Team:ACIBADEM ISTANBUL/Parts

Improve

There were over 600 different types of snake venom to choose from, but we ended up choosing Crotalus atrox (Otherwise known as the Western diamondback snake) as our reference snake. Crotalus atrox is responsible for the greatest number of snake bites in the United States of America and the majority of snakebite fatalities in Northern Mexico.
Additionally, C.atrox is a venom that has been well documented, with the majority of its enzymes available directly from well known protein databases.
Venom shouldn’t be thought of as a single entity, but rather a solution containing various types of enzymes all with different mechanisms giving the venom its characteristic deadly properties.
C.atrox is classified as a hemotoxic venom, meaning that it primarily damages the blood vessels and blood cells, leading to death via severe hemorrhage.
The zinc metalloproteinase enzyme family is responsible for the hemorrhagic, and thus hemotoxic, activity of the C.atrox venom.

Zinc Metalloproteinases

Crotalus atrox contains hemorrhagic Snake Venom Metalloproteases (SVMPs) that act by initially hydrolyzing key substrates at the Basement Membrane (BM) of the capillaries. This degradation results in the weakening of the mechanical stability of the capillary wall, which becomes distended thanks to hemodynamic biophysical forces. As a result, the capillary wall is disrupted and extravasation occurs.
SVMPs do not induce rapid toxicity to endothelial cells and the pathological effects are a result of mechanical forces. Experimental evidence suggests that type IV collagen and perhaps perlecan are the key elements in the onset of microvessel damage. (It has been demonstrated that other peptides in BM have similar degradation rate when compared with hemorrhagic vs non-hemorrhagic)

Treatments

The only way to effectively treat a venomous snake attack is through the administration of anti-venom.
The worlds first anti-venom was prepared by Albert Calmette in 1895, through the long and arduous process of antibody harvesting:

1. Venomous snakes are captured.
2. Snake venom is milked.
3. Snake venom is injected into an animal host (horses, traditionally).
4. Animal host begins reacting to the venom (antigen) and begins producing antibodies (anti-venom), which peak occurs around 8-10 weeks.
5. Antibodies (anti-venom) are purified from the animals blood. The anti-venom is now ready for use.

This process has remained the standard way of making anti-venom to this day.

The Drawbacks

The current state of anti-venom ,in this day and age, is utter disaster.

Scarcity

Currently, there is a scarcity of anti-venom in the world. This is because the demand far outweighs the supply.
High demand
The South Asia region has the highest relative incidence of venomous snake attacks, making them the region with the highest anti-venom consumption in the world as the prized target market.
Low supply
The manufacturing process of anti-venom is very long, with a minimum wait duration of at least 8-10 weeks. The use of animal hosts complicates this process even further, making the production costs of anti-venom high.
The target market for anti-venom is mainly the South Asia region, which has the highest relative incidence of venomous snake attacks. The majority of South Asian countries are still developing, meaning that they have stringent budgets reserved for anti-venom.

Cost of Treatment

The average price of a typical anti-venom costs around $1,500 to $2,200 per vial.
One snake bite requires 20-25 vials, equating to over $30,000 in pharmaceutical costs alone.
Thus, the etiology of the worlds anti-venom scarcity comes from simple economics. Manufacturing is long and expensive and the buyer has a strict budget, resulting in smaller profit margins to the manufacturer, discouraging investment.

Hypersensitivity Reactions

Interestingly, whilst anti-venom can counter the effects of snake venom, it being animal derived gives way to another problem: Hypersensitivity reactions.
In fact, administering traditional anti-venom has a 43% chance of triggering a severe Type I Hypersensitivity reaction, that will require additional treatment measures such as the administration of Epinephrine to prevent shock.

Efficacy

The overall efficacy of traditional anti-venom is low.
Traditional anti-venom takes an immunologic approach to countering the effects of venom.
The anti-venom is in this case a solution containing various antibodies against the antigens present in the venom.
It utilizes a lock-and-key mechanism, meaning that one antibody (anti-venom) can only bind to one antigen (venom). This specificity is both advantageous, as well as disadvantageous .
Advantageous because the therapy is very effective.
Disadvantageous because you need the appropriate anti-venom to neutralize the venom.

A New Hope

The Opossum (Didelphimorphia) is an animal with a very unique characteristic; it displays an outstanding resistance to venoms, snake venoms in particular. This anti-venom ability is gained through a protein; the Lethal Toxin Neutralizing Factor (LTNF). LTNF is believed to be a protease inhibitor (a metalloproteinase inhibitor to be specific).
It has been shown that LTNF might have activity against a large variety of venoms. However, LTNF is currently obtained by extracting it from the blood serum of Opossums.
Using LTNF as an alternative to traditional anti-venom is promising, but its current methodology raises some major concerns:

1. Using Opossum sourced LTNF poses a risk for triggering adverse Hypersensitivity reactions.
2. There are ethical concerns over the use of Opossums to serve as a source on LTNF for human consumption.
3. While cheaper than traditional anti-venom, the use of HPLC to separate LTNF from live Opossums is still expensive and is not economically viable.

Our Proposal

We are attempting to produce a more effective version of this anti-venom, LTNF 2.0 if you will, as a synthetic anti-venom for human use.
LTNF 2.0 will incorporate the post-translational modification process known as circularization, a process that comprises of adding cysteine amino acids to both ends of a polypeptide chain, triggering the formation of a disulphide bridge, ultimately leading to a circular structure, hence the name circularization.
Circularized proteins are known for their increased stability and efficacy, prolonging the product’s shelf life and lowering the dosage required. The resulting bioproduct will be a more efficient and effective anti-venom, which in turn will go a long way to solving the issues of insufficiency, scarcity and prohibitive cost of the current anti-venoms.
Traditional anti-venom approaches the treatment of a venomous attack from an immunologic angle, i.e using the immunoglobulins to bind to the venom (antigen) to neutralize it, LTNF takes a biochemical approach, acting as a metalloproteinase inhibitor. Metalloproteinase is the main enzyme family that gives hemotoxic venoms their hemotoxic ability. Since we are inhibiting enzymes instead of blocking antigens, LTNF has a wider therapeutic window because of its ability to target all hemotoxic venoms that utilize metalloproteinases, giving it greater efficacy.

Manufacturing

Traditional anti-venom takes months (8-10 Weeks) to produce.
LTNF 2.0’s production can be scaled to industrialization levels using either fermentation or peptide synthesis.
LTNF 2.0 can be manufactured and purified in a matter of hours.

Ethics

Traditional anti-venom uses an animal host for Immunoglobulin synthesis.
LTNF 2.0 will be produced on an industrial scale using Pischia pastoris, a yeast strain.
With LTNF 2.0, no animals will used, avoiding the previous ethical implications.

Safety

Traditional anti-venom being produced from animal hosts, poses a 43% chance of triggering a severe Type I Hypersensitivity reaction due to cross-reactions.
LTNF 2.0 being manufactured using Pichia pastors avoids the risk of cross-reactions.

Costs

A single vial of traditional, anti-venom costs around $1,500 - 2,200 per vial.
An economic analysis of LTNF 2.0’s theoretical manufacturing costs brings it down to approximately $6.00 per vial.

Readily Available

The industrialization of LTNF 2.0 shall provide readily available, high yield anti-venom to be manufactured, alleviating the worldwide anti-venom scarcity problem.
The cost-effectiveness of LTNF 2.0 allows for developing countries with limited healthcare budgets to be able to afford to pay for the high demand of anti-venom treatment in their respective countries, alleviating the scarcity problem.

PipeLine

August – HPLC Peptide stability + Auto-injector design.
September - Hemolytic + Patent application
October - Gel degradation + Biobrick part registration + iGEM giant jamboree
December – NMR templating (Ask for name of place) and PDB registration,
Modelling and MD simulation using TRUBA’s supercomputer (Dry lab 2.0)
January – NMR results publication, In vivo studies initiation, iGEM outreach program and iGEM 2019 application.
February – Publication of In vivo study findings.
March – Application for FDA testing, preparations for LTNF 3.0
April - May – Consultation for other iGEM teams, research for LTNF 3.0
June – iGEM part kits arrive, new iGEM team training, Safety form and InterLab 2019.
July – Hosting the Istanbul iGEM meetup