The application of the NUTRInity cut off project will be to use therapeutic bacteria harboring the CDI system to establish a niche in the human gut and settle permanently to assure a constant flow of secreted therapeutic compounds.
To assess the functionality of our NUTRInity cut off project we first had to define test conditions that were as close as possible to real-world conditions within the constraints of the laboratory environment. One important point in this context is to be aware of the rules and regulations for approval of therapeutic genetically modified organisms (GMOs). This will be discussed further below.
To design our real-world-mimicking experiment we took advantage of the diverse expertise on campus and interviewed medical scientists to find out more about the conditions inside the human gut. We learned that the gut is packed with bacteria and that intestinal peristaltis (or gut motility) as well as constant renewal of the epithelium ensure more or less stable conditions. To best mimic these conditions we developed a fed-batch culture protocol for which we incubated a bacterial culture at 37°C without shaking but with light swirling every 30 minutes. To imitate the occasional influx and efflux of intestinal contents, we diluted the culture to an OD of 0.2 every 2 hours.
To find out the best way to administer the bacteria we consulted a pharmaceutical scientist who supplied us with enteric-coated capsules. We could not follow his advice to freeze dry the therapeutic bacteria because we lack the facilities. Instead we centrifuged a suspension of cells and packed the cell pellets into the capsule.
The cut-off capsule containing the red fluorescent CDI+ strain was then added to the flask containing the green fluorescent E.coli strain in a calculated ratio of 1:1. We can show that this prototype works at least in laboratory environment as expected (see fig. 1).
Common treatments for undernourishment require permanent intake of drugs or dietary supplements. CDI bacteria that have established in the gut could in general produce any compound of interest continuously in the gut thus making the need for constant medication unnecessary. Additionally, the effect of CDI is locally limited. Cells need to get in contact with other cells to perform their growth inhibitory function. Antibiotics that are used to target gut bacteria will certainly spread across the whole body and can cause side effects in other organs. CDI cells can only inhibit target cells that carry BamA. This protein is conserved in gram negative bacteria but absent in gram positive ones, which make up more than two third of the microbiome and therefore, are not affected by CDI cells.
In a futuristic scenario, it could even be possible to use the CDI cells to sense any change in health indicating parameters by either signaling the deviation or counteracting by producing the corresponding suitable substance.
Next to all advantages of using CDI cells instead of common treatments, we need to think about possible disadvantages as well, especially about ethical considerations. Should we use genetically engineered bacteria to permanently modify our gut? And what would happen if the bacteria spread into the environment? The ethical issues should be discussed by politicians and lawyers together with scientists. As a solution for safety issues, we plan to implement a killswitch that ensures that none of our cells are able to live after having left the gut.
Once we prove that our prototype is not only functional in a laboratory environment but also in animal testing it is only one last but lengthy step until we are able to market our product to people all around the world and provide them with a tool to permanently improve their health conditions. This one last step is the clinical trial. By law, newly developed products have to pass four phases of clinical trials in order to get an approval.
In phase 0, the preclinical phase, the general research takes place including animal testing. In this phase, no human experiments are involved.
In phase 1, the drug or treatment is tested in a small group of healthy people. The goal of this phase is to determine whether the drug or treatment causes severe side effects and what is a safe dosage range. Analyzing the desired effectiveness is no goal of phase 1 studies.
In phase 2, the drug or treatment is tested in larger groups of patients suffering from the disease to be cured by the new drug. An essential goal of this phase is to study the effectiveness of the drug in the dosage determined in phase 1.
In phase 3, the new drug or treatment is compared to standard drugs or treatments for this disease.
Phase 4 includes every study after the drug or treatment has been introduced in the market, including long-term side effects or effects in different parts of the population.
In general, to carry out clinical trials in Europe, the criteria and requirements of the EU pharmaceutical legislation for medicinal products for human use have to be followed. This legislation ensures the safety of all trial subjects. Additionally, a vote of favor is needed by the ethics committee of the member state where the clinical trial is planned to be marketed.
If clinical trials are to be done for drugs that contain or consist of GMOs, it has to be decided if the drug leads to contained use which is defined as:
“Any activity in which organisms are genetically modified or in which such organisms are cultured, stored, transported, destroyed, disposed or used in any other way and for which specific containment and other protective measures are used to limit their contact with the general public and the environment”
or as deliberate release which are all activities not rated as contained use.
If the clinical trial is classified as deliberate release, an environmental risk assessment (ERA) has to be added to the application.
An ERA includes five points:
Identification of potential adverse effects
Estimation of the likelihood
Assessment of the overall environmental impact
The final decision for or against a product release in the European Union after a clinical trial is taken by the European commission. In the USA, the FDA is the corresponding organization. After a product is launched, further experiments are conducted as part of clinical phase 4 to observe long term side effects.
The process from a prototype in a laboratory environment to a ready to market product including approval of all governmental organisations can take decades and probably costs millions of dollars. But every billion dollar blockbuster product started at the same stage as our project - as a prototype in the lab.
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