Difference between revisions of "Team:Freiburg/Goals Approach"

“All Disease Begins in the Gut!” - Hippocrates

Some people are tough. And then there are some people, who have to fight against their own immune system- how tough is that?

In the case of ulcerative colitis patients, the mistakes made by the body’s immune system lead to cyclic inflammations and ulcers in rectum and colon. To treat this overreacted immune response, they rely on therapeutic options, which are distributed systemically. Furthermore they lead to horrible side effects, such as anemia, hysterical depression[1] and sadly, the risk for colon cancer increases enormously.
It is our goal to develop a new system of targeted drug delivery, where the drug only acts on the affected tissue and is not getting dispersed in the whole body. This approach will not harm the human body and has no negative side effects on it.

First, it is important to differ the terms prodrug and active drug in this system. A prodrug, precursor of a drug, must undergo chemical conversion by metabolic processes before becoming an active pharmacological agent, the active drug.

Standard drug delivery systems are mostly oral. Patients take the pills and most of the prodrug will be activated by enzymes in the liver and distributed throughout the entire body. Thus, the activated drug will interact with healthy tissues and organs. With the standard approach, vulnerable organs like bone marrow or brain will show symptoms like lower production of blood cells or nausea, headache etc.
The attempt to bypass the liver to avoid the systemic side effects, can be achieved by a specific activation of drugs at a specific area, i.e. the inflamed cells in the colon. We achieved this by using nanobodies, GST and the endospores of the bacterium Bacillus subtilis.
Through cloning and transformation of the Bacillus subtilis, we were able to customize the spores to our approach. We designed fusion proteins of the outer spore coat proteins dCotZ, dCotB, dCotG and dCgeA, containing a linker peptide with HA-Tag and a nanobody, which is able to bind to a specific marker. To enable the conversion of the prodrug to its active form, we also fused proteins with GST (glutathione S-transferase). Our end product is a spore combining these two functions, which is able to function as a complete new designed targeted drug delivery system, causing no negative side effects in the human body.

Our project's main target is the disease ulcerative colitis, an inflammatory bowel disease characterized by an enhanced expression of CEA (carcinoembryonic antigen) on affected tissue. Therefore, we considered it as the perfect marker. An engineered spore presents the nanobody against CEA, for the targeting of inflamed intestinal cells and the GST to locally activate the prodrug azathioprine.
The GST on the spore surface converts azathioprine in to 6-mercaptopurine. This has a toxic effect on cells in state of mitosis, because it replaces adenine and guanine in the DNA. This leads to a loss of function and therefore apoptosis of the diseased cell and the colon is able to regenerate.

Nanocillus approach

Generating functionalized spores of Bacillus subtilis for targeted drug delivery requires the production and purification of the spores and the subsequent evaluation of protein expression.
This involves transformation of competent B. subtilis cells with appropriate DNA encoding for proteins that can be displayed on the surface of the spores.

Bacillus subtilis is a microorganism that is able to stably integrate DNA strands into their genome. This ability can be used to specifically choose a region in the genome where the DNA is to be inserted. B. subtilis is a bacterium that is able to synthesize the enzyme amylase which can degrade starch.
Using the gene coding for amylase enables the easy verification of stable integration of the chosen DNA with the use of a simple starch degradation test. After the selection of successfully transformed clones, sporulation can be induced through nutrient starvation of the cells in minimal media. Our protocols can be found in our method section.

Applying genetically modified organisms to the human body involves additional safety precautions to avoid undesired and uncontrolled proliferation of introduced cells causing disturbance of the own microbiome. An unbalanced microbiome, especially in the gut, can lead to serious conditions like inflammations of the bowel tissue or colorectal cancer.

Considering this important safety aspect, we included the analysis of germination deficient strains of B. subtilis in our project.
To avoid the administration of vegetative cells, we established a purification protocol using lysozyme to hydrolyse bacterial cells by affecting the vital component peptidoglycan, which isn’t accessible on the endospores. Since not all the vegetative cells sporulate, the purification is a very important step. The vegetative cells would distort the results in further experiments.

A quality control of the generated Nanocillus was achieved by the verification of expression and proper surface localization of ectopic proteins. Chemical decoating of endospores enabled the analysis of extracted surface proteins. By including a hemagglutinin epitope tag to the introduced proteins could be detected via Western Blot.
Verification that the introduced protein is located on the surface of the endospores and is accessible, can be achieved by conjugated anti-HA antibodies. Staining of the spores with said antibodies can be used for flow cytometry analyses to demonstrate proper surface localization.

After this verification of our Nanocillus and the actual production of the spores at high scales, the capsules, serving as a carrier to the colon, need to be filled. To safely and easily manage this step, the spores were lyophilized.