Team:AUC-EGYPT/Description

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Background

Approach and Project Description

Inspiration

References

Background

Coronavirus disease 2019 (COVID-19) is an illness caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was first discovered amid an outbreak of respiratory illness cases in Wuhan City, China. Currently, COVID-19 is classified as a world health crisis and a fast-growing global pandemic. The number of confirmed cases worldwide has exceeded 34 million, and it killed over 1 million individuals [1] . COVID-19 also affects our everyday life and is associated with a tremendous economic burden due to lockdowns, Medicare expenditures, and the slowing of manufacturing. The average economic loss, only in the first 4 months of the pandemic, has significantly affected several countries with high, middle, and low income by 18%, 24%, and 22%, respectively [2] .

The virus is known for its rapid cell entry and replication. First, the coronavirus spike (S) protein attaches to angiotensin-converting enzyme 2 (ACE2) receptors that are located on the surface of many human cells, including those in the lungs permitting the virus entry. This will then activate virus fusion into the host cell and allow the viral genetic material (ssRNA) to be fully released into the cytoplasm, where the replication and transcription processes occur, relying on the host machinery. Following the virus RNA translation, replicase proteins will be generated from open reading frame 1a/b (ORF 1a/b). These proteins use the genome as a template to produce more RNAs, which subsequently serve as templates in generating addition full-length genomes and speed up the virus replication [3] .

Despite how fast medical and pharmaceutical technologies are improving, there is currently still no cure for COVID-19, and vaccines are not yet available. However, some medications are prescribed to relieve the severity of the symptoms experienced by patients, including fever, cough, and bone pain. Hypothetically, if COVID-19 affected half the world's current population over a year with a 1% fatality rate, the death toll would be 35 million, and thus increasing the number of deaths worldwide, which is already around 60 million for all causes in a typical year [4] . Therefore, scientists are in a hurry to develop an efficient and fast vaccine to save the world.

Approach and Project Description

Unlike other treatments that focus on treating the side complications and symptoms of COVID- 19, AUC-EGYPT iGEM tackles the problem from the root by developing an innovative therapeutic to stop the viral replication and thereby its invasion to the host cells.

Consequently, our project proposes a synthetic biology approach for COVID-19 treatment through a designed circuit to control the expression of the hybrid transcriptional activator GAL4-VP16, which is a potent transcriptional activator that is orthogonal to mammalian cells, using a toehold switch device that allows the translation of a downstream mRNA upon base pairing with a complementary mRNA trigger [5,6] . The switch gets activated upon sensing SARS-CoV-2 mRNA, inducing the expression of GAL4-VP16 which in turn activates the siRNA expression through binding to its upstream UAS promoter, and, thus, initiating an RNA interference (RNAi) pathway against SARS-CoV-2 mRNA [7] . This RNAi is achieved in the circuit using 2 de novo double-stranded siRNA, targeting the viral mRNA coding for replicase proteins [8, 9] . Each siRNA sequence comprises 21 nucleotides that were selected using computational inferences by siDirect software tool and were then subjected to a BLAST search to ensure the lack of homology with any other off-target genes from the host.

Having our circuit completed, it will be delivered to the targeted cells through a virus- like particle system (VLPs) which are non-replicating particles that lack the viral genome, yet can mimic the cell entry of the actual virus [10] . Here, we will have a baculovirus-mediated lentivirus vectors’ production as they have not only shown clinical success and high transduction rates in gene therapy but also a great transduction efficacy of the inserted gene of interest greater than 90% to the mammalian cell cultures [11,12] . Then we will modify those lentivirus vectors by changing them to a SARS-CoV-2 Pseudo-Virus, resulting in dealing with viruses that have biosafety level 2 instead of biosafety level 3, and allowing the specific targeting of the cells expressing the ACE2 receptor [1,4] . Consequently, we will be delivering our designed construct only to the cells that are most likely to be infected by SARS-CoV-2. We created four baculovirus donor plasmids: Two code for the essential elements for the production of the third generation, self-inactivating lentiviruses [11,12] , one codes for the SARS-CoV-2 Spike protein to replace the normal envelope glycoprotein of the lentivirus vectors [13] , and one to transfer the genes encoding for our toehold switch and siRNAs construct [12] . Through this delivery system, our designed sensing-interfering circuit will be successfully transduced into infected cells expressing the ACE2 receptor.

Inspiration

During our brainstorming sessions for the project, our team had a wide variety of ideas that coupled synthetic biology utilization in therapeutic applications of genetic engineering, which also grasped our attention because of their potential to directly not only diagnose but also treat harmful diseases. Several viral diseases were researched, including developing vaccines for HIV and Ebolavirus.

However, we wanted our project to have a maximal as well as a global impact in addition to its availability to every individual. During that time, the COVID-19 pandemic had just started, and our university (AUC) was the leading university in Egypt to emphasize the importance of quarantine and preventing access to campus. Moreover, some members of our team have lost their close ones due to the pandemic. Since then, we have realized how dangerous the novel coronavirus is, and the necessity of not downplaying it. And, that is how the idea of working on SARS-CoV-2 came to us.

Since many of us were concerned about the looming threat of the virus's rapid spread and infection among people, we thought about uprooting the virus and inhibiting its replication in the first place to eliminate its severity and invasion to neighboring cells.

Our vision became much clearer when we discovered that small interfering RNAs (siRNAs) is a highly conserved gene silencing mechanism that plays a significant role in antiviral therapeutic and prophylactic applications [14] . And, that was the first component in our circuit to knockdown the viral mRNA coding for the replicase proteins. Then, we have searched for the current treatments for COVID-19 that utilize synthetic biology platform, and we discovered that scientists from Ellington’s group can diagnose patients applying toehold circuits [15] . Thus, we agreed on attaching our toehold switch, which is turned on through the UAS promoter, to the siRNA sequences to control their activation with the aid of the hybrid transcriptional activator GAL4-VP16 if and only if they bind to a SARS-CoV-2 mRNA, and thus increase the specificity of our treatment in a cost-effective way. Together, they completed our circuit.

We designed 2 de novo siRNA sequences that play a significant role in our antiviral therapeutic approach through the knockdown of the SARS-CoV-2 mRNA coding for the replicase proteins, which are responsible for the virus replication.

Having decided on the circuit, we wanted to maximize the specificity and efficiency of our treatment. However, we have faced an obstacle when it comes to the circuit delivery inside the targeted lung cells without getting degraded from the cell environment. As a result, we want a solution to boost patients' immunity on one hand and carry our circuit safely to the cell on the

other hand. After an intensive search, this problem does no more exist when we discovered that both requirements are becoming amenable with the use of Virus-Like Particles (VLPs) that are self-assembled into a capsule in which we can load our circuit as cargo inside it [16] . Thus, we believe we have made a great advance in the eventual completion of this project, providing a specific, efficient, and cost-effective solution that helps in the struggle against COVID-19.

References

1. Coronavirus (COVID-19) Cases - Statistics and Research. 2020; Retrieved from https://ourworldindata.org/covid-cases

2. Coronavirus: the economic impact. 2020; Retrieved from https://www.unido.org/stories/coronavirus-economic-impact-10-july-2020

3. Boopathi, S., Poma, A. B., & Kolandaivel, P. (2020). Novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment. Journal of Biomolecular Structure and Dynamics, 1-10. doi: 10.1080/07391102.2020.1758788

4. Ioannidis, J. P., Axfors, C., & Contopoulos-Ioannidis, D. G. (2020). Population-level COVID- 19 mortality risk for non-elderly individuals overall and for non-elderly individuals without underlying diseases in pandemic epicenters. medRxiv. doi: 10.1101/2020.04.05.20054361

5. Green, A. A., Silver, P. A., Collins, J. J., & Yin, P. (2014). Toehold switches: de-novo- designed regulators of gene expression. Cell, 159(4), 925–939. doi: 10.1016/j.cell.2014.10.002

6. Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., Ferrante, T., Ma, D., Donghia, N., Fan, M., Daringer, N. M., Bosch, I., Dudley, D. M., O'Connor, D. H., Gehrke, L., & Collins, J. J. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell, 165(5), 1255–1266. doi:10.1016/j.cell.2016.04.059

7. Sadowski, I., Ma, J., Triezenberg, S., & Ptashne, M. (1988). GAL4-VP16 is an unusually potent transcriptional activator. Nature, 335(6190), 563–564. doi: 10.1038/335563a0

8. He, M. L., Zheng, B. J., Chen, Y., Wong, K. L., Huang, J. D., Lin, M. C., ... & Kung, H. F. (2006). Kinetics and synergistic effects of siRNAs targeting structural and replicase genes of SARS-associated coronavirus. FEBS letters, 580(10), 2414-2420. doi:10.1016/j.febslet.2006.03.066

9. Levanova, A., & Poranen, M. M. (2018). RNA interference as a prospective tool for the control of human viral infections. Frontiers in microbiology, 9, 2151. doi:10.3389/fmicb.2018.02151

10. Naskalska, A., Dabrowska, A., Nowak, P., Szczepanski, A., Jasik, K., Milewska, A., Ochman, M., Zeglen, S., Rajfur, Z., & Pyrc, K. (2018). Novel coronavirus-like particles targeting

cells lining the respiratory tract. PLoS ONE, 13(9). doi: 10.1371/journal.pone.0203489 11. Milone, M.C. & O’Doherty, U. (2018). Clinical use of lentiviral vectors. Leukemia, 32, 1529–1541. doi: 10.1038/s41375-018-0106-0

12. Lesch, H., Turpeinen, S., Niskanen, E. et al. (2008). Generation of lentivirus vectors using recombinant baculoviruses. Gene Therapy, 15, 1280–1286. doi: 10.1038/gt.2008.76

13. Crawford, K.H.D., Eguia, R., Dingens, A.S., Loes, A.N., Malone, K.D., Wolf, C.R.;, Chu, H.Y., Tortorici, M.A., Veesler, D., Murphy, M., Pettie, D., King, N.P., Balazs, A.B. & Bloom, J.D. (2020). Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses. doi: 10.3390/v12050513

14. Villegas, P. M., Ortega, E., Villa-Tanaca, L., Barrón, B. L., & Torres-Flores, J. (2018). Inhibition of dengue virus infection by small interfering RNAs that target highly conserved sequences in the NS4B or NS5 coding regions. Archives of virology, 163(5), 1331-1335. doi: 10.1007/s00705-018-3757-2

15. Shen, M., Zhou, Y., Ye, J., Al-Maskri, A. A. A., Kang, Y., Zeng, S., & Cai, S. (2020). Recent advances and perspectives of nucleic acid detection for coronavirus. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2020.02.010

16. Onodera, T., Hashi, K., Shukla, R. K., Miki, M., Takai-Todaka, R., Fujimoto, A., ... & Ato, M. (2019). Immune-focusing properties of virus-like particles improve protective IgA responses. The Journal of Immunology, 203(12), 3282-3292. doi: 10.4049/jimmunol.1900481