Decreasing lactate level and increasing polypeptide production by down regulating the expression of lactate dehydrogenase and pyruvate dehydrogenase kinase
AssigneeGenentech, Inc.
InventorsMeixia Zhou, Bradley Richard Snedecor, Chi Kin Domingos Ng, Amy Shen
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Abstract
The present invention provides methods and compositions for reducing lactate production and increasing polypeptide production in cultured cells. In one aspect, the invention provides a method comprising culturing cells expressing a) a small interfering RNA (siRNA) specific for a lactate dehydrogenase (LDH) and b) an siRNA specific for a pyruvate dehydrogenase kinase (PDHK). In another aspect, the invention provides cultured cells or vectors comprising an siRNA specific for a LDH and an siRNA specific for a PDHK.
Claims24
A vector comprising a first heterologous nucleic acid sequence encoding a small interfering RNA (siRNA) specific for a lactate dehydrogenase (LDH) and a second heterologous nucleic acid sequence encoding an siRNA specific for a pyruvate dehydrogenase kinase (PDHK), wherein the first heterologous nucleic acid sequence is operably linked to a first promoter, and wherein the second heterologous nucleic acid sequence is operably linked to a second promoter, wherein cultured cells comprising the vector have a polypeptide productivity of at least about 68% higher than cultured cells without the vector.
A vector comprising a first heterologous nucleic acid sequence encoding a small interfering RNA (siRNA) specific for a lactate dehydrogenase a (LDHa) wherein the target sequence for LDHa is CTCGATTCCGTTATCTGAT (SEQ ID NO:1), a second heterologous nucleic acid sequence encoding an siRNA specific for a pyruvate dehydrogenase kinase 1 (PDHK1) wherein the target sequence for PDHK1 is GCAGTTCCTGGACTTCGGA (SEQ ID NO:2), a second heterologous nucleic acid sequence encoding an siRNA specific for a pyruvate dehydrogenase kinase 2 (PDHK2) wherein the target sequence for PDHK2 is CATTCAGTACTTCTTGGAC (SEQ ID NO:3), and a third heterologous nucleic acid sequence encoding an siRNA specific for a pyruvate dehydrogenase kinase 3 (PDHK3) wherein the target sequence for PDHK3 is TGTAGCTGATGTCGTGAAA (SEQ ID NO:4).
A vector comprising a first heterologous nucleic acid sequence encoding a small interfering RNA (siRNA) specific for a lactate dehydrogenase a (LDHa), a second heterologous nucleic acid sequence encoding an siRNA specific for a pyruvate dehydrogenase kinase 1 (PDHK1), a second heterologous nucleic acid sequence encoding an siRNA specific for a pyruvate dehydrogenase kinase 2 (PDHK2), a third heterologous nucleic acid sequence encoding an siRNA specific for a pyruvate dehydrogenase kinase 3 (PDHK3), and a third heterologous nucleic acid sequence encoding an siRNA specific for a pyruvate dehydrogenase kinase 4 (PDHK4), wherein cultured cells comprising the vector have a polypeptide productivity of at least about 68% higher than cultured cells without the vector.
Dependencies are inferred from the claim text.