mRNA therapy for neurofibromatosis

master's thesis

• Title (english): mRNA terapija za lječenje neurofibromatoze
• Author: Laura Colar Zanjko
• Mentor: Igor Stuparević (mentor)
• Committee member: Višnja Besendorfer (predsjednik povjerenstva)
• Committee member: Dubravka Hranilović (član povjerenstva)
• Committee member: Igor Stuparević (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2023-07-03, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Messenger RNA (mRNA) presents itself as an efficient and effective therapeutical tool, especially due to its global and commercial rise in the past few years. As there are various rare and genetic disorders, the demand for novel treatments is at its highest. A promising approach to this problem is gene therapy, specifically using non-viral vectors, such as lipids, polymers and nanoparticles. One such disease is neurofibromatosis type 1 (NF1), which affects roughly 1 in 3000 people through mutations into the NF1 gene. Apart from aesthetic and physical ailments, it is characterized by benign and malignant tumors. Causing the development of NF1 related malignant tumors, but also heightening the risk of developing other types of cancers, the disease poses a serious life-threat to patients. In this project, a lipoplex made of a liposome vector and modified GRD mRNA encoding an important functional domain of Nf1 protein was used to transfect mutated Schwann cells (NF1 -/-). The GRD protein synthesized from the mRNA was analysed on a biochemical level, focusing on its functional activity, half-life and proteasome degradation. Another focus of the project was establishing a 3D culture model in the form of spheroids. As monolayer cultures cannot grasp the complexity of interactions and the metabolism of cells in physiological in vivo conditions, it is crucial to expand experiments onto 3D models like spheroids and organoids. Altogether, the experiments carried out offer insight into a possible mRNA gene therapy method. They demonstrated the functional activity of the GRD protein and how its half-life and proteasome activity influence its expression. A valuable protocol to form and transfect spheroids has been made, which will be helpful in further experiments.

Abstract (croatian)

Messenger RNA (mRNA) predstavlja se kao učinkovit terapeutski alat, posebno zbog svog globalnog i komercijalnog uspona u posljednjih nekoliko godina. Kako postoje razni rijetki i genetski poremećaji, potražnja za novim tretmanima je najveća dosad. Obećavajući pristup ovom problemu je genska terapija, posebno korištenjem nevirusnih vektora, kao što su lipidi, polimeri i nanočestice. Jedna takva bolest je neurofibromatoza tipa 1 (NF1), koja pogađa 1 od 3000 osobe kroz mutacije u genu NF1. Osim estetskog i fizičkog oboljenja, karakteriziraju ga benigni i maligni tumori. Uzrokujući razvoj NF1 povezanih malignih tumora, ali i povećavajući rizik od razvoja drugih vrsta raka, bolest predstavlja ozbiljnu prijetnju životu pacijenata. U ovom projektu lipopleks napravljen od liposoma kao vektora i modificirane GRD Mrna, koja kodira važnu funkcionalnu domenu proteina NF1, koristi se za transfekciju mutiranih Schwannovih stanica (NF1 -/-). GRD protein sintetiziran iz mRNA je analiziran na biokemijskoj razini, s fokusom na njegovu funkcionalnu aktivnost, poluživot i razgradnju proteasomom. Drugi fokus projekta bilo je uspostavljanje 3D modela kulture u obliku sferoida. Kako jednoslojne kulture ne mogu obuhvatiti složenost interakcija i metabolizam stanica u fiziološkim in vivo uvjetima, ključno je proširiti eksperimente na 3D modele poput sferoida i organoida. Sve u svemu, provedeni pokusi nude uvid u moguću metodu genske terapije mRNA. Izvedeni eksperimenti su pokazali funkcijsku aktivnost GRD proteina i kako njegov poluživot i aktivnost proteasoma utječu na njegovu ekspresiju. Napravljen je protokol za formiranje i transfekciju sferoida za daljne eksperimente.

• Keywords: neurofibromatosis type 1; lipoplex; GRD protein; spheroids
• Keywords (croatian): neurofibromatoza tipa 1; lipopleks; GRD protein; sferoidi
• Language: english
• URN:NBN: urn:nbn:hr:159:071195
• Study programme: Title: Bioprocess Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2024-04-30 09:51:46