Current Opinion in Biotechnology Vol. 7, No. 5, October 1996 Recombinant protein production in transgenic animals [Review article] Yann Echelard Current Opinion in Biotechnology 1996, 7:536-540.

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• Abstract
• Abbreviations
• Introduction
• Expression of foreign proteins in the mammary gland
• Transgenic production of blood-borne peptides
• Alternatives to pronuclear microinjection
• Conclusions
• Acknowledgements
• References and recommended reading
• Copyright

CHO—Chinese hamster ovary;

ES—embryonic stem;

HbA—hemoglobin A;

hflX—human factor IX;

hGH—human growth hormone;

hPC—human protein C;

WAP—whey acidic protein.

The commercial potential of transgenic animal bioreactors for the production of high-value proteins is appreciable. As a result, a growing body of work has focused on outlining the parameters of mammary gland expression of heterologous proteins. This review will discuss the following three subjects: milk-specific regulatory elements, post-translational modification capabilities of the mammary gland, and physiological effects of transgene expression. Recent developments in the related field of blood-borne production of recombinant proteins and peptides will also be summarized.

Pronuclear microinjection, which is still the only method routinely used to introduce foreign DNA in the germline of farm animals, is, at best, a costly, tedious, and inefficient endeavor. The last part of this review will describe some of the efforts aimed at bypassing pronuclear microinjection, either by developing pluripotent embryonic cell lines, or by developing somatic approaches similar to those currently used in gene therapy trials. These technologies could eventually improve our ability to fine tune the production characteristics of animal bioreactors.

The combination of large daily protein output, excellent post-translational processing abilities, ease of access to the recombinant protein (milking), and low capital cost of production plants (farms) as compared to industrial high-volume fermenters, has made the transgenic mammary gland an excellent candidate for the production of recombinant protein. Regulatory sequences of the major milk protein genes have been used, with variable success, to direct the expression of an array of heterologous proteins in the milk of transgenic mice (mostly), rats, rabbits, pigs, sheep, goats, and cows (for a recent review, see [1•]). Five promoters have been shown to yield high levels (>1mg ml^-1) of foreign protein in milk: sheep -lactoglobulin [2] [3] [4], goat -casein ([5] [6] [7•]; H Meade et al., abstract 110, Meeting on Mouse Molecular Genetics, Cold Spring Harbor, New York, September 1994), bovine s1-casein [8] [9], rabbit whey acidic protein (WAP) [10] [11] [12], and mouse WAP [13] [14••]. Maximum expression levels reported with the mouse WAP promoter have been lower than with other promoters [5] [13] [14••] [15] [16] [17] [18] [19]. However, this could be a function of the heterologous gene expressed. When the 4.2 kilobase (kb) mouse WAP promoter [18] was used to express a human 1-antitrypsin genomic construct in milk of transgenic mice, levels comparable to that reported with other promoters [2] [10] [19] were observed (B Wilburn, J Williams, Y Echelard, unpublished data).

In most accounts, it has been difficult to achieve protein yields superior to 1mg ml^-1 with cDNA derived constructs [3] [15] [16] [17] [19] [20] [21] [22] and sometimes the transgenes are transcriptionally silent. However, the use of a genomic construct does not always ensure high expression levels [23]. The weak performance of cDNA-containing transgenes could be caused by sensitivity to the silencing influences of chromosomal sequences surrounding the integration sites. Two groups [24] [25] have attempted to correct this situation by co-microinjecting highly expressed genomic constructs with poorly expressed cDNA constructs in order to obtain integration sites containing both silent and highly expressed transgenes. In both cases, although there was increased expression of the cDNA transgenes from co-integrated sites as compared to transgenic integration sites containing only cDNA sequences, the cDNA derived expression levels were still 2–3 orders of magnitude lower than those obtained from genomic transgenes. In another study, poorly expressed sheep -lactoglobulin driven human factor IX (hfIX) cDNA transgenes were shown to contain a cryptic 3' splice site [26••], leading to the accumulation of aberrantly spliced hfIX transcripts. With a construct eliminating the cryptic splice site, transgenic mice expressed hfIX to levels up to 60µg ml^-1. This is much higher than with unmodified hfIX transgenes and close to commercially acceptable levels if the modified transgene is expressed similarly in large animals.

Proper post-translational modification of milk-produced recombinant proteins is also an issue of concern. A recent study [27••] compares the two N-glycosylation sites of human interferon- (IFN-; Asn25 and Asn97) in recombinant protein samples obtained with three expression systems: Chinese hamster ovary (CHO) cells, baculovirus-infected SF9 cells, and the mammary gland of transgenic mice. The transgenic mouse-derived IFN- had predominantly complex sialylated biantennary N-glycans at Asn25, similar to the CHO cell-derived IFN-. An increased proportion of oligomannose at Asn97 was found in transgenic mouse-derived material as compared to the CHO cell IFN-. Increased incidence of oligomannose glycans could affect the clearance by the mannose receptor of the recombinant protein.

Transgenically produced human protein C (hPC) from both mouse and swine has also been well characterized [14••] [17] [28] [29]. Mature hPC is an extensively glycosylated heterodimer that requires the removal of prepropeptide and propeptide, -hydroxylation, as well as -carboxylation of nine glutamic acid residues, for full activity. Analysis of transgenic mice and pig mammary gland-derived hPC, as compared to plasma-derived hPC, showed reduced anticoagulant activity as well as high proportions of single chain unprocessed precursors [17] [28] [29], indicating that the mammary gland carried post-translational modifications rather inefficiently. It is possible that the high-level expression of hPC was saturating the post-translational modification machinery of the mammary gland, because mammary gland expression of lower levels of another extensively modified protein, hfIX, does not seem to give rise to a high proportion of incompletely processed protein [26••]. In an effort to ameliorate the processing of recombinant hPC, bi-transgenic mice coexpressing hPC and the human serine protease furin in their mammary gland were generated [13]. These mice secreted low amounts (<5%) of unprocessed single chain hPC in contrast to the 40–60% encountered in animals solely expressing hPC [13] [17]. Coexpression of a modification enzyme with the protein of interest could also be applied to other post-translational modifications (N- and O-glycosylation, -carboxylation). Moreover, it seems that the high efficiency of processing was obtained by using what should be a relatively inefficiently expressed human furin cDNA transgene, making this strategy even more attractive.

In another example [30••], the modification of the mouse mammary gland post-translational modification capability by the transgenic expression of a human 1,2-fucosyltransferase was exploited. Here, the objective was not to better process a recombinant protein, but to remodel the structure of milk glycoconjugates. This experiment showed that transgenic dairy animals could become an inexpensive source of otherwise hard to synthesize oligosaccharide of industrial or therapeutic value, as well as a source of recombinant pharmaceutical proteins.

The effect of the expression of the transgene on the production animal is another concern. In problem cases, the expression of the heterologous protein can either influence the physiology of the mammary gland and disrupt lactation [6] [31] [32] [33] [34] [35], or have a systemic effect on the transgenic animal, affecting its viability and reproductive performance [11] [35] [36] [37]. A number of milk proteins have been shown to be usually present in blood during lactation [38•] [39] [40]; hence the detection, in the serum, of a heterologous protein expressed in the mammary gland was not surprising when it was encountered [10] [11] [12] [19] [41] [42]. RNA analysis also showed the incidence of low-level ectopic expression of WAP-derived [10] [11] [12] [18] [23] [38•] [41] [42] [43] and -lactoglobulin-derived [44] [45] [46] [47] transgenes, whereas the expression of s1-casein and -casein transgenes (when examined) was restricted to the mammary gland [7•] [8] [9] [22] [48] [49] [50]. With the tighter regulation of s1-casein and -casein promoters, it may be argued that these sequences could be more appropriate than the current versions of the WAP and -lactoglobulin cassettes for driving the milk production of very active proteins such as human growth hormone (hGH). However, in terms of the health of the transgenic production animal, low-level ectopic expression of proteins, such as 1-antitrypsin, human serum albumin, or most monoclonal antibodies, is probably inconsequential.

The production of recombinant human hemoglobin A (HbA) has been achieved in transgenic mice and pigs [51] to levels up to 24% completely human HbA and 30% human –pig hybrid using a construct containing human -, -, -globin genes linked to a human locus control region (LCR). The key to achieving a high expression level was the construction of a fusion -globin gene containing the pig -globin promoter linked to the human -globin coding region. Previous HbA constructs containing both the human promoter and -globin coding sequences permitted the production of, at most, 10% of human HbA in transgenic pigs [52]. Animals appeared to tolerate well the high levels of human HbA in their erythrocytes. Structural analysis of the swine-derived human recombinant HbA has shown that it was equivalent to protein purified from human blood [53].

Another procedure exploiting the recombinant hemoglobin expression system, and the fact that terminally extended -globin can be functional, could become an alternative to the chemical synthesis of therapeutic peptides. Carboxy-terminal fusions of human -globin with either -endorphin (16-mer) or magainin (26-mer) were expressed in transgenic mice [54]. Fusion proteins were expressed to levels corresponding to 25% of the total hemoglobin, seemingly without ill effects on the transgenic mice. Magaining peptides were cleanly released by enterokinase digestion of purified fusion protein, using a cleavage site engineered between the two parts of the chimeric polypeptide. On the other hand, a cryptic enterokinase site caused partial degradation of -endorphin during the digestion step, showing that the release of the recombinant peptides from the chimeric protein will not always be straightforward.

Approaches combining the potential of clonal expansion offered by nuclear transfer technology, with the possibilities of gene addition and replacement achievable in embryonic stem (ES) cell lines (or ES-like cells), could be particularly beneficial to the generation of transgenic bioreactors (potential advantages are reviewed in [55]).

Pluripotent ES cells able to contribute to the germline have only been derived in mice. Recently, there have been descriptions of chimeric animals generated with rat [56] and pig [57] ES cells, but, in both cases, no evidence of germline transmission from these cells has been reported. Cow fetuses obtained following the nuclear transfer of ES cell-derived nuclei in recipient oocytes died in utero, exhibiting major defects in placental development [58•]. In a related breakthrough, two healthy phenotypically female lambs were born from embryos generated by transferring nuclei isolated from embryo-derived epithelial cells into enucleated oocytes [59••]. Microsatellites analysis demonstrated that all the nuclear transfer lambs and fetuses were derived from the cell line. It is not yet clear whether foreign DNA can be easily introduced in this type of cell line, and there are questions about the health and reproductive fitness of the recovered lambs. However, this experiment is the first demonstration that it is possible to obtain large farm animals from embryo derived-cell lines, and it is certainly a step towards the possibility of performing gene replacement in large animals.

Another group reported a strategy that bypasses entirely the transgenic route to obtain recombinant protein production in animals [60]. Replication-defective retroviral vectors carrying the hGH cDNA were used to infect the mammary gland of goats hormonally induced to lactate. Expression levels of up to 118ng of hGH per milliliter of milk were obtained on the first lactation day. By day 3, expression had stabilized to 3–12 ng ml^-1. The advantage of this method is that expression of the recombinant protein is obtained quickly, without the delay caused by the generation interval required to generate a producing transgenic animal (18 months for goats). However, production levels reported in this paper [60] are very low. In the conditions described, less than 1 mg of crude recombinant hGH would be obtained per lactation per goat. If this technology could also be applied to other potential production species (rabbit, sheep, pigs, and cows), it could permit the quick determination, for specific proteins, of which system is most appropriate for the high-level production of correctly processed polypeptides.

The expression of recombinant proteins in transgenic animals has come of age. At least two products (sheep-derived human 1-antitrypsin and goat-derived antithrombin III) will be submitted to phase I trials within two years. Transgenic animals have the ability to process highly modified proteins, although not always with total efficiency. There are species-specific and tissue-specific characteristics. However, it seems that transgenic systems are flexible and that, when needed, processing enzymes can be coexpressed with foreign products to obtain more humanized recombinant proteins. In the future, the further characterization of each bioreactor's capability, and an improved ability to introduce specific genetic modifications, should lead to more sophisticated transgenic production herds.

I wish to thank Barbara Davis for her help with manuscript preparation, and Harry Meade, Carol Ziomek, and Li-How Chen for comments and many stimulating discussions.

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