JP2014221048A - Method and composition for producing active vitamin k-dependent protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an active vitamin K-dependent protein.SOLUTION: There is provided a method for increasing the quantity of a carboxylated vitamin K-dependent protein in a cell including a step to introduce a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR) into a cell expressing a first nucleic acid encoding a vitamin K-dependent protein under a condition to express the first nucleic acid and the second nucleic acid and produce the vitamin K-dependent protein and VKOR.

Description

  This invention was made in part with grants from the National Institutes of Health grant numbers 5P01 HL06350-42 and 5-R01 HL48318. The US government has certain rights in this invention.

  The present invention relates to isolated nucleic acids, host cells comprising said isolated nucleic acids, and methods of use thereof, and identification of single nucleotide polymorphisms (SNPs) within the vitamin K epoxide reductase (VKOR) gene and against warfarin It relates to methods and compositions directed to their correlation with sensitivity.

Many protein functions require modification of multiple glutamate residues to γ-carboxyglutamate. Of these vitamin K-dependent (VKD) coagulation proteins, FIX (Christmas factor), FVII and prothrombin are the best known. The finding that gene knockout for matrix Gla protein causes calcification of mouse arteries (Luo et al. (1997) "Spontaneous calcification of arteries and cartridge in.
rice wrapping matrix GLA protein "Nature 386: 78-81) highlights the importance of the vitamin K cycle for proteins with functions other than coagulation. In addition, Gas6 and other Gla proteins of unknown function are expressed in neural tissue, and warfarin exposure in the womb results in mental retardation and facial abnormalities. This is consistent with the finding that expression of the enzyme that performs Gla modification, VKD carboxylase, is transiently tissue-specifically regulated and highly expressed in the nervous system during the early embryonic stages. Simultaneously with the carboxylation, reduced vitamin K, which is a co-substrate of the reaction, is converted to vitamin K epoxide. Since the amount of vitamin K in the human diet is limited, vitamin K epoxide must be returned to vitamin K by vitamin K epoxide reductase (VKOR) to prevent its depletion. Warfarin, the most widely used anticoagulant, targets VKOR and prevents vitamin K regeneration. The result is a reduced concentration of reduced vitamin K, which results in a reduced rate of carboxylation by γ-glutamyl carboxylase and production of a low carboxylated vitamin K dependent protein.

  In the United States alone, warfarin is prescribed to over one million patients annually, and in the Netherlands, approximately 2% of the population was reported to receive long-term warfarin treatment. Since the amount of warfarin required for anticoagulation treatment varies greatly from patient to patient, the use of warfarin carries a significant risk of side effects. For example, after the start of warfarin treatment, it has been reported that significant bleeding episodes occurred in 1-2% of patients and deaths occurred in 0.1-0.7% of patients. Despite its dangers, the use of warfarin was able to prevent 20 strokes per episode of induced bleeding and was presumed to be underutilized probably due to fear of induced bleeding .

  The present invention relates to a method for correlating single nucleotide polymorphisms in a subject with high or low susceptibility to warfarin, thereby allowing more accurate and rapid determination of warfarin treatment and maintenance doses with low risk to the subject. And overcoming the drawbacks of the prior art in the art by providing a composition.

  The present invention detects the presence of a single nucleotide polymorphism in the VKOR gene in a subject and correlates the single nucleotide polymorphism with high or low sensitivity to warfarin, thereby identifying subjects with high or low sensitivity to warfarin A method for identifying a human subject having a high or low sensitivity to warfarin.

  Further, a) correlating the presence of a single nucleotide polymorphism in the VKOR gene with a high or low susceptibility to warfarin; and b) detecting a single nucleotide polymorphism in step (a) in a subject, thereby against warfarin A method of identifying a human subject having a high or low sensitivity to warfarin is provided, comprising identifying a subject having a high or low sensitivity.

In a further embodiment, the present invention provides:
a) identifying a subject having a high or low sensitivity to warfarin;
b) detecting the presence of a single nucleotide polymorphism in the VKOR gene in a subject;
c) correlating the presence of a single nucleotide polymorphism in step (b) with a high or low sensitivity to warfarin in a subject, thereby identifying a single nucleotide polymorphism within the VKOR gene that correlates with a high or low sensitivity to warfarin. A method of identifying single nucleotide polymorphisms in the VKOR gene that correlate with high or low susceptibility to warfarin.

  In addition, the present invention includes: a) identifying a subject with high or low sensitivity to warfarin; b) determining the nucleotide sequence of the subject's VKOR gene in (a); and c) step (b). ) Comparing the nucleotide sequence of) with the wild-type nucleotide sequence of the VKOR gene; d) detecting a single nucleotide polymorphism in the nucleotide sequence of (b); and e) (d) the single nucleotide polymorphism. A method of correlating a single nucleotide polymorphism in a subject's VKOR gene with high or low susceptibility to warfarin, comprising correlating with high or low susceptibility to warfarin in a subject of (a).

  A further aspect of the invention is an isolated nucleic acid encoding vitamin K epoxide reductase (VKOR), in particular a mammalian (eg human, sheep, cow, monkey, etc.) VKOR. Examples include (a) a nucleic acid disclosed herein, such as an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9; (b) the isolated nucleic acid of (a) above or a Hybridizes to a complement (eg under stringent conditions) and / or has substantial sequence identity with the nucleic acid of (a) above (eg 80, 85, 90, 95 or 99% identical) and a nucleic acid encoding VKOR; and (c) different from the nucleic acid of (a) or (b) above due to the degeneracy of the genetic code, but depending on the nucleic acid of (a) or (b) above A nucleic acid encoding the encoded VKOR.

As used herein, the term “stringent” refers to hybridization conditions that are commonly understood in the art to define the product of a hybridization procedure. Stringency conditions can be low, high or medium, and the terms are as commonly known in the art and widely recognized by those skilled in the art. High stringency hybridization conditions that allow a homologous nucleotide sequence to hybridize to a nucleotide sequence as set forth herein are well known in the art. By way of example, hybridization of such sequences to the nucleic acid molecules disclosed herein allows for hybridization of sequences of about 60% homology to 25% formamide, 5 × SSC and 0. Can be performed at 42 ° C. in 25% formamide, 5 × SSC, 5 × Denhardt's solution and 5% dextran sulfate with 1% SDS, 42 ° C. wash conditions. Another example includes 6 × SSC, 0.1% SDS, about 45 ° C. hybridization conditions, followed by 0.2 × SSC, 0.1% SDS, 50-65 ° C. wash conditions. Another example of stringent conditions is the standard hybridization assay (SAMBROOK et al., EDS., MOLECULAR CLONING: A LABORATORY MANUAL 2d, which is incorporated herein by reference).
ed. (See Cold Spring Harbor, NY 1989)) as indicated by 0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS, 60-70 ° C wash stringency. In various embodiments, stringent conditions are, for example, highly stringent (ie, high stringency) conditions (eg, 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDT.
Hybridization to filtration-bound DNA in A at 65 ° C. and washing at 68 ° C. in 0.1 × SSC / 0.1% SDS) and / or moderately stringent (ie, moderate stringency) conditions (For example, washing at 42 ° C. in 0.2 × SSC / 0.1% SDS).

  A further aspect of the invention is a recombinant nucleic acid comprising a nucleic acid encoding a vitamin K epoxide reductase described herein operably linked to a heterologous promoter.

  A further aspect of the invention is a cell that contains and expresses a recombinant nucleic acid as described above. Suitable cells include plant, animal, mammal, insect, yeast and bacterial cells.

  A further aspect of the invention is an oligonucleotide that hybridizes to an isolated nucleic acid encoding VKOR as described herein.

  A further aspect of the invention is an isolated and purified VKOR (eg, a homogeneously purified VKOR) encoded by the nucleic acids described herein. For example, the VKOR of the present invention may comprise the amino acid sequence shown in SEQ ID NO: 10.

  A further aspect of the invention is the step of culturing a host cell that expresses a nucleic acid encoding a vitamin K-dependent protein in the presence of vitamin K and produces the vitamin K-dependent protein, and then the vitamin K-dependent from the culture. Collecting the sex protein, wherein the host cell contains and expresses a heterologous nucleic acid encoding a vitamin K-dependent carboxylase, and the host cell encodes a heterologous nucleic acid encoding a vitamin K epoxide reductase (VKOR). A method of producing a vitamin K-dependent protein further comprising and expressing and producing a VKOR as described herein. Therefore, the present invention further provides a cell comprising a heterologous nucleic acid encoding a vitamin K-dependent carboxylase and a heterologous nucleic acid encoding a vitamin K epoxide reductase. The cell can further include a nucleic acid encoding a vitamin K-dependent protein, where the nucleic acid can be heterologous to the cell or endogenous to the cell.

FIGS. 1A-1D show a comparison of warfarin dose in heterozygous and homozygous subjects for wild type, SNP vk2581, vk3294, and vk4769, and a comparison of warfarin dose in subjects heterozygous for wild type and P4502Y9. For each of the 13 siRNA pools, three T7 flasks containing A549 cells were transfected and VKOR activity was measured 72 hours later. The VKOR assay used 25 μM vitamin K epoxide. One siRNA pool specific for gene gi: 1314769 reduced VKOR activity by 64% -70% in 8 iterations. Time course of inhibition of VKOR activity by a siRNA pool specific for gene gi: 1314769 in A549 cells. VKOR activity declined continually during this period, but its mRNA levels rapidly declined to about 20% of normal values. Vitamin K epoxide 25 μM was used for this assay. siRNA did not affect the activity of VKD carboxylase or the level of lamin A / C mRNA. VKOR activity was detected when mGC_11276 was expressed in Sf9 insect cells. Approximately 1 × 10 ⁵ cells were used in this assay. The reaction was performed in buffer D using 32 μM KO for 30 minutes at 30 ° C. Blank Sf9 cells were used as negative controls and A549 cells were used as standards. Inhibition of VKOR by warfarin. The reaction was performed at 30 ° C. for 15 minutes using 1.6 mg microsomal protein made from VKOR_Sf9 cells in buffer D, 60 μM KO, and various concentrations of warfarin. Carboxylation of vitamin K-dependent protein, factor X. Control HEK293 cells that produce factor X without exogenous VKOR or VKGC. Carboxylation of vitamin K-dependent protein, factor X. HEK293 cells producing only factor X and exogenous VKGC. Carboxylation of vitamin K-dependent protein, factor X. HEK293 cells producing only factor X and exogenous VKOR. Carboxylation of vitamin K-dependent protein, factor X. HEK293 cells producing factor X and both exogenous VKOR and CKGC.

  As used herein, “a”, “an” or “the” may mean 1 or greater than 1. For example, “a” cell may mean a single cell or multiple cells.

  The invention is described in more detail below. This description is not intended to be a detailed listing of all of the various ways in which the invention may be practiced or all features that may be added to the invention. For example, features described with respect to one embodiment can be incorporated into other embodiments, and features described with respect to a particular embodiment can be deleted from that embodiment. In addition, many modifications and additions to the various embodiments suggested herein that do not depart from the invention will be apparent to those skilled in the art in light of this disclosure. As such, the following specification describes some specific embodiments of the present invention and is not intended to exhaustively identify all permutations, combinations and variations thereof.

  The “sequence listing” attached to this specification is intended to form part of this specification as if it were fully described herein.

  The present invention is based on this disclosure and is hereby incorporated by reference in its entirety, as if those disclosures were fully described herein, Stafford and Wu. Methods, ingredients and features known in the art, including but not limited to those described in U.S. Pat. No. 5,268,275 and U.S. Pat. No. 6,531,298 to Stafford and Chang It can be implemented by further utilizing

  As used herein, “nucleic acid” encompasses both RNA and DNA, including cDNA, genomic DNA, synthetic (eg, chemically synthesized) DNA and RNA and DNA chimeras. The nucleic acid can be double stranded or single stranded. If single stranded, the nucleic acid can be the sense strand or the antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (eg, inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to produce nucleic acids with altered base pairing ability or high resistance to nucleases.

An “isolated nucleic acid” is a DNA that is not directly adjacent to both of the coding sequences immediately adjacent in the naturally occurring genome of the organism from which it is derived (one at the 5 ′ end and one at the 3 ′ end) or RNA. In one embodiment, the isolated nucleic acid comprises some or all of the 5 ′ non-coding (eg, promoter) sequence immediately adjacent to the coding sequence. The term may be produced, for example, by vector, autonomously replicating plasmid or virus, or another molecule (e.g. PCR or restriction endonuclease treatment) independent of other sequences incorporated into prokaryotic or eukaryotic genomic DNA. Recombinant DNA present as a cDNA or genomic DNA fragment). The term also encompasses recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

  The term “isolated” refers substantially to cellular material, viral material, or medium (when produced by recombinant DNA techniques), or a chemical precursor or other chemical (when chemically synthesized). Nucleic acids or polypeptides not included in Furthermore, an “isolated nucleic acid fragment” is a nucleic acid fragment that does not occur naturally as a fragment and is not found in the natural state.

  The term “oligonucleotide” refers to a nucleic acid of at least about 6 to about 100 nucleotides, such as about 15 to 30 nucleotides, or about 20 to 25 nucleotides, which can be used, for example, as a primer in PCR amplification or as a probe in a hybridization assay or microarray. Refers to an array. Oligonucleotides can be natural or synthetic, such as DNA, RNA, modified backbones, and the like.

  Where a particular nucleotide sequence is said to have a particular percent identity relative to a standard nucleotide sequence, the percent identity is relative to the standard nucleotide sequence. For example, a nucleotide sequence that is 50%, 75%, 85%, 90%, 95% or 99% identical to a standard nucleotide sequence 100 bases long is 50, 75, 85, 90, 95 or 99 of the standard nucleotide sequence. It may have 50, 75, 85, 90, 95 or 99 bases that are completely identical to the sequence of nucleotides. The nucleotide sequence can also be a 100 base long nucleotide sequence that is 50%, 75%, 85%, 90%, 95% or 99% identical to the standard nucleotide sequence throughout its length. Of course, there are other nucleotide sequences that also meet the same criteria as well.

A nucleic acid sequence that is “substantially identical” to a VKOR nucleotide sequence is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9. For nucleic acid comparisons, the length of a standard nucleic acid sequence is generally at least 40 nucleotides, such as at least 60 nucleotides or more. Sequence identity is determined by sequence analysis software (eg, the Genetics Computer Group, University of Wisconsin Biotechnology).
Center, 1710 University Avenue, Madison, Wis. 53705 (Sequence Analysis Software Package).

As is known in the art, many different programs can be used to identify whether a nucleic acid or amino acid has sequence identity or similarity with a known sequence. Sequence identity or similarity is described in Smith & Waterman, Adv. Appl. Math. 2,482 (1981), a local sequence identity algorithm, Needleman & Wunsch, J. et al. Mol. Biol. 48, 443 (1970) sequence identity alignment algorithm, Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988) similarity search, computer implementation of these algorithms (The Wisconsin Genetics Software Package GAP, BESTFIT, FASTA and TFSTA, Genetics
Computer Group, 575 Science Drive, Madison, WI), Devereux et al. , Nucl. Acid Res. 12, 387-395 (1984), including, but not limited to, the Best Fit sequence program, which may be determined using standard techniques known in the art, preferably using default settings, or by examination .

  An example of a useful algorithm is PILEUP. PILEUP creates multiple sequence alignments from groups of related sequences using progressive and pairwise alignments. You can also draw a tree that shows the clustering relationships used to create the alignment. PILEUP is available from Feng & Doolittle, J.A. Mol. Evol. 35,351-360 (1987) is used. This method is similar to the method described by Higgins & Sharp, CABIOS 5: 151-153 (1989).

  Another example of a useful algorithm is Altschul et al. , J .; Mol. Biol. 215, 403-410, (1990) and Karlin et al. , Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST algorithm is the Altschul et al. , Methods in Enzymology, 266, 460-480 (1996). WU-BLAST-2 uses several search parameters, preferably set to default values. The parameters are dynamic values and are established by the program itself, depending on the composition of the particular sequence and the composition of the particular database for which the sequence of interest is searched. However, the number can be adjusted to increase sensitivity. Further useful algorithms are described in Altschul et al. Nucleic Acids Res. 25, 3389-3402 is the gap BLAST reported.

  The CLUSTAL program can also be used to determine sequence similarity. This algorithm is described in Higgins et al. (1988) Gene 73: 237; Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331.

  In addition, for sequences that include more or fewer nucleotides than the nucleic acids disclosed herein, in one embodiment, the percentage sequence identity is determined based on the number of identical nucleotides relative to the total number of nucleotide bases. Is understood. Thus, for example, sequence identity of sequences shorter than those specifically disclosed herein is determined in one embodiment using the number of nucleotide bases of the shorter sequence. In calculating percent identity, relative weights are not assigned to various manifestations of sequence changes such as insertions, deletions, substitutions, and the like.

The VKOR polypeptides of the present invention include, but are not limited to, recombinant polypeptides, synthetic peptides and natural polypeptides. The invention also encompasses nucleic acid sequences that encode forms of VKOR polypeptides in which the naturally occurring amino acid sequence has been altered or deleted. Preferred nucleic acids encode polypeptides that are soluble under normal physiological conditions. Also within the scope of the invention is a nucleic acid that encodes a fusion protein in which all or part of the VKOR is created by fusing to an irrelevant polypeptide (eg, a marker polypeptide or fusion partner). For example, the polypeptide may be a hexahistidine tag to facilitate purification of a polypeptide expressed in bacteria, or a hemagglutinin tag to facilitate purification of a polypeptide expressed in eukaryotic cells, or affinity chromatography. It can be fused to an HPC4 tag to facilitate purification of the polypeptide by graphy or immunoprecipitation. The present invention also includes isolated polypeptides (and nucleic acids encoding these polypeptides) that include a first portion and a second portion. The first portion includes, for example, all or part of the VKOR polypeptide, and the second portion includes, for example, a detection marker.

  A fusion partner can be, for example, a polypeptide that facilitates secretion, such as a secretory sequence. Such fusion polypeptides are typically referred to as preproteins. The secretory sequence can be cleaved by the cell to form the mature protein. Also within the scope of the invention is a nucleic acid encoding VKOR fused to a polypeptide sequence to produce an inactive preprotein. The preprotein can be converted to the active form of the protein by removal of the inactivating sequence.

  The present invention also includes all or one of the nucleotide sequences of SEQ ID NO: 1-6, SEQ ID NO: 8 or SEQ ID NO: 9 or their complements, for example under stringent hybridization conditions (as defined herein). A nucleic acid that hybridizes to the portion. In certain embodiments, the hybridizing portion of a hybridizing nucleic acid is typically at least 15 (eg, 20, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80%, such as at least 95%, at least 98% or 100% identical to some or all of the sequence of the nucleic acid encoding the VKOR polypeptide. Hybridizing nucleic acids of the type described herein can be used, for example, as cloning probes, primers (eg, PCR primers), or diagnostic probes. For example, inhibitory small RNAs (siRNAs) and / or antisense RNAs that inhibit the function of VKOR, as measured in activity assays described herein and known in the art, are also included within the scope of the present invention. .

  In another embodiment, the invention features a cell, eg, a transformed cell, comprising a nucleic acid of the invention. A “transformed cell” has a nucleic acid encoding all or part of a VKOR polypeptide and / or an antisense nucleic acid or siRNA introduced (or introduced into its ancestors) by recombinant nucleic acid techniques. ) A cell. Both prokaryotic and eukaryotic cells, such as bacteria, yeast, insects, mice, rats, humans, plants, etc. are included.

  The invention also features nucleic acid constructs (eg, vectors and plasmids), such as expression vectors, that include a nucleic acid of the invention operably linked to transcriptional and / or translational control elements to allow expression. “Operably linked” refers to transcription and / or transcription of a sequence such that a DNA molecule encoding a selected nucleic acid, eg, a VKOR polypeptide, can control transcription and / or translation of the selected nucleic acid. Alternatively, it means located adjacent to one or more regulatory elements that direct translation, such as a promoter.

The invention further provides fragments or oligonucleotides of the nucleic acids of the invention that can be used as primers or probes. Thus, in some embodiments, a fragment or oligonucleotide of the invention is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 of the nucleotide sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9. , 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 , 900, 1000, 1500, 2000, 2500 or 3000 contiguous nucleotides. Examples of oligonucleotides of the present invention are provided in the sequence listing included herein. Such fragments or oligonucleotides can be detectably labeled or modified to include and / or incorporate restriction enzyme cleavage sites, for example when used as primers in amplification (eg, PCR) assays.

  The present invention also includes purified or isolated VKOR polypeptides, such as a polypeptide consisting essentially of and / or consisting of the amino acid sequence of SEQ ID NO: 10, or a biologically active fragment or peptide thereof. Characterized by peptides. Such a fragment or peptide is typically at least about 10 amino acids of the amino acid sequence of SEQ ID NO: 10 (eg, 15, 20, 25, 30, 35, 40, 45, 50, 55, of the amino acid sequence of SEQ ID NO: 10, 60, 65, 75, 85, 95, 100, 125 or 150 amino acids) and can be a peptide or fragment of an amino acid adjacent to the amino acid sequence of the VKOR protein (eg, shown in SEQ ID NO: 10). The biological activity of the fragments or peptides of the invention can be measured according to the methods provided herein and as is known in the art for identifying VKOR activity. Fragments and peptides of the VKOR protein of the invention may also be active as antigens for the production of antibodies. Identification of epitopes on the fragments or peptides of the invention is performed by well-known protocols and within the skill of the artisan.

  As used herein, both “protein” and “polypeptide” mean any chain of amino acids, regardless of length or post-translational modification (eg, glycosylation, phosphorylation or N-myristylation). To do. As such, the term “VKOR polypeptide” refers to recombinant or synthetic, each corresponding to a full-length naturally-occurring VKOR protein, as well as a portion of a full-length naturally-occurring VKOR protein or a naturally-occurring or synthetic VKOR polypeptide. The polypeptide produced by

  A “purified” or “isolated” compound or polypeptide refers to a compound of interest, eg, at least a portion of a naturally occurring organism or other component of a virus, eg, a cell or viral structural component or a polypeptide thereof. A composition of at least 60% by weight of a VKOR polypeptide or antibody that is separated from or substantially free of other commonly recognized polypeptides or nucleic acids. As used herein, an “isolated” polypeptide is at least about 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 %, 99% or more (w / w) pure. Preferably, the preparation is at least 75% (eg, at least 90% or 99% by weight) of the compound of interest. Purity can be measured by any appropriate standard method, such as column chromatography, polyacrylamide gel electrophoresis or HPLC analysis.

  Preferred VKOR polypeptides comprise a sequence that is substantially identical to all or part of a naturally occurring VKOR polypeptide. A “substantially identical” polypeptide to a VKOR polypeptide sequence described herein is at least 80% or 85% (eg, 90%, 95% or 99%) identical to the amino acid sequence of the VKOR polypeptide of SEQ ID NO: 10. It has an amino acid sequence that is For comparison, the length of a standard VKOR polypeptide is generally at least 16 amino acids, such as at least 20, 25, 30, 35, 40, 45, 50, 75 or 100 amino acids.

  For polypeptides that are less than 100% identical to a standard sequence, non-identical positions are preferably, but not necessarily, conservative substitutions for the standard sequence. Conservative substitutions typically include, but are not limited to, substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; And arginine; and phenylalanine and tyrosine.

  When a particular polypeptide is said to have a certain percent identity with a standard polypeptide of a defined length, the percent identity is relative to the standard polypeptide. Thus, for example, a polypeptide that is 50%, 75%, 85%, 90%, 95% or 99% identical to a standard polypeptide of 100 amino acids in length is 50, 75, 85, 90, 95 of the standard polypeptide. Or a polypeptide of 50, 75, 85, 90, 95 or 99 amino acids that is completely identical to the 99 amino acid long portion. It can also be a 100 amino acid long polypeptide that is 50%, 75%, 85%, 90%, 95% or 99% identical to a standard polypeptide throughout its length. Of course, other polypeptides will meet the same criteria as well.

  The invention also features a purified or isolated antibody that specifically binds to a VKOR polypeptide of the invention or fragment thereof. “Specifically binds” means that the antibody recognizes and binds to a specific antigen, eg, a VKOR polypeptide, or a fragment or peptide of a VKOR polypeptide, but does not substantially bind other molecules in the sample. Does not recognize and does not bind to it. In one embodiment, the antibody is a monoclonal antibody, and in other embodiments, the antibody is a polyclonal antibody. The production of both monoclonal and polyclonal antibodies is well known in the art, including chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, antibody fragments and the like.

  In another aspect, the invention features a method for detecting a VKOR polypeptide in a sample. The method comprises contacting a sample with an antibody that specifically binds to a VKOR polypeptide or fragment thereof under conditions that allow formation of a complex between the antibody and VKOR; Detection of the polypeptide or fragment thereof includes the step of detecting the formation of a complex, if present. Such immunoassays are well known in the art and include immunoprecipitation, immunoblotting, immunolabeling assays, ELISA and the like.

  The invention further comprises contacting the sample with a nucleic acid of the invention encoding VKOR or a fragment thereof, or a complement of a nucleic acid encoding VKOR or a fragment thereof, under conditions that allow hybridization complexes to form; Detecting the nucleic acid encoding the VKOR polypeptide in the sample, comprising detecting the formation of a hybridization complex, thereby detecting the nucleic acid encoding the VKOR polypeptide in the sample. Such hybridization assays are well known in the art and include probe detection assays and nucleic acid amplification assays.

  A method for obtaining a gene related to the VKOR gene (ie, a functional homologue of the VKOR gene) is also encompassed in the present invention. Such methods include obtaining or producing a detectably labeled probe comprising an isolated nucleic acid encoding all or part of VKOR or a homolog thereof, and a nucleic acid fragment library, wherein the probe is live. A step of screening with a labeled probe, thereby forming a nucleic acid duplex, and a labeled duplex, if present, under conditions that allow hybridization to nucleic acid fragments in the library In some cases, it involves isolating and producing a full-length gene sequence from the nucleic acid fragment in the labeled duplex to obtain the gene associated with the VKOR gene.

A further aspect of the invention is the step of culturing cells expressing a nucleic acid encoding a vitamin K-dependent protein that produces vitamin K-dependent protein in the presence of vitamin K, and then the vitamin K-dependent protein from the medium. The cell comprises and expresses an exogenous nucleic acid encoding vitamin K epoxide reductase (VKOR), thereby producing VKOR, and in some embodiments, the cell is a vitamin A method of producing a vitamin K dependent protein comprising and expressing an exogenous nucleic acid encoding a K dependent carboxylase, thereby producing a vitamin K dependent carboxylase as described herein. In some embodiments, expression of a nucleic acid encoding VKOR and production of VKOR results in a higher level of vitamin K dependence in the cell than is produced in the absence of VKOR or in the absence of VKOR and carboxylase. Produce proteins and / or high levels of active (eg, fully carboxylated) vitamin K dependent proteins.

Therefore, in some embodiments, the present invention also
a) a nucleic acid encoding a vitamin K-dependent protein comprising a heterologous nucleic acid encoding a vitamin K-dependent carboxylase under conditions in which the nucleic acid is expressed and produced in the presence of vitamin K; Introducing into a cell further comprising a heterologous nucleic acid encoding vitamin K epoxide reductase;
b) optionally collecting vitamin K-dependent protein from the cell, and providing a method of producing vitamin K-dependent protein.

  The present invention also provides a cell expressing a first nucleic acid encoding a vitamin K-dependent protein with a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR), the first nucleic acid and the second nucleic acid. Provides a method for increasing the amount of carboxylated vitamin K-dependent protein in a cell comprising introducing under conditions that are each expressed to produce vitamin K-dependent protein and VKOR.

  A cell expressing a first nucleic acid encoding a vitamin K-dependent protein is expressed with a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR), and the first nucleic acid and the second nucleic acid are expressed, respectively. Further provided herein is a method for increasing carboxylation of vitamin K-dependent proteins comprising introducing under conditions that produce vitamin K-dependent proteins and VKOR.

  In addition, the present invention provides a cell expressing a first nucleic acid encoding a vitamin K-dependent protein with a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR), the first nucleic acid and the second nucleic acid. The amount of carboxylated vitamin K-dependent protein produced in the cell in the presence of VKOR is determined by the non-expressed amount of VKOR. Methods are provided for producing carboxylated (eg, fully carboxylated) vitamin K-dependent proteins in cells that are increased relative to the amount of carboxylated vitamin K-dependent proteins produced in the cells in the presence.

  Furthermore, the present invention provides a cell expressing a first nucleic acid encoding a vitamin K-dependent protein with a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR), the first nucleic acid and the second nucleic acid. 100%, 90%, 80% of the vitamin K-dependent protein produced in the cell in the presence of VKOR, comprising introducing under conditions where the nucleic acid is expressed and producing vitamin K-dependent protein and VKOR, respectively. Methods are provided for producing vitamin K-dependent proteins in cells that are 70% or 60% carboxylated (eg, fully carboxylated).

  In addition, a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR) is expressed in a cell expressing the first nucleic acid encoding a vitamin K-dependent protein, and the first nucleic acid and the second nucleic acid are expressed respectively. Also included herein is a method of producing vitamin K-dependent protein in a cell comprising introducing under conditions that have been produced to produce vitamin K-dependent protein and VKOR.

In some embodiments of the methods described above, the cell can further comprise a third nucleic acid encoding a vitamin K-dependent carboxylase, which can be, but is not limited to, bovine vitamin K-dependent carboxylase. In certain embodiments, the vitamin K-dependent carboxylase is vitamin Kγ glutamyl carboxylase (VKGC). The VKGC used in the methods of the invention can be a VKGC from any vertebrate or invertebrate species that produces VKGC, known in the art.

  In the method of the present invention for increasing the amount of carboxylated vitamin K-dependent protein in a cell in the presence of VKOR and / or VKGC, the carboxylated vitamin K-dependent protein produced in the cell in the presence of VKOR and / or VKGC Alternatively, the amount of fully carboxylated vitamin K dependent protein is compared to the amount of carboxylated vitamin K dependent protein or fully carboxylated vitamin K dependent protein produced in the cell in the absence of VKOR and / or VKGC. It can be increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or 300%.

  In some embodiments, “fully carboxylated” means that all sites (or in some embodiments, the majority of sites) on a vitamin K-dependent protein that can undergo carboxylation are carboxylated. means. In some embodiments, complete carboxylation is such that all vitamin K-dependent proteins are carboxylated to some extent and / or all vitamin K-dependent proteins are carboxylated at all or most carboxylation sites. It can mean that A carboxylated vitamin K-dependent protein or a fully carboxylated vitamin K-dependent protein is an active protein. By “active protein” is meant that a vitamin K-dependent protein has or can be active in carrying out its biological function (eg, factor IX or enzymatic activity for factor X).

  Vitamin K-dependent proteins that can be produced according to the method of the present invention include any combination of Factor VII, Factor VIIA, Factor IX, Factor X, Protein C, Activated Protein C, Protein S, Bone Gla protein ( Osteocalcin), matrix Gla protein and prothrombin, and any vitamin K-dependent protein now known or later identified per se, including, but not limited to, modified forms of such proteins as described herein possible.

  Any cell that can be transformed with the nucleic acids described herein can be used as described herein, but in some embodiments, non-human or even non-mammalian cells can be used. Thus, the cells or cell lines of the present invention can be, for example, human cells, animal cells, plant cells and / or insect cells. Nucleic acids encoding vitamin K-dependent carboxylases and nucleic acids encoding vitamin K-dependent proteins described herein are well known in the art, and their introduction into cells for expression is performed according to routine protocols. The Thus, in some embodiments, the present invention provides a cell comprising a nucleic acid encoding a vitamin K-dependent protein (endogenous or exogenous to the cell). Vitamin K-dependent proteins are produced in cells in the presence of vitamin K. The cell further comprises a heterologous (ie, exogenous) nucleic acid encoding a vitamin K epoxide reductase (VKOR) and / or a vitamin K-dependent carboxylase. The cell can be maintained under conditions known in the art in which a nucleic acid encoding VKOR and / or a vitamin K-dependent carboxylase is expressed and VKOR and / or carboxylase is produced in the cell.

Certain embodiments of the present invention are based on the inventors' discovery that a therapeutic dose of warfarin for anticoagulation treatment in a subject can be correlated with the presence of one or more single nucleotide polymorphisms in the subject's VKOR gene. As such, the present invention also includes detecting the presence of one or more single nucleotide polymorphisms (SNPs) in the VKOR gene in a subject, wherein the single nucleotide polymorphism is correlated with high or low susceptibility to warfarin, thereby Provided is a method for identifying a human subject having a high or low sensitivity to warfarin that identifies the subject as having a high or low sensitivity to warfarin.

  An example of a SNP that correlates with high susceptibility to warfarin is the G at nucleotide 2581 (SEQ ID NO: 12) of the nucleotide sequence of SEQ ID NO: 11, a standard sequence comprising the genomic sequence of SEQ ID NO: 8 and about 1000 nucleotides before and after this sequence → C change (in VKOR gene intron 2; GenBank accession number refSNP ID: rs805050894, which is hereby incorporated by reference). This sequence can have the genomic location “human chromosome 16p11.2” or be located in the physical map of the NCBI database as human chromosome 16: 31009700-31013800.

  An example of a SNP that correlates with low susceptibility to warfarin is the T → C change at nucleotide 3294 (SEQ ID NO: 13) of the nucleotide sequence of SEQ ID NO: 11 (within intron 2 of the VKOR gene; which is incorporated herein by reference. GenBank accession number refSNP ID: rs2359612), and G → A change at nucleotide 4769 (SEQ ID NO: 14) of the nucleotide sequence of SEQ ID NO: 11 (within the 3′UTR of the VKOR gene; GenBank accession number refSNP ID: rs7294), which is part of the book.

  As used herein, a subject having “high sensitivity to warfarin” carries an appropriate therapeutic or maintenance dose of warfarin carrying a SNP in the VKOR gene that gives a normal subject, ie, a high sensitivity phenotype to warfarin. Subjects who are below the therapeutic or maintenance dose of warfarin appropriate for those who do not. Conversely, as used herein, subjects with “low sensitivity to warfarin” are those within the VKOR gene where an appropriate therapeutic or maintenance dose of warfarin gives a normal subject, ie, a low sensitivity phenotype to warfarin. A subject who is above the therapeutic or maintenance dose of warfarin appropriate for a subject who does not carry a SNP. An example of a typical therapeutic dose of warfarin for normal subjects is 35 mg per week, although this amount may vary (eg, a dose range of 3.5-420 mg per week is available in Aithal et al. (1999)). Lancet 353: 717-719). A typical therapeutic dose of warfarin can be determined for a given test group according to the methods described herein, said therapeutic dose being used to identify subjects having a higher or lower therapeutic warfarin dose. And thereby identify subjects with low or high sensitivity to warfarin.

  Further, a) correlating the presence of a single nucleotide polymorphism in the VKOR gene with a high or low susceptibility to warfarin; and b) detecting a single nucleotide polymorphism in step (a) in a subject, thereby against warfarin Provided herein is a method of identifying a human subject having a high or low sensitivity to warfarin, comprising identifying a subject having a high or low sensitivity.

  In addition, the present invention comprises: a) identifying a subject having high or low sensitivity to warfarin; b) detecting the presence of a single nucleotide polymorphism in the VKOR gene in the subject; and c) step ( b) correlating the presence of a single nucleotide polymorphism with a high or low sensitivity to warfarin in a subject, thereby identifying a single nucleotide polymorphism within the VKOR gene that correlates with a high or low sensitivity to warfarin. Provided are methods for identifying single nucleotide polymorphisms in the VKOR gene that correlate with high or low sensitivity to warfarin.

A) identifying a subject having high or low sensitivity to warfarin; b) determining the nucleotide sequence of the subject's VKOR gene in (a); and c) determining the nucleotide sequence of step (b). Comparing the wild-type nucleotide sequence of the VKOR gene, d) detecting a single nucleotide polymorphism in the nucleotide sequence of (b), e) (d) the single nucleotide polymorphism, and subject (a) Also provided in the present invention is a method of correlating a single nucleotide polymorphism in a subject's VKOR gene with high or low susceptibility to warfarin, comprising correlating with high or low susceptibility to warfarin in.

  The subject is a normal subject for whom the mean therapeutic or maintenance dose of warfarin is calculated by establishing a therapeutic or maintenance dose of warfarin for anticoagulation treatment, and for that subject, according to well-known protocols. High or low susceptibility to warfarin by comparing to a therapeutic or maintenance dose of warfarin for anticoagulation treatment in a population of populations (eg, subjects who do not have a SNP in the VKOR gene that correlates with high or low susceptibility to warfarin) Is identified as having Warfarin lower than the average therapeutic or maintenance dose of warfarin (eg, the dose of warfarin that provides a therapeutic dose for a subject with a therapeutic or wild type VKOR gene, ie, without a single nucleotide polymorphism associated with warfarin sensitivity) A subject having a therapeutic or maintenance dose of is a subject identified as having high sensitivity to warfarin. A subject having a therapeutic or maintenance dose of warfarin that is higher than the average therapeutic or maintenance dose of warfarin is a subject identified as having a low sensitivity to warfarin. The average therapeutic or maintenance dose of warfarin for subjects with the wild type VKOR gene is readily determined by those skilled in the art.

  The nucleotide sequence of the subject's VKOR gene is determined according to standard methods in the art and as described in the examples provided herein. For example, genomic DNA is extracted from the subject's cells and the VKOR gene is located and sequenced according to known protocols. Single nucleotide polymorphisms within the VKOR gene are identified by comparison of the subject's sequence with a wild-type sequence known in the art (eg, a standard sequence shown herein as SEQ ID NO: 11).

  The presence of a SNP or SNPs in the VKOR gene of a subject identified as having a high or low sensitivity to warfarin, ie, having a maintenance or therapeutic dose of warfarin that is higher or lower than the average dose. And correlating with high or low susceptibility to warfarin by performing a statistical analysis of the association of SNP with high or low susceptibility to warfarin according to well-known methods of statistical analysis. An analysis that identifies a statistical association (eg, significant association) between a SNP (genotype) and high or low warfarin sensitivity (phenotype) is the presence of a SNP in a subject and the high or low to warfarin in that subject Establish a correlation between susceptibility.

  It is believed that a combination of factors, including the presence of one or more SNPs in a subject's VKOR gene, can be correlated with high or low sensitivity to warfarin in that subject. Such factors may include, but are not limited to, cytochrome p450 2C9 polymorphism, race, age, sex, smoking history and liver disease.

  Thus, in a further embodiment, the present invention provides one or more single nucleotide polymorphisms of the VKOR gene, one or more cytochrome p450 2C9 polymorphisms, race, age, sex, smoking history, liver disease and two or more of these factors Identifying in the subject the presence of a combination of factors correlated with high or low sensitivity to warfarin, selected from the group consisting of any combination of: wherein the combination of factors is correlated with high or low sensitivity to warfarin; Methods are provided for identifying human subjects with high or low sensitivity to warfarin, thereby identifying subjects with high or low sensitivity to warfarin.

Further, a) the factor is one or more single nucleotide polymorphisms of the VKOR gene, one or more cytochrome p45
0 2C9 polymorphism, race, age, sex, smoking history, liver disease and the presence of a combination of factors selected from the group consisting of any combination of two or more of these factors with high or low sensitivity to warfarin A human subject having a high or low sensitivity to warfarin comprising the step of correlating and b) detecting a combination of the factors of step (a) in the subject, thereby identifying a subject having a high or low sensitivity to warfarin. A method for identifying is provided herein.

  In addition, the present invention includes: a) identifying a subject with high or low sensitivity to warfarin; b) detecting the presence of a combination of factors in the subject; and c) the combination of factors of step (b) Correlating presence with high or low susceptibility to warfarin in a subject, thereby identifying a combination of factors that correlate with high or low susceptibility to warfarin, said factor comprising one or more single nucleotide polymorphisms of the VKOR gene Correlates with high or low susceptibility to warfarin selected from the group consisting of one or more cytochrome p450 2C9 polymorphisms, race, age, gender, smoking history, liver disease and any combination of two or more of these factors A method of identifying a combination of factors is provided.

  A) identifying a subject having high or low sensitivity to warfarin; b) identifying the presence of a combination of factors in the subject; and c) identifying the combination of factors in (b) in the subject of (a). Correlating with high or low susceptibility to warfarin, wherein the factor is one or more single nucleotide polymorphisms of the VKOR gene, one or more cytochrome p450 2C9 polymorphisms, race, age, sex, smoking history, liver disease And a method for correlating a combination of factors with a high or low susceptibility to warfarin, selected from the group consisting of any combination of two or more of these factors.

  The combination of factors described herein is high or low sensitivity to warfarin in accordance with well known methods of identifying the presence of the factor combination and statistical analysis in subjects identified as having high or low sensitivity to warfarin. By performing a statistical analysis of the association between the factor and the factor, it is correlated with a high or low sensitivity to warfarin. An analysis that identifies a statistical association (eg, significant association) between a combination of factors and a (high or low) warfarin susceptibility phenotype is the presence of the combination of factors in the subject and the high or low to warfarin in that subject Establish a correlation between susceptibility.

  Further provided herein are nucleic acids that encode VKOR and that include one or more SNPs described herein. Therefore, the present invention further provides a nucleic acid consisting essentially of and / or consisting of the nucleotide sequences shown in SEQ ID NOs: 12, 13, 14, 15 and 16. The nucleic acid can be present in the vector and the vector can be present in the cell. Furthermore, the protein encoded by the nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 12, 13, 14, 15 and 16 and the protein encoded by the nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 12, 13, 14, 15 and 16 Antibodies that specifically bind to are also included. Since numerous modifications and variations will be apparent to those skilled in the art, the invention will be described in more detail in the following examples, which are intended to be illustrative only.

Correlation between SNP and high or low susceptibility to warfarin in the VKOR gene The most common isoform of the VKOR gene is approximately 4 kb in length, has 3 exons, and has a mass of 18.4 kDa Encodes a 163 amino acid enzyme. In this study, three mutations identified as SNPs, vk2581 (G> C), vk3294 (T> C) and vk4769 (G> A) (46.9%, 46.8% and 46.3%, respectively) Of heterozygosity) in relation to their presence in subjects and the maintenance dose of warfarin required to achieve a therapeutically effective response.

1. Subject Selection Subjects were obtained from UNC Coagulation Clinic of Ambatory Care Ceter. Informed consent was obtained by an expert genetics counselor. Subjects who were not fluent in English were excluded due to lack of interpretation and the need for consent. To qualify for the study, subjects had been taking warfarin for at least 6 months and were followed up by UNC Coagulation Clinic at Ambientary Care Clinic at the age of 18 years or older.

2. Extraction of genomic DNA from whole blood Genomic DNA was extracted from the whole blood of a subject using the QIAamp DNA Blood Mini Kit (QIAGEN catalog number 51104). The DNA concentration was adjusted to 10 ng / μL.

3. Sequencing of genomic DNA samples Approximately 10 ng of DNA was used for polymerase chain reaction (PCR) assays. The primers used to amplify the VKOR gene were as follows: For the 5'-UTR and exon 1 regions, exon 1-5'CCAATCGCCGAGGTCAGAGGG (SEQ ID NO: 29) and exon 1-3 'CCCATCCCCCAGCACTGTCT (SEQ ID NO: 30); for exon 2 region, exon 2-5′AGGGGAGGATAGGGTCAGGTG (SEQ ID NO: 31) and exon 2-3′CCTGTTTAGTACCCCCCACA (SEQ ID NO: 32); and for exon 3 and 3′-UTR regions, exon 3-5 ′ ATACGTGCGTAAGCCACCAC (SEQ ID NO: 33) and exon 3-3′ACCCAGATATGCCCCCTTAG (SEQ ID NO: 34). Automated high-throughput capillary electrophoresis DNA sequencing was used to detect SNPs in the VKOR gene.

4). Detection of known SNPs using real-time PCR Assay reagents for SNP genotyping were from the Assay-by-Design ™ service (Applied Biosystems, catalog number 4332072). Primers and probes (FAM ™ and VIC ™ dye labels) were designed using Primer Express software and synthesized on an Applied Biosystems synthesizer. The primer pair for each SNP is located upstream / downstream of the SNP site and can generate DNA fragments of less than 100 bp in the PCR reaction. FAM ™ and VIC ™ dye-labeled probes were designed to cover SNP sites with a length of 15-16 nucleotides. The primer and probe sequences for each VKOR SNP are shown in Table 2.

  2X TaqMan ™ Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, catalog number 4324018) was used in the PCR reaction. Forty cycles of real-time PCR were performed in an Opticon II (MJ Research) instrument. Pre-heating at 95 ° C. for 10 minutes, followed by treatment at 92 ° C. for 15 minutes and 60 ° C. for 1 minute, after which the plate was read. The results were read according to the FAM and VIC dye signal values.

5. Statistical analysis The mean dose differences between the various genotypes were compared by analysis of variance (ANOVA) using SAS version 8.0 (SAS, Inc., Cary, NC). Two-sided p-values less than 0.05 were considered significant. Examination of the distribution and residual for the mean dose of treatment in the SNP group indicated that a logarithmic transformation was required to meet the assumption of uniformity of variance.

6). Correlation between warfarin dose and SNP Five SNPs were identified in the VKOR gene by direct genomic DNA sequencing and SNP real-time PCR detection: 1 in 5′-UTR, 2 in intron II, in coding region And one in the 3′-UTR (Table 1).

  Among these SNPs, the vk563 and vk4501 SNP alleles were carried by only one of 58 subjects in the study (triple heterozygotes that also carry the 3′-UTR SNP allele) Other SNPs were identified in 17-25 heterozygous patients.

Each marker was initially analyzed independently. FIG. 1A shows that the average warfarin dose for patients with the vk2581 wild type allele was 50.19 ± 3.20 mg (n = 26) per week, while heterozygous and homozygous for this polymorphism. One patient shows 35.19 ± 3.73 (n = 17) and 31.14 ± 6.2 mg (n = 15), respectively, per week. FIG. 1B shows that the average warfarin dose for patients with the wild type vk3294 allele was 25.29 ± 3.05 mg (n = 11) per week while carrying heterozygous and homozygous alleles Patients are shown to be 41.68 ± 4.92 (n = 25) and 47.73 ± 2.75 mg (n = 22), respectively, per week. FIG. 1C shows that the average warfarin dose for patients with the vk4769 SNP wild type was 35.35 ± 4.01 mg (n = 27) per week, while patients with heterozygous and homozygous alleles , 44.48 ± 4.80 (n = 19) and 47.56 ± 3.86 mg (n = 12) were required per week, respectively. In addition, P450 2C9 ^* 3 has been reported (Joffe et al. (
2004) “Warfarin dosing and cytochrome P450 2C9 polymorphisms” Thromb Haemost 91: 1123-1128), a significant effect on warfarin dose was observed (FIG. 1D). The average warfarin dose for patients with P450 2C9 ^* 1 (wild type) is
While 43.82 ± 2.75 mg per week (n = 50), patients who were heterozygous for this allele required 22.4 ± 4.34 mg per week (n = 8).

7). The relevance of SNP and Log _e statistical analysis VKOR the gene (warfarin average dosage) (LnDose) was examined by analysis of variance (ANOVA). To perform an iterative procedure to examine a series of factors (race, gender, smoking history, liver disease, SNPs in the cytochrome P450 2Y9 gene, etc.) to identify factors that can affect dose, excluding VKOR SNP SAS was used. P450 2C9 ^* 3 was significantly associated with the average dose of warfarin. This was therefore included as a covariate for further analysis. Analysis indicated that when including this covariate, the three VKOR SNPs were still significantly associated with the warfarin dose per week (vk2581, P <0.0001; vk3294, P <0.0001; and vk4769, P = 0.0044).

In order to test in detail whether these three SNPs of VKOR are independently related to the warfarin dose, the analysis involving two SNPs in the VKOR gene as covariates for other SNPs was repeated. The three VKOR SNPs are located at a distance of 2 kb from each other and are expected to be closely related. At least for the Caucasians, one haplotype (A = vk2581 guanine and a = vk2581 cytosine; B = vk3294 thymine and b = vk3924 cytosine; C = vk4769 guanine and c = vk4769 adenine) is AAbbcc, and another It was clear from examination that it was aaBBCC. The distribution of individual SNPs in the patient was found to be significantly correlated with the other distributions (R = 0.63 to 0.87, p <0.001). In fact, subjects with haplotype AAbbcc (n = 7) (warfarin dose = 48.98 (3.93) have haplotype aa
A significantly higher dose of warfarin was required compared to patients with BBCC (25.29 (3.05; p <0.001)).

siRNA Design and Synthesis siRNAs were selected using an improved version of a rational design algorithm (Reynolds et al. (2004) “Rational siRNA design for RNA interference” Nature Biotechnology 22: 326-330). For each of the 13 genes, the 4 siRNAs duplexes with the highest scores were selected and BLAST searches were performed using the Human EST database. To minimize the potential for off-target silencing effects, only sequence targets with 4 or more mismatches to irrelevant sequences were selected (Jackson et al. (2003) “Expression profiling reviews off- target gene regulation by RNAi "Nat Biotechnol 21: 635-7). All duplexes were modified using 2′-ACE chemistry modification method (Scaringe (2000) “Advanced 5′-silyl-2′-orthoester approach to RNA oligonucleotide synthesis” Methods Enzymol 317: U-18). Synthesized with Dharmacon (Lafayette, CO) as a terminal 21-mer, and the AS chain was chemically phosphorylated to ensure maximum activity (Martinez et al. (2002) “Single-stranded antisense siRNAs guide target RNA”. cleavage in RNAi "Cell 110: 563-74).

siRNA Transfection Transfection was essentially as described above with minor modifications (Harborth et al. (2001) "Identification of essential genes in cultured mammarian".
cells using small interfering RNAs "J Cell Sci 114: 4557-65).

VKOR activity assay siRNA transfected A549 cells were trypsinized and washed twice with cold PBS. For each VKOR assay, 1.5 × 10 ⁷ cells were harvested. Buffer D
200 μL (250 mM Na ₂ HPO ₄ —NaH ₂ PO ₄ , 500 mM KCl, 20% glycerol and 0.75% CHAPS, pH 7.4) was added to the cell pellet and then the cell lysate was sonicated. For the assay of solubilized microsomes, as described (Lin et al. (2002) “The putative vitalin K-dependent gamma-glutamine deb ente pe pe te pe te p e te p e p e s e p e p e n e p e n e p e p e n e p e n e p e n e p e n e p e n e n e p e n e n e p e n e p e n e p e n e p e n e p e p e n e p e n e p e n e p e n e p e n e p e n e p e n e p e n e p e n e p e n e p e n e p e n e p e n e p e n e? 28581-91) Microsomes were prepared from 2 × 10 ⁹ cells; 10-50 μL of solubilized microsomes were used for each assay. Vitamin K epoxide was added to the concentration indicated in the footnote in the figure and DTT was added to 4 mM to initiate the reaction. The reaction mixture was incubated in yellow light at 30 ° C. for 30 minutes and 0.05 M AgNO3 was added.
₃ : 500 μL of isopropanol (5: 9) was added to stop. Hexane 500μL
Was added and the mixture was vortexed vigorously for 1 minute to extract vitamin K and vitamin KO. After centrifugation for 5 minutes, transferred to an upper organic layer in 5mL brown vial and dried with N _2. 150 μL of HPLC buffer, acetonitrile: isopropanol: water (100: 7: 2) was added to dissolve vitamin K and vitamin KO, and the sample was analyzed by HPLC on an AC-18 column (Vydac, catalog number 218TP54). did.

RT-qPCR (reverse transcriptase quantitative PCR)
Wash 1 × 10 ⁶ cells twice with PBS and total RNA according to manufacturer's protocol (In
Vitrogen) isolated with Trizol reagent. 1 μg of RNA was digested with RQ1 DNase 1 (Promega) and heat-inactivated. First strand cDNA was prepared with M-MLV reverse transcriptase (Invitrogen). cDNA was mixed with DyNAmo SYBR Green qPCR pre-mix (Finzymes), and real-time PCR was performed on an Opticon II PCR thermal cycler (MJ Research). The following primers were used:
13124769-5 ′ (F): (TCCAACAGCATATTCGGTTGC, SEQ ID NO: 1);
13124769-3 (R) ′: (TTCTTGGACCTCTCGAGAACT, SEQ ID NO: 2);
GAPDH-F: (GAAGGTGAAGGTCGGGAGTC, SEQ ID NO: 3);
GAPDH-R: (GAAGATGGTGATGGGATTTC, SEQ ID NO: 4);
Lamin-RT-F: (CTAGGTGAGGCCAAGAAGCAA, SEQ ID NO: 5) and Lamin-RT-R: (CGTTTCCTCTCAGCAGAACTGC, SEQ ID NO: 6).

Overexpression of VKOR in the Sf9 insect cell line The cDNA for the mGC11276 coding region was cloned into pVL1392 (Pharmingen) at the amino terminus with the HPC4 tag (EDQVDPRLIDGK, SEQ ID NO: 7) and as described (Li et al. 2000) “Identification of a Drosophila vitamin K-dependent gamma-glutamyl carboylase” J Biol Chem 275: 18291-6) was expressed in Sf9 cells.

Gene selection Searches for the VKOR gene were conducted intensively on human chromosome 16 between the markers D16S3131 and D16S419. This region corresponds to chromosome 16 at 50 cM to 65 cM on the genetic map and 26 to 46.3 Mb on the physical map. 190 predicted coding sequences within this region were analyzed by BLASTX search of the NCBI non-redundant protein database. Orthologs from their human genes and related species with known functions were excluded. Since VKOR appears to be a transmembrane protein (Carlisle & Suttie (1980) “Vitamin K dependent carboxylase: subcellular localization of the carboxylase and enzymes in vivo evolved in vivo
metabolism in rat river "Biochemistry 19: 1161-7), the remaining genes are translated according to the cDNA sequence in the NCBI database and the programs TMHMM and TMAP (to predict those with a transmembrane domain)
Biology WorkBench, San Diego Supercomputer System). Thirteen genes predicted to encode integral membrane proteins were selected for further analysis.

Cell line screening strategy for VKOR activity The strategy was to identify cell lines that express relatively high amounts of VKOR activity and use siRNA to systematically knock down all 13 candidate genes. SiRNA, a 21-23 nucleotide double stranded RNA, has been shown to cause specific RNA degradation in cell culture (Hara et al. (2002) “Raptor, a binding partner of target of rapamycin (TOR), medias TOR action "Cell 110: 177-89; Krichevsky & Kosik (2002)" RNAi functions in cultured maman neurons "Proc Natl Acad Sci USA 99: 11926-9ur. Gene Snk / Plk2 Leads to Mitotic Cat strophe in Paclitaxel (Taxol) -Exposed Cells "Mol Cell Biol 23: 5556-71). However, the application of siRNA for large-scale screening in mammalian cells has not been previously reported due to the difficulty in identifying functional targets for specific mammalian cell mRNAs (Holen et al. ( 2003) “Similar behavel of single-strand and double-strand siRNAs suggests the act through a common RNAi pathway” (Nucleic Acids 240). The development of rational selection algorithms for siRNA design (Reynolds et al.) raises the possibility of developing specific siRNAs. In addition, the probability of success can be increased by pooling 4 reasonably selected siRNAs. Using siRNA to search for previously unspecified genes means that the assay measures the loss of enzyme activity, even if VKOR activity requires the product of more than one gene for activity So screening has the advantage that it should still be effective.

  Fifteen cell lines were screened and the human lung cancer line, A549, was identified as showing sufficient warfarin sensitive VKOR activity for easy measurement. A second human colorectal adenocarcinoma cell line, HT29, which expresses negligible VKOR activity was used as a standard.

SiRNA inhibition of VKOR activity in A549 cells Each of the 13 pools of siRNA was transfected into A549 cells in triplicate and assayed for VKOR activity after 72 hours. One siRNA pool specific for gene gi: 1314769 reduced VKOR activity by 64% -70% in 8 separate assays (FIG. 2).

One possible reason that VKOR activity was inhibited to only about 35% of its initial activity after 72 hours is that the half-life of mammalian proteins varies greatly (minutes to days) (Zhang et al. (1996) "The major calpain isozymes are long-lived proteins. Design of an antisense sensation for Bio18 depletion in culture 82.
iCity and Intracellular Degradation of Proteins "Biol Chem 377: 425-35; Dice & Goldberg (1975)" Relationship between in vivo
"degradative rates and isoelectric points of proteins" Proc Natl Acad Sci USA 72: 3893-7), mRNA translation is inhibited rather than enzyme activity. Therefore, the cells were allowed to survive for 11 days and their VKOR activity was followed. FIG. 3 shows that the level of mRNA for gi: 1314769 mRNA decreased rapidly to about 20% of normal value, while VKOR activity decreased continuously during this period. Since VKD carboxylase activity and lamin A / C mRNA levels remained constant, this reduction in activity is not a result of the general effects of siRNA or cell death. Furthermore, the level of gi: 132124769 mRNA is 4 times lower in HT-29 cells with low VKOR activity than in A549 cells that exhibit high VKOR activity. These data indicate that gi: 1314769 corresponds to the VKOR gene.

Identification of the gene encoding VKOR The gene IMAGE 3455200 (gi: 1314769, SEQ ID NO: 8), identified here as encoding VKOR, is located on human chromosome 16p11.2, mouse chromosome 7F3 and rat chromosome 1: 180.8 Mb . There are 338 cDNA clones in the NCBI database showing 7 different splicing patterns (NCBI)
AceView program). These consist of all or part of 2 to 4 exons. Of these, the most common isoform, mGC11276, has 3 exons and is expressed at high levels in lung and hepatocytes. The three exon transcripts (SEQ ID NO: 9) encode a predicted protein of 163 amino acids (SEQ ID NO: 10) having a mass of 18.2 kDa. This is a putative N-myristylated endoplasmic reticulum protein with 1 to 3 transmembrane domains, depending on the program used for prediction. It has seven cysteine residues and is consistent with the finding that enzyme activity is dependent on thiol reagents (Thijssen et al. (1994) “Microsomal lipoproteins replicates biomolecules 27”. 80). Five of the seven cysteine residues are conserved among humans, mice, rats, zebrafish, Xenopus (Xenopus) and Anopheles (Anopheles).

  To confirm that the VKOR gene was identified, the most common form of the enzyme (3 exons) was expressed in Spodoptera frugiperda, Sf9 cells. Sf9 cells do not have measurable VKOR activity, but show warfarin-sensitive activity when transfected with mGC11276 cDNA (FIG. 4). VKOR activity is observed at either their amino or carboxyl terminus from constructs with an epitope tag. This tag should help purify VKOR.

  VKOR should show warfarin sensitivity, so microsomes were generated from Sf9 cells expressing VKOR and tested for warfarin sensitivity. VKOR activity is warfarin sensitive (Figure 5).

In summary, the present invention provides the first example of using siRNA in mammalian cells to identify unknown genes. The identity of the VKOR gene was confirmed by its expression in insect cells. The VKOR gene encodes several isoforms. It is important to characterize the activity and expression pattern of each isoform. Millions of people around the world use warfarin to inhibit clotting. Therefore, further characterization of VKOR is important because it can lead to more accurate dose determination or safer and more effective anticoagulant design.

Tests for Factor X Carboxylation Post-translational modification of glutamic acid to gamma carboxyglutamic acid requires the activity of many proteins, most of which are associated with coagulation. Some of these have become useful tools for treating various bleeding disorders. For example, recombinant human factor IX currently accounts for the majority of factor IX used to treat hemophilia B patients. In addition, Factor VIIa is widely used to treat patients with autoantibodies (inhibitors) to either Factor IX or Factor VIII and for bleeding resulting from general trauma. Another Gla protein, activated protein C, is used for the treatment of sepsis. These vitamin K-dependent proteins can be produced in cell culture using cells such as Chinese hamster ovary (CHO), baby hamster kidney cells (BHK) and human embryonic kidney cells (HEK293). A common problem for all of these cell lines is that if significant overproduction is achieved, a significant fraction of the recombinant protein produced is hypocarboxylated. Initially, the limiting factor in carboxylation was thought to be vitamin K-dependent γ-glutamyl carboxylase. However, after its purification and cloning, co-expression of factor IX and carboxylase failed to improve the degree of carboxylation of factor IX in CHO cell lines overexpressing human factor IX. The percentage of carboxylated factor X in the HEK293 cell line can be increased by decreasing the affinity of the factor X propeptide. However, if the expression level of factor X carrying the prothrombin propeptide is sufficiently high, the level of expression still exceeds the ability of the cell to achieve full post-translational modification. This study shows that co-expression of vitamin K epoxide reductase (together with prothrombin propeptide) to the extent that only about 50% of factor X is carboxylated in cell lines overexpressing factor X It reveals that it causes carboxylation.

Test material. All restriction enzymes were obtained from New England Biolabs. Pfu DNA polymerase was obtained from Stratagene. Lipofectamine, hygromycin B and pcDNA3.1 / Hygro vector were from Invitrogen. Trypsin-EDTA, fetal bovine serum and Dulbecco's phosphate buffered saline were from Sigma. Antibiotic-antimycotic, G418 (Geneticin) and DMEM F-12 were from GIBCO. The puromycin and pIRESpuro3 vectors were from BD Biosciences. Human factor X was from Enzyme Research Laboratories. Goat anti-human factor X (affinity purified IgG) and rabbit anti-human factor X (IgG-peroxidase complex) were from Affinity Biologicals Corporation. Peroxidase conjugated Affini Pure rabbit anti-goat IgG was from Jackson ImmunoResearch Laboratories INC. Q-Sepharose ™ Fast Flow was obtained from Amersham Pharmacia Biotech. Calcium-dependent monoclonal human FX antibody [MoAb, 4G3] is available from Dr. Harold James, University of
Obtained from Texas, Tyler, TX. Bio-Scale CHT5-I hydroxyapatite was from Bio-Rad Laboratories.

Construction of mammalian cell expression vectors containing VKOR. Two primers were designed to amplify the VKOR cDNA. Kozak sequence (underlined) and primer 1: 5′-CCCGGAATTC GCCGCCACC ATGGGCAGCACCTGGGGGAGCCCTGGCTGGGTGCGG (SEQ ID NO: 35) introduced into the 5 ′ EcoRI site. Primer 2: 5′-CGGGCGGCCGCTCATGGCCTCTTAGCCCTTGCC (SEQ ID NO: 36) introduced into the NotI site at the 3 ′ end of the cDNA. After PCR amplification and digestion with EcoRI and NotI, the PCR product was inserted into pIRESpuro3, which has a CMV virus major early promoter / enhancer and confers puromycin resistance to the transformed cells.

Construction of mammalian cell expression vectors containing HGC. Two primers were designed to amplify HGC cDNA. Primer 3 introduced into the Bam H1 site and the Kozak sequence (underlined) at the 5 ′ end: 5′- CGCGGGATCC GCCGCCCACC ATGGCGGGTGTCGCCCGGGCCCTCGCGCGACTCTGCAGCTGAC Number 38). After digestion with Bam HI and Not I, the PCR product was inserted into pcDNA3.1 / Hygro, which has a CMV promoter and confers hygromycin resistance to transformed cells.

  A stable cell line expressing human VKOR. A cell line expressing a mutated factor X (HEK293-FXI16L) that produces about 10-12 mg / L of factor X (half of which is fully carboxylated) was used. HEK293-FXI16L was made as described (Camire, 2000) and selected with the neomycin analog, G418. HEK293-FXI16L was transfected with plasmid pIRESpuro3-VKOR using Lipofectin (Invitrogen) according to the manufacturer's protocol. Selection was performed with 450 μl / ml G418 and 1.75 μl / ml puromycin. Resistant colonies were picked and screened for VKOR activity. Colonies with the highest VKOR activity were selected for further analysis.

  A stable cell line expressing human GGCX. HEK293-FXI16L was transfected with the plasmid pcDNA3.1 / Hygro-HGGCX using lipofectin. Transformed colonies were selected with hygromycin 300 μg / ml and G418 450 μg / ml and 18 clones were selected for assay of GGCX activity with the small peptide substrate REEEL (SEQ ID NO: 39). Colonies with the highest GGCX activity were selected for further testing.

  A stable cell line that co-expresses human VKOR and HGC. To obtain a HEK293-FXI16L cell line that overexpresses both VKOR and GGCX, HEK293-FXI16L-VKOR was transfected with the plasmid pcDNA3.1 / Hygro-HGGCX and 18 resistant colonies were selected for analysis. HEK293-FXI16L-HGGCX was also transfected with HEK293-FXI16L-VKOR, and only one resistant colony was obtained from this selection. HEK293-FXI16L was transfected with both pIRESpuro3-VKOR and pcDNA3.1 / Hygro-HGC, resulting in 10 resistant colonies. Next, 29 isolated colonies were assayed for both VKOR and GGCX activity. The clone with the highest level of both activities was selected for further analysis.

Levels of FXI16L production by each cell line. For the sandwich ELISA antibody assay, goat anti-human factor X (affinity purified IgG) IgG-peroxidase complex was used as the capture antibody and rabbit anti-human factor X was used as the detection antibody. P-OD was used as a substrate for color development. Human factor X was used to generate a standard curve. HEK293-FXI16L, HEK293-FXI16L-VKOR, HE
K293-FXI16L-HGGCX and HEK293-FXI16L-VKOR-HGGCX were grown to confluence in T25 flasks, after which the medium was replaced with serum-free medium containing vitamin K1. Serum-free medium was changed at 12 hours and conditioned medium was collected after 24 hours and analyzed for expression of FXI16L.

  Expression of FXI16L from each cell line in roller bottles. Four stable cell lines, HEK293-FXI16L, HEK293-FXI16L-VKOR, HEK293-FXI16L-HGGCX and HEK293-FXI16L-VKOR-HGGCX were grown to confluence in T-225 flasks and transferred to roller bottles. At 24 and 36 hours, the medium was replaced with serum-free medium containing vitamin K1. Medium was collected from each cell line every 24 hours until a total of 3 liters was obtained.

Purification of FXI16L from each cell line. Conditioned medium was thawed and passed through a 0.45 μm HVLP filter. EDTA was then added to 5 mM and 0.25 ml of 100 × stock protease inhibitor cocktail was added per liter of conditioned medium. Conditioned medium was loaded onto a Q-sepharose ™ Fast Flow column equilibrated with 20 mM Tris (pH 7.2) / 60 mM NaCl / 5 mM EDTA, and the column was washed with the same buffer until the baseline was steady. 20 mM Tris (pH 7.2) / 700 mM NaCl was used to elute FXI16L from the column. Protein containing fractions were pooled and dialyzed against 8 mM Tris (pH 7.4) / 60 mM NaCl. Each sample was made 2 mM CaCl ₂ and applied to an immunoaffinity (4G3) column that had been equilibrated with 8 mM Tris (pH 7.4) / 60 mM NaCl / 2 mM CaCl ₂ . After washing with the same buffer, the eluted factor X was eluted with a linear gradient of 0-8 mM EDTA in the same buffer. Fractions containing protein were pooled and dialyzed overnight against 1 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ (pH 6.8). The dialyzed sample was applied to a Bio-Scale CHT5-I hydroxyapatite column pre-equilibrated with starting buffer. A linear gradient of 1 to 400 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ (pH 6.8) was used to separate carboxylated and uncarboxylated factor X.

  Western blotting of samples after Q-sepharose and SDS-PAGE of samples after 4G3. After purification with Q-Sepharose ™ Fast Flow, fractions from 4 cell lines (HEK293-FXI16L, HEK293-FXI16L-VKOR, HEK293-FXI16L-HGC and HEK293-FXI16L-VKOR-HGC) were obtained by Western blotting. Identified. ECL substrate was used for color development using goat anti-human factor X (affinity purified IgG) as the first antibody, peroxidase conjugated affinipure rabbit anti-goat IgG as the second antibody. After purification by affinity antibody chromatography, some samples were examined for purity.

Analysis of VKOR, HGC and FXI16L mRNA expression levels between each cell line using real-time Q-PCR. A total of 1 × 10 ⁶ cells were seeded in 12-well plates for each cell line (HEK293-FXI16L, HEK293-FXI16L-VKOR, HEK293-FXI16L-HGC and HEK293-FXI16L-VKOR-HGC). Total RNA was extracted from each cell line.

VKOR and HGC activity for each cell line (HEK293-FXI16L, HEK293-FXI16L-VKOR, HEK293-FXI16L-HGC and HEK293-FXI16L-VKOR-HGC). pIRESpuro3-VKOR was transfected into HEK293-FXI16L and selected with 1.75 μg / ml puromycin and 450 μg / ml G418. Eighteen single clones were screened for VKOR activity. A single clone containing a very high level of VKOR activity in a stable cell line,
Held as HEK293-FXI16L-VKOR. After transfecting pcDNA3.1 / Hygro-HGC into HEK293-FXI16L, transfectants can be selected with 300 μg / ml hygromycin and 450 μg / ml G418. A total of 18 single clones were screened for HGC activity. A single clone containing very high levels of HGC activity was maintained as a stable cell line, HEK293-FXI16L-HGC.

  Three methods were used to generate stable cell lines containing high levels of both VKOR and HGC activities. A total of 29 single clones were screened for VKOR and HGC activity. A single clone containing high levels of both VKOR and HGC was maintained as a stable cell line, HEK293-FXI16L-VKOR-HGC.

  Production of FXI16L in each of the cell lines. HEK293-FXI16L-VKOR, HEK293-FXI16L-HGC and HEK293-FXI16L-VKOR-HGC all expressed FXI16L at least as high as the host cells. This experiment was performed in a 25 ml T flask for comparison, and the level of expression was lower than when the protein was prepared in roller bottles. These experiments indicate that selecting cells that overproduce carboxylase or VKOR did not result in loss of factor X expression.

  Three liters of media were collected from the cells grown in roller bottles and factor X from each cell line was purified by Q-sepharose and factor X antibody affinity chromatography as described above.

Analysis of the change in the rate of carboxylation of rFXI16L between each cell line by using hydroxyapatite chromatography. 1 mM Na ₂ HPO ₄ / NaH ₂ P
After dialysis against O ₄ (pH 6.8), the fraction after 4G3 was subjected to Bio-Scale CHT5-I.
Applied to hydroxyapatite column. The column was eluted using a 0-100% linear gradient of 400 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ (pH 6.8). A total of 2 pools can be obtained from each sample. The first pool is composed of uncarboxylated human FXI16L and the second pool is composed of fully gamma carboxylated human FXI16L. For each cell line, the amount of fully gamma carboxylated human FXI16L is divided by the total amount of carboxylated human FXI16L to obtain a ratio. The carboxylation rate for the host cell line HEK293-FXI16L is 52% [4.5 mg / (4.13 mg + 4.5 mg) = 52%]. The carboxylation rates for the other three cell lines (HEK293-FXI16L-VKOR, HEK293-FXI16L-HGC and HEK293-FXI16L-VKOR-HGC) were 92% [10.5 mg / (0.9 mg + 10.5 mg) = 92, respectively. %], 57% [6.4 mg / (4.78 mg + 6.4 mg) = 57%] and ˜100% [2.4 mg / 2.4 mg = 100%].

The large difference in the carboxylation rate between the cell lines HEK293-FXI16L and HEK293-FXI16L-VKOR indicates that VKOR dramatically improves the gamma carboxylation reaction in vivo. The smaller difference in the carboxylation rate between the cell lines HEK293-FXI16L and HEK293-FXI16L-HGC indicates that HGC catalyzes the carboxylation reaction, but is not a limiting factor in the carboxylation reaction in vivo, only in vivo. It shows that the carboxylation reaction can be improved. Nearly 100% carboxylation rate in the cell line HEK293-FXI16L-VKOR-HGC indicates that VKOR may be a limiting factor in the carboxylation reaction in vivo. VKOR not only reduces vitamin K epoxide (KO) to vitamin K, but also reduces vitamin K to reduced vitamin K (KH ₂
). If the there is no second function that can be reduced to KH ₂ K, vitamin K can not be reused in carboxylation system in vivo.

  In summary, this test shows that when a nucleic acid encoding vitamin K epoxide reductase (VKOR) is transfected with a vitamin K-dependent protein such as factor X and transfected into a cell that produces vitamin K-dependent protein. Reveals the production of vitamin K-dependent proteins with high carboxylation rates, thereby increasing the amount of active vitamin K-dependent proteins in the cells.

  To perform these experiments, a human embryonic kidney (HEK) cell line (along with the prothrombin propeptide) expressing about 12-14 mg / L of mutant factor X was used. This factor X has been previously modified by replacing its propeptide with a prothrombin propeptide (Camire et al. “Enhanced gamma-carboxylation of recombinant factor th e s s ine pi s s s s s s s t” 46): 14322-9 (2000)), overexpressing factor X blood clotting factor to the extent that only -50% of factor X is carboxylated.

  This cell line producing about 12-14 mg / L Factor X was used for the starting and control cells. At this level of expression, HEK cells were unable to fully carboxylate factor X, replacing normal factor X propeptide with prothrombin peptide. HEK293 cells expressing about 12-14 mg / L of factor X are either 1) vitamin K epoxide reductase (VKOR), 2) vitamin Kγ glutamyl carboxylase, or 3) vitamin K epoxide reductase and vitamin Kγ glutamyl carboxylase (VKGC). Both were transfected with. Several cell lines have been selected that have been shown to produce large amounts of carboxylase, VKOR or both VKOR and carboxylase. In each of these selected cell lines, the expression level of factor X was at least as high as the starting cell line (within experimental limits). The results of these experiments are shown in FIGS. The comparison in all cases is to the original factor X expressing cell line that expresses factor X which is about 50% carboxylated.

Three liters of medium were collected from each of the experimental cell lines and Factor X was purified on a QAE sephadex, a Factor X antibody column and finally a hydroxylapatite column. The figure shown relates to a hydroxylapatite column. It has been shown previously that the first peak is uncarboxylated factor X and the second peak is fully carboxylated factor X (Camire et al.). FIG. 6A shows the results of carboxylation of factor X in the original cell line without exogenous VKOR or VKGC. The second peak (concentrated around fraction 26) is a fully carboxylated peak. In terms of area, 52% of factor X is fully carboxylated. FIG. 6B shows that addition of carboxylase alone to a factor X expressing cell line did not significantly increase the percentage of carboxylated factor X. The degree of complete carboxylation barely increases to 57% complete carboxylation. In this case, the complete carboxylation peak is concentrated around the fraction 25. FIG. 6C shows that cells transfected with VKOR alone showed dramatically higher levels of fully carboxylated factor X. In this case, the complete carboxylation peak (concentrated around fraction 26) and the degree of complete carboxylation rise to 92% of the total factor X produced. FIG. 6D shows that 100% of factor X is fully carboxylated when cells are transfected with VKOR and VKGC. In this situation, VKOR gene expression is a major determinant of complete carboxylation of vitamin K-dependent proteins. In other situations where substrate turnover is lower, i.e. when the propeptide binds prothrombin propeptide much more closely than factor X and the overexpression of factor X is very high,
Carboxylase gene expression is also considered to be limiting. These results can be extended to all vitamin K-dependent proteins in addition to factor X.

  These results reveal that VKOR (and possibly VKGC) promotes the production of fully carboxylated vitamin K-dependent proteins. This provides a mechanism to increase the efficiency of producing a fully active modified protein.

  The foregoing is a description of the present invention and should not be construed as limiting the invention. The invention is defined by the following claims and the equivalents of the claims contained therein.

  All publications, patent applications, patents, patent publications and other references cited herein are hereby incorporated by reference in their entirety for teachings relating to the sentences and / or paragraphs presented by the reference. Shall be made.

Claims (10)

  A cell expressing a first nucleic acid encoding a vitamin K-dependent protein is expressed with a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR), and the first nucleic acid and the second nucleic acid are expressed, respectively. A method for increasing the amount of carboxylated vitamin K-dependent protein in a cell comprising introducing under conditions that produce vitamin K-dependent protein and VKOR.   A cell expressing a first nucleic acid encoding a vitamin K-dependent protein is expressed in a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR), and the first nucleic acid and the second nucleic acid are expressed respectively. A method of increasing carboxylation of vitamin K-dependent protein comprising introducing under conditions that produce K-dependent protein and VKOR.   A cell expressing a first nucleic acid encoding a vitamin K-dependent protein is expressed in a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR), and the first nucleic acid and the second nucleic acid are expressed respectively. The amount of carboxylated vitamin K-dependent protein produced in the cell in the presence of VKOR is produced in the cell in the absence of VKOR, including introducing under conditions that produce K-dependent protein and VKOR A method for producing a carboxylated vitamin K-dependent protein in a cell that is increased relative to the amount of carboxylated vitamin K-dependent protein.   The method according to any one of claims 1 to 3, wherein the cell further comprises a third nucleic acid encoding a vitamin K-dependent carboxylase.   The vitamin K-dependent protein is selected from the group consisting of Factor VII, Factor IX, Factor X, protein C, protein S, prothrombin and any combination thereof. The method described.   The method according to claim 1, wherein the cell is a plant cell.   6. The method according to any one of claims 1 to 5, wherein the cell is an insect cell.   The method according to claim 1, wherein the cell is an animal cell.   The method according to any one of claims 1 to 8, wherein the vitamin K-dependent carboxylase is a bovine vitamin K-dependent carboxylase.   The method according to any one of claims 1 to 8, wherein the vitamin K-dependent carboxylase is vitamin Kγ glutamyl carboxylase (VKGC).

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