The Problem

The inspiration behind The E.ncapsulator was the inherent difficulty in delivering protein pharmaceuticals to the gut. Due to the delicate nature of proteins and the highly acidic environment present in the stomach, protein molecules are readily broken down - making oral drug delivery of protein pharmaceuticals very difficult .

There are several diseases that are treated using oral delivery of peptides. Among these are: Malnutrition (world hunger) and Phenylketonuria (PKU), a disease associated with mental retardation due to insufficient ability to metabolise phenylalanine.

In this project, we chose several different potential applications to illustrate the versatility of The E.ncapsulator. Firstly, we chose to synthesise phenylalanine hydroxylase (PAH), an enzyme responsible for metabolism of phenylalanine - and an enzyme that is deficient in PKU patients. Secondly we chose to synthesise the enzyme cellulase, in order to derive better nutritional value from food consumed to aleviate malnutrition around the world. Finally we chose to synthesise a small bioactive pentapeptide, opiorphin. This small molecule acts very efficiently as a pain killer and anti-depressant, whilst due to its mechanism of action, remaining non-addictive.

The E.ncapsulator system is a generic drug production and delivery platform. The project great offers potential for any problem that requires the delivery of polypeptide biopharmaceuticals to the lower GI tract.

Some statistics

In the table below we have provided some statistics of disorders requiring administration of polypeptide drugs. This highlights the need for a cost-effective and implementable solution, which is what we is offered by The E.ncapsulator.

Project specifications

• Synthesis: The system is generic and potentially able to manufacture any type of polypeptide.
• Purification: In The E.ncapsulator, protein production (module 1) and encapsulation (module 2) of the cells occur in the same place. This dual production and delivery platform means that there is no need for expensive downstream purification processes.
• Storage: Freeze drying in the encapsulation phase and secondary encapsulation allow for storage of The E.ncapsulator.
• Quality Control: If one cell fails to produce the protein or polypeptide of interest, it will have minimal impact on the whole system.
• Safety: In order to reduce risks associated with our product, we have chosen a GRAS (generally recognised as safe) chassis. Furthermore, all the genetic material is destroyed prior ingestion to prevent any risks of horizontal gene transfer after colonisation by the cell.

Our Solution

The E.ncapsulator is a novel protein manufacture and delivery platform designed to overcome these difficulties. Hover over the different parts of the image below, to learn more about each module of the system:

Growth

Growth

The cells are grown to a critical cell density, before the system is started. It allows the culture to reach a sufficient cell number before the the cells are triggered to begin protein production. This is because protein production can slow cell growth.

Module 1: Protein Production

Module 1: Protein Production

The first module is induced with IPTG, which triggers the production of the protein of interest. As part of this project we have looked into two proteins and a peptide of interest.

Module 2: Encapsulation

Module 2: Encapsulation

The second module is where the cell, after having produced the peptide of interest, produces colanic acid. This creates a protecting layer around teh bacterium to shelter it from the acidity of the stomach.

Module 3: Genome deletion

Module 3: Genome deletion

Module 3 occurs after encapsulation of the cell containing the produced peptide of interest. This module makes the bacterium non-viable. It does so by over-expressing restriction enzymes which subsequently cleave the genomic DNA into small fragments. The cell is thus unable to produce any proteins and therefore unable to survive.

Secondary Encapsulation

Secondary Encapsulation

Several manufacturing considerations regarding the post-processing of the culture have been investigated. Post-processing of the culture allows the polypeptide filled cells to be converted into a pharmaceutical tablet, that can be taken orally.

Chemoinduction

Module Integration: Chemoinduction

Module 1 is induced by the addition of a compound, IPTG. This allows the user to 'kickstart' the system once the culture has reached a sufficiently high cell density.

Autoinduction

Module Integration: Autoinduction

Module 2 is triggered by a switch from glucose consumption to a secondary carbon source consumption. When the initial preferential carbon source (glucose) is exhausted, the system will metabolise the secondary carbon source that is available. This switch triggers the promoter that controls the start of Module 2. By knowing the initial concentrations of each carbon source, this acts as a programmable time delay system for the activation of encapsulation.

Thermoinduction

Module Integration: Thermoinduction

Module 3 is initiated upon an increase in temperature. The system is initially grown at 28°C, at which point Module 3 is repressed. When the temperature is raised to 42°C, this repression is blocked, triggering the start of Module 3. This temperature sensitive system was chosen as after encapsulation, chemical induction may be less effective due to limited diffusion.

References

[1] [http://www.americanheart.org/presenter.jhtml?identifier=4551 Diabetes Mellitus statistics]
[2] [http://www.wrongdiagnosis.com/h/hemophilia/stats.htm Hemophilia statistics]
[3] [http://www.worldhunger.org/articles/Learn/world%20hunger%20facts%202002.htm World Hunger statistics]
[4] [http://www.wrongdiagnosis.com/p/phenylketonuria/stats.htm Phenylketonuria in the USA]
[5] [http://www.espku.org/3.0/index.php?option=com_content&task=view&id=1&Itemid=2 ESPKU (European statistics)]
[6] [http://www.wrongdiagnosis.com/p/phenylketonuria/stats-country.htm More PKU statistics (browse per country)]
[7] [http://www.chm.davidson.edu/erstevens/Michaelis/Michaelis.html Michaelis-Menten kinetics]

Project Tour

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These links may be useful to browse our results and achievements:

Major Results

Submitted Parts

Achievements

Pill Manufacture

Genealogy