Preparation of erythropoietin by endogenous gene activation
AssigneeRoche Diagnostics GmbH
InventorsAnne Stern, Michael Brandt, Konrad Honold, Johannes Auer, Hans Koll
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Abstract
The invention relates to human cells which are capable, on the basis of an activation of the endogenous human EPO gene, of producing EPO in a sufficient amount and purity to make possible a cost-effective production of human EPO as a pharmaceutical preparation. The invention furthermore relates to a method for the preparation of such human EPO-producing cells, DNA constructs for the activation of the endogenous EPO in human cells, and a method for the large technical production of EPO in human cells.
Claims58
Human cell, characterized in that
- it contains a copy of an endogenous EPO gene in operable linkage with a heterologous promoter active in the human cell and is capable of the production of at least 200 ng EPO/106 cells/24 h.
Human cell according to any one of claims 1 to 6, characterized in that
- it is selected from an HT 1080 cell, an HeLa S3 cell, a Namalwa cell or a cell derived therefrom.
DNA construct for activating an endogenous EPO gene in a human cell, comprising:
- (i) two flanking DNA sequences which are homologous to regions of the human gene locus selected from 5′-untranslated sequences, exon 1 and intron 1, in order to permit a homologous recombination, a modified sequence being present in the range of exon 1, coding for the amino acids:
- Met-X1-X2-X3
- wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X2-X3 is not the sequence Gly-Val-His,
- (ii) a positive selection marker gene,
- (iii) a heterologous expression control sequence which is active in a human cell, and
- (iv) an amplification gene, if desired.
DNA construct for activating an endogenous EPO gene in a human cell, comprising:
- (i) two flanking DNA sequences which are homologous with regions of the human EPO gene locus selected from 5′-untranslated sequences, exon 1 and intron 1, in order to permit a homologous recombination,
- (ii) a positive selection marker gene,
- (iii) a heterologous expression control sequence which is active in a human cell, the distance between the heterologous expression control sequence and the translation start of the EPO gene being not greater than 1100 bp, and
- (iv) an amplification gene if desired.
Plasmid p189 (DSM 11661) or a plasmid derived therefrom.
29 Method according to either one of claims 27 or 28, characterized in that
- the cells are cultured in suspension.
Method according to any one of claims 27 to 42, characterized in that
- a human EPO is obtained with α-2,3- and α-2,6-linked sialic acid residues.
Isolated DNA which codes for a human EPO with a sequence modified in the region of the first 4 amino acids of the signal peptide, which is selected from:
- Met-X1-X2-X3
- wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, on the condition that X1-X2-X3 is not the sequence Gly-Val-His.
Dependencies are inferred from the claim text.