{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block lead %}Our success in the Engineering Design Cycle{% endblock %}
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<h3><i>Our Engineering Success</i></h3>
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<h4><i>Design</i></h4>
<p>
Our team started brainstorming ideas in November 2023 the topic of hemophilia caught our interest early on, vesicle delivery was also an early concept, but the exact design of our project changed a lot during our literature research as we were learning more and more about hemophilia and the different types of vesicle delivery.
Our project design started to crystallize at the beginning of march when we decided to use ionizable lipid nanoparticles (LNPs) for mRNA delivery.
This method has already been well-researched in the last decades.
Our design is largely based on a paper called “Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver” by Kim et al. 2021
This article gives instructions for formulating LNPs that can deliver siRNA as well as mRNA to specifically the liver sinusoidal endothelial cells, which was our original target, where factor VIII is normally produced.
These LNPs contain Ionizable lipid (synthesized from 1,4-Bis(3-aminopropyl) piperazine and 1,2-epoxydecane) , DOPE, cholesterol, and DSPE PEG C16 ceramide in a molar ratio of 26.5:20:52:1.5.
To specifically target LSECs, the LNPs include conjugated DSPE PEG-manose lipids that interact with the scavenger receptors on LSECs.
Our goal was to recreate these LNPs with Factor VIII mRNA. In addition, we wanted to implement these LNPs for intra nasal delivery.
Because the Factor VIII protein coding sequence was not yet in the registry, we chose to buy a plasmid by Robert Peters containing the full sequence for the human FVIII protein from addgene.
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<h4><i>Build</i></h4>
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To achieve our goal, we needed two things: the Factor VIII mRNA and the LNPs. We worked on these in parallel.
The goal was to recreate the LNPs described by Kim et al. However, due to the cost of some components, we used a model LNP made out of cheaper alternatives. DSPE PEG C16 ceramide and DSPE PEG-manose were both substituted with the more affordable DSPE PEG-2000.
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Producing the mRNA in a chassis organism was not feasible, so the plan was to synthesize the mRNA for Factor VIII using in vitro transcription and synthetically modifying it with a 5’-cap and 3’ poly(A)-tail.
Amplifying the Factor VIII coding sequence before transcription proved to be difficult because of its length.
After trying PCR with multiple sets of primers, the whole plasmid was amplified in E.coli to then be linearised.
In our multiple tries of in vitro transcription, we managed to finetune our protocol and produce enough RNA to make LNPs.
Before the RNA could be used in LNPs, it had to be purified, and modified with a 5’-cap and a poly-A tail.
Purification was done with phenol-chloroform extraction, which also gave us some difficulties and it took us some time to troubleshoot the protocol.
Working with Factor VIII RNA proved more difficult than expected, because the length made it prone to degradation and none of our team members had previous experience working with RNA.
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<h4><i>Test</i></h4>
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The LNPs by Kim et al. specifically targeted the sinusoidal endothelial cells, but several factors, including cost and availability, made it hard for us to test our LNPs on these cells. As an alternative, we used the HEK293T/17 cell line.
To test our LNPs we tried to deliver mRNA encoding for the eGFP protein. We
We tested the properties of our synthesized mRNA without LNPs by transfecting them into HEK cells to compare with the delivery of RNA with our LNPs.
Analysis of the LNPs was done using dynamic light scattering (DLS) and transmission electron microscopy (TEM) to determine the size and zeta potential of the LNPs. The encapsulation efficiency was determined with the use of
The concentration of RNA was also frequently measured using a NanoDrop spectrophotometer.
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<h4><i>Learn</i></h4>
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During our building stage, we were already confronted with some problems that made us reconsider our approach and rethink our design.
The length of the Factor VIII RNA forced us to rethink our method of gene amplification.
We also changed the concentration of the PBS buffer after measuring the zeta potential of our first batch of LNPs.
Due to the cost of LSECs and some LNP components, we were forced to construct an affordable model that would still be able to prove our concept.
We decided to use a generic HEK cell line and because there would be no specific targeting possible with these cells, we could also replace the expensive PEG-manose lipid with a more affordable option.
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<h4><i>Improve</i></h4>
<p>
During our building stage, we were already confronted with some problems that made us reconsider our approach and rethink our design.
The length of the Factor VIII RNA forced us to rethink our method of gene amplification.
We also changed the concentration of the PBS buffer after measuring the zeta potential of our first batch of LNPs.
Due to the cost of LSECs and some LNP components, we were forced to construct an affordable model that would still be able to prove our concept.
We decided to use a generic HEK cell line and because there would be no specific targeting possible with these cells, we could also replace the expensive PEG-manose lipid with a more affordable option.
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<h4>References:</h4>
<p>Kim M, Jeong M, Hur S, Cho Y, Park J, Jung H, et al. Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver. Science Advances. 2021 Feb 26;7(9)</p>
<p>pCDNA4/Full length FVIII from Robert Peters (Addgene plasmid # 41036 ; http://n2t.net/addgene:41036 ; RRID:Addgene_41036)</p>
<h4>Current HemA treatment consists of regular injections with Factor VIII protein.</h4>
<p>
And, even though this approach has its benefits: it can be done by the patient themselves and there are possibilities for an implantable port for easy administration, there are still some limitations.
And, even though this approach has its benefits - it can be done by the patient themselves and there are possibilities for an implantable port for easy administration - there are still some limitations.
On top of that, a Haemophilia patient has to make frequent injections - every 48 hours - to keep their FVIII levels on a normal level.
The frequent injections are in part the result of the high clearance of factor VIII from the circulation with a half-life of 10-12 hours.
This causes the factor VIII concentration in the blood to fluctuate a lot, spiking up after injection, then quickly going down until the next injection.
Currently, there are multiple engineered recombinant factor VIII products available, but these are way more expensive than the cheapest variety of FVIII, which is purified from blood products and already has an annual cost of $300,000!
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When the nanoparticle reaches its target in the body, the cells will take it up into a lysosome and break the LNP preserving the genetic sequence inside.
After that, the cell produces the protein factor VIII from the mRNA and releases it into the blood.
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<p>
Ionizable lipids are crucial for LNP preparation, and they provide effective mRNA encapsulation, as well as safe LNP delivery in the body.
Their role is crucial, since they change their charge due to protonation under acidic pH (become positively charged), or deprotonation under the neutral pH (become not charged).
This feature makes it possible to encapsulate the mRNA more efficiently, as well as their safe delivery in a blood neutral pH environment.
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<p>
The LNPs are delivered via the nasal spray delivery system by the use of hydrogel, composed of poloxamer 407, poloxamer 188, NA-CMC, and almotriptan.
This hydrogel provides a biphasic release pattern: the quick release for 30 minutes (this provides an acute release of a medicine upon administration) and a sustained release for a 5 hour period (this maintains a loading dose of the medicine).
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LNPs are administered into the bloodstream to travel to the liver and target LSECs with the help of surface proteins.
Once inside the cells, the LNPs will release the mRNAs, leading to the production of coagulation factor VIII.
This approach enables patients to produce their own coagulation factor VIII, eliminating the need for frequent injections of plasma-derived or recombinant proteins.
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<imgsrc="https://static.igem.wiki/teams/5342/images/home/infinityf-method.webp"alt="Diagram that explains the InfinityF delivery process"class="main-img">
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The factor VIII mRNA is produced with in vitro transcription using factor VIII pcDNA as a template.
Our ionizable lipid nanoparticles contain synthetic lipids as well as an ionizable lipid, which is essential for the release of the mRNA in the cells.
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<!--<h5>Frequent Injections</h5>-->
<h5>Frequent Injections</h5>
<p>
While the current Hemophilia medicine has a high clearance with a half-life of 10-12 hours, thanks to the biphasic release pattern of hydrogel, applied in InfinitiF∞, patient can rely on the temporarily release of the medicine, prolonging the time of its circulation in the bloodstream, and providing patient with a longer duration of effect of the medicine.
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<h5>Concentration Spikes</h5>
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Production of protein from mRNA occurs gradually and not in all cells at the same time, spreading out the high concentration spike that occurs after injection of FVIII. €
Production of protein from mRNA occurs gradually and not in all cells at the same time, spreading out the high concentration spike that occurs after injection of FVIII.
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<!--<h5>High Clearance</h5>-->
<h5>Cost</h5>
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Costs are very important when speaking about medical treatment.
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<h3>We are the first iGEM team in Radboud’s History</h3>
<h4>We are the Radboud iGEM Team!</h4>
<h4>We are the FIRST Radboud iGEM Team!</h4>
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The Radboud iGEM team was started by a group of enthusiastic students that were looking to broaden their horizon and start something new.
We hope to introduce the iGEM competition to Radboud university while also putting Radboud university on the map within the iGEM competition.
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With our efforts, we hope to pave the way for future Radboud iGEM teams.
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We are beyond excited to showcase InfinityF as the first team to represent Radboud University within the iGEM competition, and it is with passion and determination that we say:
We are beyond excited to showcase InfinityF∞ as the first team to represent Radboud University within the iGEM competition, and it is with passion and determination that we say:
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<h4style="font-weight: bold; font-style:italic; text-align:center; color:#e3000b">Let’s synthesize a better future together!</h4>
<li>Chen, Chun-Yu, et al. “Treatment of Hemophilia a Using Factor VIII Messenger RNA Lipid Nanoparticles.” Molecular Therapy - Nucleic Acids, vol. 20, 5 June 2020, pp. 534–544, www.sciencedirect.com/science/article/pii/S2162253120301062, https://doi.org/10.1016/j.omtn.2020.03.015.</li>
<li>Salen, Philip, and Hani M. Babiker. “Hemophilia A.” PubMed, StatPearls Publishing, 2024, www.ncbi.nlm.nih.gov/books/NBK470265/#:~:text=Hemophilia%20A%20is%20the%20most.</li>
