Safety

Overview

During our progress in the project, safety is our inevitable companion along the project milestones. This year, we were inspired by the safety approach of the NMU-China iGEM team in 2023. Following iGEM Responsibility guidelines, it is an important objective to prevent harm to ourselves, colleagues, and the environment. It was crucial to predict and prevent potential accidents during our lab experiments. So, we anticipated as many risks as we could and provided solutions for them. Moreover, it is of utmost importance to adhere to lab safety standards and follow the regulations and laws during our experiments in the lab. We have put in consideration not only the lab safety but also our design safety in the form of synthetic specific receptor and mRNA-based switch. This, in return, saves the consumer from any unwanted side effects caused by our SONG-H.

Figure(1). This figure shows our Safety Goals

Safety checklist

Contributing to Biosafety

Through designing 2 systems (TID switch – dCas9 Syn-RTK) for providing strict control over the proliferation of both MSCs (while promoting its regenerative function) and viable cells. Moreover, we have implemented a new approach to control the mRNA translation which can be adapted in all synthetic biology projects. We also shared our lab safety equipment in addition to our lab safety guidebook and the requirements to ensure safety for other teams.

Contributing to Biosecurity

To prevent theft and loss of any data or the misuse of any materials provided within our project, we have implemented data security measures to protect the stored material and limit any unauthorized access.

Validation of our design

Through modelling the switch for regulation of YAP-1 expression within the viable cells of the wound in addition to providing a model for the dCas9 Syn-RTK receptor activation for expression of YAP-1 within the MSCs.

Background

Relying on a strong base of extensive literature search in addition to reviewing the approaches of previous iGEM efforts concerning our design

Potential risks

Discussing the potential risks of our project in addition to future application risk, misuse risk, experimental risks then providing the appropriate responses to these concerns.

Beyond iGEM

We addressed the use of synthetic biology not only within the competition but also beyond it through reviewing any future application risks, discussing policy analysis such as the informed consent from the patients in addition to biosafety of our product.

By providing a solution to the longstanding problem of limited sensitivity and specificity, this safety approach opens up new possibilities for groundbreaking research and clinical applications in regenerative medicine which is not only limited to other iGEM teams but also to anyone with interest in the field of regenerative medicine.

Project Design Safety

Possible Hazards

Addressing our project's safety in relation to the parts which are used in the genetic circuit.

Figure(2). This figure shows Possible Hazards

First problem

1- Our project utilizes engineered MSCs to improve wound healing , and after a lot of research, we found some literature mentioning that cell-based therapy using MSC increases the chance of developing tumors or worsens the case of already presented ones, especially connective tissue tumors.

Solution :

In order to solve this risk , we used a suicidal gene called iCaspase 9 (IC9) system into our engineered MSCs. IC9 is a suicide system that will give us the ability to terminate the life of our MSC selectively through apoptosis. The apoptotic death and suicide of the MSC will occur in case of the liability of uncontrolled proliferation and the possibility of the conversion of the MSC into cancerous cells. The IC9 system is composed of 2 main inactive-domains that are activated upon exposure to a chemical inducer of dimerization (CID). CID is an external non-toxic element that activates the IC9 system and induces our MSC apoptosis terminating its effect on the patient’s immune system.

Figure(3). This figure shows how our suicidal gene IC9 acts on MSCs in case of conversion into cancer cells by the external activation of CID injection

Second Problem

We recognized that promoting YAP-1 expression may carry relative risks, as some studies reported that YAP-1 over-expression is associated with hypertrophic tissue changes that may progress finally into tumor formation. Therefore, we had to take confident steps toward the regulation of YAP-1 gene induction,thus we determined the level in which YAP-1 may perform pathologic hypertrophic effects.

Relying on previous studies analyzing the role of YAP-1 in organ size and hypertrophy ,they have induced the expression of YAP-1 in different conditions and magnitudes and they found that YAP-1 pathological effects start to occur when its concentration is upregulated to more than fifty fold over normal in equivalent conditions.

Solution :

To establish strict control over YAP-1 induction within our engineered MSCs and our modified version of YAP-1 loaded within MSCs’ exosomes that would transfect YAP-1 into the neighboring viable cells within the wound microenvironment.

We incorporated two interacting innovative devices that would guarantee safe, and specified therapeutic effects of YAP-1 within our genetically modified MSCs and the exosome recipient cells .

Fig(4). This figure illustrates the two main two elements of our platform.

Subsequently, our first component is a new modality of synthetic receptors known as dCas9 Syn-RTK that can repurpose the external domain of native tyrosine kinase receptors (RTK) by ingrafting dCas9 within the internal domain of the receptor’s chain. As a result, this receptor could modulate the expression levels of endogenous genes in response to natural ligands.

Regarding our design, our version of the receptor was engineered to induce YAP-1 conditionally in response to VEGF-1 which is a pro-inflammatory biomarker that was found to reflect the magnitude of tissue injury.

In other words, we rewired the normal pathway of vascular endothelial growth factor receptor (VEGFR) to promote safe, effective and dose-dependent expression of YAP-1.

This Video how we conditioned our dCas (N/C) Syn-RTK .

Simultaneously, our second component is a novel approach for transgene regulation known as translation initiation device (TID) which has a great potential for pushing the boundaries of cell based gene therapy to new levels. This method relies on conditioning the translation of transgenes to specific intracellular biomarkers.

Meanwhile, we implemented TID in our design to restrict YAP-1 mRNA translation into effector proteins unless the initiator biomarker (MMP9) presents in high concentration. We have chosen MMP-9 as the biomarker of interest as it reflects cellular damage. Moreover, the expression of TID is linked to the activity of our synthetic receptor to ensure optimum regulation of our platform .

Furthermore, TID/(YAP-1) transcript will be equipped within MSCs’ exosomes through a specific RNA loading system.

Fig.(5) This figure illustrates the off/ON state of TID that initiates the translation of YAP-1.

Consequently, the exosomes carrying our therapeutic cargo will transport TID/(YAP-1) to the neighboring cells within the wound microenvironment enhancing their regenerative capabilities .

Fig.(6) This figure illustrates the transport of TID to the neighboring cells through exosomes.

Tuning of our platform therapeutic effect :

To enhance the sensitivity of our platform we integrated new techniques that would provide better concentration sensitive response.

Regarding dCas9 Syn-RTK we found that changing the sensitivity of the receptor could be mediated by altering the binding affinity of Tobacco Etch Virus (TEV) protease to its specific peptide motif TEV cleavage site (TCS). This was accomplished by performing a single point mutation in TCS that would modulate the grade of TEV recognition to TCS, creating a wide range of receptor sensitivity according to the grafted TCS version within the receptor chains. The sensitivity of the receptor will be customized based on the minimal required concentration of the biomarker of interest to activate the receptor.

Furthermore, we took the sensitivity of TID to a higher level , as we can tailor the switch responsiveness to the initiator biomarker (MMP9) by changing the rate of TID components assembly.Tuning of TID was mediated through altering the MS2 copy number repeats ,thus it was found that MS2 copy number repeats is directly proportional to TID’s sensitivity .

Third problem

Despite the high potential of CRISPR dCas9 technology in regulating the transcription level of the gene of interest. This technology still misses target specificity that may interfere with the biological safety measures required for any biological drug. The cause behind the previously mentioned off targeting event may be due to the low specificity of gRNA that may mediate target gene mismatches resulting in unwanted genes modulation that may threat the viability of the cells.

Figure(7). This figure shows dCas9 off-targeting .

Solution :

To ensure target gene specificity of our signal transduction model dCas9 we took in consideration the role of gRNA stability and sensitivity in limiting the off targeting effects of CRISPR systems. As a result we generated 58 variants of gRNA by using CRISPR ON online software tool. gRNAs are designed according to Nanog enhancer sequence which is upstream to YAP-1 encoding region located within the 11th chromosome.

Moreover we tested the off targeting potential and sensitivity for each gRNA using CRISPR OFF beside calculating the thermodynamic qualities that would affect the binding stability with the target gene.

These measurements were done by using online software vienna RNA packages including: RNAfold, RNAup, and RNAeval

Finally, these domains will unite and transfer to the nucleus under the guidance of NLS. In contrast, in absence of VEGF, the N-dcas-9 domain separates away from the C-dcas-9 domain under the guidance of NES (extra-nuclear) and NLS (intra-nuclear),respectively.In other words, we selected the best three candidates of gRNA to be implemented in the upcoming experimental validation trails to ensure specific ,safe and effective signal transduction from our platform dCas Syn-RTK.

In other words, we selected the best three candidates of gRNA to be implemented in the upcoming experimental validation trails to ensure specific ,safe and effective signal transduction from our platform dCas Syn-RTK.

Figure(8). This figure illustrates the secondary structure of our gRNA by using RNA Fold online software tool .

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References

[1]Ramos CA, Asgari Z, Liu E, Yvon E, Heslop HE, Rooney CM, Brenner MK, Dotti G. An inducible caspase 9 suicide gene to improve the safety of mesenchymal stromal cell therapies. Stem Cells. 2010 Jun;28(6):1107-15. doi: 10.1002/stem.433. PMID: 20506146; PMCID: PMC329039.

[2]Jin Y, Li S, Yu Q, Chen T, Liu D. Application of stem cells in regeneration medicine. MedComm (2020). 2023 Jun 17;4(4):e291. doi: 10.1002/mco2.291. PMID: 37337579; PMCID: PMC10276889.

[3]Wei Y, Hui VLZ, Chen Y, Han R, Han X, Guo Y. YAP/TAZ: Molecular pathway and disease therapy. MedComm (2020). 2023 Aug 9;4(4):e340. doi: 10.1002/mco2.340. PMID: 37576865; PMCID: PMC10412783.

[4]Krawczyk, Krzysztof, et al. "Rewiring of endogenous signaling pathways to genomic targets for therapeutic cell reprogramming." Nature communications 11.1 (2020): 608.

[5]Wagh K, Ishikawa M, Garcia DA, Stavreva DA, Upadhyaya A, Hager GL. Mechanical Regulation of Transcription: Recent Advances. Trends Cell Biol. 2021 Jun;31(6):457-472. doi: 10.1016/j.tcb.2021.02.008. Epub 2021 Mar 9. PMID: 33712293; PMCID: PMC8221528.

[6]oh, P.J.Y., Sudol, M. & Saunders, T.E. Optogenetic control of YAP can enhance the rate of wound healing. Cell Mol Biol Lett 28, 39 (2023).

[7]Ramos CA, Asgari Z, Liu E, Yvon E, Heslop HE, Rooney CM, Brenner MK, Dotti G. An inducible caspase 9 suicide gene to improve the safety of mesenchymal stromal cell therapies. Stem Cells. 2010 Jun;28(6):1107-15. doi: 10.1002/stem.433. PMID: 20506146; PMCID: PMC329039

[8]Oakes, B.L., Nadler, D.C., Flamholz, A., Fellmann, C., Staahl, B.T., Doudna, J.A. and Savage, D.F., 2016. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch. Nature biotechnology, 34(6), pp.646-651

[9]Alessio N, Stellavato A, Squillaro T, Del Gaudio S, Di Bernardo G, Peluso G, De Rosa M, Schiraldi C, Galderisi U. Hybrid complexes of high and low molecular weight hyaluronan delay in vitro replicative senescence of mesenchymal stromal cells: a pilot study for future therapeutic application. Aging (Albany NY). 2018 Jul 12;10(7):1575-1585. doi: 10.18632/aging.101493. PMID: 30001217; PMCID: PMC6075440.

[10]McCarty, N.S., Graham, A.E., Studená, L. et al. Multiplexed CRISPR technologies for gene editing and transcriptional regulation. Nat Commun 11, 1281 (2020).

Overview

During our progress in the project, safety is our inevitable companion along the project milestones. This year, we were inspired by the safety approach of the NMU-China iGEM team in 2023. Following iGEM Responsibility guidelines, it is an important objective to prevent harm to ourselves, colleagues, and the environment. It was crucial to predict and prevent potential accidents during our lab experiments. So, we anticipated as many risks as we could and provided solutions for them. Moreover, it is of utmost importance to adhere to lab safety standards and follow the regulations and laws during our experiments in the lab. We have put in consideration not only the lab safety but also our design safety in the form of synthetic specific receptor and mRNA-based switch. This, in return, saves the consumer from any unwanted side effects caused by our SONG-H.

Figure(1). This figure shows our Safety Goals

Safety checklist

Contributing to Biosafety

Through designing 2 systems (TID switch – dCas9 Syn-RTK) for providing strict control over the proliferation of both MSCs (while promoting its regenerative function) and viable cells. Moreover, we have implemented a new approach to control the mRNA translation which can be adapted in all synthetic biology projects. We also shared our lab safety equipment in addition to our lab safety guidebook and the requirements to ensure safety for other teams.

Contributing to Biosecurity

To prevent theft and loss of any data or the misuse of any materials provided within our project, we have implemented data security measures to protect the stored material and limit any unauthorized access.

Validation of our design

Through modelling the switch for regulation of YAP-1 expression within the viable cells of the wound in addition to providing a model for the dCas9 Syn-RTK receptor activation for expression of YAP-1 within the MSCs.

Background

Relying on a strong base of extensive literature search in addition to reviewing the approaches of previous iGEM efforts concerning our design

Potential risks

Discussing the potential risks of our project in addition to future application risk, misuse risk, experimental risks then providing the appropriate responses to these concerns.

Beyond iGEM

We addressed the use of synthetic biology not only within the competition but also beyond it through reviewing any future application risks, discussing policy analysis such as the informed consent from the patients in addition to biosafety of our product.

By providing a solution to the longstanding problem of limited sensitivity and specificity, this safety approach opens up new possibilities for groundbreaking research and clinical applications in regenerative medicine which is not only limited to other iGEM teams but also to anyone with interest in the field of regenerative medicine.

Risk identification and prevention

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 cool water, dress the affected area with dry gauze, 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.

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.


Figure(2). This figure shows the different responses in case of exposure to chemicals.

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.

  Figure(3). Electricity control panel

  

  Figure(4). Fire fighting equipment

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(5).Handling with Biological spillage

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.

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 .(1)


Figure(6). 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.


Figure(7). dCas9 off targeting

• 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 .(2)
• Based on previous publications reports, we identified that NSP3 could interfere with host cell protein synthesis by inhibiting the translation of host polyadenylated mRNAs.

  In contrast, recent studies, analyzing the effect of NSP3 on polyadenylated mRNA translation, found the opposite effect.

  Unlike old studies, they concluded that NSP3 improves the host cell’s poly(A) mRNA translation rather than inhibiting it

  we decided to avoid the debate discussing the effect of NSP3A on protein synthesis. Thus, we have used a truncated mutated variant of NSP3A which is excised from the conserved 3′-terminal region of viral mRNAs that would alter the normal function of NSP3A while preserving its affinity to the translation initiation protein eIF4G .

  In addition , we conditioned the expression of NSP3A by mediating it through a Promoter called pTET. This promoter would offer a conditioned expression of NSP3A in response to tetracycline to ensure a safe and sufficient expression of NSP3A .

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.
• We also concerned about our lab security so we ptotected our lab work by Code locked door to prevent any unauthorized access .


Figure(8). Code looked door

Identification of future application

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

Long-Term Effects of YAP-1 Up-regulation

The effect of uncontrolled expression of YAP-1 is a terrible issue as it causes an uncontrolled cell proliferation but we got a solution for this issue in our project through conditioned release of YAP-1 by the receptor and our TID switch .(3)

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

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 raise 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:

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References

1. Guo C, Ma X, Gao F, Guo Y. Off-target effects in CRISPR/Cas9 gene editing. Front Bioeng Biotechnol. 2023 Mar 9;11:1143157. doi: 10.3389/fbioe.2023.1143157. PMID: 36970624; PMCID: PMC10034092.
2. Ewaisha R, Anderson KS. Immunogenicity of CRISPR therapeutics-Critical considerations for clinical translation. Front Bioeng Biotechnol. 2023 Feb 16;11:1138596. doi: 10.3389/fbioe.2023.1138596. PMID: 36873375; PMCID: PMC9978118.
3. Fernandez-L A, Northcott PA, Dalton J, Fraga C, Ellison D, Angers S, Taylor MD, Kenney AM. YAP is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation. Genes Dev. 2009 Dec 1;23(23):2729-41. doi: 10.1101/gad.1824509. PMID: 19952108; PMCID: PMC2788333.
4. Huang Y, Wu Q, Tam PKH. Immunomodulatory Mechanisms of Mesenchymal Stem Cells and Their Potential Clinical Applications. Int J Mol Sci. 2022 Sep 2;23(17):10023. doi: 10.3390/ijms231710023. PMID: 36077421; PMCID: PMC9456387.
5. Neri S. Genetic Stability of Mesenchymal Stromal Cells for Regenerative Medicine Applications: A Fundamental Biosafety Aspect. Int J Mol Sci. 2019 May 15;20(10):2406. doi: 10.3390/ijms20102406. PMID: 31096604; PMCID: PMC6566307.