Utjecaj utišavanja kaveolina-1 na uspješnost transdukcije adenoviralnim vektorima tipa 5, 26 i 35

master's thesis

• Title (english): Effect of silencing caveolin-1 on transduction efficiency of adenoviral vectors 5, 26 and 35
• Author: Danijel Švec
• Mentor: Andreja Ambriović Ristov (mentor)
• Committee member: Kristina Radošević (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2020-02-28, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Genska terapija predstavlja prijenos genskog materijala u stanicu s ciljem postizanja terapijskog učinka, bilo ispravljanjem prisutnog poremećaja, bilo unosom u stanicu nove funkcije. Najčešće korišteni vektori u genskoj terapiji su adenovirusi, no prisustvo neutralizacijskih protutijela prema učestalim serotipovima, poput adenovirusa tipa 5 (Ad5), može biti ograničavajući faktor u njihovoj primjeni, te je korištenje rijetkih adenovirusnih tipova poput adenovirusa tipa 26 (Ad26) ili 35 (Ad35), moguće rješenje. Adenovirusi za ulazak u stanicu koriste endocitozu te se klatrinom posredovana endocitoza smatra glavnim putem njihove internalizacije. Osim navedene, endocitoza putem kaveola važna je za ulazak nekih virusa. Do sada nije poznato koja je uloga kaveola u uspješnosti transdukcije Ad5, Ad26 i Ad35. U ovom radu ispitana je uloga kaveolina-1 prilikom endocitoze Ad5, Ad26 i Ad35. Stanice adenokarcinoma pluća čovjeka (A549) transfecirane su plazmidom koji sadrži short hairpin RNA za kaveolin-1 te su izdvojeni stabilni transfektanti sa smanjenom ekspresijom kaveolina-1 na kojima je ispitana uspješnost transdukcije Ad5, Ad26 i Ad35. Smanjenje kaveolina-1 smanjilo je površinsku ekspresiju adenoviralnih receptora CAR, CD46 i αvβ5 integrina, te povećalo ekspresiju integrina αvβ3. U skladu s promjenom količine receptora, utišavanje kaveolina-1 je smanjilo internalizaciju Ad5 i Ad26, smanjilo uspješnost transdukcije Ad5, no nije imalo značajnu ulogu na uspješnost transdukcije Ad26 i Ad35. Dobiveni rezultati upućuju na zaključak da kaveolin-1 ima ulogu u endocitozi Ad5 i Ad26 u A549 stanice.

Abstract (english)

Gene therapy refers to the delivery of foreign gene material into the target cell with the purpose of therapeutic effect, in order to correct a particular disorder, or introduce a new function into the cell. The most used vectors in gene therapy are adenoviruses, but the presence of neutralizing antibodies against frequent serotypes like adenovirus type 5 (Ad5), can be a limiting factor in their application. Thus, using rare adenovirus serotypes like adenovirus type 26 (Ad26) or adenovirus type 35 (Ad35) may be the solution. Adenoviruses use endocytosis to enter the cell and it seems that clathrin mediated endocytosis is major path of their internalization. Besides that, caveolar endocytosis plays important role in internalization of some viruses. It's not known if caveolae have a part in Ad5, Ad26 and Ad35 transduction efficiency. The aim of this study is to investigate the role of caveolin-1 in Ad5, Ad26 and Ad35 endocytosis. Human lung adenocarcinoma cells (A549) have been transfected with plasmid containing short hairpin RNA for caveolin-1. Stabile transfectants with supressed levels of caveolin-1 have been selected and enroled in analysis of transduction efficiency analysis with Ad5, Ad26 and Ad35. Lower levels of caveolin-1 caused lower surface expression of adenoviral receptors CAR, C46 i αvβ5 and higher expression of αvβ3. In correlation with changes in expression levels of adenoviral receptors, caveolin-1 silencing led to lower internalization of Ad5 and Ad26, and reduced transduction efficiency of Ad5, but did not cause any significant change in transduction efficiency of Ad26 and Ad35. Results of this study indicate that caveolin-1 might play important role in Ad5 and Ad26 endocytosis in A549 cells.

• Keywords: adenovirus tipa 5; 26; 35; kaveolin; endocitoza
• Keywords (english): adenovirus type 5; 26; 35; caveolin; endocytosis
• Language: croatian
• URN:NBN: urn:nbn:hr:159:539239
• Study programme: Title: Biotechnology; specializations in: Biochemical Engineering; Course: Biochemical Engineering; Study programme type: university; Study level: graduate; Academic / professional title: diplomirani inženjer biotehnologije; smjer biokemijsko inženjerstvo (diplomirani inženjer biotehnologije; smjer biokemijsko inženjerstvo)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-03-02 10:39:02