Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-07-17
2003-03-11
Bugaisky, Gabrielle (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S252300, C435S254110, C435S325000, C435S410000, C435S226000, C536S023200, C530S381000
Reexamination Certificate
active
06531298
ABSTRACT:
FIELD OF THE INVENTION
This invention concerns Factor IX in general, and particularly concerns Factor IX containing a mutation that enhances the clotting activity thereof. This invention also concerns DNA constructs encoding such Factor IX, along with vectors containing such constructs.
BACKGROUND OF THE INVENTION
Factor IX (FIX; also known as “Christmas Factor”) plays a key role in both the intrinsic and extrinsic coagulation pathways (E. Davie et al.,
Biochemistry
30, 10363 (1991); B. Furie and B. Furie,
Cell
53, 505 (1988)). Human Factor IX and DNA encoding the same is disclosed in U.S. Pat. No. 4,994,371 to Davie et al., and in European Patent 0107278 to Brownlee.
FIX circulates as a 415 amino acid, single chain plasma zymogen (A. Vysotchin et al.,
J. Biol. Chem.
268, 8436 (1993)). The zymogen of FIX is activated by FXIa or by the tissue factor/FVIIa complex. Specific cleavages between arginine-alanine 145-146 and arginine-valine 180-181 result in a light chain and a heavy chain linked by a single disulfide bond between cysteine 132 and cysteine 289 (S. Bajaj et al.,
Biochemistry
22, 4047 (1983)). The structural organization of FIX is similar to that of the vitamin K-dependent blood clotting proteins FVII, FX and protein C (B. Furie and B. Furie, supra). The approximately 45 amino acids of the amino terminus comprise the gamma-carboxyglutamic acid, or gla, domain. This is followed by two epidermal growth factor homology domains (EGF), an activation peptide and the catalytic “heavy chain” which is a member of the serine protease family (A. Vysotchin et al.,
J. Biol. Chem.
268, 8436 (1993); S. Spitzer et al.,
Biochemical Journal
265, 219 (1990); H. Brandstetter et al.,
Proc. Natl. Acad Sci. USA
92, 9796 (1995)).
The major physiological function of FIXa in the blood coagulation cascade is to convert FX to FXa in a process that requires a phospholipid surface, calcium ions and FIXa's protein cofactor, FVIIIa. FIXa alone is an extremely poor protease but when bound to FVIIIa to form the “tenase” complex it becomes a potent FX activator(E. Duffy et al.,
J. Biol. Chem.
267, 17006 (1992); G. van Dieijen et al.,
J. Biol. Chem.
256, 3433 (1981)). It has been proposed that, upon binding FVIIIa, FIXa may undergo a conformational change at or near the active site (V. Mutucumarana et al.,
J. Biol. Chem.
267, 17012 (1992)). The importance of FIX is exemplified by the fact that patients with defective FIX molecules suffer from hemophilia B, an X-linked, recessive bleeding disorder which is clinically indistinguishable from hemophilia A in patients who have no FVIII function.
SUMMARY OF THE INVENTION
A first aspect of the present invention is a Factor IX protein having an amino acid substitution at amino acid position 338. Factor IX of the present invention is non-naturally occuring (e.g., does not contain only an arginine to proline substitution at amino acid position 338). The FIX proteins of the present invention advantageously have increased clotting activity as compared to the corresponding wild-type molecule.
A second aspect of the present invention is a pharmaceutical formulation comprising a mammalian Factor IX as described above in combination with a pharmaceutically acceptable carrier.
A third aspect of the present invention is a method of facilitating blood clotting in a subject in need of such treatment, comprising administering to the subject a mammalian Factor IX protein as described above, in an amount sufficient to facilitate or enhance blood clotting in said patient.
A forth aspect of the present invention is an isolated nucleic acid (e.g., a DNA or an RNA) encoding a mammalian Factor IX protein as described above.
A fifth aspect of the present invention is an expression cassette containing a nucleic acid encoding a mammalian Factor IX protein as described above.
A sixth aspect of the present invention is a gene transfer vector containing an expression cassette as described above.
An illustrative nucleic acid of the present invention is provided herein as SEQ ID NO:1, and an illustrative Factor IX amino acid sequence of the present invention is provided herein as SEQ ID NO:2.
The foregoing and other objects and aspects of the present invention are explained in greater detail in the drawings herein and the specification set forth below.
REFERENCES:
patent: 4994371 (1991-02-01), Davie et al.
patent: 5521070 (1996-05-01), Meulien
patent: 0 107 278 (1989-11-01), None
patent: 0 373 012 (1995-02-01), None
Bajaj et al.; Experimental and Theoretical Evidence Supporting the Role of Gly363in Blood Coagulation Factor Ixa (Gly193in Chymotrypsin) for Proper Activation of the Proenzyme;Journal of Biological Chemistry; 265:2956-2961 (1990).
Banner et al.; The crystal structure of the complex of blood coagulation factor Vlla with soluble tissue factor;Nature380:41-46 (1996).
Bottema et al.; Missense Mutations and Evolutionary Conservation of Amino Acids: Evidence That Many of the Amino Acids in Factor IX Function as “Spacer” Elements;Am J. Hum. Genet. 49:820-838 (1991).
Chang et al.; Abstract Replacing the First Epidermal Growth Factor Like Domain of Factor IX With That of Factor VII Enhances Clotting Activity,Thrombosis and Haemostasis Abstracts; 73:p 1202 (Jun. 1995).
Evans et al.; Canine hemophilia B resulting from a point mutation with unusual consequences;Proc. Natl. Acad. Sci. USA86:10095-10099 (Dec. 1989).
Geddes et al.; A Moderate Form of Hemophilia B is Caused by a Novel Mutation in the Protease Domain of Factor IX;The Journal of Biological Chemistry264 4689-4697 (1989).
Giannelli et al.; Haemophilia B: database of point mutations and short additions and deletions;Nucleic Acids Research18:4053-4059 (1990).
Ketterling et al.; The Rates of G:C→T:A and G:C→C:G Transversion at CpG Dinucleotides in the Human Factor IX Gene American J Human Genetics 54:831-835 (1994).
Kisiel et al.; Proteolytic Inactivation of Blood Coagulation Factor IX by Thrombin;Blood66:1302-1308 (1985).
Koeberl et al.; Functionally Important Regions of the Factor IX Gene Have a Low Rate of Polymorphism and a High Rate of Mutation in the Dinucleotide CpG;Am. J. Hum. Genet. 45:448-457 (1989).
Miyata et al.; Factor IX Amagasaki: A New Mutation in the Catlytic Domain Resulting in the Loss of Both Coagulant and Esterase Activities;Biochemistry30:11286-11291 (1991).
Chang Jin Li
Stafford Darrel W.
Bugaisky Gabrielle
Myers Bigel & Sibley & Sajovec
The University of North Carolina at Chapel Hill
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