Engineering of Human Commensal Bacterium for the Controlled in vivo Delivery of Human Keratinocyte Growth Factor to Trea

Inflammatory Bowel Disease (IBD) affects 0.2% of the population in developed countries. Current therapy is restricted to drugs that suppress the body‘s immune system. These are not curative, may cause severe side effects and are likely to be needed for long periods. There is therefore a need for more targeted controlled forms of therapy. Various protein molecules have been identified as potential therapeutic factors for IBD. Due to their inherent instability and potential toxicity these factors can not be given to patients by conventional means. Recently developed genetically modified (GM) bacteria to produce and secrete various factors locally, which when administered to mouse models of IBD was as effective as steroids in preventing and treating IBD. The limitation of current bacterial delivery systems, however is that the production and release of the heterologous protein by the bacteria cannot be controlled; this is an important safety concern. Therefore, a novel approach proposed to develop a second generation of GM bacteria. This involves engineering a non pathogenic commensal bacterium to produce growth factor under the control of a naturally occurring plant product which can be included in a normal diet. We propose that the delivery of these proteins inside the bowel by GM bacteria would be safe and effective in preventing and treating IBD

Aims: Genetically modified strains of B ovatus have been produced, that secrete biologically active forms of immunomodulatory molecules under the control of xylanase promoter for the in vivo treatment of different chronic gut disorders. The aims of this study are: 1) To show that cytokine secreted by the recombinant B. ovatus is biologically active, produced at physiologically relevant concentrations and under the control of the xylanase promoter. 2) In vivo testing of the effectiveness of the recombinant strains in preventing and treating chemically induced colitis in mice.
Methodology: In vitro validation of recombinant B. ovatus strains: BO-KGF has been tested for the production of KGF. We have proved (by ELISA, results in section F4) that KGF can be produced by recombinant B.O. under the control of xylan. Bioassay of the rKGF will be done, by its proliferative effect on epithelial cells.
Demonstration that recombinant strains of B.ovatus can colonise the bowel and to determine that cytokine is produced by the recombinants in vivo Colonisation will be evaluated by culturing faecal pellets under conditions that are permissive for the growth of Bacteroides spp. or rely on colony filter hybridisation techniques (KGF cDNA as probe) to identify the recombinant strains. In vivo KGF production will be confirmed by ELISA of colonic washout and homogenates from treated animals.
To use the recombinant B.ovatus for the prevention and treatment of chemically induced colitis in mice Wild type Balb/c mice will be given recombinant B.ovatus and wild type B.ovatus. Xylan will be added to the chow before and after induction of colitis by DSS (Dextran sodium sulphate). Clinical parameters (weight change, stool score, mortality), serum assay for inflammatory markers, histological evaluation via a scoring system and Myeloperoxidase measurement will be used for the assessment of efficacy of treatment and prevention regimen. Rate of epithelial cells turnover will be evaluated by bromodeoxyuridine uptake (BrdU) assay.
Medical opportunities: This study is unique and designed to test a novel approach for drug delivery. It would have a promising role in maintaining remission and hence reduce the hospital admission rate in patients with colitis. Bowel cancer is the other target, which we hope that through this approach we can give those patients longer survival and better quality of life with home medication.