DK2297307T3 - Fremgangsmåder til fremstillingen af ips-celler under anvendelse af ikke-virale metoder - Google Patents

Fremgangsmåder til fremstillingen af ips-celler under anvendelse af ikke-virale metoder Download PDF

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DK2297307T3
DK2297307T3 DK09759392.5T DK09759392T DK2297307T3 DK 2297307 T3 DK2297307 T3 DK 2297307T3 DK 09759392 T DK09759392 T DK 09759392T DK 2297307 T3 DK2297307 T3 DK 2297307T3
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Amanda Mack
James Thomson
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Cellular Dynamics Int Inc
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Claims (37)

1. Fremgangsmåde til at tilvejebringe en cellepopulation med en ændret differentieringsstatus i forhold til en udgangscellepopulation og med celler der i det væsentlige er fri for programmeringsvektor-genetiske elementer, hvilken fremgangsmåde omfatter trinnene at: (a) opnå en udgangspopulation af celler med en første differentieringsstatus; (b) opnå én eller flere differentieringsprogrammeringsvektorer, hver vektor omfatter en replikationskilde og har én eller flere ekspressionskassetter der koder for én eller flere differentieringsprogrammeringsfaktorer der, i kombination, kan ændre differentieringsstatus af udgangscellepopulationen til en anden differentieringsstatus, hvor én eller flere af ekspressionskassetterne omfatter en nukleotidsekvens der koder for en trans-agerende faktor der binder til replikationskilden til at replikere en ekstra-kromosomal template, og/eller hvor cellerne af udgangspopulationen udtrykket en sådan trans-agerende faktor; (c) indføre differentieringsprogrammeringsvektor(er) i cellerne fra udgangspopulationen; (d) dyrke cellerne til at resultere i ekspression af den ene eller flere differentieringsprogrammeringsfaktorer sådan at træk der er konsistente med den anden differentieringsstatus opstår i mindst en del af cellerne i de dyrkede celler; og (e) yderligere dyrke cellerne med trækkene for et tilstrækkeligt antal generationer til at frembringe en målcellepopulation der omfatter celler med den anden differentieringsstatus men hvilke celler er i det væsentlige fri for programmeringsvektorgenetiske elementer.
2. Fremgangsmåden ifølge krav 1, endvidere omfattende et yderligere trin valgt fra gruppen bestående af at (a) udvælge celler fra de dyrkede celler i trin d) eller e), hvilke celler er i det væsentlige fri for differentierings-programmeringsvektorgenetiske elementer, og (b) udvælge celler fra de dyrkede celler i trin d) eller e), hvilke celler er i det væsentlige fri for en selektionsmarkør omfattet i differentieringsprogrammeringsvektoren.
3. Fremgangsmåden ifølge krav 2(b), hvor selektionsmarkøren er herpes simplex virus-thymidinkinase, en antibiotika-resistensfaktor, eller et fluorescerende protein.
4. Fremgangsmåden ifølge et hvilket som helst af kravene 1 til 3, hvor ændringen af differentierings-status er valgt fra gruppen bestående af reprogrammering, differentiering, og transdifferentiering.
5. Fremgangsmåden ifølge et hvilket som helst af kravene 1 til 4, hvor ændringen af differentierings-status er reprogrammering og hvor udgangscellerne er valgt fra gruppen bestående af (a) en somatisk celle og trækkene er defineret som én eller flere karakteristikker af embryoniske stamceller, og (b) en fibroblast, en keratinocyt, en hematopoietisk celle, en mesenchymal celle, en levercelle, en mavecelle, eller en β-celle.
6. Fremgangsmåden ifølge krav 5, endvidere omfattende et trin til differentiering af målcellepopulationen.
7. Fremgangsmåden ifølge et hvilket som helst af kravene 1 til 5, hvor ændringen af differentierings-status er reprogrammering og hvor differentieringsprogrammeringsfaktorerne yderligere er defineret som reprogrammeringsfaktorer omfattende Sox-2 og Oct-4.
8. Fremgangsmåden ifølge krav 7, hvor reprogrammeringsfaktorerne yderligere omfatter Nanog, Lin28, Klf4, eller c-Myc.
9. Fremgangsmåden ifølge krav 4, hvor ændringen af differentierings-status er differentiering og hvor udgangscellen er en embryonisk stamcelle, en induceret pluripotent stamcelle, en hæmatopoietisk stamcelle, en nervestamcelle, en mesenchymal stamcelle, en hæmatopoietisk progenitor, en endoderm progenitor, en pankreatisk progenitor, eller en endotelial progenitor.
10. Fremgangsmåden ifølge krav 4, hvor ændringen af differentierings-status er transdifferentiering og hvor den første og den anden differentierings-status er terminalt differentierede.
11. Fremgangsmåden ifølge et hvilket som helst af kravene 1 til 10, hvor replikationskilden er en replikationskilde af et lymfotropisk herpes virus, et adenovirus, SV40, et bovint Papillomavirus, eller en gær.
12. Fremgangsmåden ifølge krav 11, hvor replikationskilden er en replikationskilde af et lymfotropisk herpes virus og svarer til oriP af EBV.
13. Fremgangsmåden ifølge krav 11, hvor det lymfotropiske herpes virus er Epstein Barr virus (EBV), Kaposi's sarcom herpes virus (KSHV), Herpes virus saimiri (HS), eller Marek's sygdomsvirus (MDV).
14. Fremgangsmåden ifølge et hvilket som helst af kravene 1 til 13, hvor den trans-virkende faktor (a) svarer til EBNA-1 af EBV, (b) er et derivat af et vild-type protein svarende til EBNA-1 af EBV, derivat har en nedsat evne til at aktivere transskription fra en integreret template sammenlignet med vild-type EBNA-1, (c) er et derivat af et vild-type protein svarende til EBNA-1 af EBV, hvilket derivat aktiverer transskription ved niveauer på mindst 5% af det tilsvarende vild-type protein fra en ekstra-kromosomal template efter derivatet binder replikationskilden, eller (d) er et derivat af vild-typeprotein svarende til EBNA-1 af EBV, hvilket derivat har en deletion af grupper svarende til grupperne 65 til 89 af EBNA-1, og/eller en deletion af grupper svarende til grupperne 90 til 328 af EBNA-1.
15. Fremgangsmåden ifølge krav 14(b), eller 14(c), hvor derivatet der mangler sekvenser der er til stede i vild-type-EBNA-1 protein der aktiverer transskription fra en integreret template.
16. Fremgangsmåden ifølge krav 14(b), eller 14(c), hvor derivatet har en deletion af grupper svarende til grupperne 65 til 89 af EBNA-1 og/eller en deletion af grupper svarende til grupperne 90 til 328 af EBNA-1.
17. Fremgangsmåden ifølge krav 14(b), eller 14(c), hvor derivatet koder for et protein med mindst 80% aminosyresekvensidentitet til grupperne 1 til 40 og grupperne 328 til 641 af EBNA-1.
18. Fremgangsmåden ifølge et hvilket som helst af kravene 1 til 17, hvor ekspressionskassetter er operativt bundet til et transskriptionelt regulatorelement.
19. Fremgangsmåden ifølge et hvilket som helst af kravene 1 til 18, hvor flere gener effektivt udtrykkes under anvendelse af en enkelt promotor/forstærker til at transskribere en enkelt besked.
20. Fremgangsmåden ifølge krav 19, hvor flere åbne læserammer transskriberes sammen, hver separeret afen IRES, danner polycistroniske beskeder.
21. Differentierings-programmingsvektor omfattende en replikationskilde, og har én eller flere ekspressionskassetter der koder for en trans-virkende faktor der binder til replikationskilden til at replikere vektoren ekstra-kromosomalt; og én eller flere differentierings-programmeringsfaktorer.
22. Differentierings-programmeringsvektoren ifølge krav 21, hvor den transvirkende faktor er et derivat af et vild-type protein svarende til EBNA-1 af EBV, hvilket derivat aktiverer transskription på mindst 5% af det tilsvarende vild-type protein fra en ekstra-kromosomal template efter binding til replikationskilden og har en nedsat evne til at aktivere transskription fra en integreret template sammenlignet med vild-type EBNA-1.
23. Differentierings-programmeringsvektoren ifølge krav 22, hvor derivatet omfatter en første nukleotidsekvens der koder for grupperne 1 til 40 af den tilsvarende vild-type EBNA-1 og en anden nukleotidsekvens der koder for grupperne 328 til 641 af den tilsvarende vild-type EBNA-1.
24. Differentierings-programmeringsvektoren ifølge et hvilket som helst af kravene 21 til 23, hvor differentierings-programmeringsfaktorerne vælges fra gruppen bestående af Sox-2, Sox-7, Sox-17, Oct-4, Nanog, Lin-28, c-Myc, Klf4, Esrrb, EBF1, C/EBPa, C/ΕΒΡβ, Ngn3, Pdx og Mafa.
25. Differentierings-programmeringsvektoren ifølge et hvilket som helst af kravene 21 til 23, yderligere defineret som en reprogrammeringsvektor omfattende et Sox-familiemedlem og et Oct-familiemedlem.
26. Differentierings-programmeringsvektoren ifølge krav 25, hvor differentieringsprogrammeringsfaktorerne yderligere omfatter én eller flere valgt fra gruppen bestående af Nanog, Lin-28, Klf4, og c-Myc.
27. Differentierings-programmeringsvektoren ifølge krav 21, hvor differentieringsprogrammeringsvektoren mangler evnen til at blive integreret i et værtscellegenom.
28. Differentierings-programmeringsvektoren ifølge et hvilket som helst af kravene 21 til 27, hvor replikationskilden er en replikationskilde af et lymfotropisk herpes virus, et adenovirus, SV40, et bovint papillomavirus, eller en gær.
29. Differentierings-programmeringsvektoren ifølge krav 28, hvor replikationskilden er en replikationskilde af et lymfotropisk herpes virus og svarer til oriP af EBV.
30. Differentierings-programmeringsvektoren ifølge krav 28, hvor det lymfotropiske herpes virus er Epstein Barr virus (EBV), Kaposi's sarcom herpes virus (KSHV), Herpes virus saimiri (HS), eller Marek's sygdomsvirus (MDV).
31. Differentierings-programmeringsvektoren ifølge et hvilket som helst af kravene 21 til 30, hvor den trans-virkende faktor (a) svarer til EBNA-1 af EBV, (b) er et derivat af et vild-type protein svarende til EBNA-1 af EBV, hvilket derivat har en nedsat evne til at aktivere transskription fra en integreret template sammenlignet med vild-type EBNA-1, (c) er et derivat af et vild-type protein svarende til EBNA-1 af EBV, hvilket derivat aktiverer transskription ved niveauer på mindst 5% af det tilsvarende vild-type protein fra en ekstra-kromosomal template efter derivatet binder replikationskilden, eller (d) er et derivat af vild-typeprotein svarende til EBNA-1 af EBV, hvilket derivat har en deletion af grupper svarende til grupperne 65 til 89 af EBNA-1, og/eller en deletion af grupper svarende til grupperne 90 til 328 af EBNA-1.
32. Differentierings-programmeringsvektoren ifølge krav 31(b), 31(c), eller 22, hvor derivatet mangler sekvenser der er til stede i vild-type-EBNA-l-proteinet der aktiverer transskription fra en integreret template.
33. Differentierings-programmeringsvektoren ifølge krav 31(b), 31(c), eller 22, hvor derivatet har en deletion af grupper svarende til grupperne 65 til 89 af EBNA-1 og/eller en deletion af grupper svarende til grupperne 90 til 328 af EBNA-1.
34. Differentierings-programmeringsvektoren ifølge krav 31(b), 31(c), eller 22, hvor derivatet koder for et protein med mindst 80% aminosyresekvensidentitet til grupperne 1 til 40 og grupperne 328 til 641 af EBNA-1.
35. Differentierings-programmeringsvektoren ifølge et hvilket som helst af kravene 21 til 34, hvor ekspressionskassetter er operativt bundet til et transskriptionelt regulatorelement.
36. Differentierings-programmeringsvektoren ifølge et hvilket som helst af kravene 21 til 35, hvor flere gener effektivt udtrykkes under anvendelse af en enkelt promotor/forstærker til at transskribere en enkelt besked.
37. Differentierings-programmeringsvektoren ifølge krav 36, hvor flere åbne læserammer er transskriberet sammen, hver er separeret af en IRES, danner polycistroniske beskeder.
DK09759392.5T 2008-06-04 2009-06-04 Fremgangsmåder til fremstillingen af ips-celler under anvendelse af ikke-virale metoder DK2297307T3 (da)

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US5885808P 2008-06-04 2008-06-04
US16058409P 2009-03-16 2009-03-16
PCT/US2009/046209 WO2009149233A1 (en) 2008-06-04 2009-06-04 Methods for the production of ips cells using non-viral approach

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