DK2951309T3 - Forøget transgenekspression og -processering - Google Patents
Forøget transgenekspression og -processering Download PDFInfo
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- DK2951309T3 DK2951309T3 DK14708935.3T DK14708935T DK2951309T3 DK 2951309 T3 DK2951309 T3 DK 2951309T3 DK 14708935 T DK14708935 T DK 14708935T DK 2951309 T3 DK2951309 T3 DK 2951309T3
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Claims (14)
1. Rekombinant nukleinsyremolekyle omfattende: en 5' og en 3' transposon-specifik inverteret terminal repeat (ITR), mindst én nukleinsyresekvens, der koder for et TEP (transgene expression processing)-protein eller en TEP-funktionel RNA, placeret mellem 5' og 3' ITR'ene, og som er under en promoters kontrol, og Mindst ét MAR (matrix attachment region)-element placeret mellem 5' og 3' ITR'ene; hvor TEP-proteinet er en eller flere af følgende aminosyresekvenser af proteiner fra proteinsekretionsvejen: hSRP14 med SEQ ID NO: 13, hSec61al med SEQ ID NO: 15, hSec6ip med SEQ ID NO: 17, hSec61y med SEQ ID NO: 19, hSRP54 med SEQ ID NO: 21, hSRP9 med SEQ ID NO: 23, hSRPRa med SEQ ID NO: 25, hSRPP med SEQ ID NO: 27 og hCANX med SEQ ID NO: 29 og aminosyresekvenser med mere end 80 %, 90 %, 95 % eller 98 % sekvensidentitet med de angivne sekvenser, hvilken TEP-funktionel RNA, fortrinsvis en miRNA eller en shRNA, interfererer med ekspressionen af ét protein i en rekombinationsvej, der er udvalgt fra Rad51, Rad51C, Rad51D, Xrcc2, Xrcc3, Rad52, Rad54, Brcal, Cyclin Dl, Bardi eller Mre 11, og hvor MAR-elementet er udvalgt fra SEQ ID NO: 1 (MAR 1-68), 2 (MAR 1_6), 3 (MAR X_S29), 4 (MAR S4), 5 (hønselysozym- MAR), eller er en konstrueret, særligt omarrangeret, sidestykke, der har mindst 80 %, 90 %, 95 %, 98 %, 99 % eller 100 % sekvensidentitet med en hvilken som helst af SEQ ID NO: 1 til 5, og hvor TEP'en eller den TEP-funktionelle RNA, der udtrykkes via nukleinsyremolekylet, og MAR-elementet er i stand til at øge ekspressionen af et transgen i en pattedyrcelle med mindst 10 %.
2. Rekombinant nukleinsyremolekyle ifølge krav 1, der endvidere omfatter mindst ét transgen, der er placeret mellem 5' og 3' ITR'ene, hvilket transgen er under en transgenpromoters kontrol.
3. Vektor omfattende det rekombinante nukleinsyremolekyle ifølge krav 1 eller 2.
4. Vektor ifølge krav 3, hvor vektoren omfatter et enkelt MAR-element eller to eller flere MAR-elementer, hvor elementet (elementerne) er placeret mellem 5' og 3' ITR'ene.
5. Vektor ifølge krav 3, hvor vektoren omfatter to MAR-elementer, hvor et første MAR-element er anbragt opstrøms for TEP'et eller den TEP-funktionelle RNA, og et andet MAR-element er anbragt nedstrøms for TEP'et eller den TEP-funktionelle RNA, hvor det første MAR-element omfatter et MAR l_6-element eller et element, der har mindst 80 %, 90 %, 95 %, 98 %, 99 % eller 100 % sekvensidentitet med SEQ ID NO. 2, særligt en omarrangeret MARs baseret på MAR 1-6, mere særligt elementer, der har mindst 80 %, 90 %, 95 %, 98 %, 99 % eller 100 % sekvensidentitet med SEQ ID NO 8 (MARs 1_6R2) og det andet MAR-element omfatter et MAR 1-68-element eller et element, der har mindst 80 %, 90 %, 95 %, 98 %, 99 % eller 100 % sekvensidentitet med SEQ ID NO. 1.
6. Vektor ifølge krav 3, hvor vektoren omfatter det enkelte MAR-element, hvor det enkelte MAR-element er anbragt nedstrøms for TEP'et eller den TEP-funktionelle RNA, hvor det enkelte MAR-element er et MAR 1-68- eller et MAR X-29-element eller et element, der har mindst 80 %, 90 %, 95 %, 98 %, 99 % eller 100 % sekvensidentitet med SEQ ID NO. 1 eller 3, særligt et omarrangeret MAR baseret på MAR 1-68 eller et MAR X-29, særligt et element, der har mindst 80 %, 90 %, 95 %, 98 %, 99 % eller 100 % sekvensidentitet med SEQ ID NO: 6, 7 eller 10 (MAR 1_68R, 1_68R2 eller X_29R3) eller 9, og fortrinsvis er et MAR X-29-element eller et element, der har mindst 80 %, 90 %, 95 %, 98 %, 99 % eller 100 % sekvensidentitet med SEQ ID NO. 3.
7. Fremgangsmåde til ekspression af et transgen og et TEP (transgene expression processing)-protein eller en TEP-funktionel RNA in vitro omfattende: tilvejebringelse af en rekombinant pattedyrcelle omfattende et transgen og indføring af en vektor ifølge et hvilket som helst af kravene 3 til 6 i den rekombinante pattedyrcelle, som udtrykker transgenet og TEP'et eller den TEP- funktionelle RNA, hvor TEP'et eller den TEP-funktionelle RNA, der udtrykkes via vektoren, eventuelt øger en ekspression af et transgen i pattedyrcellen med mindst 10 %, mindst 20 %, mindst 30 %, mindst 40 %, mindst 50 %, mindst 60 % eller mindst 70 %.
8. Fremgangsmåde ifølge krav 7, hvor vektoren omfatter et enkelt MAR X-29-element eller en nukleinsyresekvens med mindst 80 %, 90 %, 95 %, 98 %, 99 % eller 100 % sekvensidentitet med SEQ ID NO. 3, og hvor TEP'et eller den TEP-funktionelle RNA, der udtrykkes via vektoren, efter mere end 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 eller 14 ugers dyrkning øger en ekspression af et gen af interesse med mindst 10 %, mindst 20 %, mindst 30 %, mindst 40 %, mindst 50 %, mindst 60 % eller mindst 70 %.
9. Rekombinant pattedyrcelle omfattende højst 20, 15, 10 eller 5 rekombinante nukleinsyremolekyler ifølge krav 1 eller 2, fortrinsvis integreret i cellens genom som enkelte kopier.
10. Rekombinant pattedyrcelle omfattende: a) en nukleinsyre ifølge krav 1, hvor nukleinsyren er integreret i pattedyrcellens genom, og b) et rekombinant nukleinsyremolekyle omfattende: (i) mindst ét transgen af interesse og (ii) et MAR-element, hvor MAR-elementet er udvalgt fra SEQ ID NO: 1 (MAR 1-68), 2 (MAR 1_6), 3 (MAR X_S29), 4 (MAR S4), 5 (hønselysozym-MAR), eller er et konstrueret, delvis omarrangeret modstykke, der har mindst 80 %, 90 %, 95 %, 98 %, 99 % eller 100 % sekvensidentitet med en hvilken som helst af SEQ ID NO: 1 til 5.
11. Fremgangsmåde til transfektion in vitro af pattedyrceller, særligt hamsterceller, omfattende: transfektion, eventuelt i en første transfektion, af pattedyrcellerne med mindst ét, mindst to, mindst tre eller mindst fire rekombinante nukleinsyremolekyler ifølge krav 1 eller 2.
12. Fremgangsmåde til transfektion in vitro af pattedyrceller ifølge krav 11, der endvidere omfatter: mindst én, mindst to, mindst tre eller mindst fire isolerede TEP (transgene expression processing)-funktionelle RNA og mindst ét transgen, som eventuelt er en del af et rekombinant nukleinsyremolekyle, som eventuelt er transficeret i en anden efterfølgende transfektion, eventuelt sammen med en isoleret nukleinsyre eller mRNA, der udtrykker en transposase, som genkender 5' og 3' ITR'ene.
13. Kit omfattende i en beholder mindst én vektor, der omfatter et rekombinant nukleinsyremolekyle ifølge krav 1 eller 2 og i en anden eventuel beholder en vektor, der koder for en kompatibel transposase og i en yderligere beholder anvisning i at anvende vektoren eller vektorerne.
14. Anvendelse in vitro af en rekombinant nukleinsyre ifølge krav 1 eller 2 og/eller en rekombinant pattedyrcelle ifølge krav 10 eller 11 til forøgelse af transgenintegration og/eller -ekspression.
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