Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-05-19
2001-07-31
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S007000, C435S325000, C435S320100, C536S024100
Reexamination Certificate
active
06268545
ABSTRACT:
The present invention relates to a process for the preparation of a protein of interest in the milk of a transgenic animal. It also relates to the constructs which make it possible to obtain these animals, the animals obtained as well as the cells containing the constructs which permit the expression of a heterologous protein.
Several routes have been pursued in order to obtain proteins of biological, therapeutic or industrial interest, and which are naturally produced in small quantities or in a form difficult to purify.
It has thus been possible to produce proteins using genetic recombination techniques, by microorganisms such as bacteria or yeasts. Nevertheless, most of the proteins require, after their synthesis, a maturation stage consisting in chemical modifications of certain reacting groups, glycosylation, and the like. Prokaryotic cells do not have the adequate enzymatic content for carrying out this maturation, hence the production of inactive proteins and/or proteins with high antigenicity.
It is therefore preferable to synthesize these proteins in eukaryotic cells, which will perform the appropriate enzymatic conversions. Nevertheless, the large-scale culture of tissue cells poses a number of technical and economical difficulties.
Another approach therefore consists in causing these proteins to be produced by cells in vivo, using transgenic animals. It is desirable that the system used permits the production of proteins in large quantities and which are easily recoverable. It is therefore advantageous that the recombinant protein is produced in the mammary gland of transgenic animals, and excreted in the milk. It is indeed a biological fluid which can be easily collected, having a relatively limited complexity and a low proteolytic activity; in addition, the processes of maturation of the recombinant proteins will be probably ensured (glycosylation, phosphorylation, cleavage and the like).
Mouse or ewe mammary gland has thus been successfully made to synthesize milk proteins of another species or proteins normally absent from milk (Ref. 1 to 15).
However, the level of proteins thus produced is extremely variable. It is different from one transgenic animal to another, since it is a function of the process of integration of the transgene which is itself variable from one animal to another. The nature of the gene constructs is also essential, the elements regulating the expression of the milk protein genes being possibly many and situated at various points of the promoter region and the transcribed portion of the gene. Thus, the promoters of ovine beta-lactoglobulin, of rat WAP and of rat beta-casein are capable of causing these proteins to be synthesized in transgenic mice. The levels are, however, systematically high only in the case of beta-lactoglobulin. Likewise, the beta-lactoglobulin promoter directs the synthesis of human alpha
1
-antitrypsin which reaches the value of 7 mg/ml of milk in the milk of a transgenic mouse. The alpha
S1
-casein promoter permits the synthesis of human urokinase, but the promoters of the rat beta-casein and rabbit beta-casein genes used up until now are of a limited activity. The promoter of the mouse WAP gene directs the synthesis of several foreign proteins (plasminogen activator, CD4) which are secreted in the milk of transgenic mice. The quantities of proteins obtained with this promoter are however relatively small.
Furthermore, it may happen that the specificity of the promoter is modified by its association with a foreign gene. In this way Gunzburg et al. (Molecular Endocrinology 1991) obtain the secretion of growth hormone by means of a recombinant DNA under the dependence of the mouse WAP promoter, in transgenic animals; but the growth hormone is, in this case, also produced in the cerebellum, in Bergman glial cells. Such phenomena can result in toxicity and in the premature death of the animal.
The present invention is based on the demonstration of the special interest of the promoter of the rabbit WAP gene. Indeed, the rabbit WAP (“whey acidic protein”) is a relatively abundant rabbit milk protein (15 mg/ml of milk), and rabbits are potentially transgenic animals which can be used on a large scale.
The present invention relates to a process for the preparation of a heterologous protein of interest in mature form or in fused form in the milk of a mammalian female, in which process:
said female is bred and,
the milk is recovered and said protein is recovered from it and is separated if necessary, characterized in that said female is a transgenic animal in the genome of which has been integrated a sequence encoding said protein of interest under the control of at least one sequence present among the elements for expression of the rabbit WAP protein and situated on the fragment having a length of at least 3 Kb from the 3′ end of the complete WAP promoter.
Preferably, the present invention relates to a process in which the sequence controlling the expression of the protein of interest comprises, in addition, expression elements situated on the fragment between 3 Kb and 6.3 Kb from the 3′ end of the WAP promoter.
The production of transgenic animals is known and has been widely described with similar constructs in the documents mentioned above, but also in Günzburg et al., Hennighausen et al., Burdon et al., Reddy et al. as well as in Patent WO 90 05188.
The detailed description of the methods of transgenesis will not be repeated in detail, the above documents are incorporated in the present description by direct reference.
“Heterologous protein of interest” is understood to essentially designate a protein which is not naturally under the control of the rabbit WAP promoter.
In the process according to the invention, the mammalian female used is preferably a rabbit, but these constructs are also effective in other mammalians, for example mice.
The milk obtained contains the protein of interest which can be isolated or otherwise, and then, according to whether it is in mature of fused form, it can be subjected to a chemical or enzymatic cleavage if necessary.
The DNA constructs used are preferably introduced by microinjection into fertilized egg at the one cell up to the 8 cell stage, and then the animals corresponding to the criteria described above, that is to say transgenic and expressing said protein in the milk, are selected.
The promoter region of this WAP gene, grafted on to the reporter gene for bacterial chloramphenicol acetyl transferase (CAT) contains the elements sensitive to the two most important lactogenic hormones, prolactin and glucocorticoids. These hormones intensely stimulate the expression of the CAT gene when the hybrid gene is transfected into rabbit mammary primary epithelial cells. The hormone response depends on the length of the promoter used. The promoter of the rabbit WAP gene is therefore much more effective than the promoters of the mouse and rat WAP genes used up until now.
In particular, in the process according to the present invention, the sequence encoding the protein of interest can be preceded toward its 5′ end, by a sequence corresponding to the promoter of the complete rabbit WAP gene, or by an equivalent sequence, which ensures the function of the promoter. It is even possible, in this case, to use the entire WAP gene and a WAP promoter of said gene or of an equivalent sequence. The
FIG. 5
accompanying the present application represents said rabbit WAP gene.
“Equivalent sequence” is understood to preferably designate a sequence having at least a length of 3 Kb from the 3′ end of the WAP promoter and in particular comprising the expression elements situated on the fragment with a length of at least 6.3 Kb from the 3′ end of the complete rabbit WAP promoter, especially situated between the HindIII and BamHI sites (FIG.
1
).
The promoter may contain a 17-Kb sequence between the HindIII and EcoRI sites, or a sequence containing the expression elements situated on this fragment. The essential elements of the constructs according to the invention are situated on
Devinoy Eve
Houdebine Louis-Marie
Thepot Dominique
Crouch Deborah
Foley & Lardner
Institut National de la Recherche Agronomique
LandOfFree
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