WO2001022810A2

WO2001022810A2 - Transgenic animals expressing von willebrand factor (vwf) and vwf-related polypeptides

Info

Publication number: WO2001022810A2
Application number: PCT/US2000/026546
Authority: WO
Inventors: William Hugold Velander
Original assignee: Us Transgenics, Inc.
Priority date: 1999-09-28
Filing date: 2000-09-28
Publication date: 2001-04-05
Also published as: AU7619500A; WO2001022810A3

Abstract

The invention provides, among other things, transgenic vWF ('TrG-vWF') and vWF-related ('TrG-vWF*Rel') polypeptides and proteins, compositions comprising TrG-vWF and/or TrG-vWF*Rel or both, transgenic organisms for making TrG-vWF or TrG-vWF*Rel, methods for making the transgenic organisms, methods for making TrG-vWF and/or TrG-vWF*Rel-comprising compostions and for further purifying TrG-vWF or TrG-vWF*Rel- from the compositions. Illustrative embodiments of the invention particularly provide transgenic mammals that express an exogenous gene for vWF and excrete the vWF encoded by the gene into their milk. In a highly particular illustrative embodiment in this regard the invention provides transgenic female pigs that express vWF in their milk. In this regard, the invention relates particularly to female pigs having stably incorporated in their genomes a DNA comprising a region that encodes vWF operably linked to a mammary gland-specific promoter. Further in this regard the invention relates to the milk containing the TrG-vWF or TrG-vWF*Rel and to TrG-vWF and/or IrG-vWF*Rel containing compositions derived from the milk. It also relates to novel TrG-vWF or TrG-vWF*Rel polypeptides and proteins, and compositions comprising the same, obtained by expression of genes encoding vWF and vWF-related polypeptides in transgenic organisms. In addition to providing TrG-vWF or TrG-vWF*Rel, it relates to using TrG-vWF and TrG-vWF*Rel and to methods and compositions therefor.

Description

TRANSGENIC vWF and vWF-RELATED POLYPEPTIDES,

TRANSGENICS, METHODS, COMPOSITIONS, USES

AND THE LIKE RELATING THERETO

This application claims the benefit of U.S. Provisional Application No. 0/157,134, filed September 28, 1999.

BACKGROUND

Physiological Activity and Functions of vWF von Willebrand factor ("vWF") is an important plasma glycoprotein that plays two important roles in hemostasis. It links platelet membrane receptors to components of the sub-endothelial connective tissue, such as collagen, and in this regard is essential for platelet adhesion and accretion at sites of vascular damage. In addition, vWF associates non-covalently with factor VIII, forming a complex that protects factor VIII, from degradation in the circulation and helps to localize factor VIII at sites of injury. (For review see, for instance, Sadler, J. Biol. Chem. 266(34): 22777-22780 (1991) and TEXTBOOK OF HEMATOLOGY, 2^nd Ed.; S. McKenzie, Williams & Wilkins,

Baltimore (1996) particularly in this regard, pages 553 and 564-570, each of which is incorporated herein by reference in its entirety in parts pertinent to vWF, particularly as to general aspects of vWF structure, activities, functions, physiological functioning, and roles in health and disease).

VWF Dysfunction and Disease vWF is required for platelet adhesion to sites of injury and for persistence and proper localization of circulating factor VIII. Defects of vWF structure or metabolism contribute to a variety of disorders and diseases. The most severe forms of vWF deficiency are life-threatening or fatal. Disorders involving vWF reflect its different physiological activities. Disorders of vWF metabolism thus generally are characterized by prolonged bleeding times, adhesion-defective platelets, and deficient factor VIII activities. These underlying molecular and cellular deficiencies produce the characteristic clinical manifestations of vWF -related diseases. The impairment of hemostasis, for instance, typically manifests as mucosal bleeding, epistaxis, and, in women, hemorrhagia. The impairment of Factor VIII function associated with vWF disease typically presents as postoperative soft tissue bleeding and joint bleeding. (See for instance Schwarz et al, Thromb. Haemost. 21(6): 571-576 (1997) and Lethagen et al., Ann. Med. 21: 641-651 (1995) each of which is incorporated herein by reference in its entirely particularly in parts pertinent to etiology, clinical manifestations and treatments of disorders in which vWF plays a causative or contributory role).

Aberrations in vWF are detected in approximately 1% of the population; but, only about 125 people per million exhibit symptoms that currently are accepted as clinically significant manifestations of an underlying vWF deficiency. Clinically significant manifestations of vWF deficiency have been broadly classified as Types I, II and III, recently re-categorized into Types 1, 2 and 3. (See Sadler et al., Blood & : 676- 679 (1994).

Type 1 is the most common type. It occurs in about 100 people per million. It is characterized by quantitative vWF deficiency, rather than deficiency of patients' vWF per se. It is an autosomal dominant trait with incomplete penetrance. Type 2 vWF disease is characterized by normal levels of vWF but decreased vWF function because of abnormalities in vWF itself, that typically result in a lack of high molecular weight multimers. Type 2 vWF disease, like Type 1, is an autosomal dominant trait that exhibits incomplete penetrance. Specific mutations responsible for Type 2 vWF disease include SNPs in the vWF promoter that cause under-expression of the vWF gene. (See Keightley et al., Blood 91(12): 4277-4283 (1999) which is herein incorporated by reference in its entirety particularly in parts pertinent to variations in the vWF gene locus associated with abnormal vWF plasma Ag levels). Type 3 vWF is the most severe form of vWF deficiency. It is characterized by an almost complete lack of vWF in the blood. It is an autosomal recessive inherited disorder found in only 0.5 to 5 persons per million. Homozygotes have severely impaired hemostatic function and are afflicted by bleeding episodes that can be fatal, and must be treated with blood, vWF-cryoprecipitate or vWF-containing plasma fraction to replace the missing vWF. Heterozygous individuals generally appear normal except for reduced levels of circulating vWF.

The same three types of vWF deficiencies occur in other mammals, such as dogs. (See PCT/US97/12606, International Publication Number WO 98/03683 of Venta et al. for DNA Encoding Canine vWF and Methods of Use which is incorporated herein by reference in its entirety particularly in parts pertinent to Canine vWF and to vWF in dogs and other mammals.)

Although the three types of vWF disease constitute all of the vWF deficiencies that currently are accepted as clinically significant, it has been suggested that undiagnosed vWF deficiencies contribute to or cause many other health problems. A prominent example in this regard is unexplained hemorrhagia. If only a fraction of unexplained hemorrhagia is due to an underlying vWF deficiency, then the prevalence of clinically significant vWF deficiencies in the population is much higher then that estimated cumulatively for vWF disease Types 1, 2 and 3. (See, for instance, Sadler, J.

Biol. Chem. 266(34): 22777-22780 (1991) and TEXTBOOK OF HEMATOLOGY, 2^nd Ed.; S. McKenzie, Williams & Wilkins, Baltimore (1996) particularly in this regard, pages 553 and 564-570 and Schwarz et al, Thromb. Haemost. 11(6): 571-576 (1997) each of which is incorporated herein by reference in its entirety in parts pertinent to vWF particularly as to clinically significant manifestations of vWF deficiencies.)

Therapies for vWF-Related Disorders

Development of vWF disease-specific therapeutic agents has not progressed as quickly or as far as the development of agents to treat other, more commonly recognized, bleeding disorders, such as Factor VIII deficiencies. Presently, mild vWF disease typically is treated with desmopressin (also referred to as DDAVP), an analog of vasopressin. Severe deficiencies of vWF are treated by replacement therapy. The complexity of vWF itself, its scarcity and complexation with Factor VIII in natural sources, and uncertainty about other factors necessary for its in vivo activities have hindered development of therapeutic agents based on purified vWF from blood or other physiological sources. The size and genetic complexity of vWF genomic and cDNA, as well as its complicated post-translational processing have prevented development of agents based on pure vWF produced using recombinant DNA techniques. The replacement preparations presently used to treat vWF thus are limited to cryoprecipitates and fractionated blood products. Moreover, the concentrates used to treat severe vWF disease in the United States and elsewhere typically are made to treat factor VIII deficiencies, not deficiencies of vWF, and may not even be approved by regulatory authorities specifically for treating vWF disease. The plasma-derived products vary greatly in constitution and in quality. Moreover, they often pose a significant risk of contamination with pathogens, such as HIV and hepatitis, particularly the untreated cryoprecipitates still in use in some parts of the world. The complexity of serum-derived products, and their disadvantages, has engendered controversy over their use and uncertainty about the properties necessary for a safe and effective vWF replacement therapeutic that remains unsettled. (See for instance Menache and Aronson, Thromb. Haemost. 11(6): 566-570 (1997), Aledort, Thromb. Haemost. 11(6): 562-565 (1997) and Schwarz et al, Thromb. Haemost. 11(6): 571-576 (1997) each of which is incorporated herein by reference in its entirety in parts pertinent to vWF particularly as to treatments of vWF-related disorders.)

Synthesis and Physiology of vWF vWF in humans ordinarily is synthesized by vascular endothelial cells and megakaryocytes, initially as a single glycosylated polypeptide of 2813 amino acids with a molecular weight of about 360,000 Daltons. The full length polypeptide generally is referred to as pre-pro-vWF.

The "pre" portion of pre-pro-vWF is formed by the first 22 amino-terminal amino acids, which act as a signal peptide and are removed upon translocation of the newly synthesized pre-pro-vWF across the endoplasmic reticulum. The remaining pro- vWF, also called vWF antigen II, totals 2791 amino acids in length, made up of the 741 amino terminal amino acids that form the "pro" peptide, and the 2050 amino acids that make up the mature vWF polypeptide. pro-vWF undergoes a complex process of post-translational processing after entering the endoplasmic reticulum. For one, pro-vWF is extensively glycosylated by both Asn-linked and Thr/Ser-linked glycans. And, some of the Asn-linked oligosaccharides are sulfated.

In addition, vWF not only undergoes further proteolysis but also is polymerized. First, pairs of pro-vWF polypeptides are joined into dimers by disulfide bonds between cysteine residues near their carboxyl termini. Two or more pro-vWF dimers then are joined into multimers held together by one or more disulfide bonds between the cysteine residues located between amino acids 459 and 464 of mature vWF. The 741 amino acid pro-peptide is cleaved from some, but apparently not all, of the pro-vWF polypeptides in the multimers. vWF thus normally is synthesized as a mixture of multimers ranging in size from about 500 kDa (the dimer of two mature vWF polypeptides) to over 20 million Daltons (multimers of 40 or more dimers).

Circulating vWF exhibits great molecular weight diversity, attributable directly to numerical variation in the number of dimers in vWF multimers, to the differing composition of vWF and pro-vWF monomers among the multimers of a given multiplicity, and to variation in the type and extent of glycosylation.

It appears that the higher molecular weight multimers are required for normal physiological activity of vWF. It is likely that this is partly due to the increased number of ligand binding sites in larger multimers. It also is likely that the extensible nature of high molecular weight vWF multimer is essential to vWF-dependent platelet binding at the high shear rates characteristic of microcirculatory blood flow. In fact, vWF-dependent binding is not characteristic of platelets under other conditions.

(For review see, for instance, Sadler, J. Biol. Chem. 266(34): 22777-22780

(1991) and TEXTBOOK OF HEMATOLOGY, 2^nd Ed.; S. McKenzie, Williams & Wilkins, Baltimore (1996) particularly in this regard, pages 553 and 564-570, each of which is incorporated herein by reference in its entirety in parts pertinent to vWF, particularly as to synthesis and physiology of vWF.)

Structure of vWF Genomic and cDNAs

The gene for human vWF is located on chromosome 12. 178,000 bases of DNA containing the human vWF gene, roughly 0.1% of the chromosome, has been cloned and sequenced. The coding region of the gene is divided into 52 exons, only some of which correspond to the distinct domains of the protein. The extraordinary size of the vWF gene thus far has presented a difficult obstacle to its use for routine expression of vWF, for any purpose. cDNAs for human and canine vWF have been cloned and sequenced. Even the cDNAs are quite large. The human vWF cDNA sequences of Sadler et al. and Pannekoek et al. and the canine vWF sequence of Venta et al. are very similar and all are nearly 9,000 bases long. The length of the cDNAs make manipulation and expression of vWF difficult; although, they do not pose the same difficulty as the genomic clones. (See Sadler et al, Cold Spring Harb. Symp. Quant. Biol. LI: 515-521

(1986), European Patent Application 0 197 592 Al of Pannekoek et al. on "Preparation of human von Willebrand factor by recombinant DNA" and PCT US97/12606, International Publication Number WO 98/03683 of Venta et al. for DNA Encoding Canine vWF and Methods of Use each of which is incorporated herein by reference in its entirety as to the foregoing particularly in parts pertinent to cloned vWF and vWF- related genomic DNAs and cDNAs.)

Limitations and Problems of Current vWF Therapeutics

The large size of vWF, its structural and functional complexity, its post- translational and dimer-polymer heterogeneity, and its scarcity have made it hard to obtain vWF from natural sources in the amounts required for replacement therapy. The same factors, compounded by the large sizes of cDNAs and genomic DNA necessary to encode vWF, to date have been an insurmountable barrier to recombinant expression of physiologically active vWF and purification of recombinantly produced vWF in clinically useful amounts for replacement therapy. In consequence, patients afflicted with vWF disease requiring replacement vWF for survival must suffer the vagaries and accept the risks of current vWF replacement therapeutics derived from plasma. Those who do not require replacement vWF for survival but derive from it significant health benefits also must accept the deficiencies of currently available vWF-containing plasma concentrates. And many others who might derive substantial but more discretionary health and quality of life benefits presently must forego replacement or supplementary vWF treatment because the deficiencies and risks of current vWF-containing therapeutics outweigh the potential benefits, even though they are substantial and in some cases profound. There therefore clearly exists a need for improved methods for making and using vWF and vWF -related polypeptides and for improved vWF and vWF-related polypeptides, compositions of the same for treating vWF deficiencies, and methods for treating vWF deficiencies utilizing vWF, among other things.

DESCRIPTION OF THE INVENTION

Among other things, the invention described herein in certain illustrative and preferred embodiments is directed to the following.

The invention is directed to a transgenic organism comprising an introduced genetic construct that engenders production of a vWF or vWF-related polypeptide. The invention is also directed to a transgenic organism as above, wherein the construct engenders production of the vWF or vWF-related polypeptide in specific cells.

The invention is also directed to a transgenic organism as above, wherein the vWF or vWF-related polypeptide accumulates in a specific tissue or bodily compartment.

The invention is also directed to a transgenic organism as above, wherein the vWF or vWF-related polypeptide accumulates in a bodily fluid.

The invention is also directed to a transgenic organism as above, wherein the organism is a non-human mammal.

The invention is also directed to a transgenic organism as above, wherein the mammal is mouse, rat, hamster, rabbit, pig, sheep, goat, cow or horse.

The invention is also directed to a transgenic organism as above, wherein the organism is mouse, pig, sheep, goat or cow. The invention is also directed to a transgenic organism as above, wherein the organism is pig.

The invention is also directed to a transgenic organism as above, wherein the vWF or vWF -related polypeptide accumulates in the milk of females.

The invention is also directed to a transgenic organism as above, wherein the vWF or vWF-related polypeptide produced in the organism when isolated and purified has specific activity 50% to 75% of that of purified human vWF.

The invention is also directed to a transgenic organism as above, wherein the specific activity is 70% to 85% of that of purified human vWF from pooled human plasma. The invention is also directed to a transgenic organism as above, wherein the specific activity is 80% to 95% of that of purified human vWF from pooled human plasma.

The invention is also directed to a transgenic organism as above, wherein the specific activity is 85% to 98% of that of purified human vWF from pooled human plasma.

The invention is also directed to a transgenic organism as above, wherein the specific activity is 90% to 105% of that of purified human vWF from pooled human plasma. The invention is also directed to a transgenic organism as above, wherein the specific activity is 75% to 125% of that of purified human vWF from pooled human plasma.

The invention is also directed to a transgenic organism as above, wherein the specific activity is 50% to 110%) of that of purified human vWF from pooled human plasma.

The invention is also directed to a transgenic organism as above, wherein the specific activity is greater than that of purified human vWF from pooled human plasma.

The invention is also directed to a transgenic organism as above, wherein activity is determined by a platelet binding assay.

The invention is also directed to a transgenic organism as above, wherein activity is determined by a Factor VIII binding assay.

The invention is also directed to a transgenic organism as above, wherein activity is determined by both a platelet binding assay and a Factor VIII binding assay. The invention is also directed to a transgenic organism as above, wherein activity is determined by one or a combination of the following assays, a collagen binding assay, a collagen binding assay, a heparin binding assay, a sulfatide binding assay, a GPlb, binding assay, a botrocetin binding assay and a GPIIb-IIIa binding assay. The invention is also directed to a transgenic organism as above, wherein the purified human vWF is calibrated against an accepted international vWF activity standard.

The invention is also directed to a transgenic organism as above, wherein the vWF or vWF related polypeptide comprises a region having an amino acid sequence 80% to 100% identical to that of a mature mammalian vWF.

The invention is also directed to a transgenic organism as above, wherein the vWF or vWF related polypeptide comprises a region having an amino acid sequence 90% to 100% identical to that of a mature mammalian vWF.

The invention is also directed to a transgenic organism as above, wherein the vWF or vWF related polypeptide comprises a region having an amino acid sequence

95% to 100%) identical to that of a mature mammalian vWF. The invention is also directed to a transgenic organism as above, wherein the vWF or vWF related polypeptide comprises a region having an amino acid sequence 98%) to 100%) identical to that of a mature mammalian vWF.

The invention is also directed to a transgenic organism as above, wherein the mature mammalian vWF is mature human vWF.

The invention is also directed to a transgenic organism as above, wherein the vWF or vWF -related polypeptide comprises a region having the amino acid sequence of mature human vWF.

80% to 100%) identical to that of a mature mammalian vWF.

The invention is also directed to a transgenic organism as above, wherein the vWF or vWF related polypeptide comprises a region having an amino acid sequence 90% to 100%) identical to that of a mature mammalian vWF. The invention is also directed to a transgenic organism as above, wherein the vWF or vWF related polypeptide comprises a region having an amino acid sequence 95% to 100% identical to that of a mature mammalian vWF.

The invention is also directed to a transgenic organism as above, wherein the mammalian vWF is mature human vWF. The invention is also directed to a transgenic organism as above, wherein the vWF or vWF -related polypeptide comprises a region having the amino acid sequence of mature human vWF.

The invention is also directed to a transgenic organism as above, wherein the vWF or vWF-related polypeptide is human pre-pro-vWF, human pro-vWF or mature human vWF.

The invention is also directed to a transgenic organism as above, wherein the introduced genetic construct comprises a promoter operatively linked to the region encoding vWF or a vWF-related polypeptide, wherein further the promoter is selected from the group consisting of the promoters of whey acidic protein genes, casein genes, lactalbumin genes and .beta.lactoglobulin genes.

The invention is also directed to a transgenic organism as above, wherein the promoter is a whey acidic protein promoter or a beta.lactoglobulin promoter. The invention is also directed to a transgenic organism as above, wherein the promoter is a whey acidic protein promoter.

The invention is also directed to a transgenic organism as above, wherein the promoter is the mouse whey acidic protein promoter or the rat whey acidic protein promoter.

The invention is also directed to a transgenic organism as above, wherein the promoter is a long whey acidic protein promoter.

The invention is also directed to a transgenic organism as above, wherein the promoter is the mouse long whey acidic protein promoter. The invention is also directed to a composition comprising vWF or a vWF- related polypeptide produced in a transgenic organism as above mentioned.

The invention is also directed to a composition as above, wherein the vWF or vWF -related polypeptide produced in milk of a non-human transgenic female mammal.

The invention is also directed to a composition as above, wherein the composition is milk of the transgenic mammal.

The invention is also directed to a composition as above, wherein the composition is derived from milk of the transgenic mammal.

The invention is also directed to a vWF or vWF-related polypeptide isolated from a transgenic organism as above mentioned. The invention is also directed to a vWF or vWF-related polypeptide isolated from a transgenic organism as above, that differs in its post-translational modification from that of naturally occurring human vWF.

The invention is also directed to a human vWF polypeptide isolated from a transgenic organism as above, that differs in post-translational modification from human vWF isolated from natural sources but that has the same platelet binding activity.

The invention is also directed to a human vWF polypeptide isolated from a transgenic organism as above, that differs in post-translational modification from human vWF isolated from natural sources but that has the same Factor VIII binding activity. The invention is also directed to a vWF or vWF-related polypeptide isolated from a transgenic organism as above, that differs in its glycosylation from that of human vWF isolated from natural sources.

The invention is also directed to a vWF or vWF-related polypeptide isolated from a transgenic organism as above, that differs in its glycosylation from that of human vWF isolated from natural sources but that has the same platelet binding activity.

The invention is also directed to a vWF or vWF-related polypeptide isolated from a transgenic organism as above, that differs in its glycosylation from that of human vWF isolated from natural sources but that has the same Factor VIII binding activity.

The invention is also directed to a composition for treating a patient suffering from vWF deficiency comprising vWF or a vWF-related polypeptide as above mentioned. The invention is also directed to a method for treating a patient suffering from a vWF deficiency comprising a step of administering to the patient a composition comprising a vWF or vWF-related polypeptide as above mentioned.

The invention is also directed to a method for producing vWF or a vWF-related polypeptide comprising the step of producing the vWF or vWF-related polypeptide in a transgenic organism as above mentioned.

Notwithstanding the apparent disadvantages of currently available methods for obtaining vWF preparations that provide vWF activity for clinical and other uses, the present invention provides, among other things, transgenic organisms that express TrG- vWF and TrG-vWF*Rel that provide useful vWF activity. A "vWF-related" polypeptide is one that provides a source of vWF as disclosed herein. Such a molecule may be inactive or have lower activity and can be activated, for example, by processing or multimerization to produce a polypeptide structure having the activity of vWF as disclosed herein. The person of ordinary skill in the art would be aware that the invention provides a sequence with vWF activity, which sequence is active or can be activated to provide this activity from a vWF-related molecule by any of the mechanisms by which vWF is modified to produce an active (or more active) vWF. Furthermore, it would be understood that such a vWF-related sequence could be manufactured so as to provide an activation mechanism differing from those known in the art and/or normally occurring in nature but which, nevertheless, provides an active vWF. Related peptides also include sequence variants, such as allelic variants, homologs, and orthologs, having vWF activities, as well as chemically synthesized and recombinantly-made non-natural variants. The invention also provides, among other things, methods for obtaining TrG-vWF and TrG-vWF*Rel from the transgenic organisms, compositions comprising TrG-vWF and TrG-vWF*Rel, and uses thereof, to name a few. These and other aspects of the invention are illustrated by way of illustrative, non-limiting general and specific examples described herein below.

Methods for Making Transgenic Organisms Transgenic organisms that express TrG-vWF and TrG-vWF*Rel may be produced in accordance with the invention as described herein using a wide variety of well-known techniques, such as those described in GENETIC ENGINEERING OF ANIMALS, Ed. A. Puhler, VCH Publishers, New York (1993) and in more detail in Volume 18 in Methods in Molecular Biology: TRANSGENESIS TECHNIQUES, Eds. D. Murphy and D. A. Carter, Humana Press, Totowa, New Jersey (1993) both of which are incorporated herein by reference in their entireties, particularly as to the foregoing in parts pertinent to methods for making transgenic organisms that express TrG-vWF and TrG-vWF*Rel, especially in milk. (See also for instance Lubon et al, Transfusion Medicine Reviews X(2): 131-141 (1996) which is incorporated herein by reference in its entirety, particularly as to the foregoing in parts pertinent to methods for making transgenic organisms. A transgenic organism, in mature and embryonic forms, comprises cells containing an introduced genetic construct, e.g., as illustrated by the embodiments herein described.

In particular, transgenic mammals, such as mice and pigs, that express TrG- vWF and TrG-vWF*Rel in accordance with certain preferred embodiments of the invention, can be produced using methods described in MANIPULATING THE MOUSE EMBRYO, Hogan et al, Cold Spring Harbor Press (1986); Krimpenfort et al, Bio/Technology 9_: S44 et seq. (1991); Palmiter et al, Cell 2: 343 et se<7. (1985); GENETIC MANIPULATION OF THE EARLY MAMMALIAN EMBRYO, Kraemer et al, Cold Spring Harbor Press, Cold Spring Harbor, NY (1985); Hammer et al,

Nature 1L5_: 680 et seq. (1985); U.S. Patent number 4,873,191 of Wagner et al. for Genetic Transformation ofZygotes, and U.S. Patent number 5,175,384 of Krimpenfort et al. for Transgenic Mice Depleted in Mature T-Cells and Methods for Making Transgenic Mice, each of which is incorporated herein by reference in its entirety, particularly as to the foregoing in parts pertinent to producing transgenic mammals by introducing DNA constructs for polypeptide expression into cells or embryos. DNA:RNA constructs also may be used in for transgenic expression in this regard, in much the same way as DNA constructs. The use of DNA:RNA constructs suitable to this end is particularly is described in for instance U.S. Patent number 5,565,350 of Kmiec for Compounds and Methods for Site Directed Mutations in Eukaryotic Cells and U.S. Patent number 5,756,325 of Kmiec also for Compounds and Methods for Site Directed Mutations in Eukaryotic Cells each of which is incorporated by reference herein in its entirety particularly as to the foregoing in parts pertinent to targeted genetic manipulations useful to produce transgenic organisms that express TrG-vWF and TrG-vWF*Rel.

For example, transgenic organisms of the present invention can be produced by introducing into eggs or developing embryos one or more genetic constructs that engender expression of TrG-vWF and TrG-vWF*Rel as described herein. In certain preferred embodiments of the invention in this regard, DNAs that comprise czs-acting transcription controls for expressing vWF are operably linked to a region encoding vWF are highly preferred. RNA-DNA hybrids similarly are preferced in some embodiments in this regard. Also useful in this regard are constructs that engender non-natural expression of one or more endogenous genes that engender production of TrG-vWF or TrG-vWF*Rel, particularly an endogenous gene for vWF. One or more DNA or RNA:DNA hybrids or the like may be used alone or together in this regard, to make transgenic organisms useful in the invention. Also especially preferred in this regard are constructs that are stably incorporated in the genome of germ line cells of the mature organism and inherited in normal, Mendelian fashion by reproduction thereof. Constructs that comprise operable signal sequences that effectuate transport of the TrG- vWF or TrG-vWF*Rel into a targeted compartment of an organism, such as a tissue or fluid, are further preferred in certain embodiments in this regard. Standard techniques, as well as unusual and new techniques for making transgenic organisms generally can be used to make transgenic organisms that express TrG-vWF and or TrG-vWF*Rel in accordance with the invention. Useful techniques in this regard include those that introduce genetic constructs by injection, infection, transfection, such as calcium phosphate transfection, using cationic reagents, using sperm or sperm heads or the like, lipofection, liposome fusion, electroporation, and ballistic bombardment, to name just a few known techniques. Useful techniques include those that involve homologous recombination, such as those that can be employed to achieve targeted integration, and those that do not involve homologous recombination, such as disclosed below. Constructs can be introduced, using these and other methods, into pluripotent cells, totipotent cells, germ line cells, eggs, embryos at the one cell stage, and embryos at several cell stages, among others, to make transgenic organisms of the invention. In these regards, among others, they may be introduced into pronuclei, nuclei, cytoplasm or other cell compartments, or into extracellular compartments of multicellular systems, such as multicellular, developing embryos, to make transgenic organisms of the invention.

In a preferred method, developing embryos can be infected with retroviral vectors and transgenic animals can be formed from the infected embryos. In a particularly preferred method DNAs in accordance with the invention are injected into embryos, preferably at the single-cell stage. In some particularly preferred embodiments in this regard, DNA is injected in the pronucleus of a one-cell embryo. In other preferred embodiments in this regard, DNA is injected into the cytoplasm of a one cell embryo. In yet another particularly preferred embodiment in this regard, DNA is injected into an early stage, several cell embryo In these regards, in like manner, in yet other preferred embodiment, one or more DNA-RNA hybrids is injected into an embryo, particularly a single-cell embryo, the pronucleus or the cytoplasm of a fertilized egg or single-cell embryo or into an early stage multi-cell embryo, either into the cells or into the embryo extracellularly.

Constructs for Transgenic Expression

Certain aspects of the invention relate to the introduction into organisms of genetic constructs that engender expression of TrG-vWF and TrG-vWF*Rel. Among those that are useful in the invention in this regard are polynucleotide constructs that provide a DNA sequence encoding TrG-vWF and TrG-vWF*Rel of the invention operably linked to cts-acting signals necessary for expression in a transgenic organism and, in certain preferred embodiments, for transport of a translation product encoded by the construct into a particular compartment of the organism. Among preferred polynucleotides for constructs in preferred embodiments of the invention are DNA or RNA.DNA hybrids. Among particularly preferred embodiments in this regard are DNA polynucleotides. The genetic constructs may be a single polynucleotide or several polynucleotides when introduced into a cell or embryo or the like to form a transgenic animal in accordance with the invention. Particularly preferred are single chain, double-stranded DNA polynucleotides in this regard. Also preferred are DNA-RNA hybrid polynucleotides. When more than one polynucleotide is used in this regard, they generally combine with one another and/ or with endogenous genetic elements of the host organism, as a result of in vitro or in vivo processes, to form a construct that then engenders transgenic expression of TrG-vWF and TrG-vWF*Rel in the host organism.

In certain particularly preferred embodiments of the invention, preferred constructs provide a polynucleotide sequence encoding TrG-vWF and TrG-vWF*Rel of the invention, operably linked to the cw-acting signals necessary for expression in mammary gland cells and for secretion into milk of a non-human female transgenic mammal. Particularly highly preferred in this regard are cw-acting signals that provide efficient expression and or for expression and secretion into milk of highly active TrG- vWF and TrG-vWF*Rel with little or no expression elsewhere in the organism. DNA and RNA:DNA hybrids are particularly preferred polynucleotides in this regard. DNA is especially prefened.

VWF and vWF-Related Polypeptides

The transgenic organisms of the present invention comprises an introduced genetic construct that engenders production of a vWF or vWF-related polypeptide.

Accordingly, the invention encompasses transgenic organisms containing variants and fragments of the native human enzyme, said variants and fragments retaining one or more of the structural and/or functional properties of the enzyme, and specifically those structures and functions especially specific to human vWF. Thus, the structure and various functions, including assays for assessing such functions, are described in detail below. Structure

In a particular aspect the invention provides human vWF and vWF-related polypeptides produced in transgenic organisms, referred to herein as TrG-vWF and TrG-vWF*Rel. Particular preferred embodiments in this regard provide TrG-vWF having the amino acid sequence of naturally occurring human vWF. The invention further provides vWF -related polypeptide, proteins and protein preparations that provide vWF activity. Preferred embodiments in this regard provide vWF and vWF- related polypeptides that comprise mature vWF having the amino acid sequence of naturally occurring mature vWF. Especially preferred embodiments in this regard provide vWF -related polypeptides that upon processing or treatment in vitro provide dimers and multimers similar in both structure and distribution to naturally occurring human vWF. In another aspect, among such preferred embodiments are vWF and vWF-related polypeptides that provide TrG-vWF and TrG-vWF*Rel-more resistant to protease degradation in mammary epithelial cells and milk than human vWF itself. Preferred embodiments of the invention in this regard in particular provide TrG- vWF and TrG-vWF*Rel that are homologous to human vWF and can form dimers and multimers that provide physiological activity of human vWF. Particularly preferred polypeptides in this regard comprise a region that is 70% or more, especially 80% or more, more especially 90% or more, yet more especially 95% or more, particularly 97% or more, more particularly 98% or more, yet more particularly 99% or more identical in amino acid sequence to the amino acid sequence of naturally occurring human vWF.

Identity in this regard can be determined using a variety of well known and readily available amino acid sequence analysis software. Preferred software include those that implement the Smith- Waterman algorithms, considered a satisfactory solution to the problem of searching and aligning sequences. Other algorithms also may be employed, particularly where speed is an important consideration. Commonly employed programs for alignment and homology searching DNAs, RNAs and polypeptides that can be used in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE and MPSRCH, the latter being an implementation of the Smith- Waterman algorithm for execution on massively parallel processors made by MasPar. The BLASTN, BLASTX and BLASTP programs are among preferred programs for homology determinations, the former for polynucleotide sequence comparisons and the latter two for polypeptide sequence comparisons — BLASTX for comparison of the polypeptide sequences from all three reading frames of polynucleotide sequence and BLASTP for a single polypeptide sequence. BLAST provides several user definable parameters that are set before implementing a comparison, including the following. (1 ) A value is set for E to establish the number of High Scoring Segment Pairs expected by chance. (2) A value is set for S to establish the cut-off score for reporting a High Scoring Segment Pair, i.e., for listing a segment pair as a significant match. Usually S is calculated from E.

The values of E and S calculated for a given search string will be different on different databases. Accordingly, the values chosen for E and for the S cut off often are different for different databases. To normalize between different databases a parameter called Z is used. While the use of sophisticated techniques for setting E and S are entirely consistent with the present invention, a presently preferred method for determining similarity and homology or sequences using BLAST is to set S to the default value (10) and to calculate E from the default value of S using the default setting in the BLAST programs being employed.

Identity and homology determining methods are discussed in, for instance, GUIDE TO HUMAN GENOME COMPUTING, Ed. Martin J. Bishop, Academic

Press, Harcourt Brace & Company Publishers, New York (1994), which is incorporated herein by reference in its entirety with regard to the foregoing particularly in parts pertinent determining identity and or homology of amino acid or polynucleotide sequences, especially Chapter 7. The BLAST programs are described in Altschul et al., "Basic Local Alignment Research Tool", J Mol Biol 211: 403-410 (1990), which is incorporated by reference herein in its entirety. Additional information concerning sequence analysis and homology and identity determinations are provided in, among many other references well known and readily available to those skilled in the art: NUCLEIC ACID AND PROTEIN SEQUENCE ANALYSIS: A PRACTICAL APPROACH, Eds. M. J. Bishop and C. J. Rawings, IRL Press, Oxford, UK (1987);

PROTEIN STRUCTURE: A PRACTICAL APPROACH, Ed. T. E. Creighton, IRL Press, Oxford, UK (1989); Doolittle, R. F.: "Searching through sequence databases" Met Enz. 183_: 99-110 (1990); Meyers and Miller: "Optimal alignments in linear space" Comput. Applica. in Biosci 4: 11-17 (1988); Needleman and Wunsch: "A general method applicable to the search for similarities in amino acid sequence of two proteins" JMol Biol 4Α: 443-453 (1970) and Smith and Waterman "Identification of common molecular subsequences" J Mol Biol 142: 1950 et seq. (1981), each of which is incorporated herein by reference in its entirety with reference to the foregoing particularly in parts pertinent to sequence comparison and identity and homology determinations.

Orthologs, homologs, and allelic variants and other homologous sequences and fragments thereof can be identified using methods well known in the art. These variants can readily be identified as being able to hybridize under stringent conditions, to a vWF nucleotide sequence or a fragment of the sequence. It is understood that stringent hybridization does not indicate substantial homology where it is due to general homology, such as poly A sequences, or sequences common to all or most proteins or all natural vWF variants. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a polypeptide at least about 60-65%) homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%) or more identical to each other remain hybridized to one another. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference. One example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1 % SDS at 50-65°C. In another non-limiting example, nucleic acid molecules are allowed to hybridize in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more low stringency washes in 0.2X SSC/0.1% SDS at room temperature, or by one or more moderate stringency washes in 0.2X SSC/0.1% SDS at 42°C, or washed in 0.2X SSC/0.1% SDS at 65°C for high stringency. In one embodiment, an isolated nucleic acid molecule that hybridizes under stringent conditions to the sequence of SEQ ID NO 1 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

As understood by those of ordinary skill, the exact conditions can be determined empirically and depend on ionic strength, temperature and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS. Other factors considered in determining the desired hybridization conditions include the length of the nucleic acid sequences, base composition, percent mismatch between the hybridizing sequences and the frequency of occurrence of subsets of the sequences within other non-identical sequences. Thus, equivalent conditions can be determined by varying one or more of these parameters while maintaining a similar degree of identity or similarity between the two nucleic acid molecules.

Activity

In another aspect, preferred embodiments of the present invention relate to transgenic vWF and vWF-related polypeptides that have vWF activities, particularly vWF specific activities. vWF specific activity commonly is expressed as the ratio of the activity of sample in a vWF activity assay to the amount of vWF or vWF-related polypeptide in the sample.

Assay of vWF, vWF Reference Preparations The amount of vWF in a sample can be determined by a number of well known methods. Among preferred methods are those that determine the amount of vWF by vWF-specific immunoassay. Among particularly preferred methods in this regard are vWF-specific immunosorbent assays, particularly enzyme-linked immunosorbent assays ("ELISAs"). Among especially preferred ELISAs in this regard are widely employed ELISAs for measuring plasma vWF-Ag; i.e., the amount of vWF in plasma determined as the amount of antigen in a sample that binds to a vWF-specific antibody. A specific preferred assay of this type is a commercial assay available as a kit that employs vWF-Ag ELISA that employs a polyclonal goat anti-human vWF antibody to specifically bind to human vWF in the sample (Affinity Biologicals, Hamilton, Ontario, Canada).

The amount of vWF in the sample is determined by comparison of the results of the ELISA carried out on the sample with the results of the same assay carried out on a reference preparation. The results obtained using reference preparations provide a standard curve for interpolating the amount of vWF in a sample from the amount of the signal generated by the ELISA assay. Among preferred reference preparations for determining the amount of vWF in a sample is CryoCheck™, a commercial preparation readily available for purchase from Precision Biologicals, Dartmouth, Nova Scotia, Canada. CryoCheck™ is used widely to assay cryoprecipitates to assess their content and quality. Hemostatic parameters of the preparations in the CryoCheck™ reference standard are quantitatively determined and calibrated against the WHO International Standard for vWF Ag (recently, Lot 91/666). Like other very highly preferred embodiments in this regard, results obtained using CryoCheck™ and calibrated by the International Standard can be compared to other results that also have been calibrated against he International Standard.

One unit of vWF-Ag, vWF commonly is defined for convenience the amount present in 1 ml of a reference preparation of pooled normal plasma. For an example of vWF-Ag measurement using the aforementioned ELISA and standard preparation see for instance Keightley et al, Blood 22(12): 4277-4283 (1999) which is incorporated herein by reference in its entirety particularly in parts pertinent to determining the amount of vWF in plasma and other samples.

Similar assay systems and kits, and reference standards are available from other venders, as well, such as the Aserachrom vWF assay from Boehringer Mannheim. (For measurement of vWF produced from natural sources and in CHO cells using this assay see for instance Fischer et al, Cell. Molec. Life Sci. 51: 943-950 (1997) which is incorporated herein by reference in its entirety particularly in parts pertinent to determining the amount of vWF in preparations derived from plasma and from cells expressing vWF by means of recombinant DNA techniques). Determination of vWF by goat anti-vWF ELISA from Affinity Biologicals,

(Hamilton, Ontario, Canada) calibrated against the CryoCheck™ standard sold by Precision Biologicals (Dartmouth, Nova Scotia, Canada) is presented illustratively in somewhat greater detail in Example 8. The Example also exemplified determining vWF using the Aserachrom kit from Boehringer Mannheim. Results for both systems are calibrated against a reference preparation in the example and then, by calculation, they are calibrated by indirect comparison with the International Standard vWF.

Among the vWF activities of preferred embodiments of the present invention are those set out below.

Platelet Binding Assay C'PBA")

Binding of TrG-vWF to platelets is determined using paraformaldehyde-fixed platelets, centrifugation and gel electrophoresis, much as described in Fischer et al., Cell. Molec. Life Set 51: 943-950 (1997) which is incorporated by reference herein in its entirety particularly in parts pertinent to assaying vWF platelet binding. One unit of PBA commonly is defined for convenience as the amount present in 1 ml of a reference preparation of pooled normal plasma.

Specific activity of vWF PBA generally is the ratio of vWF-CBA activity to vWF-Ag in the assay.

Collagen Binding Activity Assay ( BA")

Collagen binding activity ("CBA") of TrG-vWF and TrG-vWF*Rel, and preparations of non-transgenic vWF as well, can be determined by any of several CBA assays. Preferred CBA assays are those that provide accurate and reliable results.

A convenient and commonly used definition of a unit (i.e., one unit) of CBA activity, as determined by any CBA assay is the activity present in 1 ml of a reference preparation of pooled normal plasma, such as the commercially available reference preparations of vWF described elsewhere herein.

Specific activity of vWF CBA generally is the ratio of vWF-CBA activity to vWF-Ag in the assay. The latter can be determined by methods described herein and illustrated in application in Example 7 below.

Particularly preferred assays of CBA in this regard are those that are calibrated against a reference standard vWF preparation, in particular one of the vWF reference preparations described elsewhere herein.

Among particular, illustrative preferred CBA assays in this regard are those described by Siekmann et al, Thromb. Haemost. 11: 1160 et seq. (1995), which is incorporated herein by reference in its entirety especially in parts pertinent to performing vWF collagen-binding assays. An illustrative preferred embodiment of the CBA assay described by Siekmann et al, is disclosed herein in somewhat greater detail in Example 9 calibrated against a reference standard vWF prepared from human serum.

Ristocetin cofactor activity ("RCA")

Ristocetin cofactor activity ("RCA") of TrG-vWF can be determined in accordance with the invention using commercially available kits or individual components such as commercially available stabilized platelets, among other methods.

One unit of RCA commonly is defined for convenience as the amount present in 1 ml of a reference preparation of pooled normal plasma.

Specific activity of vWF RCA generally is the ratio of vWF-RCA activity to vWF-Ag in the assay.

Ristocetin cofactor activity assays are carried out according to the manufacturer's instructions using RCA reagents and vWF standards from Behringwerke. TrG-vWF is obtained as described above and assayed alongside the reference preparations of human vWF. The results for TrG-vWF preparations routinely is as good as or better than that for the reference preparations.

An illustrative Ristocetin cofactor activity assay is described in somewhat greater detail in Example 10.

vWF Multimer Distribution Assay

The multimer composition of vWF preparations can be determined by a variety of methods, including but not limited to methods that utilize 1-D or 2-D polyacrylaminde gel electrophoresis to separate different multimers from one another, radioactivity or dye staining detect the multimers in the gel and scanning densitometry to quantity the amount of each such multimer form that is detected in the gel. Other techniques that can be used toward the same end include but are not limited to capillary zone electrophoresis, particularly using dynamic light scattering for detecting and quantifying the CZE-separated multimers. Also useful in this regard is Biomolecular Interaction Analysis ("BIA"). BIA does not separate or measure structure er se. Rather it provides an absorptive kinetic signature that is singularly indicative of functional binding characteristics. In certain embodiments where the kinetic signature is indicative and/or predictive of the activity reasonably to be expected in vivo, BIA Is highly prefened for characterizing and comparing preparations of TrG-vWF and TrG- vWF*Rel polypeptides.

In a certain preferred embodiment in this regard, multimer distributions of vWF bound to platelets, heparin, collagen or Factor VIII is determined, such as, for example vWF bound to platelets, heparin, collagen and or Factor VIII in accordance with assays for the same described elsewhere herein can be determined using such methods.

A preferred method for determining the multimer distribution of vWF, TrG- vWF, or TrG-vWF*Rel is illustrated in somewhat greater detail in Example 11 herein. Among preferred embodiments in this regard are those that provide vWF and vWT-related polypeptides, proteins and protein preparations that provide high vWF activity, especially high vWF activity as determined by one or more of a Factor VIII binding assay, a platelet binding assay, a GPlb binding assay, a GPIIb binding assay, a GPIIb-IIIa binding assay, a collagen binding assay, a botrocetin binding assay, a heparin binding assay and a sulfatide binding assay, among others; particularly using a standard human plasma-derived vWF preparation for comparison.

Especially preferred in this regard are vWF and vWF-related polypeptides (TrG-vWF and TrG-vWF*Rei) comprising a region having an amino acid sequence with an aforementioned degree of identity to the amino acid sequence of naturally occurring mature human vWF. Among the most especially preferred in this regard are those comprising a region having the amino acid sequence of mature human vWF. Especially preferred in this regard are vWF polypeptides having the amino acid sequence of naturally occurring human mature or human pro-vWF.

In this regard especially preferred embodiments are those that have 50% or more of the activity of a standard reference preparation of active human plasma-derived vWF, as measured by a platelet binding assay, a collagen binding assay, a heparin- binding assay, a Factor VIII binding assay, a ristocetin cofactor assay or other in vitro assay of vWF activity or activities. Particularly preferred in this regard are platelet- binding, collagen-binding, heparin-binding and Factor Vlll-binding assays. In this regard platelet-binding, heparin-binding and collagen-binding assays are especially preferred, of which platelet-binding assays are especially particularly preferred. Highly preferred in these regards, moreover, are the particular platelet-binding, collagen- binding, heparin-binding and Factor Vlll-binding assays described in greater detail in examples herein. Among prefened in vivo assays useful in this regard are assays of vWF circulating half life, particularly half-life assays earned out in dogs.

Particularly highly prefened embodiments in this regard have 65% or more of the activity of the aforementioned reference preparation. Yet more highly prefened embodiments in this regard have 75%> or more of the activity of the reference, preferably 85% or more, yet more preferably 90% or more, still yet more preferably 95% or more.

Other particularly prefened embodiments in this regard have 50% to 150% of the activity of the aforementioned reference preparation. Particularly highly prefened embodiments in this regard have 60% to 125% of the activity of the reference preparation. Yet more highly prefened embodiments have 75% to 1 10% of the activity of the reference preparation. Still more highly prefened embodiments have 85% to 125% the activity of the reference. Still more highly prefened embodiments have 90% to 110% of the activity of the reference. Among particularly prefened embodiments in this regard are those that provide vWF and vWF -related polypeptides, proteins and/or protein preparations (TrG-vWF or TrG-vWF*Rel polypeptide, protein or protein preparations) that have high specific vWF activity, particularly high specific activity as determined by one or more assays of vWF activity described above, together with an assay of vWF protein, particularly an assay of vWF antigen, especially an assay of vWF antigen as described in the examples herein. Especially prefened in this regard are TrG-vWF and TrG-vWF*Rel polypeptides comprising a region having an amino acid sequence with an aforementioned degree of identity to the amino acid sequence of naturally occurring mature human vWF. Among the most especially prefened in this regard are those comprising a region having the amino acid sequence of mature human vWF.

Especially prefened in this regard are TrG-vWF and TrG-vWF*Rel having the amino acid sequence of naturally occurring mature human vWF and/or human pro vWF

In this regard especially prefened embodiments are those that have 50% or more of the activity of a standard reference preparation of vWF, particularly as measured by one or more assays of vWF activity described above, especially using a standard human plasma preparation for comparison, particularly a vWF reference described in the examples herein. Particularly highly prefened embodiments in this regard have 65% or more of the activity of the aforementioned reference preparation. Yet more highly prefened embodiments in this regard have 75% or more of the activity of the reference, preferably 85% or more, yet more preferably 90% or more, still yet more preferably 95% or more of the activity of the reference preparation

Other particularly prefened embodiments in this regard have 50% to 150% of the activity of the aforementioned reference preparation Particularly highly prefened embodiments in this regard have 60% to 125% of the activity of the reference preparation Yet more highly prefened embodiments have 75% to 1 10% of the activity of the reference preparation Still more highly prefened embodiments have 85% to 125% the activity of the reference Still more highly prefened embodiments have 90% to 110% of the activity of the reference

Further prefened embodiments in this regard provide derivatives of the aforementioned vWF and vWF-related polypeptides, proteins and protein preparations (TrG-vWF and TrG-vWF*Rel polypeptides, proteins and protein preparations) that have high specific activity, particularly high specific activity as determined measured by one or more assays of vWF specific activity descnbed above, especially using a standard human plasma preparation for companson, particularly a vWF reference descnbed in the examples herein Especially prefened in this regard are derivatives of vWF and vWF-related polypeptides comprising a region having an ammo acid sequence with a degree an aforementioned degree of identity to the amino acid sequence of naturally occurnng mature human vWF Among the most especially prefened in this regard are those compnsing a region having the ammo acid sequence of mature human vWF Especially prefened m this regard are vWF and vWF -related polypeptides ( (TrG-vWF and TrG-vWF*Rel polypeptides) having the amino acid sequence of naturally occurnng mature human vWF In this regard especially prefened embodiments are those that have 50% or more of the specific activity of a standard reference preparation of active human plasma-denved vWF, particularly as measured by one or more assays of vWF activity described above, especially using a standard human plasma preparation for comparison, particularly a vWF reference descnbed in the examples herein Particularly highly prefened embodiments in this regard have 65% or more of the specific activity of the aforementioned reference preparation Yet more highly prefened embodiments in this regard have 75% or more of the specific activity of the reference, preferably 85% or more, yet more preferably 90% or more, still yet more preferably 95% or more of the specific activity of the reference preparation. Also among highly particularly prefened embodiments in this regard are those with a higher specific activity than that of the reference preparation, particularly those with a substantially higher specific activity. Other particularly prefened embodiments in this regard have 50% to 150% of the specific activity of the aforementioned reference preparation. Particularly highly prefened embodiments in this regard have 60% to 125% of the specific activity of the reference preparation. Yet more highly prefened embodiments have 75% to 110% of the specific activity of the reference preparation. Still more highly prefened embodiments have 85% to 125%) the specific activity of the reference, still more highly prefened embodiments have 90% to 110% of the specific activity of the reference.

Among prefened embodiments in this regard are derivatives that differ in one or more post-translational modifications from that found in human vWF prepared from natural sources. Especially prefened in this regard are differences that do not interfere with or deleteriously affect therapeutic activities of the TrG-vWF and TrG-vWF*Rel polypeptides, proteins and or protein preparations of the invention and or do not cause contraindications, toxicity or other adverse or unpleasant effects when administered to animals or humans for therapeutic or other purposes.

Also among particularly prefened embodiments in this regard are derivatives that have a lower or a higher fucose content than that of normal human vWF isolated from sera, particularly normal sera and that typical of normal human vWF; but, that also are substantially indistinguishable functionally from it, particularly as determined by USFDA regulatory practice, particularly those that have a higher content.

Additionally among particularly prefened embodiments in this regard are derivatives that have a lower or a higher N-acetylgalatosamine content than that of normal human vWF isolated from sera, particularly that of normal sera and that typical of normal human vWF, but that also are substantially indistinguishable functionally from it, particularly as determined by USFDA regulatory practice, particularly those that have a higher content.

Further among particularly prefened embodiments in this regard are derivatives that have two or more of (1) a lower or higher fucose content, (2) a lower or higher N- acetylgalactosamine content or (3) a lower or a higher content or different pattern of .glycan sulfation than that of human vWF isolated from sera, particularly that of normal sera and that typical of normal human vWF, but that also are substantially indistinguishable functionally from it, particularly as determined by USFDA regulatory practice.

As to all of the aforementioned derivatives relating to fucose, -acetylgalatosamine, and sulfation, prefened activities, in particular, are those percents and ranges set out herein above.

Also in these and other aspects and embodiments relating to structure and activity of TrG-vWF and TrG-vWF*Rel in accordance with the invention, prefened embodiments include TrG-vWF and TrG-vWF*Rel polypeptides that form multimers akin to the multimers characteristic of normal vWF expressed in situ and circulating in blood of a healthy individual. Also prefened in this regard are TrG-vWF and TrG- vWF*Rel polypeptides that form multimers differently and or that differ from normal vWF but that have the same function as vWF as measured by one or more of the measures of vWF activity described elsewhere herein, or that have enhanced function by one or more of the assays. Also prefened in this regard are TrG-vWF and TrG- vWF*Rel polypeptides that form multimers differently and or that differ from normal vWF but that are desirably different in stability such as stability to storage, to contaminants, to destabilizing and or degrading temperatures, to pathogen contaminations, to pathogen inactivation treatments, particularly virus mactivation treatments and immunogenicity, including greater immunogenicity for producing antibodies or inducing an immune response including an immune response for prophylactic purposes, such as a vaccine, and decreased immunogenicity, such as for decreasing antibody response to therapeutic compositions comprising one or more TrG- vWF and or TrG-vWF*Rel polypeptides, such as decreasing adverse immuno-reactions in patients administered compositions comprising one or more TrG-vWF and or TrG- vWF*Rel polypeptides in accordance with the invention.

DNAs Encoding TrG-vWF and TrG-vWF*Rel

Genetic constructs that encode TrG-vWF and TrG-vWF*Rel for use in making transgenic organisms in accordance with the invention can be obtained using standard molecular biology techniques, including but not limited to techniques for cloning, synthesizing and modifying DNAs, RNAs, PNAs and combinations thereof, among others. Both genomic and cDNAs are particularly prefened in this regard. Genetic constructs, such as genomic or cDNAs, encoding TrG-vWF and TrG- vWF*Rel from a variety of organisms may be used in the invention in this regard. For instance, cloned non-human vWF genes that can be used in the invention include among others vWF genes of mammals, particularly mouse, rat, pig, sheep, goat and cow. Also prefened are vWF genes of primates, especially chimpanzees. Most highly prefened are vWF genes of humans.

Particularly prefened genetic constructs for use in the present invention are those that engender expression of human vWF and vWF -related polypeptides, especially those that encode vWF itself. Genomic and cDNAs are prefened in some embodiments in this regard. Genomic DNAs that encode human vWF can be obtained, for instance, from libraries of human genomic DNA using probes based on the published DNA sequences of vWF and standard library screening and cloning techniques. Human cDNAs encoding vWF, for another example, can be obtained from cDNA libraries made from vascular epithelial cells or megakaryocytes, using much the same screening techniques and much the same probes as for human genomic DNAs.

Cloned genes for human vWF suitable for use in the invention include those described in Sadler et al, Cold Spring Harb. Symp. Quant. Biol. LI: 515-521 (1986) and European Patent Application 0 197 592 Al of Pannekoek et al. on "Preparation of human von Willebrand factor by recombinant DNA" each of which is incorporated herein by reference in its entirety as to the foregoing particularly in parts pertinent to cloned vWF and vWF-related genomic DNAs and cDNAs.

Genetic constructs that engender production of naturally occurring vWF and vWF-derived and/or related polypeptides are highly particularly prefened in some aspects and prefened embodiments of the invention, genetic constructs that engender production of altered, mutated, and/or modified forms of vWF and/or vWF-derived and/or related polypeptides are prefened in other aspects and prefened embodiments of the invention.

Modifications can be introduced into naturally occurring vWF genes and polypeptides encoded thereby by techniques well known to the art, such as the synthesis of modified genes by ligation of overlapping oligonucleotides, and by introducing mutations directly into cloned genes, as by oligonucleotide mediated mutagenesis, inter alia. Particularly prefened modifications in this context include but are not limited to those that alter post-translational processing as discussed above, that alter size, that fuse portions of other proteins to those of vWF, that alter the active sites of the vWF, such as the binding sites for, among others platelet membrane proteins involved in vWF- platelet adhesion, the binding site for factor VIII, binding sites for collagen, heparin, sulfatide, GPlb, botrocetin, GPIIb-IIIa, that alter the sites involved in dimerization or multimerization, that stabilize the TrG-vWF and or TrG-vWF*Rel, that control transport and/or secretion of the TrG-vWF and/or TrG-vWF*Rel, that alter, augment, multiply, decrease or eliminate physiological activities of the TrG-vWF and/or TrG- vWF*Rel. Guidance relating to certain specific aspects of embodiments in this regard are set out in greater detail below in the section on structure - function relationships in vWF.

For instance, among modifications prefened in this regard are those that alter or affect the natural series of proteolytic cleavages that occur during physiological processing of vWF, such as alteration to the sites of cleavage of the pre and the pro- peptides. Further guidance relating to certain specific aspects of embodiments in this regard are set out in greater detail below in the section on structure - function relationships in vWF.

Further prefened embodiments in this regard relate to modifications that affect, alter, add to, or eliminate one or more other post-translational modifications of TrG- vWF and TrG-vWF*Rel of the invention. Certain particularly prefened embodiments in this regard relate to modifications that alter physiological functions and provide improved performance, such as improved platelet or factor VIII binding or both, or improved binding to other factors, such as connective tissue components, such as collagen, or that provide improved stability of TrG-vWF and TrG-vWF*Rel, including physiological stability, processing stability, storage stability and stability to other components of compositions in which it will occur in the transgenic organism, during isolation and purification from the organism, during storage or shipment, in formulations for particular uses, and in use, particularly in use in human patients, improved properties for purification, improved physiological persistence, among others. Further guidance relating to certain specific aspects of embodiments in this regard are set out in greater detail below in the section on structure-function relationships in vWF. Certain prefened embodiments in this regard relate to addition, deletion or alteration of sites to change glycosylation of polypeptides of the invention. Particularly prefened embodiments in this regard relate to alterations to N-linked glycosylation sites that match the consensus Asn-X-Ser/Thr sequence. Further guidance relating to certain specific aspects of embodiments in this regard are set out in greater detail below in the section on structure - function relationships in vWF.

Particularly prefened embodiments in this regard are those that improve glycosylation-dependent activities of TrG-vWF and TrG-vWF*Rel of the invention, such as physiological vWF function and effects, including but not limited to enzymatic activity, substrate preferences, binding to cofactors and other moieties, complex formation, thermal stability, resistance to proteases and physiological persistence among other things. Further guidance relating to certain specific aspects of embodiments in this regard are set out in greater detail below in the section on structure - function relationships in vWF.

Structure-Function Relationships in vWF

The amino acid sequence of human vWF was first determined by direct sequencing of purified human vWF. Subsequently, the amino acid sequenced determined directly has been confirmed and augmented by sequencing of cloned human vWF complementary and genomic DNAs. (For the original amino acid sequence see Titani et al, Biochemistry 25_: 3171-3184 (1986), for cDNA and genomic sequences see

Sadler et al, Cold Spring Harb. Symp. Quant. Biol. LI: 515-523 (1986) and European Patent Application 86200518.8, Publication number 0 197 592 Al of Pannekoek et al. for Preparation of human vWF by recombinant DNA, each of which is incorporated herein by reference in its entirety particularly in parts pertinent to vWF amino acid, cDNA and genomic DNA sequences and to structure-function relationships in vWF). vWF is unusually rich in cysteine, and is unusual in the clustering of the cysteine residues in the amino and carboxyl-terminal portions of the polypeptide. Cysteine is the most abundant amino acid in human vWF, accounting for 8.3% of the amino acids in the protein. vWF also contains four unrelated types of repeated sequences, refened to as A,

B, C and D, which constitute distinct domains in the protein. The domains occur in the amino acid sequence of human vWF in the sequence D1-D2-D'-D3-A1-A2-A3-D4-B1 - B2-B3-C1-C2 , going from amino terminus to carboxyl terminus. The repeats of a given type vary in sequence identity from 23% to 43%. The three repeats of the A domain, Al, A2 and A3, differ from one another somewhat in sequence and range from 193 to 220 amino acids long. They are all disposed tandemly between amino acids 497 and l l l l of mature vWF. The three B domains, Bl, B2 and B3, are 25-35 amino acids long and lie tandemly between 1533 and 1636. The two copies of the C domain are 116 and 119 amino acids and are located in tandem between residues 1637 and 1899 of mature vWF. There are five D domains, DI, D2, D', D3 and D4. Each of the five D domains is between 270 to 289 amino acids long. Unlike the A, B and C domains, the D domains are separated from one another in the vWF sequence. Two of the D domains, DI and D2, occur in the propeptide and form a very large part of the 763 amino acid propeptide sequence. Another D domain, D', spans the junction between the end of the propeptide and the beginning of mature vWF. D3 and D4 lie adjacent to the region of vWF containing the A domains. The two E domains each are 46 amino acids long. El is located near the N terminus of mature vWF. E2 is located between

D4 and Bl, and the two E repeats thus straddle a region 1383 residues long that contains all the A and D domains in mature vWF. (See for instance Ruggeri, Thombosis and Haemostasis £2(2): 576-584 (1999) which is incorporated herein by reference in its entirety as to the foregoing in parts pertinent the domain structure). The "pre' signal sequence is required for transport of nascent and newly synthesized vWF into the endoplasmic reticulum.

The pro-peptide appears to be required for multimerization. vWF deletions that lack the pro-peptide do not form multimers. However, mutations that prevent normal cleavage of the pro-peptide do not interfere with multimer formation. As noted above, there is one collagen binding site and an RGD sequence in the vWF pro-peptide, both of uncertain significance. vWF -mediates platelet thrombus formation by sequentially binding (A) to collagen (and, possibly, other extracellular matrix constituents and then (B) to two platelet membrane receptor complexes: (1) GPIb-IX-V and (2) GPIb-IIIa. Regions of vWF involved in binding collagen and the glycoprotein receptor complexes have been mapped as a set of discrete domains in the primary sequence. Some of these are discussed below. (See for instance Sadler, J. Biol. Chem. 266(34): 22777-22780 (1991),Ruggeri, Thombosis and Haemostasis 82(2): 576-584 (1999) and TEXTBOOK OF HEMATOLOGY, 2^nd Ed.; S. McKenzie, Williams & Wilkins, Baltimore (1996), particularly pages 553 and 564-570 each of which is incorporated herein by reference in its entirety in parts pertinent to the foregoing discussion of structure-function relationships in vWF particularly as to relationships between vWF primary structure, particularly repeats, domains and other motifs and vWF functions.) vWF directly binds to collagen, in particular, collagen types I, III and VI.

Higher molecular weight multimers bind to collagen better than low molecular weight forms of vWF. Collagen binds directly to the Al domain and the A3 domain in mature vWF, respectively amino acids 449-728 and 91 1-1365 of the mature polypeptide sequence. In additional, collagen binds a site in the D2 domain in the vWF propeptide. A3 is the major domain for collagen binding. Collagen interaction with the D2 domain in the propeptide and the Al domain in the mature peptide does not play more than a subsidiary or minor role in physiologically significant vWF-collagen binding. (Regarding binding to type VI collagen see for instance Rand et al, Thromb. Haemost. 11(6): 566-570 (1997) and the immediately foregoing references on vWF structure function relationships each of which is incorporated herein by reference in its entirety in parts pertinent to collagen binding by vWF, especially collagen VI binding.)

Heparin and heparin-like molecules, which inhibit platelet binding to vWF but do not inhibit collagen binding, bind to the D' domain in the amino terminus of mature vWF and also to Al domain (amino acids 449-728 of mature vWF). Since heparin inhibits vWF binding to platelets but not to collagen, the binding site(s) in Al for heparin and for collagen apparently are different. (Regarding vWF - heparin binding see for instance the immediately foregoing references on vWF structure function relationships each of which is incorporated herein by reference in its entirety in parts pertinent to heparin binding by vWF.) Sulfatides and botrocetin also bind the vWF Al domain. (Regarding vWF sulfatide or botrocetin binding see for instance the immediately foregoing references on vWF structure function relationships each of which is incorporated herein by reference in its entirety in parts pertinent to binding of sulfatides and botrocetin by vWF.)

The glycoprotein GPlb± in the GPlb-IX-V complex in the platelet membrane binds the Al domain in mature vWF. (Regarding binding between vWF and the

GPlb± in the GPlb-IX-V complex in platelets see for instance the immediately foregoing references on vWF structure function relationships each of which is incorporated herein by reference in its entirety in parts pertinent to binding between this platelet complex and vWF.)

The Al domain of human vWF contains binding sites for GPlb. Binding of GPlb to vWF is dependent on vWF binding to connective tissue. Botrocetin, a pit viper venom that induces vWF-GPlb binding, also binds to the Al domain, within a disulfide loop. The Al region also binds heparin, and sulfatides. Heparin also binds vWF at a site n the D domain. The GPIB binding domain of vWF and vWF polypeptides that bind GPIB, as well as methods for making, assaying and using them, inter alia, are described for instance in U.S. patent Nos 5,837,488, 5,849,536 and 5,849,702, all to Garfinkel et al. for Cloning and Production of Human vWF GP 1 B

Binding Domain Polypeptides and Methods of Using Same, each of which is incorporated herein by reference in its entirety as to the foregoing in parts pertinent to GPIB binding domain of vWF and vWF polypeptides, such as fragments of vWF, that bind GPIB. The GPIIb-IIIa complex found on activated platelets binds to a four amino acid sequence, Arg-Gly-Asp-Ser ("RGDS"), located near the C-terminal end of the Cl domain in the C-terminal region of vWF. Another RGD sequence in vWF, in the propeptide; apparently does not bind the GPIIb-IIIa complex. (Regarding binding between vWF and the GPIIb-IIIa complex found on activated platelets, see, for instance, the immediately foregoing references on vWF structure/function relationships, each of which is incorporated herein by reference in its entirety in parts pertinent to binding between this platelet complex and vWF.)

Factor VIII binds to a site in the amino terminal 272 amino acids of the mature vWF polypeptide, between amino acids 1 and 272. Residues 78 and 96 are particularly important to vWF - Factor VIII binding. Intrachain disulfide linkages in the region also are important to vWF - Factor VIII binding. Specific substitutions at residues 19, 28, 53, 54 and 91 all decrease vWF binding to Factor VIII and cause an autosomal inherited form of vWF disease with symptoms that resemble hemophilia A. (See for instance Ruggeri, Thombosis and Haemostasis £2(2): 576-584 (1999) which is incorporated herein by reference in its entirety as to the foregoing in parts pertinent to vWF -Factor VIII binding.) Specific vWF fragments that bind Factor VIII have been described in, for instance, U.S. patent No. 5,597,711 of Zimmerman et al. for Factor VIII Binding Domain of von Willebrand Factor which is incorporated herein by reference in its entirety as to the foregoing in parts pertinent to the Factor VIII binding domain of vWF and vWF fragments that bind Factor VIII.

Estimates of vWF carbohydrate content as a per cent of total mass range from 10% to 19%). Sequences for N-linked glycosylation occur in the human vWF amino acid sequence at asparagines 94, 384 468, 752, 811 , 1460, 1527, 1594, 1637, 1783,

1822 and 2027. Sequences for O-linked glycosylation occur in human vWF at serines 500 and 723 and at threonines 485, 492, 493, 705, 714, 724, 916 and 1535. See for instance Titani et al., Biochemistry 21: 3171-3184 (1986) which is incorporated herein by reference in its entirety as to the foregoing in parts pertinent to sites in human vWF for N-linked and O-linked glycosylation.

N-linked glycosylation of mature vWF has been shown to be important to dimer formation and multimerization, as described in Wagner et al, J. Cell. Biol 102: 1320- 1324 (1986). While exact determination of glycosylation structures is difficult or not practically possible for the most art, the general distribution of glycosylation structures in a protein can be determined by FTIR. FTIR studies of vWF have found that the glycans are mainly in solvent exposed regions or turns. (As described in for instance Perkins et al, J. Mol. Biol. 23U: 104-119 (1994) which is incorporated herein by reference in its entirety as to the foregoing in parts pertinent to glycosylation of proteins, particularly vWF and to determination of aspects of glycosylation structure and distribution in vWF by FTIR.)

For reviews of vWF structure and function see, for instance, Sadler, J. Biol Chem. 266(34): 22777-22780 (1991) and TEXTBOOK OF HEMATOLOGY, 2^nd Ed.; S. McKenzie, Williams & Wilkins, Baltimore (1996) particularly in this regard, pages 553 and 564-570. See also Ruggeri, Thombosis and Haemostasis £2(2): 576-584 (1999) and other previously cited references relating to amino acids, cDNA and genomic sequences of vWF and to structure - function relationships thereof, each of which is incorporated herein by reference in its entirety in parts pertinent to vWF, particularly as to structure- function relationship in vWF.

Cis-Acting Sequences for Transgenic Expression A wide variety of genes have been expressed in a wide variety of transgenic organisms. Many blood proteins in particular have been expressed in animals. Moreover, transgenic expression of blood proteins has been targeted to specific compartments. The cw-acting controls used in the past to express blood proteins in transgenic organisms also can be used, in many cases, to express vWF or vWF-related polypeptides in transgenic organisms in accordance with the present invention. Examples in this regard are described in Lubon et al. , Transfusion Medicine Reviews X(2): 131-141 (1996) which is incorporated by reference herein in its entirety. Some prefened embodiments relating to expression-regulatory regions for transgenic expression of vWF and or vWF -related polypeptides are described in further detail below.

In situ activation In certain instances, endogenous vWF sequences (such as those introduced into a transgenic animal) can be activated in situ in the genome by the introduction of exogenous sequences having an effect on the expression of the sequences already present in the genome. Such coding sequences can be native or can have been introduced previously and have become an integral part of the genome. Thus, homologously recombinant host cells can be produced that allow the in situ alteration of endogenous polynucleotide sequences in a host cell genome. This technology is more fully described in WO 93/09222, WO 91/12650, and US 5,641,670. Briefly, specific polynucleotide sequences conesponding to the vWF polynucleotides or sequences proximal or distal to a vWF sequence are allowed to integrate into a host cell genome by homologous recombination where expression of the gene can be affected.

In one embodiment, regulatory sequences are introduced that either increase or decrease expression of an endogenous sequence. Accordingly, a vWF protein can be produced in a cell not normally producing it, or increased expression of the protein can result in a cell producing the protein at a specific level. Still further, specific mutations can be introduced into any desired region of the gene to produce mutant proteins. Such mutations can be introduced, for example, into the specific functional regions of the protein, including but not limited to, those disclosed herein.

In one embodiment, a human vWF sequence can be introduced into a transgenic animal and subsequently modified therein. Alternatively, a cell can be produced in vitro to express a vWF protein and subsequently the cell can be introduced into the mammary tissue of an animal so that the vWF protein is secreted into the milk of the animal. Alternatively, a human cell derived, for example, from human mammary tissue, and compatible with growth and expression in animal mammary tissue, can be modified by means of homologous recombination in situ to express the vWF gene, a gene not normally expressed in such human cells. Such a gene can be further modified in situ to express a more highly beneficial or desirable vWF sequence mutant according to the present invention. Thus, in one embodiment, the host cell can be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal containing the vWF sequence. Alternatively, the host cell can be a stem cell or other early tissue precursor that gives rise to a specific subset of cells that can be used to produce transgenic tissues in an animal.

Promoters and related sequences

The cis-acting regulatory regions useful in the invention include the promoter used to drive expression of a gene in a transgenic organism effective for the production in the organism of vWF and vWF-related polypeptides. Prefened in this regard are regulatory regions that engender the production of significant amounts of vWF and or related polypeptides that can be recovered from the organism and purified. The term

"engender" refers to the case in which the regulatory regions are operably linked to the sequences to be expressed (coding sequences) prior to introduction into a cell. The term also encompasses the case in which an exogenous regulatory sequence is introduced into a cell, which sequence then integrates into the genome by homologous or nonhomologous recombination in such a manner as to become operably linked to an endogenous expressible sequence, such as a vWF-related coding sequence, and cause expression of or contribute to causing the expression of (as by an enhancer sequence) a desired endogenous (preexisting in the genome prior to introduction of the exogenous sequence) sequence. By "significant" is meant that the vWF and or vWF-related polypeptides can be recovered from the transgenic organism in amounts useful for research or for commerce or both. Prefened concentration ranges of the vWF-related polypeptide in milk, especially useful for purification for various purposes, extends from approximately 0.1-5g/L. A prefened subrange includes from approximately 1- 5g/L. An even more prefened range includes from approximately 0.5-2.5g/L. It is understood, however, that the concentration range in milk useful for purification will depend upon, for example, the animal in which the protein is produced. Accordingly, these ranges are not intended to be limiting but to provide guidance to prefened parameters.

Particularly prefened are regulatory regions that provide for the production of significant amounts of vWF and or vWF-related polypeptides in specific compartments of an organism. Particularly prefened compartments in this regard are compartments that accumulate and or store proteins. Also among prefened specific compartments are particular tissues or organs, including but not limited to liver, kidney, spleen, lymph node, peritoneum and small intestines. Especially prefened compartments in this regard are bodily fluids, such as lymph, saliva, blood and milk. Particularly especially prefened are blood and milk, most particularly milk.

Particularly useful regulatory regions for expression in milk are promoters that are active in mammary tissue, especially those that are specifically active in cells of mammary tissue, i.e., are more active in mammary tissue than in other tissues under physiological conditions where milk is synthesized. Most prefened are promoters that are both specific to and efficient in cells of mammary tissue. By "efficient" is meant that the promoters are strong promoters in mammary tissue that engender the synthesis of large amounts of protein, particularly for secretion into milk, especially milk of pigs.

Orders of magnitude of expression in mammary tissue in comparison with non- mammary tissue can range from complete lack of expression in non-mammary tissue to orders of magnitude from 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or greater in mammary compared to non-mammary tissue.

Promoters and methods for producing proteins in milk of transgenic mammals that can be used in accordance with prefened embodiments of the invention in this regard are described in, for instance, U.S. Patent number 4,873,316 of Meade et al on Isolation of Exogenous Recombinant Proteins From The Milk of Transgenic Mammals;

U.S. Patent number 5,880,327 of Lubon et al. on Transgenic Mammals Expressing Human Coagulation Factor VIII; and U.S. Patent number 5,831,141 of Lubon et al on Expression of a Heterologous Polypeptide in Mammary Tissue of Transgenic Nonhuman Mammals Using a Long Whey Acidic Protein Promoter, each of which is incorporated herein by reference in its entirety regarding the foregoing particularly in parts pertinent to transgenic production of polypeptides in milk of transgenic non- human mammals. Whey acidic protein (refened to as "WAP") promoters are among the most highly prefened promoters in this regard. Regulatory elements of the murine WAP gene are entered in GenBank (U38816) and cloned WAP gene DNAs are available from the ATCC. A variety of transcription-promoting WAP fragments, vectors, cloning methods and the like are described on the NTH mammary expression website at: http://mammary.nih.gov/tools/molecular/Wagner001/WAP_vectors.htm and in, among others, Campbell et al, Nucleic Acids Res. 12: 8685 (1984); Burdon et al, J. Biol. Chem. 266: 6909-14 (1991); Lakso et al, Proc. Nat'l Acad. Sci., USA £2: 6232-26 (1992); McKnight et al, Molec. Endocrin. 9: 717-724 (1995); Orban et al,

Proc. Nat'l Acad. Sci., USA £2: 6861-65 (1992); Lubon et al, U.S. Patent No. 5,880,327, Transgenic Mammals Expressing Human Coagulation Factor VIII; and Lubon et al, U.S. Patent number 5,831,141, Expression of a Heterologous Polypeptide in Mammary Tissue of Transgenic Nonhuman Mammals Using a Long Whey Acidic Protein Promoter, all of which are herein incorporated by reference in their entirety, particularly regarding the foregoing in parts pertinent to WAP c/s-acting transcription elements useful for the production of polypeptides in transgenic organisms, particularly in mammary gland cells and milk of transgenic non-human female mammals. Among the most prefened promoters are those that regulate a whey acidic protein (WAP) gene, particularly, the murine and the rat WAP promoter. Especially prefened in this regard is the mouse "long" WAP promoter.

Promoters of casein, lactalbumin and lactoglobulin genes also are prefened in certain embodiments of the invention in this regard, including, but not limited to the a-, β-, K-, and γ-, -casein promoters and the α-lactalbumin and β-lactoglobulin promoters (BLG promoters), and derivatives thereof Prefened among these promoters are those from rodents, especially mice and rats, and from pigs and sheep, especially the rat β- casein promoter and the sheep β-lactoglobulin promoter.

It is understood that the invention encompasses promoters that may be constitutive in mammary tissue or which may be inducible by, for example, lactation. This is the case for, for example, the WAP and many other milk protein-specific gene promoters. For example, prolactin can be used to induce lactation in many organisms. It is understood, however, that the invention encompasses a further level of inducibility, such as by combining the regulatory sequences that provide for constitutive or mammary-specific inducible expression, with sequences that can be induced by a further exogenous signal introduced into the animal. This includes hormone-responsive elements, metal-inducible elements, and the like, which elements are well known to those of ordinary skill in the art and can be found in, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2^nd Ed., Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, NY (1989).

Among the sequences that regulate transcription that are useful in the invention, in addition to the promoter sequences discussed above, are enhancers, splice signals, transcription termination signals and polyadenylation sites, among others. Particularly useful regulatory sequences include those that increase the efficiency of expression of vWF and or vWF-related polypeptides in transgenic organisms. Also particularly prefened in this regard are those that increase the specificity of expression of vWF and or vWF-related polypeptides in targeted compartments of an organisms. Among highly particularly prefened regulatory regions in this regard are those that increase the efficiency, the specificity or both the efficiency and the specificity of expression of vWF or vWF -related polypeptides in mammary glands and in the milk of transgenic non-human mammals.

Especially useful in this regard are the other transcription regulatory sequences of genes expressed at high levels in mammary cells, such as those mentioned above, including but not limited to WAP genes, -, β- and γ-casein genes, α-lactalbumin genes and β-lactoglobulin genes. Prefened sources for regulatory sequences in this regard include rodents (such as mice and rats), pigs and sheep.

Exemplary of additional prefened regulatory sequences are those associated with the mouse and rat WAP genes, rat β-casein gene and the sheep β-lactoglobulin gene, respectively. The regulatory sequences most prefened for use in the present invention in this regard are those associated with whey acidic protein genes. Particularly prefened in this context are regulatory sequences of the murine whey acidic protein gene.

.? ' Untranslated Sequences Also among regulatory sequences prefened in certain embodiments of the invention are sequences comprised in the 3' untranslated portion of genes that increase expression of transgenically-encoded product particularly in mammary gland cells of transgenic non-human mammals, especially those that increase the amount of the product secreted into its milk. Among highly prefened particular sequences in this regard are those that apparently stabilize mRNA transcribed from transgenes. Among prefened embodiments in this regard are sequences that comprise a polyadenylation signal. Among prefened regions of this type are those derived from the genes for proteins that are expressed at high levels in mammary gland cells and or encode proteins that are found are high concentrations in milk. Especially prefened in this regard are sequences of the 3' untranslated region of whey acid protein genes, particularly the mouse and rat whey acid protein genes. Highly especially prefened is this regard are sequences of the long mouse and rat whey promoter constructs.

Trafficking and translational signals

Also important to the invention are signal peptide sequences that direct secretion of proteins into the milk of the transgenic animal. In this regard, both endogenous and heterologous signal sequences are useful in the invention. Generally, the signal peptides of proteins normally secreted into milk are useful in the invention.

The signal sequences of proteins that occur in high concentration in milk are particularly prefened, such as the signal peptides of the whey acid proteins, caseins, lactalbumins and lactoglobulins, including, but not limited to the signal peptides of the -, β- and γ-caseins and -lactalbumin and β-lactoglobulin. The signal sequences of whey acidic protein are particularly prefened in this regard, especially the signal sequences of murine whey acidic proteins and the signal sequences of rat whey acid protein genes. Also among prefened signal sequences in this regard are the signal peptides of secreted coagulation factors. Particularly prefened in this regard are the signal peptides of vWFs and t-PAs, especially secretion signal sequence of human vWF.

Among the sequences that regulate translation and transport, in addition to the signal sequences discussed above, are ribosome binding sites and sequences that augment the stability of vWF mRNA. Especially useful are the translation regulatory sequences of genes expressed at high levels in mammary cells. For instance, the regulatory sequences of the α-, β- and γ-casein genes and the α-lactalbumin and β- lactoglobulin genes are prefened, especially those from rodents (mice and rats), pigs and sheep. Also particularly prefened are the regulatory sequences of rat ²-casein and the sheep actoglobulin genes.

Among the most prefened translational regulatory sequences of the invention are those of the whey acidic protein and those of vWF genes. And the most particularly prefened regulatory sequences are those of the murine whey acidic protein and human vWF, including human genomic vWF and human vWF cDNA constructs, and including human vWF cDNA constructs that contain intron sequences. Especially useful in the present invention are sequences that advantageously modulate post- translational modifications of vWF, such that the vWF produced in the transgenic animals of the invention is active. Genomic sequences of the human vWF gene are particularly prefened in this regard.

Purification of Constructs

Constructs for producing vWF and related polypeptides in accordance with the invention, such as double-stranded DNA and DNA:RNA hybrid constructs, can be prepared by any of a wide variety of methods that produce polynucleotide constructs of requisite purity and amounts. DNAs in double-stranded form may be manipulated by conventional methods to provide constructs having the structures and properties set out above and elsewhere herein for expression of vWF and related polypeptides in transgenic organisms. For DNA:RNA hybrids, well known vectors that contain bacteriophage promoters, such as the T3 and T7 promoter can be used to produce RNA for DNA:RNA hybrids and well known vectors that produce single-stranded DNA may be used to produce single-stranded DNA for DNA:RNA hybrids.

Constructs can be amplified by conventional techniques for cloning and propagation in a host organism such as a bacterial host, a yeast host, an insect cell host, or a mammalian cell host. Constructs also can be amplified by in vitro methods such as PCR. Constructs can be derived from natural, cloned or synthesized DNA or RNA in whole or in part. Polynucleotide constructs may contain modified bases as well as the bases that occur naturally in DNA and RNA. Often constructs for making transgenic organisms in accordance with the invention are manipulated or propagated joined to or in the presence of other polynucleotides. These extraneous polynucleotides can be removed prior to using a construct to produce a transgenic organism. For instance, a construct that was propagated and amplified in a cloning typically can be separated from the vector by restriction enzyme cleavage and then purified.

Constructs for introduction into cells to make transgenic organisms in accordance with the invention can be purified by well known techniques. For instance, among other well know techniques that can be used, constructs can be purified by agarose gel electrophoresis and electroelution, by HPLC, by ultracentrifugation through a sucrose gradient, by ultracentrifugation through an NaCl gradient or, in certain particularly prefened embodiments in this regard, by combination of two or more of electroelution, HPLC, sucrose gradient centrifugation and NaCl gradient centrifugation.

Organisms

A wide variety of hosts can be used for transgenic production of vWF and related polypeptides in accordance with the present invention. Particularly prefened are those that provide vWF and or related polypeptides with the post-translational modifications required for physiological activity. Especially prefened in this regard are those that provide high specific activity vWF and those that provide high yields of vWF. Most especially prefened in this regard are those that provide high yields of high specific activity vWF and/or vWF related polypeptides. Organisms that do not suffer adverse effects of transgenesis and or transgene expression are similarly prefened, as are those that do not suffer adverse effects of production, accumulation or harvesting of transgenically expressed vWF and/or related polypeptides.

All lactating animals, that is, all mammals, particularly are prefened in this regard. Prefened mammals in this regard include domesticated mammals, particularly livestock animals. Particularly prefened mammals include mice, rats, hamsters, rabbits, pigs, sheep, goats, cows and horses. More particularly, mice, pigs, sheep and cows are prefened. Among the most prefened mammals at present are mice, pigs and sheep. Of these, pigs are especially particularly prefened.

Harvesting and Purification

Any technique suitable to the purpose may be employed to isolate and purify vWF and vWF-related polypeptides from transgenic organisms in accordance with the invention. Some such methods in this regard are illustrated by specific embodiments relating to purification of TrG-vWF from milk of TrG-vWF-expressing transgenic non- human female mammals. TrG-vWF can be purified from milk of such transgenic mammals using a variety of well know methods generally useful for purifying proteins from milk, together with well established methods for purifying vWF from natural sources and or from cell culture. Illustrative embodiments in this regard are described immediately below and further illustrated by way of specific examples.

Obtaining milk from a transgenic animal within the present invention can be accomplished by a variety of well know methods, such as those described in, among others, Burney et al, J. Lab. Clin. Med. 64: 485 et seq. (1964) and Velander et al, Proc. Nat'l Acad. Sci. USA £2: 12003 et seq. (1992) each of which is herein incoφorated by reference in its entirety particularly regarding the foregoing in parts pertinent to obtaining milk from transgenic animals.

The vWF or vWF-related polypeptides contained in such milk can be purified by known means without unduly affecting activity. Generally, it is prefened that vWF or related polypeptides in milk produced pursuant to the present invention should be isolated as soon as possible after the milk is obtained from the transgenic mammal, thereby to mitigate any deleterious effect(s) of milk components on the structure, properties or activities of the protein.

For the most part the known purification methods are employed conventionally to purify TrG-vWF from transgenic milk. Representative methods in this regard are described in, among others, Bringe et al, J. Diary Res. 56: 543 et seq. (1989) which is incoφorated herein by reference in parts pertinent to methods that can be used in whole or part to purify vWF or vWF-related polypeptides from transgenic milk.

Where necessary or desirable, EDTA and or other milk clarifying agents can be used to resolubilize TrG-vWF from complexes it may form in milk, such as complexes with milk proteins, in particular caseins and casein micelles.

Prefened methods for purifying TrG-vWF include those that use one or more of the following to purify the vWF or vWF-related polypeptide from milk or whey. Affinity chromatography, particularly affinity chromatography using combinatorial affinity ligands, such as peptides, which has proven particularly useful for purifying large molecules like Factor VIII, fibrinogen and vWF, to name a few. The combinatorial affinity ligand may a single linear peptide or an arcay of different ligands, such as an anay of peptides. Ligand anays, particularly peptide-ligand anays, formed using poly-lysine core chemistry are particularly prefened in this regard. Also prefened are lows solids content matrixes, particularly low solids content matrixes in which intramatrix transport occurs that enhances absoφtive capacity for large molecules like vWF. Also among prefened methods are those that utilize custom affinity dyes or dye-like ligands for chromatographic vWF adsoφtion. There are proteases in milk that may degrade proteins, such as transgenically expressed vWF and vWF -related proteins. The main proteases in milk thus far identified are alkaline proteases with tryptic and or chymotryptic activities, a serine protein, a chymotrypsin- like enzyme, an aminopeptidase and an acid protease. Methods thus may be employed for isolation and purification that prevent proteolytic degradation of transgene products by endogenous milk proteases such as these. Among prefened methods in this regard are rapid processing of whole milk, the use of low temperatures that inhibit protease activity and or decrease degradation of transgene products in milk and the use of proteases inhibitors. Specific inhibitors that may be useful in this regard are well known to those of skill in the are widely available from commercial reagent suppliers such as Sigma Chemical Company.

Yield vWF and vWF-related polypeptides expressed in the transgenic organisms in prefened embodiments of the invention has a high percentage of active protein, as measured by conventional assays of vWF activity. Particularly, in prefened embodiments of the invention in this regard, not only do the vWF and vWF-related polypeptides expressed in and or obtained from the organisms contain a high percentage of protein having vWF activity in in vitro assays, a high percentage of protein also is physiologically active in either factor VIII binding, platelet binding or both, among others. In prefened embodiments in this regard the activities are determined as described above, and prefened activities moreover are as described above. vWF and vWF-related polypeptides expressed in the mammary tissue and secreted into the milk of a transgenic mammal obtained in this manner in prefened embodiments of the invention has a high percentage of active protein, as measured by conventional assays of vWF activity. Particularly, in prefened embodiments of the invention in this regard, not only do the vWF and vWF-related polypeptides secreted into the milk contain a high percentage of protein having vWF activity in vitro, a high percentage of protein has physiological activity, preferably fVIII binding activity, platelet binding activity or both, as well as other physiological activities. In prefened embodiments in this regard the activities are determined as described above, and prefened activities moreover are as described above.

Yields of polypeptides of the invention in this regard in prefened embodiments are sufficiently high for recovery of useful amounts of the polypeptides. In particularly prefened embodiments the yields are substantially better than those previously achieved by other methods, either as to concentration, total amount of polypeptide obtained, activity, specific activity or homogeneity, including homogeneity of activity, specific activity, physiological activity, general or specific post-translational modification, including but not limited to glycosylation, or a combination of one or more of any of the foregoing.

For milk of transgenic non-human mammals prefened embodiments of the invention in this regard relate to yields in the range of 0.05 to 5.0 g/L, especially 0.1 to

3 g/L, as well as activities, specific activities and the like of the preferred embodiments described in detail above.

Uses vWF and vWF related polypeptides of the invention have many uses, including both clinical and non-clinical applications; that is, medically related uses, including medical related uses for both non-human and human subjects, and uses that are not medically related.

Among clinically important prefened applications in this regard are prevention and treatment of mild and severe vWF diseases. In this regard, prophylactic administration of vWF through parenteral methods, including but not limited to, infusion by pump, is prefened in certain embodiments of the invention to assure stable plasma level of vWF in treating any form of vWF deficiency. Infusion is prefened in this regard particularly for administration of TrG- vWF before, during or after surgery. The present invention is further described by reference to the following, illustrative examples. EXAMPLE 1 DNAs Useful for Transgenic Expression of VWFs in Milk

As illustrated below, DNAs, vectors and expression constructs for use in accordance with the invention can be made using standard recombinant DNA techniques, such as those set forth m MOLECULAR CLONING, A LABORATORY

MANUAL, Vol 1 - 3, Sambrook et al , Cold Spnng Harbor Press (1989), which is incoφorated herein by reference in its entirety

The czs-actmg expression signals of the mouse "long WAP" promoter construct are operatively fused to DNAs encoding human vWF for introduction into and expression in transgenic mice and pigs WAP genes and promoters therefrom are obtained and are as descnbed in the foregoing references on WAP genes and promoter sequences, particularly U S. Patent No. 5,880,327 of Lubon et al for Transgenic Mammals Expressing Human Coagulation Factor VIII, and U S Patent number 5,831,141 of Lubon et al for Expression of a Heterologous Polypeptide in Mamman Tissue of Transgenic Nonhuman Mammals Using a Long Whey Acidic Protein

Promoter, each of which are herein incoφorated by reference in their entirety, as to the foregoing particularly in parts pertinent to obtaining the murine WAP gene, especially a long WAP promoter-containing fragment for expressing a vWF or a vWF-related polypeptide in milk of a transgenic mammal In particular, the vector MCS of pUC 19 is replaced by a Notl site A mouse genomic fragment containing the WAP promoter and extending upstream about 4 6 kb from a point near but upstream of the WAP translation start site is obtained A second mouse genomic fragment containing about 1 3 kb of the WAP gene immediately downstream of the translation stop site also is obtained The two fragments are joined to form a unique Kpnl at the fusion site The resulting fragment is cloned mto the Notl site in the MCS-replaced pUC19 vector VWF-encoding DNAs are inserted into the

A human vWF sequence in the GenBank database (Accession Number GenBank Accession Number X04385 (Bonthron et al , Nuc Acids Res 14 7125-7128 (1 86)) is used to design probes that can be used to obtain DNAs encoding human vWF by conventional means by screening a vascular endothehal cell or megakaryoctye cDNA library or a human genomic DNA library and punfymg therefrom a full-length vWF cDNA using methods much the same as those described in, for instance, Sadler et al, Cold Spring Harb. Symp. Quant. Biol. LI: 515-521 (1986) and European Patent Application 0 197 592 Al of Pannekoek et al. on "Preparation of human von Willebrand factor by recombinant DNA" each of which is incoφorated herein by reference in its entirety as to the foregoing particularly in parts pertinent to cloning human vWF-encoding DNAs such as human cDNAs and human genomic DNAs and to the clones obtained thereby and their use.

In addition, the dbEST database is searched for sequences that match the probes to identify human vWF-encoding cDNA clones in the IMAGE consortium library. Matches are picked from the IMAGE library and streaked to obtain single colonies.

Individual colonies are picked and verified as vWF DNA-containing clones by hybridization with labeled human vWF-specific probe oligonucleotides. cDNA is isolated from positives and sequenced. Those that match the known sequence of human vWF are used in constructs for transgenic expression of human vWF.

(A) WAP promoter cassette

The WAP6 cassette is used as a generic vector for constructing long murine WAP promoter - transgenes. The WAP6 cassette has a single Kpnl site in the untranslated region immediately downstream of the long WAP promoter sequences, which lie further upstream. The site provides for transcription of inserted sequences from the 5' long WAP promoter and has been used to express Factor IV, protein C and fibrinogen (among plasma protein genes). Generally, as was the case for these three plasma proteins, the start and stop sequences of the inserted construct must be altered to work with control sequences in the cassette.

(1) WAP-hu-vWF-cOI A DNA construct called WAP-hu-vWF-cOI is made by inserting into the human vWF cDNA into the unique Kpnl site of the murine long WAP DNA, 24 base pairs 3' to the transcriptional start site. The WAP-vWF product is then ligated into a Bluescribe vector (Stratagene) to facilitate further manipulation. (2) WAP-hu-vWF-c02

Another DNA construct called WAP-hu-vWF-c02 is made using similar methods. It is much the same as WAP-hu-vWF-01, comprising the same murine WAP and human vWF DNAs, but lacking sequences artifactually present in WAP-huPT-01 as a result of cloning procedures.

(3) WAP-hn-vWF-g I

Similar methods are used to obtain a full length genomic clone for human vWF. However, the coding region of the genomic sequence is made up of 52 exons distributed over a length of more than 175 kb (about 178 kb). The exons range from 97 bp to 19.9 bp, moreover. A mini gene genomic construct containing only some of the natural vWF introns might be advantageously employed to reduce the overall size of an effective genomic expression construct. The length of the gene makes it generally advantageous, perhaps mandatory, to employ techniques developed especially for manipulating long DNA in making genomic expression constructs, whether full length or mini-gene. Such techniques also may be applied advantageously to building cDNA vWF expression constructs.

Genomic (or cDNA) expression constructs may be assembled, in particular in this regard, using in vivo recombination mechanisms to join properly two or more separate fragments introduced together into a host, such as a cultured cell, a fetal tissue cell line, an egg or an embryo. Provided the fragments' end-sequences overlap sufficiently, endogenous recombination activity will join the separate fragments into the desired vWF construct in the host cell.

Using in vitro methods similar to those described above for cDNA expression generally requires an effective strategy for generating a Kpnl-ended genomic fragment of interest despite internal Kpnl sites in the full length vWF gene. Typically, internal

Kpnl sites can be removed by standard mutagenesis techniques and a full length Kpnl- ended fragment with internal Kpnl sties can be produced by partially digesting a genomic clone with Kpnl, resolving the largest fragments from one another by size, such as by slab gel electrophoresis or by CE and then isolating the full length genomic fragment, which should have Kpnl ends. The recovered Kpnl-ended genomic fragment thus recovered is inserted into the Kpnl site of the long WAP promoter construct. Accordingly, vWF-encoding human genomic DNA is identified in a Cal Tech human BAC library, and the vWF-encoding region is verified by PCR and direct sequencing. A single fragment containing the vWF-encoding region is isolated, modified as noted above to remove the internal Kpnl site and to contain Kpnl ends, cloned for stability in pBeloBACl 1 and, either directly or after recovery form pBeloBACl 1, cloned into the Kpnl site of the long WAP promoter construct. In addition, the genomic DNA is recovered intact, joined to adaptors at both ends, partially digested with Kpnl and the Kpnl fragment containing the intact gene is cloned into the WAP promoter construct at the Kpnl site. A two fragment strategy also is used in which the two Kpnl genomic fragments are generated by cleavage at the internal Kpnl site, manipulated separately and then operatively recombined in conect orientation with one another in the Kpnl site of the long WAP promoter construct.

EXAMPLE 2

Preparation of DNAs for Microinjection

The WAP-human vWF cDNA fragment for microinjection is prepared from WAP-hu-vWF-c02 as follows. The DNA for injection is severed intact from other parts of WAP-hu-vWF-c02 by restriction enzyme cleavage. The solution containing the WAP-hu-vWF DNA is brought to 10 mM magnesium, 20 mM EDTA and 0.1 %

SDS and extracted with phenol/chloroform. The DNA then is precipitated from the aqueous layer with 2.5 volumes of ethanol in the presence of 0.3 M sodium acetate at - 20° C overnight. After centrifugation, the pellet is washed with 70% ethanol, dried, and resuspended in sterile distilled water. The WAP-hu-vWF DNA then is further purified by sucrose gradient centrifugation using standard procedures. DNA concentrations are determined by agarose gel electrophoresis by staining with ethidium bromide and comparing the fluorescent intensity of an aliquot of the DNA with the intensity of standards. Samples are adjusted to 10 'Λg/ml and stored at -20° C prior to microinjection. EXAMPLE S Transgenic Animal Production

(1) Mice

Transgenic mice that express human vWF are produced by pronuclear microinjection using standard techniques as described below.

Glass needles for micro-injection are prepared using a micropipet puller and micro forge. Injections are performed using a Nikon microscope having Hoffman Modulation Contrast optics, with micromanipulators and a pico-injector driven by N₂ (Narashigi). Fertilized mouse embryos are surgically removed from the oviducts of super- ovulated female CD-I mice and placed into M2 medium. Cumulus cells are removed from the embryos by treatment with 300 'Λg/ml hyaluronidase. The embryos are rinsed after treatment in fresh M2 medium, transfened into Ml 6 medium and stored at 37° C prior to injection. Female mice are made pseudo-pregnant by mating with vasectomized males.

DNA is injected into the male pronucleus of embryos prepared as described above. The injected embryos are implanted into avertin-anesthetized pseudo-pregnant recipient females. Embryos are allowed to come to term and newborn mice are delivered. The newborn mice are analyzed for the presence and integration of the injected DNA.

(2) Pigs

DNAs and injection equipment and supplies are prepared much the same as described for mice. Embryos are recovered from oviducts obtained from healthy female pigs. They are placed into a 1.5 ml microfuge tube containing approximately 0.5 ml embryo transfer media (phosphate buffered saline + 10% fetal calf serum, Gibco BRL) and centrifuged for 12 minutes at 16,000 x g RCF (13,450 RPM) in a microcentrifuge (Allied Instruments, model 235 C). The embryos are removed from the microfuge tube with a drawn and polished Pasteur pipette and placed into a 35 mm petri dish for examination. If the cytoplasm is still opaque with lipid such that pronuclei are not visible, the embryos are centrifuged again for 15 minutes. Embryos to be microinjected are placed into a microdrop of media (approximately 100 VA) in the center of the lid of a 100 mm petri dish. Silicone oil is used to cover the microdrop and fill the lid to prevent media from evaporating. The petri dish lid containing the embryos is set onto an inverted microscope (Carl Zeiss) equipped with both a heated stage and Hoffman Modulation Contrast optics (200 x final magnification). A finely drawn (Kopf Vertical Pipette Puller, model 720) and polished (Narishige micro forge, model MF-35) micropipette is used to stabilize the embryos while about 1 - 2 picoliters of purified DNA solution containing approximately 200-500 copies of DNA construct is delivered into the male pronucleus with another finely drawn micropipette. Embryos surviving the microinjection process as judged by moφhological observation are loaded into a polypropylene tube (2 mm ID) for transfer into a recipient pseudo pregnant female pig.

EXAMPLE 4 Assessing Construct Integration

(1) Preparation of DNA from transgenic from mice and pigs

A 5 mm piece of mouse tail is removed from young, potentially transgenic mice at weaning (3 weeks of age), minced, and treated with proteinase K and SDS at 37° C overnight. The mixture then is incubated with DNase-free RNase at 37° C for 1-2 hours. In some cases the mixture is extracted extensively with phenol/chloroform. DNA is precipitated from the mixture with sodium acetate and ethanol at -20° C overnight, collected by centrifugation, washed in 70% ethanol and then is dried. The dried DNA pellet is used directly for PCR.

A similar procedure is used to prepare DNA from pigs.

(2) Oligonucleotide probes for PCR assay

Oligonucleotide pairs are used to prime polymerase chain reactions to detect WAP-hu-vWF constructs in the transgenic animals. Oligonucleotide pairs that bridge the WAP-hu-vWF DNA are used to detect the exogenously-derived vWF-encoding

DNA in cells of the transgenic organisms.

A probe pair that targets a region in the WAP sequence 5' of the Kpnl site and a region in the endogenous mouse WAP sequence that lies 3' of the Kpnl site is used to provide a positive control in PCR assays of mice DNA. (3) PCR reaction conditions and product analysis

PCR reactions are performed using 40 cycles in an automated temperature cycler (M.J. Research). An annealing temperature of 58° C, a denaturation temperature of 94° C, and an extension temperature of 72° C. 100 ng of oligo primers and 50 ng of (genomic) template DNA are used per PCR reaction. Products of the PCR reactions are analyzed by agarose gel electrophoresis. Fragments sizes are estimated by migration relative to molecular weight standards and compared with the sizes expected for the injected constructs.

(4) Results of PCR analysis of transgenic animals PCR analysis of potentially transgenic mice and pigs that developed from embryos microinjected with the expression constructs described above shows that injected constructs frequently are integrated into the embryonic genomes of both mice and pigs. PCR analysis of offspring shows Mendelian transmission of integrated transgenes in mice and pigs that are initially shown to have integrated the xenogenetic DNA constructs.

EXAMPLE 5 Preparation of Milk and Whey

(1) Mice

Lactating mice are milked an average of 3 times per week. The mice are first separated from their young for approximately 5 hours. Then they are anesthetized by injection of 0.4 ml avertin at 2.5% (I.M.). 0.2 ml oxytocin is administered at 2.5 IU/ml (I.P.) to stimulate the release of milk. A milking device consisting of a vacuum pump (2.5 psi) and syringe with an eppendorf tip is used to express milk from the animals and direct it into an eppendorf tube. The milk is kept on ice throughout the collection process.

The milk is kept at 4 °C to prevent cryoprecipitation or it is frozen if it is not being used immediately. Cryoprecipitate resulting from freezing, which may contain vWF, is resolubilized using surfactants, EDTA and thermal renaturation, alone or in combinations with one another. To prepare whey the collected milk is diluted 1 : 1 (vo vol) with TS buffer (0.03 M Tris pH 7.4; 0.06 NaCl) and centrifuged in a TLA- 100 rotor in a Beckman TL-100 table top ultracentrifuge at 51,000 rpm (89,000 x g) for 30 minutes at 4° C. After centrifugation the tubes are placed on ice. Whey is collected from the chilled tubes using an 18 gauge needle. Care is taken to leave the casein pellet and the upper cream layer undisturbed in the tube. Any solids or cream that co-transfer during the initial recovery are removed from the initial whey fraction by centrifugation 12,000 φm for 30 minutes at 4° C in a TMA-4 rotor in a Tomy MTX-150 centrifuge. Thereafter, the whey-containing tubes are place on ice and the whey is again recovered using a fresh 18 gauge needle.

(2) Pigs

Similar methods are employed to prepare milk and whey from pigs, in accordance with standard practice for obtaining and working with milk from normal pigs.

EXAMPLE 6

Purification of TrG-vWF from Milk

Milk containing TrG-vWF is obtained from vWF-transgenic pigs that express TrG-vWF in their milk. The milk is treated as described in the foregoing example and TrG-vWF is purified as follows from the resulting TrG-vWF-containing, milk-derived composition.

The material is suspended in 20 mm Tris-HCl, pH 7.4 and purified by anion- exchange and heparin affinity chromatography. In particular, the suspended material is applied to a Fractogel EMD-TMAE, washed with 180 mm NaCl in the same buffer, and TrG-vWF is eluted with 280 mm NaCl. The TrG-vWF-containing fractions are pooled, are diluted to 90 mm NaCl in the same buffer, and are applied to Fractogel EMD- heparin column. The column is washed with 100 mm NaCl in Tris-HCl buffer and TrG-vWF is eluted in three step with, in succession, 160 mm, 230 mm and 280 mm NaCl in 20 mm Tris-HCl, pH 7.4.

The procedures for purification of TrG-vWF from the milk extract are canied out much as described in Fischer et al, Cell. Molec. Life Sci. 51: 943-950 (1997) which is incoφorated herein by reference in its entirety particularly in parts pertinent to methods for purifying vWF.

Yield, purity and properties of vWF that is obtained by the immediately foregoing purification method are further described in examples below.

EXAMPLE 7

Yield of TrG-vWF Determined hy vWF-Ag ELISA

Amounts of TrG-vWF are quantified as vWF antigen determined by goat anti- vWF ELISA (Affinity Biologicals, Hamilton, Ontario, Canada) using CryoCheck⁰ as standard. (Precision Biologicals, Dartmouth, Nova Scotia, Canada. Alternatively vWF Ag is determined using Aserachrom vWF from Boehringer Mannheim. Similar results are obtained with the two systems when determinations are calibrated against the reference preparation and, by calculation, to the International Standard.

EXAMPLE S Platelet Binding Activity of TrG-vWF

Binding of TrG-vWF to platelets is determined using paraformaldehyde-fixed platelets, centrifugation and gel electrophoresis, much as described in Fischer et al , Cell Molec. Life Sci. 51: 943-950 (1997) which is incoφorated by reference herein in its entirety particularly in parts pertinent to assaying vWF platelet binding. In brief, TrG-vWF at vWF-Ag concentrations from 1 to 10 ! g/ml in 20 mm Tris-HCl, 150 mm NaCl, pH 7.4 is incubated with 250 VA of paraformaldehyde-fixed human platelets and 50 VA of 15 mg/ml Ristocetin for 15 minutes at 23 °C. The platelets, and bound TrG-vWF then is pelleted by centrifugation for 10 minutes in an Eppendorf mini-centrifuge. After removing the supernatents, the pellets are resuspended and washed followed by re-centrifugation. After removing the final wash supernatant, bound TrG-vWF is determined and compared with the amount of TrG-vWF in the starting material.

In both cases the results are compared with those characteristic of normal human vWF preparations. The TrG-vWF exhibits substantially the same or better platelet binding activity as the reference normal human preparations. EXAMPLE 9 Collagen Binding Activity C'CBA") of TrG-vWF

Collagen binding activity ("CBA") of purified TrG-vWF is determined using the method described by Siekmann et al, Thromb. Haemost. 11: 1160 et seq. (1995), which is incoφorated herein by reference in its entirety especially in parts pertinent to performing vWF collagen-binding assays. In brief, TrG-vWF at various dilutions is incubated with Type III collagen-coated micro-titer plates. After incubation the plates are washed and then incubated with anti-vWF antibody conjugated to horseradish peroxidase ("HRP"). Unbound antibody is washed away and the amount of bound antibody and collagen-bound TrG-vWF is determined by colorimetric ELISA based on bound HRP activity.

The results are compared with those characteristic of normal human vWF preparations. The TrG-vWF exhibits substantially the same or better collagen binding activity as the reference normal human preparations.

EXAMPLE 10

Ristocetin Cofactor Activity of TrG-vWF

Ristocetin cofactor activity ("RCA") of TrG-vWF can be determined in accordance with the invention using commercially available kits or individual components such as commercially available stabilized platelets. Ristocetin cofactor activity assays are carried out according to the manufacturer's instructions using RCA reagents and vWF standards from Behringwerke. TrG-vWF is obtained as described above and assayed alongside the reference preparations of human vWF. The results for TrG-vWF preparations routinely is as good as or better than that for the reference preparations.

EXAMPLE 11

Multimer Distribution of TrG-vWF

The multimer composition of vWF preparations can be determined by a variety of methods, including but not limited to methods that utilize 1-D or 2-D polyacrylaminde gel electrophoresis to separate different multimers from one another, radioactivity or dye staining detect the multimers in the gel and scanning densitometry to quantity the amount of each such multimer form that is detected in the gel. Other techniques that can be used toward the same end include but are not limited to capillary zone electrophoresis, particularly using dynamic light scattering for detecting and quantifying the CZE-separated multimers. Also useful in this regard is Biomolecular Interaction Analysis ("BIA"). BIA does not separate or measure structure per se. Rather it provides an absoφtive kinetic signature that is singularly indicative of functional binding characteristics. In certain embodiments where the kinetic signature is indicative and or predictive of the activity reasonably to be expected in vivo, BIA Is highly prefened for characterizing and comparing preparations of TrG-vWF and TrG- vWF*Rel polypeptides.

The types and distribution of multimers in the bound TrG-vWF is determined after removing the final wash supernatant, the platelets are resuspended in SDS-loading buffer, loaded onto a 1%> agarose-SDS gel and subjected to electrophoresis to resolve multimers from one another by size. The multimer distribution in the TrG-vWF preparation is determined by western blotting using a primary anti-vWF antibody and a secondary antibody specific for the first antibody conjugated to alkaline phosphatase ("AP"). binding of AP-antibody conjugate to western blot was visualized by AP activity using nitroblue tetrazolium chloride / bromochloroindolyl phosphate as chromogenic substrate. The procedures used were essentially the same as those described by Fischer et al, FEBS Lett. 211: 345-348 (1994) and Fischer et al, FEBS Lett. Ill: 259-262 (1995) each of which is herein incoφorated by reference in its entirety in parts pertinent to assay of vWF multimer distribution. In each case the results are compared with those typical for preparations of normal human vWF. The TrG-vWF exhibits substantially the same or better distribution of multimers as the reference normal human preparations.

EXAMPLE 12

Factor VIII Binding Activity of TrG-vWF

Binding of TrG-vWF to purified Factor VIII is determined by several methods as follows. (1) Affinity chromatography assay

Except for reversing the stationary and mobile states of the Factor VIII and vWF, affinity chromatography assays are carried out much the same as described in U.S. patent No. 5,597,711 of Zimmerman et al for Factor VIII Binding Domain of vWF, which is incoφorated herein by reference in its entirety as to the foregoing, particularly in parts pertinent to affinity chromatography assay of vWF - Factor VIII binding interaction.

In brief, Factor VIII affinity resin for the affinity assay is prepared by incubating polystyrene beads (Pierce Chemical Company) with purified Factor VIII in PBS (i.e., 0.01 M PO₄, 0.14 NaCl, 0.02% NaN₃ pH 7.3) for approximately 2 hours and then blocking remaining protein-binding sites on the beads by incubation with PBS containing 3% human serum albumin and 0.05% Tween-20 for 1 hour. Both steps are carried out at room temperature. The beads are used immediately or are stored for up to about two weeks in the blocking solution. Immediately before use to assay vWF - Factor VIII binding, the Factor VIII - polystyrene affinity beads are washed three times for about 30 minutes, each wash with PBS containing 0.05% Tween-20. The affinity beads then are incubated with a TrG- vWF sample and a competitor of vWF - Factor VIII binding. Generally, each TrG- vWF sample is assayed without inhibitor and in the presence of several concentration of inhibitor. Competitive binding to the Factor VIII beads is canied out in 0.05 M imidazole, 0.15 M NaCl, 0.02% NaN₃ 3 mm CaCl pH 7.0 for about 1.5 hours at room temperature. The beads thereafter are washed five times at room temperature with PBS containing 0.05% Tween-20, several minutes each wash. vWF - Factor VIII binding is detected by the inhibition of Factor VIII binding of an ^I25I-labeled anti-Factor VIII monoclonal antibody specific for the vWF binding site in Factor VIII. Following incubation with the sample or standard and washing, beads are incubated with labeled antibody in PBS containing 0.5% bovine serum albumin and 0.05% Tween-20 for 1.5 hours at room temperature. Thereafter the beads are washed twice with PBS containing 0.05% Tween-20, transfened to fresh containers and washed with the same solution four more times. After the last wash, the remaining radioactivity is determined for each dilution of the sample and the standard. The standard is used to construct a dose-response curve and the amount of interaction between the vWF sample and Factor VIII relative to the standard is determined from the curve.

A similar, non-competitive assay also is performed, using an affinity sorbent matrix prepared much the same as described above. The matrix-bound Factor VIII is loaded into columns. vWF from sample or standard is bound to the columns, which then are washed to remove transiently bound material. After the wash, columns are developed by a buffer gradient of increasing ionic strength, either discontinuous or continuous. The affinity of TrG-vWF preparations for Factor VIII relative to standard preparations is determined by the relative ionic strength at which they elute. TrG-vWF preparations prepared as described above generally are found by these assays to bind Factor VIII much the same as reference preparations of human vWF.

(2) Biomolecular interaction analysis vWF - Factor VIII binding also is assayed by Biomolecular Interaction Analysis ("BIA"). For these assays Factor VIII is bound to the assay surface of Biacore cartridges in accordance with the manufacturer's directions. Assays then are carried out by flowing reference vWF and TrG-vWF-containing solutions through the Biacore cells as prescribed by the manufacturer. The perturbation of surface plasmon resonance caused by sample or reference vWF binding to immobilized Factor VIII provides a real time kinetic signature of the affinity of the vWF in each sample and reference preparation for Factor VIII.

TrG-vWF preparations in these assays generally show much the same kinetic Factor VIII binding profiles as reference preparations of human vWF in reference preparations and standards.

Claims

1. A transgenic organism comprising an introduced genetic construct that engenders production in said organism of a vWF or vWF-related polypeptide.

2. A transgenic organism according to claim 1, wherein the construct engenders production of the vWF or vWF-related polypeptide in specific cells.

3. A transgenic organism according to claim 2, wherein the vWF or vWF- related polypeptide accumulates in a specific tissue or bodily compartment.

4. A transgenic organism according to claim 3, wherein the vWF or vWF- related polypeptide accumulates in a bodily fluid.

5. A transgenic organism according to any of claims 1 to 4, wherein the organism is a non-human mammal.

6. A transgenic organism according to claim 5, wherein the mammal is mouse, rat, hamster, rabbit, pig, sheep, goat, cow or horse.

7. A transgenic organism according to claim 6, wherein the organism is mouse, pig, sheep, goat or cow.

8. A transgenic organism according to claim 7, wherein the organism is Pig-

9. A transgenic organism according to any of claims 3-8, wherein the vWF or vWF -related polypeptide accumulates in the milk of females.

10. A transgenic organism according to any of claims 1 to 9 , wherein the specific activity of the vWF or vWF-related polypeptide is 50% to 110% of that of purified human vWF from pooled human plasma.

11. A transgenic organism according to any of claims 1 to 9, wherein the specific activity of the vWF or vWF -related polypeptide is greater than that of purified human vWF from pooled human plasma.

12. A transgenic organism according to claims 10 or 11 , wherein activity is determined by a platelet binding assay.

13. A transgenic organism according to claims 10 or 1 1, wherein activity is determined by a Factor VIII binding assay.

14. A transgenic organism according to claims 10 or 11 , wherein activity is determined by both a platelet binding assay and a Factor VIII binding assay.

15. A transgenic organism according to claims 10 or 11, wherein activity is determined by one or a combination of the following assays, a collagen binding assay, a collagen binding assay, a heparin binding assay, a sulfatide binding assay, a GPlb, binding assay, a botrocetin binding assay and a GPIIb-IIIa binding assay.

16. A transgenic organism according to any of claims 10 to 15, wherein the purified human vWF is calibrated against an accepted international vWF activity standard.

17. A transgenic organism according to any of claims 1 to 16 , wherein the vWF or vWF related polypeptide comprises a region having an amino acid sequence 90% to 100% identical to that of a mature mammalian vWF.

18. A transgenic organism according to claim 17, wherein the mature mammalian vWF is mature human vWF.

19. A transgenic organism according to claim 18, wherein the vWF or vWF- related polypeptide is human pre-pro-vWF, human pro-vWF or mature human vWF.

20. A transgenic organism according to any of claims 1-19, wherein the introduced genetic construct comprises a promoter operatively linked to the region encoding vWF or a vWF-related polypeptide, wherein further the promoter is selected from the group consisting of the promoters of whey acidic protein genes, casein genes, lactalbumin genes and .beta.lactoglobulin genes.

21. A transgenic organism according to claim 20, wherein the promoter is a whey acidic protein promoter or a beta.lactoglobulin promoter.

22. A transgenic organism according to claim 21 , wherein the promoter is a whey acidic protein promoter.

23. A transgenic organism according to claim 22, wherein the promoter is the mouse whey acidic protein promoter or the rat whey acidic protein promoter.

24. A transgenic organism according to claim 23, wherein the promoter is a long whey acidic protein promoter.

25. A transgenic organism according to claim 24, wherein the promoter is the mouse long whey acidic protein promoter.

26. A composition comprising a vWF or a vWF-related polypeptide produced by an introduced genetic construct in a transgenic organism.

27. A composition according to claim 26, comprising a vWF or vWF-related polypeptide produced in milk of a non-human transgenic female mammal.

28. A composition according to claim 27, wherein the composition is milk of the transgenic mammal.

29. A composition according to claim 28, wherein the composition is derived from milk of the transgenic mammal.

30. A vWF or vWF-related polypeptide isolated from a transgenic organism according to any of claims 1 to 25.

31. A human vWF or vWF -related polypeptide with post-translational modification different from that of naturally occurring human vWF.

32. A human vWF or vWF -related polypeptide according to claim 31 that has the platelet binding activity of human vWF isolated from natural sources.

33. A human vWF or vWF -related polypeptide according to claim 32 that has the Factor VIII binding activity of human vWF isolated from natural sources.

34. A human vWF or vWF-related polypeptide isolated from a transgenic organism according to any of claims 1 to 25 that differs in its glycosylation from that of human vWF isolated from natural sources.

35. A human vWF or vWF -related polypeptide according to claim 34 that has the platelet binding activity of human vWF isolated from natural sources.

36. A human vWF or vWF-related polypeptide according to claim 34 that has the Factor VIII binding activity of human vWF isolated from natural sources.

37. A method for producing a vWF or a vWF -related polypeptide comprising the step of producing the vWF or vWF-related polypeptide in a transgenic organism by expression of an introduced genetic construct.

38. A method according to claim 37, wherein the transgenic organism is an organism according to any of claims 1 to 25.