Modified factor VIII cDNA

Chemistry: molecular biology and microbiology – Vector – per se

Reexamination Certificate

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C435S069100, C435S069600, C435S455000, C435S461000, C435S325000, C536S023100, C536S023500

Reexamination Certificate

active

06271025

ABSTRACT:

This invention is directed to a modified Factor VIII cDNA and its use for the improvement of the Factor VIII production.
Factor VIII (FVIII) is a plasma coagulation cofactor implicated in the activation of Factor X (FX). A decrease in the presence or activity of Factor VIII in blood stream leads to hemophilia A. The level of the decrease in Factor VIII activity is directly proportional to the severity of the disease (Foster and T. S., 1989; Kaufman, 1992; Vlot et al., 1998). The current treatment of hemophilia A consists of the replacement of the missing protein by plasma-derived or recombinant FVIII. Recombinant FVIII is produced in CHO or BHK cells after selection of the best producing clones and amplification of the FVIII cDNA copy number.
Several studies have stressed the low FVIII production level in different cellular systems: Biosynthesis of FVIII was shown to be regulated in at least three different levels. First, among the FVIII cDNA sequence two nucleotides stretches, localized in the A2 coding domain, were demonstrated to act as transcriptional silencers (Fallaux et al, 1996; Hoeben et al., 1995; Koeberl et al., 1995; Lynch et al., 1993). Second, FVIII protein synthesis is tightly regulated by several reticulum endoplasmic chaperones (BiP; Calreticulin; Calnexin; ERGIC-53). Many of these interactions retain FVIII in the cell and direct it through the cellular degradation machinery (Dorner et al., 1987; Nichols et al., 1998; Pipe et al., 1998). Third, once secreted FVIII is sensitive to protease degradation and needs to be protected by von Willebrand Factor (vWF) (Kaufman et al., 1989).
It is therefore a problem to develop improved processes which result in higher yields of FVIII. The present invention offers a solution to this problem by a modified FVIII cDNA.
SUMMARY OF THE INVENTION
According to this invention, a modified FVIII cDNA is available comprising a truncated FIX intron 1 inserted in at least one location of the FVIII cDNA, in which the B-domain of the wild-type FVIII cDNA has been deleted. In addition, the B-domain of the wild-type cDNA has been replaced by four arginines.


REFERENCES:
patent: 2 763 959 (1998-12-01), None
patent: WO 92/16557 (1992-10-01), None
patent: WO 94/19471 (1994-12-01), None
J. L. Plantier, et al., “A Combination of Truncated Factor IX Intron I Highly Improves FVIII Production,” Blood, vol. 94, No. 10 Supp. 1 Part 1, p. 454a, Forty -first Annual Meeting of the American Society of Hematology. Abstract No. 2021, (1999).
Abstract, WO 98/55639 (Dec. 10, 1998).
Kozak, M. “Recognition of AUG and Alternative Initiator Codons is Augmented by G in Position +4 but is not Generally Affected by the Nucleotides in Positions +5 and +6,”EMBO Journal,vol. 16, pp. 2482-2492 (1997).
Kurachi S., et al. “Role of Intron 1 in the Expression of the Human Factor IX Gene,”Journal of Biological Chemistry,vol. 270, pp. 5276-5281 (1995).
Lind, P., et al., “Novel Forms of B-domain-deleted Recombinant Factor VIII Molecules. Construction and Biochemical Characterization”,European Journal of Biochemsistry,vol. 232, pp. 19-27 (1995).
Lynch, C.M., et al. “Sequences in the Coding Region of Clotting Factor VIII Act as Dominant Inhibitors of RNA Accumulaton and Protein Production,”Human Gene Therapy,vol. 4, pp. 259-272 (1993).
Nichols, W.C., et al. “Mutations in the Er-Golgi Intermediate Compartment Protein ERGIC-53 Cause Combined Deficiency of Coagulation Factors V and VIII,”Cell,vol. 93, pp. 61-70 (1998).
Pipe, S.W., et al. “Differential Interaction of Coagulation Factor VIII and Factor V with Protein Chaperones Calnexin and Calreticulin,”Journal of Biological Chemistry,vol. 273, pp. 8537-8544 (1998).
Pittman, D.D., et al. “Biochemical, Immunological, and In Vivo Function Characterization of B-domain-deleted Factor VIII,”Blood,vol. 81, pp. 2925-2935 (1993).
Pittman, D.D., et al. “Role of the B-domain for Factor VIII and Factor V Expression and Function,”Blood,vol. 84, pp. 4214-4225 (1994).
Vlot, A.J., et al. “Factor VIII and von Willebrand Factor,”Thromb. Haemost.,vol. 79, pp. 456-465 (1998).
Dorner, A.J., et al. “The Relationship of N-Linked Glycosylation and Heavy Chain-binding Protein Association with the Secretion of Glycoproteins,”Journal of Cell Biology,vol. 105, pp. 2665-2674 (1987).
Fallaux, F.J., et al. “The Human Clotting Factor VIII cDNA Contains an Autonomously Replicating Sequence Consensus—and Matrix Attachment Region-like Sequence that Binds a Nuclear Factor, Represses Heterologuos Gene Expression, and Mediates the Transcriptional Effects of Sodium Butyrate,”Mol. Cell. Biol.,vol. 16, pp. 4264-4272 (1996).
Foster, P.A., et al. “Factor VIII Structure and Function,”Blood Reviews,vol. 3, pp. 180-191 (1989).
Hoeben, R.C. et al. “Expression of the Blood-Clotting Factor-VIII cDNA is Repressed by a Transcriptional Silencer Located in its Coding Region,”Blood,vol. 85, pp. 2447-2454 (1995).
Kaufman, R.J. “Biological regulation of Factor VIII Activity,”Annual Reviews of Medicine,vol. 43, pp. 325-339 (1992).
Kaufman, R.J., et al. “Effects of von Willebrand Factor Coexpression on the Synthesis and Secretion of Factor VIII in Chinese Hamster Ovary Cells,”Mol. Cell. Biol.,vol. 9, pp. 1233-1242 (1989).
Koeberl, D.D., et al. “Sequence within the Coding Regions of Clotting Factor VIII and CFTR Block Transcriptional Elongation,”Human Gene Therapy,vol. 6, pp. 469-479 (1995).

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