Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-06-09
2003-05-13
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S358000
Reexamination Certificate
active
06562588
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to sialidase activity, in particular isolated sialidase enzyme and recombinant cell lines having modified sialidase activity.
BACKGROUND OF THE INVENTION
Sialidases are a family of glycohydrolytic enzymes which cleave sialic acid residues from the oligosaccharide components of glycoproteins and glycolipids. Viral and bacterial enzymes have been studied, for example the influenza sialidases in particular (Air, G. M. and Laver, W. G. (1989)
Proteins: Struct. Func. Genet.,
6:341), but mammalian sialidases have not been well characterized. For the most part, studies of mammalian sialidases have been confined to investigation of substrate specificities and kinetic analysis using partially purified preparations, although a sialidase from rat liver and muscle has been purified to homogeneity (Miyagi, T. and Tsuiki, S. (1985)
J. Biol. Chem.,
260:6710). Sialidases have been identified in a number of cellular organelles: the plasma membrane (Schengrund, C., Rosenberg, A., and Repman, M. A. (1976)
J. Cell. Biol.,
70:555), the lysosomes and the cytosol (Tulsiani, D. R. P., and Carubelli, R., (1970)
J. Biol. Chem.,
245:1821).
Glycoproteins are often produced by expression of encoding genes in recombinant host cells in vitro, the cells having the normal enzyme components of cellular glycosylation machinery. Sialic acid in the oligosaccharide component of a glycoprotein is involved in mediation of clearance from the serum and affects the physical, chemical and immunogenic properties of the protein molecule. It is therefore important to maintain the sialic acid content of glycoproteins, particularly of those proteins intended for use as therapeutics.
SUMMARY OF THE INVENTION
The present invention is based on modification in a recombinant cell line of the constitutive expression of genes which encode enzymes which are involved in the destruction or production of the oligosaccharide portions of glycoproteins. In particular, the modification to the recombinant cell line may be such as to ensure that the gene or genes of interest are not functionally expressed. Of particular interest is a sialidase gene within a recombinant cell line, especially a gene encoding a cytosolic sialidase. Example cell lines are those derived from Chinese hamster ovaries and human embryonic kidneys.
In the recombinant cell line functional gene expression may be disrupted by mutation, addition or deletion of one or more nucleotides. Such mutation, addition or deletion may be by any of the methods known to the person skilled in the art, for instance homologous recombination between the genomic gene and a differing but largely homologous nucleic acid sequence introduced into the cells. The gene may be deleted altogether.
The gene may be not functionally expressed by virtue of disruption of the gene function by regulation of its transcription or translation, eg using antisense RNA.
The present invention also provides a substantially homogeneous sialidase which can be obtained from cell culture fluid of a Chinese hamster ovary cell line. Characteristics of such a sialidase are described and discussed infra.
It also provides an oligonucleotide probe which is useful in obtaining a sialidase-encoding gene and a nucleic acid sequence obtained by a process comprising hybridizing the probe with nucleic acid in a mammalian DNA library to form hybrids which can be isolated. The nucleic acid may be used for expression of sialidase. It may be modified in all manner of ways, eg by mutation, addition or deletion of one or more nucleotides, amplification, cleavage and tailoring.
In a preferred embodiment of the present invention a sialidase gene of a cell line is disrupted so that it is not functionally expressed, the level of functional sialidase produced by the cells being such that sialic acid residues in the carbohydrate side-chains of glycoprotein produced by the cells are not cleaved, or are not cleaved to an extent which affects the function of the glycoprotein. Such cells are useful as host cells for the expression of recombinant glycoproteins from nucleic acid transformed into the cells under appropriate conditions. Glycoproteins produced by expression of encoding nucleic acid introduced into these cells should have intact, functional carbohydrate side chains.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Recombinant Cell Line
This expression refers to cells established in ex vivo culture and which have some genetic modification from the original parent cells from which they are derived. Such genetic modification may be the result of introduction of a heterologous gene for expression of the gene product, or it may be by the introduction of a gene, possibly with promoter elements, for production within the cells of antisense RNA to regulate expression of another gene. Equally, the genetic modification may be the result of mutation, addition or deletion of one or more nucleotides of a gene or even deletion of a gene altogether, by any mechanism. Cells of a recombinant cell line used in the production of a desired protein product have the means for glycosylating proteins by addition of oligosaccharide side chains. Such cells also have the capability to remove and/or modify enzymatically part or all of the oligosaccharide side chains of glycoproteins.
Functional Expression, and Grammatically Related Terms
Functional expression of a gene refers to production of the protein product encoded by the gene in a form or to the extent required for the product to perform its normal function within the cell environment. Thus, a gene encoding an enzyme involved in protein glycosylation, or deglycosylation, is functionally expressed when enough of the enzyme is produced in a working form to glycosylate, or deglycosylate, at a normal level protein produced in the cell. Functional expression of a gene may be disrupted by modification of the nucleotide sequence of the gene so that protein product of the gene is defective in its function, or by deletion or modification of part or all of promoter sequences associated with the gene and involved in transcription of the gene, or by deletion of the gene itself from the genome of the cell, or by interference with translation of mRNA transcribed from the gene eg interference by antisense RNA, or by any combination of any of these with each other or with any other means known to the person skilled in the art for disrupting gene function.
The terms “DNA sequence encoding”, “DNA encoding” and “nucleic acid encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide chain. The DNA sequence thus codes for the amino acid sequence.
The terms “replicable expression vector” and “expression vector” refer to a piece of DNA, usually double-stranded, which may have inserted into it a piece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its insert (foreign) DNA may be generated. In addition, the vector contains the necessary elements that permit translating the foreign DNA into a polypeptide. Many molecules of the polypeptide encoded by the foreign DNA can thus be rapidly synthesized.
The terms “transformed host cell” and “transformed” refer to the introduction of DNA into a cell. The cell is termed a “host cell”, and it may be a prokaryotic or a eukaryotic cell. Typical prokaryotic host cells include various strains of
E. coli.
Typical eukaryotic host cells are mammalian, such as Chinese hamster ovary cells or human embryonic kidney 293 cells. The introduced DNA is usually in the form of a vector containing an inserted piece of DNA. The introduced DNA is usually in the form of a vector containing an inserted piece of DNA. The introduced DN
Sliwkowski Mary B.
Warner Thomas G.
Genentech Inc.
Ketter James
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