Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-01-27
2001-03-13
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S004000, C435S069100
Reexamination Certificate
active
06201167
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to the production of recombinant proteins in animal's semen using the seminal gland as a bioreactor. Particularly, this invention relates to an expression system which comprises at least a semen-specific protein promoter operatively linked to a DNA sequence coding for a signal peptide and a desired recombinant protein product. When such a system is transgenically incorporated into an animal, the recombinant protein is expressed in the semen of the animal. This invention also relates to the transgenic animal that produces the desired recombinant product in its semen. Recombinant products produced by the expression systems and transgenically altered animals of this invention can be produced at significantly less cost than by conventional recombinant protein production techniques. There is also a potential to alter specific characteristics related to sperm viability and potential storage systems.
(b) Description of Prior Art
Recombinant DNA technology has enabled the cloning and expression of genes encoding medically and agriculturally important proteins and glycoproteins. Such products include, “for example, insulin, growth hormone, growth hormone releasing factor, somatostatin, tissue plasminogen activator, tumor necrosis factor, lipocortin, coagulation factors VIII and IX, erythropoietin, the interferons, colony stimulating factor, the interleukins and urokinase, antibodies.
Many of these important proteins, however, are large (molecular weights in excess of 30 Kd), secreted, require sulfhydryl bonds to maintain proper folding, are glycosylated and are sensitive to proteases. As a result, the recombinant production of such products in prokaryotic cells has proven to be less than satisfactory because the desired recombinant proteins are incorrectly processed, lack proper glycosylation or are improperly formed. Accordingly, resort has been had to the production of those recombinant proteins in cultured eukaryotic cells. This technique has proven to be both expensive and often unreliable due the variability of cell culture methods. For example, average yields are 10 mg of recombinant protein per liter of culture media, with the resulting cost typically for exceeding one thousand dollars per gram of recombinant protein. Accordingly, resort has been had to the production of those recombinant proteins in cultured eukaryotic cells. It is believed that the use of the genital tract as a tissue for expression overcomes, either wholly or to a satisfactory degree, this potential source of difficulty. Several examples using mammary glands of transgenic mammals as bioreactors have demonstrated their potential to produce recombinant protein products.
Harvesting from body fluids as opposed to solid tissue is desirable, because such routes, are by and large renewable, and most proteins of biomedical importance are themselves secreted into body fluids. Secretion into the bloodstream is a possible route, either from liver or B lymphocytes, but the coagulating properties of blood and the presence of biologically active peptides and antigenic molecules may prove a hindrance to subsequent downstream processing.
It would be highly desirable to be provided with a means to produce recombinant proteins in large quantities.
SUMMARY OF THE INVENTION
The above difficulties may be overcome in accordance with the present invention, as it is the case, for example, for the production of recombinant protein milk, by the use of the genital tract as a tissue of expression. Semen is readily collected, available in large quantities in several animal species and well characterized biochemically. Further, several proteins are present at high concentrations in this body fluid.
The present invention is a new method to solve such problems by providing an efficient means of producing large quantities of recombinant protein products in the semen of transgenically altered animals.
According to one embodiment of the present invention, a DNA sequence coding for a desired protein is operatively linked in an expression system to a genital tract-specific protein promoter, or any promoter sequence specifically activated in male genital tissue, through a DNA sequence coding for a signal peptide that permits secretion and maturation of the desired protein n the genital tract tissue. More preferably, the expression system also includes a 3′ untranslated region downstream of the DNA sequence coding for the desired recombinant protein. This untranslated region may stabilize the rDNA transcript of the expression system. Optionally, the expression system also includes a 5′ untranslated region upstream of the DNA sequence coding for the signal peptide.
The expression system is transgenetically introduced into a host genome. As a result, one or more copies of the construct or system become incorporated into the genome of the transgenic animal. The presence of the expression system will permit the male species to produce and to secrete the recombinant protein product, into or along with its semen. Such method permits the low cost, high level production of the desired proteins.
The expression “operatively linked” as used herein is intended to mean the linking of a genital tract-specific promoter or a promoter specifically activated in genital tract tissue to a DNA sequence coding for a desired protein so as to permit and control expression of that DNA sequence and production of that protein.
The expression “recombinant protein” as used herein is intended to mean a protein or peptide coded for by a DNA sequence which is not endogenous to the native genome of the animal in whose semen it is produced in accordance with this invention or a protein or peptide coded for by a DNA sequence which is endogenous to the native genome of the animal in whose semen it is produced does not lead to the production of that protein or peptide in its semen at the same level that the transgenic animal of this invention produces that protein in its semen.
The expression “genital tract” as used herein is intended to mean the reproductive anatomical male system whole or in part involving the prostate gland, the seminal vesicle, epididymis, seminiferous tubules, ampule, vas deferens, and the bulbourethral gland.
In accordance with the present invention there is provided a method for the production and secretion into a non-human animal's semen of an exogenous recombinant protein comprising the steps of:
a) producing a non-human transgenic animal characterized by an expression system comprising a promoter specific for the genital tract or accessory glands operatively linked to an exogenous DNA sequence coding for the recombinant protein through a DNA sequence coding for a signal peptide effective in secreting and maturing the recombinant protein in genital tract tissue;
b) collecting semen produced by the non-human transgenic animal; and
c) isolating the exogenous recombinant protein from the semen.
The expression system used in accordance with the present invention may also include a 3′ untranslated region downstream of the DNA sequence coding for the recombinant protein or a 5′ untranslated region between the promoter and the DNA sequences coding for the signal peptide.
In accordance with another embodiment of the present invention, the promoter may be selected from the group consisting of p12, p25, kallikreins, PSA, SBP-C and secretory protein IV promoters.
In accordance with another embodiment of the present invention, the recombinant protein may be selected from the group consisting of mono- and bi-specific antibodies, immunoglobulins, cytokines, coagulation factors, tissue plasminogen activator, GM-CSF, erythropoietin, thrombopoietin, alpha-1 anti-trypsin, animal growth hormones, cell surface proteins, insulin, interferons, lipases, antiviral protein, antibacterial protein, bacteriocins, peptide hormones, lipocortins and epidermal growth factor.
In accordance with another embodiment of the present invention, there is provided a method to increase spe
Côté France
Crouch Deborah
Sawbey Ogilvy Renault
Universite Laval
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