Use of factor XIIa for treatment of hemophilia A and B and prevention of bleeding

Abstract

The present invention is directed to a Factor XIIa-coated solid support comprising a recombinant Factor XIIa polypeptide or a Factor XIIa-expressing cell comprising an expression vector construct containing a gene encoding a Factor XII polypeptide. The invention is also directed to methods of treating a subject with a coagulation protein. Additionally, the invention is directed to methods of treating or preventing bleeding. The present invention is also directed towards a kit.

Description

FIELD OF THE INVENTION

[0002] The invention relates to the use of Factor XII polypeptides, whether recombinant or naturally occurring, in truncated or mutated forms, for conversion of inactive Factor VII to its active form in the treatment of Hemophilia A and B. The invention also relates to the use of the same in the prevention of bleeding in a subject and a kit incorporating the same.

BACKGROUND OF THE INVENTION

[0003] Hemophilia A is a coagulation disorder caused by a deficiency in Factor VIII. In particular, the defect is located in Factor VIII:C (which is used interchangeably with the term “Factor VIII” herein) (Bloom, 1991; Rosendaal et al, 1991; Thompson, 1991; Handin et al, 1994). Factor VIII:C is the moiety of Factor VIII that induces coagulation. Hemophilia B is a coagulation disorder caused by a deficiency of Factor IX. Most U.S. and European hemophiliacs are undertreated due to the extraordinarily high cost of recombinant Factor VIII:C, and also due to the high incidence of “inhibitors” or auto-antibodies to the Factor VIII:C moiety in patients (Aledort, 1998). Not only do these antibodies inhibit Factor VIII activity, but they also have a catalytic destructive effect on themselves (Lacroix-Desmazes, et al, 1999). Between 10% (Yee, et al, 1999) and 25% (Prescott et al, 1997) of hemophiliacs have such inhibitors, and these hemophiliacs cost 10-fold more than the average hemophiliac to treat because more frequent hospitalizations are required (Goudemand, 1998). Attempts to induce immune tolerance to Factor VIII:C are not often successful. Initially, such antibody development was not as frequently reported for recombinant Factor VIII:C as for plasma-derived Factor VIII:C (see Seremetis et al, 1999). However, Prescott et al (1997) from the American Red Cross report that there is no significant difference between the two classes of patients. Given financial limitations, priorities have been established for access to therapy, in which HIV-positive and/or hepatitis C-positive hemophiliacs are less likely to be treated optimally (Giangrande, 1997).

[0004] Hemophilia A is conventionally treated by replacement with either donor plasma-derived or recombinant Factor VIII:C (Bloom, 1992; Sultan and Algiman, 1992; Nilsson, 1992; Hedner and Glazer, 1992; Kasper, 1991). Similar approaches are used for the less common hemophilia B. The most common and debilitating consequences of hemophilia A are joint destruction and intracranial bleeds. The frequency of these catastrophic events can be reduced by prophylactic administration of Factor VIII:C. However, as stated previously, recombinant Factor VIII:C is extraordinarily expensive, and it is usually given whenever a bleeding episode occurs, rather than continuously on a true prophylactic basis. Indeed, a therapeutic level of recombinant Factor VIII:C is said to have been achieved when coagulation has returned to 5% of “normal.” A 1% level is also said by some to be therapeutic. Aledort (1996) has concluded that “financial underwriting of care for this chronic disease remains the major uncertainty.” Two years later, Aledort (1998) further concluded “programs for prophylaxis or immune tolerance induction are impossible for most patients.”

[0005] The Hemophilia Foundation supports the concept that continuous prophylactic use is best, but agrees that the current economics makes this difficult for all but the most advantaged (Skolnick, 1994). Yet, even the data on true continuous prophylaxis with the best reagents available for the so-called “most advantaged” are far from encouraging. For example, a case has been made that continuous prophylaxis with recombinant Factor VIII:C yields a more favorable outcome than the treatment that is rendered whenever a bleeding episode occurs (Miners et al, 1998). However, the term “favorable” in the latter case means that of 179 patients studied in London, the number of bleeds per year only decreased from a mean of 23.5 (range=1-107) to a mean of 14 (range=0-45). As a consequence of the expense of recombinant Factor VIII:C, governments and other health-related organizations have argued against its prophylactic use on the basis of cost vs. benefit analysis (Smith et al, 1996; Miners et al, 1997; Green and Akehurst, 1998; Szucs et al, 1998).

[0006] It is known that the administration of “activated prothrombin complex” (APC, or FEIBAd, ImmunoAG, Vienna, Austria) (Abildgaard et al, 1988; Kelly and Penner, 1976; Kurczynski and Penner, 1974; Lusher et al, 1980) can be used to achieve coagulation in subjects with hemophilia. The improvement in coagulation state is believed to be due to high levels of Factor VIIa (Giles, 1987; Stewart et al, 1998). In 1983 purified Factor VIIa was used to treat two patients with Hemophilia A (Hedner and Kisiel, 1983; Hedner et al, 1989). Extensive clinical studies of recombinant Factor VIIa, manufactured by NOVO Nordisk (Bagsvaerd, Denmark), have verified the therapeutic value of this reagent for both Hemophilia A (Bell et al, 1993; Hedner, 1991; Schulman, 1992) and Hemophilia B (Schmidt et al, 1991; Brinkhous et al, 1989; Macik et al, 1993; Telgt et al, 1989). The mechanism by which Factor VIIa bypasses the Factor VIII:C and Factor IX steps in coagulation are not known. It was hypothesized that the Factor VIIa bypass activity occurred by Factor VIIa activation of the extrinsic pathway by complexing with Tissue Factor (Edgington et al, 1991). However, while this reaction certainly occurs, it turns out not to be the basis of the bypass reaction.

[0007] It is also known that an additional advantage of Factor VIIa therapy is that many hemophiliacs have high levels of anti-Factor VIII:C antibodies, which act to inhibit wild type or recombinant Factor VIII given therapeutically. The bypass activity provided by Factor VIIa is completely unaffected by these anti-Factor VIII:C antibodies. However, despite the attractiveness of the Factor VIIa approach to therapy of Hemophilia A, the use of recombinant Factor VIIa has been clinically compromised. First, cost considerations have made it available only to the most financially well off. Secondly, and perhaps more importantly, recombinant Factor VIIa has an extremely short half life in vivo (ca. 2 hours: Thomsen, et al, 1993; Hedner et al, 1993). The consequence is that in one of many reported recent examples of its use, 10 of 12 Hemophilia A patients survived catastrophic intracranial hemorrhages by receiving an average of 97 injections each of recombinant Factor VIIa (“NovoSeven”) over a 15 day period (Arkin et al, 1998). Factor VIIa may offer advantages to the hemophiliac patient. However, the present outstanding need for a more efficacious and less costly form of recombinant factor VIIa mitigates against its practical use in the widespread treatment of hemophilia.

[0008] The most commonly used source of Factor VIII:C is from donor plasma. The plasma is collected and the Factor VIII:C is purified by affinity chromatography on an anti-Factor VIII:C column. The product is also tested for viruses such as HIV, hepatitis, parvovirus B19 and others, and the viruses, real or potential, then inactivated by a combination of heat, detergent and solvent treatments. As a byproduct of these treatments the Factor VIII:C activity is substantially decreased. Nonetheless, the probability of infection due to transfusion with the final product still can not be reduced to zero (Altman, 1996).

[0009] There is a large amount of Factor VII present in human plasma, and this amount of Factor VII can be used as a continuous source of activated Factor VII, called Factor VIIa, to treat Hemophilia A (and B as well). However, until recently no method was available to catalyze the conversion of Factor VII into Factor VIIa in vivo. It was known that Factor VII could be converted in vitro into Factor VIIa by using either activated Factor X, called Factor Xa (Giles et al, 19880; Osterud, 1990) or activated Factor XII, called Factor XIIa (Hageman Factor, Factor XIIa; Kisiel et al, 1977; Brose and Majerus, 1980). The disadvantage of using Factor Xa is that phospholipids such as phosphatidylserine are needed as cofactors for the reaction. However, Factor XIIa is able to produce Factor VIIa from Factor VII without phospholipids. The resulting Factor VIIa then bypasses the hemophilia A or B defect and allows coagulation to commence (Ton-That et al, 2000).

[0010] Studies from the inventor's laboratory have validated these findings in vitro. The inventors have also extended the study with Factor XIIa into in vivo conditions (Ton-That et al, 2000). In the latter studies, bypass activity around a human anti-Factor VIII:C antibody were achieved for up to one month. It is noted that such an advance or discovery has not been disclosed in the art.

SUMMARY OF THE INVENTION

[0011] The present invention is based on the fact that hemophilia patients have normal levels of unactivated Factor VII, and therefore the patient's own blood contains a potential source of continuous and endogenous Factor VIIa. In order to access the patient's own Factor VII and convert it to VIIa in an on-line manner, the present invention places an exogenous activator of Factor VII, in or near the blood stream, where it can activate Factor VII to VIIa, but in a manner which limits formation of thrombin.

[0012] Therefore, an object of the invention is a recombinant Factor XIIa-coated bead.

[0013] A further object of the invention is a Factor XIIa-expressing cell generated by transfection with an expression vector construct containing a gene encoding a Factor XIIa protein, or a truncated or mutant form thereof with Factor VII activating activity.

[0014] Another object of the invention is an implant containing a Factor XIIa-expressing cell which is capable of producing Factor VIIa from endogenous Factor VII.

[0015] Another object of the invention is a method of treatment comprising administering a recombinant Factor XII polypeptide to a recipient in need of such treatment. This method comprises administering (1) a recombinant Factor XIIa-coated bead, (2) an implant containing a recombinant Factor XIIa-coated bead, (3) a cell expressing recombinant Factor XIIa, (4) an implant containing a cell expressing recombinant Factor XIIa, (5) a naked DNA vector containing a gene encoding a Factor XIIa polypeptide or a truncated or mutated form thereof, and/or (5) a liposome encapsulated DNA vector containing a gene encoding a Factor XIIa polypeptide or a truncated or mutated form thereof.

[0016] Another object of the invention is a method for treating a subject with a congenital deficiency for Factor XIIa comprising administering an expression vector containing a gene encoding a Factor XIIa polypeptide, or a truncated or mutated form thereof.

[0017] Another object of the invention is a method of treating bleeding in a subject with an antibleeding effective amount of a coagulation agent that comprises Factor XIIa polypeptide.

[0018] Another object of the invention is a kit comprising an applicator and Factor XIIa polypeptide.

[0019] Further objects and advantages of the present invention will be clear from the description as follows.

BRIEF DESCRIPTION OF THE FIGURES

[0020] FIG. 1. Ex-Vivo Gene Therapy. The Factor XIIa gene, or a modified form of this gene (1) is prepared either as the naked DNA, the recombinant viral form or as a lipid adduct (2). One of these forms is transferred or transfected into a recipient cell, and the cell isolated in a form expressing recombinant Factor XIIa (3). The cell is then injected into a recipient organism, where it expresses Factor XIIa in the circulation.

[0021] FIG. 2. Ex-Vivo Gene Therapy with Encapsulated Recombinant Cell. The Factor XIIa gene, or a modified form of this gene (1) is prepared either as the naked DNA, the recombinant viral form or as a lipid adduct (2). One of these forms is transferred or transfected into a cell, where it expresses Factor XIIa (3). The cell is placed in a porous chamber (4). The chamber is implanted in a recipient where Factor XIIa is expressed in the circulation (5).

[0022] FIG. 3. In-Vivo Gene Therapy. The Factor XIIa gene, or a modified form of this gene (1) is prepared either as the naked DNA, the recombinant viral form or as a lipid adduct (2). One of these forms is transferred or transfected into a recipient organism, where it expresses Factor XIIa in the circulation.

[0023] FIG. 4. Full length or modified recombinant Factor XIIa as a source of immobilized Factor XIIa inside an implant. Factor XIIa or modified Factor XIIa gene (1) is prepared for transfer to a cell for expression (2). The gene is transferred to an expression system (3), and the recombinant Factor XIIa species is assembled into a porous chamber (4). A porous chamber containing recombinant Factor XIIa is implanted into a recipient (5).

[0024] FIG. 5. A chart detailing bypass activity in rhesus monkeys implanted peritoneally with a Factor XIIa chamber.

DETAILED DESCRIPTION OF THE INVENTION

[0025] In the present invention, the activator of Factor VII is a recombinant coagulation protein, preferably recombinant Factor XIIa. The ability of Factor XIIa to activate Factor VII and thereby convert it into VIIa is known and is described in a textbook of hematology (Hemostasis and Thrombosis, Robert W. Colman, Jack Hirsh, Victor J. Marder, and Edwin W. Salzman, eds. J.B. Lippincott Co. Philadelphia and Toronto, 4th ed., page 9, 2000).

[0026] Human Factor XII was originally cloned and sequenced by Cool et al (1985) and by Que et al (1986) (SEQ ID No. 1). The protein comprised an N-terminal protease domain, a proline-rich hinge region, and five C-terminal regulatory domains (SEQ ID No. 2). Citarella et al (1992) reported that a full length recombinant Factor XII species of 80kDa could be cloned into a vaccinia expression vector and expressed in HepG2 cells. This full-length species was similar to native Factor XII in all ways tested, and, upon activation, promoted coagulation with high efficiency.

[0027] Citarella et al (1992) also prepared a truncated Factor XIIa containing only the protease region and a portion of the proline-rich hinge domain (“FXII.lpc”; amino acids residues 1-196 of SEQ ID No. 2). This 32kDa species was described as unexpectedly displaying marked Factor XIIa-like protease activity, without need for preliminary activation.

[0028] The present invention concerns vector or plasmid constructs containing a gene encoding a full length polypeptide for Factor XIIa, or a truncated or a mutated form thereof. Most preferably, the protease activity is retained for a recombinant Factor XIIa polypeptide. A recombinant Factor XIIa polypeptide can range in size from the 80 kDa full length form of the protein (SEQ ID No. 2) to the 32 kDa form described by Citarella. Preferably, the Factor XIIa polypeptide comprises a sequence of amino acid residues 1 to 615, 1 to 515, 1 to 415, 1 to 315 or 1 to 215 of SEQ ID No. 2, and more preferably, a sequence of amino acid residues of about 1 to 196 of SEQ ID No. 2. The term “active fragment” refers to at least one of these sequences of amino acid residues.

[0029] The term “gene” refers to a nucleic acid or a polynucleotide, which encodes a peptide, prepeptide, protein or marker, or to a vector or plasmid containing such a nucleic acid or polynucleotide.

[0030] A “mutant” or “modified” gene or peptide refers to a gene having a sequence in that one or more bases or residues are deleted, substituted or added at any position therein, including either terminus.

[0031] An object of this invention is the delivery of Factor XIIa-related sequences as in vivo or ex vivo gene therapies for treatment of Hemophilia A and B.

[0032] The drawback of much gene therapy today is delivery of a therapeutic gene to a cell in vitro, more particularly to a cell in a living organism, and the level and duration of expression of the therapeutic gene (Kaufman, 1999). Vectors that have been used in gene therapy applications related to hemophilias include adenovirus, retrovirus, adeno-associated virus (AAV), and naked DNA transfer (Pipe and Kaufman, 2000).

[0033] The gene that is to be introduced by the vector constructs can be in the form of a nucleic acid containing the Factor XIIa gene or a fragment thereof, or a mutant thereof which, if necessary, is provided with the appropriate regulatory regions such as promoters.

[0034] Preferably, the DNA construct contains a promoter to facilitate expression of the DNA of interest within a host cell. Preferably the promoter is a strong, eukaryotic promoter. Exemplary eukaryotic promoters for facilitating transcription in a eukaryotic cell include promoters from cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), and adenovirus. More specifically, exemplary promoters include the promoter from the immediate early gene of human CMV (Boshart et al., Cell 41:521-530, 1985) and the promoter from the long terminal repeat (LTR) of RSV (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777-6781, 1982). Of these two promoters, the CMV promoter is preferred as it provides for higher levels of expression than the RSV promoter.

[0035] For eukaryotic expression (e.g., in an endothelial cell), the construct preferably comprises at least a eukaryotic promoter operably linked to a DNA of interest, which is in turn operably linked to a polyadenylation sequence. The polyadenylation signal sequence may be selected from any of a variety of polyadenylation signal sequences known in the art. Preferably, the polyadenylation signal sequence is the SV40 late polyadenylation signal sequence. The construct may also include sequences in addition to promoters which enhance expression in host cells (e.g., enhancer sequences, introns). For example, the construct can include one or more introns, which can increase levels of expression of the DNA of interest, particularly where the DNA of interest is a cDNA (e.g., contains no introns of the naturally-occurring sequence). Any of a variety of introns known in the art may be used. Preferably, the intron is the human beta-globin intron and inserted in the construct at a position 5′ to the DNA of interest.

[0036] Other components such as a marker (e.g., an antibiotic resistance gene (such as an ampicillin resistance gene) or beta-galactosidase) to aid in selection of cells containing and/or expressing the construct (e.g., during the process of vector construction), an origin of replication for stable replication of the construct in a bacterial cell (preferably, a high copy number origin of replication), a nuclear localization signal, or other elements which facilitate production of the DNA construct, the protein encoded thereby, or both.

[0037] Techniques for production of nucleic acid constructs for expression of exogenous DNA or RNA sequences in a host are known in the art (see, for example, Kormal et al., Proc. Natl. Acad. Sci. USA, 84:2150-2154, 1987; Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; each of which is hereby incorporated by reference in its entirety.

[0038] Another object of the invention is a transfected host cell containing a stably integrated vector construct containing a gene encoding a full length polypeptide for Factor XIIa, or a truncated or a mutated form thereof. In this case, the source of Factor XIIa is a host cell genetically engineered to express a proteolytically active form of Factor XIIa.

[0039] Examples of suitable host cells include eukaryotic cells, preferably mammalian cells, more preferably human cells, and most preferably cells obtained from the same subject or individual. Specific kinds of eukaryotic cells include but are not limited to blood cells, hepatocytes, fibroblasts, and endothelial cells.

[0040] The term “mammal” refers to a human being or an animal.

[0041] A non-viral vector construct can be transfected under in vitro conditions into a host cell by any one of the following methods including but not limited to calcium phosphate or DEAE-dextran precipitation, microinjection, electroporation, cationic liposomes, pH sensitive or negatively-charged liposomes, or ballistic DNA injection.

[0042] The activity for an Factor XIIa expressing cell can be measured by any clinically recognized assay, more preferably a COATEST assay.

[0043] An ex-vivo transfected host cell expressing a proteolytically active form of Factor XIIa can be administered to a recipient by intravenous, intraarterial, intraportal, intracranial, intrapleural, intraperitoneal or local introduction. The concentration of the recombinant protein expressed by the transfected cells is an effective amount whereby any inactive Factor VII contacting Factor XIIa is protealyzed into active Factor VIIa.

[0044] Cells expressing a recombinant protein, preferably Factor XIIa, which have been attached to a solid support, may also be placed in porous chambers as described in U.S. Pat. No. 5,908,399 and U.S. Pat. No. 6,174,299 (both of which are incorporated by reference in their entirety), and implanted in hemophiliac patients. The porous chamber comprises a permeable membrane having one or more walls defining a hollow chamber and a plurality of holes extending through the walls of the membrane and permitting fluid to enter and exit the chamber of the membrane. Each of the holes is sized so that it is large enough to permit inactive Factor VII to enter the chamber of the membrane and activated Factor VIIa to exit the chamber of the membrane, but small enough to prevent fibrinogen from entering the chamber of the membrane.

[0045] The porous chamber should possess a region that allows the chamber to be affixed to the abdominal wall, also having a design such as a diaphragm that would allow penetration of a syringe for removal or addition of materials therefrom, including but not limited to replacement of a solid support, a cell-coated support or a coagulation protein-coated support.

[0046] The solid support may comprise any solid matrix or particle such as a microcarrier bead, more preferably, a CYTODEX™ bead.

[0047] The permeable membrane may comprise any container having the property of being biologically compatible, and having holes being sized to permit inactive Factor VII to enter the chamber of the membrane and activated Factor VIIa to exit the chamber. Examples of membranes include but are not limited to a dialysis bag or a dialysis membrane, a geometric filter comprising polymethacrylate or polycarbonate, or a statistical hole filter comprising cellulose acetate. Preferably, the size of each of the holes in the membrane is less than about 100 angstroms, and most preferably 40-50 angstroms, which is suitable for a protein of about 50,000 daltons.

[0048] In the methods of the present invention, the membrane is implanted so that it is in contact with the subject's blood stream. In this regard, the membrane is filled with a solid support being bound to a recombinant coagulation protein, more preferably a Factor VII activator, or to a cell expressing a Factor VII activator, more preferably, Factor XIIa, sterilized and implanted using conventional medical equipment and procedures into the femoral vein, or intraperitoneally. The membrane may be removed by similar surgical procedures. Alternatively, a bypass device may be installed into the subject, and circulation allowed through an arteriovenous bypass (extracorporeal therapy).

[0049] The concentration of the recombinant protein expressed by the attached cells is an effective amount whereby any inactive Factor VII passing through the chamber is protealyzed into active Factor VIIa.

[0050] Purified, recombinant Factor XIIa-related sequences including the full-length protein, or a truncated or mutated form thereof, can be coupled to a solid support, which is then placed in the porous chamber or administered directly to a recipient.

[0051] The activity for a Factor XIIa coated-bead can be measured by any clinically recognized assay, more preferably a COATEST assay.

[0052] A solid support may comprise any material widely used for similar purposes in protein chemistry including but not limited to a 4% cross-linked beaded agarose, CNBr-activated Sepharose 4B, beaded acrylamide and porous glass beads.

[0053] A recombinant Factor XIIa protein can be purified by chemistries involving protein separation including fraction or immunoadsorption.

[0054] In a preferred embodiment, a purified coagulation protein, preferably a purified recombinant Factor XIIa, is coupled to beads by mixing the protein with 750 μl of Affigel-10 beads (Pierce) in desalting buffer (0.1 M HEPES/NaOH buffer, pH7.4 and 0.3 M NaCl) adjusted to 80 mM CaCl2 for 12 hr at 4° C. The beads are centrifuged at 12,000 g for 10 sec and washed three times in protein-free, calcium-free desalting medium. The unreacted sites are blocked by resuspending the beads for 24 hr at 4° C. in 1M glycine in desalting buffer followed by washing in phosphate buffered saline.

[0055] Non-viral vector constructs can be employed as a pharmaceutical or a constituent of a pharmaceutical, with the vectors preferably being used for preparing a pharmaceutical for the intravenous, intraarterial, intraportal, intracranial, intrapleural, intraperitoneal or local introduction of a desired gene into specific target cells. Pharmaceutically acceptable carriers for the novel vectors are those listed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1988, hereby incorporated by reference.

[0056] The simplicity of injecting a naked plasmid DNA ranging in size from 2-20 kilobases into a target organ, tissue or cell with a syringe has greatly influenced many aspects of gene therapy research. Tissues which exhibit transgene expression following naked plasmid DNA injection include but are not limited to thymus, skin, cardiac muscle and skeletal muscle. Of these tissues, long-term transgene expression has been observed in striated muscle.

[0057] The number of injections influences the effectiveness of gene transfer by plasmid DNA. Multiple injections may improve the overall expression by a single target organ or tissue, but can also result in deleterious side effects such as induction of host-mediated immune response. Fewer injections are preferable and can range from 1-20, 1-15, 1-10 or 1-5.

[0058] The non-viral carriers for the gene are preferably those compounds that are known to have a long half-life in the blood. As a result of this relatively long half-life in the blood, the target cell is exposed for as long as possible to as high a concentration of the vector as possible in order thereby to achieve the maximum possible binding of vectors to the target cells by means of the target cell-specific ligands. In a particularly preferred embodiment, these non-viral carriers are cationized to enable them to complex with the negatively charged DNA.

[0059] The non-viral carriers for the gene, that can be employed in accordance with the invention, are known in the art (for reviews, see Cotten et al., Curr. Biol., 4: 705 (1993); Behr, Acc. Chem. Res., 26: 274 (1993); Feigner, Adv. Deliv. Rev., 5: 163 (1990); Behr, Bioconjugate Chem., 5: 382 (1994); Ledley, Hum. Gene Ther. 6: 1129 (1995) all of which are hereby incorporated by reference). Those that are preferably employed within the context of the present invention are liposomes, cationic liposomes that are prepared using cationic lipids such as stearyl amines in a mixture with neutral phospholipids, dioctadecyldimethylammonium bromide (DDA) in a mixture with neutral phospholipids, N-1(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium bromide(DOTMA), 3β-N-(N′,N′-dimethylaminoethane)carbamoyl cholesterol (DC-Chol), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), dimethyidioctadecylammonium bromide (DDAB) and 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP).

[0060] Cationic polypeptides and proteins, such as polylysine, protamine sulfates, histones, polyornithine and polyarginine, are also suitable as non-viral carriers, as are cationic amphiphilic lipopolyamines such as dioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanolamidospermine (DPPES), N-t-butyl-N′-tetradecyl-3-tretradecylaminopropionamide (diC14-amidine), DOTB, ChoTB, DoSC, ChoSC, LPLL, DEBDA, DTAB, TTAB, CTAB or TMAG, or cationic polysaccharides such as diethylaminoethyldextran, and also cationic organic polymers such as POLYBRENE™.

[0061] In a preferred embodiment, liposomes are prepared from phosphatidyl serine where stock lipids are dried onto the surface of a plastic dish and allowed to dry. Ten ml of 0.3 M sucrose is layered, and the liposomes allowed to bubble into the bulk phase over a 1 hour period. The sucrose solution is recovered and mixed 1:1 with 10 ml of PBS followed by centrifugation at 15,000 rpm for 20 min at 4° C. The liposome pellet is resuspended in a 1 ml volume of 150 mM NaCl and 25 mM Tris-HCl, pH 7.5. The concentration of the final liposome solution is determined by phosphate determination and adjusted accordingly.

[0062] There are a number of natural activators and inhibitors of Factor XIIa, which may influence the intrinsic activity of Factor XIIa when present in a subject for an extended period of time. The physiological activation of Factor XII occurs by exposure to anionic surfaces, such as found on cut vascular elements (Griffin and Cochrane, 1976), or by subendothelial collagen. However, collagens III, IV and V inhibit Factor XII activation (Koenig et al, 1991). The activation of Factor XII to XIIa is blocked by amyloid precursor protein (APP) isoforms, with and without the Kunitz protease inhibitor domain (Niwano et al, 1995). These forms of APP are secreted by activated platelets, T-lymphocytes and leukocytes. Antithombin III is a plasma inhibitor of Factor XIIa (Chan et al, 1977). However, Pixley et al (1985) report that antithrombin III only contributes 2-3% to the total inhibition of Factor XIIa by plasma, and that the predominant inhibitor is C1 inhibitor. Estrogens also enhance Factor XII levels in the plasma, and may be a reason for a hypercoagulable state induced in some patients on estrogen therapy (Citarella et al, 1996). Bismuth subgallate, a known activator of Factor XII to XIIa, can reduce the clotting time of plasma deficient in Factor VII (Thorisdottir et al, 1988).

[0063] A primary object is to provide a method of gene therapy where the expressed protein is secreted directly into the blood stream to obtain therapeutically effective amounts of the protein thereby treating the subject in need of the protein.

[0064] The term “effective amount” means a sufficient amount of compound, e.g. nucleic acid and its corresponding recombinant protein delivered to produce an adequate level of the Factor XIIa, i.e., levels capable of converting VII to VIIa. Thus, the important aspect is the level of Factor XIIa expressed. Accordingly, one can use multiple transcripts or one can have the gene under the control of a promoter that will result in high levels of expression. In an alternative embodiment, the gene would be under the control of a factor that results in extremely high levels of expression.

[0065] Coagulation activity in the blood plasma of a recipient is determined by bypass activity following administration of a recombinant Factor XIIa-coated bead, an implant containing a recombinant Factor XIIa-coated bead, a cell expressing recombinant Factor XIIa, an implant containing a cell expressing recombinant Factor XIIa, a naked DNA vector containing a gene encoding a Factor XIIa polypeptide or a truncated or mutated form thereof, or a liposome encapsulated DNA vector containing a gene encoding a Factor XIIa polypeptide or a truncated or mutated form thereof.

[0066] Bypass activity is measured by the activated partial thromboplastin time (aPPT) assay. An aPPT assay for the quantification of Factor VII bypass activity is performed by preincubation of a mixture containing 100 μl of Factor VIII-deficient plasma, 100 μl of aPPT reagent (Sigma), and 20 μl of test samples for 3 min at 37° C. in a Fibrometer fibro-cup (B&L Fibro systems, Cockeysville, Mo.). The reaction is initiated by the addition of 100 μl of 25 mN CaCl2. The time taken for formation of the clot is determined, and a value in units s−1 is calculated from the reciprocal of the clotting time. Control measurements are made from pooled normal plasma.

[0067] The present invention is also directed towards a method of treating bleeding in a subject in which the method comprises administering to the patient an anti-bleeding effective amount of a coagulation agent. The coagulation agent preferably comprises at least one type of Factor XIIa polypeptide. The Factor XIIa polypeptides may be recombinant and may be either a complete Factor XIIa polypeptide or an active fragment of a Factor XIIa polypeptide. Examples of active fragments of Factor XII have been set forth above. It is noted that the active fragment of the Factor XII polypeptide provides an added advantage over the full peptide in that the active fragment renders the subject's body's control pathways inaccessible. In other words, the active fragment lacks the binding sites for the subject's immune system to attack.

[0068] Additionally, the coagulation agent may contain one or more supplemental coagulation agents. These agents are selected from stearic acid, bismuth subgallate, ellagic acid, and nanoparticles coated with stearic acid. The combination of the coagulation agent with the supplemental coagulation agents would be especially beneficial in cases of severe bleeding. Such cases would include gunshot or knife wounds, head wounds, severed limbs, shrapnel wounds, etc.

[0069] The subject of the method may suffer from Factor VIII or IX deficiency hemophilia and the subject is preferably a mammal, and, more preferably, a human. The method of administering the coagulation agent includes, but is not limited to, topical, intraperitoneal, anal, vaginal, parenteral, nasal, topical through the fingernails and toenails, through injection, through the eyes, through the ears, into the lungs, through aerosol application, and intravenously.

[0070] An IV preparation can be administered as a sterile buffered saline solution, while the oral preparation will be prepared with the standard pharmaceutically acceptable excipients through standard pharmaceutical manufacturing techniques.

[0071] For preparing pharmaceutical compositions containing the coagulation agent, inert, pharmaceutically acceptable carriers are used. The pharmaceutical carrier can be either solid or liquid. Solid form preparations of the coagulation agent include, for example, powders, tablets, dispersible granules, capsules, and cachets.

[0072] A solid carrier can be one or more substances which can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material. In powders, the carrier is generally a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active compound is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0073] Powders and tablets preferably contain between about 5% to about 70% by weight of the coagulation agent. However, this amount may be increased or decreased, if necessary. Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter and the like.

[0074] The pharmaceutical compositions can also include the formulation of the coagulation agent with encapsulating material as a carrier providing a capsule in which the coagulation agent (with or without other carriers) is surrounded by a carrier, which is thus in association with it. In a similar manner, cachets are also included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.

[0075] Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, or suspensions, and emulsions suitable for oral administration. Sterile water solutions of the coagulation agent or sterile solutions of the coagulation agent in solvents comprising water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration.

[0076] Sterile solutions can be prepared by dissolving the coagulation agent in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the coagulation agent in a previously sterilized solvent under sterile conditions.

[0077] Aqueous solutions for oral administration can be prepared by dissolving the coagulation agent in water or other appropriate solvents and adding suitable flavorants, coloring agents, stabilizers, and thickening agents as desired. Aqueous suspensions for oral use can be made by dispersing the finely divided coagulation agent in water together with a viscous material such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other suspending agents known to the pharmaceutical formulation art.

[0078] Preferably, a pharmaceutical composition containing the coagulation agent is in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the coagulation agent. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, packeted tablets, capsules, and powders in vials or ampules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms.

[0079] The effective amount would be easily determined by one of ordinary skill upon reviewing the extent of the bleeding, the age, sex, and weight of the subject. However, it is preferred that the effective amount be in the range of 0.0001 mg to 100 mg. It is more preferred if the range is between 0.001 and 10 mg. It is even more preferred that the range be between 0.01 and 10 mg. It is further preferred that the range be between 0.1 and 10 mg. It is even further preferred that the range be between 1 and 5 mg and it is most preferred that the effective amount be 1 mg.

[0080] Preferred frequency of dosage is twice a day. However, the dosage may be increased or decreased on a case by case basis. This decision would be based on age and sex of the victim, the severity of the bleeding, and the presence or absence of any genetic clotting deficiencies.

[0081] The invention is also directed towards a kit that comprises an applicator and an effective amount of the coagulation agent. Examples of the applicator include bandages, an immobilized surface, a spray bottle, a chamber, and a shaker to contain powdered coagulation agent.

[0082] The invention is also directed to a method of preventing bleeding in a subject. The method comprises administering to a subject, preferably a mammal, and, more preferably, a human, a therapeutically effective amount of the coagulation agent. For example, the coagulation agent can be administered in a time release capsule or in an implant device that slowly releases the coagulation agent into the subject over time, thereby placing the subject's body in a state of readiness to close any wounds that may occur.

[0083] The present invention is described in further detail in the following experimental details section, which sets forth specific examples to aid in understanding the invention and should not be construed to limit in any way the invention as defined in the claims which follow thereafter.

EXAMPLE 1

[0084] Ex-vivo and in vivo gene therapy systems for delivery of recombinant, full length or modified Factor XIIa.

[0085] As an alternative to introducing natural abundance or recombinant Factor XIIa within a porous chamber implant, it is also possible to introduce full length or modified Factor XIIa directly into the recipient using all the same gene therapy systems traditionally employed for gene therapy of individual Factor VIII or IX genes. Factor XIIa gene therapy is a universal therapeutic for both Hemophilia A and B.

[0086] Immobilized, recombinant Factor XIIa was introduced directly into a recipient, e.g., a guinea pig, and the bypass activity generated over time was measured by the aPPT assay. Guinea pigs were obtained, housed and sampled as described (Nature Biotechnology 18:289-295, Mar. 2000) except that a 0.5 ml sample of Factor XIIa beads (50% slurry in PB) was injected, intraperitoneally, into the guinea pigs. Glycine beads were used a negative control. Samples of blood were obtained by cardiac puncture one day prior to the injections in the guinea pigs. Thereafter, blood samples were obtained by the same route at 24, 48, and 72 hours post injection. A peak of bypass activity was noted at 24 hours post injection in the guinea pigs injected with Factor XIIa beads, but not in the glycine bead negative control guinea pigs. Bypass activity was back to baseline by 48 hours in the guinea pigs injected with Factor XIIa beads (data not shown). No changes in activity were noted at other times under either condition.

[0087] Introduction of Factor XIIa into the peritoneal cavity, unprotected by the membrane-enclosed porous chamber, was transiently therapeutic. It follows that a method of sustainedly expressing Factor XIIa activity might also prove therapeutic over a sustained period of time. The advantages of such a method would be (i) that therapy would be effective for both Hemophilia A and B; (ii) that antibodies against Factor XIIa would be unlikely since the factor is present at some level in most hemophiliacs; (iii) that antibodies against Factor VIII would not be generated; and (iv) that biological strategies to decrease immune responses would not be needed.

EXAMPLE 2

[0088] Treatment of Congenital Factor XIIa Deficiency

[0089] It has been reported that some patients with either Hemophilia A or B also have a congenital partial lack of Factor XIIa (Barthels et al, 1982). Indeed, there is a significant prevalence of moderate to severe Factor XIIa deficiency among the general European population, amounting to 2.3% (Halbmayer et al, 1994). Fortunately, the consequence of Factor XIIa deficiency alone is not profound. A case of a Japanese girl has also been reported with both hemophilia A and heterozygous Factor XIIa deficiency (Matsushita et al, 1992). The incidence of heterozygous Factor XIIa deficiency is not known in any population, nor was the genotype reported by Halbmayer et al (1994). Accordingly, any one or a combination of the aforementioned embodiments for presenting a recombinant FXIIa polypeptide can be applied in gene therapy related methods for treating patients with congenital deficiencies for FXIIa.

REFERENCES

[0090] The following references are all incorporated by reference in their entirety.

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Claims

1. A Factor XIIa-coated solid support comprising a recombinant Factor XIIa polypeptide.

2. The Factor XIIa-coated solid support of claim 1, wherein the polypeptide is a truncated polypeptide.

3. The Factor XIIa-coated solid support of claim 2, wherein the truncated polypeptide comprises a sequence of amino acid residues 1-215 of SEQ ID No. 2.

4. The Factor XIIa-coated solid support of claim 2, wherein the truncated polypeptide comprises a sequence of amino acid residues of about 1-196 of SEQ ID No. 2.

5. The Factor XIIa-coated solid support of claim 1, wherein the solid support is a bead.

6. A Factor XIIa-expressing cell comprising an expression vector construct containing a gene encoding a Factor XII polypeptide.

7. The Factor XIIa-expressing cell of claim 6, wherein the polypeptide is a truncated polypeptide.

8. The Factor XIIa-expressing cell of claim 7, wherein the truncated polypeptide comprises a sequence of amino acid residues 1-215 of SEQ ID No. 2.

9. The Factor XIIa-expressing cell of claim 7, wherein the truncated polypeptide comprises a sequence of amino acid residues of about 1-196 of SEQ ID No. 2.

10. The Factor XIIa-expressing cell of claim 6, wherein the cell comprises a blood cell, a hepatocyte, a fibroblast, and an endothelial cell.

11. A method of treating a subject with a coagulation protein comprising providing a gene prepared as a naked DNA vector and administering the vector to the subject in a therapeutically effective amount, wherein the gene encodes a Factor XIIa polypeptide.

12. A method of treating a subject with a coagulation protein comprising the steps of a) providing a gene for a coagulation protein prepared as an expression vector construct; b) transfecting host cells with the construct; c) adding cells of step b) expressing the coagulation protein to a porous chamber; and d) implanting the chamber into the subject, wherein the chamber is implanted in fluid communication with the bloodstream and inactive Factor VII in the bloodstream passing through the chamber becomes activated upon contact with the coagulation protein.

13. The method of claim 12, wherein the coagulation protein is a Factor XIIa polypeptide.

14. The method of claim 13, wherein the polypeptide is a truncated polypeptide.

15. The method of claim 14, wherein the truncated polypeptide comprises a sequence of amino acid residues of about 1-196 of SEQ ID No. 2.

16. The method of claim 12, wherein the chamber comprises a permeable membrane having one or more walls, defining a hollow chamber therewithin, a plurality of holes extending through the membrane and permitting fluid to enter and exit the membrane, each of the holes being sized to permit only Factor VII to enter the chamber of the membrane, and activated Factor VIIa to exit the chamber, and an effective amount of the coagulation protein or a source of the coagulation protein being expressed by the cells.

17. The method of claim 16, wherein the permeable membrane is a dialysis membrane formatted as a geometric filter comprising polymethacrylate or polycarbonate, or a statistical hole filter comprising cellulose acetate.

18. The method of claim 12, wherein the chamber comprises a region for affixing the chamber to an abdominal wall and for removing or adding materials.

19. The method of claim 12, wherein the expression vector construct of a) is a naked DNA.

20. The method of claim 12, wherein the expression vector construct of a) is encapsulated by a liposome.

21. The method of claim 12, wherein the expression vector construct of a) is a viral vector.

22. A method of treating bleeding, wherein said method comprises administering to a subject suffering from bleeding, a coagulation agent, wherein said coagulation agent comprises a therapeutically effective amount of a Factor XIIa polypeptide, and wherein said coagulation agent is administered in an amount effective to treat bleeding.

23. The method of claim 22, wherein the Factor XIIa polypeptide is a recombinant Factor XIIa polypeptide.

24. The method of claim 22, wherein the Factor XIIa polypeptide is administered topically, intraperitoneally, anally, vaginally, nasally, through the ear canal, parenterally, via inhalation, or intravenously.

25. The method of claim 24, wherein the topical administration of the Factor XIIa polypeptide is through the fingernails or toenails.

26. The method of claim 24, wherein the Factor XIIa polypeptide is injected in the eyes.

27. The method of claim 22, wherein the amount effective to reduce bleeding is 0.0001 mg to 100 mg.

28. The method of claim 22, wherein the subject is a mammal.

29. The method of claim 28, wherein the subject is human.

30. The method of claim 22, wherein the subject suffers from Factor VIII or Factor IX deficiency hemophilia.

31. The method of claim 22, wherein the Factor XIIa polypeptide is comprised of at least one of a complete Factor XIIa polypeptide or an active fragment of a Factor XIIa polypeptide.

32. A kit comprising an applicator and a coagulation agent, wherein said coagulation agent comprises a therapeutically effective amount of a Factor XIIa polypeptide.

33. The kit of claim 32, wherein the applicator is selected from the group consisting of a bandage, an immobilized surface, a chamber, a shaker, and a spray bottle

34. The method of claim 22, wherein the coagulation agent further comprises at least one of stearic acid, bismuth subgallate, ellagic acid, and nanoparticles coated with stearic acid.

35. A method of preventing bleeding, wherein said method comprises administering to a subject, a coagulation agent, wherein said coagulation agent comprises a therapeutically effective amount of a Factor XIIa polypeptide, and wherein said coagulation agent is administered in an amount effective to prevent bleeding.

36. The method of claim 35, wherein the coagulation agent further comprises at least one of stearic acid, bismuth subgallate, ellagic acid, and nanoparticles coated with stearic acid.

37. The kit of claim 32, wherein the coagulation agent further comprises at least one of stearic acid, bismuth subgallate, ellagic acid, and nanoparticles coated with stearic acid.