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Biologie Studijní Obor Univerzita Karlova Přírodovědecká fakulta Studijní program: Biologie Studijní obor: Genetika, molekulární biologie a virologie Bc. Barbora Číhařová Studium účinku modifikace virových částic polyhistidinem na jejich intracelulární lokalizaci a dopravu genů do jádra Effect of polyhistidine modification of viral particles on their intracellular localization and gene delivery to the nucleus Diplomová práce Školitel: RNDr. Hana Španielová, Ph.D. Praha, 2021 Prohlášení Prohlašuji, že jsem závěrečnou práci zpracovala samostatně a že jsem uvedla všechny použité informační zdroje a literaturu. Tato práce ani její podstatní část nebyla předložena k získaní jiného nebo stejného akademického titulu. V Praze, 26.4. 2021 ……..….…………………………………. Barbora Číhařová Poděkovaní Moc ráda bych poděkovala všem, kteří mě během mého studia a při psaní diplomové práce podporovali, věnovali mi svůj čas a vytvářeli příjemné prostředí pro dokončení tohoto projektu. Jmenovitě moc děkuji mé školitelce paní doktorce Haně Španielové za inspirativní přístup k vědě, trpělivost, spoustu rad a především za její čas strávený nad plánováním tohoto projektu a následnou kontrolou diplomové práce. Také děkuji magistře Janě Váňové, která zajišťuje každodenní úsměv všem členům laboratoře, za neuvěřitelnou trpělivost, zaučení v laboratoři a neocenitelnou pomoc v průběhu celého studia. Moc děkuji také Mgr. et Mgr. Alžbětě Hejtmánkové za řadu důležitých rad, přátelský přístup a zaučení při práci s elektronovým mikroskopem. Paní docentce Forstové děkuji za přijetí do laboratoře a možnost naučit se techniky molekulární biologie. Velký dík také patří všem současným i bývalým členům laboratoře, kteří vytvářeli přátelské prostředí, ve kterém byla radost pracovat. Mnohokrát děkuji mé rodině za nesmírnou podporu, poskytnutí zázemí a pochopení po celou dobu studia. V neposlední řadě moc děkuji Bc. Danielu Žuchovi za podporu nejen při psaní diplomové práce, ale i za pomoc se statistickým vyhodnocením dat a zpracováním grafů. Tato práce vznikla za finanční podpory Grantové agentury České republiky (GAČR 17-11397S). Abstrakt Virové vektory odvozené od myšího polyomaviru jsou vhodným nástrojem pro studium přístupů, které se používají v nanomedicíně pro dosažení efektivního přenosu terapeutických látek do buněk, do míst jejich cílového účinku. Vektory odvozené od myšího polyomaviru jsou stejně jako mnohé nanočástice často zachyceny v endosomu a následně degradovány. Diplomová práce se proto zabývá možností modifikace takovýchto vektorů, která by zajistila efektivnější přenos do cytosolu či až do jádra. Tato práce přinesla poznatky o zefektivnění přenosu částic modifikovaných pomocí membránově aktivních peptidů kovalentně vázaných ke kapsidovému proteinu VP3 lokalizovanému uvnitř částice. Pro kovalentní genetické modifikace VP3 proteinu byl využit polyhistidinový peptid KH27K a jeho potenciál ke zvýšení efektivity transdukce modifikovaných virových částic byl srovnáván s modifikací pomocí peptidů LAH4 nebo R8. Výsledky transdukčního testu ukázaly, že kovalentně navázaný peptid R8 je schopen proti nemodifikovaným částicím několikanásobně zvýšit účinnost dopravy do jádra. Modifikace LAH4 se naopak ukázala být účinnou pouze v případě, kdy byly částice s LAH4 asociovány pouze nekovalentně, v takovém případě byl přenos do jádra proti kontrolním částicím až 40- násobně efektivnější. Modifikace polyhistidinem, který by měl dle literatury zlepšit únik endocytovaného materiálu z endosomu pomocí tzv. efektu protonové houby, odhalila, že u částic modifikovaných KH27K k přenosu do cytosolu skutečně dochází. Práce tak svými výsledky přispěla k lepšímu pochopení účinku vybraných peptidů penetrujících membrány na transport částic. Klíčová slova: polyomavirus, VLP, únik z endosomu, polyhistidin, doprava genů, peptidy penetrující membrány, CPP Abstract Viral vectors derived from mouse polyomavirus are a convenient tool for studying the targeted delivery of therapeutical agents into the cells and cellular organelles. Vectors derived from mouse polyomavirus face difficulties similar to other nanoparticles, as they often end up trapped inside an endosome where they are subsequently degraded. This diploma explored the potential of vector modifications, which have the potential to make the transport to the nucleus or cytosol more effective. This work had particularly focused on increasing the transduction efficiency by modifying particle’s internally localized VP3 capsid protein with covalently bound membrane-penetrating peptides. Primary covalent genetic modification to the VP3 protein was the polyhistidine peptide KH27K. Its potential of improving the transduction effectivity was compared with two other peptide modifications – LAH4 and R8. The results of the transduction test showed that covalently bound R8 peptide had many-fold improved the transport to the nucleus when compared to the unmodified particles. The modification with LAH4 peptide had been regarded more effective only when was associated with the particles non-covalently. In such scenario the transduction efficiency rose 40-times when compared with unmodified particles. Polyhistidine modification, which should aid the endosomal escape via the proton sponge mechanism, had indeed enabled particles to escape to the cytosol. To conclude, this work elucidates the effect cell-penetrating peptides have on a transport of particles. Keywords: polyomavirus, VLP, endosome escape, polyhistidine, gene delivery, cell-penetrating peptides OBSAH 1 ÚVOD ................................................................................................................................................... 11 2 LITERÁRNÍ PŘEHLED ......................................................................................................................... 13 2.1 Polyomaviry ................................................................................................................................ 13 2.2 Myší polyomavirus ..................................................................................................................... 13 2.2.1 Struktura virové částice ........................................................................................................15 2.2.2 Vstup viru do buňky a následný transport .......................................................................... 17 2.3 Viru podobné částice a jejich možné využití .............................................................................. 18 2.4 VLP a pseudoviriony odvozené od myšího polyomaviru .......................................................... 19 2.5 Problém degradace a uvěznění materiálu v endosomu ............................................................ 21 2.6 Peptidy penetrující membrány................................................................................................... 22 2.6.1 Peptidy bohaté na arginin ................................................................................................... 24 2.6.2 Peptidy bohaté na histidin ................................................................................................... 24 2.6.3 LAH4 .................................................................................................................................... 26 2.7 Endosomální únik ....................................................................................................................... 26 3 CÍLE PRÁCE ........................................................................................................................................ 30 4 MATERIÁL A METODY ........................................................................................................................ 31 4.1 Materiál ....................................................................................................................................... 31 4.1.1 Antibiotika ............................................................................................................................ 31 4.1.2 Buněčné kultury ................................................................................................................... 31 4.1.3 Často používané roztoky a chemikálie ................................................................................ 32 4.1.4 Enzymy ................................................................................................................................ 34 4.1.5 Expresní systémy ................................................................................................................. 34 4.1.6 Komerční soupravy a produkty ........................................................................................... 34 4.1.7 Kultivační média .................................................................................................................. 35 4.1.8 Markery molekulových hmotností ...................................................................................... 37 4.1.9 Peptidy penetrující membrány ............................................................................................ 38 4.1.10 Primery ................................................................................................................................ 38 4.1.11 Protilátky .............................................................................................................................. 38 4.1.12 Software ............................................................................................................................... 39 4.1.13 Vektory ................................................................................................................................
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