Our project BCAID aims to provide a new type of treatment for large wound healing, and our ultimate goal is to apply this system to the clinical treatment of large human wounds. So in the project design, we considered the multiple effects that BCAID may have on the human body, and used a fully safe implementation scheme in the project design to ensure that BCAID will not adversely affect the human body. In addition, the safety of the experimenter in the experimental operation and other safety problems in the project also need multiple guarantees. We will demonstrate the safety and reliability of BCAID from multiple perspectives.

Adeno-associated virus (type 6, AAV6) is a type of non-enveloped virus that can be engineered to deliver DNA to target cells. AAV6 vectors have been proved to be safe and effective in preclinical and clinical environments. Because of the unique biological characteristics, they can be widely used in clinical applications of various diseases, and have become one type of the leading gene delivery carriers in clinical development.

Through continuous development and research, the safety and clinical value of AAV6 have been recognized by the industry. First, the AAV6 immunogenicity is very low, which means that the use of AAV6 vectors will not cause serious immune reaction, and the retained viral structure does not have pathogenicity, which greatly improves the safety of AAV6 vectors. Secondly, the currently used AAV6 vectors have lost the ability of wild AAV6 to integrate into the genome. The introduced DNA fragment exists in the nucleus in the form of head to tail circular DNA, which is called episomal DNA, would further improve the safety of AAV6 vectors. In addition, we use subcutaneous injection to inject AAV6 into the wound. This is an in situ injection, which allows AAV6 to rapidly infect the target tissue and rarely spread to the whole body, thus reducing the impact of AAV6 on other normal tissues and organs. Finally, unlike retroviruses, which are of murine origin, the adeno-associated viral transfer systems are derived from human viruses and therefore may be more feasible for human gene therapy.

In conclusion, the safety of AAV6 vectors has been repeatedly verified and the clinical value of them has also been continuously developed. AAV6 vectors are safe and reliable gene delivery vectors.

Lentiviral vectors are a type of gene therapy vectors developed on the basis of HIV-1 (human immunodeficiency virus type I). These vectors have the ability to stably integrate into the genome of target cells and can lead to the continuous expression of genes of interest. Lentiviral vector technology has been widely used in basic biology and translation research and rapidly commercialized. Its commercial availability has accelerated the rapid expansion of lentiviral vector technology.

First, lentiviral vectors have low immunogenicity and the advantage of being able to transduce non-dividing cells. They have a wider tissue orientation and a potentially safer distribution of integration sites. These characteristics enable them to be safely used in clinical trials. Secondly, the lentiviral vectors have been transformed for many generations to eliminate the toxic genes and constructed self inactivated versions. In this way, even if all the viral proteins are present, RNA cannot be transcribed, and most of the genes of the original HIV genome are replaced, which enhances the safety.

In conclusion, lentiviral vectors have evolved into safe and effective gene delivery vectors, and their safety has been verified and rapidly commercialized.

In this project, we will transfer the cellulase gene into human fibroblasts with lentivirus vectors. Under the irradiation of blue light, cellulase will be continuously expressed and secreted until the BC membranes are completely degraded. In order to prevent the continuous expression and secretion of cellulase after the completion of BC degradation, we plan to introduce the REDMAP system into the modified cell line expressing cellulase by using lentivirus as the vector. After wound healing, we initiate self-apoptosis by red light to stimulate the secretion of MazF by engineered cells, so as to avoid tissue adverse reactions caused by continuous expression of cellulase. At the same time, this switch can also remove the foreign genes and proteins we have introduced, avoiding the possible risk of gene diffusion.

As a light-controlled cell suicide switch, it is likely to start when the shading conditions are not ideal. As we do not want it to kill our engineered cells ahead of time, the switch should be activated after the engineered cells have completed their tasks. We will achieve this by adding PCB from outside. PCB is a necessary factor for the operation of the REDMAP system. And we will add PCB by oral administration or injection at the right time, which is equivalent to providing double insurance for our suicide switch. It should be noted that PCB is a unique natural pigment of algae with special spectral properties, which has been widely used in various studies, and has been proved to be no adverse effects on the human body.

Cellulose, cellobiose and glucose will be produced during the degradation of cellulose. These sugars will persist since the beginning of the degradation process, and may form higher sugar concentrations in a certain period of time. In our design, most of the sugars will spread with the blood, while some of the glucose can be absorbed and utilized by the surrounding cells as an energy source. In addition, in order to reduce the inhibitory effects on the upstream pathway caused by the mass production of sugars, we will use the method of bioinformatics to reasonably match the mixed proportion and quantity of the three cell lines, and combine the experimental results to ensure the stable degration of cellulose without burdening the injury.

Laboratory safety rules are principles that every experimenter must keep in mind and abide by, which are vital to the safety of experimenters and the smooth development of experiments.

Before all experiments were carried out, we conducted laboratory training for each team member, including experimental skills, safety training, and the uses of each experimental equipment. And in the early stage of the experiment, we made sure there were two people work in groups to supervise each other and ensure the safety of the experiment. Teachers will regularly check the safety of the laboratory and standardize the behavior of team members in the laboratory.

We have two types of laboratories, molecular laboratory and cell laboratory. When entering the laboratories for experiments, we must abide by the laboratory rules, and wear experimental clothes and gloves. All laboratories are equipped with complete safety emergency equipments. Large-scale instruments are affixed with eye-catching and complete safety signs. When entering the cell laboratory, we must wear additional shoes and headgear. The cell laboratory needs to be disinfected regularly by UV to ensure pollution-free. The operations of cell biology related experiments have been safely taught and carried out in the ultra clean platform to reduce the possibility of our contact with viruses and cells, prevent pollution and ensure safety.

We have experienced instructors who gave us guidance and specifications on experimental operations and safety managements, so that team members can have a safer experimental environments and smoother experimental processes.

The maintenance of laboratory safety is inseparable from the efforts of each team member. The formulation and compliance of laboratory rules isa necessary condition for the smooth operation of the experiment.

Fig.1 Tidy and safe laboratory

Fig.2 Our team members are using the bechtop

Fig.3 Safety rules posted in the laboratory

For comprehensive human practice, we paid special attention to the safety of the participants. Under the influence of COVID-19, all parts of the country were in a state of normal prevention and control. During the visits and interviews, our members wear masks, actively cooperated with local epidemic prevention and control measures, and went to activities on the premise of confirming their own health.

In Integrated human practice, we received information and suggestions from people of all ages and identities, and we ensured that the personal privacy of participants were not be disclosed and individual rights were not be violated. In interviews, we had obtained the permission of the interviewees if recordings or screencaps were required, and ensured that the interview content would not be applied outside the project. At the same time, if the identity of the interviewee was special, we also took the form of anonymity to fully protected the personal privacy and rights of the interviewees through telephone, text communication and other forms. In addition, all our activities have made a commitment to the protection of personal information with participants.