A new biological strategy for high productivity of recombinant proteins inanimal cells by the use of hypoxia-response enhancer

Oxygen supply is one of the major problems in the production of useful prot eins by cultured animal cells and therefore it is of importance to devise a system by which a-high-productivity of human therapeutic recombinant prote ins can be maintained or enhanced under law oxygen concentrations. A number of hypoxia-inducible genes have been found in animal cells and the inducti on in most: cases is due to hypoxic activation of the gene transcription. A consensus sequence (HRE = hypoxia-response enhancer) responsible for the h ypoxic activation exists in these genes and the binding of a protein,which is widely distributed in animal cells, to this sequence responding to hypox ia activates the promoter activity. The promoter of lactate dehydrogenase A gene is: active in Chinese hamster ovary (CHO) cells and the vicinal HRE s timulates the promoter activity efficiently in hypoxia; We have prepared a number of permanent CHO cell lines producing recombinant human erythropoiet in (Epo) under control of this promoter/HRE. Epo production was highly hypo xia-inducible when the wild-type of HRE was used but uninducible when the m utant HRE was used. There was little difference in the in vitro and in vivo activities,:and glycosylation between Epo produced by the cells cultured i n 21% and 2% oxygen. Furthermore, forced expression of hypoxia-inducible fa ctor-1 alpha (HIF-1 alpha) enhanced Epo production in all oxygen concentrat ions. These results indicate that a biological strategy based on the hypoxi c induction of gene transcription provides a novel:System:wh ich guarantees a high productivity even uner low oxygen concentrations. (C) 2000 John Wil ey & Sons, Inc.