DK2576580T3 - Sænkning af lactatniveau og øgning af polypeptidproduktion ved nedregulering af ekspressionen af lactatdehydrogenase og pyruvatdehydrogenasekinase - Google Patents

Sænkning af lactatniveau og øgning af polypeptidproduktion ved nedregulering af ekspressionen af lactatdehydrogenase og pyruvatdehydrogenasekinase Download PDF

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DK2576580T3
DK2576580T3 DK11787435.4T DK11787435T DK2576580T3 DK 2576580 T3 DK2576580 T3 DK 2576580T3 DK 11787435 T DK11787435 T DK 11787435T DK 2576580 T3 DK2576580 T3 DK 2576580T3
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Meixia Zhou
Bradley Richard Snedecor
Chi Kin Domingos Ng
Amy Shen
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Hoffmann La Roche
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Claims (43)

1. Fremgangsmåde til sænkning af lactatproduktion i dyrkede celler, hvilken fremgangsmåde omfatter dyrkning af cellerne omfattende en første heterolog nukleinsyresekvens der koder for et lille interfererende RNA (siRNA) der er specifik for en lactatdehydrogenase (LDH) og en anden heterolog nukleinsyresekvens der koder for en siRNA der er specifik for en pyruvat-dehydrogenase-kinase (PDHK), hvor den første heterologe nukleinsyresekvens er operabelt koblet til en første promotor, og hvor den anden heterologe nukleinsyresekvens er operabelt koblet til en anden promotor, hvor siRNA'erne inaktiverer eller nedregulerer gen-transskriptionen af LDH'en og PDHK'en.
2. Fremgangsmåden ifølge krav 1, hvor LDH'en er LDHa.
3. Fremgangsmåden ifølge krav 1, hvor de dyrkede celler yderligere omfatter en tredje heterolog nukleinsyresekvens der koder for en siRNA der er specifik for en anden PDHK og hvor den tredje heterologe nukleinsyresekvens er operabelt koblet til en tredje promotor.
4. Fremgangsmåden ifølge krav 3, hvor de dyrkede celler yderligere omfatter en fjerde heterolog nukleinsyresekvens der koder for en siRNA der er specifik for en tredje PDHK og hvor den fjerde heterologe nukleinsyresekvens er operabelt koblet til en fjerde promotor.
5. Fremgangsmåden ifølge krav 4, hvor de dyrkede celler yderligere omfatter en femte heterolog nukleinsyresekvens der koder for en siRNA der er specifik for en femte PDHK og hvor den femte heterologe nukleinsyresekvens er operabelt koblet til en femte promotor.
6. Fremgangsmåden ifølge et hvilket som helst af kravene 1, 3, 4, og 5, hvor PDHK'en er valgt fra gruppen bestående af PDHK1, PDHK2, PDHK3, og PDHK4.
7. Fremgangsmåden ifølge et hvilket som helst af kravene 1, 3, og 4, hvor PDHK'en er valgt fra gruppen bestående af PDHK1, PDHK2, og PDHK3.
8. Fremgangsmåden ifølge krav 1 eller 3, hvor PDHK'en er valgt fra gruppen bestående af PDHK1 og PDHK2.
9. Fremgangsmåden ifølge krav 1 eller 3, hvor PDHK'en er valgt fra gruppen bestående af PDHK1 og PDHK3.
10. Fremgangsmåden ifølge krav 1 eller 3, hvor PDHK'en er valgt fra gruppen bestående af PDHK2 og PDHK3.
11. Fremgangsmåden ifølge krav 4, hvor LDH'en er LDHa, hvor den første PDHK er PDHK1, hvor den anden PDHK er PDHK2, og hvor den tredje PDHK er PDHK3.
12. Fremgangsmåden ifølge et hvilket som helst af kravene 1, 3, 4, og 5, hvor de dyrkede celler producerer et heterologt polypeptid.
13. Fremgangsmåden ifølge krav 12, hvor det heterologe polypeptid er er et antistof.
14. Fremgangsmåden ifølge krav 4, hvor en gennemsnits-lactatproduktions-hastighed af de dyrkede celler er mindre end negativ 0,02 mg/106 celler/dag.
15. Fremgangsmåden ifølge krav 4, hvor de dyrkede celler har en Specifik Produktivitet på mindst 75% større end de dyrkede celler uden den heterologe nukleinsyresekvens der koder for en siRNA der er specifik for PDHK'erne og LDH'en.
16. Fremgangsmåden ifølge krav 4, hvor de dyrkede celler har en osmolalitet på mindre end 300 mOsm.
17. Fremgangsmåden ifølge krav 4, hvor de dyrkede celler har en polypeptid-produktivitet på mindst 68% større end de dyrkede celler uden den heterologe nukleinsyresekvens omfattende kodning for en siRNA der er specifik for PDHK'erne og LDH'en.
18. Fremgangsmåden ifølge krav 12 eller 13, hvor de dyrkede celler er pattedyrceller.
19. Fremgangsmåde of inaktivering eller nedregulering af lactatdehydrogenase (LDH) og pyruvatdehydrogenasekinase (PDHK) transskription i en dyrket celle omfattende at: indføre i cellen en vektor omfattende en første heterolog nukleinsyresekvens der koder for et lille interfererende RNA (siRNA) der er specifik for LDH'en og en anden heterolog nukleinsyresekvens der koder for en siRNA der er specifik for PDHK'en, hvor den første heterologe nukleinsyresekvens er operabelt koblet til en første promotor, og hvor den anden heterologe nukleinsyresekvens er operabelt koblet til en anden promotor, hvor siRNA'erne udtrykkes, derved inaktiverer eller nedregulerer gen-transskriptionen af LDH'en og PDHK'en.
20. Fremgangsmåde til fremstilling af en celle der udviser nedsat lactatproduktion i kultur, omfattende indføring i cellen en vektor omfattende en første heterolog nukleinsyresekvens der koder for et lille interfererende RNA (siRNA) der er specifik for LDH'en og en anden heterolog nukleinsyresekvens der koder for en siRNA der er specifik for PDHK'en, hvor den første heterologe nukleinsyresekvens er operabelt koblet til en første promotor, og hvor den anden heterologe nukleinsyresekvens er operabelt koblet til en anden promotor, hvor siRNA'erne udtrykkes, derved inaktiverer eller nedregulerer gen-transskription af LDH'en og PDHK'en.
21. Celler i kultur omfattende en første heterolog nukleinsyresekvens der koder for et første lille interfererende RNA (siRNA) der er specifik for en lactatdehydrogenase (LDH) og en anden heterolog nukleinsyresekvens der koder for en anden siRNA der er specifik for en pyruvat-dehydrogenasekinase (PDHK), hvor den første heterologe nukleinsyresekvens er operabelt koblet til en første promotor, og hvor den anden heterologe nukleinsyresekvens er operabelt koblet til en anden promotor, hvor siRNA'erne inaktiverer eller nedregulerer gen-transskriptionen af LDH'en og PDHK'en.
22. Cellerne ifølge krav 21, hvor cellerne yderligere omfatter en tredje heterolog nukleinsyresekvens der koder for en siRNA der er specifik for en anden PDHK og hvor den tredje heterologe nukleinsyresekvens er operabelt koblet til en tredje promotor.
23. Cellerne ifølge krav 22, hvor cellerne yderligere omfatter en fjerde heterolog nukleinsyresekvens der koder for en siRNA der er specifik for en tredje PDHK og hvor den fjerde heterologe nukleinsyresekvens er operabelt koblet til en fjerde promotor.
24. Cellerne ifølge krav 23, hvor cellerne yderligere omfatter en femte heterolog nukleinsyresekvens der koder for en siRNA der er specifik for en femte PDHK og hvor den femte heterologe nukleinsyresekvens er operabelt koblet til a femte promotor.
25. Cellerne ifølge et hvilket som helst af kravene 21, 22, 23, og 24, hvor PDHK'en er valgt fra gruppen bestående af PDHK1, PDHK2, PDHK3, og PDHK4.
26. Cellerne ifølge et hvilket som helst af kravene 21, 22 og 23, hvor PDHK'en er valgt fra gruppen bestående af PDHK1, PDHK2, og PDHK3.
27. Cellerne ifølge krav 21 eller 22, hvor PDHK'en er valgt fra gruppen bestående af PDHK1 og PDHK2.
28. Cellerne ifølge krav 21 eller 22, hvor PDHK'en er valgt fra gruppen bestående af PDHK1 og PDHK3.
29. Cellerne ifølge krav 21 eller 22, hvor PDHK'en er valgt fra gruppen bestående af PDHK2 og PDHK3.
30. Cellerne ifølge krav 23, hvor the LDH'en er LDHa, hvor den første PDHK er PDHK1, hvor den anden PDHK er PDHK2, og den hvor den tredje PDHK er PDHK3.
31. Cellerne ifølge krav 21, hvor cellerne producerer et heterologt polypeptid.
32. Cellerne ifølge krav 31, hvor det heterologe polypeptid er er et antistof.
33. Cellerne ifølge krav 23, hvor cellerne haren gennemsnits-lactatproduktions-hastighed på mindre end negative 0,02 mg/106 celler/dag.
34. Cellerne ifølge krav 23, hvor cellerne har en Specifik Produktivitet på mindst 75% større end cellerne uden den heterologe nukleinsyresekvens omfattende LDH og PDHK'erne.
35. Cellerne ifølge krav 23, hvor cellerne har en osmolalitet på mindre end 300 mOsm.
36. Cellerne ifølge krav 23, hvor cellerne haren polypeptidproduktivitet på mindst 68% højere end cellerne uden den heterologe nukleinsyresekvens omfattende LDH'en og PDHK'erne.
37. Cellerne ifølge krav 31 eller 32, hvor cellerne er pattedyrceller.
38. Vektor omfattende en første heterolog nukleinsyresekvens der koder for et lille interfererende RNA (siRNA) der er specifik for en lactatdehydrogenase (LDH) og en anden heterolog nukleinsyresekvens der koder for en siRNA der er specifik for en pyruvat-dehydrogenasekinase (PDHK), hvor den første heterologe nukleinsyresekvens er operabelt koblet til en første promotor, og hvor den anden heterologe nukleinsyresekvens er operabelt koblet til en anden promotor.
39. Fremgangsmåde til sænkning af lactatproduktion i dyrkede celler, hvilken fremgangsmåde omfatter dyrkning af celler udtrykkende a) et lille interfererende RNA (siRNA) der er specifikt for en lactatdehydrogenase (LDH) og b) en siRNA der er specifik for en pyruvat-dehydrogenasekinase (PDHK), hvor siRNA'erne udtrykkes, derved inaktiverer eller nedregulerer gen-transskriptionen af LDH'en og PDHK'en.
40. Fremgangsmåden ifølge krav 39, hvor de dyrkede celler yderligere udtrykker en siRNA der er specifik for en anden PDHK.
41. Fremgangsmåden ifølge krav 40, hvor de dyrkede celler yderligere udtrykker en siRNA der er specifik for en tredje PDHK.
42. Fremgangsmåden ifølge krav 40, hvor de dyrkede celler yderligere udtrykker en siRNA der er specifik for en fjerde PDHK.
43. Fremgangsmåden ifølge krav 41, hvor de dyrkede celler har en polypeptidproduktivitet på mindst 68% større end de dyrkede celler uden siRNA'erne der er specifikke for LDH'en og PDHK'erne.
DK11787435.4T 2010-05-28 2011-05-26 Sænkning af lactatniveau og øgning af polypeptidproduktion ved nedregulering af ekspressionen af lactatdehydrogenase og pyruvatdehydrogenasekinase DK2576580T3 (da)

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US34972710P 2010-05-28 2010-05-28
PCT/US2011/038191 WO2011150241A2 (en) 2010-05-28 2011-05-26 Decreasing lactate level and increasing polypeptide production by downregulating the expression of lactate dehydrogenase and pyruvate dehydrogenase kinase

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