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Hamady, Zaed Zoher Raouf

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Engineering of a human commensal bacterium for the controlled in vivo delivery of immunomodulatory proteins

Inflammatory bowel disease (IBD) is a significant public health problem in western societies, affecting nearly 1 in 1000 individuals. The aetiology is not fully understood although immune system dysfunction and hypersensitivity to the intestinal microbiota plays a significant role in the pathogenesis of IBD. Several immunoregulatory agents are being used in the treatment of human IBD to control dysregulated immune responses. Growth factors such as keratinocyte growth factor (KGF-2) and transforming growth factor (TGF-β) are an important immunoregulatory and epithelial growth factor and potential therapeutic proteins for IBD. However, due to protein instability in the upper gastrointestinal tract it is difficult to achieve therapeutic levels of these proteins in the injured colon when given orally. Furthermore, short half-life necessitates repeated dosage with large amounts of the growth factor that may have dangerous side effects, such as vascularisation of non-target tissues or growth of tumours. Hence, the importance of temporal and spatial control of growth factor delivery. The work described in this thesis have overcome these issues by engineering the human commensal gut bacterium, Bacteroides ovatus to produce human KGF-2 or TGF-β1 (BO-KGF or BO-TGF) in a regulated manner in response to the dietary plant polysaccharide, xylan. The successful application of BO-KGF or BO-TGF to the treatment and prevention of dextran sodium sulphate (DSS) induced murine colitis is presented here as well. Continuous administration of xylan in drinking water to BO-KGF or BO-TGF treated mice resulted in a significant improvement of DSS-induced colitis; reducing weight loss, improving stool consistency, reducing rectal bleeding, accelerating healing of damaged colonic epithelium, reducing inflammatory cell and neutrophil infiltration, reducing expression of pro-inflammatory cytokines and promoting production of mucin-rich goblet cells in colonic crypts. These beneficial effects are comparable and in most cases superior to that achieved by conventional steroid therapy. This novel drug delivery system also had a significant prophylactic effect, limiting the development of intestinal inflammation both clinically and histopathologically. The ability to regulate heterologous protein production by B. ovatus using xylan is both unique and an important safety feature of this drug delivery system. An added advantage of using B. ovatus as a drug delivery vehicle is its anaerobic nature that provides a natural inbuilt biosafety feature that is lacking in other recombinant drug delivery bacteria. The use of genetically engineered B. ovatus for the controlled and localized delivery of epithelial growth promoting and immunomodulatory proteins has potential clinical applications for the treatment of various diseases targeting the colon including IBD

Engineering of a human commensal bacterium for the controlled in vivo delivery of immunomodulatory proteins

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