Acta Scientiae Veterinariae . 38(Supl 2): s615-s626, 2010.
ISSN 1678-0345 (Print)
ISSN 1679-9216 (Online)
All roads lead to milk: Transgenic and non-transgenic approaches for
expression of recombinant proteins in the mammary gland
Fidel Ovidio Castro1, Jorge Roberto Toledo2, Oliberto Sánchez2 & Lleretny Rodríguez1
ABSTRACT
Bakcground: With the advent of transgenic technology to farm animals, it became possible to express recombinant
proteins of high complexity in the body compartments and fluids of these animals; the term “pharming” was coined.
This was a tremendous achievement taking into consideration the high costs associated with conventional (cellbased) production methods and the incapacity of lower organisms to adequately process complex proteins. The
mammary gland had been the organ of choice and milk the appropriate vector for successful expression of many
recombinant drugs of high added value.
Review: While theoretically the mammary gland is able to carry out all thecomplex post-translational changes related
with glycosylation or others, in the practice, not all proteins can be actually processed in a way that closely remembers
the wild protein, thus making difficult the production of some proteins in full biologically active form. This is especially
true for complex (branched) forms of glycosylation as it is the case of human erythropoietin (hEPO), or gamma
carboxylation of blood clotting factors, to mention a few examples. These cases are discussed in this review, with
special emphasis in the glycosylation of hEPO. In spite of the imperfectness of the mammary gland to accurately add
some sugar residues, it continues to be the most desirable organ to which target gene expression, due to its potent
biosynthetic machinery and the possibilities to amend the said incapacity to glycosylate appropriately all kinds of
proteins. In line with this, the European Medicines Agency first (in 2006) and the Food and Drug Administration later
(2009) approved the first milk-derived recombinant protein for human use, (ATryn; human anti-thrombin-III) after more
than two decades of thorough reviews and test, thus opening the way for future massive production of blockbuster
drugs using the mammary gland as bioreactor. In this job we reviewed briefly the state of the art of mammary glandbased production of recombinant proteins with emphasis in two different systems to target it. In the first approach, a
transgenic mammal carrying appropriate mammary specific gene promoter linked to a transgene is made, then
grown, mated, its milk tested for the presence of the protein, if expression levels and biological activity of the proteins
meet the requirements, then a production flock is created from the founder(s) and milk collected and processed. In
this way, most of the recombinant proteins produced in the milk had been created, including the leading drug ATryn.
We developed an alternative method for transient viral vectors-mediated transduction of the mammary gland, using
constitutive viral promoters linked to the transgene, thus producing very quickly high amounts of the desired protein.
The drawback of this method is its transient nature; the advantage is the fastness and easiness to produce grams of
recombinant proteins in the milk of otherwise non-transgenic mammals. In this way several drugs had been produced.
Notably one of them, the E2 antigen of classic swine fever (CSF) had been secreted in biologically active form at high
levels in goats´ milk; a veterinary vaccine formulation was established and tested successfully in clinical trails that
included viral challenging with CSF. It is foreseen that this vaccine could be in the market this year and became the
first recombinant drug produced in the milk of non-transgenic animals to get regulatory approval. In this article, we
also reviewed the state of the art of different body fluids as vectors for recombinant protein production
Conclusions: At least for the next coming years, all the animal-based recombinant protein production ways will lead
to the milk.
Keywords: transgenic, milk, adenovirus, pharming, lentivirus, glycosylation.
1
Department of Animal Science. Faculty of Veterinary Sciences. Universidad de Concepción, Avenida Vicente Méndez 595, 3801061,
Chillán, Chile.
2
Department of Physiopathology. Faculty of Veterinary Sciences. Universidad de Concepción. CORRESPONDENCE: F.O. Castro.
[[email protected] or [email protected]].
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Castro FO, Toledo JR, Sánchez O, Rodríguez L. 2010. All roads lead to milk: Transgenic and non-transgenic approaches
for expression of recombinant proteins in the mammary gland.
Acta Scientiae Veterinariae. 38 (Supl 2): s615-s626
I. INTRODUCTION
II. MAMMARY SPECIFIC EXPRESSION OF RECOMBINANT PROTEINS. THE CASE OF
HUMAN EPO: NOT REALLY A “PAVED” WAY
III. GLYCOSYLATION IN THE MAMMARY GLAND: THE NEED FOR A SMOOTH ROAD
IV. ADENOVIRAL DELIVERY OF RECOMBINANT PROTEINS TO THE MAMMARY
GLAND: TAKING A BYPASS
V. NON-PRECISE POST-TRANSLATIONAL PROCESSING OR, DO ALL THE ROAD
REALLY LEAD TO MILK?
VI. HIGHWAYS FOR HUMAN DRUGS, SIDE ROADS FOR VETERINARY
TECHNOLOGIES
VII. BUILDING ALTERNATIVE ROADS: LENTIVIRAL TRANSGENESIS; AN
IMPORTANT AND WELCOME NEWCOMER
VIII. CONCLUSIONS
I. INTRODUCTION
From an historical point of view of mankind, 25 years stands like a small grain of sand in the desert;
however in modern biology, this can make the difference between an idea and colossal achievements. This period of
time, i.e. 25 years is exactly the elapsed time since the first transgenic farm animals were generated in 1985 [13]. So,
historically animal farm transgenesis is in its very early infancy, however important breakthroughs have been made
in the transgenic technology. Among those are: the creation of knock out animals [29], pigs for the production of
organs intended for xenotransplantation to humans [25], farm animal species secreting complex drugs in their milk
[38], the birth of transgenic cloned ruminants from differentiated adult or fetal cells through nuclear transfer [3,36],
highly efficient lentiviral transgenesis in mammals and birds [21] and more recently induced pluripotent stem cells
capable of generating live animals upon nucleus transfer [55].
Among the areas in which transgenic technology is expected to exert a powerful influence, the expression
of recombinant protein genes in the milk of transgenic livestock is undoubtedly one of the most developed at present.
In 1987, Gordon and co-workers showed for the first time that transgenic mice could appropriately process a complex
human protein gene like the tissue plasminogen activator gene [12]. This finding opened the avenues for prospecting
the mammary gland as potential site for the production of highly complex proteins and also started the search for
alternative ways to produce such proteins, these included: blood, urine, saliva, egg yolk and white, seminal fluid, and
solid tissues [reviewed in 20].
The rationale behind all the mentioned productive transgenic systems was to achieve fully functional proteins
otherwise impossible to product as complex molecules in simpler expression systems, at cost-effective ways [31].
Despite the reported examples of complex proteins successfully produced using all the above mentioned
expression systems, none of them warrants correct processing of all kinds of protein and it is still subject of research.
Arguments against using blood as expression system rely mainly on three drawbacks: 1) circulating
biologically active proteins may impair the health status of the transgenic animal, 2) blood proteins might be unstable
upon time and 3) collecting high volumes of blood on a constant basis can be detrimental for the animal. Additionally
some concerns rose about possible cross contamination of the final product with animal proteins and DNA or pathogens
[22]. The use of urine and saliva to express transgenic proteins would probably fail due to the complexity of the
collection procedures and the relatively scarcity of proteins in these fluids [6]. Seminal plasma can be used for
transgene expression only in pigs, whose ejaculate volumes are large enough as to merit further processing. Although
acceptable levels (0.5 mg/ml) of hGH was reported using seminal fluid of pigs as expression system [9], it has not
been proven yet from an economical perspective and the system does not appear to be as flexible as required for the
production of all kinds of therapeutic proteins.
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Castro FO, Toledo JR, Sánchez O, Rodríguez L. 2010. All roads lead to milk: Transgenic and non-transgenic approaches
for expression of recombinant proteins in the mammary gland.
Acta Scientiae Veterinariae. 38 (Supl 2): s615-s626
With the advent of lentiviral transgenesis in farm animals [19,18], transgenic chicken are now produced relatively
easy at least when compared to previous conventional methods [21]. This, in combination with documented glycosylation
advantages of the egg biosynthetic machinery [18,41] made chicken a very attractive model for the expression of complex,
fully active recombinant proteins in both yolk and white fractions of the chicken eggs. Synageva BioPharma (formerly
AviGenics, Inc.) has developed proprietary technologies for humanization of the glycosylation pattern of recombinant
proteins expressed in the white of chicken eggs. Said technologies are based upon lentiviral transgenesis [21] on early the
(X-stages) of embryonic development. In this way erythropoietin, interferon Beta and several monoclonal antibodies have
been produced in eggs.The current general status of chicken egg expression system, recalls very closely early developments
in mammary gland expression system, but so far no such product is still even close to market. Lack of stability of the
transgenes, as well as position effects still hamper the wide introduction of this technology [21].
At present only transgenic milk-derived products are in or close to the market place. Several tens of lines
of transgenic mice expressing recombinant proteins in their milk have been produced; however, mice have served
only as a predictive model for the generation of transgenic farm animals, and the choice criteria for selecting the most
suitable species for gene farming are usually based on the quantity of protein needed per year. A simplified rule is: the
production of a protein in tons should be carried out by cows, in hundreds of kg by sheep or goats and in kgs per year
by rabbits. Only recently EMEA and later FDA approved for human use, the first pharmaceutical protein expressed in
the milk, ATryn, the trademark for Genzyme´s human anti thrombin III (ATryn) secreted in transgenic goats´ milk.
Concomitantly with this, many of the patents on milk promoters and transgenic systems are expiring or close to
obsolescence. The same is true for several blockbuster drugs, like erythropoietin, growth factors, insulin. This is
opening the way for the so called biogeneric drugs. Taking into account all the previous discussion about pros and
cons of different expression systems, for producing these drugs, the milk of transgenic animals will undoubtedly play
an important role. This lead us to a allegory with human history: in ancient Europe, Roman civilization and further
Roman Empire established itself as one of the most successful model of social development, this included not only
the roots of modern democracy based on Greek principles, but also a vast network of relatively well paved ways
interconnecting the entire Empire with the capital city. “All ways lead to Rome” is a very antique aphorism that
acknowledged the importance of that city in all aspects of life in those days. In a way, taking into consideration the
little time elapsed between the onset of transgenic technologies and the imminent commercialization of milk derived
pharmaceuticals, and after discussing all alternative ways for the transgenic production of biogenerics or new drugs,
it can be stated that as far as transgenic production concerns, so far “all ways lead to milk”.
II. MAMMARY SPECIFIC EXPRESSION OF RECOMBINANT PROTEINS. THE CASE OF
HUMAN EPO: NOT REALLY A “PAVED” WAY
We and others have used transgenic technology to produce recombinant proteins in the milk of several
laboratory and commercial species [reviewed in 6]. The choice of the species in which a given gene will be expressed
depends of course on the quantity required of said protein. Even though, not all genes will produce the expected
results in a given species, in spite of the correct calculations of protein and milk yield, and of having a clear or proven
strategy for downstream treatment of the transgenic milk. There are other factors that can impinge upon an appropriate
choice of the species; one of the most important is the biological activity of the expressed protein and the possible
physiological effect of eventual leakage of the transgene to the blood stream of the host animal. In addition, the
biological activity and shelf life of a recombinant protein in most cases depends on its glycosylation pattern.
In order to set up a really feasible production unit for a given transgenic protein with a complex glycosylation
pattern, as for example, erythropoietin (EPO), the study of the biochemical and biological properties of the recombinant
proteins is crucial. This is not always easy to perform; transgenic mouse or even rabbit models are expensive, time
consuming and not at all times as predictive as required. Our attempts to express human EPO in transgenic mice and
rabbits, constantly failed to produce enough pure EPO as to conduct glycosylation studies of EPO-derived milk
[4,7,32], this in spite of testing six different gene constructs bearing more than 6 kb of an otherwise tested rabbit whey
acidic protein (WAP) gene promoter [27] and chromosomal, cDNA or synthetic fragments of the human EPO.
In the experiments described above, there was also a decrease in the efficiency of generation of transgenic
animals, when compared to historical data of the laboratory. Our interpretation of the results was that EPO was
expressed ectopically during early gestation and further leaked into blood as a result of inappropriate regulation of the
rabbit WAP promoter. These events lead to premature death of EPO expressing foetuses and thus to a phenomenon
of counter selection. That deregulated expression of the WAP promoter, was responsible for timely deregulated EPO
expression was evident after in vivo and mammary gland explants analysis [1,5]. We could clearly detect ectopic
WAP and hEPO expression even during oestrus cycle and early pregnancy of the transgenic female rabbits. Interestingly
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Castro FO, Toledo JR, Sánchez O, Rodríguez L. 2010. All roads lead to milk: Transgenic and non-transgenic approaches
for expression of recombinant proteins in the mammary gland.
Acta Scientiae Veterinariae. 38 (Supl 2): s615-s626
others also find similar behaviour of EPO transgenes in mice and rabbits [24]. Similarly, we and other found differential
biological activity of milk-derived EPO in comparison with CHO produced [24,32]. A resume is provided in Table 1.
III. GLYCOSYLATION IN THE MAMMARY GLAND: THE NEED FOR A SMOOTH ROAD
Data presented above indicate that dealing with expression of complex proteins in a heterologous environment
can set hurdles for their appropriate post-translational processing and biological activity. Additional complexity is added
when proteins such as EPO can exist under different glycoforms. This is also the case for antithrombin-III, the only milkderived transgenic protein currently in the market. Plasmatic human anti-thrombin III (phAT) has four potential Nglycosylation sites with a low heterogeneity of branching per site. The most common glycosylation processing adds
complex bi-antennary sugars (without galactosamine and fucose) with NANA capping in both antennas [10]. However,
when expressed in transgenic goats’ milk, the recombinant AT (rhAT) contained fucose, rare in mammals oligomannosidic
structures such as man3-man9, less syalilated structures and both NANA and NGNA residues [10]. Nevertheless ATryn
got the approval of EMEA and FDA, since its biological activity was not diminished nor had a deleterious effect on
patients due to the presence of the N-glycosylneuraminic acid (NGNA) glycoform. The take away message is that
recombinant proteins do not have to be perfect matches of the wild type, furthermore, this is highly unlikely to happen,
but rather it must meet satisfactory clinical and bio safety criteria on a case by case basis [20].
Table 1. Expression of human erythropoietin in the milk of transgenic and non transgenic animals.
Human EPO Gene Species
Method
# of
Maximal
Biological Ref
expressing expression activity
F0/tested level in
milk (ng/ml)
Synthetic
rabbits
microinjection
0/3
0
NA
7
cDNA
Mice
microinjection
2/3
10
NA
32
cDNA
rabbits
microinjection
1/1
25
500000
32
U/mg(in vitro)
Chrom
Mice
microinjection
1/1
44
NA
Chrom
rabbits
microinjection
2/2
8
450000
6
U/mg (in vitro)
Chrom
Mice
microinjection
7/12
50000
ND
*
Chrom
rabbits
microinjection
1/1
800
ND
*
cDNA
rabbits
microinjection
1/1
50000
ND
*
cDNA
mice
Adenoviraltransduction
10/10
2800000
Yes in vitro **
cDNA
goats
Adenoviral transduction
6/6
2000000
Yes in vitro 30
7
*Adapted from reference 7; ** this review
NA= not assayed; ND= no data; Chrom= chromosomal gene.
IV. ADENOVIRAL DELIVERY OF RECOMBINANT PROTEINS TO THE MAMMARY
GLAND: TAKING A BYPASS
As stated previously, assaying the biological activity of a milk-derived protein, and more over, to study its
glycosylation pattern is not an obvious task, especially if the final goal is to produce said protein in ruminants or other
species with long reproductive intervals, transgenic intermediate models (mice or rabbits) are still expensive. To avoid
this and also to generate fast enough milk-derived proteins we developed an adenovirus-based method for transduction
of the mammary gland of mice and goats [35]. The approach is rather simple and takes advantage of the Coxsackie-
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Castro FO, Toledo JR, Sánchez O, Rodríguez L. 2010. All roads lead to milk: Transgenic and non-transgenic approaches
for expression of recombinant proteins in the mammary gland.
Acta Scientiae Veterinariae. 38 (Supl 2): s615-s626
adenovirus receptor (CAR) present in the basolateral surface of the mammary epithelial cells. For this, the mammary
gland is flushed with saline or similar solution and thereafter filled with culture medium containing high titters of
replication deficient adenovirus bearing the transgene. The foreign DNA gets access into the mammary epithelial cells
where it is transcribed and converted into protein and secreted through milk. In the first attempts, only non-lactating
goats could be successfully transduced after induction of lactation [35], this was of course a handicap in terms of
productivity and because of the need of hormonal induction of lactation. In subsequent experiments, the tight junctions
between epithelial cells in actively lactating goats were temporarily disrupted with EGTA just to allow a window for the
exposure of CAR receptors to viral particles and then resealed for proper lactation to continue [30,44,45].
Using this approach we were able to target efficiently the mammary gland of lactating mice and goats and
several recombinant proteins have been secreted at high levels (>1gr/lt) in their milk, which allowed for detailed
characterization of glycosylation patterns as for EPO [30,44,45], or for a protective vaccine against classic swine
fever based on the expression of the viral E2 antigen [43]. The system is absolutely flexible and in principle any
recombinant protein can be obtained quickly in a cost-effective way in mice or ruminants, thus avoiding the making
of transgenic models. The two major drawbacks of the system are related with its transient nature: limited temporal
expression (around one week) of the transgene and the need to transduce each animal each time.
A few examples of the potential of adenoviral delivery for the expression of recombinant proteins in the
mammary gland are provided below.
V. NON-PRECISE POST-TRANSLATIONAL PROCESSING OR, DO ALL THE ROAD
REALLY LEAD TO MILK?
In the middle to late nineties we and other pointed to the fact that the mammary gland might not properly
process all kind of proteins, these observations were based mainly on the differential biological activity of milkderived EPO [4,32,7] and more precisely to an incorrect glycosylation of this very complex molecule [24] as well as
in the incorrect processing of recombinant protein C in the mammary gland of transgenic mice and pigs [8,28].
Furthermore for the first time it was proposed by Bill Velander and colleagues in 1996, [40] that there were constraints
in posttranslational processing of protein C by the mammary gland of transgenic animals due to a rate limitation in
gamma-carboxylation at expression levels above 20 micrograms/ml in mice and 500 micrograms/ml in pigs. It has
been suggested that the mammary gland has rate limiting capacity for complete and correct glycosylation at expression
levels higher than 1 mg/ml [20] as it is the case for ATryn [17] and for protein C1 inhibitor [23]. Interestingly, these
limitations are not only inherent to the mammary gland incompetence to carry out complex post-translational processing
in certain cases, but it has also been show that antibodies expressed in chicken white lack sialic acid residues, thus
limiting their full potential of action [18].
EPO was the leading blockbuster recombinant drug in worldwide sales for almost 20 years until re-creation
of naturally existing proteins became replaced by second-generation drugs and custom designed molecules such as
monoclonal antibodies with new specificities [26]. Nevertheless its market is still in the range of billions of dollars per
annum, so its production is in the focus of many biotechnological companies. Conventional cell culture production is
still the choice for EPO production, what leads to elevated costs. Thus a milk-based alternative would be very
welcome by the biotechnology industry. However as discussed earlier, it is a great challenge for the mammary gland
biosynthetic machinery to fully and correctly process the complex glycosylation pattern of EPO, especially at high
expression levels. Using adenoviral delivery system we were able to transiently produce human EPO at > 2 gr/Lt in
the milk of mice and goats [45]. Not surprisingly EPO was inappropriately glycosylated both in vivo in goats [30] and
in vitro in a proprietary goat mammary epithelial cell line GMGE and in mouse primary cultures derived from adenovirus
transduced mammary gland [34,44]. In goats’ milk, EPO displayed lower molecular weight and was essentially
monosialylated biantennary, with unusual termination motive of N-glycans and with diminished in vivo activity, while
in the in vitro experiments the most prominent findings were polyfucosylation and low sialylation leading to significant
differences with CHO-derived EPO (Figure 1). Taken together these data and those retrieved from the similar patterns
of N-glycans found for inhibitor of protein C1 [23] and anti-thrombin III [17] in rabbits and goats respectively, it is
tempting to speculate that final N-glycosylation and antennal branching in the mammary gland is dependent on the
combined action of diverse glycosyltransferases enzymatic activities.
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for expression of recombinant proteins in the mammary gland.
Acta Scientiae Veterinariae. 38 (Supl 2): s615-s626
Figure 1. Electrophoretic migration and composition of isoforms of human EPO expressed in mammary epithelial goat cells in vitro and in
vivo. A: SDS-PAGE (12.5%) stained with Comassie blue. Lanes: 1) EPO-goats milk (GM); 2) EPO-GMGE (in vitro); 3) EPO-CHO
(commercial); 4) molecular weight markers. B: SDS-PAGE (12.5%) stained with Comassie blue. Lanes: 1, 4 and 7, EPO-CHO, EPO-GMGE
and EPO-GM respectively; 2, 5 and 8, desialylated EPO-CHO, EPO-GMGE and EPO-GM. Samples were treated with the enzyme syalyse
from S. typhimurium before loading; 3, 6 and 9, EPO-CHO, EPO-GMGE and EPO-GM, treated with the endoglycosidase enzyme PNGase
F. C: Isoelectrofocus and Western blotting analysis using antibody against áhEPO-HRP of samples showed in A. The run was performed in
a previously hydrated polyacrilamide gel and in a 2.5-5.0 pH gradient. Lanes: 1 EPO-goats milk (GM); 2) EPO-GMGE (in vitro); 3) EPO-CHO
(commercial).
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Acta Scientiae Veterinariae. 38 (Supl 2): s615-s626
From the issues discussed above it can be concluded that the mammary gland is by no means able to
provide biomolecules with complex branched antennary glycosylated structures, in other words, that not all the roads
lead to milk. However is this really the case? Can we mistakenly conclude that? In the mammary gland, naturally
occurring glycosylation is restricted to some proteins and no extended branching of sugar residues are common, thus
to express in this gland complex branched glycosylated structures is a hard task. Genetic engineering offers the
means to solve this problem and it can be foreseen in the next models, a complex transgenic (or transiently transduced)
mammary gland expressing both the enzymes defective in the glycosyltransferase pathway and EPO or other
proteins with branched N-glycans mandatory for biological activity and half-life in vivo.
Our pioneer experimental model has been adapted by other groups. In this sense human nerve growth
factor (NGF), lactoferrin and hGH have been expressed at acceptable levels by others [please refer to Table 2,
references 14,16,52,54]. In addition, we and others have expressed several pharmaceutical proteins in the milk of
experimental models using direct infusion of adenovirus into their mammary glands. In this way, hGH and hEPO and
were expressed in mouse milk by our group [35 and unpublished], and human nerve growth factor and lactoferrin in
rabbits milk [15,53].
VI. HIGHWAYS FOR HUMAN DRUGS, SIDE ROADS FOR VETERINARY
In the biotechnology industry, costs matter, then, are veterinary products attractive for milk expression?
The answer to this question is neither easy nor obvious. In a primary approach it is tempting to think that only proteins
of high added value, such as blockbuster drugs of human use are of interest to the pharmaceutical industry, due to the
ratio between production costs and sales prices. In this sense, the majority of the milk-produced drugs that are in, or
close to the market are intended for human use (please refer to Table 3). However, intensive productive systems in
agriculture such as porcine or poultry, or aquaculture systems such as salmon production relay in high densities of
animals per square meters thus requiring vaccinations and use of prophylactic or therapeutic drugs. Many of these
drugs are produced by biotechnological means, but not always adequate post-translational changes are made in a
cost-effective manner thus, alternative production systems are welcome.
Table 2. Expression of recombinant proteins in the mammary gland of transduced goats.
Gene (inoculum)
Expressing/
inoculated
animals
Average* expression
level (highest)
Biological activity
Ref
hGH(1x109 GTU/ml)
3/3
0.2 g/L (0.3 g/Lt)
NA
35
Hepo(1x109 GTU/ml)
6/6
0.67 (2.0 g/Lt)
Yes in vitro assays
30,44
E2-his(5x109 GTU/ml)
4/4
0.48 (1.2 g/Lt)
Protective in vivo in
challenge trials
43
hGH (1x109 GTU/ml)
ND
16
3/3
0.6 (2.4 g/Lt)
9
NGF(1x10 GTU/ml)
3/3
0.011- 0.165 (0.196 g/Lt) Similar to commercial,
52
in vitro assayed in PC12
cells
Lactoferrin
(0.4-2.0 x109GTU/ml)
3/3
0.68 (2.6 g/Lt)
ND
14
Anti-thrombinIII
(1x1010 GTU/ml)
2/2
ND (2.8 g/Lt)
Equivalent to pAT-III in
an in vivo model
54
*As reported or calculated from seven continuous expressing days
NA= not assayed; ND= no data
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Table 3. Summary of some recombinant proteins produced in milk and their status in advanced or finished
clinical trials.
Product
Species/method
Clinical status
Company
a-1 Antitrypsin(rhATT)
Sheep/transgenics
Phase III
Bayer-PPL ARC
Anti-thrombin III(ATrynR)
Goat/transgenics
Approved by EMEA
(2006) and FDA (2009)
GTC Biotherapeutics
C1 inhibitor (esterase inhibitor)
Goat/transgenics
Phase III
Pharming BV
Humanalpha-glucosidase
Rabbits/transgenics
Phase III (orphan drug)
Pharming Bv
E2-his
Goats/non
transgenics
Expanded final field
tests
Heber Biotec
The data cited in the table are not comprehensive and were adapted from companies’ websites, and from data presented in this
review.
In this sense, Toledo et al [43] made an important advance when expressed up to 1.2 mg/ml of E2
glycoprotein of the classical swine fever virus in the milk of transiently transduced caprine mammary gland using
adenoviral vectors. Said protein was tagged with poli histidines to ease purification. E2-his was glycosylated slightly
differently than the normal viral protein as showed by the presence of oligomannoside, hybrid and complex type Nglycans attached to it. This is in agreement with previous discussion about differential glycosylation pattern of several
recombinant proteins in transgenic milk, as it was the case for EPO, ATryn and lactoferrin [47].
In spite of these minor changes in glycosilation, the capacity of goat milk-derived E2 antigen to protect pigs
from both classical swine fever clinical signs and viral infection was assessed in a vaccination and challenge trial
[43]. One of the major advantages of this approach is that sufficient quantities of recombinant proteins can be
produced in a minimal time, thus economizing long reproductive intervals and transgene testing. At present expanded
clinical trials are in progress in production flocks in Cuba, and the appropriate regulatory permits for commercial
exploitation of this vaccine are to be granted in 2010, thus converting the E2-his based vaccine in the first mammary
gland derived in the veterinary market. Undoubtedly, the combined use of adenoviral rapid testing and minute production
with lentiviral transgenesis or somatic cell nucleus transfer will pave the way for the development and challenge
testing of other veterinary or aquaculture vaccines.
VII. BUILDING ALTERNATIVE ROADS: LENTIVIRAL TRANSGENESIS; AN
IMPORTANT AND WELCOME NEWCOMER
The fact that mammary gland can be targeted using retroviral vectors was shown for rats in 1991 by Wang
et al. [48] and by Thompson et al., 1998 [42]. In both cases, the authors used replicant-incompetent retroviral vectors
that carried oncogenes and caused experimental tumoriginesis of the targeted mammary gland. Later hGH transgene
was expressed in the milk of goats transduced with a retroviral vector [2]. This approach yielded very low levels of of
hGH in the milk at the onset of lactation, and its expression dropped to almost undetectable levels (below 20 ng/ml)
until the end of the experiment.
Recently with the development of pseudotyped lentiviral vectors, transgenic mammals and birds have
been developed with astonishingly high efficiencies [reviewed in 51]. Lentiviruses are unique retrovirus, with the
remarkable feature that they infect non-dividing as well as dividing cells, thus overcoming a major drawback associated
with the use of most other retroviruses, which usually very poorly infect non-dividing cells. After genetic engineering
of the viral vector with the desire gene construct, the next step is to produce high viral titters in helper cells, usually
293 T or FT cells, and subsequent microinjection of viral suspensions in the previtelline space of an egg or zygote,
causing very little distress to the embryos and thus yielding high efficient rates of transgenesis [19,18]. Alternatively,
zona-free embryos can be infected by co-culture with the said viral vector [11]. The technique is simple and easy to
master being the viral engineering the most complex and limiting step.
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Recently direct transduction of the mammary gland using lentiviral vectors was achieved [50]. For that
intraductal delivery of lentiviral particles carrying EGFP led to sporadic infection throughout the ductal tree of mice,
causing 4% of the mammary cells to produce EGFP [50]. In another experiment an elegant approach was used to
infect in vitro total primary mammary epithelial cells in suspension with high titter lentiviruses. The transgenic cells
were then replaced into cleared mammary fat pad and gave rise via clonal outgrowth to all the epithelial populations
of the mammary gland [49]. Authors went further and used lentiviral-mediated Wnt-1 over expression to replicate
MMTV-Wnt-1 mammary phenotypes. For the first time it was created a phenocopy of a mammary gland genotype
without making transgenic animals.
The implications of this finding are huge and might imply a complete new
shift of paradigms in approaching mammary gland transgenesis. This is in concordance with recent advances in
mammary gland stem cell biology, where it was demonstrated that a single mammary stem cell is capable of
repopulating entirely an otherwise cell depleted (cleared) mammary gland in vivo [37,39,46].
As far as transgenic mammary gland gene expression concerns, no reports using lentiviral vectors have
been published to our knowledge. Only very recently the group of Dr. Sánchez, at that time working in Havana, Cuba
[Oliberto Sánchez; personal communication and article submitted] succeeded in expressing the classical swine
fever E2 glycoprotein fused to poli histidine tag in the milk of transgenic mice after subzonal lentiviral injections of one
cell embryos and subsequent embryo transfer. Authors created a dual promoter viral vector encompassing a classic
SV40 promoter linked to EGFP in order to select for transgenic (green) embryos as well as a short (1.5 kb áS1-casein
promoter from water buffalo) driving the expression of the E2 glycoprotein. Out of 28 founder mice, 24 (85%) carried
the transgene; however none of them expressed the GFP gene. This is in contrast with otherwise 55% of F0 pups
expressing GFP, when only SV40-EGFP lentiviral construct was injected into one cell embryos. Apparently there was
interference between the two promoters that impeded the pre-screening of transgenic embryos based on GFP expression.
Nevertheless, four out of six founders tested expressed transgenic E2-his protein in their milk as judged by a specific
ELISA test. Expression levels ranged from 4 to 422 ìg/ml. Interestingly the founder mice carrying higher copy number
of the transgene (n=8) was the highest expressing animal. In addition, as demonstrated by SDS-PAGE under nonreducing conditions, E2his showed the expected molecular weight corresponding to the polypeptide homodimeric
form of the glycosylated E2his protein. This finding was in agreement with the previously observed N-glycosylation
pattern of E2 protein transiently expressed in goats mammary gland after adenoviral transduction [43]. This is first
report of the expression of a recombinant protein in the milk of transgenic animals generated by lentitransgenesis.
Further authors created one transgenic cow using the same gene construct; [Oliberto Sánchez; personal communication
and article submitted] however no data about expression of the transgene were available at the time of writing of this
review.
Undoubtedly the combined use of lenti transgenesis with somatic cell nuclear transfer (SCNT) will render a
substantial improvement in simplicity and speed of making mammary gland bioreactors. Very recently we created
transgenic cloned bovine embryos after a similar gene construct, as that described above for mouse lentitransgenesis,
was used to stably transfect an adult skin fibroblast of proven clonogenic potential in vivo [33]. Working in small
culture vessels and with low cell numbers, a few days after viral exposition, all the cells were positive for GFP
expression and were used for SCNT. Green blastocysts were transferred to females and thus we did the initial steps
toward the use of lentiviral transgenesis for the expression of recombinant proteins in the milk of farm animals
[Rodríguez-Alvarez et al., unpublished data].
VIII. CONCLUSIONS
Transgenic and non-transgenic production of recombinant proteins is almost state of the art, after more
than two decades, regulatory as well as technical issues had been surmounted and the first drug produced in a
transgenic mammal is already in the market for human use. Veterinary applications of these technologies are foreseen
to enter in the market sooner than later, thus the advent of a new era in the production of recombinant proteins is here.
Obsolescence of several on processes and drugs is opening market space for “biogeneric” drugs. Many of these
drugs would be produced using living bioreactors, both transgenic or not. Different body compartments and fluids of
the animals can be used as target for recombinant protein expression. Nevertheless, to the question: in which organ
to express a recombinant protein using an animal? The answer is clearly: in the mammary gland. New developments
in lentiviral transgenesis and the use of eggs as bioreactors would probably make transgenic chicken a very useful
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tool for producing blockbuster drugs and antibodies. New technologies to transiently transduce the mammary gland
are available and big amounts of recombinant proteins can be produced in a very short period of time. Whether
regulatory issues related to the use of viral vectors will be solved for human use is still to be seen; however veterinary
vaccines or drugs as well as human and veterinary diagnostic means can be produced in non-transgenic mammal’s
milk. In conclusion, as in the ancient times all ways leaded to Rome, today as far as using animals as bioreactors for
the production of recombinant proteins, all ways lead to milk, for now…
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