Glycobiology vol. 9 no. 9 pp. 841–850, 1999
MINI REVIEW
Enhancing therapeutic glycoprotein production in Chinese hamster ovary cells by
metabolic engineering endogenous gene control with antisense DNA and gene targeting
Thomas G.Warner1
Received on December 9, 1998; revised on February 17, 1999; accepted on
March 1, 1999
1Correspondence
should be addressed to the author at: 541 Wellington Drive,
San Carlos, CA 94070; or E-mail at [email protected]
Recombinant glycoprotein therapeutics have proven to be invaluable pharmaceuticals for the treatment of chronic and
life-threatening diseases. Although these molecules are extraordinarly efficacious, many diseases have high dosage requirements of several hundred milligrams of protein for each
administration. Multiple doses at this level are often required
for treatment. One of the major challenges currently facing
the biotechnology industry is the development of large-scale,
cost-effective production and manufacturing processes of
these biologically synthesized molecules. Metabolic engineering of animal cell expression hosts promises to address this
challenge by substantially enhancing recombinant protein
quality, productivity, and biological activity. In this report, we
describe a novel approach to metabolic engineering in Chinese hamster ovary cells by control of endogenous gene expression. Analysis of the advantages and limitations of using
antisense DNA and gene targeting as a means of control are
discussed and several gene candidates for regulation with
these techniques are identified. Practical considerations for
using these technologies to reduce the levels of the CHO cell
sialidase (Warner et al., Glycobiology, 3, 455–463, 1993) as a
model gene system for regulation are also presented.
Key words: sialidase, recombinant protein/Chinese hamster
ovary cells/gene targeting/homologous recombination/
antisense/protease/apoptosis/caspase/metabolic engineering
Introduction
Commercially viable processes for the production of recombinant
protein therapeutics in animal cells have mandated the development of both highly efficient expression vector systems and host
cell lines with the metabolic capacity for robust protein biosynthesis. To address these needs, a number of laboratories over the past
10 years have made considerable progress devising highly
effective mammalian expression plasmids. Nucleotide sequences
of promoter/enhancer regions have been carefully scrutinized and
various construct designs have been meticulously evaluated in
order to maximize the levels of recombinant protein biosynthesis
and secretion (Bradley, 1990; Yarranton, 1990; Chisholm et al.,
1990; Lin et al., 1994; Lucas et al., 1996). In remarkable contrast,
similar efforts to optimize the expression host itself have been
sorely lacking. Presently, ideal candidate production cell lines, such
as Chinese hamster ovary cells (CHO cells) or baby hamster kidney
cells (BHK cells) are identified empirically, after the gene coding
 1999 Oxford University Press
for the recombinant product is introduced into the cell. Large
populations of transfected cells, grown under the selective pressure
of an amplifiable marker, e.g., methotrexate, are screened for
optimal protein secretor status and growth properties. Although the
methodology has proven successful in many cases, it is tedious and
not without pitfalls. Some production cell lines identified in this
way have subsequently been discovered, late in the development
process, to be lacking the metabolic capacity to give complete
glycosylation of the protein, to give aberrant glycosylation as a
result of genetic mutations, or to express high levels of hydrolytic
enzymes which degrade the recombinant product. In all of these
cases, the result is a heterogeneous mixture of recombinant protein
isoforms of compromised quality, reduced bioactivity, and significantly lower yield.
As a result of these experiences, it has become apparent that
there is pressing need to enhance, through genetic means, the
metabolic machinery of animal cells used as expression hosts.
This awareness has led to the development of a new field of study
called ”metabolic cellular engineering” (Bailey, 1991; Stephanopoulos, 1994; Jacobsen and Khosla, 1998). Although the
definitions of metabolic engineering vary widely (Cameron and
Tong, 1993), the overall notion is to genetically modify the
metabolic pathways of an organism in order to increase the
production of a particular metabolite. In our case the metabolite
of interest is recombinantly expressed protein. The addition of
genes to the host cell that will improve product, yield, quality, or
consistency of production are manipulations that are in keeping
with the metabolic engineering concept.
Chinese hamster ovary cells are an attractive mammalian
expression host that have been extensively employed for the
production of recombinant protein therapeutics. These cells offer
the advantages that they are easily genetically manipulated, can
be adapted for large-scale suspension culture, and most importantly, can give rise to proteins with glycans that are similar,
although not identical, to those found on human glycoproteins.
Even though CHO cells have many attractive properties for
recombinant protein production, improvements in the cellular
machinery have been made. For example, the rat α-2,6 sialyltransferase, a gene that is normally nonfunctional in CHO cells,
has been coexpressed along with the human recombinant tissue
plasminogen activator factor, tPA. The resulting tPA molecule
contains 2,6 linked sialic acid residues on its glycans. In this way,
the metabolic machinery of the cell has been augmented by the
addition of the rat sialyltransferase gene, and the quality of the
therapeutic product is improved because it is presumably more
like the naturally occurring human molecule which contains sialic
acid in these linkages (Minch et al., 1995). Recently, elegant
metabolic engineering of CHO cells has been achieved by Bailey
and co-workers who have introduced a single tricistronic
construct encoding a model recombinant protein, alkaline
phosphatase, and two additional genes, p21 and the differentiation factor CCAAT/enhancer-binding protein alpha that effec841
T.G.Warner
tively arrests cell proliferation of growing CHO cell cultures. The
resulting engineered cell line gave a 10- to 15-fold increase in
recombinant protein production compared with an isogenic
control cell line (Fussenegger et al., 1998 ). Overexpression of
other genes such as cycline E which stimulates CHO cell
proliferation in protein-free cultures (Renner et al., 1995) or the
overexpression of the proinsulin gene which minimizes the
insulin requirements of animal cell cultures (Groskreutz et al.,
1994) are additional examples of modifying metabolic processes
with the expectation of improving the recombinant product.
An alternate avenue for metabolic engineering initiated by our
laboratory, which does not employ gene overexpression, focuses
on the direct manipulation of the host cell genome or intercession
in transcriptional and translational processes utilizing contemporary molecular techniques such as gene targeting or antisense
DNA or ribozyme-antisense RNA. These approaches may be a
valuable means to: (1) control or delete deleterious endogenous
genes whose gene products may adversely affect the expressed
recombinant protein or (2) enhance expression of endogenous
protein(s) that may contribute to improved product quality or
enhance cell productivity or longevity.
In this review an analysis of the advantages, disadvantages,
and limitations of these approaches for altering the genetic
machinery of the host cell is considered in the context of
metabolic engineering Chinese hamster ovary cells with the
intent to devise high yield, high quality production processes for
recombinant proteins. Examples of our own experiences along
these lines are discussed and target genes that are potential
candidates for manipulation are also identified.
Gene candidates targeted for control in CHO cells
Loci within the CHO cell genome whose regulation or control
may give improved recombinant protein production are listed in
Table I. A detailed discussion of these targets and the rationale for
these specific selections are discussed in detail here.
Sialidase. This glycohydrolytic enzyme was discovered at high
levels in the culture fluid of CHO cell lines expressing recombinant
DNase (Sliwkowski et al., 1992). Under some culture conditions,
particularly in cultures without pH control, the enzyme cleaved
significant amounts of sialic acid from the protein, giving a
heterogeneous mixture of DNase isoforms. The sialidase has also
been found to degrade the glycans of several other recombinant
proteins in CHO cell cultures (Gramer et al., 1995). In these cases,
the action of the sialidase could not be controlled by modifying the
culture conditions and this manifested in lower yields of recombinant product with completely capped oligosaccharide side chains.
As might be expected, the molecule prepared with this process was
rapidly cleared from the plasma when evaluated in preclinical
studies. In order to achieve the desired therapeutic effectiveness,
augmentation of the dosage would be required, significantly
raising production levels and manufacturing costs.
Thus, considerable effort has been expended to devise genetic
means to control the activity of this enzyme with some success.
The sialidase has been purified to homogeneity, its cDNA
isolated, and its gene structure elucidated (Warner et al., 1993;
Ferrari et al., 1994, 1996). Recently, sialidase expressing
antisense cell lines have been made (Ferrari et al., 1998). The
sialidase serves as a good model for gene control for other
enzymes or proteins that have a deleterious effect on the quality
of recombinantly expressed protein.
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Table I. CHO cell gene candidates for control with antisense RNA or gene
disruption
Gene
Method of control
Objective
Soluble sialidase
Antisense/targeting
Reduce desialylation and
glycoform heterogeneity,
improve plasma residence
time of the therapeutic
Extracellular
proteases
Antisense/targeting
Reduce clipping, improve
bioactivity of recombinant
product
CMP-sialic acid
hydroxylase
Antisense/targeting
Eliminate a potential
immunogenetic component
of glycoproteins
Minute virus of mice
receptor
Antisense/targeting
Eliminate or reduce the
potential of virus infection
Endogenous CHO
proteins homologous
with recombinant
product
Targeting
Improve product purity and
quality, reduce rodent
contaminating protein
Rescue of silent
alleles/enhance
expression of
endogenous genes
Targeting
Improve glycosylation by
enhancing expression of
glycosyltransferase/resurrect
the 2,6 sialyltransferase
Genes involved in
cell death or growth
Antisense/targeting
Improve cell culture
productivity
Proteases. Although sialidase degradation of recombinant therapeutics is an important issue to be resolved, its impact is minor
compared with the devastating effects that CHO cell derived
proteases exert on recombinant proteins. In some cases, proteolytic cleavage results in profoundly reduced product yield, decreased product quality and, with some molecules, diminished
biological activity. Furthermore, proteolytic degradation affects
all proteins, including glycoproteins that do not contain sialic
acid, e.g., antibodies. Extensive proteolytic clipping of both,
recombinant interferon gamma and human nerve growth factor
expressed in CHO cells has been documented (Eng et al., 1997;
Goldman et al., 1997). Several proteases such as serine proteases,
elastase, collagenase, and plasminogen activator have been
suspected to be secreted by CHO cells (Tsuji and Miyama, 1992).
Thus, unlike sialidase which is a single gene locus, protease
regulation requires manipulation of multiple genetic loci, magnifying immensely the problem of gene control. And, unlike
sialidase which is primarily released into the culture fluid by cell
lysis, many proteases are actively secreted into the culture fluid
and therefore their presence is independent of cell viability or
time in culture. Whereas sialidase action on recombinant proteins
can be minimized by maintaining highly viable cultures at near
neutral pH, protease degradation cannot be similarly controlled.
CMP-sialic acid hydroxylase. Enzyme catalyzed oxidation of the
acetyl C-2 carbon of N-acetylneuraminic acid gives rise to the
N-glycoyl derivative of this carbohydrate. The enzyme is actively
expressed in nearly all animals with the notable exceptions of
humans and chickens (Dzulynska et al., 1966). Glycoconjugates
containing N-glycoylneuraminic acid, including glycoproteins
and glycolipids, are extremely antigenic in chickens and high
titers of antibodies specifically directed against the carbohydrate
have been raised in the serum of these animals and their eggs
(Fujii et al., 1982; Warner, unpublished observations). It has long
been suspected, although never proven conclusively, that a
similar immunogenic response may be elicited in humans if they
are exposed to glycoconjugates bearing this epitope (Higashi and
Metabolic engineering in CHO cells
Naiki, 1977). Usually, recombinant proteins produced in CHO
cells have low levels (∼2–3%) of NGNA as a component of their
sialic acids (Hokke et al., 1990). However, we have observed that
with some proteins such as human recombinant DNase, NGNA
levels can be substantial, as high as 14% of the sialic acid species
(Quan et al., 1997). Furthermore, some CHO cell lines have been
identified in which nearly 100% of the sialic acid species are
NGNA (Hubbard et al., 1994). The environmental factors
affecting the expression levels of the hydroxylase are unknown,
and the amounts of NGNA on cell surface glycoconjugates and
recombinant CHO cell-derived proteins varies greatly between
different clones. A modicum of success at reducing NGNA levels
by altering carbon dioxide partial pressures in CHO cell cultures
has been achieved. Unfortunately, the culture conditions also lead
to a reduction in the yield of recombinant protein (Kimura and
Miller, 1997). It thus seems cogent to devise a means to control
or eliminate the hydroxylase gene from the CHO cell genome and
thereby avoid the potentially catastrophic situation of developing
a therapeutic which initiates an immunogenic reaction in patients.
Minute virus of mice receptor. Minute virus of mice, MVM, is an
adventitious parvovirus harbored in rodent hosts (Siegl, 1976).
Because the virus is believed to be ubiquitous it can be expected to
be an insidious contaminant of the raw materials utilized as media
components in the cultures of CHO cells used for recombinant
protein production. Contamination of large scale cultures with the
virus can be devastating, resulting in the loss of costly reagents and
substantial delays in product manufacture. Several episodes of viral
contamination of production facilities using CHO cells have been
reported (Garnick, 1998). This has prompted the implementation of
physical barriers to prevent infection as well as the institution of
screening protocols to insure viral clearance during down stream
processing (Chang et al., 1997).
It has clearly been demonstrated that cultured rodent cells
express defined binding sites for MVM at the cell surface (Linser
et al., 1977). Interestingly, a specific derivative of sialic acid is
suspected to be a component of the viral receptor (Barbis et al.,
1992; Liu et al., 1997). It is therefore highly probable that the
receptor may be the product of a single gene locus which would
facilitate its disruption by gene targeting techniques. Elimination
of the receptor would provide an extremely valuable CHO cell
line that would be impervious to virus infection. Such genetic
manipulation would supplement physical protective barriers,
acting as a safety net, preventing viral contamination of all phases
of culture development including, the research laboratory, cell
banking, seed culture preparation, as well as manufacturing
facilities. At present, the physical barrier approach provides
protection only at the level of the production facilities
Endogenous CHO cell proteins homologous with the expressed
therapeutic protein. Although it has not yet been documented, the
host cell itself may secrete an endogenous protein that is a homolog
to the recombinant human protein therapeutic. Separation of the
rodent material from the human protein may be extremely difficult
since they both may have very similar chemical and physical
properties. Construction of a CHO cell line, by gene disruption of
the homologous protein would eliminate the contaminating rodent
material from the therapeutic protein.
Enhancing expression of endogenous glycosyltransferase genes
or gene rescue of silent alleles. It is well known that complete
extension of the oligosaccharides side chains on glycoproteins is
rarely observed and a heterogeneous mixture of partially
truncated structures at any particular glycosylation site on the
polypeptide chain of the protein is common. More complete
glycosylation of recombinantly expressed proteins may be
feasible if the activities of endogenous CHO cell glycosyl
transferases (particularly galactosyl and sialyl transferase) are
augmented above normal levels. This may be possible by
exchanging endogenous promoters with more potent viral
sequences using gene targeting. Similarly, repair of the silent 2,6
sialyltransferase gene in CHO cells by analogous gene rescue
strategies may result in increased sialylation of oligosaccharide
chains, and in this case, give the added advantage of creating a
more ‘human like’ recombinant protein. Directed gene targeting
has proven to be a valuable means to resurrect silent alleles or
correct gene defects by repairing altered nucleotide sequences in
coding regions or in untranslated promoter sequences (Adair et
al., 1989; Jasin et al. 1989; Narin et al., 1993).
Regulating genes involved in apoptosis and cell growth. Apoptosis is scripted cell death precipitated by a cascade of enzymatic
degradative events (Mundle et al., 1996; O’Connor, 1998). In
CHO cell cultures, the primary mechanism of cell death is by
means of apoptotic processes. A family of related cytosolic
cystine proteases, called caspases, play a key role in executing
apoptosis (Stennicke and Slavesen, 1998). Control of the activity
of these enzymes by the use of antisense or gene disruption may
provide a valuable mechanism to delay or protract cell death by
intercession in the apoptotic cascade. With CHO cell cultures,
inhibiting or reducing these proteolytic events may prolong cell
viability, allowing for extended culture times and greater
recombinant protein productivity. It has recently been demonstrated that inclusion of apopain, an inhibitor of caspase 3, in
CHO cell cultures significantly reduced apoptosis. These results
support the notion of augmenting cell productivity by delaying
the onset of cell death (Lee et al., 1998).
Antisense control of gene expression in CHO cells
Antisense (AS) control of gene expression in cultured cells has
proven to be a valuable tool for determining the functional role
and significance of many enzymes involved in a wide variety of
cell processes (Stout and Caskey, 1987; Helene and Toulme,
1990; Takayma and Inouye, 1990; Nellen and Sczakiel, 1996). As
yet, two reports of using antisense as a means to enhance
recombinant protein production have appeared (Dorner et al.,
1988; Ferrari et al., 1995, 1998). Some of the advantages as well
as the limitations of AS technology in the context of genetically
altering cell lines for improving recombinant protein productivity
are summarized in Table II. One of the major advantages of
antisense, over other methods of gene regulation such as gene
knockouts, is that complete elimination of gene expression is
rarely observed. This feature could be especially valuable if
inhibition of the targeted gene was lethal, but its partial reduction
could be tolerated by the cell and lead to an improvement in
recombinant protein quality or productivity. Alternatively, if
complete reduction of gene expression is desired, then AS
inhibition may not be the method of choice. An additional
advantage of AS is that the construction of vectors is straightforward, requiring only cDNA or segments of the gene of interest,
and the use of conventional eukaryotic expression plasmids.
However, one of the major concerns with AS is the long term
stability in the CHO cell genome. Different gene segments or
construct designs may influence longevity of cell retention and
expression. Fortunately, this issue can be resolved and genetic
stability studies of AS cell lines can be easily carried out.
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T.G.Warner
Table II. Comparative advantages of antisense and gene targeting
Method of gene control
Advantages
Disadvantages/limitations
Construct a host cell line with constitutively
expressed AS
Universal host for many products, easily
constructed requires only cDNA, may be useful
for growth-sensitive genes since complete
inhibition is not likely
Long-term stability is uncertain, stability after
product amplification unknown, complete
inhibition of expression is not likely
Add constitutively expressed AS to an existing
production cell line
AS is added after product amplification
eliminating chance of altering the AS gene
Not versatile, must be repeated with each product
Use AS under control of an inducer
May permit gene inhibition at later stages in
culture
Increases process costs, may require extensive
downstream clearance validation of the inducer
Ribozyme/AS constitutively expressed
May be more effective at inhibition than antisense
alone
Constructs more difficult, effectiveness must be
determined empirically
Gene disruption with replacement constructs using
positive and negative markers
Complete elimination of gene expression is
possible and permanent, has been used
successfully with CHO cells
Requires extensive characterization of genomic
material, more time-consuming than AS
Gene disruption with insertional constructs
In some cases, may be more efficient at targeting
than replacement constructs
Not recommended for production process cell
lines since gene rearrangement can resurrect active
gene
Gene disruption with promoterless marker
constructs
Has proven very effective with somatic cells
Genomic material must be characterized in detail,
vector construction is time consuming
Constructing a universal host cell line which constitutively
expresses antisense RNA. Two basic strategies for the construction of AS expressing cell lines can be devised. First, a universal
host cell line which expresses antisense RNA, designed to
specifically inhibit the gene of interest, can be developed through
conventional transfection and selection techniques. Once the AS
cell line is established the product expression vector can then be
introduced with a second transfectional event. A concern with this
approach is that the mutagenic process (e.g., adaptation to
increasing levels of methotrexate) utilized to augment product
productivity may adversely alter expression levels of the existing
antisense gene.
Recently, in an effort to prevent enzymatic cleavage of sialic
acid residues on recombinant proteins produced in CHO cells, we
constructed a universal host CHO cell line which constitutively
expressed a 474 base pair AS coding segment of the CHO cell
sialidase gene. The intercellular sialidase activity in the AS cell
line was reduced 60–70% of the parental host cell line (Ferrari et
al., 1998). In order to test if this level of enzyme reduction gave
an improvement in sialic acid content of a recombinant product,
the gene coding for human DNase, which served as a model
glycoprotein, was subsequently introduced into the AS cell line.
Sialidase released into the culture fluid from the DNase/antisense
cell line was considerably lower throughout the entire culture
period when compared with the control cell line expressing
similar levels of DNase but without AS, Figure 1. The amount of
sialic acid on the purified DNase molecule produced by the AS
cell line increased to about 3.9 ± 0.1 mol sialic acid/mole protein,
compared with about 3.0 ± 0.1 mol sialic acid/mol protein on
DNase produced in the control cell line without AS. Although a
∼1 mol sialic acid/mol protein increase may not seem to be
significant, small changes of this magnitude have been demonstrated to have a profound effect on plasma residence time for
some proteins (Thotakura et al., 1991; Cole et al., 1993).
Introducing antisense RNA into a product expressing cell line. A
second approach for constructing an AS cell line is to introduce
the AS expression vector into an existing product-expressing host
after productivity and cell growth parameters have been optimized. In contrast to the previous strategy, where the product vector
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Fig. 1. Sialidase activity in suspension cultures of DNase wild-type cells
(circles) and two cell lines coexpressing DNase and a 474 bp antisense
segment of the sialidase gene (squares, triangles) The levels of sialidase both
in the culture fluid and in cell homogenates were similar with the activity
reduced by about 60–70% in the antisense expressing lines (taken from
Ferrari, et al, 1998, with permission).
is added to the existing AS line, reversing the sequence eliminates
the possibility that the AS DNA will be altered during cell and
product optimization. An additional advantage of modifying an
established production cell line is that enhancement in product
productivity, recombinant protein quality, or cell growth rates that
result from the inhibitory action of the antisense RNA can be
readily evaluated by comparison with the product produced by
the unmodified production host cell, which serves as a well
characterized control. The major disadvantage of introducing the
AS into a product expressing host is that the overall process lacks
versatility and must be repeated with each new therapeutic.
Metabolic engineering in CHO cells
Table III. Gene targeting in somatic cells
Freq. of targeting
Targeting approach
Cell line
Locus targeted
(targeted/random integration)
Reference
Replacement construct—PNS
CHO
Dihydrofolate reductase
1/42–1/330
Zheng and Wilson, 1990
Replacement & insertional
constructs—gene rescue
CHO
Adenine phosphoribosyltransferase
1/1800 & 1/1600
Narin et al., 1993
Replacement construct—gene
rescue
CHO
Adenine phosphoribosyltransferase
1/4000
Adair et al., 1989
Replacement construct—PNS
3T3-L1
Insulin receptor
1/700
Accili and Taylor, 1991
Replacement construct—epitope
addition
Human T-cells
CD4
1/900
Jasin et al., 1990
Promoterless neomycin construct
3T3-L1
Polyoma middle T antigen
1/10,000
Hanson and Sedivy, 1995
Promoterless neomycin/
hygromycin constructs
TGR-1 fibroblast
c-myc gene
N.D.
Sedivy and Sharp, 1989
Promoterless neomycin construct
Murine myoblasts
Interferon gamma receptor
1/800–1/4000
Argones et al., 1994
Promoterless neomycin construct
Human fibroblasts
p21CIP1/WAF1
N.D.
Brown et al., 1997
N.D., Not determined.
Utilizing antisense with ribozymes for gene control in protein
expressing cell lines. Ribozyme is an RNA molecule possessing
catalytic enzyme activity that cleaves RNA in a highly sequencespecific manner, thus inactivating the RNA substrate. When the
ribozyme is flanked by antisense nucleotide sequence it can be
specifically directed to a unique complementary RNA target
(Zaia et al., 1990; Rossi, 1995; Sczakiel and Nedbal, 1995).
Although it is generally believed that ribozymes-antisense
constructs are more efficient at inactivating RNA than antisense
RNA alone, in controlled experiments a comparison of the
efficacy of both types of structures has given mixed results in
some systems (Cantor et al., 1996). Comparative kinetic modeling of ribozymes and antisense RNA has led to the hypothesis that
ribozymes are more effective when directed against abundant
RNA, whereas antisense RNA is predicted to be more effective
when targeted toward less abundant RNA or molecules with rapid
turnover (Woolf, 1995).
Clearly, ribozymes may be valuable for inhibiting gene
expression in some of the systems here but the relative efficiency
compared with antisense RNA will most likely have to be
determined empirically by testing both types of vector constructions with each gene in detail.
Antisense under the control of inducible promoters. Although
inducer-controlled AS gene expression has proven to be an
invaluable tool for elucidating functional roles of a number of
cellular processes (Ding et al., 1992; Higgins et al., 1993; Qu et
al., 1994; Weinstein et al., 1994; Smulson et al., 1995), this
approach may not be cost effective for a commercial process
setting unless there is a compelling advantage to do so. Many
small molecular weight inducers are potent antibiotics or
hormones such as tetracycline, dexamethasone, glucocoidsteroids, or they are heavy metal ions such as Cu or Zn (Ding et al.,
1992; Smulson et al., 1995; Hoshikawa et al., 1998). Their
presence in cultures of recombinant therapeutics at process scale
would require extensive downstream analysis and clearance
validations which dramatically increases process costs and
complexity. Thus, usage should be limited to those systems where
there is a clearly defined necessity for the application. An
excellent example of the use of the tetracycline inducible
promoter in a metabolically engineered CHO cell systems comes
from the work of Bailey and co-workers (discussed earlier) who
have employed inducer controlled expression of p21 and
CCAAT/enhancer binding protein alpha. The use of the inducer
allowed cultures to grow unabated until they reached maximal
densities, after which gene induction was initiated, resulting in the
arrest of further cell proliferation and ultimately greater protein
productivity (Fussenegger et al., 1998).
Control of gene expression in CHO cells with gene targeting
Gene targeting in pluripotent embryonic stem (ES) cells has
proven to be an exciting and extremely valuable tool for
addressing complex biological issues (Capecchi, 1989; Thomas
and Capecchi, 1990). Introducing specific gene mutations in
genomic DNA sequences in ES cells to produce animals with
altered phenotypic characteristics has been the most common
application of gene disruption technology. Functionality of many
novel genes has been revealed by this powerful approach
(Waldman, 1991; Zimmer, 1992).
In contrast to the enormous amount of experimentation that
has been carried out with ES cells and transgenic knock-out mice,
only recently have gene targeting efforts been directed toward
somatic cell systems (Table III). As yet, the application of this
technology for the purposes of enhancing recombinant protein
production in mammalian cells has not been reported. In this
context, the method holds great promise as a means to regulate the
expression of many of the genes which have been discussed in this
review. The advantages and limitations of gene targeting
compared to AS RNA inhibition are summarized in Table II.
One of the major advantages of gene targeting compared with
AS RNA inhibition is that, with the appropriate vectors, gene
disruption is permanent. Since the target DNA sequence is
replaced by vector sequence, resurrection of an active gene is not
possible. Also, unlike AS RNA which, in many cases gives only
a partial reduction of gene inhibition, disruption of both alleles by
targeting will give complete elimination of gene expression.
Unlike AS RNA, construction of targeting vectors is more
complex and it requires well characterized genomic DNA. Thus, the
overall process is comparatively more time consuming and
involved. An additional caveat to be considered, is that targeting
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T.G.Warner
frequencies for each loci are highly variable, and that some targeting
events may be below the detection limits of current technologies.
Although there has been limited success of targeting somatic cells,
only three reports describing targeting in highly mutagenized animal
cells (e.g., CHO cells) have appeared (Adair et al., 1989; Zheng and
Wilson, 1990; Narin et al., 1993). And, as will be discussed in a later
section of this review, within the CHO cell family targeting success
or frequencies may be cell line dependent.
We discuss in the following section considerations of using
gene targeting that specifically relate to its application with CHO
cells and recombinant protein production. We also describe some
of the practical considerations for targeting in a mutagenized cell
line, using the sialidase locus to illustrate the unique issues
involved in this cell system.
Targeting vectors and strategies
Considerations for the design of effective targeting constructs
have been reviewed in detail elsewhere (Waldman, 1992;
Zimmer, 1992). In brief, there are two basic configurations of
vectors for homologous recombination. These are insertional
constructs and replacement constructs.
Insertional constructs. The insertional construct contains a
segment of DNA sequence which is homologous to the target and
a marker gene outside the region of homology that allows for
selection of integrated events. The homologous region of the
targeting vector is linearized with a unique internal restriction
site. Homologous recombination results in the insertion of the
vector sequence into the homologous region of the target,
interrupting the gene and positioning the selectable marker within
the disrupted sequence. Since it is possible to regenerate a normal
gene from the disrupted gene by intrachromasomal recombination, this approach is not recommended for cell lines destined for
a production process as concern over the long term genetic
stability would limit its application.
Replacement constructs with positive and negative selectable, PNS,
elements. Replacement constructs are by far the most commonly
employed constructs. Vectors of this type contain two DNA
segments of gene homology that flank a positive selectable marker
gene (e.g., neomycin or neo). Homologous recombination occurs
when a double crossover event takes place and the positive
selectable marker gene replaces a segment of the target gene
sequence. The resulting disrupted gene is non functional and it
cannot be regenerated in an active form since genetic material has
been deleted. This is clearly the method of choice for the metabolic
engineering of process cell lines for recombinant protein production.
After transfection, genetically unstable cell lines without
integrated plasmid are removed from the population by growth in
the presence of drugs such as G418, a lethal substrate that is
inactivated in cells expressing the neomycin gene. When the vector
DNA is introduced into the CHO cell, the most predominant event
is random integration of the DNA into the host genome. In marked
contrast, gene targeting is an extremely rare event and it is
estimated (based on experiences with ES cells) to occur with a
frequency relative to random insertion of about 1:30 to more than
1:100,000, with the frequency for an average gene of about 1:1000
(Sedivy and Sharp, 1989). Even lower targeting frequencies have
been predicted for somatic cells (Hanson and Sedivy, 1995).
In order to specifically identify the relatively infrequent
targeted cells in the immense background of random integration
events, a second drug marker (e.g., hsv-thymidine kinase,
846
HSV-TK) is introduced into the targeting vector. But HSV-TK is
positioned outside the regions of homology that flank the
negative selectable marker gene. A true targeting or crossover
event will result not only in the loss of DNA sequence of the target
gene; also, segments of the vector sequence will be deleted,
notably, the HSV-thimidine kinase gene. Random integration of
the vector gives rise to cells with an active thimidine kinase gene,
creating a population of cells that will be sensitive to the presence
of the drugs FIAU or gancyclovir. The random integration events
in the population will thereby be eliminated in the presence of the
thymidine kinase substrates. The remaining population will be
enriched in, but not exclusively limited to, homologous recombination events. The HSV-TK gene, under some conditions, is
inactivated by means other than homologous recombination
(Mansour et al., 1988). Therefore the remaining population of
cells must be screened to identify true homologous recombination
events. The degree of enrichment of targeted events from the pool
of random integration events provided by the HSV-TK gene is a
critical element of this approach that is required to successfully
identify cells with homologous recombination. The more robust
the expression of HSV-TK in the transfected cells the more
effective the elimination of random integration events, because
higher concentration of the toxic derivatives of gangcylovir or
FIAU will be produced by elevated levels of the kinase.
Research from many laboratories has established that recombinant gene expression in CHO cells is dramatically influenced by the specific DNA sequences of promoter/enhancer
elements from various sources (Lin et al., 1994). It may therefore
be valuable to evaluate several targeting constructs with HSV-TK
under the control of different promoter/enhancer elements for
their effectiveness at reducing the population of random integration events using different CHO cell lines.
Replacement constructs with promoterless selectable marker
genes. This recent method of targeting has proved to be especially
valuable for disrupting loci in somatic cells because it provides
significantly greater enhancements than those achieved with the
conventional PNS approach (Table III). In this method, the
neomycin gene lacking a promoter or translation start site is fused
in frame within the gene of interest. Expression of the fused gene
would only occur if it correctly targeted the intended locus which
supplies the endogenous gene promoter and confers G418
resistance to the targeted clones. Using this approach with rat
fibroblasts, Sedivy and Sharp (1989) identified targeting at the
c-myc gene with a reasonably high frequency of about 1 out of
100 of G418 resistant cells. In other experiments, however, the
enhancement was substantially less, and only a single targeted
locus was identified out of 3000 G418 clones. Recently, others
groups, using a similar strategy have been successful at disrupting
the γ-interferon gene in mouse myoblasts with nearly 12–17% of
the G418 clones identified as positive targeting events.(Arbones
et al., 1994).
The major disadvantage of this approach is that is requires well
characterized genomic DNA, with detailed focus on the 5′
untranslated region of the targeted gene. It is essential that the
hybridizing DNA in the vector lack active or cryptic promoter
motifs. In addition, preparation of constructs is somewhat more
time consuming than conventional PNS plasmids.
Practical considerations for gene targeting in CHO
cells—experiences with the sialidase locus
Gene disruption experiments of the sialidase locus in Chinese
hamster ovary cells have recently been carried out using the
Metabolic engineering in CHO cells
replacement PNS construct design (Ferrari et al., unpublished
observations). These experiments serve as an excellent paradigm
for many of the other CHO cell genes that are candidates for
inhibition or disruption. Some of the general considerations that
were employed for targeting the sialidase locus are summarized
here.
The mutagenized nature of CHO cells and its implications for
gene targeting. CHO cell lines are highly mutagenized and have
profound chromosomal rearrangements. This is dramatically
demonstrated by comparing meta-phase chromosome analysis of
CHO cells with those of ES cells (Figure 2A,B). ES cells have a
normal complement of both maternal and paternal alleles which
are located at defined chromosome positions invariant in all cells
(Figure 2A). In CHO cells however, as a result of their highly
mutagenized nature, the ploidy at any specific locus is questionable (Figure 2B). It is important to address the issue of gene copy
number before extensive resources have been expended in the
development of a targeting construct, because with current
technology it is not practical to carry out gene disruption at more
than two alleles. In our effort to target the CHO sialidase gene, we
answered this question by carrying out in situ hybridization
studies (FISH analysis) of metaphase chromosomes employing a
sialidase probe staining nuclei from two different DHFR– CHO
cell lines. Signals from the probe were counted in about 200
nuclei from the CHO K1 (Urlaub and Chasin, 1980) and DG 44
(Urlaub et al., 1986) cell lines (Table IV).
Table IV. Sialidase copy number detected with in situ hybridization
Cell line
Number of nuclei giving signals with sialidase probe
Monoploid
Diploid
Triploid
Multiploid
CHO K1
77
113
10
0
DG 44
47
146
5
2
aMetaphase
chromosome spreads were prepared as described previously
(Peterson et al., 1979). In situ hybridization was carried out using a labeling
kit from VYSIS, Inc. (Downers Grove, IL). The cells were hybridized with a
nick-translated sialidase genomic probe modified with spectrum orange
dUTP according to the manufacturer’s instructions. After 48 h, the
hybridization signals were analyzed by visual inspection and the number of
orange spots in 200 nuclei was determined by counting.
With this analysis each signal is presumed to represent a single
gene copy of the sialidase locus. The results indicate that the
majority of the cells, about 56%, in both cell lines, are diploid.
Only a small percentage are multiploidy, and about 38% appear
to be hemizygous.
These results were especially encouraging not only because
they verify that sialidase gene duplication is minimal, but also
because they revealed that a high percentage of the cells contained
only a single gene copy. Thus, in these cell populations there is
a 38% chance that a single targeting event will give complete
elimination of sialidase expression.
Although it is not requisite for targeting, identifying the
chromosome location of the target allele may be insightful for
interpreting the results of a targeting experiment. This is an
important consideration because chromosome location and rates
of gene transcription may influence targeting frequencies (Waldman, 1991). If the target gene is positioned at different
chromosome locations in different cell lines, then an unsuccessful
gene targeting experiment in one cell line may not be predictive
of the outcome if carried out in another CHO cell line. Moreover,
Fig. 2. Comparison of chromosome content and rearrangements in mouse
embryonic stem cells (A) and the highly mutagenized CHO cells line DG 44
(B). Chromosomes were identified based on the Giemsa banding pattern.
each of the individual alleles themselves may have different
targeting frequencies if they are in different chromosome
locations.
In order to investigate the chromosome location and the
distribution of the CHO cell sialidase gene, we carried out in situ
hybridization analysis of metaphase chromosome spreads in
CHO K1 and DG 44 cell lines using a sialidase gene probe (Figure
3). In CHO K 1 cells, one allele of the sialidase gene was localized
on the terminus of chromosome 2. The second allele, however,
was localized on a marker chromosome M1, which appears as a
fragmented derivative of chromosome 2. In contrast, in DG 44
cells, no intact chromosome 2 was identified and one allele of the
sialidase gene was found on marker chromosome M 1. The other
allele was located on marker chromosome M16 q+ which appears
as a fusion of M 1 with chromosome 5.
These results dramatically demonstrate the complexity of
targeting in mutagenized cells. Model targeting systems using
mouse fibroblast cell lines containing a defective thymidine
kinase gene as an artificial targeting locus have shown that
targeting frequencies varied dramatically between closely related
cell lines. Only 1 out of 10 cell lines containing the defective
HSV-TK gene was susceptible to gene targeting. This has led to
the speculation that the site of integration of the target gene
greatly influences the targeting rate (Lin et al., 1985). Thus, the
847
T.G.Warner
Fig. 3. Chromosome location of the sialidase gene in CHO-K1 and DG 44
cells (noted by the arrow) determined by FISH analysis using a sialidase
genomic probe. No chromosome 2 was detected in DG 44 cells. The marker
chromosome M16q+ appears to be fusion of marker chromosome M1 and
chromosome 5.
DG 44 and CHO non K1 cell lines, and other similarly
CHO-derived cell lines, should be considered as distinct cell
types, each with their own unique genotype and, correspondingly,
distinct recombination frequencies for identical allelic targets.
Significant differences in targeting frequencies of the sialidase
locus can be expected not only between related CHO cell lines,
but also at each allele within a specific cell line genome.
Targeting vector selection. A replacement vector construct with
positive and negative selectable makers that is especially
attractive for the purposes here, where multiple genes of a single
host cell line may be targets for disruption, has been assembled
by Mortensen and co-workers (1992; Figure 4). This vector
contains a modified neomycin gene that allows for the selection
of homozygous targeted alleles (double knockouts) at high G418
concentrations (presumably driven by a mitotic nondisjunctive
process; Mortensen et al., 1992). Thus, only a single targeting
construct may be needed to disrupt both sialidase alleles.
Homozygous mutant ES cells have been produced with this single
construct approach; however, similar results have not been
obtained with some somatic cells (Arbones et al., 1994). The
marker has not, as yet, been tested in CHO cells.
An additional unique feature of this knockout construct is that
the neomycin gene has been ingeniously positioned in the vector
so that it is flanked by two lox sites which are recognition
sequences for the bacterial cre recombinase. In this way the vector
is ideally suited for disrupting more than one target in a single host
because the marker can be removed from the cell and recycled.
After stable transfectants are isolated under G418 pressure,
transient transfection with the cre-recombinase will allow the
neomycin gene to be excised by enzyme mediated recombination.
Another round of targeting at a different locus can then be carried
out using the appropriate vector containing the same mutant
neomycin marker. Theoretically, the marker could be recycled
indefinitely.
Summary
Metabolic engineering of animal cell expression hosts for the
purposes of enhancing recombinant protein production is currently a young and growing field. Given the biological complexities
of cell growth, metabolism, and death along with the intricacies
of protein biosynthesis, folding, intracellular transport, secretion,
and posttranslation modifications, the cellular parameters and
pathways that can be potentially altered are enormous. We have
focused, here, on identifying technologies that may have general
848
Fig. 4. Sialidase targeting plasmid pSTKLNCL. The host plasmid,
pTKLNCL was prepared by Richard Mortensen, Harvard University.
Plasmid is shown with sialidase gene inserts (Ferrari et al., unpublished
observations). A 3.3.kb insert, containing exons 1 and 2 and a stretch of 5′
untranslated region flank the mutant neomycin gene followed by a small,
0.9 kb segment containing exon 3.
application for modifying or controlling selected gene candidates
that will give significantly improved therapeutics based on the
experiences we have had with a number of recombinant protein
processes. Although some success has been achieved using
antisense DNA to inhibit deleterious gene expression, a more
detailed and thorough investigation of this approach is certainly
warranted since it is clear that the method holds great potential
based on the encouraging results obtained thus far. With
continued development more effective gene inhibition will
undoubtedly be achieved. Similarly, gene disruption as a means
of controlling gene expression is an equally attractive technology
since it makes possible the directed manipulation of the host cell
genome in a very specific manner. Given the genetic variations
between cell lines and each gene locus, it is reasonable to carry
out multiple targeting experiments with a variety of CHO cell
lines or different targeting strategies.
Clearly, there remains a great deal of adaptation and optimization of both of these technologies in order for them to be highly
effective with the unique cell systems and production environments that are currently employed for therapeutic protein
manufacturing. Efforts at pursuing these approaches of metabolic
engineering are clearly justified in view of market demands for
more cost effective, high productivity processes for recombinant
therapeutics. Prompting these needs are the development and
successful implementation of recent immunotherapies for the
treatment of Her-2 mediated breast cancer and anti CD-20 for
treatment of non-Hodgkins lymphoma. Dosage requirements are
high for these and other immunotherapeutics, on the order of
several hundred milligrams of protein, often with multiple
administrations over extended time periods. Optimization of
expression hosts with metabolic engineering promises a means to
Metabolic engineering in CHO cells
enhance productivity levels and minimize costs of goods and
ultimately costs of therapeutics to patients.
Acknowledgments
I acknowledge the support of Genentech, Inc., for a portion of this
work. I thank Lydia Santell and Jeff Ferrari for their outstanding
technical assistance and their invaluable contributions to the
project. I also thank Richard Mortensen, Harvard University, for
his generous gift of his targeting plasmids, pTKLNCL and
pNTK; and Tim Stewart, Genentech, Inc., for his plasmid
pRKTK. I also appreciate the advice and support of John Wilson,
Baylor University; Jamey Marth, U.C. San Diego; Hui Zheng,
Merck, Inc.; Mary B.Sliwkowski, Vannessa Chisholm, Craig
Crowley, Lynne Krummen, Chris Petropoulos, and Joy ManaghasMolony, Genentech, Inc. I thank B.Hukku, Children′s Hospital of
Michigan for carrying out FISH analysis of the sialidase gene.
Abbreviations
CHO, Chinese hamster ovary; AS; antisense; NGNA, N-glycoylneuraminic acid; MVM, minute virus of mice; tPA, tissue plasminogen activator; ES, embryonic stem; PNS, positive-negative
selection; HSV-TK, herpes simplex virus-thymidine kinase, FIAU,
[1-(2′-deoxyl-2′-fluoro-1-β-D-arabinofuranosyl-5-iodo)uracil];
FISH, fluorescent in situ hybridization.
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