two plasmids, we achieved the desired effect that the loss of either plasmid would result in the death of the engineered bacteria. This ensures the simultaneous presence of these two plasmids and the prevention of the loss of the killing module, whichfurther enhancing the safety of our project.
In the design of the artificial suicide switch, antitoxin (Sok RNA) will be steadily transcribed to protect engineered bacteria from toxin (Hok protein). When the patients’ condition improves after tumor regression, they can take doxycycline (Dox) orally to repress the expression of Sok gene through the “logical suicide circuit”, while the expression of Hok gene remains affected. Ultimately, this leads to the death of engineered bacteria. Dox is a tetracycline analog that is well-tolerated and can be absorbed almost completely by the digestive system. It has a wide range of therapeutic properties2,3, including controlling invasive and metastatic cancer cells, inhibiting tumor growth, and preventing cancer metastasize4. Studies have also shown its potential as an anti-tumor drug for the treatment of cancer patients4. Furthermore, clinical trials have demonstrated the use of oral doxycycline for the treatment of other diseases5. Therefore, we believe that our oral doxycycline program is safe and feasible.
To maintain the presence of both plasmids in the engineered bacteria, we utilized the different half-lives of hok protein and sok RNA in the “logical suicide circuit”. Through the intricate interaction between the gene circuit contained in the two plasmids, we achieved the desired effect that the loss of either plasmid would result in the death of the engineered bacteria. This ensures the simultaneous presence of these two plasmids and the prevention of the loss of the killing module, whichfurther enhancing the safety of our project.
Laboratory safety
Overview
When working on synthetic biology projects, we need to focus on safety issues at every step. Our team
takes the safety of our members and the environment as an important prerequisite. Consequently, we are
dedicated to taking all necessary precautions to avoid personal or environmental injury. Our team has
improved laboratory safety guidelines, standardized experimental operations, and conducted systematic
experimental operation training to jointly ensure experimental safety from various aspects.
Lab Safety
(i) Universal safety in the laboratory
First, we clarified the rules regarding personal protection when entering the laboratory. Team members need to wear lab coats, gloves, masks, and wear goggles when necessary. Second, we isolated laboratories and rest areas. It is strictly forbidden to bring food into the laboratory and take things away from the laboratory at the same time. Third, we provided explicit guidance on chemical safety. It is necessary to achieve classified and standardized storage. Irritating volatile drugs must be operated in a fume hood. Our laboratory is equipped with perfect fire prevention and emergency equipment, such as fire blankets, fire extinguishers, smoke alarms, emergency showers, eyewashes, and so on.
(ii) Special laboratory safety of our project
We conducted experiments in two types of laboratories, molecular and microbiology laboratory and cell biology laboratory. When entering the molecular and microbiology laboratory, it is necessary to follow the laboratory rules and wear a lab coat and gloves. It is momentous to avoid direct contact with microorganisms, pay attention to aseptic operation in biological safety cabinets and clean tables, and use alcohol lamps safely. Upon entering the Cell Biology Laboratory, additional safety measures are required, including the use of masks, head covers, and shoe covers. Cell biology laboratory is regularly disinfected with ultraviolet light to ensure contamination free. Besides, personnel are arranged to clean and disinfect the laboratory regularly.
Our project involved attenuated Salmonella. Although there is a proof that attenuated engineered bacteria is safe enough, we still required team members to wear gloves and masks when performing relevant operations to ensure safety. At the same time, we performed operations such as bacterial inoculation in specific biosafety cabinets and culture in different incubators to avoid contamination and infection.
Additionally, we benefited from the guidance and expertise of experienced instructors. These instructors provided comprehensive instructions for experimental procedures and safety protocols. This concerted effort guaranteed not only a secure experimental environment but also a smoother workflow for all team members.
First, we clarified the rules regarding personal protection when entering the laboratory. Team members need to wear lab coats, gloves, masks, and wear goggles when necessary. Second, we isolated laboratories and rest areas. It is strictly forbidden to bring food into the laboratory and take things away from the laboratory at the same time. Third, we provided explicit guidance on chemical safety. It is necessary to achieve classified and standardized storage. Irritating volatile drugs must be operated in a fume hood. Our laboratory is equipped with perfect fire prevention and emergency equipment, such as fire blankets, fire extinguishers, smoke alarms, emergency showers, eyewashes, and so on.
Figure 1. Laboratory safety information board and emergency equipment
We conducted experiments in two types of laboratories, molecular and microbiology laboratory and cell biology laboratory. When entering the molecular and microbiology laboratory, it is necessary to follow the laboratory rules and wear a lab coat and gloves. It is momentous to avoid direct contact with microorganisms, pay attention to aseptic operation in biological safety cabinets and clean tables, and use alcohol lamps safely. Upon entering the Cell Biology Laboratory, additional safety measures are required, including the use of masks, head covers, and shoe covers. Cell biology laboratory is regularly disinfected with ultraviolet light to ensure contamination free. Besides, personnel are arranged to clean and disinfect the laboratory regularly.
Our project involved attenuated Salmonella. Although there is a proof that attenuated engineered bacteria is safe enough, we still required team members to wear gloves and masks when performing relevant operations to ensure safety. At the same time, we performed operations such as bacterial inoculation in specific biosafety cabinets and culture in different incubators to avoid contamination and infection.
Additionally, we benefited from the guidance and expertise of experienced instructors. These instructors provided comprehensive instructions for experimental procedures and safety protocols. This concerted effort guaranteed not only a secure experimental environment but also a smoother workflow for all team members.
Training and Enforcement
Before all experiments are conducted, we conducted laboratory training for all team members, including
experimental skills, safety training, and the operation of related experimental equipment. In the early
stage of the experiment, we made sure that two people work in pairs to supervise each other and ensure the
safety of the experiment. Teachers regularly check the safety of the laboratory and regulate the behavior
of the laboratory team members. Prominent and comprehensive safety signs are prominently displayed on
large instruments.
(i) Assessment of cell biology experiments
The members of the previous iGEM team conducted comprehensive training of the four basic cell experiments (cell resuscitation, counting, passage and cryopreservation) for the new team members. We require experimenters to change their shoes in the buffer room before entering the cell culture room, and they should try to avoid walking or staying in the buffer room. During the training period, a maximum of four students can use the cell culture room at the same time, and they should try to avoid lingering or engaging in conversations inside. Following this training, an assessment was conducted. Only team members who passed the assessment can carry out subsequent cellular experiments.
(ii) Training on the operation of electroporator
Our project required an electroporator to transfer the constructed plasmid into attenuated Salmonella. Therefore, we invited relevant technicians to train us on the use of the electroporator and precautions, and printd and pasted the relevant operation steps next to the electroporator.
(iii) Training on the operation of Biological safety cabinet
We required team members to perform experiments related on attenuated Salmonella in a biosafety cabinet. Before the start of the experiment, we conducted training on the use of biological safety cabinets, emphasizing the differences between biological safety cabinets and ultra-clean workbenches. The latter mainly protect samples in the work area from external environmental pollution, but do not effectively protect operators. When using a biosafety cabinet, avoid placing too many items in the cabinet, so as not to affect the airflow and safety performance of the safety cabinet. Before and after the use of the safety cabinet, we need to follow the procedure to sterilize the surface of the safety cabinet.
(iv) Training on the operation of large instruments
The training content includes the use of large instruments such as high-speed centrifuges, autoclaves, inverted fluorescence microscopes and so on. The training details the principles of action of these large instruments and highlights the precautions. The use of high-speed centrifuges requires high attention to strict trim. We should check the liquid level before each use of the autoclave and promptly remove items post-use. Inverted fluorescence microscope needs to be observed in a dark space. We should pay attention to wait for the eyes to adapt to the dark environment before observing and at the same time we need to wear protective glasses when adjusting the light source to avoid ultraviolet rays damage to the eyes.
Figure 2. Safety marking of large instrument
The members of the previous iGEM team conducted comprehensive training of the four basic cell experiments (cell resuscitation, counting, passage and cryopreservation) for the new team members. We require experimenters to change their shoes in the buffer room before entering the cell culture room, and they should try to avoid walking or staying in the buffer room. During the training period, a maximum of four students can use the cell culture room at the same time, and they should try to avoid lingering or engaging in conversations inside. Following this training, an assessment was conducted. Only team members who passed the assessment can carry out subsequent cellular experiments.
Figure 3. Cell biology experiment training in the buffer room
(ii) Training on the operation of electroporator
Our project required an electroporator to transfer the constructed plasmid into attenuated Salmonella. Therefore, we invited relevant technicians to train us on the use of the electroporator and precautions, and printd and pasted the relevant operation steps next to the electroporator.
Figure 4. Training of electroporator operation
(iii) Training on the operation of Biological safety cabinet
We required team members to perform experiments related on attenuated Salmonella in a biosafety cabinet. Before the start of the experiment, we conducted training on the use of biological safety cabinets, emphasizing the differences between biological safety cabinets and ultra-clean workbenches. The latter mainly protect samples in the work area from external environmental pollution, but do not effectively protect operators. When using a biosafety cabinet, avoid placing too many items in the cabinet, so as not to affect the airflow and safety performance of the safety cabinet. Before and after the use of the safety cabinet, we need to follow the procedure to sterilize the surface of the safety cabinet.
Figure 5. Biosafety cabinets used in this project
(iv) Training on the operation of large instruments
The training content includes the use of large instruments such as high-speed centrifuges, autoclaves, inverted fluorescence microscopes and so on. The training details the principles of action of these large instruments and highlights the precautions. The use of high-speed centrifuges requires high attention to strict trim. We should check the liquid level before each use of the autoclave and promptly remove items post-use. Inverted fluorescence microscope needs to be observed in a dark space. We should pay attention to wait for the eyes to adapt to the dark environment before observing and at the same time we need to wear protective glasses when adjusting the light source to avoid ultraviolet rays damage to the eyes.
Figure 6. Training on the use of autoclave
Safety in Integrated Human Practices
Personnel safety
During the post-pandemic era, our members steadfastly placed the personnel safety of all participants at
the top of the list by consistently following basic protective measures while carrying out activities.
Furthermore, prior to entering the laboratory and engaging in activities such as bacteria painting and lab
tour, we took great care in educating visitors about laboratory safety rules. We emphasized the importance
of wearing protective coverings , including masks and gloves, as a strict requirement.
Personal privacy safety
In the integrated human practice, we ensured the personal privacy of every participant and their
individual rights. Specifically, when collecting survey information, we employed an anonymous approach and
committed to not using the gathered data for any other purposes. During interviews, if audio or video
recording was required, we obtained prior consent from the interviewees. If we intended to publish the
interview content online, we sought approval from the interviewees in advance for review. In essence, we
made every effort to guarantee the personal privacy of our participants.
References
1 TOSO J F, GILL V J, HWU P, et al. Phase I study of the intravenous administration of attenuated
Salmonella typhimurium to patients with metastatic melanoma [J]. Journal of clinical oncology:
official journal of the American Society of Clinical Oncology, 2002, 20(1): 142.
2 CROSS R, LING C, DAY N P, et al. Revisiting doxycycline in pregnancy and early childhood--time to rebuild its reputation? [J]. Expert Opin Drug Saf, 2016, 15(3): 367-82.
3 SHEN L C, CHEN Y K, LIN L M, et al. Anti-invasion and anti-tumor growth effect of doxycycline treatment for human oral squamous-cell carcinoma--in vitro and in vivo studies [J]. Oral Oncol, 2010, 46(3): 178-84.
4 MARKOWSKA A, KAYSIEWICZ J, MARKOWSKA J, et al. Doxycycline, salinomycin, monensin and ivermectin repositioned as cancer drugs [J]. Bioorg Med Chem Lett, 2019, 29(13): 1549-54.
5 AMORES-MARTIN E, MELE-NINOT G, DEL ALCAZAR VILADOMIU E, et al. Successful Treatment of White Sponge Nevus With Oral Doxycycline: A Case Report and Review of the Literature [J]. Actas Dermosifiliogr (Engl Ed), 2021, 112(5): 463-6.
2 CROSS R, LING C, DAY N P, et al. Revisiting doxycycline in pregnancy and early childhood--time to rebuild its reputation? [J]. Expert Opin Drug Saf, 2016, 15(3): 367-82.
3 SHEN L C, CHEN Y K, LIN L M, et al. Anti-invasion and anti-tumor growth effect of doxycycline treatment for human oral squamous-cell carcinoma--in vitro and in vivo studies [J]. Oral Oncol, 2010, 46(3): 178-84.
4 MARKOWSKA A, KAYSIEWICZ J, MARKOWSKA J, et al. Doxycycline, salinomycin, monensin and ivermectin repositioned as cancer drugs [J]. Bioorg Med Chem Lett, 2019, 29(13): 1549-54.
5 AMORES-MARTIN E, MELE-NINOT G, DEL ALCAZAR VILADOMIU E, et al. Successful Treatment of White Sponge Nevus With Oral Doxycycline: A Case Report and Review of the Literature [J]. Actas Dermosifiliogr (Engl Ed), 2021, 112(5): 463-6.
