Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-09-15
2002-02-05
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S007000, C800S015000, C800S016000, C800S017000, C800S018000, C435S069600, C435S325000
Reexamination Certificate
active
06344596
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the production of natural and modified forms of Factor IX. In particular, the invention relates to a transgenic animal containing, stably incorporated in its genomic DNA, an exogenous Factor IX gene that is expressed specifically in mammary tissue, such that Factor IX is secreted into milk produced by the animal. In particular, the invention relates to the production of human Factor IX in the milk of a transgenic non-human mammal using a DNA molecule that comprises a whey acidic protein promoter gene, 5′ regulatory sequences containing the promoter, human Factor IX cDNA that lacks at least a portion of the complete or any portion of or the complete the 3′-untranslated region of the native human Factor IX gene, but contains the 5′ and 3-untranslated region of the mouse whey acidic protein. gene.
2. Background
Human Factor IX, or “Christmas factor,” is encoded by a single-copy gene residing on the X-chromosome at q27.1. For a review of Factor IX gene structure and expression, see High et al., “Factor IX,” in MOLECULAR BASIS OF THROMBOSIS AND HEMOSTASIS, High (ed.), pages 215-237 (Dekker 1995); Kurachi et al.,
Thromb. Haemost
. 73:333 (1995). The Factor IX gene is at least 34 kilobase (kb) pairs in size, and it is composed of eight exons. The major transcription start site of the Factor IX gene in human liver is located at about nucleotide-176. The human Factor IX mRNA is composed of 205 bases for the 5′ untranslated region, 1383 bases for the prepro Factor IX, a stop codon and 1392 bases for the 3′ untranslated region.
Factor IX is synthesized as a prepropolypetide chain composed of three domains: a signal peptide of 29 amino acids, a propeptide of 17 amino acids, which is required for &ggr;-carboxylation of glutamic acid residues, and a mature Factor IX protein of 415 amino acid residues. The Factor IX zymogen undergoes three types of post-translational modifications before it is secreted into the blood: a vitamin K-dependent conversion of glutamic acid residues to carboxyglutamic acids, addition of hydrocarbon chains, and &bgr;-hydroxylation of an aspartic acid. Mature Factor IX protein contains 12 &ggr;-carboxylated glutamic acid (Gla) residues. Due to the requirement of vitamin K by &ggr;-carboxylase, Factor IX is one of several vitamin K-dependent blood coagulation factors.
The activation of Factor IX is achieved by a two-step removal of the activation peptide (Ala
146
-Arg
180
) from the molecule. Bajaj et al., “Human factor IX and factor IXa,” in METHODS IN ENZYMOLOGY (1993). The first cleavage is made at the Arg
145
-Ala
146
site by either Factor XIa or Factor VIIa/tissue factor. The second, and rate limiting cleavage is made at Arg
180
-Val
181
. The activation pathways involving Factor XIa and Factor VIIa/tissue factor are both calcium-dependent. However, the Factor VIIa/tissue factor pathway requires tissue factor that is released from damaged endothelial cells. Activated human Factor IX thus exists as a disulfide linked heterodimer of the heavy chain and light chain. For full biological activity, human Factor IX must also have the propeptide removed and must be fully &ggr;-carboxylated. Kurachi et al.,
Blood Coagulation and Fibrinolysis
4:953 (1993).
Factor IX is the precursor of a serine protease required for blood clotting by the intrinsic clotting pathway. Defects in Factor IX synthesis result in hemophilia B (or Christmas disease), an X-linked disorder that occurs in about one in 30,000 males. Patients with hemophilia B are treated with Factor IX obtained from pooled plasma from normal individuals. Martinowitz et al.,
Acta Haematol
94(
Suppl
. 1):35 (1995). Such Factor IX preparations, however, may be pyrogenic and may be contaminated with pathogenic agents or viruses. Accordingly, it would be advantageous to develop a means to prepare purified Factor IX that did not require extraction from human plasma.
In the past, therapeutic proteins have been produced in
E. coli
. However, limitations in secretion and post-translational modification which occur in all living cells has rendered recombinant protein production a highly species, tissue and cell specific phenomena. In an example of recombinant FIX expression in mammalian cells, the populations of recombinant FIX produced in baby hamster kidney cells are not the same protein products as FIX produced in Chinese hamster ovary cells (Busby et al.,
Nature
316:684-686 (1985); Kaufman et al.,
J. Biol. Chem
. 261: 9622-9628 (1986)). These proteins have profound differences in &ggr;-carboxylation and propeptide removal and these differences have been established as being very important in determining biological activity. Most importantly, only less than about 40 milliunits/hr/ml of active rFIX were detected in CHO cells even after coexpression of the propeptide cleaving enzyme PACE, coexpression of the carboxylase enzyme, and extensive gene amplification with methotrexate in an attempt to increase expression level and activity (Wasley et al.
J. Biol. Chem
. 268: 8458-8465 (1993); Rehemtulla et al.,
Proc. Natl. Acad. Sci
. (
USA
), 90: 4611-4615 (1993)). Researchers concluded that multiple limitations in the secretion of active rFIX exist in mammalian cells (Rehemtulla et al., 1993) and that the problem of gene transcription was secondary and indeed trivial with respect to post-translational processing of biologically active rFIX in mammalian cells. Thus, FIX mRNA splicing is a species specific effect occurring in mice and perhaps sheep, but not pigs. Although one might hypothesize that a FIX could be expressed, one could not predict with any certainty whether such product would be a clinically acceptable, practical, recombinant therapeutic FIX product for a given hemophiliac indication.
Production of recombinant Factor IX in mammalian cell culture (HepG2, mouse fibroblast, mouse hepatoma, rat hepatoma, BHK, CHO cells) repeatedly has been shown to be recalcitrant and cell-system specific with respect to intracellular restrictions on secretion and proteolytic processing, post-translational modification, expression levels, biological activity, downstream recovery from production media, and substantiation of circulation half-life (Busby et al., (1985); de la Salle et al.
Nature
316: 268-270 (1985); Anson et al.,
Nature
315: 684-686 (1985); Rehemtulla et al., 1993; Wasley, et al., (1993); Kaufman et al., (1986); Jallat et al.,
EMBO J
. 9: 3295-3301 (1990)). Importantly, the aforementioned works concluded that nontrivial improvements in these combined criteria are needed if a practical prophylactic FIX therapeutic product is to be made available from any recombinant mammalian cell production source. For example, attempts to increase the specific activity of rFIX produced by CHO cells by rectifying problems with under-carboxylation by co-expression of the vitamin K-dependent carboxylase enzyme resulted in no improvement in &ggr;-carboxylation or biological activity (Rehemtulla et al., (1993), implying that multiple rate limitations in this post-translational modification exist.
Similar difficulties in the production of significant amounts of biologically active rFIX in the mammary epithelial cells of transgenic animals also has been documented in the literature. Although WO-A-90/05188 and WO-A-91-08216 predict that production of rFIX should be possible in their production systems, no data are presented in WO-A-91-08216, and only very low levels of secreted rFIX (25 ng/ml) with no biological activity were reported in transgenic sheep in WO-A 90/05188 and in related publications (Clark et al.,
Bio/Technology
7: 487-4992 (1989)). Higher expression levels have recently been reported in the milk of sheep (5 &mgr;g/ml), but again, the product had no biological activity (Colman,
IBC Third International Symposium on Exploiting Transgenic Technology for Commercial Development
, San Diego, Calif. (1995)). This demonstrates that the polypeptides produced in WO-A-90/05188, Clark et al. (1989), and Colman
Drohan William N.
Johnson John L.
Lubon Henryk
Velander William H.
American Red Cross
Crouch Deborah
Foley & Lardner
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