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 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 [alpha]-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 effectively 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.
Table I.
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 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.
Table II.
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
Figure 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). 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 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.
Table III.
Targeting approach
Cell line
Locus targeted
Freq. of targeting
(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
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 sequence-specific 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 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, 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 [gamma]-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[prime] 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 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
Figure 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.
Table IV.
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
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, 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
Figure 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.
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 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
Figure 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[prime] untranslated region flank the mutant neomycin gene followed by a small, 0.9 kb segment containing exon 3.
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.
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 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 enhance productivity levels and minimize costs of goods and ultimately costs of therapeutics to patients.
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 Managhas-Molony, Genentech, Inc. I thank B.Hukku, Children[prime]s Hospital of Michigan for carrying out FISH analysis of the sialidase gene.
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[prime]-deoxyl-2[prime]-fluoro-1-[beta]-d-arabinofuranosyl-5-iodo)uracil]; FISH, fluorescent in situ hybridization.
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