Browsing by Subject "Living therapeutics"
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Item Open Access A bacterial living therapeutics with engineered protein secretion circuits to eliminate breast cancer cells(Bilkent University, 2024-01) Binte Shahid, GozeelCancer therapy often faces limitations due to potential side effects, prompting scientific interest in bacteria-based living cancer treatments. Yet, the complete utilization of bacteria in therapeutic applications confronts engineering hurdles. This thesis focuses on introducing a novel bacterial mechanism specifically intended to target and eliminate breast cancer cells. Our innovative approach involves modifying Escherichia coli (E. coli) to secrete a Shiga toxin called HlyE, a pore-forming protein that binds to HER2 receptors found on breast cancer cells. This binding process is facilitated by a nanobody expressed on the bacterial surface through the Ag43 autotransporter protein system. Our research demonstrates the effective binding of the nanobody to HER2+ cells in laboratory conditions (in vitro). Utilizing the YebF secretion system, we successfully leverage the secretion of HlyE, leading to the eradication of the targeted cancer cells. These outcomes emphasize the significant potential of our engineered bacteria as an innovative and promising strategy for breast cancer treatment. This pioneering approach represents a groundbreaking development in the field of cancer therapeutics. By harnessing the unique properties of bacteria and utilizing advanced engineering techniques, we've succeeded in creating a targeted and potent system capable of attacking breast cancer cells specifically marked by the HER2 receptor. Our study lays a robust foundation for future exploration and development in the realm of bacterial-based cancer therapies, offering potential solutions to the challenges encountered in traditional cancer treatment methods.Item Embargo Biotechnological drug platforms(Bilkent University, 2023-11) Ahan, Recep ErdemBiopharmaceuticals, also known as biotechnological drugs, have revolutionized the treatment of many diseases by providing access to new mechanisms of action that can target the underlying biological processes behind the diseases. Technological advancement in biological sciences opens new paths to uncover new biopharmaceutical modalities in nature as well as to augment the existing modalities new functions. The implementation of engineering principles i.e., synthetic biology approaches have been transforming biopharmaceutical research wherein “smart” therapeutics are developed and deployed for treatment of previously intractable diseases. However, there are still unmet clinical needs that require novel and advanced biopharmaceuticals. In this thesis, I explored different biopharmaceuticals to characterize and/or advance their capabilities for diverse indications. Firstly, we have developed a prophylactic agent from the lectin protein, griffithsin, as for ancestral and the emerged strains of SARS-CoV-2. Secondly, we have advanced genetic technologies to engineer the probiotic Escherichia coli (E. coli) strain, Nissle 1917 (EcN), for therapeutical purposes. We developed a stable recombinant DNA transfer system based on cryptic plasmids of EcN. Furthermore, a synthetic protein secretion system was envisioned and functionally validated in EcN to shuttle therapeutical proteins to diseases site. Finally, peptide tags for extracellular protein secretion as well as a cell surface protein display system were developed for Lachnospiraceae species which are parts of the healthy human gut microbiome. The technologies and methodologies described herein will pay the way for inventing and/or discovering novel biopharmaceuticals to treat current and future diseases.Item Open Access A cellular device to target cancer cells(Bilkent University, 2021-10) Ostaku, JulianCancer is the second leading cause of death globally, affecting one out of three people during their lifetime. Due to its severity and high incidence, numerous treatment methods have been implemented, with the Chimeric Antigen Receptor T-Cell (CAR-T) therapy remaining the most promising one. Other therapy options such as surgery, chemotherapy and radiation therapy remain the backbone of cancer treatment, however these therapies are not effective enough as they do not discriminate among the healthy and cancerous tissues. Therefore, there is an imperative need in developing novel cancer treatment therapies that offer precise localization and on target therapeutics release. In this study, we aim to develop an engineered bacterial device, that can sense Jimt1 breast cancer cells, which are characterized by overexpression of human epidermal growth factor receptor 2 (HER2). 2Rs15d, a nanobody that binds to HER2 receptor, is expressed on the surface of Escherichia coli BL21 (DE3) via Ag43 autotransporter protein. Upon localization in the tumor site, a therapeutic agent will be released on the outer surface. By creating this platform, we aim to target the main problems of the existing cancer therapies.Item Open Access Engineering of probiotic Saccharomyces boulardii as a host for living therapeutic applications(Bilkent University, 2022-09) Kurt, Orhan NedimTherapeutic molecules or biologically active agents used in the treatments of disorders have been manufactured employing chemical synthesis methods with high costs and limited accessibility. Even though this approach to producing therapeutic molecules has enabled life-changing substances to be produced, there are still countless diseases waiting for a cure. Therefore, it would not be possible to meet the treatment needs via just conventional manufacturing approaches. Living therapeutics are programmed cells for the production and direct delivery of therapeutic molecules to the human body. Indeed, biologically engineered cells have been used for a long time to manufacture biomolecules in industrial settings. Also, there have been many studies that designed and programmed living organisms to cure diseases such as cancer, diabetes, inflammatory bowel syndrome, ulcer, and colitis. accharomyces boulardii CNCM I-745 is a yeast strain used as a probiotic for a very long time in humans. S. boulardii is the first and the only yeast strain approved to be used in human medicine so far. It has been shown that S.boulardii has many benefits to its host, including restoring the microbiome and competing with pathogens. Also, S.boulardii is a promising organism to be engineered and programmed as a living therapeutic factory due to its great compatibility with the human body and its metabolic features enabling us to manufacture complex molecules. Aiming for this, we created a TRP1 auxotroph S.boulardii strain using synthetic biology tools to eliminate the use of antibiotic resistance genes in the further steps since it would be problematic for the spread of antibiotic resistance. To do that, we employed the CRISPR CAS-9 system and a donor DNA harboring a stop codon to be inserted into the target gene (TRP1). After verifying the construction of the TRP1 auxotroph strain, we designed an episomal expression vector comprising a strong, constitutive promoter, extracellular signal sequence (Alpha sequence), and a terminator (CYC1). Next, we inserted the Intrinsic Factor protein (the key protein responsible for vitamin B12 absorption) sequence into the vector to recombinantly produce it and secrete it into the gut. However, according to our preliminary data, we could not observe an expression of the protein of interest, suggesting that the system needs further optimization and investigation to work properly. Considering the lack of auxotrophic yeast strains available to be engineered for therapeutic purposes, our engineered S.boulardii strain can be employed in yeast-based living therapeutics applications against different disorders waiting for their treatment.Item Open Access A theranostic bio-device for biomedical applications(Bilkent University, 2019-08) Hacıosmanoğlu, NedimBiological systems are programmable by their nature. With using the abilities of these systems, scientists have designed, engineered and repurposed living machines for various tasks including biological sensing, recording of cellular events, drug production and disease treatment. Compared to the current methodology for these tasks, engineering biological systems provide a promising tool for the future of medicine, especially in the case of disease treatment. Type II Diabetes Mellitus (T2DM) is a medical condition which occurs by the deficiency of insulinotropic hormones inside the body, and affects nearly half billion people worldwide. Treatment strategies for this disease includes monitoring patient for blood glucose levels, fine production of insulinotropic hormones and providing dose-controlled treatment for the patients. All these operations increase the cost of the treatment and cause a global problem for both medical professionals and the patients. In this thesis, we propose novel systems for developing theranostic strategies for T2DM by using synthetic biology principles and genetically controlled sense-and-response cascades inside living cells. Proposed systems include a whole-cell glucose biosensor module, which can detect glucose concentrations by using internal glycolysis machinery of a probiotic Escherichia coli (E. coli) bacteria, and a release module, which can controllably secrete therapeutic molecules from the E. coli cell surface. To do that, we engineered an enzyme based biosensor module which takes the pyruvate synthesized as a result of glycolysis and turns that molecule into hydrogen peroxide via SpxB pyruvate oxidase enzyme to later detect that signal with an optimized hydrogen peroxide biosensor. In order to later incorporate this biosensor with a release mechanism, we designed and engineered an Antigen-43 (Ag43) autotransporter based peptide release system. In that system, we used Ag43 autotransporter fused GLP-1 peptide, an insulinotropic hormone for the type II diabetes treatment that is controllably displayed on the cell surface. Another Ag43 fused protein, TEV protease, with a different control mechanism is also cooperated in the system to release GLP-1 from the surface by cutting the peptide from its recognition site. Taking the ability of glucose sensing and the successfully engineered release mechanisms, our proposed system has a huge potential to be used as an alternative system for treatment of the T2DM.