Risk identification and prevention

1. Lab risk scenario and response plans

Despite undergoing comprehensive laboratory training, unexpected dangers can still arise beyond our control, such as equipment malfunctions or accidental mishandling of experiments. It is crucial to identify potential risks and implement effective plans to maintain a safe working environment. Here are our general risk management procedures:

Burns

If scalded while using an alcohol lamp, autoclaving, or handling agarose gel, immediately rinse with cold water, soak the affected area, and apply appropriate burn medication.

Cuts

In case of cuts from sharp instruments while working with agarose gel or other materials, clean the wound immediately, disinfect it, and apply a bandage to prevent infection. Seek medical attention promptly for serious injuries.



(Figure 1) Burn and Cut wounds management steps

Chemical Exposure

• Skin contact:We Immediately flush the affected area with plenty of water for at least 15 minutes in addition to Removing any contaminated clothing and shoes.
• Eye contact:Immediately flush eyes with plenty of water for at least 15 minutes,We occasionally lift the upper and lower eyelids.
• Inhalation:Moving the victim to fresh air. If he/she is not breathing, We give artificial respiration.
• Ingestion:Never induce vomiting,but give plenty of water to drink, and immediately seek medical attention.

  (Figure2) Types of chemical exposures

Fire and Major Accidents

• For small incidents within our capacity to manage, Use appropriate firefighting equipment and emergency measures after disconnecting power sources.
• For larger incidents, evacuate the area using designated fire escape routes and immediately contact emergency services.

Spillage of Biological Substances

If there is an accidental spill of engineered bacteria or other biologically active substances:

1. Immediately decontaminate the affected area thoroughly.
2. Disinfect hands and any exposed skin promptly.

Hopefully, by adhering strictly to these risk management procedures, we aim to minimize potential hazards and ensure the safety of all personnel involved in our experiments.



(Figure 3) Biological spillage

2. Dual-use Identification

When the recombinant DNA technology emerged it was a breakthrough in the field of synthetic biology which led to its rapid growth and the increases in its influence on other fields.However,This breakthrough can be used for evil purposes and of course this depends on the intentions of the one holding this technology itself. While the misuse of such advancements can pose significant risks, we firmly believe that these challenges should not dissuade us. Instead, we advocate approaching synthetic biology with a steadfast commitment to rigorous risk assessment. This approach empowers us to design and conduct experiments with meticulous care, ensuring that our endeavors uphold the highest standards of safety and efficacy.

3. Misuse risk

Potential risks of the project

• We mentioned the oncogenic potential of YAP-1 which if overexpressed ; it would lead to uncontrolled cell growth and tumor formation.


(Figure 4) YAP-1 overexpression risk

• While high specificity of CRISPR/dCas9 is present , it can still have off-target effects , causing the miss expression of the intended genes.
• Introducing a foreign genetic material to our body as the CRISPR/dCas9 system can induce an immune response that leads to rejection of the MSCs.

Potential hazards of information and technology used in this project

• There is a risk of data breaching or unauthorized access to the database, so it is a threat to our data privacy and security, so we stored the data with limited access (authorized personnel) where the survey data is stored using MySQL. There are a limited number of people who gain access to the server.

4. Identification of future application risks

While the project design addresses many safety concerns, there are still potential risks to consider for future applications:

1. Long-Term Effects of YAP-1 Up-regulation

Our project focuses on short-term YAP-1 expression to accelerate wound closure. However, the long-term effects of even transient YAP-1 up regulation are not fully understood. It's crucial to investigate if YAP-1 could contribute to scar formation, abnormal tissue growth, or even cancer development in the long run.

2. Off-Target Effects of dCas9

Surely, after using dCas9, the risk of DNA cleavage was decreased but off targeting risk is still present so we need to ensure that the dCas9 doesn't accidentally activate or repress any genes (or even unwanted ones) through repeated extensive testing

3. Unexpected Immune Response

Introducing engineered MSCs might trigger unforeseen immune responses in some patients. Close monitoring and potential immunosuppressive therapy might be necessary during initial applications.

4. Stability and Control of Engineered MSCs

There is a need to evaluate the stability of genetic modifications within the engineered MSCs due to The risk of genetic machinery malfunctioning or becoming uncontrollable over time. Additionally, mechanisms for controlling or eliminating the engineered MSCs after wound healing need to be established.

5. Ethical Considerations

Genetically modifying cells for therapeutic purposes raises ethical concerns. Informed consent, potential risks versus benefits, and long-term consequences for patients need careful consideration and open communication.

We conducted a survey with burn patients along with doctors (plastic and orthopedics) in addition to online surveys but before that they signed informed consent implying that they accepted entering the survey voluntarily.

The informed consent is found below:



(Figure 5) Informed Consent

Content of page 1

hhullo

• Silman AJ, MacGregor AJ, Thomson W, Holligan S, Carthy D, Farhan A, Ollier WE. Twin concordance rates for rheumatoid arthritis: results from a nationwide study. Br J Rheumatol. 1993 Oct;32(10):903-7.
• Qiu M, Zhou XM, Liu L. Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection. J Anal Test. 2022;6(1):44-52. doi: 10.1007/s41664-022-00212-4. Epub 2022 Feb 28. PMID: 35251748; PMCID: PMC8883004.