WO2012075138A1

WO2012075138A1 - Method of producing biologically active vitamin k dependent proteins in transgenic animals

Info

Publication number: WO2012075138A1
Application number: PCT/US2011/062641
Authority: WO
Inventors: Stephen P. Butler; Julian D. Cooper
Original assignee: Progenetics Llc
Priority date: 2010-11-30
Filing date: 2011-11-30
Publication date: 2012-06-07

Abstract

A method is disclosed of increasing the biological activity of a Vitamin K dependent protein produced in a transgenic animal, cell or cell line comprising feeding a transgenic animal or cell /cell line expressing a gene encoding the Vitamin K dependent protein an animal feed or media comprising an increased amount of vitamin K over that of standard animal feed containing a standard amount of vitamin K. The Vitamin K dependent protein expressed in the transgenic animal possesses an increased biological activity over a Vitamin K dependent protein expressed in a transgenic animal that is fed standard animal feed or standard media containing a standard amount of Vitamin K where the biological activity is increased as a result of an increase of gamma carboxylation of the Vitamin K dependent protein.

Description

METHOD OF PRODUCING BIOLOGICALLY ACTIVE

VITAMIN K DEPENDENT PROTEINS IN TRANSGENIC ANIMALS FED LARGE QUANTITIES OF VITAMIN K IN THEIR DIETS BACKGROUND OF THE INVENTION To obtain larger quantities of Vitamin K Dependent proteins (VKD proteins) that cannot be obtained from natural sources, these proteins have been produced and recovered from several different transformed cell types and from the tissues of transgenic animals.

However, for a VKD protein to be fully functional it needs to be gamma carboxylated at specific glutamic acid residues on the protein by gamma-glutamyl carboxylase. When VKD proteins are produced at low levels, the resulting proteins are usually fully carboxylated but as synthesis rates of a VKD proteins increase in a cell type or tissue, under gamma carboxylation of the VKD proteins occurs, resulting in reduced activity or no activity of the produced VKD proteins. The currently known VKD proteins include the following: Prothrombin, Factor VII, Factor IX (FIX), Factor X, Protein C, Protein S, Protein Z, matrix Gal protein, osteocalcin, Gas6, various receptor tyrosine kinase ligand proteins, connexin 26, connexin 32, PRGP1, PRGP2, TmG3, TmG4, small peptides found in the venom of the marine snail Conus and the Gamma Glutamyl Carboxylase itself (which are disclosed in Oldenburg et al., Antioxidants & Redox Signaling, Vol.8, 3-4: 347-353(2006)). SUMMARY OF THE INVENTION

In one embodiment, the present invention is directed to a method of increasing the biological activity of a Vitamin K dependent protein produced in a transgenic animal comprising feeding a transgenic animal expressing a gene encoding the Vitamin K dependent protein an animal feed comprising an increased amount of vitamin K over that of standard animal feed containing a standard amount of vitamin K. The Vitamin K dependent protein expressed in the transgenic animal fed the vitamin K supplemented feed possesses an increased biological activity over a Vitamin K dependent protein expressed in a transgenic animal that is fed standard animal feed containing a standard amount of Vitamin K. This method further includes that the increased biological activity of the Vitamin K dependent protein is the result of increased gamma carboxylation of the Vitamin K dependent protein. The present invention also includes a method of increasing the gamma carboxylation of a Vitamin K dependent protein produced in a transgenic animal comprising feeding a transgenic animal expressing a gene encoding the Vitamin K dependent protein an animal feed comprising an increased amount of vitamin K over that of standard animal feed containing a standard amount of vitamin K, wherein the Vitamin K dependent protein expressed in the transgenic animal contains a greater level of gamma

carboxylation to glutamate residues on the protein than a Vitamin K dependent protein expressed in a transgenic animal that is fed standard animal feed containing a standard amount of Vitamin K. The present invention is also directed to increasing the availability of vitamin K in animals or cell by supplementing the diet/intake of animals/cells with much higher levels of vitamin K than the normally recommended daily/hourly rate to facilitate the production of VKD proteins that have increased biological activity over animals/cells that are feed with a diet or intake of normal or standard vitamin K. The present invention is further directed to a method of delivery of large quantities of vitamin K to animals by food and avoids the negative limitations of delivering vitamin K to animals by injection. The present invention is further directed to supplementing above normal levels of vitamin K to non-human mammals or to cells or cell lines, that may be transgenic or non- transgenic for carrying a foreign gene not normally carried by the mammals, cells or cell lines. The cell lines may be used to transfer the gamma-carboxylation enzymic pathway into perhaps, a plant cell. The present invention also is further directed to supplementing above normal levels of vitamin K using different kinds of vitamin K, for examples K1, K2, K3 and their side chain derivates or a combination thereof. Further, the present invention can further be directed to delivering vitamin K in a hydrophobic carrier, including an edible oil or digestible oil suitable for maintaining tissue culture cells. Additionally, the present invention may also include supplementing above normal levels of vitamin K to transgenic non-human animals, cell cultures or cell lines, and optionally in quantities of more than mg/kg/day in non-human animals. For example, supplementing more than once per day for non-human animals may be useful to maintain the life of the vitamin K. The present invention may also include a method of controlling or managing the diet of a non-human animal (and transgenic animals) to increase production of a protein or a foreign protein introduced into the animal Such management may include managing the diet of non-human animals to maintain a low body fat of the animal to increase the effectiveness of vitamin K supplementation. The methods of the present invention are generally directed to producing high levels of biologically active VKD proteins by increasing the effectiveness of supplementation with vitamin K in the animal or cell line. The cell lines may be obtained from cells obtained from the transgenic animals. In addition to producing these proteins, these proteins may be harvested from a body fluid of the animal, and purified to obtain an active VKD protein by purifying the active VKD protein from inactive and other proteins.

Additionally, the purified VKD protein can be formulated with pharmaceutically acceptable carriers or any acceptable excipient to use for the treatment of humans and animals. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to producing and ultimately obtaining transgenically produced biologically active VKD proteins from the fluids and/or tissue of a transgenic animal that has been transformed to contain genes that express the VKD protein by increasing vitamin K in the diet of the animal. Preferably, the transgenic animal is a transgenic non-human mammal. More specifically, the present invention is directed to increasing gamma carboxylation of VKD proteins that are produced in transgenic animals or transgenic cells that have been engineered to produce increased quantities of biologically active VKD proteins by increasing vitamin K in the diet of the animal. The inventors observed that once the production of the VKD proteins in the transgenic animal or transgenic cells was over a certain level of production, the VKD proteins were biologically less active. The inventors believed that the VKD proteins produced in these hosts were not properly gamma carboxylated. Basically, it is believed that the post- translational processing enzyme gamma-glutamyl carboxylase, did not have sufficient amounts of vitamin K substrate in the cells or tissue in sufficient amounts to properly gamma carboxylate the VKD proteins that are produced in larger amounts in these transgenic animals and cells. Transgenic animals and transgenic cells are known to be good sources for producing important pharmaceutical products. These transgenic animals and cells are genetically modified by introducing genes encoding these pharmaceutical products into these animals and cells so that they express these introduced genes in their body fluids, tissues and cells. But with the increased levels of production of specific proteins comes the concerns for overburdening the endogenous protein expression and processing systems of these transgenic animals and cells to produce larger amounts of biologically active expressed VKD proteins. To produce and obtain increased amounts of biologically active VKD proteins, the present invention provides a method of providing more Vitamin K to the transgenic animals and cells producing larger quantities of the VKD proteins by increasing the amount of vitamin K available to the transgenic animals’ tissues of interest or cells in which the VKD protein is synthesized and/or processed through specific dietary manipulations including feeding high levels of vitamin K (100 times of that found in standard preparations of animal feed). The RDI (recommended daily allowance) for specific animals has not been established. So, Vitamin K is supplemented in feed as a preventive measure to avoid the onset of coagulation deficiencies. Table 1 lists the amounts added to commercially prepared or academic formulated diets that have been shown to avoid the onset of vitamin K deficiency. These suggested values do not include the use of a lipophilic carrier for the vitamin K and with or without caloric reduction of the diet. The present invention considers that previous reports disclosed that when a VKD protein is expressed in mammary tissue of a transgenic, non primate animal, where secretion is low (e.g., 200 μg/ml or less produced in mammary gland cell and secreted into milk), then most of the VKD protein was properly gamma carboxylated resulting in the majority of the protein being biologically active. See Van Cott et al., 1996 and VanCott et al., 1999, referenced below. However, when the VKD protein is expressed at concentrations greater than approximately 200 μg/ml, then there was a lower percentage of biologically active VKD protein that was produced because it was not completely gamma glutamyl carboxylated. A similar result of under gamma carboxylation is seen when VKD protein secreting transformed cells are grown in culture as detailed by Kaufman et al,. 1986 and others (Sun et al., 2005). Kaufman et al. 1986 could not correct the under gamma glutamyl carboxylation of a VKD protein that was overexpressed in Chinese Hampster Ovary (CHO) cells by simply adding more vitamin K1 or vitamin K3 to the culture media. The amount of fully gamma glutamyl carboxylated VKD proteins that are produced may vary depending on the specific VKD protein produced. But one skilled in the art can determine sufficient amounts of vitamin K that needs to be fed to a transgenic animal or included in media for transgenic cells to increase the biological activity of the

transgenically produced VKD proteins. The present invention is directed to increasing the total amount of fully gamma carboxylated , biologically active VKD proteins in the total amount of the VKD protein produced in a transgenic animal genetically modified to contain the gene expressing the VKD protein. This increase in biologically active VKD proteins is the result of adding vitamin K to the transgenic animal’s diet which is compared to the amount of VKD proteins that are produced in a transgenic animal genetically modified to contain the gene expressing the VKD protein that is fed a standard diet containing the standard or normal amount of vitamin K in commercial feed or specially produced feed. Since transgenic animals can secrete proteins into milk at gram/liter amounts, the present inventors considered that it would be beneficial to use a method that would increase the amount of gamma glutamyl carboxylation of the VKD proteins produced in the transgenic animal when VKD proteins are expressed in amounts over 200 μg/ml in fluid of the animal, such as milk or urine. Vitamin K is a lipophilic molecule characterized by a 1,4-naphthoquinone usually containing a methyl group at the 2 position an associated liphatic side chain at the 3 position. Vitamin K1 (Phylloquinone) is principally acquired in the human diet by the consumption of dark green leafy vegetables. The U.S. dietary recommended intake for humans is approximately is 90 μg for adult females and 120 μg for adult males per day (Olson, American Journal of Clinical Nutrition, 45, 687-692(1987)). Vitamin K1 is absorbed in the intestines and transported to non-hepatic tissue by a poorly defined mechanism while a certain percentage is converted in the liver to Vitamin K2 (Menaquinone) through changing the number of isoprene units in the liphatic side chain. Menaquinones of varying side chain lengths are also synthesized by bacterial flora in the large intestine. Up to 85% absorption of Vitamin K was observed when subjects were fed 1 mg of vitamin K with a light meal and the transport of Vitamin K to peripheral tissues is postulated via the lymph in chylomicrons and carried by chylomicron remnants to the plasma in association with lipoproteins (Shearer et al., 1974). Absorbed Vitamin K has been found distributed in lung, adrenal gland, kidney and bone marrow (Shearer et al.1974). Animal studies indicate absorption of Vitamin K is in the small intestine and that overall body store turn over is rapid. (Olsen, 1984). Menadione Vitamin K3 and Menadiol Vitamin K4 are commercially available in their water soluble forms and are readily absorbed after oral administration (Basu and

Dickerson, 1996). They are characterized by a lack of an isoprene side chain at the 3 position of 1,4-naphthoquinone. Oral toxicity (LD 50) for menadione is 0.5g /kg b.w. in mice, rats and chickens. (Molitor, H. and Robinson, H.J. (1940). Human Factor IX along with many other blood plasma proteins fall into a grouping commonly referred to as Vitamin K Dependent proteins. These proteins require post- translational gamma carboxylation modifications to specific glutamate residues for proper functionality where the modifying enzyme gamma glutamyl carboxylase requires Vitamin K as a cofactor in processing. Synthesis of VKD blood proteins occurs mainly in the liver, however, their production has been demonstrated using recombinant methodologies in transgenic animal, and specifically in animal tissues, such as the mammary gland, resulting in the secretion of the proteins into the milk of the animal. See, for example, U.S. Patent No.5,880,327 (Factor VIII); U.S. Patent Nos: 6,344,596 and 7,419,948 (Factor IX); and U.S. Patent No.5,589,604 (Protein C). Numerous publications and issued patents disclose the expression of VKD proteins in various transgenic animal species and cell cultures with the goal of these expression systems to obtain large quantities of biologically active VKD proteins in amounts sufficient for pharmaceutical use. VKD proteins are not limited to blood proteins but include others that function in bone metabolism, arterial calcification and signal transduction (Oldenburg et al., 2006). Also, Gamma-Glutamyl Carboxylation (GGC) has been found in other organs besides liver and in non-liver cell lines, such as 293 and BHK, thus demonstrating the tissue diversity that GGC is available to operate in. (Wajih et al.2005) and (Van Cott et al., 1996). The base model for gamma glutamyl carboxylase of a VKD protein involves secretion of the peptide into the endoplasmic reticulum (ER) followed by recognition of an approximate 18 amino acid propeptide sequence by the gamma glutamyl carboxylase. Upon binding, gamma glutamyl carboxylase starts the gamma glutamyl carboxylation process by using the reduced quione form of Vitamin K (H₂VK) as a cofactor for fixing CO₂ to the glu residues present on the peptide chain (K. Berkner 2005). The resulting gamma glutamyl residue (gla) is released along with oxidized Vitamin K (VKO) and the cycle is repeated with the carboxylase still attached to the peptide until (as with Factor IX) 12 residues have been converted (Hallgren et al., 2002). After release, the recognition peptide can be cleaved by furin and the carboxylated protein subjected to other post-translational events. For full carboxylation of a VKD protein to occur, gamma glutamyl carboxylase needs to have an ample supply of reduced Vitamin K to prevent a postulated processing hesitation and premature release from the target peptide resulting in the VKD protein being in an under gamma carboxylated state. Intracellular pools of H₂VK are thought to be limited as this compound is rapidly oxidized in an oxygen rich environment; therefore a reductase has been suggested to play a critical role in providing enough H₂VK for full carboxylation to occur (Sun. Y. et al., 2005). To increase the full carboxylation of a VKD protein, the present invention increases the intracellular pools of H₂VK by feeding Vitamin K in amounts greater than the normal daily requirements for that animal species resulting in improved gamma glutamyl carboxylation of the VKD protein. Swine diets usually provide 1 mg per 100 pounds of body weight for an approximate total of 4 mg per animal (Borden and Carlson). The present invention feeds the transgenic animals more than a 100 fold the standard dietary intake to provide greater amounts of vitamin K to the transgenic animal resulting in improved gamma-glutamyl carboxylation of the VKD protein. The method of the invention utilizes increased amount of vitamin K in the feed comprises in the range of approximately 1 to 60g per day, preferably approximately 5 to 50 g per day, more preferably approximately 10-49 g per day and most preferably approximately 15– 35 g per day. The method of the invention employs the ratio of increased amount of vitamin K as compared to the standard amount of vitamin K in the feed comprises in the range of 1:100 to 1:5000 ,and preferably 1:1750 to 1:3200. Further, the method of the present invention , wherein the biological activity of the Vitamin K dependent protein produced in an animal feed increased amounts of vitamin K in the feed as compared to the biological activity of a Vitamin K dependent protein produced in an animal feed with standard amounts of vitamin comprises an increased activity of 100 to 10,000 percent of units per ml, and preferably 2000 to 2500 percent of units per ml. In addition to increasing the dietary vitamin K to the transgenic animal, the present invention can also utilize dietary feed restriction to enhance the utilization of dietary supplemented vitamin K through as of yet a poorly defined mechanism. Caloric reduction of the diet where body weight is reduced on a daily basis up to 0.5% per day is useful for increasing the animal’s utilization of the Vitamin K in the diet that has been supplemented with vitamin K. The animals may vary in size but the caloric reduction in diet can be controlled so that the animal’s body weight is reduced on a daily basis up to 0.5% per day. Note: More that 0.5% daily weight loss is not veterinary recommended. For most (if not all) animals, a recommended daily intake of vitamin K has not been established. However, animal feed diets have been formulated that incorporate adequate amounts of vitamin K by the manufacturers, as shown in Table 1, such as those that produce the various animal feeds, that contain sufficient amounts of vitamin K so that symptoms of vitamin K deficiency are not observed in the animals. A summary of Vitamin K amounts in various prepared standard diets containing standard acceptable amounts of vitamin K in relation to species and the dietary intake amount on a daily basis when known is listed below.

Table 1:

a University of Missouri

b Kansas State University

c Nutrient requirements of laboratory animals, 4^th ed.1995

d Harlan laboratory animal diets

These animal feeds contain in the general range of 1-5 mg of vitamin K to 1 kg of feed with outliers outside that range for cats and ruminates. More specifically standard vitamin K supplemented swine feed contains between 2.2 - 4.4mg vitamin K to 1 kg of feed. The present invention’s method is applicable to the production of any VKD protein, and the currently known VKD proteins, include: Prothrombin, Factor VII, Factor IX, Factor X, Protein C, Protein S, Protein Z, matrix Gal protein, osteocalcin, Gas6, various receptor tyrosine kinase ligand proteins, connexin 26, connexin 32, PRGP1, PRGP2, TmG3, TmG4, small peptides found in the venom of the marine snail Conus and the Gamma Glutamyl Carboxylase itself (Oldenburg et al., 2006). Applicants recognize that a transgenic non-primate mammal can produce and/or secrete VKD proteins that are under gamma carboxylated and that it would be useful to develop a method of animal husbandry practice that would increase production and/or secretion of properly gamma carboxylated VKD proteins. Applicants further recognize that reducing caloric intake during times of high energy demand, such as growth and lactation, is counter intuitive, because it is common practice in the art to increase caloric consumption to maintain or increase body weight during growth and lactation. Applicants have determined that feeding high levels of vitamin K to a transgenic animal increases the amount of gamma carboxylation occuring in a tissue of a transgenic animal whereas Kaufman et al. 1986 could not alleviate under gamma carboxylation of a VKD protein being overexpressed in transformed CHO cells by simply adding more vitamin K1 or vitamin K3 to the culture media. The specific problem that the present invention addresses had been recognized by Van Cott et al., 1996 and VanCott et al., 1999. When a VKD protein is expressed in mammary tissue of a transgenic, non primate animal, where secretion is low (200 μg/ml or less in milk) then most of the VKD protein can be properly gamma carboxylated. When the VKD protein is expressed at concentrations beyond 200 μg/ml then most if not all of the VKD protein being produced is under gamma carboxylated. Since transgenic animals can secrete proteins into milk at g/l amounts it would be beneficial to use a method that would increase the amount of gamma carboxylation occuring when VKDs are expressed over 200 μg/ml in milk. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. EXAMPLES The Activated Partial Thromboplastin Time Test (APTT) is a common central laboratory test, typically performed using a coagulometer, for example MLA Electra Coag-a-Mate 750, or another coagulometer produced by this company or otherwise known in the art. The test is performed using a diluted milk sample in imidazole buffer, mixed with Factor IX deficient plasma, incubated with Alexin reagent (cephalin from rabbit brain), then clot formation is initiated by Ca²⁺ addition. Activity of the sample is related to the time it takes for clot formation as measured by the coagulometer. Activity for a sample is calculated from a standard curve generated when dilutions of a known verified reference plasma pool are measured and plotted using ln of units verses ln of clotting time, where “ln” is a natural log function.1 unit is the amount of FIX activity found in 1 ml of pooled verified reference plasma. 1 unit of activity is produced from 5 μg of fully active FIX. Pigs carrying the human Factor IX (FIX) gene and its expression in milk is defined in detail by VanCott et al., 1999, and in U.S. Patent Nos: 6,344,596 and 7,419,948 , which are herein incorporated by reference. Milk was collected by separating the piglets from the sow for a period of 1 hour followed by administering 60 units of oxytocin

intramuscular to the ham of the sow. Approximately 8-10 minutes after injection teats were washed with standard teat dip, dried and milked by hand. Aliquots were frozen and stored at -40^oC until analysis. The term“vitamin K” refers to any 1,4-naphthoquinone which may or may not contain substitutions at the 2 and 3 position of the molecule and is used by the Gamma-glutamyl carboxylase as a cofactor in the gamma carboxylation of proteins. The term“vitamin K” also refers to any 1,4-naphthoquinonol (1,4-naphthodihydroquinone) which may or may not contain substitutions at the 2 and 3 position of the molecule and is used by the gamma-glutamyl carboxylase as a cofactor in the gamma carboxylation proteins.

The term“Vitamin K₁” refers to Phylloquinone, 2-methyl-3-phytyl-1,4-naphthoquinone. The term " Vitamin K2" refers to Menatetrenone, 2-Methyl-3-(3,7,11,15-tetramethyl- 2,6,10,14-hexadecatetraenyl)-1,4-naphthoquinone. The term“Vitamin K₃” refers to Menadione, 2-methyl-1,4-naphtoquinone. The term“Vitamin K4” refers to Menadiol. Carrier oil is defined as a general lipid that can be ingested and absorbed by the animal and possesses the ability to solvate vitamin K. Examples include but are not limited to unsaturated lipids, monosaturated lipids, polysaturated lipids and various mixtures of unsaturated, monosaturated or polysaturated lipids. These lipid molecules can also be linked into more complex lipids such as triglycerides. Examples include but not limited to vegetable oils such as canola, corn, sesame, flax, palm, sunflower, olive, safflower, walnut, soybean, coconut and cottonseed. Examples include but not limited to animal fats and oils such as lard, cream and fish oil. Vitamin K is formulated with the carrier oil before application to feed. Formulation consists of adding carrier oil to the vitamin K or the vitamin K to carrier oil followed by simple mixing. The amount of carrier oil to vitamin K can be varied. The lower limit of carrier oil added equals the vitamin K solubility in that oil. In the case of menadione, 1.26 gram is soluble in 50 ml of vegetable oil. Example 1: Adding different types of vitamin K at a high level to the diet of a transgenic animal carrying a Factor IX (FIX) gene that results in more gamma glutamyl carboxylated human FIX being produced as measured by high FIX activity in milk. Animal was producing 2000 μg/ml of FIX in milk. One transgenic sow expressing human F IX in her milk was fed a standard diet initially containing 4 mg of vitamin K that was supplemented with either Vitamin K₃ or Vitamin K₁ formulated with canola oil. Vitamin K₃ was formulated at a ratio of 1.26g per 50 ml of canola oil. Vitamin K₁ was formulated at either 5g per 45ml of canola oil or 25g per 75 ml of canola oil. Supplementation consisted of pouring the formulation over the feed and mixing until the formulation is evenly distributed in the food. Milk was collected from different days of lactation and multiple milkings were performed on a given day. Milks were then subjected to analysis using APTT. Results from feeding high levels of different vitamin Ks over the course of a single lactation is summarized in Table 2. In this experiment both vitamin K₁ and vitamin K₃ were able to be absorbed by the animal and utilized by the transgenic mammary gland to facilitate gamma carboxylation of human Factor IX.

Table 2:

.

Example 2:

Adding varying amounts of vitamin K1 and K3 to the diet of transgenic swine results in increased production of gamma carboxylated human FIX being produced as measured by APTT. Five transgenic sows that express human Factor IX in their milk (2000 μg/ml) were fed a standard diet containing 4 mg of vitamin K that was supplemented with either Vitamin K₃ or Vitamin K₁ formulated with canola oil or fed the standard diet alone with out supplementation. Vitamin K₃ was formulated at a ratio of 1.26g per 50 ml of canola oil. Another animal was fed a standard diet containing only the 4mg of Vitamin K. Vitamin K₁ was formulated at either 5g per 45ml of canola oil, 10g per 35 ml, 15g per 35 ml, 25g per 75 ml or 30g per 70 ml of canola oil. Supplementation consist of pouring the formulation over the diet and mixing until the formulation was evenly distributed in the food. Milk was collected from different days of lactation. Milks were then subjected to analysis using APTT. Results from feeding high levels of different vitamin Ks and different amounts over the course of a single lactation is summarized in Table 3. In this experiment vitamin K₁ and vitamin K₃ were able to be absorbed by the animal and utilized by the transgenic mammary gland to facilitate gamma carboxylation of human Factor IX when given at different amounts. The minimal fold increase with vitamin K supplementation was 5 fold with the highest being a 50 fold increase over that observed when one animal was feed a standard diet without vitamin K supplementation.

Table 3:

*Vitamin K added to the standard diet to maintain health

Example 3

Reducing caloric consumption of transgenic swine while supplementing with vitamin K in high doses results in increased production of gamma carboxylated human FIX being produced in milk as measured by APTT. Four transgenic sows that express human Factor IX in their milk (2000 μg/ml) were fed a standard diet containing 4 mg of vitamin K that was supplemented with Vitamin K₁ formulated with canola oil. Vitamin K₁ was formulated at either 10g per 35ml or 30g per 70 ml of canola oil. Supplementation consist of pouring the formulation over the diet and mixing until the formulation was evenly distributed in the food. Milk was collected from different days of lactation. Milks were then subjected to analysis using APTT. Results from feeding differing amounts of high levels of vitamin K₁ with and without caloric reduction over the course of a single lactation is summarized in Table 4. In this experiment different amounts of vitamin K₁ were able to be absorbed by the animal and utilized by the transgenic mammary gland to facilitate gamma carboxylation of human Factor IX. When the dietary caloric intake was reduced by reducing the amount of food, the activity observed in milk increased. This observation holds true for different amounts of vitamin K₁ supplemented to the diet.

Table 4:

Example 4: The ratio of carrier oil to amount of vitamin K fed is able to maintain utilization of vitamin K over a 10 fold range. Five transgenic sows that express human Factor IX in their milk (2000 μg/ml) were fed a standard diet containing 4 mg of vitamin K that was supplemented with either Vitamin K₃ or Vitamin K₁ formulated with canola oil or fed the standard diet alone with out supplementation. Vitamin K₃ was formulated at a ratio of 1.26g per 50 ml of canola oil or a ratio of 1:39.6. Vitamin K₁ was formulated at either 5g per 45ml of canola oil (1:10), 10g per 35 ml (1:5), 15g per 35 ml (1:3.33) or 25g per 75 ml (1:4) of canola oil.

Supplementation consist of pouring the formulation over the diet and mixing until the formulation was evenly distributed in the food. Milk was collected from different days of lactation. Milks were then subjected to analysis using APTT. Results from feeding high levels of different vitamin Ks and different amounts over the course of a single lactation is summarized in Table 5. In this experiment vitamin K₁ and vitamin K₃ were able to be absorbed by the animal and utilized by the transgenic mammary gland to facilitate gamma carboxylation of human Factor IX when formulated with different ratios of canola oil.

Table 5:

The functionality of the invention is illustrated by the data in Table 6 where an individual animal was fed no additional vitamin K during lactation resulting in low activity (< 10 units/ml of milk). This same animal was then fed vitamin K1 at 30 to 60g a day over different lactations resulting in a marked increase in unit activity. Table 6. Milk activity results from different Vitamin K feeding regiments using the same animal.

7 < 6 76 n/a 70

8 8 n/a n/a 70

9 < 6 n/a n/a 75

10 < 6 n/a 37 89 50μg/kg yield 275 nmol/liter in plasma at t=6

Olson, J.A. (1987) Recommended dietary intakes (RDI) of vitamin K in humans.

American Journal of Clinical Nutrition, 45, 687-692. Complete citations of the references cited above are provided below and are incorporated by reference in their entirety : Kaufman et al. Expression, purification, and characterization of recombinant gamma- carboxyated factor IX synthesizecd in chinese hamster ovary cells.(1986) J Biol Chem vol.261, 21: 9622-9628 VanCott et al. Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland. (1999) Genetic Analysis 15, 155-160. Shearer, M.J. et al. (1974) Studies on the absorption and metabolism of phylloquinone (vitamin K1) in man. Vitamins and Hormones, 32, 513-542. Olsen, R.E. (1984) The function and metabolism of vitamin K. Annual Review of Nutrition, 4, 281-337 Basu, T.K. and Dickerson, J.W.T. (1996) In: vitamins in Human Health and disease, Cab International, Oxford, UK, pp 228-239 QP771 .B371996. Molitor, H. and Robinson, H.J. (1940) Oral and parenteral toxicity of vitamin K1, phthicol and 2-mehtyl-1,4-naphthoquinone. Proceedings of the society for experimental Biology and Medicine, 43, 125-128. QP1 .S8 in storage cited by others Wajih N. et. al., Increased Production of Functional Recombinate Human Clotting Factor IX by Baby Hamster Kidney Cells Engineered to Overexpress VKORC1, The Vitamin K2,3-Epoxided-reducing enzyme of the Vitamin K Cycle (2005), JBC 280: 10540-10547 Van Cott K.E. et. al. Affinity purification of biologically active and inactive forms of recombinant human Protein C produced in porcine mammary gland. J Mol Recognition (1996);9: 407-41. K. Berkner, The Vitamin K-dependent carboxylase (2005) Annu. Rev. Nutr 25:127-149. Hallgren K.W. et al. Carboxylase overexpression Effects Full Carboxylation but poor release and secretion of Factor IX: Implications for the release of vitamin K dependent proteins (2002) Biochemistry 41:15045-15055 Sun. Y. et.al. Vitamin K epoxide reductase significantly improves carboxylation in a cell line overexpressing factor X. (2005) Blood, 106:3811-3815 Olson, J.A. (1987) Recommended dietary intakes (RDI) of vitamin K in humans.

American Journal of Clinical Nutrition, 45, 687-692. Oldenburg, J. et al. (2006) Vitamin K epoxide reductase complex subunit 1 (VKORC1): the key protein of the vitamin K cycle. Antioxidants & redox signaling, vol.8, 3-4: 347- 353. Breeding Herd Recommendations for swine, (MF 2302). Kansas State University, Oct. 2007 Boren, C and Carlson, M. Nutrient requirements of swine and recommendations for Missouri. (G2320) MU Extension, University of Missouri-Columbia.

Claims

Claims:

1. A method of increasing the biological activity of a Vitamin K dependent protein produced in a transgenic animal comprising

feeding a transgenic animal expressing a gene encoding the Vitamin K dependent protein an animal feed comprising an increased amount of vitamin K over that of standard animal feed containing a standard amount of vitamin K.

2. The method of claim 1, wherein the Vitamin K dependent protein expressed in the transgenic animal possesses an increased biological activity over a Vitamin K dependent protein expressed in a transgenic animal that is fed standard animal feed containing a standard amount of Vitamin K.

3. The method of claim 1 or claim 2, wherein the increased biological activity of the Vitamin K dependent protein is the result of increased gamma carboxylation of the Vitamin K dependent protein.

4. A method of increasing the gamma carboxylation of a Vitamin K dependent protein produced in a transgenic animal comprising of feeding a transgenic animal expressing a gene encoding the Vitamin K dependent protein an animal feed comprising an increased amount of vitamin K over that of standard animal feed containing a standard amount of vitamin K, wherein the Vitamin K dependent protein expressed in the transgenic animal contains a greater level of gamma carboxylation to glutamate residues on the protein than a Vitamin K dependent protein expressed in a transgenic animal that is fed standard animal feed containing a standard amount of Vitamin K.

5. The method of any one of claims 1-4, wherein the increased amount of vitamin K in the feed comprises in the range of approximately 1 to 60g per day, preferably approximately 5 to 50 g per day, more preferably approximately 10-49 g per day and most preferably approximately 15– 35 g per day.

6. The method of any one of claims 1-4, wherein the ratio of increased amount of vitamin K as compared to the standard amount of vitamin K in the feed comprises in the range of 1:100 to 1:5000 ,and preferably 1:1750 to 1:3200.

7. The method of any one of claims 1-4, wherein the biological activity of the Vitamin K dependent protein produced in an animal feed increased amounts of vitamin K in the feed as compared to the biological activity of a Vitamin K dependent protein produced in an animal feed with standard amounts of vitamin comprises an increased activity of 100 to 10,000 percent of units per ml, and preferably 2000 to 2500 percent of units per ml.

8. The method of any one of claims 1-7, wherein the Vitamin K dependent protein is selected from the group consisting of Prothrombin, Factor VII, Factor IX (FIX), Factor X, Protein C, Protein S, Protein Z, matrix Gal protein, osteocalcin, Gas6, a receptor tyrosine kinase ligand protein, connexin 26, connexin 32, PRGP1, PRGP2, TmG3, TmG4, peptides from the venom of the marine snail Conus and Gamma Glutamyl Carboxylase.

9. The method of any one of claims 1-8, wherein the calories in the feed is reduced on a daily basis up to 0.5% per day of the body weight of non-human transgenic animal to increase the animal’s utilization of the Vitamin K in the diet..

10. The method of any one of claims 1-9, wherein the Vitamin K protein is produced in the animal and obtained or harvested from the animal’s tissues or fluids.

11. The method of any one of claims 1-10, wherein the vitamin K comprises Vitamin K₁, K₂, K₃, K₄ , their derivatives or a combination thereof.

12. A method of increasing the biological activity of a Vitamin K dependent protein produced in a transgenic cell or cell line comprising of contacting or feeding a transgenic cell or cell line expressing a gene encoding the Vitamin K dependent protein a nutrient media comprising an increased amount of vitamin K over that of standard nutrient media containing a standard amount of vitamin K, wherein the Vitamin K dependent protein expressed in the transgenic cell or cell line possesses an increased biological activity over a Vitamin K dependent protein expressed in a transgenic cell or cell line that is fed standard nutrient media containing a standard amount of Vitamin K.

13. The method of claim 12, wherein the increased biological activity of the Vitamin K dependent protein is the result of increased gamma carboxylation of the Vitamin K dependent protein.