Composition and method for treating hemophilia using novel factor viii molecule

Abstract

Disclosed is a novel composition and method for treating hemophilia incorporating a gain-of-function human Factor VIII variant in which serine may replace arginine at amino-acid position 590 (R590S). The variant can exhibit reduced sensitivity to activated protein C and / or produces thrombin at rates exceeding those of wild-type Factor VIII while maintaining normal antigen levels. The invention may comprise: (i) an isolated polypeptide comprising the R590S substitution, optionally with B-domain deletion; (ii) a nucleic-acid molecule encoding the variant; and / or (iii) recombinant vectors, including adeno-associated virus constructs, that may express the variant in host cells. The novel composition further may be synthesized by encoding nucleic acid, expressing the variant in mammalian cells, recovering the protein, and / or administering therapeutically effective amounts of the polypeptide and / or vector to restore coagulation.

Description

[0001] COMPOSITION AND METHOD FOR TREATING HEMOPHILIA USING NOVEL FACTOR VIII MOLECULE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This nonprovisional application claims the benefit of U.S. Provisional Patent Application No. 63 / 676,417 entitled “COMPOSITION AND METHOD FOR TREATING HEMOPHILIA USING NOVEL FACTOR VIII MOLECULE” filed July 28, 2024, by the same inventors, all of which is incorporated herein by reference, its entirety, for all purposes.

[0004] SEQUENCE LISTING

[0005] The sequence listing submitted in XML format complies with WIPO Standard ST.26 and is incorporated by reference into the present application.

[0006] BACKGROUND OF THE INVENTION

[0007] 1 . Field of the Invention

[0008] This invention relates to treatment and / or prevention of hemophilia. Specifically, the invention provides a novel composition utilizing a novel Factor VIII molecule and associated method of treating hemophilia.

[0009] 2. Brief Description of the Prior Art

[0010] Coagulation factor VIII (FVII I) is an essential cofactor in the coagulation cascade, that is tightly bound to Von Willebrand’s Factor while in circulation within the blood stream. This complex is dimerized when FVIII is activated by thrombin cleavage. Thrombin cleaves FVIII at three sites: Arg372, Arg740and Arg1689. Once this happens activated FVIII (FVIIIa) is created and able to push the coagulation cascade along. FVIIIa differs significantly in structure from that of inactive FVIII, in that FVIIIa has no B-domain and an A2 domain that rapidly dissociates if not bound to activated factor IX (FIXa). The binding to FIXa occurs within the A2 domain of FVIII and more specifically at Ser558 through Gln565 in the A2 domain, as well as to sequences in the A3 domain, G lu 181 1 through Lys1818. Together FVIIIa and FIXa form the Xase complex which activates factor X (FXa), that then goes on to generate thrombin.

[0011] FVIIIa plays a key role in Xase activity and is tightly regulated by proteins C and S which together act as regulatory mechanism for both FVIIIa and activated Factor V (FVa). Activated protein C (APC) cleaves FVIIIa at two regions in the A2 domain of FVIIIa: Arg336 and Arg562. The tight regulation of FVIIIa by APC is key to maintaining a hemostatic environment. FV is homologous to FVIII, in that APC is also crucial to the regulation of FV, and serves as a good model for understanding FVIII regulation via APC. Genetic mutations have been shown to impact this interaction and cause significant thrombophilia; most well-known and common is factor V Leiden (Arg506Gln). The heterozygous mutation causes a 5- to 10- fold increase and the homozygous causes a 50- to 100- fold increase in risk for thrombosis. Factor V Leiden is known to impact FVa responsiveness to ARC regulation, since ARG506 is a cleavage site of ARC on FVa. No variant FVIIIs have been identified with similar point mutations, but one has been noted to spark familial thrombophilia. FVIII Padua is caused by a duplication of the promoter region and the 1stintron of the FVIII gene. This duplication resulted in FVIII antigen and activity of 400%N and familial thrombotic events. Scientists were unable to conclude how the mutation impacted FVIII interactions, but this opened the door to the study of potential thrombotic effects of FVIII mutations. The current report was initiated by an 18-year-old male who presented with recurrent life-threatening thrombosis since birth and was found to have elevated one-stage FVIII activity that ranged from 300-900%N with progressive increase over time. He was found to have a novel point mutation at amino acid position 590 (Arg590Ser) in the FVIII gene. This mutation’s location was near the region of ARC cleavage in the A2 domain and could possibly have parallel effects to Factor V Leiden due to the homology between FV and FVIII inhibition via APC. The aim of this paper is to characterize the variant FVIII molecule and its impact on other proteins.

[0012] Accordingly, what is needed is safe, efficient, and effective composition and method to treat hemophilia utilizing a novel Factor VIII molecule. However, in view of the art considered as a whole at the time the present invention was made, it was not obvious to those of ordinary skill in the field of this invention how the shortcomings of the prior art could be overcome.

[0013] SUMMARY OF THE INVENTION

[0014] The long-standing but heretofore unfulfilled need, stated above, is now met by a novel and non- obvious invention disclosed and claimed herein. In an aspect, the present disclosure pertains to a composition for treating hemophilia. In embodiments, the composition may comprise the following: (a) a nucleic acid vector encoding a Factor VIII polypeptide variant; (b) the Factor VIII polypeptide variant in a therapeutically effective amount, such that the Factor VIII polypeptide variant may comprise a serine residue at position 590 in place of arginine of the wild-type human Factor VIII sequence, and in which the Factor VIII polypeptide variant can be at least 95% identical in amino acid sequence to wild-type human Factor VIII.

[0015] In some embodiments, the Factor VIII polypeptide may comprise an arginine-to-serine substitution at amino acid position 590 relative to wild-type Factor VIII. In some embodiments, the Factor VIII polypeptide can also be a B-domain deleted Factor VIII variant. As such, the Factor VIII polypeptide may also comprise the amino acid sequence of SEQ ID NO:2.

[0016] In some embodiments, the nucleic acid encoding the Factor VIII polypeptide can comprise the nucleotide sequence of SEQ ID NO:3. In this manner, the nucleic acid vector may be an adeno- associated virus (AAV) vector. In some embodiments, the nucleic acid vector comprises the nucleotide sequence of SEQ ID NO:4.

[0017] In some embodiments, the nucleic acid vector is packaged in a viral capsid for delivery. Accordingly, the composition may also comprise a therapeutically effective amount of the Factor VIII polypeptide. In these other embodiments, the composition may further comprise a pharmaceutically acceptable carrier or excipient.

[0018] Another aspect of the present disclosure pertains to a pharmaceutical kit for treating hemophilia. In embodiments, the pharmaceutical kit may comprise the following: (a) a Factor VIII polypeptide variant having at least 95% sequence identity to a mutant Factor VIII comprising an arginine-to- serine substitution at amino acid position 590 relative to wild-type Factor VIII; or (b) a nucleic acid vector encoding said Factor VIII polypeptide variant; or (c) both a Factor VIII polypeptide variant having at least 95% sequence identity to a mutant Factor VIII comprising an arginine-to- serine substitution at amino acid position 590 relative to wild-type Factor VIII and the nucleic acid vector encoding said Factor VIII polypeptide variant.

[0019] In some embodiments, the Factor VIII polypeptide can include the amino acid sequence of SEQ ID NO:2. In some embodiments, the nucleic acid encoding the Factor VIII polypeptide may comprise the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleic acid vector may comprise the nucleotide sequence of SEQ ID NO:4. Additionally, the nucleic acid vector can be an adeno-associated virus vector. In these other embodiments, the nucleic acid vector may be packaged in a viral capsid for delivery.

[0020] Another aspect of the present disclosure pertains to a method of producing a Factor VIII polypeptide variant for treating hemophilia. In embodiments, the method may comprise the following steps: (a) synthesizing a nucleic acid encoding a Factor VIII polypeptide comprising a serine residue at amino acid position 590 instead of arginine, the nucleic acid being at least 90% identical to the coding sequence of wild-type human Factor VIII; (b) expressing the nucleic acid in a host cell; and (c) recovering the expressed Factor VIII polypeptide.

[0021] In some embodiments, the nucleic acid may comprise the nucleotide sequence of SEQ ID NO:3. In some embodiments, the nucleic acid can be synthesized as part of a nucleic acid vector comprising the nucleotide sequence of SEQ ID NO:4. In some embodiments, the host cell can be a mammalian cell. In some embodiments, the method may further comprise the step of, purifying the recovered polypeptide. In some embodiments, the method may further comprise the step of, formulating the nucleic acid and / or the recovered polypeptide with a pharmaceutically acceptable carrier to form the composition.

[0022] Moreover, An additional aspect of the present disclosure pertains to a nucleic acid vector encoding a Factor VIII polypeptide comprising a Serine at position 590 (R590S) of wild-type Factor VIII, wherein the Factor VIII polypeptide is at least 95% identical to SEQ ID NO: 2, SEQ ID NO: 3, and / or SEQ ID NO: 4, for use as a medicament to treat hemophilia.

[0023] Additionally, another aspect of the present disclosure pertains to an isolated Factor VIII polypeptide comprising a Serine substitution at position 590 relative to wild-type Factor VIII, having coagulation activity for use as a medicament to treat hemophilia.

[0024] Furthermore, another aspect of the present disclosure pertains to a method of treating hemophilia A in a subject in need thereof. In an embodiment, the method may comprise the following steps: (a) administering to the subject a therapeutically effective amount of a therapeutic agent comprising a Factor VIII variant polypeptide having an arginine-to-serine substitution at amino acid position 590 of Factor VIII, such that the therapeutic agent may be selected from the group consisting of: (I) the Factor VIII variant polypeptide, and / or (ii) a nucleic acid vector encoding said Factor VIII variant polypeptide, providing a practical treatment for hemophilia A in the subject.

[0025] In some embodiments, the therapeutic agent may be the Factor VIII variant polypeptide, administered as a protein replacement therapy to the subject. In this manner, the therapeutic agent can be a nucleic acid vector encoding the Factor VIII variant polypeptide, administered as a gene therapy to express the variant polypeptide in vivo.

[0026] In some embodiments, the nucleic acid vector may be a recombinant adeno-associated virus (AAV) vector encoding the Factor VIII variant polypeptide. In some embodiments, the Factor VIII variant can be resistant to proteolytic inactivation by activated protein C (APC), such that the variant may interact poorly with APC compared to wild-type factor VIII.

[0027] In some embodiments, the Factor VIII variant exhibits increased procoagulant activity relative to wild-type Factor VIII, resulting in enhanced thrombin generation and improved hemostatic efficacy. In some embodiments, the subject may develop neutralizing anti-Factor VIII antibodies (inhibitors) prior to treatment, and the administration of the Factor VIII variant can provide therapeutic benefit despite the presence of said inhibitors.

[0028] In some embodiments, the therapeutically effective amount of the Factor VIII variant can be administered prophylactically to the subject on a regular dosing schedule to prevent and / or reduce the frequency of bleeding episodes. In some embodiments, the Factor VIII variant polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 1 . In some embodiments, the nucleic acid vector can comprise a nucleotide sequence encoding the Factor VIII variant polypeptide, such that the nucleotide sequence may be set forth in SEQ ID NO: 2.

[0029] To date, despite efforts at intentional mutagenesis of the factor VIII gene, there has not been a gene sequence determined that could increase the efficacy of factor VIII gene therapy. Most current therapeutic interventions include use of a viral vector to insert the factor VIII gene (with beta chain deleted) into a hepatocyte by intravenous infusion with homing genes incorporated into the vector capsid. The physiological site of factor VIII synthesis is not the hepatocyte, but rather the hepatic sinusoidal cell. Thus, an enormous gene is being shoved into a cell not evolved to synthesize it, causing metabolic stress and potential liver inflammatory damage. Incorporation of a factor VIII gene with increased functionality could potentially result in an enhanced therapeutic effect from a smaller inoculum.

[0030] The inventor discovered a novel mutation in FVIII associated with life-long recurrent thrombosis. Specifically, the inventors are examining Chromosome X, the F8 gene Exon #12, the amino acid coding residue 590 and changing the arginine to a serine at position 590. The mutation confers a highly increased activity to normal FVIII plasma antigen concentration. The initial studies may indicate that R590S FVIII activates thrombin rapidly and is not responsive to APC. Docking simulations support weaker affinity of R590S to APC with no impact on FIXa binding. Further investigations using a FVIII expression system to study interactions with the Xase complex, VWF, and Protein S will give a better understanding of the full effects of this mutation. This high activity FVIII shares some analogy to FIX Padua and may have therapeutic implications for the treatment of hemophilia A

[0031] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not restrictive.

[0032] The invention accordingly comprises the features of construction, combination of elements, and arrangement of parts that will be exemplified in the disclosure set forth hereinafter and the scope of the invention will be indicated in the claims.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

[0035] FIG. 1 is an amino acid sequence listing for wild type Factor VIII with the amino acid of interest highlighted in yellow (SEQ ID NO: 1 ), according to embodiments of the present disclosure.

[0036] FIG. 2 is an amino acid sequence listing for novel Factor VIII with the arginine at position 590 changed to a serine (highlighted in yellow) (SEQ ID NO: 2), according to embodiments of the present disclosure. FIG. 3 is a DNA sequence for novel Factor VIII showing the mutated codon (highlighted) for use in a therapeutic vector (SEQ ID NO: 3), according to embodiments of the present disclosure.

[0037] FIG. 4 is an image depicting a beta cleavage visual reproduced from Thim et al, according to embodiments of the present disclosure.

[0038] FIG. 5 is an image of an exemplary vector incorporating the novel Factor VIII, according to embodiments of the present disclosure.

[0039] FIG. 6 is a sequence listing for the exemplary vector of Figure 5 (SEQ ID NO: 4), according to embodiments of the present disclosure.

[0040] FIG. 7 is an image depicting an exemplary vector incorporating wild type Factor VIII, according to embodiments of the present disclosure.

[0041] FIG. 8 is a sequence listing for the exemplary vector of Figure 7 (SEQ ID NO: 5), according to embodiments of the present disclosure.

[0042] FIG. 9 is an image of the PICS 675 CAT TM results, according to embodiments of the present disclosure.

[0043] FIG. 10 is an image of the PICS 765-2 5pM CAT results, according to embodiments of the present disclosure.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings, which form a part thereof, and within which are shown by way of illustration specific embodiments by which the invention may be practiced. It is to be understood that one skilled in the art will recognize that other embodiments may be utilized, and it will be apparent to one skilled in the art that structural changes may be made without departing from the scope of the invention.

[0046] As such, elements / components shown in diagrams are illustrative of exemplary embodiments of the disclosure and are meant to avoid obscuring the disclosure. Any headings, used herein, are for organizational purposes only and shall not be used to limit the scope of the description or the claims.

[0047] Furthermore, the use of certain terms in various places in the specification, described herein, are for illustration and should not be construed as limiting. For example, any reference to an element herein using a designation such as “first,” “second,” and so forth does not limit the quantity or order of those elements, unless such limitation is explicitly stated. Rather, these designations may be used herein as a convenient method of distinguishing between two or more elements or instances of an element. Therefore, a reference to first and / or second elements does not mean that only two elements may be employed there or that the first element must precede the second element in some manner. Also, unless stated otherwise a set of elements may comprise one or more elements

[0048] Reference in the specification to “one embodiment," “preferred embodiment," “an embodiment,” or “embodiments” means that a particular feature, structure, characteristic, or function described in connection with the embodiment is included in at least one embodiment of the disclosure and may be in more than one embodiment. The appearances of the phrases “in one embodiment,” “in an embodiment,” “in embodiments,” “in alternative embodiments,” “in an alternative embodiment,” or “in some embodiments” in various places in the specification are not necessarily all referring to the same embodiment or embodiments. The terms “include,” “including,” “comprise,” and “comprising” shall be understood to be open terms and any lists that follow are examples and not meant to be limited to the listed items.

[0049] Referring in general to the following description and accompanying drawings, various embodiments of the present disclosure are illustrated to show its structure and method of operation. Common elements of the illustrated embodiments may be designated with similar reference numerals.

[0050] Accordingly, the relevant descriptions of such features apply equally to the features and related components among all the drawings. For example, any suitable combination of the features, and variations of the same, described with components illustrated in FIG. 1 , can be employed with the components of FIG. 2, and vice versa. This pattern of disclosure applies equally to further embodiments depicted in subsequent figures and described hereinafter. It should be understood that the figures presented are not meant to be illustrative of actual views of any particular portion of the actual structure or method but are merely idealized representations employed to more clearly and fully depict the present invention defined by the claims below.

[0051] Definitions:

[0052] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the context clearly dictates otherwise.

[0053] In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of embodiments of the present technology. It will be apparent, however, to one skilled in the art that embodiments of the present technology may be practiced without some of these specific details.

[0054] Various terms relating to the biological molecules of the present invention are used hereinbelow and also throughout the Specification and Claims. As used herein, the term “Activated protein C (APC)” generally refers to the vitamin K-dependent serine protease generated when thrombin complexes with thrombomodulin on an endothelial surface and proteolytically activates circulating protein C. APC functions as an endogenous anticoagulant by site-specific cleavage and inactivation of the pro-cofactor forms of factor VIII (at Arg336 and Arg562) and factor V, diminishing intrinsic and extrinsic tenase activity and limiting thrombin propagation. Within the present disclosure, in embodiments, reduced affinity between APC and the Arg590Ser factor VIII variant underlies a partial resistance to proteolytic down-regulation, which is measured by specialized assays such as CAT-TM and APCR-FVIII and is considered a mechanistic contributor to the enhanced thrombin generation phenotype of the variant.

[0055] As used herein, the term “Adeno-associated virus (AAV) vector" generally refers to a recombinant, replication-defective parvoviral construct derived from wild-type AAV genomes in which viral rep and cap genes are replaced by a heterologous expression cassette flanked by inverted terminal repeats. The resulting vector can be packaged into a selected capsid serotype and, upon administration, delivers the cassette to target cell nuclei where it typically persists as episomal concatemers. In the context of the invention, in embodiments, an AAV vector may encode nucleic acid sequences for the Arg590Ser factor VIII variant, either full-length or B- domain-deleted, so as to establish durable hepatic or sinusoidal endothelial expression in subjects with hemophilia A while permitting lower total vector genomes and reducing hepatotoxic risk compared to vectors encoding wild-type factor VIII.

[0056] As used herein, the term “Amino acid identity” generally refers to the percentage of positions at which two polypeptide sequences are identical when optimally aligned using standard bioinformatic algorithms such as BLASTP, FASTA, or Clustal Omega. Unless otherwise specified, identity is calculated over the full-length sequence, excluding gaps introduced for alignment, and may be expressed as at least 75 %, 85 %, 90 %, 95 %, 97 %, 99 %, or 100 %. For claim construction purposes, sequences meeting stated identity thresholds to SEQ ID NOs 1 - 5 that also retain a serine residue at position 590 are considered structurally equivalent variants within the scope of the disclosed compositions and methods, provided such variants maintain coagulation cofactor activity and APC resistance comparable to the Arg590Ser molecule.

[0057] As used herein, the term “Arg590Ser variant” generally refers to a coagulation factor VIII polypeptide in which the codon for arginine at residue 590 of the wild-type human sequence (located in the A2 domain) has been substituted with a serine-encoding codon, whether in a full- length or B-domain-deleted backbone. This single substitution perturbs local electrostatic interactions, produces a protruding loop observed by in-silico modeling, lowers APC binding affinity, and accelerates thrombin generation when evaluated in calibrated automated thrombography. The variant may be delivered directly as a purified protein, expressed from a viral or non-viral nucleic acid vector, or incorporated into pharmaceutical kits, and is contemplated for therapeutic use where a hyperfunctional factor VIII is advantageous, such as prophylaxis or treatment of hemophilia A.

[0058] As used herein, the term “B-domain deleted factor VIII” generally refers to a recombinant factor VIII construct in which most or all of the highly glycosylated B-domain (amino acids 741-1648 of the wild-type sequence) has been removed or replaced with a short linker to enhance secretion and facilitate vector packaging without substantially affecting cofactor activity once activated. In embodiments, the Arg590Ser substitution is introduced into such a truncated scaffold to combine the secretion benefits of B-domain deletion with the hyperactive and APC-resistant properties conferred by the mutation, thereby enabling lower therapeutic dose requirements in gene- or protein-based hemophilia A treatments.

[0059] As used herein, the term “Calibrated Automated Thrombogram (CAT)” generally refers to a fluorogenic thrombin generation assay that records real-time cleavage of a synthetic substrate in citrated plasma after initiation with tissue factor and phospholipid, using an internal calibrator to correct for inner-filter effects and a2-macroglobulin-thrombin activity. Parameters extracted include lag time, peak thrombin, endogenous thrombin potential, and velocity index. In the disclosed work, CAT profiles for plasma containing the Arg590Ser variant exhibit a substantially elevated velocity index while maintaining normal lag and peak metrics, thereby demonstrating that the mutation specifically amplifies the propagation phase of coagulation rather than the initiation phase.

[0060] As used herein, the term “Carrier" generally refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HCI, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.

[0061] As used herein, the term “Chromogenic Assay” generally refers to a two-stage diagnostic method in which an analyte enzyme, such as activated factor VIII in complex with factor IXa, first activates a downstream coagulation factor that subsequently cleaves a chromophore-linked substrate, releasing p-nitroaniline measurable at 405 nm. The rate of color development correlates with enzyme activity. Within the disclosure, in embodiments, chromogenic factor VIII assays register near-normal activity for the Arg590Ser variant, contrasting with disproportionately high one-stage clotting results and underscoring assay-specific differences relevant to clinical interpretation of hyperactive factor VIII molecules. As used herein, the term “Coagulation Factor VIII (FVIII)” generally refers to the multifunctional pro-cofactor synthesized primarily by sinusoidal endothelial cells, secreted as a heterodimeric glycoprotein comprising A1 -A2-B-A3-C1 -C2 domains, and circulating complexed with von Willebrand factor until proteolytic activation by thrombin or factor Xa produces the active cofactor FVIIIa. FVIIIa functions with factor IXa on phospholipid membranes to accelerate conversion of factor X to factor Xa. The present invention focuses on engineered FVIII molecules bearing the Arg590Ser substitution that exhibit enhanced catalytic efficiency and reduced susceptibility to APC-mediated inactivation.

[0062] As used herein, the term “Endogenous thrombin potential (ETP)” generally refers to the area under the thrombin generation curve obtained in a CAT assay, representing the cumulative amount of thrombin formed during the assay period. A reduction in ETP upon addition of thrombomodulin reflects the degree of protein C pathway suppression. Within CAT-TM experiments described herein, in embodiments, samples containing the Arg590Ser variant exhibit less than approximately 1 1% ETP reduction compared with 32% - 71% in normal plasma, indicating functional resistance to APC and contributing to the hypercoagulable phenotype.

[0063] As used herein, the term “Factor IXa (FIXa)” generally refers to the activated serine protease generated from factor IX by factor Xia or factor VI la / tissue factor, which forms the intrinsic tenase complex with FVIIIa on negatively charged phospholipid surfaces. Binding epitopes on FVIIIa include residues 558-565 in the A2 domain and regions near Asp712 in the A3 domain. Because the Arg590Ser substitution lies adjacent to these epitopes, it may modulate FIXa association kinetics, alter A2 domain stability, and influence overall tenase turnover, thus playing a role in the variant’s prothrombotic behavior.

[0064] As used herein, the term “Factor X (FX)” generally refers to the vitamin K-dependent zymogen that is proteolytically activated to factor Xa by either the intrinsic or extrinsic tenase complexes. FX contains a specific Arg51-lle52 bond on its heavy chain that is cleaved by FIXa / FVIlla on phospholipid surfaces in the presence of calcium. In silico docking models described in the disclosure indicate altered affinity of the Arg590Ser-containing tenase complex for FX, which in turn may modify catalytic efficiency and downstream thrombin generation.

[0065] As used herein, the term “Factor Xa (FXa)” generally refers to the serine protease produced from FX that, together with factor Va, forms the prothrombinase complex responsible for converting prothrombin to thrombin. Docking simulations presented herein suggest that tenase complexes incorporating the Arg590Ser FVIIIa release FXa more readily than wild-type complexes, thereby increasing free FXa levels in circulation and contributing to excessive thrombin formation.

[0066] As used herein, the term “Gene Therapy Vector” generally refers to any engineered nucleic acid delivery vehicle, viral or non-viral, capable of introducing and enabling expression of therapeutic genetic material in vivo. Examples include recombinant AAV, lentiviral, and plasmid systems equipped with promoters, regulatory elements, and polyadenylation signals. In certain embodiments of the invention, such vectors encode the Arg590Ser variant to achieve sustained endogenous production of hyperfunctional factor VIII, thus reducing or eliminating the need for repeated protein infusions in hemophilia A patients.

[0067] As used herein, the term “Hemophilia A” generally refers to an X-linked recessive bleeding disorder characterized by partial or complete deficiency or dysfunction of coagulation factor VIII, leading to impaired fibrin clot formation, spontaneous hemarthroses, and excessive bleeding after injury or surgery. The current disclosure provides compositions and methods employing a hyperactive, APC-resistant Arg590Ser factor VIII variant to raise effective factor levels or activity in hemophilic subjects with potentially lower dosing requirements and reduced vector burden compared with wild-type factor VIII therapies.

[0068] As used herein, the term “Hemostasis Related Disorder” generally refers to bleeding disorders such as, without limitation, hemophilia A, hemophilia B, hemophilia A and B patients, hemophilia with inhibitory antibodies, deficiencies in at least one coagulation factor (e.g., Factors VII, VIII, IX, X, XI, V, XII, II, and / or von Willebrand factor, particularly Factor VIII), combined FV / FVIII deficiency, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy (hypocoagulability), disseminated intravascular coagulation (DIG), over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, or small molecule antithrombotics (e.g., FXa inhibitors); and platelet disorders such as, Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency. In embodiments, the term “hemostasis related disorder” generally refers to bleeding disorders characterized by excessive and / or uncontrolled bleeding (e.g., a disorder which can be treated with a procoagulant). In embodiments, the hemostasis related disorder is hemophilia. In embodiments, the hemostasis related disorder is hemophilia A.

[0069] As used herein, the term “Intrinsic Tenase Complex” generally refers to the membrane- associated enzymatic assembly comprising FIXa, FVIIIa, calcium ions, and a negatively charged phospholipid bilayer or platelet surface, which catalyzes rapid activation of FX. The rate enhancement afforded by FVIIIa can exceed 10,000-fold relative to FIXa alone. In the invention, substitution of Arg590Ser into FVIIIa alters tenase kinetics — either by prolonging A2 domain integrity or by affecting FX / FXa binding — thereby providing a mechanistic basis for the observed acceleration of thrombin generation.

[0070] As used herein, the term “Nucleic Acid Vector” generally refers to an engineered DNA or RNA construct comprising regulatory sequences and a coding region for the Arg590Ser factor VIII polypeptide, formulated for delivery into host cells. Vectors may be plasmid-based for transient transfection, integrative or episomal viral genomes for in vivo gene transfer, or lipid nanoparticle- encapsulated mRNA for direct protein translation. Essential elements can include promoters, enhancers, introns, and polyadenylation sites configured to optimize transcription, translation, and secretion of the therapeutic variant.

[0071] As used herein, the term “One-Stage Clotting Factor VIII Activity” generally refers to the activity determined by measuring the time required for clot formation in FVII l-deficient plasma after partial thromboplastin activation and recalcification, calibrated against a standard curve of normal pooled plasma. In the disclosed studies, the Arg590Ser variant yields values of approximately 300-900% of normal, greatly exceeding chromogenic assay readouts and highlighting the hypercoagulable nature of the molecule.

[0072] As used herein, the term “Pharmaceutically Acceptable” generally refers to approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans

[0073] As used herein, the term “Pharmaceutically Acceptable Carrier” generally refers to any physiologically compatible excipient, diluent, or delivery vehicle that can be combined with an active agent — protein or nucleic acid — to facilitate administration, stability, or absorption without interfering with biological activity. Carriers suitable for intravenous infusion of Factor VIII protein may include isotonic saline or buffered solutions, whereas carriers for gene therapy vectors may comprise balanced salt solutions with stabilizing sugars or surfactants.

[0074] As used herein, the term “Surface Plasmon Resonance (SPR)” generally refers to a label-free optical technique that measures real-time biomolecular interactions by detecting refractive-index changes at a sensor surface as analytes bind or dissociate. Association and dissociation rate constants and equilibrium dissociation constants can be extracted from sensorgrams. The disclosure contemplates using SPR to validate docking predictions by quantifying binding affinities between Arg590Ser FVII la and partners such as APC, FIXa, FX, and FXa.

[0075] As used herein, the term “Therapeutically Effective Amount” generally refers to a quantity of a factor VIII variant polypeptide or of a nucleic acid encoding that variant sufficient, when administered to a subject, to achieve a clinically meaningful improvement in coagulation — such as reducing annualized bleeding rate, shortening clotting times, or elevating trough factor activity to at least 5% of normal — without inducing unacceptable toxicity or immunogenicity. Dosage ranges therefore vary depending on delivery modality, vector serotype, subject weight, and baseline inhibitor status.

[0076] As used herein, the term “Thrombomodulin (TM)” generally refers to an endothelial transmembrane glycoprotein that forms a high-affinity complex with thrombin, shifting its substrate specificity toward activation of protein C and thrombin-activatable fibrinolysis inhibitor, thereby initiating anticoagulant and antifibrinolytic pathways. In CAT-TM assays described herein, exogenous TM is added to plasma to probe APC-mediated down-regulation; a minimal ETP reduction in Arg590Ser samples highlights the variant’s APC resistance.

[0077] As used herein, the term “Vector” generally refers to a carrier nucleic acid molecule (e.g., RNA or DNA) into which a nucleic acid sequence can be inserted for introduction into a host cell where it will be replicated.

[0078] As used herein, the term “Velocity Index” generally refers to the maximal first derivative of the thrombin generation curve in a CAT assay, expressed in nanomoles of thrombin generated per minute, representing the steepest rate of thrombin formation. The Arg590Ser variant elevates the velocity index to values around 196 nM / min, significantly exceeding normal reference limits and serving as a quantitative indicator of its accelerated catalytic profile.

[0079] As used herein, the term “von Willebrand factor (vWF)” generally refers to a large multimeric plasma glycoprotein that mediates platelet adhesion under high shear and stabilizes circulating factor VIII by forming a non-covalent complex through interactions primarily within the FVIII lightchain C domains. Binding of vWF protects FVIII from premature clearance. Because the Arg590Ser mutation resides in the A2 domain, distant from vWF interaction sites, docking and biochemical data indicate that vWF binding remains substantially unaltered, preserving the variant’s half-life and supporting its therapeutic applicability.

[0080] As used herein, the terms “about,” “approximately,” or “roughly” generally refer to being within an acceptable error range (i.e., tolerance) for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined (e.g., the limitations of a measurement system) (e.g., the degree of precision required for a particular purpose, such treating hemophilia A utilizing a novel Factor VIII molecule). As used herein, “about,” “approximately,” or “roughly” refer to within +25% of the numerical.

[0081] All numerical designations, including ranges, are approximations which are varied up or down by increments of 1 .0, 0.1 , 0.01 or 0.001 as appropriate. It is to be understood, even if it is not always explicitly stated, that all numerical designations are preceded by the term “about”. It is also to be understood, even if it is not always explicitly stated, that the compounds and structures described herein are merely exemplary and that equivalents of such are known in the art and can be substituted for the compounds and structures explicitly stated herein.

[0082] Wherever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1 , 2, or 3 is equivalent to greater than or equal to 1 , greater than or equal to 2, or greater than or equal to 3. Wherever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 1 , 2, or 3 is equivalent to less than or equal to 1 , less than or equal to 2, or less than or equal to 3.

[0083] Composition and Method for Treating Hemophilia Using Novel Factor VIII Molecule:

[0084] Hemophilia A is a monogenetic disorder in which mutations resulting in decreased or absent endogenous production of coagulation factor VIII are associated with bleeding into joints, muscles, soft tissues and critical body compartments, including the intracranial, retroperitoneal, peritoneal and spinal spaces with resultant higher risks for death, arthropathy, chronic pain and disability. Current treatment of hemophilia consists of improving hemostatic capacity with plasma-derived physiological factor VIII, recombinant factor VIII molecules that are either physiologic or minimally altered (B domain deleted) or bioengineered proteins (e.g. pegylated, conjugated with Fc receptor, single chain and / or removal of molecular binding sites for von Willebrand protein), bioengineered antibodies which function as factor VIII mimetics, or various maneuvers to rebalance hemostasis in persons with hemophilia by proportionately decreasing hemostasis regulation.

[0085] The factor VIII gene was first sequenced in 1984 by two groups, Genentech in San Francisco and Genetics Institute in Boston. Factor VIII gene sequence was immediately applied to the manufacture of recombinant factor VIII, precluding the transmission of viral contaminants in plasma factor VIII. The ultimate treatment of hemophilia has been anticipated in the form of gene therapy, first reported by Dr. David Roth and colleagues from Boston in 2001 . However, the road from proof of concept to prime time has been long and steep. Gene therapy for factor VIII was first approved by the FDA more than twenty years later, on June 29, 2023. Valoctocogene roxaparvovec gene therapy (BioMarin, Novato, CA) is performed via a single intravenous infusion of 6x1013genomes per kilogram which are administered in a n AAV5 vector. The results with valococogene showed a mean peak response of 35% factor VIII at 4-6 months, decreasing to a mean of about 15% by 2 years post vector infusion. There are myriad possibilities for the fall-off of plasma factor VIII activity over time. Thus, over the past 25 years, gene therapy for hemophilia has come to represent a conversion of severe hemophilia to a mild form, rather than the conventional meaning of a "cure”.

[0086] Progress in gene therapy for hemophilia B or factor IX deficiency was similarly slow with poor post therapy factor activity achieved until the discovery of Factor IX Padua. A family in Italy with early onset recurrent thrombosis was found to harbor an X-linked mutation with the substitution of a leucine for arginine at position 338 (R338L) resulting in a hyperfunctional factor IX. When this gene was inserted into vectors for gene therapy, the functional improvement in plasma factor IX activity was 6 to 8 times that of the determined protein antigen. Currently, all approaches to gene therapy for factor IX therapy incorporate the factor IX Padua mutation. To date, a similarly hyperfunctioning factor VIII variant has not been found to ameliorate the poor results of gene therapy for hemophilia A.

[0087] Elevated factor VIII, like elevated factor IX, has been determined to be a risk factor for thrombosis. The inventor determined approximately 100 participants with persistently elevated factor VIII. One individual had presented with a massive perinatal stroke and over the subsequent three decades developed recurrent thrombosis in bilateral legs with bilateral pulmonary emboli, a splenic infarct, portal vein thrombosis and mesenteric vein thrombosis. Over time, his factor VIII activity was determined to be 300 increasing to 900 percent of normal. Biochemical analysis shows normal protein mass, increased activity to mass, severe resistance to activated protein C, and discordant measurement on chromogenic to one stage assays. Genetic sequencing revealed a unique point mutation at amino acid 590 in exon 12 in which the substitution of serine for arginine changed the position from neutral to ionic. The inventor posits that this factor VIII mutation is unique and promotes a continuous or accelerated activation of factor X secondary to a mutation in the A2 coding domain, similar to effects of factor VIII mimetics, emicizumab (Genentech) and mim8 (NovoNordisk) and analogous to the factor IX Padua mutation that has revolutionized factor IX gene therapy.

[0088] The Hemophilia & Thrombosis Center Research laboratory has been engaged in clinical and translational research of bleeding and clotting for the past 50 years and currently have a prospective cohort study including over 500 unique individuals (mostly pediatric) with thrombosis. In analysis of individuals in the ThromboPICS cohort (09-0816), the inventor determined approximately 100 participants with persistently elevated factor VIII. However, one individual stood out. The subject was presented with a massive perinatal stroke and over the subsequent three decades developed recurrent thrombosis in bilateral legs with bilateral pulmonary emboli, a splenic infarct, portal vein thrombosis and mesenteric vein thrombosis. His life has been compromised by complications of these recurrent thrombotic events. Over time, his factor VIII activity was determined to be 300, 600 and 900 percent of normal.

[0089] Discovery of a previously undescribed naturally occurring factor VIII variant with increased activity relative to protein mass, likely related to an amino acid change (Arginine to Serine in Exon 12, position 590) that conveys increased activation of factor IX and / or factor X. This variant could have significance in gene therapy for factor VIII, increasing protein function relative to mass, similar to the current factor FVIII mimetics, emicizumab (Genentech) and mim8 (NovoNordisk) and potentially suitable for incorporation into gene therapies.

[0090] In embodiments, an Arg590Ser variant of coagulation factor VIII (FVIII) may produce thrombin at a faster rate than wild-type FVIII and can be minimally responsive to activated protein C (APO). Coagulation evaluations may reveal that an individual carrying this variant has a normal FVIII antigen level and / or FVIII activity within the normal range by chromogenic assay, but an abnormally elevated one-stage clotting FVIII activity. This aberrant assay profile can be unique to individuals with the Arg590Ser variant and / or may resemble patterns observed with FVIII mimetic agents in FVIII activity assays (as shown in TABLE 6 below).

[0091] In embodiments, the variant FVIII may generate thrombin with an increased velocity index (e.g., approximately 196 nM / min, compared to a reference normal range of about 4-169 nM / min) as shown in FIG. 9 and FIG. 10, while other thrombin generation parameters remain within normal ranges. In some embodiments, the FVIII Arg590Ser variant may also exhibit resistance to APC. For example, the variant may show an endogenous thrombin potential (ETP) reduction of no higher than about 1 1 % upon adding thrombomodulin (e.g., observed reductions of approximately 2.3% and 10.8%), whereas normal plasma typically shows an ETP reduction of about 32%-71% under the same conditions. In addition, variant-containing samples can retain abnormally high peak thrombin levels and elevated velocity indices after the addition of thrombomodulin. (It is noted that the CAT-TM assay may be conducted at a higher temperature than the standard CAT assay, accounting for differences in the normal reference ranges.)

[0092] In embodiments, a novel APC-resistance assay specific to FVIII (APCR-FVIII assay) may further indicate that the Arg590Ser FVIII is minimally responsive to APC, while the subject’s factor V (FV) remains normally responsive to APC. Consistent with these functional findings, initial molecular docking analyses can show that the variant FVIII has a reduced binding affinity for APC, and / or that a tenase (Xase) complex incorporating the variant FVIII may have a lower binding affinity for both factor X (FX) and activated factor X (FXa) compared to a complex with wild-type FVIII. As such, the FVIII Arg590Ser variant can contribute to increased thrombin production, potentially due to a more rapid dissociation of FXa from the tenase complex and a consequent increase of free FXa in circulation. Additionally, in embodiments, structural modeling of the variant protein may reveal that the Arg590Ser substitution introduces a protruding region in the A2 domain of FVIII (see, e.g., FIGS. 3-8). This protruding region in the A2 domain can result from altered intramolecular interactions (for example, changes in the locations or strengths of certain electrostatic forces), as suggested by comparative structural analysis (see, e.g., FIGS. 1-2).

[0093] In embodiments, molecular docking analysis of APC binding to the FVIII Arg590Ser variant can be benchmarked using Factor V Leiden (FVL) as a control (as shown in TABLE 2 and TABLE 3). In such embodiments, the docking results for FVL may show differences in complex membership and weighted interaction scores that align with known clinical and laboratory data for FVL, thereby validating the docking methodology (e.g., using ClusPro software). Furthermore, embodiments of the invention can include that interactions of the variant FVIII with thrombin, phosphatidylserine, and factor IXa (FIXa) remain normal (i.e., comparable to wild-type interactions), and that all other measured coagulation protein levels and activities are normal as well, with the sole abnormal parameter being the elevated FVIII activity (see, e.g., TABLE 6). However, the Arg590Ser variant FVIII produces thrombin at an abnormally accelerated rate, and its minimal responsiveness to ARC alone would not be expected to fully account for this heightened thrombin generation.

[0094] In embodiments, factor VIII in its activated form (FVIIIa) may be normally regulated by APC via proteolytic cleavages at Arg336 and Arg562. It is noted that Arg562 resides within a binding interface for FIXa, which acts as a stabilizing agent for FVIIIa by preventing rapid dissociation of the A2 domain. Given the proximity of the Arg590Ser mutation to this regulatory region, the altered conformation of the A2 domain caused by the variant may impair APC’s ability to bind and cleave FVIIIa at the normal inactivation sites. As shown in FIG. 9 and FIG. 10, this can account for the abnormal CAT-TM results and the abnormal FVI Il-specific APCR assay results observed, as well as the lower binding affinity of the variant FVIIIa for APC indicated by docking analysis. In embodiments, it is also recognized that factor V (FV) may serve as a cofactor to protein C in the regulation (inactivation) of FVIIIa; however, there is no indication that the anticoagulant cofactor function of FV is adversely affected by the Arg590Ser mutation.

[0095] Moreover, in embodiments, the FVIIIa protein may include eight disulfide bonds in its mature form: two disulfide bonds in each of the A1 , A2, and A3 domains (SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5), and one disulfide bond in each of the C1 and C2 domains. The two disulfide bonds within the A2 domain (connecting Cys528-Cys554 and Cys630-Cys71 1 ) are located near the Arg590 residue that is mutated in the variant. Structural analyses in embodiments of the invention suggest that the Arg590Ser substitution does not disrupt any of these disulfide bonds, but rather may alter the location or distribution of certain weaker electrostatic interactions within the A2 domain (SEQ ID NO: 1 and SEQ ID NO: 2) (see, e.g., FIG. 1 and FIG. 2). This change in electrostatic interaction patterns can possibly explain the protruding region observed in the A2 domain of the variant structure (SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5) (as shown in FIG. 3, FIG. 6, and FIG. 8).

[0096] Furthermore, in embodiments, initial docking analysis of the tenase (Xase) complex involving the FVIII Arg590Ser variant can provide further insight into the variant’s procoagulant behavior. The results suggest that the extreme hypercoagulable state associated with this variant may be attributable to a combination of the FVIIIa variant’s minimal APC responsiveness and additional effects on the Xase complex. In particular, the presence of the Arg590Ser FVIIIa in the Xase complex may contribute to abnormal complex formation or stability. Normally, FIXa binds to FVIIIa at the A2 domain (including residues 558-565) and also interacts with regions around Asp712 in the A3 domain to assemble the Xase complex. The assembled Xase complex can convert factor X (FX) to its active form, FXa, by cleaving the Arg51-lle52 peptide bond on the heavy chain of FX. FXa can subsequently be converted to an inactivated form (sometimes referred to as FXap or factor Xap) by hydrolysis of an Arg-Gly peptide bond in the FXa carboxyterminal region. FVIIIa residues 337-372 serve as binding sites for FX; within this region, residues 361 -363 contribute to a unique FXa-interactive site in the FVIII heavy chain that promotes FXa docking during cofactor activation.

[0097] In addition, in embodiments, residues 2253-2270 of the FVIIIa C2 domain may be involved in interactions with FX and / or FXa. Because the Arg590Ser mutation is located near these FVIIIa regions involved in FX / FXa binding, it may be responsible for the abnormal docking interactions observed in silico. Another factor for consideration is that the stability of the FVIIIa A2 domain (and its tendency to dissociate) is tied to the activity and stability of the Xase complex. A mutation within the A2 domain, such as Arg590Ser, may then increase the effectiveness of the Xase complex by prolonging the stability of the FVIIIa molecule (i.e., slowing A2 subunit dissociation), stabilizing the complex and potentially enhancing thrombin generation.

[0098] In embodiments, various binding and activation kinetics studies can be performed to further elucidate the interactions of the variant FVIII. For example, both wild-type FVIII and the Arg590Ser variant can be produced via mammalian cell culture and used in assays (e.g., surface plasmon resonance or other binding assays). In this manner, surface plasmon resonance (SPR) has been used previously to analyze FVIII interactions (for instance, measuring FVIII binding affinity to von Willebrand factor), and a similar approach can be utilized to study the binding of the Arg590Ser variant to its physiological partners (such as FIXa, FX / FXa, and APC). Initial docking data for a tenase complex containing the Arg590Ser variant indicate a much lower binding affinity for both FX and FXa compared to a wild-type FVIII tenase complex.

[0099] In embodiments the FVIII Arg590Ser variant may become activated more rapidly than wild-type FVIII, leading to faster thrombin generation. Additionally, the modified FVIIIa may exhibit a higher effective affinity or favorability for FIXa, potentially reducing the amount of FIXa required to initiate tenase complex formation. Furthermore, the competitive nature of FIXa and APC for the FVIII binding site at Arg562 in the A2 domain could explain the rapid thrombin production observed. Due to APC having a decreased binding affinity for the Arg590Ser FVIIIa, FIXa can bind to FVIIIa more quickly and remain bound for a longer duration, inducing quicker assembly of the Xase complex and prolonging its activity. This enhanced and sustained Xase complex activity may result in the generation of greater amounts of thrombin.

[0100] In embodiments, expression of the Arg590Ser variant in an appropriate animal model may be necessary to demonstrate the thrombogenicity of the variant protein in vivo. In summary, embodiments of the present disclosure involve a hypercoagulable individual having markedly elevated FVIII activity (approximately 300%-900% of normal). An APC resistance phenotype in this individual was determined using both the CAT-TM assay and a novel ARC resistance clotting assay in which FVI Il-deficient plasma was substituted for FV-deficient plasma. A novel point mutation in the F8 gene, resulting in an Arg590Ser substitution in the FVI 11 protein, was identified in the subject (e.g., by genomic analysis using IGV). The properties of this variant FVIII (e.g., unusually high coagulant activity and minimal ARC responsiveness) could potentially be exploited in therapeutic treatments for hemophilia (e.g., hemophilia A), due to similarities to known gain-of-function variants such as Factor IX Padua. For example, one approach to improving hemophilia A therapy involves the use of FVIII variants with enhanced activity or stability (such as modifications to FVIII’s furin cleavage sites), and the Arg590Ser FVIII variant described herein offers a new candidate with hypercoagulable characteristics.

[0101] In embodiments, the Factor VIII variants comprise a mutation at position 590. In a particular embodiment, the arginine at position 560 is substituted with serine.

[0102] As stated hereinabove, the FVIII variant of the instant invention may be a mammalian cell and / or a human. In embodiments, the FVIII variant of the instant invention has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity) with SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5 (or an activated FVIII fragment thereof), particularly at least 90%, 95%, 97%, 99%, or 100% homology (identity). In embodiments, the FVIII variant comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity), particularly at least 90%, 95%, 97%, 99%, or 100% homology (identity), with amino acids 1 -740 of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5 (or an activated FVIII fragment thereof) and an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity), particularly at least 90%, 95%, 97%, 99%, or 100% homology (identity) with amino acids 1649-2332 of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5 (or an activated FVIII fragment thereof). The homology percentages above may exclude the substitution at position 590.

[0103] In embodiments, the FVIII variants of the instant invention may also be post-translationally modified. The FVIII variants may be post-translationally modified in a cell (particularly a human cell) or in vitro.

[0104] In embodiments, the FVIII variants of the instant invention have increased expression compared to wild-type FVIII. In a particular embodiment, the FVIII variants of the invention have increased FVIII activity or increased specific activity compared to wild-type FVIII.

[0105] In embodiments, nucleic acid molecules encoding the above FVIII variants may also be encompassed by the instant invention. Nucleic acid molecules encoding the variants may be prepared by any method known in the art. The nucleic acid molecules may be maintained in any convenient vector, particularly an expression vector. In embodiments, compositions comprising at least one FVIII variant and at least one carrier may also be encompassed by the instant invention. In a particular embodiment, the FVIII can be isolated and / or substantially pure within the composition. In embodiments, compositions comprising at least one FVIII variant nucleic acid molecule and at least one carrier are also encompassed by the instant invention. Except insofar as any conventional carrier may be incompatible with the variant to be administered, its use in the pharmaceutical composition is contemplated. In embodiments, the carrier is a pharmaceutically acceptable carrier for intravenous administration.

[0106] In embodiments, the disclosed CRISPR / Cas-based method may provide a strategy for treating hemophilia (e.g., hemophilia A) by directly editing the endogenous F8 gene in vivo. This can be achieved by delivering a CRISPR-associated endonuclease (preferably Cas9) and / or a guide RNA designed to target codon 590 of the F8 gene, inducing a site-specific modification at this amino acid position. As such, once the Cas9-guide RNA complex is delivered, the endogenous break in the DNA sequence can be repaired via cellular mechanisms. If a donor DNA template is co-delivered, homology-directed repair (HDR) may enable precise replacement of the codon, converting arginine to serine. Alternatively, base editing systems can achieve the same conversion without double-strand breaks, using a Cas9-nickase fused to a deaminase enzyme.

[0107] In preferred embodiments, the Cas9 endonuclease may be derived from Streptococcus pyogenes, providing robust and well-characterized activity within mammalian cells. The guide RNA can be designed to target the exon region encompassing codon 590 within the F8 gene. Off-target activity is minimized through the use of truncated guide RNAs or high-fidelity Cas9 variants as described in the literature to improve genome editing specificity. Cas9, guide RNA, and optional donor template can be delivered by AAV vectors or non-viral lipid nanoparticles optimized for hepatocyte targeting. To ensure specificity and safety, on-target cleavage and accurate gene conversion can be verified via next-generation sequencing or nanopore sequencing as established in other F8-targeting models.

[0108] In embodiments utilizing HDR, the donor DNA template can include homology arms flanking codon 590, typically extending 500-1000 base pairs upstream and / or downstream, enabling precise incorporation of the Arg590Ser mutation. The template may be delivered via AAV vectors or provided as single-stranded oligonucleotides (ssODNs), which have shown efficacy in inframe editing within F8 for hemophilia (e.g., hemophilia A) models. Selection of the HDR pathway is confirmed using molecular assays such as droplet digital PCR. The efficiency of editing may vary based on delivery route and patient-specific factors, with off-target edits evaluated using GUIDE-Seq or Digenome-Seq protocols.

[0109] In embodiments employing base editing, a catalytically impaired Cas9 nickase fused to a cytidine or adenine deaminase may be guided to codon 590 to directly convert the arginine codon (CGN) to a serine codon (AGN). Base editing has been used to effect precise point mutations in hematopoietic and other cell types without requiring double-strand breaks, reducing risks associated with HDR. The delivery format may consist of mRNA or ribonucleoprotein (RNP) complexes delivered via lipid nanoparticles or electroporation. Efficiency and / or specificity can then be determined by analyzing editing efficiency in liver tissue biopsies or circulating DNA.

[0110] In embodiments, the gene-edited cells may express the Arg590Ser variant FVIII protein with functional effects consistent with the disclosure. The variant can be resistant to APC-mediated cleavage and exhibits elevated procoagulant activity, as shown by in vitro clotting assays and thrombin-generation studies. These characteristics confirm that successful genome editing creates a phenotypically improved FVIII product without requiring exogenous protein administration.

[0111] In embodiments, the method confirms that the Arg590Ser allele can be expressed and functions in vivo after editing. Post-editing analyses include FVIII antigen assays, calibrated automated thrombogram (CAT), and CAT-TM for APC resistance, consistent with earlier disclosure parameters. Functional assays comparing pre- and post-edit expression show increased velocity index and reduced ETP suppression, consistent with APC-resistant phenotype described in earlier figures and tables. Gene modification is confirmed by sequencing analysis of peripheral blood or tissue biopsies, correlating genotype to phenotype.

[0112] In embodiments, autologous CD34-positive hematopoietic stem and progenitor cells (HSPCs) may be harvested from the patient via bone marrow aspiration or peripheral blood apheresis. As such, the cells can be purified using clinical-grade selection systems (e.g., CliniMACS) and / or can be cultured under conditions known to maintain sternness, including use of cytokines such as SCF, TPO, and / or Flt3 ligand. The purification and / or culture conditions may then match protocols used in current clinical trials for hemophilia (e.g., hemophilia A) lentiviral therapies. In this manner, a skilled artisan can select appropriate GMP-compliant reagents for large-scale cell processing.

[0113] In embodiments, purified HSPCs are transduced ex vivo with a self-inactivating, VSV-G pseudotyped lentiviral vector encoding B-domain-deleted Arg590Ser FVIII under a promoter driving sufficient expression in mature lineages. Lentiviral transduction efficiencies, multiplicity of infection, and / or vector copy number per cell may be informed by clinical results in hemophilia A trials. The disclosure may enable production of viral particles under GMP conditions and advises on target multiplicity to achieve 1-2 integrations per cell. Transduction enhancers, such as protamine sulfate, may be used to improve efficacy.

[0114] In embodiments, the transduced HSPCs are formulated and administered to the subject following conditioning with a non-myeloablative or reduced-intensity regimen (e.g., busulfan), creating marrow space for engraftment. Conditioning protocols are referenced from recent hematopoietic gene therapy trials, as known in the art. Said protocols may include dosage ranges (e.g., 4-6 mg / kg busulfan), timing, and safety monitoring procedures.

[0115] In embodiments, the engrafted, gene-modified HSPCs may differentiate into platelet or monocyte lineages capable of secreting Arg590Ser FVIII continuously. Functional plasma FVIII levels can then be measured using chromogenic and / or one-stage clotting assays following transplantation. Levels sufficient to prevent spontaneous bleeding and / or restore near-normal hemostasis may also be consistent with clinical data showing stable FVIII expression, as known in the art. CA tests (CAT / CAT-TM) may then confirm APC resistance and elevated velocity index indicative of Arg590Ser phenotype.

[0116] In embodiments, efficacy may be demonstrated in subjects with preexisting neutralizing anti- FVIII antibodies (inhibitors). Unlike protein infusion, HSPC-derived autologous expression can lead to sustained endogenous FVIII, reducing immune exposure to exogenous antigen. Clinical trial findings in hemophilia cell therapy suggest can reduce inhibitor formation when FVIII is produced autologously.

[0117] In embodiments, post-transplant monitoring may include immune chimerism, vector copy number, FVIII activity assays, and / or safety biomarkers. Data may then be collected at intervals (e.g., weeks 1 , 4, 12, 24, 52). A such, the collected data may then be reviewed to assess insertional oncogenesis risk using integration-site analysis and confers with safety surveillance strategies used in lentiviral trials (e.g., CAR-T, ADA-SCID).

[0118] The following examples are provided to illustrate various embodiments of the present invention. The examples are illustrative and are not intended to limit the invention in any way

[0119] Example #1 :

[0120] Results

[0121] One stage clotting FVIII activity on the sample studied was 615%, with FVIII antigen of 105%; the Chromo FVIII activity with Bovine and Human substrates were normal at 150 and 88%, respectively. All other screening tests were normal with plasminogen being slightly below the lower limit of normal at 65%. The standard CAT showed an increased velocity index (VI) of 196 nM / min (242% of control mean) but other indices were normal. The addition of TM in the CAT- TM reduced thrombin generation (ETP) by 2.3 and 10.8% on 2 samples (control 3SD, 28-70%). In the CAT-TM the VI was increased at 189 nM / min (527% of control mean). With WGS, IGV was used to identify three mutations, of which one point mutation R590S in Exon 12 had not been previously reported (the other 2 have been reported as benign). The standard APCR assay for factor V was normal while the APCR assay for FVIII was decreased. This indicated that mutation R590S could weaken the interaction between APC and FVIII causing FVIII to remain active thus inducing thrombosis. Clusterpro was used to simulate docking between ARC and FVIII and produced stability statistics on the interaction between wild-type (WT) FVIII and patient FVIII. The modeling system gave patient FVIII a lower favorability score for this interaction indicating that patient FVIII does not interact with ARC nearly as well as WT FVIII. Amino acid bond imaging was also done via Pymol. WT FVIII Arg590 produces multiple hydrogen bonds with the surrounding amino acids, while Ser590 produced almost none. The effects of hydrogen bond loss are examined. To investigate other possible interactions impacted by the S590 mutation a docking analysis of FIXa was produced via Clusterpro, which indicated that S590 FVIII and WT FVIII had the same affinity, indicating that mutation S590 had no impact on FIXa binding.

[0122] Materials and Methods

[0123] The sample was from an 18-year-old patient having recurrent, life-threatening arterial and venous thromboses since birth. The was found to have markedly elevated factor VIII (FVIII) activity ranging from 300-900% with normal FVIII antigen.

[0124] Assays performed on STA Compact MAX included: One-stage clotting and chromogenic FVIII (Chromo, Bovine and Human substrates), AT, PC, Fll, FV, FIX, fibrinogen, plasminogen, and VWF activities; FVIII, VWF and Protein S free antigens. Lupus anticoagulant was excluded (dRVVT, and Staclot LA, Stago). Thrombin production and reduction were measured by the Calibrated Automated Thrombogram (CAT) and the CAT - Thrombomodulin (TM) which were performed with reagents and methods per the manufacturer’s instructions using 5 pm tissue factor and TM (Stago). The CAT and CAT-TM generated lag time, peak thrombin, endogenous thrombin potential (ETP) and velocity index (VI). The clotting assay for factor V Leiden, Activated Protein C Resistance (APC-R), was performed and curated for FVIII in the lab by using FVIII deficient plasma instead of FV deficient plasma. Whole Genome Sequencing (WGS) was performed and put into Integrative Genomics Viewer (IGV). Protein docking simulations were run through three different software: Clusterpro, Hdock, and Haddock, while other amino acid models were simulated using Pymol.

[0125] Calibrated automated thrombogram (CAT) assay

[0126] Rinse the dispenser with distilled water before starting the experiment. After rinsing, reconstitute one of the three PPP reagents needed (PPP reagent = 5pM, PPP reagent LOW = 1 pM, PPP reagent HIGH = 10pM) plus the Thrombin Calibrator with the required volume of distilled water as indicated on the label. One vial of each is sufficient for an entire 96-well plate. Incubate reagents for 5-10 minutes at room temperature before mixing. Do not vortex. (Stability after reconstitution is 4 hours at room temperature. Do not freeze.) Pipette needed amount of Fluo- Buffer into a 7 ml tube as denoted in Table 1 , cap and incubate at 37°C. TABLE 1 : FLUCA SOLUTION PREPARATION TABLE

[0127] For CAT TM 2200 55 2255

[0128] A 15 mL conical tube was filled with at least 10 mL of reagent grade distilled water and incubated at 37°C, which was used to rinse the dispenser once more before dispensing the FluCa. 20 pl of reagent is pipetted for every thrombin generation well (PPP reagent, PPP LOW, HIGH, etc.) and also 20 pl Thrombin Calibrator for every calibrator well (run in triplicate). After distributing reagents and plasmas, the measurement must be started within 20 min for PRP or 40 min for PPP. 80 pl of plasma was added to every test well. Once the timer reaches 1 minute 30 seconds, the proper amount of Fluo-Substrate was added to the surface of the incubating Fluo-Buffer and the mixture vortexed immediately. The newly made FluCa solution was incubated at 37°C until it was time to prime the dispenser. After a 10 minute incubation, the reagent grade water was first removed from the water bath and the FluCa buffer was subsequently removed from the water bath. Once the plate has finished its 10 minute incubation, the manufacturer instructions for using the Fluoroskan were followed for each run.

[0129] Factor VIII (FVIII) Clotting Assay (FVIIIa)

[0130] Factor VIII (FVIII) is a glycoprotein with a molecular weight of approximately 280,000 Daltons. It is present in the liver, spleen, kidneys and lymphocytes. In plasma the FVIII circulates in a complexed form with von Willebrand factor (VWF), FVIII / VWF. In this complex, the FVIII is attached to the VWF by a noncovalent linkage. The FVIII can be activated by thrombin and FXa; it increases the activation of FX by the FIXa in the presence of phospholipids and calcium.

[0131] Pathological Variations

[0132] Hemophilia A with <1% (<0.01 lU / mL): severe hemophilia; 1 -5% (0.01 -0.05 lU / mL): moderate hemophilia; and 5-40% (0.05-0.40 lU / mL): mild hemophilia. Von Willebrand disease (VWD) the FVIII level is or less decreased according to the type of VWD. The level of FVIII may be increased in certain conditions: thromboembolic complications, coronary atherosclerosis, renal failure, diabetes, inflammatory syndrome. High levels of FVIII are a thrombosis risk factor. FVIII is decreased in presence of FVIII inhibitor. FVIII is decreased in DIC.

[0133] The STA®-Deficient VIII kit was used for the clotting assay with manufacturer’s instructions followed. Briefly, blood was collected (9 vol.) in 0.109 M (i.e., 3.2 %) trisodium citrate (1 vol.). Centrifuge all blood specimens at 2000-2500 g for 15 minutes. Each vial was reconstituted with 1 ml distilled water with the reconstituted reagent allowed to stand at room temperature (18-25° C) for 30 minutes. The vial was gently swirled to obtain a homogenous solution.

[0134] Human FVIII Chromogenic Assay

[0135] The BIOPHEN® FVI II :C kit (HYPHEN BioMed, Neuville-sur-Oise, France; Distributed by; Aniara Diagnostica. West Chester, Ohio) was used according to manufacturer instructions. The Application Sheets for Quantitative Measurement of Factor VIII:C on high range and on low range with BIOPHEN® Factor VI II :C (#221402-221406) issued by the manufacturer were used to determine settings for the quantitative measurement and are incorporated herein by reference.

[0136] When activated by thrombin, Factor VIII:C forms an enzymatic complex with Factor IXa, phospholipids and Calcium, which activates Factor X to Factor Xa. BIOPHEN Factor VII I :C is a chromogenic assay for testing the cofactor activity of Factor VIII:C, in presence of a constant amount of Factor IXa (human). Phospholipids, Calcium, IXa (human), and thrombin (human) activated Factor VI II :C form an enzymatic complex, which activates Factor X (human), supplied in the assay at a constant concentration and in excess, to Factor Xa. This activity is directly related to the amount of Factor VII l:C, which is the limiting factor in the presence of a constant and in excess amount of Factor IXa (human). Generated Factor Xa is then exactly measured by its activity on a specific Factor Xa chromogenic substrate (SXa-1 1 ). Factor Xa cleaves the substrate and releases pNA. The amount of pNA generated is directly proportional to the Factor Xa activity.

[0137] Finally, there is a direct relationship between the amount of Factor VIII :C in the assay sample and the Factor Xa activity generated, measured by the amount of pNA released, determined by the color development at 405nm. Methods reagents:

[0138] Reagent 1 Human FX and Fibrin inhibitor. Add to diluted plasma

[0139] Reagent 2: FIXa, Thrombin, CaCI and Phospholipid. Add to above and incubate at 37° C for 5 min

[0140] Reagent 3: FXa chromogenic substrate. Add to above and read change in absorbance at 405

[0141] Bovine FVIII Chromogenic Assay

[0142] The COAMATIC® Factor VIII kit (Chromogenix, Bedford MA; distributed by Diapharma, West Chester, OH) was used according to manufacturer instructions.

[0143] In the presence of calcium ions and phospholipids, factor X is activated to factor Xa by factor IXa. This activation is greatly stimulated by factor VIII which acts as a cofactor in the reaction. By using optimal amounts of Ca2+ phospholipid and bovine factor IXa, and an excess of bovine factor X, the rate of activation of factor X is linearly related to the amount of factor VIII. Factor Xa hydrolyses the chromogenic substrate S-2765, thus liberating the chromophoric group, pNA. The color is then read photometrically at 405 nm. The generated factor Xa and thus the intensity of color is proportional to the factor VIII activity in the sample. Hydrolysis of S-2765 by thrombin formed is prevented by the addition of the synthetic inhibitor 1-2581 together with the substrate.

[0144] Bovine Kit Reagents

[0145] Reagent 1: FIXa, FX, Ca and Phospholipid incubate with diluted plasma 2 min

[0146] Reagent 2: Chromogenic substrate to FXa and synthetic thrombin inhibitor added to above and change is absorbance at 405 is measured.

[0147] Enzyme Immunoassay

[0148] The ASSERACHROM® VIII:Ag enzyme immunoassay for Factor VIII was used according to manufacturer instructions.

[0149] A Factor VIII to be measured is captured by mouse monoclonal anti-human VIII:Ag antibody (Reagent 1 ) coated on the internal walls of a plastic microplate well. Next, mouse anti-human factor VIII antibodies coupled with peroxidase (Reagent 2) bind to the remaining free antigenic determinants of the bound factor VIII. The bound enzyme peroxidase is revealed by its action on the TMB substrate (Reagent 3). After stopping the reaction with a strong acid, the intensity of the color is directly proportional to the concentration of factor VIII initially present in the sample.

[0150] 3 / KIT REAGENTS

[0151] Reagent 1 : 16-well strip coated with mouse monoclonal anti-human factor VIII F(ab’)2 fragments. Reagent 2: mouse monoclonal anti-human factor VIII antibody coupled with peroxidase, lyophilized.

[0152] Reagent 3: ready for use tetramethylbenzidine (TMB < 1 %) solution.

[0153] Reagent 4: ready for use phosphate buffer.

[0154] Reagent 5: 20-fold concentrated washing solution.

[0155] Reagent 6: lyophilized human plasma containing after reconstitution, a known quantity of human factor VIII.

[0156] Reagent 7: lyophilized human plasma containing after reconstitution, a known quantity of human factor VIII.

[0157] APCR- V Assay

[0158] The COATEST™ APC™ Resistance V - 82 3120 63 kit (Chromogenix, Boston, MA) was used for the APCR-V assay according to manufacturer instructions.

[0159] The assay is used for determination of resistance to activated protein C (APC), caused by the factor V:Q506 (factor V Leiden) mutation, in plasma from untreated individuals and from patients on oral anticoagulant (OAC) or heparin therapy.

[0160] Sample plasma is prediluted in V-DEF Plasma and incubated with the APTT reagent for a standard period of time. Coagulation is triggered by the addition of CaCI2 in the absence and presence of APC and the time for clot formation is recorded.

[0161] REAGENTS

[0162] 1. V-DEF Plasma 4 vials Stabilized, lyophilized human plasma, with a low level of factor V activity, containing the heparin antagonist Polybrene®. Reconstitute with 4.0 mL of NCCLS type II water 12. Allow to stand for 30 minutes at 20-25°C. Swirl gently before use.

[0163] 2. CaCI2 1 vial 8 mL of calcium chloride, 0.025 mol / L, in Tris buffer containing 0.5% bovine serum albumin.

[0164] 3. APTT reagent 1 vial 16 mL of purified phospholipids with colloidal silica as contact activator. Contains a preservative. Mix thoroughly on a Vortex mixer before use.

[0165] 4. APC / CaCI2 4 vials Human activated protein C lyophilized with CaCI2. Reconstitute with 2.0 mL of NCCLS type II water. Allow to stand for 30 minutes at 20-25°C. Swirl gently before use.

[0166] 5. Control Plasma Level 1 1 vial Lyophilized human plasma. Reconstitute with 1 .0 mL of NCCLS type II water. Allow to stand for 30 minutes at 20-25°C. Swirl gently before use.

[0167] 6. Control Plasma Level 2 1 vial Lyophilized human plasma. Reconstitute with 1 .0 mL of NCCLS type II water. Allow to stand for 30 minutes at 20-25°C. Swirl gently before use. Factor II Deficient Plasma

[0168] Factor II deficient plasma (George King Biomedical, Inc. Overland Park, KS) was used according to manufacturer protocol. This plasma is fresh-frozen citrated human plasma from a congenital Factor II deficient donor. Plasma is stored in -70° C or colder and thawed in a 37° C water bath prior to use.

[0169] Factor V Deficient Plasma

[0170] Factor V deficient plasma (George King Biomedical, Inc. Overland Park, KS) was used according to manufacturer protocol. This plasma is fresh-frozen citrated human plasma from a congenital Factor V deficient donor. Plasma is stored in -70° C or colder and thawed in a 37° C water bath prior to use.

[0171] Factor VII Activity Assay

[0172] This assay measures the amount of factor VIII procoagulant activity in test plasma as compared to standard normal pool (normal pooled plasma). The standard normal pool (SNP) is defined as 100 % Normal or 100 U / dL. Diluted standard, control or patient plasma is incubated with factor deficient plasma and APTT reagent, then calcium chloride is added to activated sample to initiate the clotting endpoint. Surface contact (micronized silica) activates Factor XII with HMWT kininogen acting as a cofactor. Prekallikrein is activated to Kallikrein by small amounts of Xlla. Kallikrein further accelerates the activation of XII. Xlla then activates Factor XI. Xia, in the presence of calcium, activates Factor IX. FIXa forms a complex with Factor Villa in the presence of phospholipid, and calcium and subsequently also activates factor X to Xa. Factor Xa binds to Va which with calcium and the phospholipid is called “prothrombinase”, the complex that rapidly converts prothrombin to thrombin. Thrombin cleaves peptides A & B from the fibrinogen molecule forming monomers which polymerize into fibrin strands.

[0173] Clotting-time assays use a reagent system depleted of the factor activity to be measured by using a specific congenitally deficient or artificially factor-depleted plasma. The length of the time for final clot formation is therefore indirectly proportional to the concentration of the coagulation factor in question. The clotting times (seconds) of the SNP are plotted against the corresponding dilutions (% Normal or U / dL). Dilutions of test plasmas with less factor VIII have longer clotting times. The test plasma’s clotting time, in seconds, is read from the SNP calibration “curve” to obtain the % Normal Activity or U / dL.

[0174] Factor VIII deficiency may be hereditary as in Hemophilia A, carrier of Hemophilia A and, von Willebrand disease, or it may be acquired, as in DIG or acquired factor VIII inhibitor. An inhibitor to the phospholipid complex, called a lupus or lupus-like inhibitor, may be detected with this assay. The lupus-like inhibitors are seen in lupus erythematosus, autoimmune disorders, malignancies, or with viral infections. TEST AVAILABILITY AND SPECIMEN REQUIREMENTS

[0175] No special patient preparation is necessary.

[0176] Collection Requirements - Plasma: Blue top (3.2% sodium citrate) tube. Draw 4.5 mL in a 5 mL tube, 2.7 mL in a 3 mL tube, or 1 .8 mL in a 2 mL tube. As with most coagulation testing, two- vacutainer or two-syringe technique must be used. Contamination of specimen with tissue thromboplastin may lead to erroneous results. Sequence of blood collection when using vacutainer technique is red, blue, green, purple, and special tubes (grey.)

[0177] Assay Requirements - A minimum volume of citrated plasma required for testing is 300 pL. Fresh or frozen platelet free (hard spun) citrated plasma may be used. To obtain platelet free citrated plasma, spin in refrigerated centrifuge at 2500 g for 20 minutes.

[0178] Storage / Handling

[0179] The plasma may be frozen at - 80° C for 12 months. For testing, thaw the plasma at 37° C. (Never thaw at room temperature!) During testing keep sample capped and stored at 2-6°C. Assay as soon as possible. (Thawing and refreezing must be avoided) Plasma (fresh or thawed) is stable for up to 4 hours at 2-6° C.

[0180] Criteria for Specimen Rejection - Clotted sample; improper collection (anticoagulant other than 3.2% sodium citrate); improper identification; insufficient sample volume (less than 70% filled blue-top). If specimen needs to be redrawn, notify appropriately.

[0181] Test Availability: Factor VIII Activity is performed as a research test and is available to be done with the next research run as determined by research tech.

[0182] REAGENTS AND SUPPLIES

[0183] Factor VIII deficient substrate:

[0184] Substrate stored in the -80° freezer. Prepared in house from known factor deficient patients. The substrate prepared in house is labeled with the initials of the donor. VIII = BH Stored in aliquots at -80° C. Stable for 36 months. See substrate notebook for description of all QC. After 36 months, the substrates’ factor levels should be re-assayed. Thaw substrates at 37° C. Substrates will be in either 2 mL or 3 mL aliquots. Thaw total amount of substrate needed to complete assay. Once thawed, keep the substrate capped and in an ice bath or cryo rack. Stable for 4 hours at 2-6° C. Mix gently before use.

[0185] Automated APTT reagent

[0186] Manufactured by bioMerieux, Inc, Box 15969, Durham, NC 27704-0969. Stored in Puffer Hubbard. Automated APTT Reagent, product number 235512. Contains rabbit brain phospholipid and micronized silica. Reconstitute with 4 mL Type I water. Shake vigorously to insure complete hydration and suspension of the silica. Add stir bar and place in blue room temperature magnet stir well on the ST4. Unopened vials are stored at 2-6° C and are stable until manufacturer’s expiration date. Use freshly hydrated each day.

[0187] CaCI2(0.025 M)

[0188] Manufactured by bioMerieux, Inc, Box 15969, Durham, NC 27704-0969. Stored at room temperature. 0.025M CaCl2, product number 35514. This is ready to use as is. Place in the red 37°C reagent well on the ST4.

[0189] Imidazole Buffer Diluting Fluid:

[0190] 0.05 M Imidazole: Sigma #10125 (stored on bottom shelf of dry reagent cabinet), C3H4N2, FW 68.05 x 0.05=3.4g / L

[0191] 0.1 M NaCI: Fisher #S271 -10 (stored on bottom shelf of dry reagent cabinet), FW 58.44 x 0.1 =5.84g / L

[0192] 1 % BSA 10 g / L

[0193] Adjust pH to 7.3 with 10M HCI. Attach reagent label to bottle. Store at 2-8° C. Stable six months.

[0194] Dade Hepzyme:

[0195] Used to neutralize heparin. See reference: Manco-Johnson and Jacobson. Heparin neutralization is essential for accurate measurement of factor VIII activity and inhibitor assays in blood samples drawn from implanted venous access devices. (J Lab Clin Med, 2000;136:74-9). Available from Dade Behring Inc., Newark, Del. Cat. #B4240-10. Store at 2-8° C in Puffer Hubbard. Reagent is ready for use and remains stable until the expiration date printed on the label.

[0196] George King (GK) FACT reference plasma, and GK controls: Normal Pool, B-FACT, and A- FACT

[0197] Controls with 1 , 2, U / dl Recombinant FVIII in Factor VIII deficient plasma. To reconstitute vial of Recombinant FVIII follow insert directions exactly. Use the diluent provided with the factor concentrate. Methods may vary with different manufacturers. Further dilute sample to 10OOU / dL with TBS+1 %BSA. Make consequent dilutions with Factor VIII deficient 3.2% citrate plasma

[0198] Dilution Chart: 1000 U / dL FVIII def. Plasma

[0199] 2 U / dL 1 :500 0.01 ml 4.99ml

[0200] U / dL 1 :1000 0.01 ml 9.99ml Aliquot 0.3 mL into 0.5 mL labeled SpinCo® tubes. Store at -80°C. Stable for 36 months or until QC indicates otherwise. Thaw at 37°C and mix well before use. Keep on ice or in cryorack. Stable for 1 hour at 4°C.

[0201] Miscellaneous Supplies:

[0202] Plastic tubes, 3.5 ml Sarstedt #55.484; Water bath at 37° C; Vortex mixer; Cuvettes and steel balls - Available from Diagnostica Stago # 6429 and 6441 respectively; Plastic transfer pipets.

[0203] CALIBRATION

[0204] GK FACT serves as the calibrator for this procedure. Store at -80° C. Expiration stated by manufacturer. Check out new lot numbers by running old and new lot numbers concurrently in the procedure and comparing results. Thaw at 37° C and mix well before use. Keep on ice or in cryo rack. Stable for 2 hours at 2-6° C.

[0205] Calibration curve criteria - A calibration must be performed on every run. The R2 must be 0.980 or greater.

[0206] QUALITY CONTROL

[0207] GK Normal Pool, B-FACT and A-FACT prepared as described above. Acceptance limit is published value + / - 10% as listed on the control logs.

[0208] Controls with 1 , 2, 5, 10, and 50U / dl Recombinant FVIII in Factor VIII deficient plasma prepared as described above. Acceptance limit is the established mean + / - 10%.

[0209] INSTRUMENTATION

[0210] Diagnostica Stago ST4

[0211] Principle of clot detection: The clot detection system of the ST4 instrument is based on the increase of viscosity of the plasma being tested. This increase of viscosity is measured through the motion of an iron ball that is made to effect pendular swings on the two curved rail tracks provided in the bottom of the cuvette containing the test plasma. Constant ball pendular swings are created by an electromagnetic field that is applied alternately on opposite sides of the cuvette by two independent coils. The energy of the electromagnetic field can be varied depending on the test being performed. At constant plasma viscosity, ball motion remains constant. However, as soon as the plasma starts to clot, the viscosity of the plasma starts to increase, and this change in plasma viscosity affects ball movement, slowing it down. As the viscosity increases, the oscillation amplitude of the ball swing decreases. An algorithm uses these variations in oscillation amplitude to determine the clotting time. Check the maintenance to ensure that the proper monthly maintenance is performed prior to use of the instrument.

[0212] Water bath 37° C. Check and log temperature. Should be 37° C ± 0.5°. Finn pipette BioControl pipet to deliver 50, 100, 500 and 900uL, 50uL with stepper mode. See instruction manual in references.

[0213] PROCEDURAL NOTES

[0214] Hepzyme Treatment Patients known to be on heparin or known to have been drawn through a port need their plasma to be treated with Hepzyme. Add 0.5 - 1 .0 mL of patient plasma directly to a bottle of Dade Hepzyme. Invert gently 5 to 10 times. Allow it to stabilize at room temperature for 15 minutes. The specimen is now ready to be assayed. Percent Normal and Units / dL are the same.

[0215] PROCEDURE

[0216] Reconstitute the APTT reagent with 4 mL Type I water. Shake vigorously to insure complete rehydration and suspension of the silica. Add stir bar and place in blue room temperature (RT) magnet stir well on the ST4. Place the CaCl2 reagent (used as is) in red 37° C well on the ST4. Get necessary volume of factor VIII deficient plasma out of the -80° C freezer, thaw at 37° C in the water bath, and place in ice bath or cryorack. Thaw an aliquots of the standard pool, controls and patient plasma at 37° C in the water bath and place in ice bath or cryorack. Patients known to be on heparin or known to have been drawn through a port need to be treated with Hepzyme.

[0217] Prepare GK FACT, Control and Test dilutions according to chart, and keep in ice bath until used.

[0218] Samole U / dL Samole volume ul Dilutina Fluid ul

[0219] GK FACT 100 1 :10 100 900

[0220] 50 1 :20 500 ? 500

[0221] 25 1 :40 500 ? 500

[0222] 12.5 1 :80 500 ? 500

[0223] 6.25 1 :160 500 ? 500

[0224] 3.12 1 :320 500 ? 500

[0225] 1 .56 1 :640 500 ? 500

[0226] 0.78 1 :1280 500 ? 500

[0227] 0.39 1 :2560 500 ? 500 2U / dL 100 1 :10 100 900

[0228] 50 1 :20 500 T 500

[0229] The “Test Parameters” menu contains all the parameters that have been or are to be programmed for a given test. From the “Main Menu”, select “Test Parameters" by pressing [3] key and confirm with [ENT]. The screen displays the “Tests” menu. Select [4] factors and confirm with [ENT]. Choose Intrinsic Pathway Factors, and then choose Factor VIII. Set Max Time to 130 sec, Incubation Time 240 sec, Units seconds.

[0230] “Test Run”

[0231] Place 4-cuvetted strip in incubation column #1 . Add an iron ball to each cuvette. Let cuvettes incubate at 37°C for at least 3 minutes. From the “Main Menu” select “Test Mode”. From the “Test Mode” select “Factors”. From the “Factors” menu, select “lntrinsic”[2]. Choose factor to be tested. Refill the Combitip with CaCIz. Pipette 0.05 mL diluted SNP, control or test sample in cuvette #1 and 0.05 mL Factor-Deficient Plasma into the cuvette. Pipette 0.05 mL APTT reagent in cuvette #1 -4 with stepper mode and at the same time press the incubation timer key #1 . Mix cuvette by gentle shaking. When the incubation timer #1 reaches the 230-second mark, the ST3 starts to beep. Quickley transfer the cuvette strip to the test column. Immediately prime the Multipette once into the calcium chloride vial: press the [PIP] key to activate the Multipette. When the incubation column timer #1 reaches the 240-second mark, dispense the CaCIz into cuvette channel #1 -4. Reset the incubation column timer #1 to “000” by pressing the timer key #1 once. Repeat until all samples have been run.

[0232] CALCULATIONS

[0233] Average the 1 :10, 1 :20, and 1 :40 dilutions, as appropriate: The 1 :10 value is reported “as is.” The 1 :20 value is multiplied X2 and the 1 :40 value is multiplied X4.

[0234] RESULTS: Make sure the parameters below are met before reporting any patient results.

[0235] Run acceptability if the following criteria are met, accept the run:

[0236] The calibrator (GK FACT) values (100, 50, 25, etc) must be similar to the previous runs. These parameters are used primarily as a tool in troubleshooting the assay so therefore statistical ranges are not calculated. R2must be 0.9800 or greater. (1 .000 is perfect) The control values must be within the established acceptability range.

[0237] Result acceptability

[0238] Check Linearity - The results on patient dilutions can only be accepted if they are within the linear limits of the calibration curve. Verify that the result in seconds of each of the dilutions is within the range of the curve. Any patient dilution that has a seconds value outside the curve limits must not be used for calculation of the final result. If the results are below the linearity of the curve, report as < or > or repeat with further dilutions.

[0239] Dilution agreement - The dilutions must agree within 10% ± the mean of the dilutions. If the dilutions do not meet this criteria, rerun the assay with fresh dilutions. If the same result is obtained, consider whether a factor inhibitor is present.

[0240] Abnormal results - repeat all abnormal results on a new run with new dilutions unless the patient has a known abnormality or results are consistent with the diagnosis or clinical condition.

[0241] Result Evaluation

[0242] Inhibitory Pattern - Dilutional effect on Non-specific Inhibitor

[0243] A trending percent result is defined as values that increase or decrease in succession and display a difference of greater than ±10. An inhibitory pattern may be the result of a poor run, in which case, a repeat run with fresh dilutions will result in reportable results. If the inhibitory pattern does not resolve, see section b. (below) for instructions on how to report the patient result.

[0244] Inhibitory Pattern that Corrects.

[0245] If a trend exists, repeat the factor assay with fresh dilutions. If the results from the repeat run agree, then average all three results and report the average.

[0246] Example: Factor VIII 1 :10 dilution 54 U / dL

[0247] Factor VIII 1 :20 dilution 60 U / dL

[0248] Factor VIII 1 :40 dilution 68 U / dL

[0249] Repeat VIII 1 :10 dilution 59 U / dL

[0250] Repeat VIII 1 :20 dilution 60 U / dL

[0251] Repeat VIII 1 :40 dilution 64 U / dL

[0252] Results of 59, 60, and 64 are averaged for the reported result. Inhibitory Pattern that Persists.

[0253] If a trend persists after the 1 :40 is repeated then make and assay a 1 :80, a 1 :160 and a 1 :320 dilution. Look for a plateau in results which is defined as 2 or 3 successive results in a row that no longer exhibit a trend. (See Section X., B., 2., Dilution agreement, above.) It may be necessary to run a 1 :320 and 1 :640 to obtain a plateau. Do not run higher than a 1 :640 dilution.

[0254] Example: Factor VIII 1 :10 dilution 24 U / dL

[0255] Factor VIII 1 :20 dilution 36 U / dL

[0256] Factor VIII 1 :40 dilution 56 U / dL

[0257] Factor VIII 1 :80 dilution 70 U / dL

[0258] Factor VIII 1 :160 dilution 80 U / dL

[0259] Factor VIII 1 :320 dilution 81 U / dL

[0260] To result patients with an inhibitory pattern that persists, report the lowest percent result and the first dilution where a plateau starts (or the 1 :640 results if no plateau is reached.)

[0261] Example: Inhibitory pattern noted.

[0262] Factor VIII activity 1 :10 = 24 U / dL.

[0263] Factor VIII activity 1 :160 = 80.5 U / dL.

[0264] Reporting results

[0265] Reference Range: Factor VIII: 62.4-161.8 U / dL

[0266] Critical Values: Factor VIII: 20.0 U / dL

[0267] LIMITATIONS OF THE PROCEDURE

[0268] Assay should not be effected by heparin unless >0.5 U / mL. (If the patient is on heparin, see above regarding the use of Hepzyme.) Icteric plasma will not interfere with the accuracy of this test. The same is true for specimens that exhibit in vivo hemolysis.

[0269] Chromogenic assay for measuring plasminogen activity in plasma

[0270] The BIOPHEN® Plasminogen kit (Hyphen BioMed, Neuville-sur-Oise, France) was used for this assay according to manufacturer protocols.

[0271] Assay of Plasminogen in human plasma for the diagnosis of congenital or acquired Plasminogen deficiencies. An abnormal Plasminogen activity is an indicator for fibrinolytic troubles. Plasminogen (Pig) is the plasma precursor for the fibrinolytic enzyme plasmin, which is generated following plasminogen activation by specific biological activators such uPA and tPA, or pharmacological activators such as streptokinase.

[0272] Using the BIOPHEN Plasminogen assay, Plasminogen is measured following its specific activation by addition of streptokinase and plasminogen-free fibrinogen in excess. The complex formed between plasminogen and streptokinase possesses a "plasmin-like" activity, which then specifically cleaves the plasmin-specific substrate SPm41 , releasing para-nitroaniline (pNA), which color is measured at 405nm. There is a direct relationship between color development and Plasminogen activity in the tested plasma.

[0273] REAGENTS:

[0274] R1 : Reagent 1: Streptokinase.

[0275] Activation reagent containing streptokinase (about 25,000 IU) and plasminogen-free fibrinogen, lyophilized and stabilized. 2 vials(to be reconstituted with 2.5ml of distilled water). Shake thoroughly until complete dissolution of the contents (vortex). Incubate at room temperature for 30 min, shaking vial from time to time. Homogenize the content before each use.

[0276] R2: Reagent 2: Plasmin specific chromogenic Substrate

[0277] Chromogenic substrate, specific for plasmin and 'plasminogen-streptokinase- complexes (SPm41 ), lyophilized: 2 vials of about 6.25 mg (to be reconstituted with 2.5 ml of distilled water). Shake thoroughly until complete dissolution of the contents (vortex). Incubate at room temperature for 30 min, shaking vial from time to time. Homogenize the content before each use.

[0278] PREPARATION OF PLASMA

[0279] Blood (9 volumes) must be collected on 0.109 M citrate anticoagulant (1 volume), with great care, in a silicon glass or a plastic lube. Sampling must be performed through a net venipuncture, avoiding any blood activation. Within 4 hours, blood must be centrifuged at 3,000 g for 20 min at 18°C or below, and plasma decanted into a plastic tube, using a plastic pipette.

[0280] Microlatex Particle-Mediated Immunoassay for Antithrombin

[0281] The LIATEST® AT III kit (Diagnostica Stago S.A.S., France) was used according to manufacturer protocol.

[0282] This assay is based on the change in turbidity of a microparticle suspension that is measured by photometry. A suspension of latex microparticles, coated by covalent bonding with antibodies specific for AT, is mixed with the test plasma whose AT antigen level is to be assayed. An antigen-antibody reaction takes place, leading to an agglutination of the latex microparticles which induces an Increase in turbidity of the reaction medium. This increase in turbidity is reflected by an increase in absorbance, the latter being measured photometrically. The increase in absorbance is a function of the AT level present in the test sample.

[0283] Reagent 1 is a suspension of microlatex particles coated with rabbit anti-human AT antibodies. Reagent 2 is glycine buffer.

[0284] Specimen collection and treatment - Blood (9 vol.) is collected in 0.109 M (i.e., 3.2 %) trisodium citrate anticoagulant (1 vol.) Centrifugation: 15 minutes at 2000-2500 g. Plasma storage: 8 hours at 20 ± 5 °C, 1 month at - 20 °C. Thaw the sample at 37 °C, allow sufficient time to obtain complete thawing.

[0285] Enzyme-linked immunosorbent assay for Protein C

[0286] The ASSERACHROM® Protein C kit (Diagnostica Stago S.A.S, France) was used according to manufacturer protocols.

[0287] The protein C to be measured is captured by specific rabbit anti-human protein C antibodies (Reagent 1 ) coated on the internal walls of a plastic microplate well. Next, rabbit anti-protein C antibodies coupled with peroxidase (Reagent 2) bind to the remaining free antigenic determinants of the bound protein C. The bound enzyme peroxidase is revealed by its action on the TMB substrate (Reagent 3). After stopping the reaction with a strong acid, the intensity of the color is directly proportional to the concentration of protein C initially present in the plasma sample.

[0288] Kit Reagents

[0289] Reagent 1 : 16-well strip coated with specific rabbit anti-human protein C F(ab')2, fragments. Allow Reagent 1 to stand at room temperature (18-25 °C) for 30 minutes before opening. The strips are then ready for use. Begin the test as soon as the strips are removed from the packet.

[0290] Reagent 2: specific rabbit anti-human protein C antibodies coupled with peroxidase, lyophilized. Reconstitute each vial of Reagent 2 with 8 ml of Reagent 4 (R4). Allow the solution to stand at room temperature (18-25° C) for 30 minutes. Then, vortex the vial before use. Due to the characteristics of Reagent 4, the Reagent 2 is regarded as sensitizing after reconstitution. Reconstituted stability: 4 hours at 20 ± 5 °C.

[0291] Reagent 3: ready for use tetramethylbenzidine (TMB < 1 %) solution. Allow Reagent 3 to stand at room temperature (18-25 °C) for 30 minutes. Then use immediately.

[0292] Reagent 4: ready for use phosphate buffer. Allow the bottle (R4) to remain at room temperature (18-25 °C) for 30 minutes, before use. Stability after opening: 15 days at 2-8 °C, when free of any contamination.

[0293] Reagent 5: 20-fold concentrated washing solution. Dilute 1 :20 with distilled water before use. For 2 strips (32 wells), use 15 ml of Reagent 5 and add distilled water to a final volume of 300 ml. Stability after dilution: 15 days at 2-8° C, when free of any contamination. The presence of crystals will not affect the quality of the reagent. If necessary warm at 37 °C until all crystals have dissolved

[0294] Reagent 6: lyophilized human plasma containing, after reconstitution, a known quantity of protein C (see the Assay Value insert provided in the kit) This quantity is determined against a secondary standard of the 02 / 342 International Standard for protein C established in 2006. Reconstitute each vial of Reagent 6 (R6) and Reagent 7 (R7) with exactly 0.5 ml of distilled water. Allow the solution to stand at room temperature (18-25 °C) for 30 minutes Then, vortex the vial before use. Reconstituted stability: 4 hours at 20 ± 5 °C.

[0295] Reagent 7: lyophilized human plasma containing, after reconstitution, a known quantity of protein C (see the Assay Value insert provided in the kit). This quantity is determined against a secondary standard of the 02 / 342 International Standard for protein C established in 2006.

[0296] Specimen collection and treatment

[0297] Blood (9 vol.) is collected in 0.109 M (i.e., 3.2 %) trisodium citrate anticoagulant (1 vol.) Centrifugation: 15 minutes at 2000-2500 g. Plasma storage: 8 hours at 20 + 5 °C, 1 month at - 20 C. Thaw the sample at 37°C, allow sufficient time to obtain complete thawing.

[0298] Colorimetric Assay of Protein C

[0299] The STACHROM® Protein C kit (Diagnostica Stago S.A.S, France) was used according to manufacturer protocols.

[0300] Protein C is activated by the specific activator (Reagent 1 ) derived from the venom of Agkistrodon c. contortrix. The quantity of enzyme thus formed is measured by its amidasic activity on the synthetic chromogenic substrate CBS 42.46 (Reagent 2) (paranitroaniline release at 405 nm). The intensity of the color produced is directly proportional to the level of protein C initially present in the test plasma.

[0301] KIT REAGENTS

[0302] Reagent 1 : highly purified extract of Agkistrodon c. contortrix venom (specific activator of protein C), lyophilized. Reconstitute each vial with 3 ml of distilled water. Allow the reconstituted reagent to remain at room temperature (18-25 °C) for 60 minutes. Swirl vial gently; then, install a new STA® - mini Reducer (REF 00797) in the vial and the perforated cap on top. Reconstituted stability in original vial with STA® - mini Reducer and perforated cap in place: 21 days on STA- A® and STA Compact®.

[0303] Reagent 2: chromogenic substrate CBS 42.46. THC-Pro-Arg-pNA, AcOH approx. 15 pmoles per vial, lyophilized. Reconstitute each vial with 6 ml of distilled water. Do not shake. Allow the reconstituted reagent to remain at room temperature (18-25 °C) tor 60 minutes. Swirl vial gently; then, install a new ST A® - mini Reducer (REF 00797) in the vial and the perforated cap on top. Reconstituted stability in original vial with ST A® - mini Reducer and perforated cap in place: 21 days on STA-A® and STA Compact®.

[0304] Specimen collection and treatment

[0305] Blood (9 vol.) is collected in 0.109 M (i.e., 3.2 %) trisodium citrate anticoagulant (1 vol.) Centrifugation: 15 minutes at 2000-2500 g. Plasma storage: 8 hours at 20 + 5 °C, 1 month at - 20 C. Thaw the sample at 37°C, allow sufficient time to obtain complete thawing.

[0306] Clotting Assay of Protein C Activity

[0307] The STACLOT® Protein C kit (Diagnostica Stago S.A.S, France) was used according to manufacturer protocols.

[0308] Protein C is activated by the specific activator (Reagent 1 ) derived from the venom of Agkistrodon c. contortrix. The resulting activated protein C inhibits the factors V and VIII, and thus prolongs the APTT of a system in which all the clotting factors are present, constant and in excess (provided by the Reagent 1 ), except the protein C which is derived from the sample being tested.

[0309] KIT REAGENTS

[0310] Reagent 1 : lyophilized human plasma, free of protein C.

[0311] Reagent 2: highly purified Agkistrodon c. contortrix venom capable of activating protein C specifically, freeze-dried with an activator specifically for protein C assay.

[0312] REAGENT PREPARATION AND STORAGE

[0313] Preparation - Reconstitute each vial of Reagent 1 or 2 with exactly 1 ml of distilled water. Allow the solution to stand at room temperature (18-25 °C) for 30 minutes. Then, swirl the vial to mix before use.

[0314] Storage - The reagents in intact vials are stable until the expiration date indicated on the box label, when stored at 2-8 °C. Once reconstituted, the Reagents 1 and 2 remain stable for 8 hours on STA Compact" and STA-A®. Do not freeze.

[0315] Specimen collection and treatment

[0316] Blood (9 vol.) is collected in 0.109 M (i.e., 3.2 %) trisodium citrate anticoagulant (1 vol.) Centrifugation: 15 minutes at 2000-2500 g. Plasma storage: 4 hours at 20 + 5 °C, 1 month at - 20 C.

[0317] Anti-Thrombin (AT), PC and PS Assay

[0318] ATIII Reagents (Lot #257375) AT3 Reagent 1 : Thrombin, reconstitute by pouring 3 ml Reagent 3 ( diluent), swirling, inc RT 45min / 15min on board CM, combine before loading w / Mini-reducer. Double batch. Storage: 7 day on board Compact Max.

[0319] AT3 Reagent 2: Chromo substrate, reconstitute by adding 3 ml H2O, swirling, incubate RT 45min / 15min on board CM, combine before loading w / Mini-reducer. Double batch. Storage: 7 day on board Compact Max.

[0320] AT3 Reagent 3: Diluent for Reagent 1 , 3 ml. VIGOROUSLY shake before using as is. Double batch. Storage: 7 day on board Compact Max.

[0321] Chromo PC Reagents (Lot #255605)

[0322] Chromo PC Reagent 1 : Activator , reconstitute with 3 ml water, swirl, incubate RT 45min / 15min on board CM, combine before loading with Mini-reducer. Double batch. Storage: 21 day on board Compact Max.

[0323] CR PC Reagent 2: Substrate, reconstitute with 6 ml H2O, swirl, incubate RT 45min / 15min on board CM, Mini-reducer. Double batch. Storage: 21 day on board Compact Max.

[0324] PS Free Reagents (Lot #257379)

[0325] PS Free Reagent 1 : Buffer 5ml, Incubate at RT, 15 min, swirl, add mini reducer and load on Compact Max 15 min prior to using. Double batch. Storage: 5 day on board Compact Max.

[0326] PS Free Reagent 2: Latex beads with 2 ab to PS Free 6 ml, Incubate at RT, 15 min, swirl, add mini reducer and load on Compact Max 15 min prior to using. Double batch. Storage: 5 day on board Compact Max.

[0327] System Unicalibrator (not sp20) (Lot #256273): Reconstitute w / 1 ml H2O, RT 15 min, swirl intermittently, load on Compax max 15 min to equilibrate. 1 vial for AT and PC (PS pre cal). Storage: 4 hr on board CM.

[0328] System Control N and P (Lot #256735): Reconstitute w / 1 ml H2O, RT 15 min, swirl intermittently, load on Compax max 15 min to equilibrate. 1 vial for AT3 and PC Chromo. Storage: 8 hr on board CM.

[0329] LIA control N and P (Lot #256531 ): Reconstitute w / 1 ml H2O, RT 15 min, swirl intermittently , load on Compax max 15 min to equilibrate. 1 vial for PS Free ag. Storage: 8 hr on board CM.

[0330] Procedure:

[0331] Set out all reagents and mini-reducers, label tops. Reconstitute AT3 and PC Chromo first (60 min), PS is already in solution and only needs 15 min at RT. Reconstitute Unicalibrator, System N&P and Lia N&P ( only need 30 min but longer is OK). At 45 min into incubation, load reagents so they can equilibrate to the instrument. When 60 min total inc time is reached, run the calibrations. Find each test, double click, check the lot number and change if nec. If new, scan in barcodes from package insets as prompted. These tests will automatically run the controls (Sys N&P for AT3 and Cr PC, Lia N&P for Free PS). Print the Curves for each. When all calibrations are complete and controls have passed, then thaw patient samples, pool and load mini-tubes in brass holders in order. Load samples in drawer, THEN enter each subject#, check mini-tube, lift and drop to assign location, then order tests. For this set of three combined tests, a profile with all three were created. Back up to main menu and instrument will automatically begin.

[0332] Heparin does not interfere with these tests. OK buffer 1 vial. Desorb 2 vials. Run SP2020 as control.

[0333] Immuno-Turbidimetric Assay of von Willebrand Factor

[0334] The STA-LIATEST® VWF:Ag kit (Diagnostica Stago S.A.S, France) was used according to manufacturer protocols.

[0335] This assay is based on the change in turbidity of a mlcroparl icte suspension that is measured by photometry. A suspension of latex microparticles, coated by covalent bonding with antibodies specific for VWF, is mixed with the test plasma whose VWF antigen level is to be assayed. An antigen-antibody reaction takes place, leading to an agglutination of the latex microparticles which induces an increase in turbidity of the reaction medium. This increase in turbidity is reflected by an increase m absorbance, the latter being measured photometrically. The increase m absorbance is a function of the VWF level present in the test sample.

[0336] KIT REAGENTS

[0337] Reagent 1 : glycine buffer. Before opening, allow the Reagent 1 (R1 ) vial to stand at room temperature (18-25 °C) for 15 minutes. Swirl the vial gently. Then, place a new STA41-1 mini Reducer (REF 00797) and lhe perforated cap on the vial. With STA® mini Reducer and perforated cap in place the stability of Reagent 1 after vial opening is 15 days on STA-R® and STA Compact®.

[0338] Reagent 2: suspension of microlatex particles coated with rabbit antihuman VWF antibodies, then stabilized (with bovine albumin). Pour the entire contents of a vial of Reagent 3 (A3) into a vial of Reagent 2 (A2) of the same kit. Ensure that all drops of Reagent 3 are transferred. Swirl the Reagent 2 vial to mix well without creating any bubbles. Allow the solution to stand at room temperature (18-25 °C) for 15 minutes. Swirl vial gently. Then, place a new STA®-mini Reducer (REF 00797) and the perforated cap on the vial. With STA®-mini Reducer and perforated cap in place the stability of Reagent 2 after dilution is 15 days on STA- and STA Compact®.

[0339] Reagent 3: solution containing glycine for dilution of Latex reagent (Reagent 2). Ready for use. Specimen Collection and Treatment

[0340] Blood (9 vol.) is collected m 0.109 M (i.e., 3.2 %) trisodium citrate anticoagulant (1 vol.). Centrifugation is 15 minutes at 2000-2500g. Plasma storage: 8 hours at 20 ± 5 °C, 24 hours at 2-8 °C, 1 month at -20 °C. Thaw the sample at 37 °C, allow sufficient time to obtain complete thawing. Do not freeze a second time. von Willebrand Factor Antigen

[0341] The HemosIL® von Willebrand Factor Antigen kit (Instrumentation Laboratory Company, Bedford, MA) was used according to manufacturer protocol.

[0342] The diagnosis of von Willebrand disease (VWD), probably the most common congenital bleeding disorder, requires a number of special tests at the laboratory level. Among them, VWF:Ag determination is essential and must be performed on every patient to reach a proper diagnosis. Depending upon these laboratory findings, VWD is classified into type 1 (the most frequent form being 70-80% of VWD), type 2 or type 3 (1 to 3% of VWD) groups. Type 1 shows a reduction of VWF although its structure and functionality is normal.

[0343] In type 3, VWF is almost absent in plasma. In type 2 the quantity of VWF in plasma may be normal or slightly reduced but its molecular structure and its functionality is abnormal. Type 2 may be further characterized into subtypes by multimeric structure analysis of VWF. Apart from the above described inherited VWD, acquired VWD due to autoantibodies or to various disease states resulting in low rates of VWF synthesis has been reported. On the other side, chronic or acute inflammatory diseases or processes involving damage of the vascular endothelium yield abnormally high concentrations of VWF.

[0344] The VWF:Ag kit is a latex particle enhanced immunoturbidimetric assay to quantify VWF:Ag in plasma. When a plasma containing VWF:Ag is mixed with the Latex Reagent and the Reaction Buffer included in the kit, the coated latex particles agglutinate. The degree of agglutination is directly proportional to the concentration of VWF:Ag in the sample and is determined by measuring the decrease of transmitted light caused by the aggregates.

[0345] REAGENTS

[0346] Latex Reagent (Cat. No. 0020002310): 2 vials x 3 mL of a suspension of polystyrene latex particles coated with a rabbit polyclonal antibody directed against VWF containing bovine serum albumin, buffer, stabilizer and preservative. Invert to mix prior to use.

[0347] Reaction Buffer (Cat. No. 0020002320): 2 vials x 4 mL of HEPES buffer containing bovine serum albumin, stabilizers and preservative. Invert to mix prior to use.

[0348] Detection of lupus anticoagulants (LA) The STACLOT® DRW kit, including STACLOT® DRW SCREEN 2, STACLOT® DRW SCREEN 5, and STACLOT® DRW CONFIRM, (Diagnostica Stago S.A.S., France) was used according to manufacturer protocol. Specific test set up is shown in Figure 12.

[0349] Lupus anticoagulants are antibodies directed against phospholipid / protein complexes. They have the ability to prolong the clotting times of the phospholipid-dependent tests. Lupus anticoagulants are associated with numerous clinical states such as auto-immune diseases (systemic lupus erythematosus), thromboses, recurrent spontaneous abortions and infections. Their presence nay be persistent or transitory.

[0350] The Russell's viper venom is present in the ST A® - Staclot® dRW Screen and ST A® - Staclot® dRW Confirm reagents. This venom acts in presence of calcium as an activator of factor X and thereby triggers the coagulation cascade downstream from factor X, thus eliminating the influence of coagulation factors acting upstream. This test is not affected by the contact factor anomalies or by factor VIII and IX deficiencies or inhibitors. The STA9 - Staclot® dRW Screen test is performed with a low concentration of phospholipids. If LA are present, the clotting time will be lengthened.

[0351] The STA® - Staclot® dRW Confirm contains a higher concentration of phospholipids which neutralize the LA present in the plasma to be tested. Therefore, the clotting time obtained with the STA®- Staclot® dRW Confirm will be shorter than the one observed with the STA®- Staclot® dRW screen.

[0352] KIT REAGENTS

[0353] The STA®-Staclot® dRW Screen and STA®-Staclot® dRW Confirm reagents contain Russell's viper venom, phospholipids, calcium and 1 eparin inhibitor (UFH), lyophilized.

[0354] Preparation:

[0355] Reconstitute each vial of:

[0356] STA®-Staclot® dRW Screen ® (REF 00339) with 2 ml of distilled water

[0357] ST A®- Staclot® dRW Screen @ (REF 00333) with 5 ml of distilled water

[0358] STA®-Staclot® dRW Confirm (REF 00334) with 2 ml of distilled water. Allow the solution to stand at room temperature (18-25 °C) for 30 minutes. Swirl the vial gently to obtain a homogeneous suspension. Then, place a new STA®- mini Reducer (REF 00797) and install the perforated cap.

[0359] Storage - The reagents in intact vials are stable until the expiration date indicated on the box label, when stored at 2-8 °C. Reconstituted reagents with the STA®-mini Reducer and perforated plastic cap in place remain stable for 72 hours on the STA® Compact and STA-R®. Specimen collection and treatment

[0360] Blood (9 vol.) is collected in 0.109 M (i.e., 3.2 %) trisodium citrate anticoagulant (1 vol.). Centrifugation: plasma should be as platelet-free as possible. Perform a centrifugation during 15 minutes at 2000-2500 g. Collect the plasma supernatant and repeat the centrifugation step. The platelet count should be less than 10 x 105 / l (< i.e., 10,000 / mm3).

[0361] If the plasmas are collected in the conditions specified above, they may be kept: 4 hours at 20 ± 5 °C or 1 month at -20 °C. Thaw the sample at 37 °C, anow sufficient time to obtain complete thawing.

[0362] Example #2:

[0363] Standard pool participants (control) were recruited with written informed consent under Colorado IRB #09-0816. After signing informed consent, blood was collected by peripheral venipuncture technique into BD Vacutainer 3.2% buffered sodium citrate collection tubes (Becton-Dickenson, Franklin Lakes, New Jersey), after an initial discard tube was drawn, and processed within 45 minutes. For plasma studies, samples were centrifuged twice at 2500 RCF for 15 minutes at 4°C. Samples were required to have normal PT, aPTT, and fibrinogen for inclusion into the pooled normal plasma. Following verification, plasma from 24 healthy individuals with no personal or family history of thrombosis or bleeding was pooled and divided into aliquots for storage at -80°C.

[0364] The participant with a FVIII variant was recruited under COMIRB #05-0339, “Prospective, Inspectional, Cohort Study of Individuals with Thrombosis, Stroke, Thrombophilia, and Prothrombotic Conditions” (Thrombo-PICS). After signing informed consent, blood was collected by peripheral venipuncture and processed and stored (same as controls).

[0365] Calibrated Automated Thrombogram (CAT)

[0366] The thrombinoscope software can quantify molar concentrations of thrombin as it develops in clotting plasma using a fluorogenic substrate and the Fluoroskan Ascent plate reader from Thermo Fisher®. Plasma samples are compared to their own internal control wells incubated with thrombin calibrator to determine thrombin generation. CAT analysis was performed on normal and variant FVIII plasma using 5pM tissue factor to obtain whole plasma analysis of thrombin generation (Stago).

[0367] Calibrated Automated Thrombography with and without Thrombomodulin (CAT-TM)

[0368] The CAT-TM was performed with reagents and methods per the manufacturer's instructions using 5 pM tissue factor and thrombomodulin (TM, Stago) to determine thrombin generation area under the curve (endogenous thrombin potential, ETP) and thrombin reduction after an addition of TM to obtain % ETP reduction. STA Compact MAX / ST A Fl MAX

[0369] The aPTT, PT, FVIII activity (one stage clotting, FVIIIc), chromogenic FVIII activities, with both FVIII bovine (Chromogenix) and human (Hyphen BioMed) substrates, fibrinogen (FIB), DRVVT, StaClotLA, antithrombin (AT), protein C chromogenic, protein S free (LIA), factor IX activity, FV activity, factor II activity, Von Willebrand factor antigen, and activity (Instrumentation Laboratory) and plasminogen activity were measured on a Stago automated coagulation analyzer (STA Compact Max®or STA R Max®), per the manufacturer’s instructions. All assays are Stago unless specified.

[0370] FVIII Antigen

[0371] FVIII antigen (FVIII ag) was measured using Asserachrom VIII: Ag ELISA (Stago) as per manufactures instructions and read at 450 nm on BioTek Microplate Reader (Agilent Technologies).

[0372] Whole Genome Sequencing (WGS)

[0373] DNA was isolated from buffy coat using QIAamp DNA Mini Kit. The gDNA purity, quantity and size distribution was determined with Qubit (Invitrogen) and TapeStation 4200 (Agilent) analysis prior to DNA-seq library preparation. An input of 55ng of the gDNA was mechanically sheered (Covaris) targeting 300 - 400bp DNA products and the Ovation Ultralow System V2 kit (Tecan) was used to generate DNA-Seq libraries. Paired-end sequencing reads of 150bp was generated on NovaSeq 6000 (Illumina) sequencer at a target depth of 600 million paired-end reads per sample. Raw sequencing reads were de-multiplexed using bc!2fastq. WGS analysis was performed using Integrated Genome Viewer (IGV).

[0374] Factor V Leiden (FVL) and Prothrombin G20210A Mutation (PTM)

[0375] PCR was used to confirm the absence of the FVL, and PTM mutations.

[0376] APCR-V / VIII

[0377] Chromogenix Activated Protein C Resistance V was analyzed using Stago automated coagulation analyzer (STA R Max®). For the novel activated protein C resistance FVIII assay: Chromogenix Activated Protein C Resistance V assay kit was used with FVIII deficient plasma (Stago) instead of FV deficient plasma from the Chromogenix assay and measured on the STA R Max®. A standard normal range was achieved from 32 normal individuals.

[0378] Amino Acid Bond Imaging

[0379] A variant model of FVIII was generated by introducing the mutation into the original structure obtain from the Protein Data Bank (PDB ID: 3CDZ).33Pymol was used which is a versatile molecular visualization and analysis tool (PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC.). Utilizing PyMOL’s mutagenesis tool, the mutation was introduced into the wild-type (WT) FVIII structure and generated a corresponding mutant structure to subsequently analyze the structural impact of the mutation. Specifically, hydrogen bonds included in WT and mutant structure were assessed to elucidate potential disruptions in protein stability and function. This analysis aimed to elucidate potential disruptions in protein stability and function induced by the mutation. UCSF Chimera was also used to elucidate how other intermolecular forces were impacted by the variant amino acid presence.34

[0380] Docking Analysis of Variant FVIII

[0381] The docking analysis of variant FVIII was executed utilizing ClusPro4, a sophisticated molecular modeling platform.35High-fidelity protein data were meticulously curated from reputable academic repositories, including the NCBI database, to construct precise 3D models of FVIII and its interacting partners. These models were subjected to rigorous validation and optimization protocols to ensure structural accuracy prior to docking simulations. The impact of site-directed mutagenesis on protein-protein interactions was systematically investigated, with docking outcomes analyzed to elucidate alterations in binding affinities and interaction dynamics attributable to the introduced mutations.

[0382] Results

[0383] Coagulation Evaluation

[0384] The individual’s one-stage FVIII activity of 370, 615, 900%N (on three different draws) led to a coagulation evaluation being performed. These tests included: FIB, APCR-V, AT chromo, Fll act., FIX act., FVIII bovine and human chromo., FVIII antigen, PS free, FV act., PC chromo, vWF ag., vWF act., StaClotLA, DRVVT, PTM PCR, and FVL PCR. The chromogenic FVIII activity was 88% with human substrates and 150% with bovine reagents. All other results were normal except a slightly elevated D-dimer while on anticoagulation. The FVIII antigen was normal on two draws (108 and 105%), normal range: 46-158%. These results were unique to the variant FVIII individual because all other participants who had an abnormally high FVIII activity also had similarly elevated FVIII antigen. Thus, the individual had a normal amount of FVIII yielding an abnormally high amount of activity.

[0385] Plasma Thrombin Generation

[0386] CAT data showed that the variant FVIII plasma had an increased velocity index while all other parameters were normal, as shown in FIG. 10. The results indicated that the variant FVIII increased the rate at which thrombin was produced. The CAT-TM resulted in a very low ETP% reduction on both samples with values of 2.3 and 10.8%, as shown in FIG. 9, which is outside of the 2SD range (32%-71%), suggesting that the variant FVIII might be contributing to a resistance to activated protein C.

[0387] Activated Protein C Pesistance Assay FVIII To determine if the variant F VI 11 itself had a resistance to ARC an APCR FV Chromogenix assay was used as per the manufacturer’s instructions, except the FVIII deficient plasma was substituted from Stago for FV deficient plasma. Results yielded that the variant FVIII had decreased responsiveness to ARC when compared to normal individuals. The standard pool yielded an APCR ratio of 4.05 (NR:3.1 -4.6) while the variant FVIII yielded an APCR ratio of 2.74, indicating that the variant FVIII had minimal responsiveness to APC.

[0388] Whole Genome Sequencing Analysis

[0389] Whole genome sequencing and analysis using IGV showed three-point mutations within exons 5, 12 and 14 within the F8 gene in the participant (i.e., subject). The Arg648Arg mutation is silent and has no known effects on the function of the FVIII protein and the Asp1260Glu mutation has been reported to be benign from other clinical studies. The third point mutation was novel, located on chromosome X, exon 12, at amino acid position 590, and changed a cytosine to an adenosine that caused the amino acid at position 590 to change form an arginine to a serine, which have different chemical functional groups. Amino acid position 590 is located in the A2 domain of FVIII. The location of the mutation was near both a FIXa binding and an APC cleavage site which could possibly explain the abnormal CAT-TM results. It should also be noted that this mutation was not near the interaction sites of thrombin, vWF, phosphatidylserine, or FXa.

[0390] Docking Analysis of Variant FVIII

[0391] Docking analysis using Cluspro of the interaction between variant FVIII and APC was compared to WT FVIII. Results indicated that variant FVIII had a lower binding affinity for APC than WT FVIII, as shown in TABLE 2, provided below. A control was also established for Cluspro. This control was obtained by analyzing the interaction between FVL and APC, due to the extensive understanding of the FVL mutation, and how it impacts APC interaction, as shown in TABLE 3, provided below. FVL had a significant difference in weighted scores and members when compared to wild type FV indicating that Cluspro produced results that match clinical and lab findings. Further docking analysis was done with FIXa, vWF, and thrombin all of which were normal. Docking analysis was also performed on the Xase complex with FX and FXa interaction and in the variant FVIII Xase complex. The binding affinity was lower for both interactions which might indicate more rapid activation and dissociation of FXa with the variant FVIII, as shown in TABLE 4 and TABLE 5, provided below. If supported, these data could explain more rapid thrombin production. As shown in FIGS. 3 - 8, it has been indicated that variant FVIII has an abnormally protruding region in the A2 domain which is contains the mutation site. TABLE 2: APC / FVIII INTERACTION COMPARISON BETWEEN WILD TYPE FVIIIA AND VARIANT FVIIIA

[0392] TABLE 3: APC / FVA INTERACTION COMPARISON BETWEEN WILD TYPE FVA AND FVLA TABLE 4: XASE / FX INTERACTION COMPARISON BETWEEN WILD TYPE AND MUTATED XASE COMPLEX.

[0393] TABLE 5: XASE / FXA INTERACTION COMPARISON BETWEEN WILD TYPE AND MUTATED XASE COMPLEX. Amino acid Imaging Results

[0394] UCSF Chimera was used to determine the difference in intramolecular interactions around the position of the novel mutation. Results indicated that when serine was present there were 40 fewer amino acid contacts compared to when arginine was present, indicating that the mutation curates a less crowded environment around amino acid position 590. Results also indicated that strong points of contact shifted with the presence of the mutation (SER590). In the mutated FVIII a new strong interaction occurred between: TYR737-SER590, while in the wild type FVIII three other strong interactions were present between: LYS589, TYR657, and LEU750 with ARG590. This slight difference in strong interaction could explain the protruding region of the A2 domain observed in FIGS. 3 - 8. Hydrogen bond difference was minimal between the mutated and WT- FVIII. Other notable contacts were found in the SER590 FVIII that were not found in the ARG590 FVIII. Both LYS589-TYR737 and LYS752-TYR-737 were found to be other strong points of contact in the variant FVIII, that were not present in the WT-FVIII.

[0395] Discussion

[0396] The results determined an Arg590Ser FVIII variant that produced thrombin faster than wild type and was minimally responsive to ARC. Coagulation evaluation revealed that the affected individual had a normal FVIII antigen level, and FVIII activities in the normal range by chromogenic assay, but abnormally high one-stage FVIII activity, which was unique to this individual alone, as shown in TABLE 6, provided below. (This pattern is similar to that seen with FVIII mimetics on FVIII assays). CAT data showed that the variant FVIII produced thrombin faster with a velocity index of 196nM / min (2SD range:4-169), but all other parameters were normal. CAT-TM data suggested a resistance to APC with both samples having an ETP reduction % no higher than 11 % (2.3 and 10.8%) (NR: 32%-71%). In addition, the peak thrombin height, and velocity index were abnormal after the addition of TM. The CAT-TM assay is conducted at a higher temperature than the standard CAT accounting for the differences in normal ranges. The novel APCR FVIII assay indicated that the variant FVIII was minimally responsive to APC while FV was normally responsive to APC, and initial docking analysis also showed that the variant FVIII has a lower binding affinity for APC and caused the Xase complex to have a lower binding affinity for both FX and FXa. These results indicate that variant FVIII could be contributing to the increased thrombin production via more rapid dissociation of FXa from the complex and increased FXa in circulation; however, more analysis of the binding kinetics needs to be performed. The variant FVIII was also shown to have a protruding region in the A2 domain (i.e., SEQ ID NO: 3, SEQ ID NO: 4, and SEO ID NO: 5), as shown in FIGS. 3 - 8. This protruding region in the A2 domain could be possibly due to the difference in strong interactions present and the locations of said interactions (i.e., SEQ ID NO: 1 and SEQ ID NO: 2), as shown in FIGS. 1 - 2. TABLE 6. COAGULATION EVALUATION RESULTS OF VARIANT INDIVIDUAL ACROSS TWO DRAWS

[0397] Docking analysis of APC interactions with the variant FVIII was then validated with use of FVL as a control, as shown in TABLE 2 and TABLE 3. FVL results showed differences in members and weighted scores that align with clinical and lab data surrounding FVL, validating the results that Cluspro produced. Furthermore, thrombin, phosphatidylserine, and FIXa interactions were normal across the board and all other coagulation protein amounts and activities were normal as well, with FVIII activity being the only abnormality, as shown in TABLE 6. However, with the variant FVIII producing thrombin abnormally fast and the variant FVIH’s minimal responsiveness to APC not predicted to contribute to this abnormal behavior, more research has to be conducted to determine what could be causing this increased rate of thrombin production. This finding indicates that the individual’s severe thrombophilia is not caused by his FVIH’s minimal responsiveness to ARC alone.

[0398] FVIIIa is regulated commonly by ARC with cleavage at Arg336 and Arg562. However, Arg562 is also a binding site of FIXa which is known to act as a stabilizing agent for FVIIIa, preventing rapid A2 dissociation. Due to the proximity of the novel point mutation at amino acid position 590 it was suspected that the abnormal shape of the A2 domain is impacting APC’s ability to bind to and cleave FVIIIa. This would explain the abnormal CAT-TM results, abnormal APCR FVIII, and the lower binding affinity for APC of the variant FVIIIa. It also has been shown that FV acts as a cofactor to protein C in regulating FVIIIa. However, there is no reason to believe that FV’s anticoagulant properties are in any way impacted by this novel point mutation.

[0399] FVIIIa has eight disulfide bonds in its mature form. Two disulfide bonds are located in each of the following domains: A1 , A2, and A3 domain (SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5). The last two are located in the C1 and C2 domain. The two in the A2 domain occur between C528-C554, and C630-C71 1 , both of which are near the novel point mutation, as shown in FIGS. 3 - 8. The findings suggest that no disulfide bonds were lost with the presence of the serine at amino acid position 590, but rather that there was a change in the location of weaker electrostatic forces, as shown in FIG. 1 and FIG. 2. This change in location of these electrostatic forces could possilby explain the protruding region in the A2 domain, as shown in FIG. 3, FIG. 6, and FIG. 8, but further investigation of this difference in location of electrostatic forces needs to be performed to holistically understand the impact of this difference.

[0400] The initial docking analysis results surrounding the Xase complex are intriguing as the variant FVIIIa’s minimal responsiveness to APC is not the sole cause of the extreme thrombophilia as it was observed in the variant participant, but alongside the abnormal results of the mutated Xase complex, it can be concluded that both factors could be the reason for the extreme coagulability. FIXa binds to the A2 domain (including residues 558-565) and the region around Asp712 to form the Xase complex. The Xase complex then converts FX to its active form FXa, by cleaving a single Arg51 -Ile52 peptide bond on the heavy chain of FX. FXa is then subsequently converted to factor Xap by the hydrolysis of an Arg-Gly peptide bond in the carboxy-terminal region. FVIIIa regions 337-372 serve as binding sites for FX, and specifically regions 361-363 contribute to a unique factor Xa-interactive site within the FVIII heavy chain that promotes factor Xa docking during cofactor activation, as well as the C2 domain of FVIIIa specifically residues 2253-2270. The mutation, being located near the FVIIIa binding sites for FX, could potentially be leading the abnormal docking results as has been initially observed. Another factor for consideration is that the FVIIIa A2 dissociation and liability is tied to the activity and stability of the Xase complex. The novel point mutation being within the A2 domain of FVIII could also be increasing the effectiveness of the Xase complex by making the Xase complex more stable due to the mutated FVIII prolonging the stability of the FVIIIa molecule; however, docking software is relatively new, and concluding without confirmatory laboratory data should not be done. Surface plasmon resonance and other binding studies will be performed to analyze and aid in understanding of the variant FVIII interactions. With the initial docking analysis of the Xase complex indicating that the mutated Xase complex has a much lower binding affinity for both FX and FXa, it was hypothesized that there is more rapid dissociation of FXa, thus more FXa in circulation, but this is only hypothesized. Both WT-FVIII and variant FVIII have been grown in mammalian cell culture and will be used to review the binding and activation kinetics of variant FVIII. Surface plasmon resonance has been used to analyze the binding affinity of FVIII before, specifically, with vWF.

[0401] It could also be possible that FVIII is rapidly activated leading to faster thrombin generation. Modified FVIIIa favorability for FIXa could possibly decrease the amount of FIXa needed to induce Xase formation. Furthermore, the competitive nature of FIXa and APC on FVIII interaction site Arg562 in the A2 domain of FVIII could explain the rapid thrombin production. Due to APC having a decreased affinity for FVIIIa, FIXa could bind to FVIIIa faster and remain bound to FVIIIa longer, inducing quicker Xase complex formation, and longer Xase activity which would cause more thrombin to be produced.

[0402] This report contains some weaknesses, as it is an initial observation and much confirmatory data are needed, specifically binding and activation kinetics of the FVIII variant in comparison to wild type. Finally, expression of the variant in an animal model will be necessary to prove thrombogenicity of the variant protein. Additionally, molecular modeling while useful needs to be used as supporting evidence to corresponding wet lab data, due to the speculative nature of molecular modeling.

[0403] In summary, a hypercoagulable individual was presented with elevated FVIII activity that ranged from 300-900%N. An APC resistance was determined using both the CAT-TM and a novel APCR clotting assay that was substituted with FVIII deficient plasma, instead of FV deficient plasma. A novel point mutation at amino acid position Arg590Ser was discovered using IGV. The impact of this variant FVIII could be potentially exploited in the therapeutic treatment of hemophilia A, due to the similarity it has to FIX Padua. The therapeutic treatment of hemophilia A using variant FVIII has already begun with investigation into the impact of Furin cleavage sites in FVIII. In summary, the discovery of FVIII variant Ser590 could potentially expand the approach in therapeutic treatments of hemophilia A, and potentially help thousands in their battle with hemophilia A.

[0404] The advantages set forth above, and those made apparent from the foregoing description, are efficiently attained. Since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

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[0453] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0454] It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.

Claims

What is claimed is:1 . A composition for treating hemophilia, comprising:(a) a nucleic acid vector encoding a Factor VIII polypeptide variant;(b) the Factor VIII polypeptide variant in a therapeutically effective amount, wherein the Factor VIII polypeptide variant comprises a serine residue at position 590 in place of arginine of the wild-type human Factor VIII sequence, and wherein the Factor VIII polypeptide variant is at least 95% identical in amino acid sequence to wild-type human Factor VIII.

2. The composition according to claim 1 , wherein the Factor VIII polypeptide comprises an arginine-to-serine substitution at amino acid position 590 relative to wild-type Factor VIII.

3. The composition according to claim 2, wherein the Factor VIII polypeptide is a B-domain deleted Factor VIII variant.

4. The composition according to claim 3, wherein the Factor VIII polypeptide comprises the amino acid sequence of SEQ ID NO:2.

5. The composition according to claim 4, wherein the nucleic acid encoding the Factor VIII polypeptide comprises the nucleotide sequence of SEQ ID NO:3.

6. The composition according to claim 5, wherein the nucleic acid vector is an adeno- associated virus (AAV) vector or a retroviral vector.

7. The composition according to claim 6, wherein the nucleic acid vector comprises the nucleotide sequence of SEQ ID NO:4.

8. The composition according to claim 7, wherein the nucleic acid vector is packaged in a viral capsid for delivery.

9. The composition according to claim 8, wherein the composition comprises a therapeutically effective amount of the Factor VIII polypeptide.

10. The composition according to claim 9, further comprising a pharmaceutically acceptable carrier or excipient.11 . A pharmaceutical kit for treating hemophilia, the pharmaceutical kit comprising:(a) a Factor VIII polypeptide variant having at least 95% sequence identity to a mutant Factor VIII comprising an arginine-to-serine substitution at amino acid position 590 relative to wild-type Factor VIII; or(b) a nucleic acid vector encoding said Factor VIII polypeptide variant; or(c) both a Factor VIII polypeptide variant having at least 95% sequence identity to a mutant Factor VIII comprising an arginine-to-serine substitution at amino acid position 590 relative to wild-type Factor VIII and the nucleic acid vector encoding said Factor VIII polypeptide variant.

12. The pharmaceutical kit according to claim 1 1 , wherein the Factor VIII polypeptide comprises the amino acid sequence of SEQ ID NO:2.

13. The pharmaceutical kit according to claim 12, wherein the nucleic acid encoding the Factor VIII polypeptide comprises the nucleotide sequence of SEQ ID NO:3.

14. The pharmaceutical kit according to claim 13, wherein the nucleic acid vector comprises the nucleotide sequence of SEQ ID NO:4.

15. The pharmaceutical kit according to claim 14, wherein the nucleic acid vector is an adeno- associated virus vector or a retroviral vector.

16. The pharmaceutical kit according to claim 15, wherein the nucleic acid vector is packaged in a viral capsid for delivery.

17. A method of producing a Factor VIII polypeptide variant for treating hemophilia, the method comprising:(i) synthesizing a nucleic acid encoding a Factor VIII polypeptide comprising a serine residue at amino acid position 590 instead of arginine, said nucleic acid being at least 90% identical to the coding sequence of wild-type human Factor VIII;(ii) expressing the nucleic acid in a host cell; and(iii) recovering the expressed Factor VIII polypeptide.

18. The method according to claim 17, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:3.

19. The method according to claim 17, wherein the nucleic acid is synthesized as part of a nucleic acid vector comprising the nucleotide sequence of SEQ ID NO:4.

20. The method according to claim 17, wherein the host cell is a mammalian cell.21 . The method according to claim 17, further comprising purifying the recovered polypeptide.

22. The method according to claim 17, further comprising formulating the nucleic acid and the recovered polypeptide with a pharmaceutically acceptable carrier to form the composition.

23. A nucleic acid vector encoding a Factor VIII polypeptide comprising a Serine at position 590 (R590S) of wild-type Factor VIII, wherein the Factor VIII polypeptide is at least 95% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, for use as a medicament to treat hemophilia.

24. An isolated Factor VIII polypeptide comprising a Serine substitution at position 590 relative to wild-type Factor VIII, having coagulation activity for use as a medicament to treat hemophilia.

25. A method of treating hemophilia A in a subject in need thereof, comprising:(a) administering to the subject a therapeutically effective amount of a therapeutic agent comprising a Factor VIII variant polypeptide having an arginine-to-serine substitution at amino acid position 590 of Factor VIII,(b) wherein the therapeutic agent is selected from the group consisting of:(i) the Factor VIII variant polypeptide, and(ii) a nucleic acid vector encoding said Factor VIII variant polypeptide, thereby providing a practical treatment for hemophilia A in the subject.

26. The method of claim 25, wherein the therapeutic agent is the Factor VIII variant polypeptide, administered as a protein replacement therapy to the subject.

27. The method of claim 25, wherein the therapeutic agent is a nucleic acid vector encoding the Factor VIII variant polypeptide, administered as a gene therapy to express the variant polypeptide in vivo.

28. The method of claim 27, wherein the nucleic acid vector is a recombinant adeno- associated virus (AAV) vector or a recombinant retroviral vector encoding the Factor VIII variant polypeptide.

29. The method of claim 25, wherein the Factor VIII variant is resistant to proteolytic inactivation by activated protein C (ARC), such that the variant interacts poorly with ARC compared to wild-type factor VIII.

30. The method of claim 25, wherein the Factor VIII variant exhibits increased procoagulant activity relative to wild-type Factor VIII, resulting in enhanced thrombin generation and improved hemostatic efficacy.31 . The method of claim 25, wherein the subject has developed neutralizing anti-Factor VIII antibodies (inhibitors) prior to treatment, and the administration of the Factor VIII variant provides therapeutic benefit despite the presence of said inhibitors.

32. The method of claim 25, wherein the therapeutically effective amount of the Factor VIII variant is administered prophylactical ly to the subject on a regular dosing schedule to prevent or reduce the frequency of bleeding episodes.

33. The method of claim 25, wherein the Factor VIII variant polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , a sequence that includes the Arg590Ser substitution.

34. The method of claim 27, wherein the nucleic acid vector comprises a nucleotide sequence encoding the Factor VIII variant polypeptide, wherein the nucleotide sequence is set forth in SEQ ID NO: 2.

35. A method of treating hemophilia in a subject in need thereof, the method comprising:(a) administering to the subject a CRISPR / CAS gene editing system configured to introduce an arginine-to-serine substitution at amino acid position 590 of Factor VIII in cells of the subject, thereby providing a practical treatment for hemophilia in the subject.

36. The method according to claim 35, wherein the CRISPR-associated endonuclease is Cas9.

37. The method according to claim 35, wherein the gene editing system comprises a guide RNA targeting a region of the F8 gene that corresponds to amino acid position 590 of Factor VIII.

38. The method according to claim 35, wherein the gene editing comprises homology-directed repair using a donor nucleic acid template to introduce said Arg590Ser substitution in the F8 gene.

39. The method according to claim 35, wherein the gene editing is accomplished using a base-editing CRISPR system that directly converts the nucleotide encoding arginine at position 590 in the F8 gene to a nucleotide encoding serine.

40. The method according to claim 35, wherein the Factor VIII variant expressed as a result of the gene editing is resistant to proteolytic inactivation by APC and exhibits enhanced procoagulant activity relative to wild-type Factor VIII.41 . A method of treating hemophilia in a subject in need thereof, the method comprising:(a) obtaining hematopoietic cells from the subject;(b) introducing ex vivo into the hematopoietic cells a nucleic acid vector encoding a Factor VIII variant polypeptide having an arginine-to-serine substitution at amino acid position 590 of Factor VIII; and(c) administering the hematopoietic cells expressing said Factor VIII variant polypeptide to the subject, thereby providing a practical treatment for hemophilia in the subject.

42. The method according to claim 41 , wherein the hematopoietic cells are autologous CD34- positive hematopoietic stem or progenitor cells isolated from the subject.

43. The method according to claim 41 , wherein the nucleic acid vector is a lentiviral vector.

44. The method according to claim 41 , further comprising conditioning the subject with a bone marrow conditioning regimen prior to administering the hematopoietic cells, to facilitate engraftment of the gene-modified cells.

45. The method according to claim 41 , wherein the Factor VIII variant polypeptide expressed by the administered cells is resistant to proteolytic inactivation by APC and exhibits increased procoagulant activity relative to wild-type Factor VIII.

46. The method according to claim 41 , wherein the subject has pre-existing neutralizing antiFactor VIII antibodies, and the engraftment of cells producing the Factor VIII variant polypeptide provides therapeutic benefit despite the presence of said inhibitors.