Projects / Programmes
Protein engineering of recombinant probiotic lactic acid bacteria for treatment of irritative bowel disease
| Code |
Science |
Field |
Subfield |
| 1.05.00 |
Natural sciences and mathematics |
Biochemistry and molecular biology |
|
| Code |
Science |
Field |
| B000 |
Biomedical sciences |
|
| Code |
Science |
Field |
| 1.06 |
Natural Sciences |
Biological sciences |
Crohn disease, colitis, IBD, recombinant, probiotics, TNFalpha
Researchers (16)
Organisations (2)
Abstract
Inflammatory bowel disease (IBD) is a chronic disease of small and large intestine, which is manifested as ulcerative colitis or as Crohn’s disease. The treatment with anti-inflammatory drugs and antibiotics is symptomatic and is often not very efficient. In recent years, monoclonal antibodies against pro-inflammatory cytokine TNFα have been applied with great success. The systemic treatment with monoclonal antibodies is, however, very expensive, causes systemic side effects, requires parenteral administration and is not effective in all patients. Due to these drawbacks, new approaches for the treatment of IBD are intensively being sought.
Pro-inflammatory cytokines stimulate inflammation, and many are increased in IBD, including IL-6, IL-12, IL-15, IL-17, IL-21, IL-22, IL-23 and IL-26.
In healthy intestine, epithelial cells form a barrier which prevents passage of proteins or microbes from lumen to sub-epithelial immune cells. On the contrary, the intestinal conditions in IBD are markedly different. Intestinal epithelium is damaged, resulting in lesions and ulcers. This results in an increased exposure of immune cells to the luminal content. Considering this, it is a rational strategy to orally administer neutralizing molecules against pro-inflammatory cytokines. The biggest obstacle in the stability is the inefficient oral delivery of proteins, which are degraded or denatured in the stomach due to low pH and proteolytic activity.
Lactic acid bacteria have recently been applied as vectors for the delivery of recombinant proteins to the gastrointestinal tract. Lactic acid bacteria have accompanied human race for centuries, are used as food and have proven safety record. Some are termed probiotics, as they can exert beneficial health effects in several diseases, including IBD. Lactic acid bacteria could therefore be used as vectors for the delivery of cytokine/chemokine-binding proteins to the gastrointestinal tract.
In the proposed research project we intend to prepare lactic acid bacteria, which will have the ability to display small cytokine/chemokine-binding proteins on their surface. The cytokine/chemokine-binding bacteria will be tested in the treatment of experimental animal model of chronic enterocolitis.
We will begin the project with the engineering of the system for the display of model protein on the surface of lactic acid bacterium Lactococcus lactis. In parallel, we will develop our proprietary surface display system, by exploiting appropriate surface proteins, identified by proteomic approach, as candidate carrier proteins. Small protein binding molecules, such as affibodies, DARPins or single chain variable fragments will be used to target cytokines/chemokines. Beside, we will develop proprietary binding molecules against cytokines/chemokines of choice, using phage display. We will also prepare purified recombinant protein for surface display, and use it for binding to the surface of unmodified lactic acid bacteria from genus Lactobacillus. Such approach will avoid the use of genetically-modified lactic acid bacteria. Most promising engineered lactic acid bacteria will be used in animal experimental models of enterocolitis, either alone or in mixtures.
Chronic enterocolitis in mice will be either chemically induced or due to genetic mutation. Cytokine/chemokine binding lactic acid bacteria will be administered orally. The efficacy and safety of cytokine/chemokine-binding lactic acid bacteria will be assessed by their administration to animals without enterocolitis. Our approach introduces several levels of synergy in the treatment of IBD. It combines the probiotics with intrinsic beneficial effect in IBD with targeted delivery of cytokine-neutralizing proteins. The production of engineered lactic acid bacteria would also be cost effective in comparison to current therapy, and would enable patient-friendly oral administration. Novel therapy of IBD would have highly significant medical and industrial impac
Significance for science
The development of biological medicines, as well as medicinal products and dietary supplements based on the use of probiotics represent a huge basic research potential. On the other hand, such alternative system is recognized as safe, therefore could be commercialized in a relatively short period of time. At the time being, in the treatment with modern biological drugs-antibodies and fusion proteins (adalimumab, etanercept, infliximab, certolizumab pegol) against proinflammatory citokines (TNFalpha) represent successful first line medication in severe Crohn disease and ulcerative colitis. However, due to the systemic route of administration, some serious side effects could occur. In order to develop a safe, alternative administration of biological medicines without mentioned side effects due to the direct aplication into the gastrointestinal tract, we developed a general platform based on the expression and re-adhesion of target ligand proteins aganist proinflamamtory citokines into the GIT. Furthermore, the platform could act as a carrier for any further expressed protein of interest and will be further developed and patented based on the results obtained in the present project.
Significance for the country
Biotechnology is strategic priority field of R&D in Slovenia from year 2010 til 2014 with the special emphasis to pharmaceutical biotechnology. Both partners in the projekst, Department for Biotechnology IJS and Faculty of Pharmacy UL have been developing the tight research connections with the pharma industry in Slovenia, in particular with the LEK-SANDOZ company. Beside bilateral projekct, many post-doc researchers that work as scientist in LEK-SANDOZ had been recruiting from both partners in projest over last 3 years. Beside, we expect that at least some parts of the successfuly finished project is going to be implemented in pharma and diary industry in Slovenia and abroad. During last 3 years, some PhD disertations and many diploma works were directly disseminated from the project research. We believe that such types of projects and the scientific and pedagogical outcomes clearly contributed to the reputation of biotechnology in Slovenia and bring Slovenia into the map of advanced biotech knowledge.
Most important scientific results
Annual report
2013,
2014,
2015,
final report
Most important socioeconomically and culturally relevant results
Annual report
2013,
2014,
2015,
final report
