Safe Project Design

Lentiviruses vs. PiggyBac Vectors

We primarily engineered HEK293T cells to express various external nanobodies, which correspond with antigen-expressing bacteria, allowing the HEKs and bacteria to adhere via the nanobody-antigen pairs. We also used an anti-GFP SNIPR complex that is expressed on the outside of the HEK cells to explore whether adhesion of bacteria can directly induce signaling. When the anti-GFP SNIPR complex binds to its ligand (GFP) and experiences a sufficient mechanical force, the receptor will release Gal4, a transcription factor. The Gal4 then interacts with a UAS>tBFP sequence that we synthetically introduced into the genome, producing tBFP upon adhesion (Zhu et al., 2022). We ordered two plasmids for this system from Roybal KT on Addgene, and they came in a lentiviral vector. We chose to clone the coding region for each gene into a Piggybac backbone.

Lentiviral vectors have been a highly popular method of transduction due to their ability to integrate genes of interest into a host genome stably (Zheng et al, 2018). However, they come with several associated risks that make the use of them a BioSafety Level 2 threat. Lentiviral vectors are derived from the retroviral family and work by enveloping a gene of interest known as a transgene into an envelope protein that is able to “infect” the host cell of interest. (Zheng, 2018). Although newer classes of lentiviruses have been developed to reduce adverse side effects associated with infection, the potential for harm remains. Such risks include the potential to generate replication-competent lentiviruses during vector production, the transactivation of neighboring genomic sequences, and the spread of viral vectors to untargeted areas (Pauwels, 2009). Lentiviral vector infected cells thus have the possibility to become cancerous if the vector activates oncogenesis or inactivates tumor suppressor genes (Zhao, 2016), making the use of them non ideal.

To mitigate such risk we investigated a nonviral pathway of plasmid transfection: the PiggyBac vector. Piggybac is a DNA transposon that can incorporate large fragments of DNA sequences at random sites throughout the host genome without leaving a trace, making it a safe option for foreign DNA integration (Mirzaei et. al, 2016). Since Piggybac vectors are nonviral, meaning they contain no viral antigens, they have the potential to be less immunogenic than lentiviral vectors, and they are less costly to manufacture than conventional viral vectors (Di Matteo, 2012).

For the purposes of our research, Piggybac quickly became the prime candidate for transfection due to their ease of replication. Only requiring a BioSafety Level One cabinet for use and considering the affordability of attaining such vectors, our team favored the use of Piggybac vectors over Lentiviral vectors. It is our hope that this decision makes the “stick-and-secrete” system that we are investigating truly modular. By eliminating biosafety hazards associated with lentiviral vectors and choosing a more accessible and affordable method of engineering our mammalian cells.

Safe Lab Work

Team Princeton is strictly following Biosafety Level 1 and Level 2 cabinet protocols, as specified in the Biosafety Training and Bloodborne Pathogens for Researchers training provided by the Office of Environmental Health and Safety at Princeton University. The Level 1 lab space is designated for bacterial work, and the Level 2 cabinet is reserved for handling mammalian cells. We are working with Escherichia coli strains K-12, DH10B, and stellar competent cells, along with Homo sapiens HEK293T mammalian cells. Our objective is for the engineered bacteria to bind and interact with mammalian cells.

This project involves the use of several potentially hazardous chemicals, including carcinogens, mutagens, highly flammable substances, acids, and corrosive agents. For example, chemicals like Ethidium Bromide, MES buffer, sodium-dodecyl sulfate (SDS) running buffer, methanol, ethanol, lithium dodecyl sulfate (LDS), QIAprep Spin Miniprep Kit buffers, Anhydrous tetracycline, binding buffer NTI, and Tris buffer can be dangerous if mishandled. However, we exercise extreme care and caution when working with these chemicals. We have consulted safety data sheets to understand their risks and have communicated this information to our team members. Additionally, it's crucial to highlight that personal protective equipment, such as gloves, is always worn in the vicinity of the laboratory benches. Having easy access to our mentor and PI in the same building provides valuable support in addressing unforeseen or novel challenges that may arise in the lab. By combining these safety measures, support systems, and heightened awareness, we aim to reduce the level of risk in our project and effectively respond to any unexpected events.

Below are few photos of the lab space our team worked at this summer!

Safety and Security Award

A major focus of our summer was dedicated towards investing time in understanding relevant biosecurity and safety concerns pertinent to researchers like us. Early in the year, we brainstormed ways to emphasize the importance of biosafety in our community. Eventually, we decided to conduct a BioSafety Simulation at the beginning of the semester in which participants would be lead through a series of scenarios and group discussion to think critically about real world implications of BioSecurity.

To create this simulation, we first had to educate ourselves about what biosecurity means and why it's crucial to comprehend its global implications. This led us to conduct a Malice Analysis, guided by our graduate student mentor, Jeffrey Lee. The results of this analysis helped us generate a list of "inject proposals" that we later incorporated into our BioSecurity Simulation. These injects are brief scenarios designed to prompt people to think critically about relevant biosecurity concerns.

Possible Inject Proposals

Malice Analysis Workshop

On July 14, 2023 our team sat through a Malice Analysis Workshop led by graduate student and mentor Jeffrey Lee. An effort by the Engineering Biology Research Consortium (EBRC), a Malice Analysis workshop “train researchers and others associated with engineering biology to critically evaluate research for potential security concerns” (ERBC) by discussing the importance of biosecurity through historical case examples. Trying to engineer a modified biological system ourselves, it was important for our team to think critically about the choices we make and the implications that our project could have on future research. After the completion of the Malice Analysis Simulation our team worked on developing a report of our findings. This report allowed us to have a thorough understanding and better insight into all that we learned.

Virtual Biosecurity SImulation

Biosecurity Simulation

References

Di Matteo M, Mátrai J, Belay E, Firdissa T, Vandendriessche T, Chuah MK. PiggyBac toolbox. Methods Mol Biol. 2012;859:241-54. doi: 10.1007/978-1-61779-603-6_14. PMID: 22367876.

Mirzaei, H., Sahebkar, A., Jaafari, M. et al. PiggyBac as a novel vector in cancer gene therapy: current perspective. Cancer Gene Ther 23, 45–47 (2016).

Pauwels K, Gijsbers R, Toelen J, Schambach A, Willard-Gallo K, Verheust C, Debyser Z, Herman P. State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations. Curr Gene Ther. 2009 Dec;9(6):459-74. doi: 10.2174/156652309790031120. PMID: 20021330.

Zhao S, Jiang E, Chen S, Gu Y, Shangguan AJ, Lv T, Luo L, Yu Z. PiggyBac transposon vectors: the tools of the human gene encoding. Transl Lung Cancer Res. 2016 Feb;5(1):120-5. doi: 10.3978/j.issn.2218-6751.2016.01.05. PMID: 26958506; PMCID: PMC4758974.

Zheng CX, Wang SM, Bai YH, Luo TT, Wang JQ, Dai CQ, Guo BL, Luo SC, Wang DH, Yang YL, Wang YY. Lentiviral Vectors and Adeno-Associated Virus Vectors: Useful Tools for Gene Transfer in Pain Research. Anat Rec (Hoboken). 2018 May;301(5):825-836. doi: 10.1002/ar.23723. Epub 2018 Jan 24. PMID: 29149775; PMCID: PMC6585677.

Zhu I, Liu R, Garcia JM, Hyrenius-Wittsten A, Piraner DI, Alavi J, Israni DV, Liu B, Khalil AS, Roybal KT. Modular design of synthetic receptors for programmed gene regulation in cell therapies. Cell. 2022 Apr 14;185(8):1431-1443.e16. doi: 10.1016/j.cell.2022.03.023. PMID: 35427499; PMCID: PMC9108009.