DK2501805T3 - Gærstammer, der producerer pattedyrslignende komplekse n-glycaner - Google Patents

Gærstammer, der producerer pattedyrslignende komplekse n-glycaner Download PDF

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DK2501805T3
DK2501805T3 DK10812879.4T DK10812879T DK2501805T3 DK 2501805 T3 DK2501805 T3 DK 2501805T3 DK 10812879 T DK10812879 T DK 10812879T DK 2501805 T3 DK2501805 T3 DK 2501805T3
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nucleic acid
glycans
cell
acid encoding
transferase
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Wouter Vervecken
Steven Christian Jozef Geysens
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Oxyrane Uk Ltd
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Claims (27)

1. Fremgangsmåde til fremstilling af en Yarrowia lipolytica-celle, der er i stand til at producere proteiner omfattende GlcNAc2Man3GlcNAc2-N-glycaner, hvilken fremgangsmåde omfatter: a) tilvejebringelse af en Yarrowia lipolytica-celle, der er genmanipuleret til at producere proteiner omfattende Man3GlcNAc2-N-glycaner, hvor cellen er deficient med hensyn til ALG3-aktivitet og omfatter en nukleinsyre, der koder for en a-1,2-mannosidase, hvilken nukleinsyre omfatter en nukleotidsekvens, der koder for en targeting-sekvens til målretning af den kodede a-1,2-manno-sidase mod det endoplasmatiske reticulum, og hvor cellen yderligere omfatter en nukleinsyre, der koder for ALG6; b) indføring i cellen afen nukleinsyre, der koder for en GIcNAc-transferase I, hvor nukleinsyren omfatter en nukleotidsekvens, der koder for en targeting-sekvens til målretning af den kodede GIcNAc-transferase I til et intracellulært rum, og en nukleinsyre, der koder for en GIcNAc-transferase II, hvor nukleinsyren omfatter en nukleotidsekvens, der koder for en targeting-sekvens til målretning af den kodede GIcNAc-transferase II mod et intracellulært rum, hvor ekspression af nævnte GIcNAc-transferase I og nævnte GIcNAc-transferase II i Yarrowia lipolytica-cellen producerer proteiner omfattende GlcNAc2Man3GlcNAc2-N-glycaner.
2. Fremgangsmåde ifølge krav 1, hvor Yarrowia lipolytica-cellen, der er genmanipuleret til at producere proteiner omfattende Man3GlcNAc2-N-glycaner, endvidere er deficient med hensyn til OCFI1-aktivitet.
3. Fremgangsmåde ifølge krav 1, hvor det intracellulære rum er Golgi-appara-tet.
4. Fremgangsmåde ifølge et hvilket som helst af kravene 1-3, hvilken fremgangsmåden yderligere omfatter indføring i cellen afen nukleinsyre, der koder for a- og β-underenhederne af en Glucosidase II, hvor ekspression af a- og β-underenhederne af nævnte Glucosidase II i Yarrowia lipolytica-cellen producerer proteiner omfattende GlcNAc2Man3GlcNAc2-N-glycaner.
5. Fremgangsmåde ifølge et hvilket som helst af kravene 1-4, hvilken fremgangsmåde yderligere omfatter indføring i cellen af en nukleinsyre, der koder for et målprotein, hvor cellen producerer målproteinet, der er modificeret til at omfatte GlcNAc2Man3GlcNAc2-N-glycanerne.
6. Fremgangsmåde ifølge krav 5, hvor: - målproteinet binder til en Fc-receptor; - målproteinet er et antistof eller fragment deraf; - målproteinet er et terapeutisk glycoprotein; eller - målproteinet er lnterferon-β, GM-CSF, Interferon y, eller erythropoietin.
7. Fremgangsmåde ifølge et hvilket som helst af kravene 1-4, hvilken fremgangsmåden yderligere omfatter indføring i cellen afen nukleinsyre, der koder for en galactosyltransferase, hvor nukleinsyren, der koder for galactosyltrans-ferasen, omfatter en nukleotidsekvens, der koder for en targeting-sekvens til målretning af den kodede galactosyltransferase mod Golgi-apparatet, hvor ekspression af galactosyltransferasen i Yarrowia lipolytica-cellen producerer proteiner omfattende GalGlcNAc2Man3GlcNAc2- eller Gal2GlcNAc2Man3GlcNAc2-N-glycaner.
8. Fremgangsmåde ifølge krav 7, hvor galactosyltransferasen er en fusion af en UDP-Glc-4-epimerase og katalytisk domæne af en β-1,4-galactosyltransfe-rase I.
9. Fremgangsmåde ifølge krav 7 eller 8, hvilken fremgangsmåden yderligere omfatter indføring i cellen af en nukleinsyre, der koder for et målprotein, hvor cellen producerer målproteinet, der er modificeret til at omfatte Gal-GlcNAc2Man3GlcNAc2- eller Gal2GlcNAc2Man3GlcNAc2-N-glycanerne.
10. Fremgangsmåde til at producere et målprotein omfattende GlcNAc2Man3GlcNAc2-N-glycaner, hvilken fremgangsmåde omfatter: a) tilvejebringelse af en Yarrowia lipolytica-celle, der er genmanipuleret til at være deficient med hensyn til ALG3-aktivitet, omfattende en nukleinsyre, der koder for en a-1,2-mannosidase, hvilken nukleinsyre omfatter en nukleotidsekvens, der koder for en targeting-sekvens til målretning af den kodede a-1,2-mannosidase mod det endoplasmatiske reticulum, omfattende en nukleinsyre, der koder for ALG6, og omfattende en nukleinsyre, der koder for en GIcNAc-transferase I og en GIcNAc-transferase II, hvor nukleinsyren, der koder for nævnte GIcNAc-transferase I og nævnte GIcNAc-transferase II, omfatter nu-kleotidsekvenser, der koder for targeting-sekvenser til målretning af hvert kodede protein mod et intracellulært rum; b) indføring i cellen af en nukleinsyre, der koder for et målprotein, hvor cellen producerer målproteinet omfattende GlcNAc2Man3GlcNAc2-N-glycanerne.
11. Fremgangsmåde til at producere et målprotein omfattende Gal-GlcNAc2Man3GlcNAc2 eller Gal2GlcNAc2Man3GlcNAc2-N-glycaner, hvilken fremgangsmåde omfatter: a) tilvejebringelse af en Yarrowia lipolytica-celle, der er genmanipuleret til at være deficient med hensyn til ALG3-aktivitet, omfattende en nukleinsyre, der koder for en a-1,2-mannosidase, hvilken nukleinsyre omfatter en nukleotidse-kvens, der koder for en targeting-sekvens til målretning af den kodede a-1,2-mannosidase mod det endoplasmatiske reticulum, omfattende en nukleinsyre, der koder for ALG6, og omfattende en nukleinsyre, der koder for en GIcNAc-transferase I, en GIcNAc-transferase II og en galactosyltransferase, hvor nukleinsyren, der koder for nævnte GIcNAc-transferase I, nævnte GIcNAc-transferase II og galactosyltransferasen, omfatter nukleotidsekvenser, der koder for targeting-sekvenser til målretning af hvert kodede protein mod et intracellulært rum; b) indføring i cellen af en nukleinsyre, der koder for et målprotein, hvor cellen producerer målproteinet omfattende GalGlcNAc2Man3GlcNAc2- eller Gal2GlcNAc2Man3GlcNAc2-N-glycanerne.
12. Fremgangsmåde ifølge krav 11, hvor galactosyltransferasen er en fusion afen UDP-Glc-4-epimeraseog katalytisk domæne af en β-1,4-galactosyltrans-ferase I.
13. Fremgangsmåde ifølge et hvilket som helst af kravene 10-12, hvor Yarrowia lipolytica-cellen endvidere er deficient med hensyn til OCH1-aktivitet.
14. Fremgangsmåde ifølge krav 10 eller 13, hvor Yarrowia lipolytica-cellen endvidere omfatter en nukleinsyre, der koder for a- og β-underenhederne af en Glucosidase II, hvor ekspression af a- og β-underenhederne af nævnte Glucosidase II i Yarrowia lipolytica-cellen producerer målproteinet omfattende GlcNAc2Man3GlcNAc2-N-glycanerne.
15. Fremgangsmåde ifølge et hvilket som helst af kravene 11-13, hvor Yarrowia lipolytica-cellen endvidere omfatter en nukleinsyre, der koder for a- og β-underenhederne af en Glucosidase II, hvor ekspression af a- og β-underenhe-derne af nævnte Glucosidase II i Yarrowia lipolytica-cellen producerer målproteinet omfattende GalGlcNAc2Man3GlcNAc2- eller Gal2GlcNAc2Man3GlcNAc2-N-glycaner.
16. Isoleret Yarrowia lipolytica-celle, der er genmanipuleret til at producere proteiner omfattende GlcNAc2Man3GlcNAc2-N-glycaner, hvor Yarrowia lipoly-tica-cellen er genmanipuleret til at være deficient med hensyn til ALG3-aktivi-tet, omfatter en nukleinsyre, der koder for en a-1,2-mannosidase, hvilken nukleinsyre omfatter en nukleotidsekvens, der koder for en targeting-sekvens til målretning af den kodede a-1,2-mannosidase mod det endoplasmatiske reticulum, omfatter en nukleinsyre, der koder for ALG6, og omfatter en nukleinsyre, der koder for en GIcNAc-transferase I og en GIcNAc-transferase II, hvor nukleinsyren, der koder for nævnte GIcNAc-transferase I og nævnte GIcNAc-transferase II, omfatter nukleotidsekvenser, der koder for targeting-sekvenser til målretning af hvert kodede protein mod et intracellulært rum, hvor ekspression af nævnte GIcNAc-transferase I og nævnte GIcNAc-transferase II i Yarrowia lipolytica-cellen producerer proteiner omfattende GlcNAc2Man3GlcNAc2-N-glycaner.
17. Yarrowia lipolytica-celle ifølge krav 16, hvor den genmanipulerede Yarrowia lipolytica-celle endvidere er deficient med hensyn til OCH1-aktivitet.
18. Yarrowia lipolytica-celle ifølge krav 16 eller 17, hvilken Yarrowia lipolytica-celle endvidere omfatter en nukleinsyre, der koder for a- og β-underenhederne af en Glucosidase II, hvor ekspression af a- og β-underenhederne af nævne Glucosidase II i Yarrowia lipolytica-cellen producerer proteinet omfattende GlcNAc2Man3GlcNAc2-N-glycanerne.
19. Yarrowia lipolytica-celle ifølge et hvilket som helst af kravene 16-18, hvor den genmanipulerede Yarrowia lipolytica-celle endvidere omfatter en nukleinsyre, der koder for et målprotein, hvor cellen producerer målproteinet, der er modificeret til at omfatte GlcNAc2Man3GlcNAc2-N-glycanerne.
20. Yarrowia lipolytica-celle ifølge et hvilket som helst af kravene 16-19, hvilken Yarrowia lipolytica-celle endvidere omfatter en nukleinsyre, der koder for en galactosyltransferase, hvor nukleinsyren, der koder for galactosyltransfera-sen, omfatter en nukleotidsekvens, der koder for en targeting-sekvens til målretning af den kodede galactosyltransferase mod Golgi-apparatet, hvor ekspression af galactosyltransferasen i Yarrowia lipolytica-cellen producerer proteiner omfattende GalGlcNAc2Man3GlcNAc2- eller Gal2GlcNAc2Man3GlcNAc2-N-glycaner.
21. Yarrowia lipolytica-celle ifølge krav 20, hvor galactosyltransferasen er en fusion afen UDP-Glc-4-epimerase og katalytisk domæne afen β-1,4-galacto-syltransferase I.
22.1 det væsentlige ren kultur af Yarrowia lipolytica-celler, hvoraf et væsentligt antal er genmanipulerede til at producere glycoproteiner omfattende GlcNAc2Man3GlcNAc2-N-glycaner, hvor cellerne er genmanipulerede til at være deficiente med hensyn til ALG3-aktivitet, omfatter en nukleinsyre, der koder for en a-1,2-mannosidase, hvilken nukleinsyre omfatter en nukleotidsekvens, der koder for en targeting-sekvens til målretning af den kodede a-1,2-mannosidase mod det endoplasmatiske reticulum, omfatter en nukleinsyre, der koder for ALG6, og omfatter en nukleinsyre, der koderforen GIcNAc-trans-ferase I og en GIcNAc-transferase II, hvor nukleinsyren, der koder for nævnte GIcNAc-transferase I og nævne GIcNAc-transferase II, omfatter nukleotidse-kvenser, der koder for targeting-sekvenser til målretning af hvert kodede protein mod et intracellulært rum, hvor ekspression af nævnte GIcNAc-transferase I og nævnte GIcNAc-transferase II i cellen producerer proteiner omfattende GlcNAc2Man3GlcNAc2-N-glycaner.
23.1 det væsentlige ren kultur af Yarrowia lipolytica-celler, hvoraf et væsentligt antal er genmanipulerede til at producere glycoproteiner omfattende Gal- GlcNAc2Mari3GlcNAc2- eller Gal2GlcNAc2Mari3GlcNAc2-N-glycaner, hvor cellerne er genmanipulerede til at være deficiente med hensyn til ALG3-aktivitet, omfatter em nukleinsyre, der koder for en a-1,2-mannosidase, hvilken nukleinsyre omfatter en nukleotidsekvens, der koder for en targeting-sekvens til målretning af den kodede a-1,2-mannosidase mod det endoplasmatiske reticulum, omfatter en nukleinsyre, der koder for ALG6, og omfatter en nukleinsyre, der koder for en GIcNAc-transferase I, en GIcNAc-transferase II og en galactosyltransferase, hvor nukleinsyren, der koder for nævnte GIcNAc-transferase I, nævnte GIcNAc-transferase II og galactosyltransferasen, omfatter nu-kleotidsekvenser, der koder for targeting-sekvenser til målretning af hvert kodede protein mod et intracellulært rum, hvor ekspression af nævnte GIcNAc-transferase I, nævnte GIcNAc-transferase II og galactosyltransferasen i cellen producerer proteiner omfattende GalGlcNAc2Man3GlcNAc2- eller Gal2GlcNAc2Man3GlcNAc2-N-glycaner.
24.1 det væsentlige ren kultur ifølge krav 23, hvor galactosyltransferasen er en fusion af en UDP-Glc-4-epimerase og katalytisk domæne af en p-1,4-ga-lactosyltransferase I.
25.1 det væsentlige ren kultur ifølge et hvilket som helst af kravene 22-24, hvor cellerne endvidere er deficiente med hensyn til OCH1-aktivitet.
26.1 det væsentlige ren kultur ifølge krav 22 eller 25, hvor cellerne endvidere omfatter en nukleinsyre, der koder for a- og β-underenhederne af en Gluco-sidase II, hvor ekspression af a- og β-underenhederne af Glucosidase II i Yar-rowia lipolytica-cellen producerer målproteinet omfattende GlcNAc2Man3GlcNAc2-N-glycanerne.
27.1 det væsentlige ren kultur ifølge et hvilket som helst af kravene 23-25, hvor cellerne endvidere omfatter en nukleinsyre, der koder for a- og β-underenhe-derne af en Glucosidase II, hvor ekspression af a- og β-underenhederne af Glucosidase II i Yarrowia lipolytica-cellen producerer målproteinet omfattende GalGlcNAc2Man3GlcNAc2- eller Gal2GlcNAc2Man3GlcNAc2-N-glycaner.
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