Razvoj gensko spremenjenih mlečnokislinskih bakterij za zdravljenje kronične vnetne črevesne bolezni

Kosler, Staša (author), Štrukelj, Borut (mentor), Berlec, Aleš (comentor)

Za zdravljenje kroničnih vnetnih črevesnih bolezni uporabljamo predvsem kortikosteroide, imunosupresive in biološka zdravila proti provnetnim citokinom, zlasti TNFα, ki delujejo sistemsko. Sistemsko delovanje bioloških zdravil zmanjša koncentracije TNFα in drugih provnetnih citokinov povsod, ne samo v prebavnem traktu, kar se kaže v povečanem številu okužb in nekaterih vrst raka. V okviru doktorske naloge smo zato razvili mlečnokislinske, ki na svoji površini predstavljajo različne citokin-vezavne proteine, ki bi jih lahko uporabili za lokalno zdravljenje. Zaradi vloge sistema Th17 pri patologiji kroničnih vnetnih črevesnih bolezni, smo kot tarči izbrali citokina interlevkin 17 in interlevkin 23. Kot vezalca za interlevkin 17 in interlevkin 23 smo najprej uporabili hipervariabilne dele monoklonskih protiteles z afiniteto do interlevkina 17 (1733LH) in do interlevkina 23 (2350LH), ki smo jih kasneje v sklopu osnovne hipoteze zamenjali s interlevkin 17 vezavnim finomerom (FIN17) in s interlevkin 23 vezavnim adnektinom (ADN23), z namenom izboljšanja moči vezave. Vezalce citokinov smo na površini mlečnokislinskih bakterij seva L. lactis NZ9000 predstavili ob uporabi plazmida pSDLBA3b kot citokin vezavna proteina s C-končnim delom laktokoknega proteina AcmA (cA) in signalnim peptidom proteina Usp45. Dodatno smo interlevkin 17 in interlevkin 23vezavne fuzijske proteine, ki so jih v gojišče izločile rekombinantne bakterije seva Lactococcus lactis NZ9000, po odstranitvi celic proizvajalk preko sidrne domene proteina AcmA vezali na gensko nespremenjeni sev Lactobacillus salivarius ATCC 11741. Da bi povečali spekter vezave provnetnih citokinov, smo na površini predstavili še TNF-α vezavni konstrukt »Affibody« in nato izvedli prvo hkratno predstavitev treh citokin-vezavnih fuzijskih proteinov na površini mlečnokislinskih bakterij. Tako obloženi Lb. Salivarius ATCC 11741 bi lahko uporabili za lokalno, ciljano zdravljenje KVČB.

• Language: Slovenian
• Keywords: biotehnologija, kronična vnetna črevesna bolezen, mlečnokislinske bakterije, predstavitev na površini, vezalci citokinov
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Publisher: [S. Kosler]
• Year: 2018
• PID: 20.500.12556/RUL-102281
• UDC: 606:616.34:602.6:579.864(043.3)
• COBISS.SI-ID: 8969081
• Publication date in RUL: 10.08.2018

KOSLER, Staša, 2018, Razvoj gensko spremenjenih mlečnokislinskih bakterij za zdravljenje kronične vnetne črevesne bolezni [online]. Doctoral dissertation. S. Kosler. [Accessed 13 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=102281

Secondary language

• Language: English
• Title: Development of genetically modified lactic acid bacteria for treatment of inflammatory bowel disease

Abstract

Systemic neutralization of proinflammatory cytokines (especially TNFα) is an established strategy in the treatment of inflammatory bowel disease, however it has shown through the last years many unwanted adverse effects such as a high increase in opportunistic infections and, in some cases, of cancer. Within this doctoral dissertation we have developed recombinant lactic acid bacteria with cytokine binders displayed on their surface. We targeted interleukine17 and interleukine 23 due to the involment of Th17 immune response in inflammatory bowel disease pathology. Firstly, we used single chain variable fragments (ScFv) directed against interleukine17 (1733LH) and interleukine 23 (2350LH), which we, later on due to their poor functionality, replaced with interleukine17 binding fynomer (FIN17) and interleukine 23 binding adnectine (ADN23). Genes for binders were cloned into our proprietary surface display plasmid (pSDLBA3b) and expressed in L. lactis NZ9000 strain as fusion proteins with LysM-containing C-terminus of AcmA and Usp45 secretion signal. Additionaly we used an alternative approach in which L. lactis NZ9000 strain’s producer cells were removed, and its fusion protein-containing growth media were used for coating of genetically non-modified Lb. salivarius ATCC11741. To broaden the spectra of cytokine binders we also expressed and then used for coating a recently developed TNF-α binding »Affibody«. All three cytokine binders were then used for coating of Lb. salivarius ATCC11741 simultaneously. Simultaneous display of three different cytokine-binding proteins on the surface of lactic acid bacteria was demonstrated for the first time and suggested as a new potential treatment for inflammatory bowel disease.

• Keywords: biotechnology, inflammatory bowel disease, lactic acid bacteria, surface display, cytokine binders