Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-05-28
2001-07-17
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S015000, C800S016000, C800S017000, C800S018000, C800S007000, C536S024100
Reexamination Certificate
active
06262336
ABSTRACT:
The present invention relates to an isolated DNA sequence which regulates the expression of a heterologous gene comprising a mouse whey acidic protein (WAP) promoter having a length of greater than about 2.4 kb extending upstream from the unique KpnI site in the mouse whey acidic protein. The present invention also relates to a transgenic non-human mammal containing a DNA sequence stably integrated in its genome, wherein the DNA sequence contains the mouse whey acidic protein promoter having a length of greater than about 2.4 kb extending upstream from the unique KpnI site in the mouse whey acidic protein gene and is operably linked to a DNA sequence encoding a heterologous polypeptide and a process of producing the heterologous polypeptide in the milk of the transgenic mammal. The DNA sequence can comprise cDNA or genomic DNA encoding the heterologous protein.
The present invention also relates to the production of natural and modified forms of the human coagulation factor protein C. In particular, the invention relates to a transgenic animal containing, stably incorporated in its genomic DNA, an exogenous gene which is expressed in tissue of the animal, specifically in mammary tissue, such that protein C is secreted into a body fluid of the animal, particularly into the milk produced by the animal. In particular, the invention relates to the production of natural and modified forms of protein C in the milk of a transgenic non-human mammal using a DNA molecule that comprises, a mouse whey acid protein promoter having a length of greater than about 2.4 kb extending upstream from the unique KpnI site in the mouse whey acidic protein gene, particularly the upper range of this length, which is herein referred to as the long whey acidic protein (WAP) promoter fragment and a cDNA or genomic DNA encoding human protein C. The long whey acidic protein promoter fragment also can be used to express high levels of other genes in cells, such as mammary cells of transgenic non-human mammals.
BACKGROUND OF THE INVENTION
Protein C is an important component of the coagulation system that has strong anticoagulant activity. In its active form it is a serine protease that proteolytically inactivates Factors V
a
and VIII
a.
Human protein C (hPC) is a 62 kD, disulfide-linked heterodimer consisting of a 25 kD light chain and a 41 kD heavy chain which circulates as an inactive zymogen in plasma. At the endothelial cell surface it is activated to activated protein C (APC) by limited thrombin proteolysis in the presence of thrombomodulin; cleavage of an Arg-Leu bond in the amino terminal portion of the heavy chain releases a 12 amino acid peptide. See generally Gardiner & Griffin in PROGRESS IN HEMATOLOGY, Vol. XIII at page 265-278 (Brown, Grune and Stratton, Inc. 1983).
Several regions of the molecule have important implications for function as an anticoagulant in the regulation of hemostasis. The amino terminal portion of the light chain contains the nine y-carboxyglutamic acid (Gla) residues required for calcium-dependent membrane binding and functional activation. Another post-translational modification is &bgr;-hydroxylation of aspartic acid reside 71, possibly required for calcium-dependent membrane binding which is independent of the binding activity of the Gla regions.
There are a variety of clinical situations for which protein C may prove beneficial. It may serve as replacement therapy in homozygous deficient infants suffering from purpura fulminans neonatalis. Other conditions include patients with a previous history of warfarin-induced skin necrosis who must have additional warfarin therapy, heparin-induced thrombocytopenia, septic shock for prevention of intravascular coagulation and organ damage, and for fibrinolytic therapy, as protein C can protect tPA from plasma inhibitor proteins. Table 1 represents one estimate of the number of individual cases of several clinical syndromes which might be treated by purified protein C. Because there has not been sufficient material available from plasma for clinical trials until recently, these data are necessarily based on an incomplete assessment of the therapeutic potential for protein C.
TABLE 1
PARTIAL ESTIMATE OF U.S. CLINICAL REQUIREMENTS
FOR PROTEIN C AND ACTIVATED PROTEIN C
Estimated
Dose (mg)
Per
# Treatments
Total U.S.
Indication
Treatment
Per Year
Req. (Kg)
Septic Shock
5-50
120,000
0.6-6.0
Thrombolytic
10-100
800,000
8-80
Therapy**
Hip Replacement
10-100
200,000
2-20
Homozygous
3
100 × 365*
0.10
Deficient
Heterozygous
50
1,000
0.05
Deficient
Total
10.8-106.2
*100 individuals in U.S. × 365 treatment/year
**Referes to the use of APC, following thrombolytic therapy, to prevent the reformation of blood clots.
The gene for human protein C has been cloned and sequenced, as has bovine protein C gene. See Forster et al.,
Proc. Nat'l Acad. Sci. USA
82: 4673 (1985); U.S. Pat. No. 4,775,624. It is synthesized as an inactive precursor that undergoes several proteolytic events during the processes of secretion and activation. First, a signal sequence as proteolytically removed upon secretion. A second proteolytic event removes the dipeptide lys156 arg157, producing the inactive zymogen, a two chain disulfide bridged protein, consisting of a light chain of 155 amino acids and a heavy chain of 262 amino acids. The zymogen is activated by a final proteolytic event that removes residues 158-169, yielding active protein C, a serine protease with potent anticoagulant activity. Beckmann et al.,
Nucleic Acids Res
. 13: 5233 (1985).
In addition to proteolytic processing, human protein C undergoes several post-translation modifications. Perhaps most salient among these modifications is the &ggr;-carboxylation of the first nine glutamic acid residues in protein C, by a vitamin K dependent enzyme. DiScipio & Davie,
Biochemistry
18: 899 (1979). Gamma-carboxylation is required for anticoagulant activity, and is associated with Ca
2+
-dependent membrane binding. The anticoagulant activity of protein C varies directly with the extent of &ggr;-carboxylation, and the highest levels of activity are achieved only when &ggr;-carboxylation of the sixth and seventh glutamic acid residues is effected. Zhang & Castellino,
Biochemistry
29: 10829 (1990).
Protein C is also post-translationally modified by &bgr;-hydroxylation of aspartic acid 71. Drakenberg et al.,
Proc. Nat'l Acad. Sci. USA
80: 1802 (1983). Beta-hydroxylation may be important to protein C activity. Although its function is not known it has been suggested that it may be involved in &ggr;-carboxyglutamic acid independent Ca
2+
binding, and it may be required for full anti-coagulant activity.
Human protein C is also glycosylated. Kisiel,
J. Clin. Invest
. 64: 761 (1979). It contains four potential N-linked glycosylation sites, located at Asn97, Asn248, Asn313 and Asn329. The first three signals match the consensus Asn-X-Ser/Thr glycosylation sequences, and are actively glycosylated. There is an atypical glycosylation signal at Asn329, Asn-X-Cys-Ser. The Asn329 signal is glycosylated in bovine protein C, but it is not yet known if Asn329 is glycosylated in human protein C. Miletich et al.,
J. Biol. Chem
. 265: 11397 (1990). The pattern and extent of glycosylation can alter the physiological activity of protein C.
Until recently, human protein C for experimental and therapeutic use was obtained exclusively from human plasma. Unfortunately, the quantity of protein that can be obtained from human serum is limited. Furthermore, products derived from human serum pose difficulties of reliability, purity and safety.
The expression of therapeutic proteins by recombinant DNA technology is an attractive alternative to plasma production of protein C, in that it eliminates the risk of potential contamination with blood-borne viruses and theoretically provides an unlimited supply of product. But the complexity of the post-translational modifications, as discussed above, has rendered problematic the production of commercially useable amounts of suitably act
Drohan William N.
Hennighausen Lothar
Lubon Henryk
Velander William H.
American Red Cross
Crouch Deborah
Foley & Lardner
LandOfFree
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