Team:AUC-EGYPT/Implementation



The COVID-19 Situation

COVID-19 situation is escalating all over the world. Many countries have witnessed an increase in the number of confirmed cases starting last month. This happened in some countries whose numbers was stable until from mid-June, which encouraged them to ease the restrictions on travel and work. For example, France recorded its highest confirmed cases during the first wave in March 31 with 7578 cases. However, we can see the increasing numbers during its second wave as France recorded 52010 cases in Oct. 25. Another example is the UK, which reported its highest number during the first wave in April 10 with 7860, while during the second wave it recorded 26684 in Oct. 21. These examples are for countries which responded to the epidemiological situation from the beginning with applying preventative measures like a lock down and mandatory social distances. On the other hand, countries like India did not respond to the measures properly. We can see the ascending increase in the numbers with no borders between first and second wave, until it reaches its highest number of cases in Sept. 16 with 97859 cases.

In Egypt, the government responded to the situation with a partial lock down, mandatory social distancing, and the closure of massive gathering sites. This made the first wave passes with a maximum of 1774 cases in June 19. It started to decline since then and it became steady of 100sh cases until today. Many experts say that Egypt is going to encounter a second wave without any doubt, so the government put a plan to combat it. However, it still a matter of when.

Our Solution in Brief

This season, we are designing a targeted synbio-based therapy for SARS-CoV-2. A Sars-Cov2 like particle that is capable of targeting cells expressing ACEII receptor will deliver our therapeutic circuit. Upon circuit delivery, a toehold switch is constitutively expressed. If the cell is infected with sars-cov2, the toehold will unfold expressing a downstream GAL4BD-VP16 activator. The activator will, in turn trigger the expression of short interfering RNAs. Once expressed, they will search for their complementary targets in the replicase region of Sars-Cov2 mRNA thereby inhibiting further replication of the virus.

Milestones

Our milestones involve three stages during 3 years: proof of concept and animal testing for 8 months and funded by a research grant, clinical trials for 18 months and funded by seed funding, and finally proof of market and partnership for 6 month.

End Users: Our proposed end users are COVID-19 patients.

Market Segmentation and Penetration

Following a brief analysis of the surrounding environment and the identification of a problem, we wanted to develop a solution that would address the issue. We wanted to develop a therapeutic that could help solve the problem of the COVID-19 pandemic, but we first needed to conduct a market segmentation to identify the needs of the various segments and accordingly identify our target market. Our product follows a B2B model, so we started by segmenting the market into governmental and non-governmental entities as a first step. Then, we chose to distribute to hospitals since we value equity and fairness and we hope that our product will eventually become available and accessible to everyone regardless of socioeconomic class. So, now we knew that we wanted to target Egyptian hospitals, but we had to narrow this down to a more specific segment. Based on this, we segmented Egyptian hospitals in order to narrow down to the most suitable target market for our product. Based on an analysis of these hospitals’ firmographics, behaviors, and needs during segmentation, we have come to identify that we primarily want to target military hospitals during the first rollout phase of our drug. Our decision to select this particular segment to begin with was based on a host of reasons. First, Military hospitals have the lowest capacity out of all hospitals, which is actually a benefit to us as a new startup since our production capacity will be limited during the product’s introduction phase, and we will also not have the technological capacity at this point to benefit from economies of scale. Second, we realize the level of power that Military hospitals have on influencing the decisions of other types of hospitals. This means that although the segment is small, there is higher potential for segment growth. Finally, military hospitals market themselves as quality sensitive, and since our end goal is to be the most efficient treatment on the market, this is the most relevant segment for us to go after.

Challenges

• Lack of regulatory framework for recombinant therapies. Attempt to obtain direct approval from MOH is a proposed solution for this issue.

• Unprecedented product type. Synbio solutions are not very familiar to people. Also there is stigma against the use of genetic engineering. We think raising awareness regarding the therapeutic potential of SynBio solutions might contribute to lessening this stigma.

Other expected challenges include the risk of our therapy being distributed through the black market as illegitimate distribution tends to happen with low supply commodities in high demand, and we are currently in a global crisis with severe shortage of therapeutic solutions. This next challenge that we have identified is a problem for both our startup as well as for the wider market. We will face a challenge upon launch due to the lack of supporting facilities and production platforms. Likewise, the SynBio market will also face a challenge due to increased competition from our business. The only way to overcome this challenge is through governmental intervention and Policy making. Strict and tough laws need to be implemented to prevent exploitation of the situation.

Alternative Applications

We envision physicians using end product for therapeutic purposes to treat corona patients. However, there is a further potential for the circuit built into our product to be utilized by researchers as a diagnostic tool to identify other viruses in the future. The sensing moiety of our circuit, specifically the toehold switch, can be used to diagnose RNA viruses such as SARS-CoV-2. By simply ligating a reporter gene to the toehold switch, it can give a colorometric signal if it finds its trigger. In one study, Toeholds were designed to detect Zika virus from blood samples from patients (Pardee et al., 2016). Pardee et al. designed and charecterized a set of toehold switches that activate the expression of lacZ reporter upon binding to triggers in Zika virus mRNA. Because the level of mRNA in the samples was not sufficient to trigger the activation of the toeholds, Pardee et al. used NASBA (nucleic acid sequence based amplification) as to amplify the mRNA of Zika virus if present in the patients’ samples. NASBA has an outstanding track record in the field of diagnostics as it does not require sophisticated tools. Amplification in NASBSA occurs in isothermal steps, and, hence, there is no need for expensive thermocycler (Casper et al., 2007). The platform (Toeholds and NASBA) has several advantages over conventional PCR:

1. It is much cheaper

2. It does not require sophisticated kits

3. No need for sophisticated training.

4. Results are obtained in a shorter period of time.

5. More importantly, the platform is of similar efficacy and sensitivity as conventional PCR testing.

Compared to antibody testing, the platform is much more sensitive, and the cost is more or less similar (Pardee et al., 2016).

Safety

Delivery system safety

For the safety of our delivery system, we decided to design pseudo-viruses that express only the spike protein of SARS-CoV-2 instead of the usual virus-like-particles that need the genes coding for the spike, envelope and membrane to be assembled. To perform that we will pseudotype third generation self-inactivating lentivirus vectors. Accordingly, our delivery system will have a biosafety level 2 instead of 3. Furthermore, we carefully chose the minimal parts required for the production of our lentivirus vectors, so that they can’t replicate or form any arrangements. We included 3’ and 5’ LTRs and separated the genes coding for the vectors’ proteins into 4 plasmids. Also, only the gag, pol and rev genes were included. The tat gene was not involved and the gene coding for the envelope protein was replaced by the SARS-CoV-2 spike. Additionally, we had a baculovirus-mediation production for our lentiviruses, so that we can have safe high production of our pseudo-viruses.

Safety of siRNAs

For the safety of our delivery system, we decided to design pseudo-viruses that express only the spike protein of SARS-CoV-2 instead of the usual virus-like-particles that need the genes coding for the spike, envelope and membrane to be assembled. To perform that we will pseudotype third generation self-inactivating lentivirus vectors. Accordingly, our delivery system will have a biosafety level 2 instead of 3. Furthermore, we carefully chose the minimal parts required for the production of our lentivirus vectors, so that they can’t replicate or form any arrangements. We included 3’ and 5’ LTRs and separated the genes coding for the vectors’ proteins into 4 plasmids. Also, only the gag, pol and rev genes were included. The tat gene was not involved and the gene coding for the envelope protein was replaced by the SARS-CoV-2 spike. Additionally, we had a baculovirus-mediation production for our lentiviruses, so that we can have safe high production of our pseudo-viruses.

Orthogonality of toeholds

For the safety of our delivery system, we decided to design pseudo-viruses that express only the spike protein of SARS-CoV-2 instead of the usual virus-like-particles that need the genes coding for the spike, envelope and membrane to be assembled. To perform that we will pseudotype third generation self-inactivating lentivirus vectors. Accordingly, our delivery system will have a biosafety level 2 instead of 3. Furthermore, we carefully chose the minimal parts required for the production of our lentivirus vectors, so that they can’t replicate or form any arrangements. We included 3’ and 5’ LTRs and separated the genes coding for the vectors’ proteins into 4 plasmids. Also, only the gag, pol and rev genes were included. The tat gene was not involved and the gene coding for the envelope protein was replaced by the SARS-CoV-2 spike. Additionally, we had a baculovirus-mediation production for our lentiviruses, so that we can have safe high production of our pseudo-viruses.

References

Casper, E.T., Patterson, S.S., Bhanushali, P., Farmer, A., Smith, M., Fries, D.P., and Paul, J.H. (2007). A handheld NASBA analyzer for the field detection and quantification of Karenia brevis. Harmful Algae 6, 112–118.

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. https://doi.org/10.1016/j.cell.2016.04.059