Theses and Dissertations from UMD
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Item GENETICALLY ENGINEERED PROBIOTICS FOR DIAGNOSTICS AND DRUG DELIVERY: APPLICATIONS FOR CROHN’S DISEASE(2018) McKay, Ryan; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the history of medicine, therapies have evolved while their mode of delivery has remained largely static. Generally, the active ingredient is formulated with an excipient to confer stability, and is ultimately delivered orally or intravenously in most applications. Crohn’s disease (CD), an illness with increasing global prevalence characterized by chronic inflammation of the intestines, is commonly treated with intravenously administered biologics. When these medicines spread throughout the body, only a small percentage acts at the desired site and side effects often arise. Thus, a targeted system is desired to localize treatment at sites of colonic inflammation. There is an entire field dedicated to localized delivery that typically employs drug-laden particles or capsules that can respond to local chemical or physical cues. We believe that bacteria can be “programmed” to respond analogously, and ultimately synthesize and deliver therapeutics. Nitric oxide (NO) levels are elevated at sites of intestinal inflammation, and thus serves as a targeting molecule that can attract programmed bacteria via a process called pseudotaxis. This is achieved by rewiring the native motility circuits of bacteria to respond to high NO levels. Additionally, localized treatment is attained by an NO- specific response whereby the designed bacteria produce and secrete a human protein reported to reduce inflammation in CD patients. This system may improve CD treatment via: 1) site-specific targeting to minimize side effects and increase efficacy, 2) in situ synthesis of the therapeutic avoids payload loss in the digestive tract and manufacturing obstacles associated with biologics, 3) probiotics are reported to provide innate benefits to CD patients, and 4) oral delivery is preferred by patients versus intravenous. We have also developed probiotics that fluoresce in response to NO which may serve as an ingestible biosensor for CD. We believe these reporter probiotics can assist in the diagnosis of CD by utilizing visualization of bacteria in a stool sample to reduce the need for invasive colonoscopies and biopsies. Overall, we have developed a platform of probiotic cells that respond to NO with applications for Crohn’s disease in mind, translating to noninvasive methods for both the diagnosis and treatment of CD.Item TOWARDS A GENETICALLY-ENGINEERED BACTERIUM FOR GASTROINTESTINAL WOUND HEALING(2017) Virgile, Chelsea; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Society and physicians frequently associate the increase of antibiotic-resistant bacteria with the overuse of antibiotics. This proposes a question, “Why use antibiotics to fight bacteria and risk resistance, when one could engineer bacteria to target and kill infectious bacteria?” Bacteria are often thought of as ‘good’ bacteria (e.g., commensals, probiotics) or ‘bad’ bacteria (e.g., pathogens). Synthetic biology enables the augmentation of biosynthetic capabilities and retooling of regulatory structures in the creation of cells with unprecedented ability to make products. One can also, however, think of the cell as the product – a cell that operates in a noisy environment to execute non-native tasks. There have been several recent reports of the rewiring of bacterial cells to function as conveyors of therapeutics. The engineering and rewiring of the bacteria such as E. coli into ‘smart' bacteria potentially allows for a broad range of applications, from the treatment of wounds, the elimination of pathogenic strains, to the delivery of vaccines, particularly in the gastrointestinal (GI) tract. I have engineered smart bacteria as a therapeutic delivery vehicle for wound healing in the GI tract. The approach comprises synthetic biology and microfluidics for the creation of a biological ‘test track' for ensuring the appropriate design and testing of engineered bacteria. Bacterial motility was engineered for response to wound-generating signals such as hydrogen peroxide. Specifically, we have placed a motility enzyme CheZ under the control of the hydrogen-peroxide-responsive oxyR/S gene-promoter pair so that the ‘run’ in the tumble and run scheme of bacterial movement is externally regulated. These engineered cells exploit pseudotaxis for directional swimming towards hydrogen peroxide, a non-native signal. Additionally, the therapeutic enzyme transglutaminase plays an important role in the tissue clotting cascade. Microbial transglutaminase can crosslink fibrinogen, similar in function to human transglutaminases during the clotting cascade, but independently of calcium ions. This allows for a potentially faster, increased wound-healing response. By combining microbial transglutaminase expression with controlling motility and lysis expression using the OxyR/S system, the ‘smart’ bacteria can potentially swim towards and treat at the wound site with subsequent cell lysis. Ultimately, this strategy can lead to new bacterial therapies.