WO2016073837A1 - Methods and compositions for modified factor ix proteins - Google Patents
Methods and compositions for modified factor ix proteins Download PDFInfo
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- WO2016073837A1 WO2016073837A1 PCT/US2015/059433 US2015059433W WO2016073837A1 WO 2016073837 A1 WO2016073837 A1 WO 2016073837A1 US 2015059433 W US2015059433 W US 2015059433W WO 2016073837 A1 WO2016073837 A1 WO 2016073837A1
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention pertains to Factor IX proteins containing modifications in the amino acid sequence of the Factor IX protein, as well as nucleic acid constructs encoding the Factor IX proteins and methods of using these compositions to treat a bleeding disorder.
- FIX Factor IX
- Mononine® has the disadvantage that there is a potential to transmit disease through contamination with bacteria and viruses (such as HIV, hepatitis) that are carried through the purification procedure.
- recombinant FIX protein e.g., Benefix®
- rFactor IX recombinant Factor IX
- pd Factor IX human plasma-derived Factor IX
- the present invention provides Factor IX (FIX) proteins comprising additional glycosylation sites with and without amino acid sequence modifications and methods of their use in treating bleeding disorders.
- the Factor IX proteins of this invention have higher specific activity and a longer useful clotting function relative to Factor IX protein without the modifications described herein.
- the present invention provides an isolated Factor IX (FIX) protein comprising the amino acid sequence of SEQ ID NO:2 (FIX23):
- the present invention provides an isolated Factor IX protein comprising the amino acid sequence of SEQ ID NO:3 (FIX24):
- the present invention provides a method of treating a bleeding disorder, comprising administering to a subject (e.g., a subject in need thereof) an effective amount of the Factor IX protein o this invention, the nucleic acid molecule of this invention, the vector of this invention and/or the cell of this invention, singly or in any combination and/or order.
- a subject e.g., a subject in need thereof
- an effective amount of the Factor IX protein o this invention, the nucleic acid molecule of this invention, the vector of this invention and/or the cell of this invention singly or in any combination and/or order.
- Also provided herein is a method of increasing the bioavailablity of a Factor IX protein in a subject (e.g., a subject in need thereof) comprising administering to the subject an effective amount of the Factor IX protein of this invention, the nucleic acid molecule of this invention, the vector of this invention and/or the cell of this invention, singly or in any combination and/or order..
- a cell can mean a single cell or a multiplicity of cells.
- bioavailability is the fraction or amount of an administered dose of a biologically active drug that reaches the systemic circulation.
- bioavailability includes the usual and customary meaning but, in addition, is taken to have a broader meaning to include the extent to which the Factor IX protein is bioactive.
- Factor IX for example, one measurement of "bioavailability” is the procoagulant activity of Factor IX protein obtained in the circulation post-infusion.
- Posttranslational modification has its usual and customary meaning and includes but is not limited to removal of leader sequence, ⁇ -carboxylation of glutamic acid residues, ⁇ -hydroxylation of aspartic acid residues, N-linked glycosylation of asparagine residues, O-linked glycosylation of serine and/or threonine residues, sulfation of tyrosine residues, phosphorylation of serine residues and any combination thereof.
- biological activity is determined with reference to a standard derived, e.g., from human plasma. For Factor IX, the standard can be MONONINE ® (ZLB Behring). The biological activity of the standard is taken to be 100%.
- Factor IX protein or "FIX protein” as used herein includes wild type Factor IX protein as well as naturally occurring or man-made proteins (e.g., the T/A dimorphism in the activation peptide of human FIX at position 148 (numbering based on the mature human FIX amino acid sequence of SEQ ID NO: 1 , which shows a T at position 148) as described in Graham et al. ("The Malmo polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other FIX polymorphisms" Am. J. Hum. Genet.
- a FIX protein of this invention includes a FIX protein having the amino acid sequence of SEQ ID NO: l , wherein the amino acid at position 148 can be a T or an A and a subject can be either heterozygous or homozygous for either T or A at this site.
- a FIX protein of this invention can further include mutated forms of FIX as are known in the literature (see, e.g., Chang et al. "Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity" J Biol. Chem. 273: 12089-94 (1998); Cheung et al.
- a FIX protein of this invention further includes any other naturally occurring human FIX protein or man made human FIX protein now known or later identified, and derivatives and active fragments/active domains thereof, as are known in the art.
- a Factor IX protein of this invention further includes the pharmacologically active form of FIX, which is the molecule from which the activation peptide has been cleaved out of the protein by the action of proteases (or by engineering it out of the protein by removing it at the nucleic acid level), resulting in two non-contiguous polypeptide chains for FIX (light chain and heavy chain) folded as the functional FIX clotting factor.
- FIX is the pharmacologically active form of FIX, which is the molecule from which the activation peptide has been cleaved out of the protein by the action of proteases (or by engineering it out of the protein by removing it at the nucleic acid level), resulting in two non-contiguous polypeptide chains for FIX (light chain and heavy chain) folded as the functional FIX clotting factor.
- Factor IX proteins having a modification to increase the degree of glycosylation are specifically included in the broad term.
- half life is a broad term which includes the usual and customary meaning as well as the usual and customary meaning found in the scientific literature for Factor IX. Specifically included in this definition is a measurement of a parameter associated with Factor IX which defines the time post-infusion for a decrease from an initial value measured at infusion to half the initial value.
- the half life of FIX can be measured in blood and/or blood components using an antibody to Factor IX in a variety of immunoassays, as are well known in the art and as described herein.
- half life may be measured as a decrease in Factor IX activity using functional assays including standard clotting assays, as are well known in the art and as described herein.
- the term "recovery” as used herein includes the amount of FIX, as measured by any acceptable method including but not limited to FIX antigen levels or FIX protease or clotting activity levels, detected in a recipient animal or human subject (e.g., in the circulation) at the earliest practical time of removing a biological sample (e.g., a blood or blood product sample) for the purpose of measuring the level of FIX following its infusion, injection, delivery or administration otherwise.
- a biological sample e.g., a blood or blood product sample
- the earliest biological sampling time for measuring FIX recovery typically falls within the first 15 minutes post infusion, injection, or delivery/administration otherwise of the FIX, but it is reasonable to expect quicker sampling times as scientific and/or clinical technologies improve.
- the recovery value for FIX is meant here to represent the maximum fraction of infused, injected or otherwise delivered/administered FIX that can be measured in the recipient (e.g., in the circulation) at the earliest possible time point following infusion, injection, or other delivery to a recipient animal or patient.
- glycosylation site is a broad term that has its usual and customary meaning. In the context of the present application the term applies to both sites that potentially could accept a carbohydrate moiety, as well as sites within the protein, specifically FIX, on which a carbohydrate moiety has actually been attached and includes any amino acid sequence that could act as an acceptor for oligosaccharide and/or carbohydrate.
- isolated can refer to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).
- an "isolated fragment” is a fragment of a nucleic acid or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state.
- An “isolated cell” refers to a cell that is separated from other cells and/or tissue components with which it is normally associated in its natural state.
- an isolated cell is a cell that is part of a cell culture.
- An isolated cell can also be a cell that is administered to or introduced into a subject, e.g., to impart a therapeutic or otherwise beneficial effect.
- a "transformed” is a cell that has been transformed, transduced and/or transfected with a nucleic acid molecule encoding a Factor IX protein of this invention, including but not limited to a Factor IX protein vector constructed using recombinant DNA techniques.
- a "subject" of the invention includes any animal susceptible to a bleeding disorder or bleeding condition for which control of bleeding is needed and/or desired, which can be treated, ameliorated or prevented by administration of Factor IX to the subject.
- a subject is generally a mammalian subject (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.), a farm or commercial animal (e.g., a cow, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.).
- the subject is a primate subject, a non-human primate subject (e.g., a chimpanzee, baboon, monkey, gorilla, etc.) or a human.
- Subjects of the invention can be a subject known or believed to be at risk of a bleeding disorder or bleeding condition for which control is needed and/or desired.
- a subject according to the invention can also include a subject not previously known or suspected to be at risk of a bleeding disorder or bleeding condition for which control is needed or desired..
- Subjects include males and/or females of any age, including neonates, juvenile, mature and geriatric subjects.
- the subject can be an infant (e.g., less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months, or younger), a toddler (e.g., at least about 12, 18 or 24 months and/or less than about 36, 30 or 24 months), or a child (e.g. , at least about 1 , 2, 3, 4 or 5 years of age and/or less than about 14, 12, 10, 8, 7, 6, 5, or 4 years of age).
- the subject is a human subject that is from about 0 to 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 3 to 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 12 to 18, 24, 36, 48 or 60 months of age, from about 18 to 24, 30, 36, 48 or 60 months of age, or from about 24 to 30, 36, 48 or 60 months of age.
- treat treating or “treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
- the term “treat,”, “treating” or “treatment of (and grammatical variations thereof) refer to a reduction in the amount and/or frequency of undesirable or uncontrolled bleeding.
- a “treatment effective” amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
- prevent refers to prevention and/or delay of the onset and/or progression of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset and/or progression of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention.
- the term “prevent,” “preventing” or “prevention of” (and grammatical variations thereof) refer to prevention and/or delay of the onset and/or progression of undesirable or uncontrolled bleeding in the subject, with or without other signs of clinical disease. The prevention can be complete, e.g.
- the prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset and/or the progression is less than what would occur in the absence of the present invention.
- a "prevention effective” amount as used herein is an amount that is sufficient to prevent (as defined herein) the disease, disorder and/or clinical symptom in the subject.
- compositions of the invention can be determined by detecting a clinical improvement as indicated by a change in the subject's symptoms and/or clinical parameters as would be well known to one of skill in the art.
- the present invention provides an isolated Factor IX (FIX) protein comprising the amino acid sequence of SEQ ID NO:2 (FIX23):
- the present invention provides an isolated Factor IX protein comprising the amino acid sequence of SEQ ID NO:3 (FIX24):
- FIX24 (SEQ ID NO:3) is a Factor IX protein that has been modified to incorporate three additional glycosylation sites in the activation peptide of Factor IX and a fourth additional glycosylation site in the catalytic domain of Factor IX. These modifications have been introduced into the amino acid sequence of the Factor IX protein to produce a Factor IX protein that stays in the circulation longer.
- the lysine (Lys) at position 5 in the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 can be substituted with an arginine (Arg).
- the lysine (Lys) at position 5 in the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 can be substituted with a threonine, leucine or isoleucine, as nonlimiting examples.
- any substitution of the Lys at position 5 e.g., with an amino acid as listed in Table 1 ) that results in a Factor IX molecule that increases the affinity between Factor IX and type IV collagen (e.g., relative to the affinity of a Factor IX protein that does not have a substitution of the lysine at position 5) is an embodiment of this invention.
- the substitution at position 5 is not alanine. Identification of substitutions that result in increased affinity between Factor IX and type IV collagen can be done according to protocols described herein and as are known in the art.
- a nonlimiting example of such a protocol can comprise growing human aortic endothelial cells on a plate or on a Biocor sensor, removing the cells but leaving behind the extracellular matrix and then incubating the extracellular matrix with Factor IX (e.g., a Factor IX protein of this invention, such as a Factor IX protein that has a substitution at position 5 for which it is desired to determine if said substitution has produced a Factor IX protein of this invention with increased affinity for type IV collagen) under conditions in which binding of the Factor IX protein to the extracellular matrix can occur.
- Detection of bound Factor IX protein can be done with standard Biocor techniques and/or with an antibody to Factor IX and/or by labeling the Factor IX fluorescently in its active site. The Kd is then determined from standard algorithms.
- the phenylalanine in some embodiments of the Factor IX protein of this invention, the phenylalanine
- amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 can be substituted with any other amino acid (e.g., from the list of amino acids in Table 1 ) that is not phenyalanine..
- valine (Val) at position 10 in the amino acid sequence of FIX23 (SEQ ID NO:2) or FIX24 (SEQ ID NO:3) can be substituted with the following nonlimiting examples: leucine, isoleucine, methionine, phenylalanine, histidine or threonine.
- the glutamine (Gin) at position 11 of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 can be substituted with asparagine, lysine or arginine, as nonlimiting examples.
- the arginine (Arg) at position 389 in the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 can be substituted with any other amino acid (e.g., from the amino acids listed in Table 1) that is not arginine.
- the substituted amino acid at position 389 can be alanine or leucine.
- the FIX protein of this invention can be a FIX protein with a substitution of the lysine (Lys) at position 5 as described herein and/or a substitution of the phenylalanine (Phe) at position 9 as described herein and/or a substitution of the valine (Val) at position 10 as described herein and/or a substitution of the glutamine (Glu) at position 1 1 as described herein and/or a substitution of the arginine (Arg) at position 389 as described herein, of the amino acid sequence of FIX23 (SEQ ID NO:2) or FIX24 (SEQ ID NO: 3), singly or in any combination.
- the present invention provides an isolated nucleic acid molecule encoding the FIX protein of this invention, as well as a vector comprising the nucleic acid molecule of this invention..
- the FIX protein of this invention is human FIX protein.
- the Factor IX protein with the substitutions as described herein at positions 5, 9, 10 and 1 1 , singly or in combination can further comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) additional glycosylation sites in the amino acid sequence of FIX23 (SEQ ID NO:2) or FIX24 (SEQ ID NO:3).
- additional glycosylation sites is meant that the number of glycosylation sites in the FIX protein is greater than the number of glycosylation sites normally present in the Factor IX protein of this invention (e.g., FIX23; FIX24).
- the present invention is further directed to FIX proteins containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, etc.) additional sugar chains.
- additional sugar side chains can be present at one or more of the glycosylation sites in the FIX proteins of this invention by the methods described herein.
- the additional sugar side chains can be present at sites on the FIX protein as a result of chemical and/or enzymatic methods to introduce such sugar chains to the FIX molecule, as are well known in the art.
- “additional” or “new” sugar chains is meant that the number of sugar chains in the FIX protein is greater than the number of sugar chains normally present in a "wild type" form of Factor IX.
- sugar side chains e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50
- additional sugar side chains e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50
- At least one additional glycosylation site is in the activation peptide of Factor IX (e.g., the human FIX activation peptide having the amino acid sequence of SEQ ID NO: l).
- the FIX protein has an insertion of a peptide segment that introduces one or more glycosylation sites between position N 157 and 167 of the Factor IX amino acid sequence of SEQ ID NO : 1.
- Insertion(s) can be introduced into a FIX protein of this invention to increase the number of glycosylation sites and such insertion(s) can include from about one to about 100 amino acid residues, including any number of amino acid residues from one to 100 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90,
- the insertion can include all or at least part (e.g., at least 3,
- the glycosylation site(s) may be N-linked glycosylation site(s).
- the added glycosylation site(s) include N-linked glycosylation site(s) and the consensus sequence is NXT/S, with the proviso that X is not proline.
- glycosylation site(s) can be added to the amino acid sequence of the FIX protein of this invention.
- about 1 to about 50 glycosylation site(s) e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50
- Embodiments of the invention include FIX proteins in which a glycosylation site has been created by insertion, deletion or substitution of specific amino acids.
- the insertion, deletion and/or substitution is in the region of the activation peptide.
- the amino acid sequence of the human FIX activation peptide is provided herein as: Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu Thr He Leu Asp Asn He Thr Gin Ser Thr Gin Ser Phe Asn Asp Phe Thr Arg (SEQ ID NO: 1 1).
- the additional glycosylation sites introduced into a FIX amino acid sequence can be introduced anywhere throughout the amino acid sequence of the FIX protein.
- the additional glycosylation site or sites are introduced in the activation peptide (amino acids 146-180 of the mature human FIX amino acid sequence of SEQ ID NO: l), outside the activation peptide (e.g., before and/or after the activation peptide) or both inside the activation peptide and outside the activation peptide.
- a glycosylation attachment site can be introduced by inserting additional amino acid residues between or at any of amino acids 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25.
- a "glycosylation attachment site” or "glycosylation site” can mean a sugar attachment consensus sequence (i.e., a series of amino acids that act as a consensus sequence for attaching a sugar (mono-, oligo-, or poly-saccharide) to an amino acid sequence or it can mean the actual amino acid residue to which the sugar moiety is covalently linked.
- the sugar moiety can be a monosaccharide (simple sugar molecule), an oligosaccharide, or a polysaccharide.
- additional amino acids can be inserted between and/or substituted into any of the amino acid residues that make up the activation peptide, such as between any of amino acids 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155,
- the same insert of this invention can be introduced multiple times at the same and/or at different locations in the amino acid sequence of the FIX protein, including within the activation peptide. Also, different inserts and/or the same inserts can be introduced one or more times at the same and/or at different locations between amino acid residues throughout the amino acid sequence of the FIX protein, including within the activation peptide.
- Some proteins can support a large number of sugar side chains and the distance between N-linked glycosylation sites can be as few as three, four, five or six amino acids (see, e.g., Lundin et al. "Membrane topology of the Drosophila OR83b odorant receptor” FEBS Letters 581 :5601 -5604 (2007); Apweiler et al. "On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database” Biochimica et Biophysica Acta 1473:4-8 (19991), the entire contents of which are incorporated by reference herein).
- the FIX protein of this invention can be modi ied by mutation (e.g., substitution, addition and/or deletion of amino acids) to introduce N-linked glycosylation sites.
- mutation e.g., substitution, addition and/or deletion of amino acids
- amino acid residues on the surface of the functional FIX protein can be identified according to molecular modeling methods standard in the art that would be suitable for modification (e.g., mutation) to introduce one or more glycosylation sites.
- FIX proteins of this invention having additional glycosylation sites may be produced by recombinant methods such as site-directed mutagenesis using PGR.
- the Factor IX protein of this invention may be chemically synthesized to prepare a Factor IX protein with one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, etc.) additional glycosylation sites.
- one or more e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, etc.
- Embodiments of the invention are directed to recombinant Factor IX proteins (e.g., FIX23; FIX24) in which glycosylation sites have been added to improve the recovery and/or half-life and/or stability and/or type IV collagen binding affinity of Factor IX.
- the Factor IX proteins of this invention comprise modifications that allow for increased bioavailability of the Factor IX protein to a subject to whom the Factor IX protein of this invention has been administered.
- Increased bioavailability in some embodiments refers to the standard current thinking in hematology that the concentration of FIX in plasma is the relevant concentration.
- increased bioavailability refers to the ability of the FIX protein to stay in a subject's circulation for a longer period of time. This is because it is understood that an effective means of increasing the hemostatic effectiveness of FIX is to increase its affinity for type IV collagen rather than increase its concentration in the plasma.
- the FIX proteins described herein are modified to result in a FIX protein that has an increased affinity for type IV collagen and in some embodiments, the present invention provides a method of increasing the hemostatic effectiveness of a FIX protein in a subject, comprising administering to the subject an effective amount of the Factor IX protein of this invention, the nucleic acid molecule of this invention, the vector of this invention and/or the cell of this invention, wherein the Factor IX protein that is administered to the subject in any o these embodiments is a Factor IX protein of this invention that has increased affinity for type I V collagen.
- At least one additional glycosylation site is introduced into the FIX amino acid sequence at a site that is outside of the activation peptide.
- the at least one additional glycosylation site corresponds to a site that is glycosylated in the native form of a non-human homolog of Factor IX.
- a modification of the human FIX amino acid sequence to introduce a serine or threonine at amino acid 262 of the amino acid sequence of SEQ ID NO: 1, which is the mature (i.e., secreted) form of human FIX, would introduce an additional N-linked glycosylation site in the human protein.
- the non-human homolog is from dog, pig, cow, or mouse.
- nucleic acid comprising, consisting essentially of and/or consisting of a nucleotide sequence encoding a FIX amino acid sequence of this invention.
- nucleic acids can be present in a vector, such as an expression cassette.
- some embodiments of the invention are directed to expression cassettes designed to express a nucleotide sequence encoding any of the Factor IX proteins of this invention.
- the nucleic acids and/or vectors of this invention can be present in a cell (e.g., an isolated cell; a transformed cell; a recombinant cell, etc.).
- various embodiments of the invention are directed to recombinant host cells containing the vector (e.g., expression cassette).
- Such a cell can be isolated and/or present in a transgenic animal. Therefore, certain embodiments of the invention are further directed to a transgenic animal comprising a nucleic acid comprising a nucleotide sequence encoding any of the Factor IX proteins of the present invention.
- a comparison of the amino acid sequence of the activation peptide of human, mouse, guinea pig and platypus FIX reveals that the mouse FIX amino acid sequence has an additional nine amino acids present in its activation peptide, the guinea pig FIX amino acid sequence has an additional ten amino acid residues present in its activation peptide and the platypus has an additional 14 amino acid residues present in its activation peptide. These extra amino acids are between the two naturally occurring glycosylation sites (N 157 and N 167) in human Factor IX.
- the human and mouse FIX have essentially identical structures and the human enzyme can function in the mouse.
- this region of the Factor IX molecule can tolerate modifications within its sequence, including insertions, substitutions and/or deletions, without substantial loss in structural, biochemical, or otherwise functional integrity of the molecule.
- the inserted nine amino acids in mouse are most likely surface residues (as supported by structural studies) and therefore accessible for modification by the glycosylation enzymes.
- the two N-linked glycosylation sites are 12 and 14 amino acids distant from the amino and carboxyl cleavage sites, respectively, of the activation peptide.
- additional amino acid residues can be added between N157 and N 167 of the human Factor IX protein of SEQ ID NO: 1 in order to add glycosylation sites to improve half life and/or bioavailability.
- glycosylation sites are added by insertion, deletion and/or modification o the native sequence to include an attachment sequence for consensus sequences for N-linked glycosylation.
- the human sequence for the activation peptide starts at residue 146 of the mature protein.
- the natural glycosylation sites are at N157 and N167 (SEQ ID NO:l).
- additional amino acid residues can be inserted between the two normal glycosylation sites (between N157 and N167 in the mature sequence) to provide additional glycosylation sites.
- about 3 to about 100 additional amino acid residues are added.
- about 5 to about 50 amino acid residues are added.
- about 5 to about 20 amino acid residues are added.
- about 7 to about 15 amino acid residues are added.
- amino acid residues are chosen from the 20 biological amino acids with the proviso that proline is not used as "X" in the glycosylation site NXT/S, which is the consensus sequence for N-linked glycosylation.
- Table 1 shows 20 common biological amino acids and their abbreviations.
- endogenous N-linked attachment sequences from mouse, human and other mammalian Factor IX sequences are inserted into the activation peptide. These may be inserted individually or in combination.
- the inserted segment includes a spacer region between glycosylation sites, which can be present individually, in tandem repeats, in multiples, etc.
- a spacer region o this invention can be from one to about 100 amino acids in length (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73.
- the spacer region can be from one to about 20 amino acids. In other embodiments the spacer region can be from one to about ten amino acids. In further embodiments, the spacer region can be from one to about five amino acid residues.
- a spacer region o this invention is included between the added carbohydrate attachment sites and/or between naturally occurring glycosylation sites and added glycosylation sites to reduce or eliminate steric hindrance and provide efficient recognition by the appropriate glycosyltransferase.
- a spacer region of this invention can be comprised of any combination of amino acid residues provided that they are not cysteine or proline and provided that the amino acid sequence of the spacer does not have more than about 10% residues that are hydrophobic (e.g., tryptophan, tyrosine, phenylalanine and valine).
- NXT/S is incorporated into the inserted amino acid sequence to add one or more additional glycosylation sites.
- "X" may be any biological amino acid except that proline is disfavored.
- at least one additional glycosylation site is added to the Factor IX protein of this invention (e.g., FIX23; FIX24).
- two additional glycosylation sites are added.
- three additional glycosylation sites are added.
- four additional glycosylation sites are added.
- five additional glycosylation sites are added.
- six additional glycosylation sites are added. In other embodiments, more than six additional
- glycosylation sites are added.
- glycosylation sites are added at sites outside of the activation peptide of the Factor IX protein of this invention. These additional sites can be selected, for example, by aligning the amino acid sequence of Factor IX from human with the Factor IX amino acid sequence from other species and determining the position of glycosylation sites in non-human species. The homologous or equivalent position in the human FIX amino acid sequence is then modified to provide a glycosylation site. This method may be used to identify both potential N-glycosylation and O-glycosylation sites.
- FIX proteins according to the invention are produced and characterized by methods well known in the art and as described herein. These methods include determination of clotting time (partial thromboplastin time (PPT) assay) and
- FIX protein administration of the FIX protein to a test animal to determine recovery, half life, and bioavailability by an appropriate immunoassay and/or activity-assay, as are well known in the art.
- the Factor IX protein, nucleic acid, vector and/or cell of this invention can be included in a pharmaceutical composition.
- Some embodiments are directed to a kit which includes the Factor IX protein of this invention and/or reagents and/or instructions for using the kit, e.g., to carry out the methods of this invention.
- the Factor IX protein of this invention can be used in a method of treating a bleeding disorder by administering an effective amount of the Factor IX protein to a subject (e.g., a human patient) in need thereof.
- a subject e.g., a human patient
- the present invention also provides a method of treating a bleeding disorder comprising administering to a subject in need thereof an effective amount of the Factor IX protein, the nucleic acid molecule, the vector and/or the cell of this invention.
- Also provided herein is a method of increasing the bioavailablity of a Factor IX protein in a subject, comprising administering to the subject an effective amount of the Factor IX protein, the nucleic acid molecule and/or the cell of this invention.
- Bleeding disorders that can be treated according to the methods of this invention include, but are not limited to, a FIX deficiency, hemophilia B and Christmas disease.
- Such treatment protocols and dosing regimens for administering or delivering Factor IX protein of this invention and/or a nucleic acid molecule encoding a Factor IX protein of this invention to a subject are well known in the art.
- the dosage of a vector e.g., a viral vector or other nucleic acid vector
- the dosage of a FIX protein of this invention can be in a range of about 10 units per kilogram to about 400 units per kilogram.
- the Factor IX protein of the invention will typically be administered within about 24 hours prior to performing the intervention, and for as much as 7 days or more thereafter.
- Administration as a coagulant can be by a variety of routes as described herein.
- the pharmaceutical compositions are primarily intended for parenteral administration for prophylactic and/or therapeutic treatment.
- the pharmaceutical compositions are administered parenteral ly, i.e., intravenously, subcutaneously, or intramuscularly, or it may be administered by continuous or pulsatile infusion.
- the pharmaceutical compositions may be formulated for administration in various ways, including, but not limited to, orally, subcutaneously, intravenously, intracerebral ly, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other acceptable manner.
- compositions for parenteral administration comprise the Factor IX protein of the invention in combination with (e.g., dissolved in), a pharmaceutically acceptable carrier, preferably an aqueous carrier.
- a pharmaceutically acceptable carrier preferably an aqueous carrier.
- aqueous carriers such as water, buffered water, 0.4% saline, 0.3% glycine and the like.
- the Factor IX protein of the invention may also be formulated with compositions that prolong stability and storage, such as methionine and sucrose.
- the Factor IX protein of the invention can also be formulated into liposome preparations for delivery or targeting to the site(s) of injury. Liposome preparations are generally described in, e.g., U.S. Pat. Nos.
- compositions may be sterilized by conventional, well- known sterilization techniques.
- the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
- the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
- the compositions may also contain preservatives, isotonifiers, non-ionic surfactants or detergents, antioxidants and/or other miscellaneous additives.
- the concentration of the Factor IX protein in these formulations can vary widely, i.e., from less than about 0.5% by weight, usually at or at least about 1% by weight to as much as about 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
- a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution and 10 mg of the Factor IX protein.
- Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa. (1990).
- compositions comprising the Factor IX protein of the present invention and/or nucleic acid molecules that encode the Factor IX protein of the present invention can be administered for prophylactic and/or therapeutic treatments.
- compositions are administered to a subject already suffering from a disease, as described above, in an amount sufficient to cure, alleviate or partially arrest the disease and its complications.
- An amount adequate to accomplish this is defined as a "therapeutically effective amount.”
- amounts effective for this purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject.
- compositions containing the Factor IX polypeptide of the invention are administered to a subject susceptible to or otherwise at risk of a disease state or injury to enhance the subject's own coagulative capability. Such an amount is defined to be a "prophylactically effective dose.” In prophylactic applications, the precise amounts once again depend on the subject's state of health and weight.
- the Factor IX protein may be administered by continuous infusion using e.g., a portable pump system.
- the Factor IX protein of the present invention may also be formulated in sustained, or extended release formulations.
- sustained or extended release compositions include, but are not limited to, semi-permeable matrices of solid hydrophobic particles containing the polypeptide.
- Factor IX protein of the present invention such as, for example, topical application may be carried out, for example, by means of a spray, perfusion, double balloon catheters, stent, incorporated into vascular grafts or stents, hydrogels used to coat balloon catheters, or other well established methods.
- the pharmaceutical compositions should provide a quantity of Factor IX protein sufficient to effectively treat the subject.
- Many expression vectors can be used to create genetically engineered cells. Some expression vectors are designed to express large quantities of recombinant proteins after amplification of transfected cells under a variety of conditions that favor selected, high expressing cells. Some expression vectors are designed to express large quantities of recombinant proteins without the need for amplification under selection pressure.
- the present invention includes the production of genetically engineered cells according to methods standard in the art and is not dependent on the use of any specific expression vector or expression system.
- cells are transfected with an expression vector that contains a nucleic acid molecule (e.g., cDNA) encoding the protein.
- a nucleic acid molecule e.g., cDNA
- the FIX protein is expressed with selected co-transfected enzymes that cause proper post-translational modification of the FIX protein to occur in a given cell system.
- the cell may be selected from a variety of sources, but is otherwise a cell that may be transfected with an expression vector containing a nucleic acid molecule (e.g., a cDNA) encoding a Factor IX protein.
- a vector is a replicable DNA construct. Vectors are used herein either to amplify nucleic acid encoding Factor IX protein and/or to express nucleic acid which encodes Factor IX protein.
- An expression vector is a replicable nucleic acid construct in which a nucleotide sequence encoding a Factor IX protein is operably linked to suitable control sequences capable of effecting the expression of the nucleotide sequence to produce a Factor IX protein in a suitable host cell. The need for such control sequences will vary depending upon the host cell selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation.
- Vectors comprise plasmids, viruses (e.g., adenovirus, cytomegalovirus), phage, and integratable DNA fragments (i.e., fragments integratable into the host cell genome by recombination).
- the vector can replicates and function independently of the host cell genome (e.g., via transient expression), or can integrate into the host cell genome itself (e.g., stable integration).
- Expression vectors can contain a promoter and RNA binding sites that are operably linked to the nucleic acid molecule to be expressed and are operable in the host cell and/or organism.
- DNA regions or nucleotide sequences are operably linked or operably associated when they are functionally related to each other.
- a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation of the sequence.
- Suitable host cells include prokaryote, yeast or higher eukaryotic cells such as mammalian cells and insect cells.
- Cells derived from multicellular organisms are a particularly suitable host for recombinant Factor IX protein synthesis, and mammalian cells are particularly preferred. Propagation of such cells in cell culture has become a routine procedure (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)).
- useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, HEK 293, BH , COS-7, CV, and MDCK cell lines.
- Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the nucleotide sequence encoding Factor IX protein to be expressed and operatively associated therewith, along with a ribosome binding site, an RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence.
- expression can be carried out in Chinese Hamster Ovary (CHO) cells using the expression system of U.S. Patent No. 5,888,809, which is incorporated herein by reference in its entirety.
- transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources.
- Nonlimiting examples include promoters derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See. e.g., U.S. Pat. No. 4,599,308.
- An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV 40 or other viral (e.g., polyoma, adenovirus, VSV, or BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
- an exogenous origin such as may be derived from SV 40 or other viral (e.g., polyoma, adenovirus, VSV, or BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
- DHFR dihydrofolate reductase
- thymidine kinase thymidine kinase
- Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as the baculovirus expression vector (e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed in carrying out the present invention, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al.
- a baculovirus expression vector comprises a baculovirus genome containing the nucleotide sequence to be expressed inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedrin promoter.
- Prokaryote host cells include gram negative or gram positive organisms, for example Escherichia coli (E. coli) or bacilli, respectively.
- Higher eukaryotic cells include established cell lines of mammalian origin as described herein.
- Exemplary bacterial host cells are E. coli W31 10 (ATCC 27,325), E. coli B, E. coli XI 776 (ATCC 31 ,537) and E. coli 294 (ATCC 31,446).
- a broad variety of suitable prokaryotic and microbial vectors are available.
- E. coli is typically transformed using pBR322. Promoters most commonly used in recombinant microbial expression vectors include the betalactamase
- the promoter and Shine- Dalgarno sequence are operably linked to the nucleic acid encoding the Factor IX protein, i.e., they are positioned so as to promote transcription of Factor IX messenger RNA from DNA.
- Eukaryotic microbes such as yeast cultures may also be transformed with protein- encoding vectors (see, e.g., U.S. Pat. No. 4,745,057).
- Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available.
- Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, nucleic acid encoding Factor IX protein, sequences for polyadenylation and transcription termination, and a selection gene.
- An exemplary plasmid is YRp7,
- Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3 -phosphoglycerate kinase (Hitzeman et al. J Biol. Chem. 255:2073 ( 1980) or other glycolytic enzymes (Hess et al. J. Adv. Enzyme Reg. 7: 149 ( 1968); and Holland et al. Biochemistry 17:4900 (1978)).
- Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657.
- Cloned coding sequences of the present invention may encode FIX of a * ny species of origin, including mouse, rat, dog, opossum, rabbit, cat, pig, horse, sheep, cow, guinea pig, opossum, platypus, and human, but preferably encode Factor IX protein of human origin.
- Nucleic acid encoding Factor IX that is hybridizable with nucleic acid encoding proteins disclosed herein is also encompassed.
- Hybridization of such sequences may be carried out under conditions of reduced stringency or even stringent conditions (e.g., stringent conditions as represented by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 60°C or even 70°C) to nucleic acid encoding Factor IX protein disclosed herein in a standard in situ hybridization assay.
- stringent conditions as represented by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 60°C or even 70°C
- the FIX proteins produced according to the invention may be expressed in transgenic animals by known methods. See for example, U.S. Patent No. 6,344,596, which is incorporated herein by reference in its entirety.
- transgenic animals may include but are not limited to farm animals (e.g., pigs, goats, sheep, cows, horses, rabbits and the like) rodents (such as mice, rats and guinea pigs), and domestic pets (for example, cats and dogs).
- farm animals e.g., pigs, goats, sheep, cows, horses, rabbits and the like
- rodents such as mice, rats and guinea pigs
- domestic pets for example, cats and dogs.
- Livestock animals such as pigs, sheep, goats and cows, are particularly preferred in some embodiments.
- the transgenic animal of this invention is produced by introducing into a single cell embryo an appropriate polynucleotide that encodes a human Factor IX protein of this invention in a manner such that the polynucleotide is stably integrated into the DNA of germ line cells of the mature animal, and is inherited in normal Mendelian fashion.
- the transgenic animal of this invention would have a phenotype of producing the FIX protein in body fluids and/or tissues.
- the FIX protein would be removed from these fluids and/or tissues and processed, for example for therapeutic use. (See, e.g., Clark et al.
- DNA molecules can be introduced into embryos by a variety of means including but not limited to microinjection, calcium phosphate mediated precipitation, liposome fusion, or retroviral infection of totipotent or pluripotent stem cells.
- the transformed cells can then be introduced into embryos and incorporated therein to form transgenic animals. Methods of making transgenic animals are described, for example, in
- Transgenic animals also can be generated using methods of nuclear transfer or cloning using embryonic or adult cell lines as described for example in Campbell et al., Nature 380:64-66 (1996) and Wilmut et al., Nature 385:810-813 (1997). Further a technique utilizing cytoplasmic injection of DNA can be used as described in U.S. Pat. No. 5,523,222.
- Factor IX-producing transgenic animals can be obtained by introducing a chimeric construct comprising Factor IX-encoding sequences. Methods for obtaining transgenic animals are well-known. See, for example, Hogan et al., MANIPULATING THE
- c .s-acting regulatory regions may be used that are "active" in mammary tissue in that the promoters are more active in mammary tissue than in other tissues under physiological conditions where milk is synthesized.
- promoters include but are not limited to the short and long whey acidic protein (WAP), short and long ⁇ , ⁇ and K casein, a-lactalbumin and ⁇ -lactoglobulin ("BLG”) promoters.
- WAP short and long whey acidic protein
- BLG ⁇ -lactalbumin and ⁇ -lactoglobulin
- Signal sequences can also be used in accordance with this invention that direct the secretion of expressed proteins into other body fluids, particularly blood and urine. Examples of such sequences include the signal peptides of secreted coagulation factors including signal peptides of Factor IX, protein C, and tissue-type plasminogen activator.
- enhancers are useful sequences that regulate transcription, in addition to the promoters discussed above, and other sequences which regulate the expression of transgenes.
- the expression system or construct includes a 3' untranslated region downstream of the nucleotide sequence encoding the desired recombinant protein.
- This region can increase expression of the transgene.
- sequences that provide a poly A signal are sequences that provide a poly A signal.
- Suitable heterologous 3'-untranslated sequences can be derived, for example, from the SV40 small t antigen, the casein 3' untranslated region, or other 3' untranslated sequences well known in this art. Ribosome binding sites are also important in increasing the efficiency of expression of FIX. Likewise, sequences that regulate the post- translational modification of FIX are useful in the invention.
- nucleotide sequence encoding the amino acid sequence of FIX24 (SEQ ID NO:5):
Abstract
Factor IX proteins are described with an increase in the number of glycosylation sites and other modifications to provide Factor IX proteins that have higher specific activity and a longer useful clotting function relative to wild type or non-modified Factor IX protein.
Description
METHODS AND COMPOSITIONS FOR MODIFIED FACTOR IX PROTEINS
PRIORITY STATEMENT
This application claims the benefit, under 35 U.S.C. § 1 19(e), of U.S. Provisional Application Serial No. 62/076,951 , filed November 7, 2014, the entire contents of which are incorporated by reference herein.
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under Grant No.
5P01HL006350 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION
The invention pertains to Factor IX proteins containing modifications in the amino acid sequence of the Factor IX protein, as well as nucleic acid constructs encoding the Factor IX proteins and methods of using these compositions to treat a bleeding disorder.
BACKGROUND OF THE INVENTION
Factor IX (FIX) is commercially available as both a plasma-derived FIX product (Mononine®) and a recombinant FIX protein (Benefix®). Mononine® has the disadvantage that there is a potential to transmit disease through contamination with bacteria and viruses (such as HIV, hepatitis) that are carried through the purification procedure. The use of recombinant FIX protein (e.g., Benefix®) avoids these problems. However, the pharmacokinetic properties of recombinant Factor IX (rFactor IX, e.g., Benefix®) do not compare well with the properties of human plasma-derived Factor IX (pd Factor IX, e.g., Mononine®) after intravenous (i.v.) bolus infusion in laboratory animal model systems and in humans. Due to the less favorable pharmacokinetic properties of rFactor IX, generally 20-30% higher doses of rFactor IX are required to achieve the same procoagulant activity level as pdFactor IX (White et al. Seminars in Hematology 35(2) Suppl. 2:33-38 (1998); Roth et al. 5/oo< 98(13):3600-3606(2001)).
The present invention provides Factor IX (FIX) proteins comprising additional glycosylation sites with and without amino acid sequence modifications and methods of their use in treating bleeding disorders. The Factor IX proteins of this invention have
higher specific activity and a longer useful clotting function relative to Factor IX protein without the modifications described herein.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides an isolated Factor IX (FIX) protein comprising the amino acid sequence of SEQ ID NO:2 (FIX23):
YNSGKLEEFVQGNLERECMEE CSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYE C CPFGFEGK CELDVTCNIKNGRCEQFCKNSADNKWCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQT SKLTRAETVFPDVDYWSTEAEGSPGSGANATGPSGEGASAPSENATGPGTSGGSPANSTGGSPAEGSPG SEGTILDNITQSTQSFNDFTRWGGEDAKPGQFPWQWLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKI TWAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIF LKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYN MFCAGFHEGGRDSCQGDSGGPH VTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYV WIKE TKLT
In a further aspect, the present invention provides an isolated Factor IX protein comprising the amino acid sequence of SEQ ID NO:3 (FIX24):
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYE CWCPFGFEGKNCELDVTCNIK GRCEQFCKNSADN WCSCTEGYRLAENQ SCEPAVPFPCGRVSVSQT SKLTRAETVFPDVDYVNSTEAEGSPGSGANATGPSGEGASAPSENATGPGTSGGSPANSTGGSPAEGSPG SEGTILDNITQSTQSFNDFTRWGGEDAKPGQFP QWLNGKVDAFCGGSIVNE IVTAAHCVETGV I TWAGEHNIEETEHTEQKRNVIRIIPHHNYNA lNKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIF LKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPH VTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT
In additional aspects, the present invention provides a method of treating a bleeding disorder, comprising administering to a subject (e.g., a subject in need thereof) an effective amount of the Factor IX protein o this invention, the nucleic acid molecule of this invention, the vector of this invention and/or the cell of this invention, singly or in any combination and/or order.
Also provided herein is a method of increasing the bioavailablity of a Factor IX protein in a subject (e.g., a subject in need thereof) comprising administering to the subject an effective amount of the Factor IX protein of this invention, the nucleic acid molecule of this invention, the vector of this invention and/or the cell of this invention, singly or in any combination and/or order..
Further aspects, features and advantages of this invention will become apparent from the detailed description of the embodiments which follow.
DETAILED DESCRIPTION OF THE INVENTION
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. The terminology used in the description of the invention herein is for
the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Definitions
As used herein, "a," "an" or "the" can mean one or more than one. For example,
"a" cell can mean a single cell or a multiplicity of cells.
Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").
The term "about," as used herein when referring to a measurable value such as an amount (e.g., an amount of methylation) and the like, is meant to include variations of
±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.
As used herein, the transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461 , 463
(CCPA 1976) (emphasis in the original); see also MPEP § 21 1 1.03. Thus, the term
"consisting essentially of" when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising."
The term "pharmacokinetic properties" has its usual and customary meaning and refers to the absorption, distribution, metabolism and excretion of the Factor IX protein.
The usual and customary meaning of "bioavailability" is the fraction or amount of an administered dose of a biologically active drug that reaches the systemic circulation.
In the context of embodiments of the present invention, the term "bioavailability" includes the usual and customary meaning but, in addition, is taken to have a broader meaning to include the extent to which the Factor IX protein is bioactive. In the case of
Factor IX, for example, one measurement of "bioavailability" is the procoagulant activity of Factor IX protein obtained in the circulation post-infusion.
"Posttranslational modification" has its usual and customary meaning and includes but is not limited to removal of leader sequence, γ-carboxylation of glutamic acid residues, β-hydroxylation of aspartic acid residues, N-linked glycosylation of asparagine residues, O-linked glycosylation of serine and/or threonine residues, sulfation of tyrosine residues, phosphorylation of serine residues and any combination thereof.
As used herein, "biological activity" is determined with reference to a standard derived, e.g., from human plasma. For Factor IX, the standard can be MONONINE® (ZLB Behring). The biological activity of the standard is taken to be 100%.
The term "Factor IX protein" or "FIX protein" as used herein includes wild type Factor IX protein as well as naturally occurring or man-made proteins (e.g., the T/A dimorphism in the activation peptide of human FIX at position 148 (numbering based on the mature human FIX amino acid sequence of SEQ ID NO: 1 , which shows a T at position 148) as described in Graham et al. ("The Malmo polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other FIX polymorphisms" Am. J. Hum. Genet. 42:573- 580 ( 1988 )) Thus, in some embodiments, a FIX protein of this invention includes a FIX protein having the amino acid sequence of SEQ ID NO: l , wherein the amino acid at position 148 can be a T or an A and a subject can be either heterozygous or homozygous for either T or A at this site. A FIX protein of this invention can further include mutated forms of FIX as are known in the literature (see, e.g., Chang et al. "Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity" J Biol. Chem. 273: 12089-94 (1998); Cheung et al. "Identification of the endothelial cell binding site for factor IX" PNAS USA 93: 1 1068-73 (1996); Horst, Molecular Pathology, page 361 (458 pages) CRC Press, 1991, the entire contents of each of which are incorporated by reference herein). A FIX protein of this invention further includes any other naturally occurring human FIX protein or man made human FIX protein now known or later identified, and derivatives and active fragments/active domains thereof, as are known in the art.
A Factor IX protein of this invention further includes the pharmacologically active form of FIX, which is the molecule from which the activation peptide has been cleaved out of the protein by the action of proteases (or by engineering it out of the protein by removing it at the nucleic acid level), resulting in two non-contiguous polypeptide chains for FIX (light chain and heavy chain) folded as the functional FIX clotting factor.
Specifically, Factor IX proteins having a modification to increase the degree of glycosylation are specifically included in the broad term.
The term "half life" is a broad term which includes the usual and customary meaning as well as the usual and customary meaning found in the scientific literature for Factor IX. Specifically included in this definition is a measurement of a parameter
associated with Factor IX which defines the time post-infusion for a decrease from an initial value measured at infusion to half the initial value. In some embodiments, the half life of FIX can be measured in blood and/or blood components using an antibody to Factor IX in a variety of immunoassays, as are well known in the art and as described herein. Alternatively, half life may be measured as a decrease in Factor IX activity using functional assays including standard clotting assays, as are well known in the art and as described herein.
The term "recovery" as used herein includes the amount of FIX, as measured by any acceptable method including but not limited to FIX antigen levels or FIX protease or clotting activity levels, detected in a recipient animal or human subject (e.g., in the circulation) at the earliest practical time of removing a biological sample (e.g., a blood or blood product sample) for the purpose of measuring the level of FIX following its infusion, injection, delivery or administration otherwise. With current methodologies, the earliest biological sampling time for measuring FIX recovery typically falls within the first 15 minutes post infusion, injection, or delivery/administration otherwise of the FIX, but it is reasonable to expect quicker sampling times as scientific and/or clinical technologies improve. In essence, the recovery value for FIX is meant here to represent the maximum fraction of infused, injected or otherwise delivered/administered FIX that can be measured in the recipient (e.g., in the circulation) at the earliest possible time point following infusion, injection, or other delivery to a recipient animal or patient.
The term "glycosylation site" is a broad term that has its usual and customary meaning. In the context of the present application the term applies to both sites that potentially could accept a carbohydrate moiety, as well as sites within the protein, specifically FIX, on which a carbohydrate moiety has actually been attached and includes any amino acid sequence that could act as an acceptor for oligosaccharide and/or carbohydrate.
The term "isolated" can refer to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated fragment" is a fragment of a nucleic acid or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state.
An "isolated cell" refers to a cell that is separated from other cells and/or tissue components with which it is normally associated in its natural state. For example, an isolated cell is a cell that is part of a cell culture. An isolated cell can also be a cell that is administered to or introduced into a subject, e.g., to impart a therapeutic or otherwise beneficial effect.
As used herein, a "transformed" is a cell that has been transformed, transduced and/or transfected with a nucleic acid molecule encoding a Factor IX protein of this invention, including but not limited to a Factor IX protein vector constructed using recombinant DNA techniques.
A "subject" of the invention includes any animal susceptible to a bleeding disorder or bleeding condition for which control of bleeding is needed and/or desired, which can be treated, ameliorated or prevented by administration of Factor IX to the subject. Such a subject is generally a mammalian subject (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.), a farm or commercial animal (e.g., a cow, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.). In particular embodiments, the subject is a primate subject, a non-human primate subject (e.g., a chimpanzee, baboon, monkey, gorilla, etc.) or a human. Subjects of the invention can be a subject known or believed to be at risk of a bleeding disorder or bleeding condition for which control is needed and/or desired. Alternatively, a subject according to the invention can also include a subject not previously known or suspected to be at risk of a bleeding disorder or bleeding condition for which control is needed or desired..
Subjects include males and/or females of any age, including neonates, juvenile, mature and geriatric subjects. With respect to human subjects, in representative embodiments, the subject can be an infant (e.g., less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months, or younger), a toddler (e.g., at least about 12, 18 or 24 months and/or less than about 36, 30 or 24 months), or a child (e.g. , at least about 1 , 2, 3, 4 or 5 years of age and/or less than about 14, 12, 10, 8, 7, 6, 5, or 4 years of age). In embodiments of the invention, the subject is a human subject that is from about 0 to 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 3 to 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 12 to 18, 24, 36, 48 or 60 months of age, from about 18 to 24, 30, 36, 48 or 60 months of age, or from about 24 to 30, 36, 48 or 60 months of age.
By the term '"treat," "treating" or "treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder. In representative embodiments, the term "treat,", "treating" or "treatment of (and grammatical variations thereof) refer to a reduction in the amount and/or frequency of undesirable or uncontrolled bleeding.
A "treatment effective" amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
The term "prevent," "preventing" or "prevention of (and grammatical variations thereof) refer to prevention and/or delay of the onset and/or progression of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset and/or progression of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. In representative embodiments, the term "prevent," "preventing" or "prevention of" (and grammatical variations thereof) refer to prevention and/or delay of the onset and/or progression of undesirable or uncontrolled bleeding in the subject, with or without other signs of clinical disease. The prevention can be complete, e.g. , the total absence of the disease, disorder and/or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset and/or the progression is less than what would occur in the absence of the present invention.
A "prevention effective" amount as used herein is an amount that is sufficient to prevent (as defined herein) the disease, disorder and/or clinical symptom in the subject.
Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.
The efficacy of treating a bleeding disorder by the methods of the present invention can be determined by detecting a clinical improvement as indicated by a change in the subject's symptoms and/or clinical parameters as would be well known to one of skill in the art.
Compositions of the invention
In one aspect, the present invention provides an isolated Factor IX (FIX) protein comprising the amino acid sequence of SEQ ID NO:2 (FIX23):
YNSG LEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYE C CPFGFEGKNCELDVTCNIKNGRCEQFC NSADNKWCSCTEGYRLAENQ SCEPAVPFPCGRVSVSQT SKLTRAETVFPDVDYWSTEAEGSPGSGANATGPSGEGASAPSENATGPGTSGGSPANSTGGSPAEGSPG SEGTILDNITQSTQSFNDFTRWGGEDAKPGQFP QWLNGKVDAFCGGSIVNEK IVTAAHCVETGVKI TWAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIAPKEYTNIF LKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPH VTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVN IKEKTKLT
In a further aspect, the present invention provides an isolated Factor IX protein comprising the amino acid sequence of SEQ ID NO:3 (FIX24):
YNSG LEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYE CWCPFGFEGK CELDVTCNI GRCEQFC NSADN WCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQT SKLTRAETVFPD-VDY STEAEGSPGSGA ATGPSGEGASAPSENATGPGTSGGSPANSTGGSPAEGSPG SEGTILDNITQSTQSFNDFTRWGGEDAKPGQFPWQWLNGKVDAFCGGSIVNE WIVTAAHCVETGVKI TWAGEHNIEETEHTEQKRNVIRIIPHHNYNAtflNKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIF LKFGSGYVSG GRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPH VTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKE TKLT FIX23 (SEQ ID NO:2) is a Factor IX protein that has been modified to incorporate three additional glycosylation sites in the activation peptide of Factor IX. FIX24 (SEQ ID NO:3) is a Factor IX protein that has been modified to incorporate three additional glycosylation sites in the activation peptide of Factor IX and a fourth additional glycosylation site in the catalytic domain of Factor IX. These modifications have been introduced into the amino acid sequence of the Factor IX protein to produce a Factor IX protein that stays in the circulation longer.
In some embodiments of the Factor IX protein of this invention, the lysine (Lys) at position 5 in the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 can be substituted with an arginine (Arg). In some embodiment, the lysine (Lys) at position 5 in the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 can be substituted with a threonine, leucine or isoleucine, as nonlimiting examples. Any substitution of the Lys at position 5 (e.g., with an amino acid as listed in Table 1 ) that results in a Factor IX molecule that increases the affinity between Factor IX and type IV collagen (e.g., relative to the affinity of a Factor IX protein that does not have a substitution of the lysine at position 5) is an embodiment of this invention. In some embodiments, the substitution at position 5 is not alanine. Identification of substitutions that result in increased affinity between Factor IX and type IV collagen can be done according to protocols described herein and as are known in the art. A nonlimiting example of such a protocol can comprise growing human aortic endothelial cells on a plate or on a Biocor sensor,
removing the cells but leaving behind the extracellular matrix and then incubating the extracellular matrix with Factor IX (e.g., a Factor IX protein of this invention, such as a Factor IX protein that has a substitution at position 5 for which it is desired to determine if said substitution has produced a Factor IX protein of this invention with increased affinity for type IV collagen) under conditions in which binding of the Factor IX protein to the extracellular matrix can occur. Detection of bound Factor IX protein can be done with standard Biocor techniques and/or with an antibody to Factor IX and/or by labeling the Factor IX fluorescently in its active site. The Kd is then determined from standard algorithms.
In some embodiments of the Factor IX protein of this invention, the phenylalanine
(Phe) at position 9 of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 can be substituted with any other amino acid (e.g., from the list of amino acids in Table 1 ) that is not phenyalanine..
In further embodiments of the FIX protein of this invention, the valine (Val) at position 10 in the amino acid sequence of FIX23 (SEQ ID NO:2) or FIX24 (SEQ ID NO:3) can be substituted with the following nonlimiting examples: leucine, isoleucine, methionine, phenylalanine, histidine or threonine.
In some embodiments of the Factor IX protein of this invention, the glutamine (Gin) at position 11 of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 can be substituted with asparagine, lysine or arginine, as nonlimiting examples.
In some embodiments of the Factor IX protein of this invention, the arginine (Arg) at position 389 in the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 can be substituted with any other amino acid (e.g., from the amino acids listed in Table 1) that is not arginine. In some embodiments, the substituted amino acid at position 389 can be alanine or leucine.
In some embodiments, the FIX protein of this invention can be a FIX protein with a substitution of the lysine (Lys) at position 5 as described herein and/or a substitution of the phenylalanine (Phe) at position 9 as described herein and/or a substitution of the valine (Val) at position 10 as described herein and/or a substitution of the glutamine (Glu) at position 1 1 as described herein and/or a substitution of the arginine (Arg) at position 389 as described herein, of the amino acid sequence of FIX23 (SEQ ID NO:2) or FIX24 (SEQ ID NO: 3), singly or in any combination.
In further embodiments, the present invention provides an isolated nucleic acid molecule encoding the FIX protein of this invention, as well as a vector comprising the nucleic acid molecule of this invention..
In some embodiments, the FIX protein of this invention is human FIX protein. In further embodiments, the Factor IX protein with the substitutions as described herein at positions 5, 9, 10 and 1 1 , singly or in combination, can further comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) additional glycosylation sites in the amino acid sequence of FIX23 (SEQ ID NO:2) or FIX24 (SEQ ID NO:3).
By "additional" glycosylation sites is meant that the number of glycosylation sites in the FIX protein is greater than the number of glycosylation sites normally present in the Factor IX protein of this invention (e.g., FIX23; FIX24).
The present invention is further directed to FIX proteins containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, etc.) additional sugar chains. Such additional sugar side chains can be present at one or more of the glycosylation sites in the FIX proteins of this invention by the methods described herein. Alternatively, the additional sugar side chains can be present at sites on the FIX protein as a result of chemical and/or enzymatic methods to introduce such sugar chains to the FIX molecule, as are well known in the art. By "additional" or "new" sugar chains is meant that the number of sugar chains in the FIX protein is greater than the number of sugar chains normally present in a "wild type" form of Factor IX. In various embodiments, about 1 to about 50 additional sugar side chains (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) can be added.
In some embodiments, at least one additional glycosylation site is in the activation peptide of Factor IX (e.g., the human FIX activation peptide having the amino acid sequence of SEQ ID NO: l). In particular embodiments, the FIX protein has an insertion of a peptide segment that introduces one or more glycosylation sites between position N 157 and 167 of the Factor IX amino acid sequence of SEQ ID NO : 1.
Insertion(s) can be introduced into a FIX protein of this invention to increase the number of glycosylation sites and such insertion(s) can include from about one to about 100 amino acid residues, including any number of amino acid residues from one to 100 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25,
26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 and 100).
In some embodiments, the insertion can include all or at least part (e.g., at least 3,
4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or more amino acid residues) of a Factor IX activation peptide from a non-human species, such as mouse. This inserted peptide sequence can be further modified to introduce additional glycosylation sites according to the teachings herein.
The glycosylation site(s) may be N-linked glycosylation site(s). In some embodiments, the added glycosylation site(s) include N-linked glycosylation site(s) and the consensus sequence is NXT/S, with the proviso that X is not proline.
In some embodiments about one to about 15 glycosylation site(s) can be added to the amino acid sequence of the FIX protein of this invention. In various embodiments, about 1 to about 50 glycosylation site(s) (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50) can be added. Embodiments of the invention include FIX proteins in which a glycosylation site has been created by insertion, deletion or substitution of specific amino acids. In particular embodiments, the insertion, deletion and/or substitution is in the region of the activation peptide. The amino acid sequence of the human FIX activation peptide is provided herein as: Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu Thr He Leu Asp Asn He Thr Gin Ser Thr Gin Ser Phe Asn Asp Phe Thr Arg (SEQ ID NO: 1 1).
It is contemplated that the additional glycosylation sites introduced into a FIX amino acid sequence can be introduced anywhere throughout the amino acid sequence of the FIX protein. Thus, in some embodiments, the additional glycosylation site or sites are introduced in the activation peptide (amino acids 146-180 of the mature human FIX amino acid sequence of SEQ ID NO: l), outside the activation peptide (e.g., before and/or after the activation peptide) or both inside the activation peptide and outside the activation peptide. Thus, based on the numbering of the 415 amino acids of the amino acid sequence of the mature human FIX protein as shown in SEQ ID NO: l , a glycosylation attachment site can be introduced by inserting additional amino acid residues between or at any of amino acids 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 ,
22, 23, 24, 25. 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 1 1 , 1 12, 1 13, 114, 1 15, 1 16, 1 17, 1 18, 1 19, 120, 121 , 122,123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140,141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155,156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 21 1 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 31 1 , 312, 313, 314, 315, 316, 317, 318, 319, 320, 321 , 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397. 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 41 1 , 412, 413, 414, 415 and any combination thereof. As used herein, a "glycosylation attachment site" or "glycosylation site" can mean a sugar attachment consensus sequence (i.e., a series of amino acids that act as a consensus sequence for attaching a sugar (mono-, oligo-, or poly-saccharide) to an amino acid sequence or it can mean the actual amino acid residue to which the sugar moiety is covalently linked. The sugar moiety can be a monosaccharide (simple sugar molecule), an oligosaccharide, or a polysaccharide.
In particular embodiments, additional amino acids can be inserted between and/or substituted into any of the amino acid residues that make up the activation peptide, such as between any of amino acids 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155,
156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182 and any combination thereof. Furthermore, the same insert of this invention can be introduced multiple times at the same and/or at
different locations in the amino acid sequence of the FIX protein, including within the activation peptide. Also, different inserts and/or the same inserts can be introduced one or more times at the same and/or at different locations between amino acid residues throughout the amino acid sequence of the FIX protein, including within the activation peptide.
Some proteins can support a large number of sugar side chains and the distance between N-linked glycosylation sites can be as few as three, four, five or six amino acids (see, e.g., Lundin et al. "Membrane topology of the Drosophila OR83b odorant receptor" FEBS Letters 581 :5601 -5604 (2007); Apweiler et al. "On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database" Biochimica et Biophysica Acta 1473:4-8 (19991), the entire contents of which are incorporated by reference herein).
Furthermore, the FIX protein of this invention can be modi ied by mutation (e.g., substitution, addition and/or deletion of amino acids) to introduce N-linked glycosylation sites. For example, amino acid residues on the surface of the functional FIX protein can be identified according to molecular modeling methods standard in the art that would be suitable for modification (e.g., mutation) to introduce one or more glycosylation sites.
FIX proteins of this invention having additional glycosylation sites may be produced by recombinant methods such as site-directed mutagenesis using PGR.
Alternatively, the Factor IX protein of this invention may be chemically synthesized to prepare a Factor IX protein with one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, etc.) additional glycosylation sites.
It is within the scope of this invention and within the skill of one of ordinary skill in the art to modify any amino acid residue or residues in the mature FIX amino acid sequence according to methods well known in the art and as taught herein and to test any resulting FIX protein for activity, stability, recovery, half life, binding affinity for type 4 collagen, etc., according to well known methods and as described herein (see, e.g., Elliott et al. "Structural requirements for additional N-linked carbohydrate on recombinant human erythropoietin" J Biol. Chem. 279: 16854-62 (2004), the entire contents of which are incorporated by reference herein).
Embodiments of the invention are directed to recombinant Factor IX proteins (e.g., FIX23; FIX24) in which glycosylation sites have been added to improve the recovery and/or half-life and/or stability and/or type IV collagen binding affinity of Factor
IX. The Factor IX proteins of this invention comprise modifications that allow for increased bioavailability of the Factor IX protein to a subject to whom the Factor IX protein of this invention has been administered. Increased bioavailability in some embodiments refers to the standard current thinking in hematology that the concentration of FIX in plasma is the relevant concentration. In some embodiments of the present invention, increased bioavailability refers to the ability of the FIX protein to stay in a subject's circulation for a longer period of time. This is because it is understood that an effective means of increasing the hemostatic effectiveness of FIX is to increase its affinity for type IV collagen rather than increase its concentration in the plasma. Accordingly, in some embodiments of the present invention, the FIX proteins described herein are modified to result in a FIX protein that has an increased affinity for type IV collagen and in some embodiments, the present invention provides a method of increasing the hemostatic effectiveness of a FIX protein in a subject, comprising administering to the subject an effective amount of the Factor IX protein of this invention, the nucleic acid molecule of this invention, the vector of this invention and/or the cell of this invention, wherein the Factor IX protein that is administered to the subject in any o these embodiments is a Factor IX protein of this invention that has increased affinity for type I V collagen.
As noted herein, in some embodiments, at least one additional glycosylation site is introduced into the FIX amino acid sequence at a site that is outside of the activation peptide. In some embodiments, the at least one additional glycosylation site corresponds to a site that is glycosylated in the native form of a non-human homolog of Factor IX. A modification of the human FIX amino acid sequence to introduce a serine or threonine at amino acid 262 of the amino acid sequence of SEQ ID NO: 1, which is the mature (i.e., secreted) form of human FIX, would introduce an additional N-linked glycosylation site in the human protein. In various embodiments, the non-human homolog is from dog, pig, cow, or mouse.
Additionally provided herein is a nucleic acid comprising, consisting essentially of and/or consisting of a nucleotide sequence encoding a FIX amino acid sequence of this invention. Such nucleic acids can be present in a vector, such as an expression cassette. Thus, some embodiments of the invention are directed to expression cassettes designed to express a nucleotide sequence encoding any of the Factor IX proteins of this invention. The nucleic acids and/or vectors of this invention can be present in a cell (e.g., an isolated
cell; a transformed cell; a recombinant cell, etc.). Thus, various embodiments of the invention are directed to recombinant host cells containing the vector (e.g., expression cassette). Such a cell can be isolated and/or present in a transgenic animal. Therefore, certain embodiments of the invention are further directed to a transgenic animal comprising a nucleic acid comprising a nucleotide sequence encoding any of the Factor IX proteins of the present invention.
A comparison of the amino acid sequence of the activation peptide of human, mouse, guinea pig and platypus FIX reveals that the mouse FIX amino acid sequence has an additional nine amino acids present in its activation peptide, the guinea pig FIX amino acid sequence has an additional ten amino acid residues present in its activation peptide and the platypus has an additional 14 amino acid residues present in its activation peptide. These extra amino acids are between the two naturally occurring glycosylation sites (N 157 and N 167) in human Factor IX.
The human and mouse FIX have essentially identical structures and the human enzyme can function in the mouse. As the human FIX functions without the additional nine amino acid segment found in the mouse, this region of the Factor IX molecule can tolerate modifications within its sequence, including insertions, substitutions and/or deletions, without substantial loss in structural, biochemical, or otherwise functional integrity of the molecule. The inserted nine amino acids in mouse are most likely surface residues (as supported by structural studies) and therefore accessible for modification by the glycosylation enzymes. In native human factor IX, the two N-linked glycosylation sites are 12 and 14 amino acids distant from the amino and carboxyl cleavage sites, respectively, of the activation peptide. Thus, in some embodiments of the invention, additional amino acid residues can be added between N157 and N 167 of the human Factor IX protein of SEQ ID NO: 1 in order to add glycosylation sites to improve half life and/or bioavailability. In various embodiments, glycosylation sites are added by insertion, deletion and/or modification o the native sequence to include an attachment sequence for consensus sequences for N-linked glycosylation.
The human sequence for the activation peptide starts at residue 146 of the mature protein. The natural glycosylation sites are at N157 and N167 (SEQ ID NO:l). In some embodiments, additional amino acid residues can be inserted between the two normal glycosylation sites (between N157 and N167 in the mature sequence) to provide additional glycosylation sites. In some embodiments, about 3 to about 100 additional
amino acid residues are added. In other embodiments, about 5 to about 50 amino acid residues are added. In further embodiments, about 5 to about 20 amino acid residues are added. In yet further embodiments, about 7 to about 15 amino acid residues are added. Typically, the amino acid residues are chosen from the 20 biological amino acids with the proviso that proline is not used as "X" in the glycosylation site NXT/S, which is the consensus sequence for N-linked glycosylation. Table 1 shows 20 common biological amino acids and their abbreviations.
In some embodiments, endogenous N-linked attachment sequences from mouse, human and other mammalian Factor IX sequences are inserted into the activation peptide. These may be inserted individually or in combination. In certain embodiments, the inserted segment includes a spacer region between glycosylation sites, which can be present individually, in tandem repeats, in multiples, etc. A spacer region o this invention can be from one to about 100 amino acids in length (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73. 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 and 100). In some embodiments, for example, the spacer region can be from one to about 20 amino acids. In other embodiments the spacer region can be from one to about ten amino acids. In further embodiments, the spacer region can be from one to about five amino acid residues.
A spacer region o this invention is included between the added carbohydrate attachment sites and/or between naturally occurring glycosylation sites and added glycosylation sites to reduce or eliminate steric hindrance and provide efficient recognition by the appropriate glycosyltransferase. A spacer region of this invention can be comprised of any combination of amino acid residues provided that they are not cysteine or proline and provided that the amino acid sequence of the spacer does not have more than about 10% residues that are hydrophobic (e.g., tryptophan, tyrosine, phenylalanine and valine).
In some embodiments, NXT/S is incorporated into the inserted amino acid sequence to add one or more additional glycosylation sites. "X" may be any biological amino acid except that proline is disfavored. In some embodiments, at least one additional glycosylation site is added to the Factor IX protein of this invention (e.g., FIX23; FIX24). In other embodiments, two additional glycosylation sites are added. In
further embodiments, three additional glycosylation sites are added. In yet further embodiments, four additional glycosylation sites are added. In further embodiments, five additional glycosylation sites are added. In some embodiments, six additional glycosylation sites are added. In other embodiments, more than six additional
glycosylation sites are added.
In some embodiments, glycosylation sites are added at sites outside of the activation peptide of the Factor IX protein of this invention. These additional sites can be selected, for example, by aligning the amino acid sequence of Factor IX from human with the Factor IX amino acid sequence from other species and determining the position of glycosylation sites in non-human species. The homologous or equivalent position in the human FIX amino acid sequence is then modified to provide a glycosylation site. This method may be used to identify both potential N-glycosylation and O-glycosylation sites.
The FIX proteins according to the invention are produced and characterized by methods well known in the art and as described herein. These methods include determination of clotting time (partial thromboplastin time (PPT) assay) and
administration of the FIX protein to a test animal to determine recovery, half life, and bioavailability by an appropriate immunoassay and/or activity-assay, as are well known in the art.
The Factor IX protein, nucleic acid, vector and/or cell of this invention can be included in a pharmaceutical composition. Some embodiments are directed to a kit which includes the Factor IX protein of this invention and/or reagents and/or instructions for using the kit, e.g., to carry out the methods of this invention.
Methods of the invention
The Factor IX protein of this invention can be used in a method of treating a bleeding disorder by administering an effective amount of the Factor IX protein to a subject (e.g., a human patient) in need thereof. Thus, the present invention also provides a method of treating a bleeding disorder comprising administering to a subject in need thereof an effective amount of the Factor IX protein, the nucleic acid molecule, the vector and/or the cell of this invention.
Also provided herein is a method of increasing the bioavailablity of a Factor IX protein in a subject, comprising administering to the subject an effective amount of the Factor IX protein, the nucleic acid molecule and/or the cell of this invention.
Bleeding disorders that can be treated according to the methods of this invention include, but are not limited to, a FIX deficiency, hemophilia B and Christmas disease. Such treatment protocols and dosing regimens for administering or delivering Factor IX protein of this invention and/or a nucleic acid molecule encoding a Factor IX protein of this invention to a subject (e.g., a subject in need thereof) are well known in the art.
In embodiments of the invention, the dosage of a vector (e.g., a viral vector or other nucleic acid vector) encoding the Factor IX protein of this invention can be in an amount such that a final plasma concentration of Factor IX protein of from about 10 units per kilogram to about 400 units per kilogram is achieved. In embodiments of this invention, the dosage of a FIX protein of this invention can be in a range of about 10 units per kilogram to about 400 units per kilogram. One of skill in the art would be able to determine the optimal dose for a given subject and a given condition.
For treatment in connection with deliberate interventions, the Factor IX protein of the invention will typically be administered within about 24 hours prior to performing the intervention, and for as much as 7 days or more thereafter. Administration as a coagulant can be by a variety of routes as described herein.
The pharmaceutical compositions are primarily intended for parenteral administration for prophylactic and/or therapeutic treatment. Preferably, the pharmaceutical compositions are administered parenteral ly, i.e., intravenously, subcutaneously, or intramuscularly, or it may be administered by continuous or pulsatile infusion. Alternatively, the pharmaceutical compositions may be formulated for administration in various ways, including, but not limited to, orally, subcutaneously, intravenously, intracerebral ly, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other acceptable manner.
The compositions for parenteral administration comprise the Factor IX protein of the invention in combination with (e.g., dissolved in), a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, such as water, buffered water, 0.4% saline, 0.3% glycine and the like. The Factor IX protein of the invention may also be formulated with compositions that prolong stability and storage, such as methionine and sucrose. The Factor IX protein of the invention can also be formulated into liposome preparations for delivery or targeting to the site(s) of injury. Liposome preparations are generally described in, e.g., U.S. Pat. Nos. 4,837,028,
4,501 ,728, and 4,975,282. The compositions may be sterilized by conventional, well- known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. The compositions may also contain preservatives, isotonifiers, non-ionic surfactants or detergents, antioxidants and/or other miscellaneous additives.
The concentration of the Factor IX protein in these formulations can vary widely, i.e., from less than about 0.5% by weight, usually at or at least about 1% by weight to as much as about 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Thus, as one nonlimiting example, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution and 10 mg of the Factor IX protein. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa. (1990).
The compositions comprising the Factor IX protein of the present invention and/or nucleic acid molecules that encode the Factor IX protein of the present invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a subject already suffering from a disease, as described above, in an amount sufficient to cure, alleviate or partially arrest the disease and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective amount." As will be understood by the person skilled in the art amounts effective for this purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject.
In prophylactic applications, compositions containing the Factor IX polypeptide of the invention are administered to a subject susceptible to or otherwise at risk of a disease state or injury to enhance the subject's own coagulative capability. Such an amount is
defined to be a "prophylactically effective dose." In prophylactic applications, the precise amounts once again depend on the subject's state of health and weight.
Single or multiple administrations of the compositions can be carried out with dose levels and patterns being selected by the treating physician. For ambulatory subjects requiring daily maintenance levels, the Factor IX protein may be administered by continuous infusion using e.g., a portable pump system.
The Factor IX protein of the present invention may also be formulated in sustained, or extended release formulations. Methods of formulating sustained or extended release compositions are known in the art and include, but are not limited to, semi-permeable matrices of solid hydrophobic particles containing the polypeptide.
Local delivery of the Factor IX protein of the present invention, such as, for example, topical application may be carried out, for example, by means of a spray, perfusion, double balloon catheters, stent, incorporated into vascular grafts or stents, hydrogels used to coat balloon catheters, or other well established methods. In any event, the pharmaceutical compositions should provide a quantity of Factor IX protein sufficient to effectively treat the subject.
Production of Factor IX proteins of this invention
Many expression vectors can be used to create genetically engineered cells. Some expression vectors are designed to express large quantities of recombinant proteins after amplification of transfected cells under a variety of conditions that favor selected, high expressing cells. Some expression vectors are designed to express large quantities of recombinant proteins without the need for amplification under selection pressure. The present invention includes the production of genetically engineered cells according to methods standard in the art and is not dependent on the use of any specific expression vector or expression system.
To create a genetically engineered cell to produce large quantities of a Factor IX protein, cells are transfected with an expression vector that contains a nucleic acid molecule (e.g., cDNA) encoding the protein. In some embodiments, the FIX protein is expressed with selected co-transfected enzymes that cause proper post-translational modification of the FIX protein to occur in a given cell system.
The cell may be selected from a variety of sources, but is otherwise a cell that may be transfected with an expression vector containing a nucleic acid molecule (e.g., a cDNA) encoding a Factor IX protein.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning; A Laboratory Manual, 2nd ed. (1989); DNA Cloning, Vols. I and II (D. N Glover, ed. 1985); Oligonucleotide
Synthesis (M. J. Gait, ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J.
Higgins, eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins, eds. 1984); Animal Cell Culture (R. I. Freshney, ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the series. Methods in Enzymology (Academic Press, Inc.), particularly Vols. 154 and 155 (Wu and Grossman, and Wu, eds., respectively); Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos, eds. 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology, Mayer and Walker, eds. (Academic Press, London, 1987); Scopes, Protein Purification: Principles and Practice, 2nd ed. 1987 (Springer-Verlag, N.Y.); and Handbook of Experimental Immunology Vols I-IV (D. M. Weir and C. C. Blackwell, eds 1986). All patents, patent applications, and publications cited in the specification are incorporated herein by reference in their entireties.
Genetic Engineering Techniques
The production of cloned genes, recombinant DNA, vectors, transformed cells, proteins and protein fragments by genetic engineering is well known. See, e.g., U.S. Pat. No. 4,761 ,371 to Bell et al. at Col. 6, line 3 to Col. 9, line 65; U.S. Pat. No. 4,877,729 to Clark et al. at Col. 4, line 38 to Col. 7, line 6; U.S. Pat. No. 4,912,038 to Schilling at Col. 3, line 26 to Col. 14, line 12; and U.S. Pat. No. 4,879,224 to Wallner at Col. 6, line 8 to Col. 8, line 59.
A vector is a replicable DNA construct. Vectors are used herein either to amplify nucleic acid encoding Factor IX protein and/or to express nucleic acid which encodes Factor IX protein. An expression vector is a replicable nucleic acid construct in which a nucleotide sequence encoding a Factor IX protein is operably linked to suitable control sequences capable of effecting the expression of the nucleotide sequence to produce a Factor IX protein in a suitable host cell. The need for such control sequences will vary
depending upon the host cell selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation.
Vectors comprise plasmids, viruses (e.g., adenovirus, cytomegalovirus), phage, and integratable DNA fragments (i.e., fragments integratable into the host cell genome by recombination). The vector can replicates and function independently of the host cell genome (e.g., via transient expression), or can integrate into the host cell genome itself (e.g., stable integration). Expression vectors can contain a promoter and RNA binding sites that are operably linked to the nucleic acid molecule to be expressed and are operable in the host cell and/or organism.
DNA regions or nucleotide sequences are operably linked or operably associated when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation of the sequence.
Suitable host cells include prokaryote, yeast or higher eukaryotic cells such as mammalian cells and insect cells. Cells derived from multicellular organisms are a particularly suitable host for recombinant Factor IX protein synthesis, and mammalian cells are particularly preferred. Propagation of such cells in cell culture has become a routine procedure (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)). Examples of useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, HEK 293, BH , COS-7, CV, and MDCK cell lines.
Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the nucleotide sequence encoding Factor IX protein to be expressed and operatively associated therewith, along with a ribosome binding site, an RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence. In one embodiment, expression can be carried out in Chinese Hamster Ovary (CHO) cells using the expression system of U.S. Patent No. 5,888,809, which is incorporated herein by reference in its entirety.
The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources. Nonlimiting
examples include promoters derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See. e.g., U.S. Pat. No. 4,599,308.
An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV 40 or other viral (e.g., polyoma, adenovirus, VSV, or BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
Rather than using vectors that contain a viral origin of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and the nucleic acid molecule encoding the Factor IX protein. Nonlimiting examples of suitable selectable markers are dihydrofolate reductase (DHFR) or thymidine kinase. This method is further described in U.S. Pat. No. 4,399,216 which is incorporated by reference herein in its entirety.
Other methods suitable for adaptation to the synthesis of Factor IX protein in recombinant vertebrate cell culture include those described in Gething et al. Nature
293:620 (1981 ); Mantei et al. Nature 281 :40; and Levinson et al., EPO Application Nos. 1 17,060A and 1 17,058A, the entire contents of each of which are incorporated herein by reference.
Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as the baculovirus expression vector (e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed in carrying out the present invention, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al. In general, a baculovirus expression vector comprises a baculovirus genome containing the nucleotide sequence to be expressed inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedrin promoter.
Prokaryote host cells include gram negative or gram positive organisms, for example Escherichia coli (E. coli) or bacilli, respectively. Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Exemplary bacterial host cells are E. coli W31 10 (ATCC 27,325), E. coli B, E. coli XI 776 (ATCC 31 ,537) and E. coli 294 (ATCC 31,446). A broad variety of suitable prokaryotic and microbial vectors are available. E. coli is typically transformed using pBR322. Promoters most commonly
used in recombinant microbial expression vectors include the betalactamase
(penicillinase) and lactose promoter systems (Chang et al. Nature 275:615 (1978); and Goeddel et al. Nature 281 :544 (1979)), a tryptophan (trp) promoter system (Goeddel et al. Nucleic Acids Res. 8:4057 (1980) and EPO App. Publ. No. 36,776) and the tac promoter (De Boer et al. Proc. Natl. Acad. Set USA 80:21 (1983)). The promoter and Shine- Dalgarno sequence (for prokaryotic host expression) are operably linked to the nucleic acid encoding the Factor IX protein, i.e., they are positioned so as to promote transcription of Factor IX messenger RNA from DNA.
Eukaryotic microbes such as yeast cultures may also be transformed with protein- encoding vectors (see, e.g., U.S. Pat. No. 4,745,057). Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, nucleic acid encoding Factor IX protein, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7,
(Stinchcomb et al. Nature 282:39 (1979); Kingsman et al. Gene 7: 141 (1979); Tschemper et al. Gene 10: 157 (1980)). Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3 -phosphoglycerate kinase (Hitzeman et al. J Biol. Chem. 255:2073 ( 1980) or other glycolytic enzymes (Hess et al. J. Adv. Enzyme Reg. 7: 149 ( 1968); and Holland et al. Biochemistry 17:4900 (1978)). Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657.
Cloned coding sequences of the present invention may encode FIX of a*ny species of origin, including mouse, rat, dog, opossum, rabbit, cat, pig, horse, sheep, cow, guinea pig, opossum, platypus, and human, but preferably encode Factor IX protein of human origin. Nucleic acid encoding Factor IX that is hybridizable with nucleic acid encoding proteins disclosed herein is also encompassed. Hybridization of such sequences may be carried out under conditions of reduced stringency or even stringent conditions (e.g., stringent conditions as represented by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 60°C or even 70°C) to nucleic acid encoding Factor IX protein disclosed herein in a standard in situ hybridization assay. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor Laboratory).
The FIX proteins produced according to the invention may be expressed in transgenic animals by known methods. See for example, U.S. Patent No. 6,344,596, which is incorporated herein by reference in its entirety. In brief, transgenic animals may include but are not limited to farm animals (e.g., pigs, goats, sheep, cows, horses, rabbits and the like) rodents (such as mice, rats and guinea pigs), and domestic pets (for example, cats and dogs). Livestock animals such as pigs, sheep, goats and cows, are particularly preferred in some embodiments.
The transgenic animal of this invention is produced by introducing into a single cell embryo an appropriate polynucleotide that encodes a human Factor IX protein of this invention in a manner such that the polynucleotide is stably integrated into the DNA of germ line cells of the mature animal, and is inherited in normal Mendelian fashion. The transgenic animal of this invention would have a phenotype of producing the FIX protein in body fluids and/or tissues. The FIX protein would be removed from these fluids and/or tissues and processed, for example for therapeutic use. (See, e.g., Clark et al. "Expression of human anti-hemophilic factor IX in the milk of transgenic sheep" Bio/Technology 7:487-492 (1989); Van Cott et al. "Haemophilic factors produced by transgenic livestock: abundance can enable alternative therapies worldwide" Haemophilia 10(4):70-77 (2004), the entire contents of which are incorporated by reference herein).
DNA molecules can be introduced into embryos by a variety of means including but not limited to microinjection, calcium phosphate mediated precipitation, liposome fusion, or retroviral infection of totipotent or pluripotent stem cells. The transformed cells can then be introduced into embryos and incorporated therein to form transgenic animals. Methods of making transgenic animals are described, for example, in
Transgenic Animal Generation and Use by L. M. Houdebine, Harwood Academic Press, 1997. Transgenic animals also can be generated using methods of nuclear transfer or cloning using embryonic or adult cell lines as described for example in Campbell et al., Nature 380:64-66 (1996) and Wilmut et al., Nature 385:810-813 (1997). Further a technique utilizing cytoplasmic injection of DNA can be used as described in U.S. Pat. No. 5,523,222.
Factor IX-producing transgenic animals can be obtained by introducing a chimeric construct comprising Factor IX-encoding sequences. Methods for obtaining transgenic animals are well-known. See, for example, Hogan et al., MANIPULATING THE
MOUSE EMBRYO, (Cold Spring Harbor Press 1986); Krimpenfort et al.,
Bio/Technology 9:88 (1991); Palmiter et al, Cell 41 :343 (1985), Kraemer et al.,
GENETIC MANIPULATION OF THE EARLY MAMMALIAN EMBRYO, (Cold Spring Harbor Laboratory Press 1985); Hammer et al., Nature 315:680 (1985); Wagner et al., U.S. Pat. No. 5,175,385; Krimpenfort et al., U.S. Pat. No. 5,175,384, Janne et al., Ann. Med. 24:273 (1992), Brem et al., Chim. Oggi. 1 1 :21 (1993), Clark et al., U.S. Pat. No. 5,476,995, all incorporated by reference herein in their entireties.
In some embodiments, c .s-acting regulatory regions may be used that are "active" in mammary tissue in that the promoters are more active in mammary tissue than in other tissues under physiological conditions where milk is synthesized. Such promoters include but are not limited to the short and long whey acidic protein (WAP), short and long α, β and K casein, a-lactalbumin and β-lactoglobulin ("BLG") promoters. Signal sequences can also be used in accordance with this invention that direct the secretion of expressed proteins into other body fluids, particularly blood and urine. Examples of such sequences include the signal peptides of secreted coagulation factors including signal peptides of Factor IX, protein C, and tissue-type plasminogen activator.
Among the useful sequences that regulate transcription, in addition to the promoters discussed above, are enhancers, splice signals, transcription termination signals, polyadenylation sites, buffering sequences, RNA processing sequences and other sequences which regulate the expression of transgenes.
Preferably, the expression system or construct includes a 3' untranslated region downstream of the nucleotide sequence encoding the desired recombinant protein. This region can increase expression of the transgene. Among the 3' untranslated regions useful in this regard are sequences that provide a poly A signal.
Suitable heterologous 3'-untranslated sequences can be derived, for example, from the SV40 small t antigen, the casein 3' untranslated region, or other 3' untranslated sequences well known in this art. Ribosome binding sites are also important in increasing the efficiency of expression of FIX. Likewise, sequences that regulate the post- translational modification of FIX are useful in the invention.
It will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present invention. Therefore, it should be clearly understood that the forms of the present invention are illustrative only and are not intended to limit the scope of the present invention.
All publications, patent applications, patents, patent publications, sequences identified by GenBank* database accession numbers and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.
The invention is defined by the following claims, with equivalents of the claims to be included therein.
Exemplary nucleotide sequence encoding the amino acid sequence of FIX23 (SEQ ID NO:4):
tataacagcggcaaactggaagaatttgtgcagggcaacctggaacgcgaatgcatggaa
gaaaaatgcagctttgaagaagcgcgcgaagtgtttgaaaacaccgaacgcaccaccgaa
ttttggaaacagtatgtggatggcgatcagtgcgaaagcaacccgtgcctgaacggcggc
agctgcaaagatgatattaacagctatgaatgctggtgcccgtttggctttgaaggcaaa
aactgcgaactggatgtgacctgcaacattaaaaacggccgctgcgaacagttttgcaaa
aacagcgcggataacaaagtggtgtgcagctgcaccgaaggctatcgcctggcggaaaac
cagaaaagctgcgaaccggcggtgccgtttccgtgcggccgcgtgagcgtgagccagacc
agcaaactgacccgcgcggaaaccgtgtttccggatgtggattatgtgaacagcaccgaa
gcggaaggcagcccgggcagcggcgcgaacgcgaccggcccgagcggcgaaggcgcgagc
gcgccgagcgaaaacgcgaccggcccgggcaccagcggcggcagcccggcgaacagcacc
ggcggcagcccggcggaaggcagcccgggcagcgaaggcaccattctggataacattacc
cagagcacccagagctttaacgattttacccgcgtggtgggcggcgaagatgcgaaaccg
ggccagtttccgtggcaggtggtgctgaacggcaaagtggatgcgttttgcggcggcagc
attgtgaacgaaaaatggattgtgaccgcggcgcattgcgtggaaaccggcgtgaaaatt
accgtggtggcgggcgaacataacattgaagaaaccgaacataccgaacagaaacgcaac
gtgattcgcattattccgcatcataactataacgcggcgattaacaaatataaccatgat
attgcgctgctggaactggatgaaccgctggtgctgaacagctatgtgaccccgatttgc
attgcggataaagaatataccaacatttttctgaaatttggcagcggctatgtgagcggc
tggggccgcgtgtttcataaaggccgcagcgcgctggtgctgcagtatctgcgcgtgccg
ctggtggatcgcgcgacctgcctgcgcagcaccaaatttaccatttataacaacatgttt
tgcgcgggctttcatgaaggcggccgcgatagctgccagggcgatagcggcggcccgcat
gtgaccgaagtggaaggcaccagctttctgaccggcattattagctggggcgaagaatgc
gcgatgaaaggcaaatatggcatttataccaaagtgagccgctatgtgaactggattaaa gaaaaaaccaaactgacc
Exemplary nucleotide sequence encoding the amino acid sequence of FIX24 (SEQ ID NO:5):
cgcgaagtggaacgcagcgaaacccgcgcgaacagcctggcgaccattaactttagcccg
ccgtttgcgcatatggcgaacaccgcggcgagcgaaagcgaacaggaaaactgcgaattt
atgagcaccctgattaaagaactgtattgcgataacagcaccgcgaccgcggcgtgcgcg
ggctgcggcggctgcgcggcggcgtgcaccggcggcgcggcgggcgcggcgaccaccacc
ggcaccggctgcgcgggcggcggctgcgcggcgtgctgcaccggcggcgcggcgtgcggc
tgcggcgcggcgaccggctgcgcgaccggcggcgcggcgggcgcggcggcggcggcgacc
ggctgcgcgggctgcaccaccaccggcgcggcgggcgcggcgggctgcggctgcggctgc
ggcgcggcgggcaccggcaccaccaccggcgcggcggcggcgtgcgcgtgctgcggcgcg
gcgtgcggctgcgcgtgctgcgcgtgctgcggcgcggcgaccaccaccaccggcggcgcg
gcggcgtgcgcgggcaccgcgaccggcaccggcggcgcgaccggcggctgcggcgcgacc
tgcgcgggcaccggctgcggcgcggcggcgggctgcgcggcgtgctgctgcggcaccggc
tgctgcaccggcgcggcgtgcggcggctgcggcggctgcgcgggctgcaccggctgcgcg
gcggcgggcgcgaccggcgcgaccgcgaccaccgcggcgtgcgcgggctgcaccgcgacc
ggcgcggcgaccggctgcaccggcggcaccggctgctgctgcggcaccaccaccggcggc
tgcaccaccaccggcgcggcgggcggctgcgcggcggcggcggcgtgcaccggctgcggc
gcggcgtgcaccggcggcgcgaccggcaccggcgcgtgctgcaccggctgcgcggcgtgc
gcgaccaccgcggcggcggcggcgtgcggcggctgctgcggctgcaccggctgcggcgcg
gcgtgcgcgggcaccaccaccaccggctgcgcggcggcggcggcgtgcgcgggctgcggc
tgcggcggcgcgaccgcggcgtgcgcggcggcgggcaccggcggcaccggcaccggctgc
gcgggctgcaccggctgcgcgtgctgcggcgcggcgggcggctgcaccgcgacctgcggc
tgctgcaccggcggctgcggcggcgcggcggcggcgtgctgcgcgggcgcggcggcggcg
ggctgcaccggctgcggcgcggcgtgctgcggcggctgcggcggcaccggctgctgcggc
accaccacctgctgcggcaccggctgcggcggctgctgcggctgcggcaccggcgcgggc
tgcggcaccggcgcgggctgctgcgcgggcgcgtgctgcgcgggctgcgcggcggcgtgc
accggcgcgtgctgctgcggctgcggctgcggcggcgcggcggcgtgctgcggcaccggc
accaccacctgctgcggcggcgcgaccggcaccggcggcgcgaccaccgcgaccggcacc
ggcgcggcgtgcgcgggctgcgcgtgctgcggcgcggcgggctgcggcggcgcggcgggc
ggctgcgcgggctgctgctgcggcggcggctgcgcgggctgcggcggctgcggctgcggc
gcggcgtgcggctgcggcgcgtgctgcggcggctgctgctgcggcgcgggctgcggcggc
tgcggcgcggcgggcggctgcggctgcggcgcgggctgcggctgcggctgctgcggcgcg
ggctgcggcgcggcggcggcgtgcggctgcggcgcgtgctgcggcggctgctgctgcggc
ggcggctgcgcgtgctgcgcgggctgcggcggctgcggcggctgcgcgggctgctgctgc ggcggctgcggcgcggcgtgcgcgggctgcgcgtgctgcggcggctgcggcggctgcgcg ggctgctgctgcggcggctgcggcggcgcggcgggcggctgcgcgggctgctgctgcggc ggcggctgcgcgggctgcggcgcggcgggcggctgcgcgtgctgcgcgaccacctgcacc ggcggcgcgaccgcggcgtgcgcgaccaccgcgtgctgctgcgcgggcgcgggctgcgcg tgctgctgcgcgggcgcgggctgcaccaccaccgcggcgtgcggcgcgaccaccaccacc gcgtgctgctgcggctgcggcaccggcggcaccggcggcggctgcggcggctgcggcgcg gcgggcgcgaccggctgcggcgcggcggcgtgctgcggcggcggctgctgcgcgggcacc accacctgctgcggcaccggcggctgcgcgggcggcaccggcggcaccggctgcaccggc gcggcgtgcggcggctgcgcggcggcgggcaccggcggcgcgaccggctgcggcaccacc accaccggctgcggcggctgcggcggctgcgcgggctgcgcgaccaccggcaccggcgcg gcgtgcggcgcggcggcggcggcgaccggcggcgcgaccaccggcaccggcgcgtgctgc ggctgcggcggctgcggctgcgcgaccaccggctgcggcaccggcggcgcggcggcgtgc tgcggcggctgcggcaccggcgcggcggcggcgaccaccgcgtgctgcggcaccggcggc accggcggctgcggcggcggctgcggcgcggcgtgcgcgaccgcggcgtgcgcgaccacc ggcgcggcgggcgcggcggcgtgctgcggcgcggcgtgcgcgaccgcgtgctgcggcgcg gcgtgcgcgggcgcggcggcgtgcggctgcgcggcgtgcggcaccggcgcgaccacctgc ggctgcgcgaccaccgcgaccacctgctgcggctgcgcgacctgcgcgaccgcggcgtgc accgcgaccgcggcgtgcggctgcggcggctgcggcgcgaccaccgcggcgtgcgcggcg gcgaccgcgaccgcggcgtgctgcgcgaccggcgcgaccgcgaccaccggctgcggctgc accggctgcaccggcggcgcggcgtgcaccggcggcgcgaccggcgcggcgtgctgcggc tgcaccggcggcaccggctgcaccggcgcggcgtgcgcgggctgcaccgcgaccggcacc ggcgcgtgctgctgctgcggcgcgaccaccaccggctgcgcgaccaccggctgcggcggc gcgaccgcggcggcgggcgcggcgaccgcgaccgcgtgctgcgcggcgtgcgcgaccacc accaccacctgcaccggcgcggcggcgaccaccaccggcggctgcgcgggctgcggcggc tgcaccgcgaccggcaccggcgcgggctgcggcggctgcaccggcggcggcggctgctgc ggctgcggcaccggcaccaccacctgcgcgaccgcggcggcgggcggctgctgcggctgc gcgggctgcggctgcggctgcaccggcggcaccggctgcaccggctgcgcgggcaccgcg acctgcaccggctgcggctgcggcaccggctgctgcggctgcaccggcggcaccggcggc gcgacctgcggctgcggctgcggcgcgtgctgcaccggctgctgcaccggctgcggctgc gcgggctgcgcgtgctgcgcggcggcgaccaccaccgcgtgctgcgcgaccaccaccgcg accgcggcgtgcgcggcgtgcgcgaccggcaccaccaccaccggctgcggctgcggcggc ggctgcaccaccacctgcgcgaccggcgcggcgggcggctgcggcggctgctgcggctgc
ggcgcgaccgcgggctgcaccggctgctgcgcgggcggcggctgcggcgcgaccgcgggc tgcggcggctgcggcggctgctgctgcggctgcgcgaccggcaccggcgcgtgctgcggc
gcggcgggcaccggcggcgcggcgggcggctgcgcgtgctgcgcgggctgcaccaccacc
tgcaccggcgcgtgctgcggcggctgcgcgaccaccgcgaccaccgcgggctgcaccggc
ggcggcggctgcggcgcggcgggcgcggcgaccggctgcggctgcggcgcgaccggcgcg
gcggcgggcggctgcgcggcggcgaccgcgaccggcggctgcgcgaccaccaccgcgacc
gcgtgctgcgcggcggcgggcaccggcgcgggctgctgcggctgcaccgcgaccggcacc
ggcgcggcgtgcaccggcggcgcgaccaccgcggcggcgggcgcggcggcggcggcggcg
tgctgcgcggcggcgtgcaccggcgcgtgctgc
Factor IX amino acid sequence without propeptide sequence (SEQ ID NO: l):
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFW QYVDGDQCESN PCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVV CSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAEG SPGSGSANATGPSGEGS APSEGNATGPGTSGGSPANSTGGSPAEGSPGSEILDNITQ STQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNE WIVTAAHCVE TGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNATINKYNHDIALLELDEPLVL NSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRA TC LRSTKFTIYN MFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAM KG Y GI YTK V S R Y VN WIKEKTKLT
Factor IX sequence with propeptide sequence (SEQ ID NO: 6):
MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRRRRYNSGKLEEFV QGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKD DINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLA ENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAEGSPGSGSANAT GPSGEGSAPSEGNATGPGTSGGSPANSTGGSPAEGSPGSEILDNITQSTQSFNDFTR VVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVA GEHNIEETEHTEQKRNVIRIIPHHNYNATINKYNHDIALLELDEPLVLNSYVTPICIA DKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRA rCLRSTKFTIYN NMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTK VSRYVNWIKEKTKLT
Table 1
Amino Acids One-Letter Symbol Common Abbreviation
Alanine A Ala
Arginine R Arg
Asparagine N Asn
Aspartic acid D Asp
Cysteine C Cys
Glutamine Q Gin
Glutamic acid E Glu
Glycine G Gly llistidine H His
Isoleucine I lie
Leucine L Leu
Lysine K Lys
Phenylalanine F Phe
Proline P Pro
Serine s Ser
Threonine T Thr
Tryptophan w Trp
Tyrosine Y Tyr
Valine V Val
Claims
1. An isolated Factor IX (FIX) protein comprising the amino acid sequence of SEQ ID NO:2 (FIX23):
YNSGKLEEFVQGNLERECMEE CSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYE C CPFGFEGKNCELDVTCNIK GRCEQFC NSADNKWCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQT SKLTRAETVFPDVDYVNSTEAEGSPGSGA ATGPSGEGASAPSENATGPGTSGGSPANSTGGSPAEGSPG SEGTILDNITQSTQSFNDFTRWGGEDAKPGQFP QWLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKI TWAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIF LKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPH VTEVEGTSFLTGIISWGEECAMKGKYGIYT VSRYVN IKEKTKLT
2. An isolated Factor IX protein comprising the amino acid sequence of SEQ ID NO: 3 (FIX24):
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEF KQYVDGDQCESNPCLNGGSCKDDINSYE CWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKWCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQT SKLTRAETVFPDVDYVNSTEAEGSPGSGANATGPSGEGASAPSENATGPGTSGGSPANSTGGSPAEGSPG SEGTILDNITQSTQSFNDFTRWGGEDA PGQFPWQWLNGKVDAFCGGSIVNEKWIVTAAHCVETGV I TWAGEHNIEETEHTEQKRNVIRIIPHHNYNA lNKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIF LKFGSGYVSGWGRVFH GRSALVLQYLRVPLYDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPH VTEVEGTSFLTGIISWGEECAM GKYGIYTKVSRYV WIKEKTKLT
3. The Factor IX protein of claim 1 or claim 2, wherein the lysine (Lys) at position 5 in the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 is substituted with an arginine (Arg).
4. The Factor IX protein of any preceding claim, wherein the arginine (Arg) at position 389 is substituted with any other amino acid.
5. The Factor IX protein of claim 4, wherein the substituted amino acid at position 389 is alanine or leucine.
6. The Factor IX protein of any preceding claim, wherein the valine (Val) at position 10 in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:3 is substituted with a leucine, isoleucine, methionine or phenylalanine, histidine or threonine.
7. The Factor IX protein of any preceding claim, wherein the phenylalanine (Phe) at position 9 of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 is substituted with any other amino acid.
8. The Factor IX protein of any preceding claim, wherein the glutamine (Gin) at position 1 1 of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 is substituted with asparagine, lysine or arginine.
9. An isolated nucleic acid molecule encoding the FIX protein of any of claims 1 -
8.
10. A vector comprising the nucleic acid molecule of claim 9.
1 1. Λ transformed cell comprising the nucleic acid molecule of claim 9 and/or the vector of claim 10.
12. A transgenic animal comprising the FIX protein of any of claims 1-8, the nucleic acid molecule of claim 9, the vector o claim 10 and/or the transformed cell of claim 1 1.
13. The FIX protein of any of claims 1 -8, which is a human FIX protein.
14. A method of treating a bleeding disorder, comprising administering to a subject in need thereof an effective amount of the Factor IX protein of any of claims 1 -8, the nucleic acid molecule of claim 9, the vector of claim 10 and/or the cell of claim 11.
15. The method of claim 14, wherein the bleeding disorder is selected from the group consisting of a FIX deficiency, hemophilia B and Christmas disease.
16. A method of increasing the bioavailablity of a Factor IX protein in a subject comprising administering to the subject an effective amount of the Factor IX protein of any of claims 1-8, the nucleic acid molecule of claim 9, the vector of claim 10 and/or the cell of claim 11.
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US10413598B2 (en) | 2014-11-12 | 2019-09-17 | Ucl Business Plc | Factor IX gene therapy |
US10842885B2 (en) | 2018-08-20 | 2020-11-24 | Ucl Business Ltd | Factor IX encoding nucleotides |
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WO2014081831A1 (en) * | 2012-11-20 | 2014-05-30 | The University Of North Carolina At Chapel Hill | Methods and compositions for modified factor ix proteins |
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WO2014081831A1 (en) * | 2012-11-20 | 2014-05-30 | The University Of North Carolina At Chapel Hill | Methods and compositions for modified factor ix proteins |
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Title |
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CHEN SHI-HAN ET AL.: "Three point mutations in the factor IX genes of five hemophilia B patients. Identification strategy using localization by altered epitopes in their hemophilic proteins", J. CLIN. INVEST., vol. 84, 1989, pages 113 - 118 * |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US10413598B2 (en) | 2014-11-12 | 2019-09-17 | Ucl Business Plc | Factor IX gene therapy |
US11344608B2 (en) | 2014-11-12 | 2022-05-31 | Ucl Business Ltd | Factor IX gene therapy |
US10842885B2 (en) | 2018-08-20 | 2020-11-24 | Ucl Business Ltd | Factor IX encoding nucleotides |
US11517631B2 (en) | 2018-08-20 | 2022-12-06 | Ucl Business Ltd | Factor IX encoding nucleotides |
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