Composition and method for treating hemophilia

Abstract

The present invention relates to a method for treating hemophilia A the compositions and the use of compositions for the treatment of hemophilia A. The disclosed compositions comprises Factor VIII variants. The disclosed Factor VIII variants comprise at least one mutation at positions 570, 571, 572, and / or 707 of mature human FVIII or its variants.

Description

WSGR Docket No. 57837-747.601COMPOSITION AND METHOD FOR TREATING HEMOPHILIACROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 715,820 filed on November 4, 2024, and U.S. Provisional Application No. 63 / 766,045 filed March 3, 2025, the entirety of which is hereby incorporated by reference herein.BACKGROUND

[0002] Coagulation factor VIII (FVIII) circulates in blood tightly bound to its carrier protein. Proteolytic processing by thrombin liberates FVIII from its carrier protein and produces the active cofactor species (FVIIIa), which is a heterotrimer comprised of an A2-domain weakly associated with the metal ion-stabilized A1 / A3-C1-C2 heterodimer. FVIIIa associates with activated factor IX (FIXa) on anionic phospholipid surfaces forming the intrinsic Xase enzyme complex that activates factor X.

[0003] Hemophilia A and B are congenital bleeding disorders caused by a deficiency or complete absence of coagulation factor VIII (FVIII) or factor IX (FIX), respectively. These X- linked disorders represent the most inherited deficiencies of clotting factors, occurring in approximately one per 5000 and one per 50,000 male births, with no racial predilection. Type A, the most common type, is caused by a deficiency of factor VIII, one of the proteins that helps blood to form clots. This type is known as classic hemophilia.

[0004] Although hemophilia is usually diagnosed at birth, the disorder can also be acquired later in life if the body begins to produce antibodies that attack and destroy clotting factors. The development of inhibitory alloantibodies to FVIII can severely complicate the treatment of genetic cases. Rarely, development of autoantibodies to FVIII results in acquired hemophilia A.

[0005] A symptom of hemophilia is bleeding and depends on whether the hemophilia is the mild, moderate, or severe form of the disorder. For severe hemophilia, unprovoked (spontaneous) bleeding episodes occur often; for moderate hemophilia, prolonged bleeding tends to occur after a more significant injury; for mild hemophilia, a patient might have unusual bleeding, but only after a major injury, surgery, or trauma. Nonetheless, people with hemophilia may have any type of internal bleeding, but most often it occurs in the muscles and joints, such as the elbows, knees, hips, shoulders, and ankles. Thus, it is important to identify efficient and effective treatment for hemophilia A and hemophilia B.BRIEF SUMMARY

[0006] Currently, there is a need for the development of drugs and methods that can beWSGR Docket No. 57837-747.601 effective for treating hemophilia A. The compositions and methods disclosed herein solve the above-mentioned problems and meet the need.

[0007] Provided herein is a Factor VIII (FVIII) variant comprising at least one amino acid substitution mutation of (i) Lys at position 570, (ii) Arg at position 571, (iii) Asn at position 572, and / or (iv) Lys at position 707 of mature human FVIII or its variants. In some embodiments, the amino acid Lys (K) at position 570 is substituted with amino acid Ala (A), Asp (D), Glu (E), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the amino acid Arg (R) at position 571 is substituted with Ala (A), Cys (C) Asp (D), Glu (E), Phe (F), Gly (G), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the amino acid Asn (N) at position 572 is substituted with Ala (A), Cys (C), Glu (E), Phe (F), Gly (G), He (I), Leu (L), Met (M), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the amino acid Lys (K) at position 707 is substituted with Ala (A), Asp (D), Glu (E), Phe (F), Gly (G), His (H), lie (I), Leu (L), Met (M), Asn (N), Pro (P), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the FVIII variant comprises (i) the substitution mutation of Lys at position 570, and (ii) the substitution mutation of Arg at position 571 of mature human FVIII or its variants. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Lys at position 570 with Vai; and (2) the substitution mutation of Arg at position571 with Ser. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Lys at position 570, and (ii) the substitution mutation of Asn at position 572 of mature human FVIII or its variants. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Lys at position 570, and (ii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Arg at position 571, and (ii) the substitution mutation of Asn at position572 of mature human FVIII or its variants. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Arg at position 571, and (ii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Asn at position 572, and (ii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Arg at position 571, and (iii) the substitution mutation of Asn at position 572 of mature human FVIII or its variants. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Arg at position 571, and (iii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Lys atWSGR Docket No. 57837-747.601 position 570, (ii) the substitution mutation of Asn at position 572, and (iii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Arg at position 571, (ii) the substitution mutation of Asn at position 572, and (iii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants. In some embodiments, the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Arg at position 571, (iii) the substitution mutation of Asn at position 572, and (iv) the substitution mutation of Lys at position 707 of mature human FVIII or its variants. In some embodiments, an FVIII variant of the present disclosure further comprises at least one substitution mutation of: Pro at position 290 is substituted with Thr; Phe at position 309 is substituted with Ser; Arg at position 336 is substituted with Gin; Arg at position 562 is substituted with Gin; Lys at position 659 is substituted with Met; Lys at position 659 is substituted with Vai; or Lys at position 1813 is substituted with Ala; wherein the positions are based on the full-length mature human FVIII. In some embodiments, an FVIII variant of the present disclosure further comprises two substitution mutations selected from the group consisting of: Pro at position 290 is substituted with Thr; Phe at position 309 is substituted with Ser; Arg at position 336 is substituted with Gin; Arg at position 562 is substituted with Gin; Lys at position 659 is substituted with Met; Lys at position 659 is substituted with Vai; or Lys at position 1813 is substituted with Ala; wherein the positions are based on the full-length mature human FVIII. In some embodiments, an FVIII variant of the present disclosure further comprises three substitution mutations selected from the group consisting of: Pro at position 290 is substituted with Thr; Phe at position 309 is substituted with Ser; Arg at position 336 is substituted with Gin; Arg at position 562 is substituted with Gin; Lys at position 659 is substituted with Met; Lys at position 659 is substituted with Vai; or Lys at position 1813 is substituted with Ala; wherein the positions are based on the full-length mature human FVIII. In some embodiments, the FVIII variant is based on any of the following engineered FVIII proteins: A3-SP / DE, FVIII-AF, FVIII-CC, FVIII-ET3, VIII-HR, VIII-IR8, FVIII-N8, FVIII-OL, FVIILQ, FVIII-QQ, FVIII-RH, FVIII-SC, FVIII-SQ, FVIII- V3, FVIIL VV, FVIII-X5, or any combination thereof. In some embodiments, the FVIII variant comprises a linker sequence having at least 90% sequence identity with any one of SEQ ID NOs: 4-20, wherein a B-domain is replaced with the linker sequence. In some embodiments, the B domain consists of amino acids 741-1648 of wild type FVIII (SEQ ID NO: 2). Provided herein is a composition comprising at least one FVIII variant of the present disclosure and at least one pharmaceutically acceptable carrier. Provided herein is a method for treatment of a hemostasis- related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of an FVIII variant of the present disclosure in a pharmaceutically acceptableWSGR Docket No. 57837-747.601 carrier, wherein the FVIII variant is in a form of polypeptide, DNA or RNA. In some embodiments, the FVIII variant is a component of or a product of lentivirus-based vector, adenovirus-based vector, another viral vector, or non-viral vector. In some embodiments, a nucleic acid encoding for the FVIII variant is transfected into a cell via electroporation. In some embodiments, the FVIII variant is expressed by an engineered CD34 stem cell. In some embodiments, the hemostasis related disorder is hemophilia A. Provided herein is a polynucleotide comprising a nucleic acid encoding an FVIII variant of the present disclosure. In some embodiments, the polynucleotide is a recombinant nucleotide. In some embodiments, the polynucleotide is a synthetic polynucleotide. In some embodiments, the polynucleotide is codon optimized. In some embodiments, the nucleic acid encoding the human Factor VIII variant comprises a reduced number of CpG dinucleotides as compared to a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain. In some embodiments, the nucleic acid encoding the human Factor VIII variant has a reduced immunogenicity compared to a wildtype nucleic acid that encodes a wild-type Factor VIII peptide without a B domain. In some embodiments, the polynucleotide comprises a promoter operably connected to the nucleic acid. In some embodiments, the promoter is a liver-specific promoter. Provided herein is a recombinant adeno-associated virus (rAAV) particle comprising a polynucleotide of the present disclosure, wherein the rAAV particle comprises an AAV capsid protein selected from the group consisting of AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAVHSC, AAVDJ, LK03, NP59, KPI, or a variant thereof. In some embodiments, the rAAV particle is a single-stranded AAV (ssAAV). In some embodiments, the AAV capsid protein is AAV5 or AAV8. Provided herein is a recombinant adeno-associated virus (rAAV) particle prepared by transfecting a polynucleotide of the present disclosure into a host cell. In some embodiments, the host cell is an insect cell, a human cell, or an animal cell. In some embodiments, the insect cell is a Drosophila S2 cell or a Sf9 cell. In some embodiments, the animal cell is a fibroblasts, a Chinese hamster ovary (CHO) cell, a COS cell, a murine myeloma (NSO) cell, a HeLa cell, or a Baby Hamster Kidney (BHK) cell. In some embodiments, the human cell is a human embryonic kidney 293 (HEK293) cell, a HEK293 derivative (such as 293T), a human fibrosarcoma (HT-1080) cell, a differentiated hepatocyte-derived carcinoma (Huh-7) cell, or a PER.C6 cell. Provided herein is a method for treatment of a hemostasis related disorder in a patient in need thereof comprising administering a therapeutically effective amount of a polynucleotide of the present disclosure in a pharmaceutically acceptable carrier. In some embodiments, the hemostasis related disorder is hemophilia A. Provided herein is a method for treatment of a hemostasis related disorder in a patient in need thereof comprising administering a therapeutically effective amount of an rAAV particle of the present disclosure in a pharmaceutically acceptable carrier. In some embodiments,WSGR Docket No. 57837-747.601 the hemostasis related disorder is hemophilia A. Provided herein is a protein comprising an activated form of an FVIII variant of the present disclosure. Provided herein is a Factor VIII-SQ (FVIII-SQ) variant comprising at least one amino acid substitution mutation, wherein the at least one amino acid substitution mutation comprises substitution of Arg at position 571 with Ser, wherein the FVIII-SQ variant comprising the substitution mutation of Arg at position 571 with Ser has higher activity compared with a FVIII-SQ variant lacking a substitution mutation of Arg at position 571 with Ser. In some embodiments, a one stage clotting assay is used to measure activity.

[0008] In one aspect, the present disclosure provides a Factor VIII (FVIII) variant comprising at least one amino acid substitution mutation of (i) Lys at position 570, (ii) Arg at position 571, (iii) Asn at position 572, or (iii) Lys at position 707 of mature human FVIII or its variants.

[0009] In some embodiments, the Lys at position 570 is substituted with Ala (A), Asp (D), Glu (E), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the Arg at position 571 is substituted with Ala (A), Cys (C) Asp (D), Glu (E), Phe (F), Gly (G), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the Asn at position 572 is substituted with Ala (A), Cys (C), Glu (E), Phe (F), Gly (G), He (I), Leu (L), Met (M), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the Lys at position 707 is substituted with Ala (A), Asp (D), Glu (E), Phe (F), Gly (G), His (H), He (I), Leu (L), Met (M), Asn (N), Pro (P), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

[0010] In another aspect, the present disclosure provides a composition comprising at least one FVIII variant disclosed herein and at least one pharmaceutically acceptable carrier. In some embodiments, these compositions of the at least FVIII variants can be used for cell therapy, gene therapy and protein therapy, among other therapeutic methods. In some embodiments, these FVIII variants can be expressed in cells ex vivo and in vivo by gene delivery or gene editing. In some embodiments, these FVIII variants disclosed herein can contain additional mutations that are different from those disclosed here (e.g., additional mutations come from other variants), and that display enhanced half-life, activity, expression, and / or secretion in a subject. In some embodiments, the FVIII variants disclosed herein are used for recombinant protein therapy after been produced ex vivo and purified.

[0011] In still another aspect, the present disclosure provides a method for treatment of a hemostasis-related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of the FVIII variant disclosed herein in a pharmaceutically acceptable carrier. In some embodiments, the FVIII variants are FVIII protein variants. In someWSGR Docket No. 57837-747.601 embodiments, the FVIII variants are DNA molecules encoding the FVIII protein variants. In some embodiments, the FVIII variants are RNA molecules that can be translated / transcribed into the FVIII protein variants.

[0012] In another aspect, the present disclosure provides a polynucleotide comprising a nucleic acid encoding the FVIII variant disclosed herein.

[0013] In still another aspect, the present disclosure provides a recombinant adeno- associated virus (rAAV) particle, comprising a polynucleotide disclosed herein, wherein the rAAV particle comprises an AAV capsid protein selected from the group consisting of AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAVHSC, AAVDJ, LK03, NP59, KPI, or a variant thereof.

[0014] In another aspect, the present disclosure provides a method for treatment of a hemostasis related disorder in a patient in need thereof comprising administering a therapeutically effective amount of the polynucleotide disclosed herein in a pharmaceutically acceptable carrier.

[0015] In still another aspect, the present disclosure provides a method for treatment of a hemostasis related disorder in a patient in need thereof comprising administering a therapeutically effective amount of the rAAV particle enclosing DNA molecules encoding FVIII variants disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “FIG.” herein), of which:

[0017] FIG. 1 shows results of transient transfections for some FVIII variants disclosed herein.

[0018] FIG. 2 shows antigen, activity, and specific activity of various FVIII variants with mutations at site 707 of human FVIII relative to WT FVIII-SQ in transient transfections of Huh7 cells.

[0019] FIG. 3A shows FVIII variant antigen expression of various FVIII variants with mutations at site 570 of human FVIII relative to WT FVIII-SQ. FIG. 3B shows FVIII variant one-stage activity of various FVIII variants with mutations at site 570 of human FVIII relative to WT FVIII-SQ. FIG. 3C shows FVIII variant specific activity of various FVIII variants with mutations at site 570 of human FVIII relative to WT FVIII-SQ.WSGR Docket No. 57837-747.601

[0020] FIG. 4A shows FVIII variant antigen expression of various FVIII variants with mutations at site 571 of human FVIII relative to WT FVIII-SQ. FIG. 4B shows FVIII variant one-stage activity of various FVIII variants with mutations at site 571 of human FVIII relative to WT FVIII-SQ. FIG. 4C shows FVIII variant specific activity of various FVIII variants with mutations at site 571 of human FVIII relative to WT FVIII-SQ.

[0021] FIG. 5 shows specific activity of some FVIII variants with single amino acid mutations at different sites compared to WT FVIII-SQ.

[0022] FIG. 6 shows specific activity of some FVIII variants containing multiple mutations from multiple variants could be further increased compared to each individual variant.

[0023] FIG. 7 shows FVIII variant specific activity of various variants with mutations at site 572 of human FVIII relative to WT FVIII-SQ.

[0024] FIG. 8 shows FVIII antigen, activity and specific activity of the combo FVIII variants.

[0025] FIG. 9A shows expression levels of human FVIII antigen in mouse plasma. FIG. 9B shows FVIIII activity. FIG. 9C shows FVIII specific activity.DETAILED DESCRIPTION

[0026] Although described herein have been shown and described various embodiments of the present invention, but for obvious to the person skilled readily apparent that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art would recognize many variations, changes, and substitutions. It should be understood that the present invention may be employed herein, according to various alternatives to the next embodiment.

[0027] As discussed above, mutations in Factor VIII (FVIII) can lead to severe bleeding disorders associated with hemophilia A. Either a defective FVIII or a lack of FVIII activity may result in an inability to effectively form clots. Only about 20% of patients with hemophilia A worldwide may have received regular treatment with FVIII replacement therapy due to its high cost. Typically, the FVIII is plasma-derived or recombinantly produced. An enhanced FVIII molecule may benefit the treatment of hemophilia. Therefore, there is an obvious need for FVIII molecules with improved biological properties.

[0028] Unless otherwise stated, the practice of some embodiments disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds.); the series Methods In Enzymology (AcademicWSGR Docket No. 57837-747.601Press, Inc.), PC 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications , 6th Edition (RI Freshney, ed. (2010)).

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods, and materials are described below. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.DEFINITIONS

[0030] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term“rAAV particle” includes one or more rAAV particles.

[0031] The term “about” or “approximately” refers to within the acceptable error range of a specific value determined by a person of ordinary skill in the art, which will depend in part on how the value is measured or determined, that is, the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, “about” can mean a range of up to 20 %, up to 10 %, up to 5 %, or up to 1 % of a given value. Or, particularly for biological systems or processes, the term may represent an order of magnitude of the value, preferably within 5 times, more preferably within 2 times. Where specific values are described in the application and claims, unless otherwise stated, it should be assumed that the term "about" means within the acceptable error range of the specific value.

[0032] It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively unless the context specificallyWSGR Docket No. 57837-747.601 refers to a disjunctive use.

[0033] As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polypeptide can be linear, cyclic or branched. The polypeptide can contain modified amino acids. The polypeptide can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified, such as by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic treatment, phosphorylation, prenylation, racemization, selenization, transfer RNA- mediated addition of amino acids to proteins (such as arginylation), ubiquitination, or any other operations, such as conjugation with labeling components.

[0034] As used herein, the term “amino acid” refers to natural and / or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics. A polypeptide or amino acid sequence "derived" from a given protein refers to the origin of the polypeptide. Preferably, the polypeptide has an amino acid sequence that is substantially the same as the amino acid sequence of the polypeptide encoded in the sequence, or a part thereof, wherein the part consists of at least 10-20 amino acids or at least 20-30 amino acids or at least 30-50 amino acids. Alternatively, the polypeptide can be identified immunologically with the polypeptide encoded in the sequence. The term also includes polypeptides expressed from a designated nucleic acid sequence. As used herein, the term “domain” refers to a part of a protein that is physically or functionally distinguished from other parts of the protein or peptide. Physically defined domains include amino acid sequences that are extremely hydrophobic or hydrophilic, such as those that are membrane-bound or cytoplasmic- bound. The domain can also be defined by internal homology caused by gene duplication, for example. Functionally defined domains have different biological functions. For example, the antigen-binding domain refers to the part of the antigen-binding unit or antibody that binds to the antigen. The functionally defined domain does not need to be encoded by a continuous amino acid sequence, and the functionally defined domain may contain one or more physically defined domains.

[0035] As used herein, in the case of a polypeptide, a “sequence” is the sequence of amino acids in the polypeptide in the direction from the amino terminal to the carboxy terminal, wherein the residues adjacent to each other in the sequence are continuous in the primary structure in the polypeptide. The sequence can also be a linear sequence of a part of a polypeptide known to contain additional residues in one or two directions.

[0036] As used herein, “identity”, “homology”, or “sequence identity” refers to the similarity or interchangeability between two or more polynucleotide sequences or between twoWSGR Docket No. 57837-747.601 or more polypeptide sequences. When using programs such as BLAST, Emboss Needle or BestFit to determine the sequence identity, similarity or homology between two different amino acid sequences, the default settings can be used, or an appropriate scoring matrix can be selected, such as blosum45 or blosum80, to optimize identity, similarity, or homology score. In a preferred embodiment, the sequence identity is determined by BLAST.

[0037] With regard to the antigen-binding unit determined herein, the “percentage of sequence identity ( % )” is defined as after aligning the sequences and introducing gaps if necessary to obtain the maximum sequence identity percentage, and not removing any conservative substitutions are regarded as part of sequence identity, and the percentage of amino acid residues in the query sequence that are identical to the amino acid residues of the second, reference polypeptide sequence or part thereof. The alignment aimed at determining the percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, such as using publicly available computer software, such as BLAST, BLAST-2 (preferred), ALIGN, NEEDLE or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment over the full length of the sequence being compared. The percent identity can be measured over the length of the entire defined polypeptide sequence, or can be measured over a shorter length, for example, the length of a fragment taken from a larger, defined polypeptide sequence, such as A fragment of at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, or at least 200 consecutive residues. These lengths are exemplary only, and it should be understood that the forms herein shown in the drawings, or the sequence supported in the Sequence Listing can be used to describe any fragment length thereon may be measured with a percentage of the length.

[0038] The proteins described herein may have one or more modifications relative to the reference sequence. The modification may be deletion, insertion or addition, or substitution or substitution of amino acid residues. “Deletion” refers to a change in amino acid sequence due to the lack of one or more amino acid residues. “Insert” or “Add” means results in the addition of one or more as compared to the reference sequence amino acid residues of amino acid sequence changes. “Substitution” or “substitution” refers to the replacement of one or more amino acids with different amino acids.

[0039] As used herein, with respect to protein, the term “isolated protein” generally refers to a protein produced by expression of an isolated nucleic acid molecule of the disclosure. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would be associated (e.g., so as to exist in “substantially pure” form). “Isolated” within the protein context is not meant to exclude artificial or synthetic mixtures withWSGR Docket No. 57837-747.601 other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

[0040] As used herein, the term “sub isolated" refers to cells and other aspects of components of the separation surface, wherein in nature, polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof It is associated with it under normal circumstances. Those skilled in the art know that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not need to be “isolated” to distinguish them from their naturally occurring counterparts. In addition, “concentrated”, “isolated” or “diluted” polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are distinguishable from their naturally occurring counterparts because of the concentration or number of molecules per unit volume Greater than (“concentrated”) or less than from its naturally occurring counterpart (“isolated”). Enrichment can be measured based on absolute amounts, such as the weight of solution per unit volume, or it can be measured relative to the second, potentially interfering species present in the source mixture.

[0041] An “effective amount” as used herein refers to at least the minimum amount required to achieve a measurable improvement or prevention of a particular condition. The effective amount herein can vary with the patient's disease state, age, sex, weight and other factors. An effective amount is also an amount in which the therapeutic benefit exceeds any toxic or adverse effects of the treatment. In the treatment of cancer or tumor, the effective dose of the drug can have the following effects: reduce the number of cancer cells, reduce tumor size, inhibit the infiltration of cancer cells into peripheral organs, inhibit tumor metastasis, inhibit tumor growth to a certain extent and / or to a certain extent alleviate one or more symptoms related to the disease. The effective amount can be administered in one or more applications.

[0042] As used herein, the terms “recipient”, “individual”, “subject”, and “patient” are used interchangeably herein, and refer to those any mammalian subject that wish to be diagnosed or treated, especially human.

[0043] As used herein, the terms “treatment” and “treatment” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and / or may be therapeutic in terms of partially or completely stabilizing or curing the disease and / or adverse reactions attributed to the disease. “Treatment” as used herein encompasses any treatment of diseases in mammals, such as mice, rats, rabbits, pigs, primates, including humans and other apes, especially humans, and the term includes: (a) preventing a disease or symptom from occurring in subjects who may be susceptible to the disease or symptom but not yet diagnosed; (b) inhibitingWSGR Docket No. 57837-747.601 disease symptoms; (c) preventing the development of the disease; (d) relieving symptoms of the disease; (e) causing the disease or symptoms to subside; or any combination thereof. The term “kit” as used herein refers to a combination packaged for common use or commercially available. For example, the kit of the present disclosure may include the composition of the present disclosure, and instructions for using the composition or the kit. The term “instructions” refers to the explanatory inserts usually contained in commercial packages of therapeutic products, which contain information about indications, use, dosage, administration, combination therapy, contraindications and / or warnings about the use of such therapeutic products.

[0044] As used herein, the term “Factor VIII” generally refers to all wild-type and variant Factor VIII sequences, for example, a wild-type Factor VIII sequence or a wild-type Factor VIII without a B domain. In some embodiments, a Factor VIII comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, a Factor VIII comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, a Factor VIII comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, a Factor VIII comprises an amino acid sequence of SEQ ID NO: 3.

[0045] The phrase “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 Cl deficiency, gamma- carboxylase deficiency, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy (hypocoagulability), disseminated intravascular coagulation (DIC), over- anti coagulation 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 a particular embodiment, the term “hemostasis related disorder” refers to bleeding disorders characterized by excessive and / or uncontrolled bleeding (e.g., a disorder which can be treated with a procoagulant).

[0046] The term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.), particularly at least 75% by weight, or at least 90-99% or more by weight of the compound of interest. Purity may be measured by methods appropriate for the compound of interest (e.g. chromatographicWSGR Docket No. 57837-747.601 methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0047] 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. An “expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions (e.g., promoter) needed for expression in a host cell.HEMOPHILIA A and FVHI

[0048] The present disclosure presents FVIII variants that may be used to treat a subject suffering or at risk of suffering hemophilia A. Hemophilia A is a rare, inherited bleeding disorder in which blood cannot clot normally at the site of a wound or injury. Hemophilia A is a deficiency of FVIII and is a recessive sex-linked, X chromosome disorder that represents 80% of hemophilia cases. The standard of care for the management of hemophilia A is replacement therapy with recombinant factor VIII concentrates.

[0049] FVIII is a glycoprotein cofactor that serves as a critical component in the intrinsic blood coagulation pathway. In the circulation, FVIII is mainly complexed to von Willebrand factor. Upon activation by thrombin (Factor Ila), FVIII dissociates from the complex to interact with factor IXa in the intrinsic coagulation cascade, which, in turn, activates factor X. Once removed from the von Willebrand factor complex, activated FVIII (FVIIIa) is proteolytically inactivated by activated Protein C (APC), factor Xa, and factor IXa, and is quickly cleared from the blood stream. When complexed with normal von Willebrand factor protein, the half-life of FVIII is about 12 hours, whereas in the absence of von Willebrand factor, the half-life of FVIII is reduced to about 2 hours.

[0050] FVIII is a large multidomain glycoprotein with domain structure A1-A2-B-A3-C1- C2, wherein the heavy chain is composed of domains A1-A2-B, and the light chain of domains A3-C1-C2. The A domains are bordered by short spacers that contain clusters of aspartic acid and glutamic acid residues, called acidic regions. The large B domain is encoded by a single large exon and has no detectable homology to any other known genes. It is extensively glycosylated on asparagine, serine, and threonine residues. The C domains occur twice in the carboxy terminus of the FVIII light chain and exhibit homology to proteins that bind glycoconjugates and negatively charged phospholipids. FVIII, a divalent metal ion-dependent heterodimer, contains a single copper atom but, the role of this metal in the structure and function of the cofactor is unclear. Furthermore, association of FVIII heavy chain (A1-A2-B domains) and light chain (A3-C1-C2 domains) is metal ion-dependent with residues in the Al and A3 domains, which contain the interactive sites. Both Ca(II) and Mn(II) support thisWSGR Docket No. 57837-747.601 interaction, which is of interest since Ca(II) sites are typically formed by carboxylate moieties while Mn(II) can bind carboxylates as well as coordinated histidine residues. The divalent metal ion binding site(s) required for subunit association has (have) not been identified.

[0051] The liver is the major site of F VIII synthesis. The initial stage of secretion involves the translocation of the mature 2332 amino acids polypeptide into the lumen of the endoplasmic reticulum (ER), where glycosylation occurs. Within the ER, FVIII appears to interact with a number of chaperone proteins, including calreticulin, calnexin and the IgG-binding protein. Due to the interaction with these chaperone proteins, a significant proportion of the FVIII molecules is retained within the ER, thereby limiting the transport of FVIII to the Golgi apparatus. The mechanism responsible for the transport from the ER to the Golgi apparatus is not elucidated yet. The activation of FVIII coincides with proteolysis of both the heavy and light chain. Cleavage within the heavy chain after arginine residue 740 generates a 90 kDa polypeptide, which is subsequently cleaved after arginine residue 372 to generate polypeptides of 50 and 43 kDa, whereas, the 80 kDa light chain is cleaved after arginine residue 1689 to generate a 73 kDa polypeptide.

[0052] FVIII becomes a potent cofactor of factor IXa once it is cleaved within the heavy and light chain. It is a cofactor for factor IXa, which, in the presence of Ca2+and phospholipids, forms a complex that converts factor X to the activated form Xa. FVIII gene can also produce another alternative spliced variant, which encodes a putative small protein consisting primarily of the phospholipid binding domain of factor VIIIc.

[0053] Table 1. A list of amino acid sequences of human FVIII (hFVIII) wild-type with B domain and hFVIII variant without B domain (FVIII-SQ, also called FVIII-BDD).WSGR Docket No. 57837-747.601WSGR Docket No. 57837-747.601The bolded sequence in the full length mature human FVIII is the B domain, which can be replaced with a short peptide sequence in human FVIII-SQ variant.

[0054] In some embodiments, additional FVIII variants include, but are not limited to, F309 variant (including F309S variant), K659 variant (including K659M variant), K1813 variant (including K1813A variant), P290 variant (including P290T variant), R336 variant (including R336Q variant), R562 variant (including R562Q variant), A3-SP / DE, FVIII-AF, FVIII-CC, FVIII-ET3, VIII-HR, VIII-IR8, FVIII-N8, FVIII-OL, FVIII-Q, FVIII-QQ, FVIII-RH, FVIII-SC, FVIII-SQ, FVIII-V3, FVIII- VV, or FVIII-X5. In some embodiments, additional FVIII variants include, but are not limited to, B-domain (amino acids 741-1648 of SEQ ID NO: 2) replaced by a linker peptide having a length of from about 5 amino acids to about 50 amino acids, inWSGR Docket No. 57837-747.601 particular, a linker peptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity) with any one of SEQ ID NOs: 4-20, particularly at least 90%, 95%, 97%, 99%, or 100% homology (identity) with any one of SEQ ID NOs: 4-20 (Tables 2 and 3). In some embodiment, FVIII variant is FVIII-X5 or BDD-FVIII-X5, which is a human B-domain deleted (BDD)-FVIII variant with five substitution mutations of I86V, A108S, G132K, M147T, and L152P.

[0055] In some embodiments, FVIII variants comprise short peptide linkers substituted for the B domain as shown in Table 2.

[0056] Table 2. Peptide linkers substituted for the B domain in FVIII variantsFurin recognition motif bolded. Abbreviations: c, canine; cl, cell-line; h, human; p, porcine; r, recombinant.

[0057] In some embodiments, FVIII variants comprise short peptide linkers substituted for the B domain as shown in Table 3.

[0058] Table 3 Additional peptide linkers substituted for the B domain in FVIII variantsWSGR Docket No. 57837-747.601Preparation of F VIII Variants

[0059] Nucleic acid molecules encoding the desired FVIII variants may be prepared by using recombinant or synthetic DNA technology methods. For example, a nucleic acid sequence encoding a specific variant may be designed and isolated from appropriate biological sources using standard protocols. The nucleic acid molecules may be maintained as RNA or DNA in a cloning vector. In some embodiments, clones are maintained in a plasmid cloning / expression vector, which is propagated in a suitable A. coli host cell. In some embodiments, the nucleic acids can be maintained in a vector suitable for an expression in a mammalian cell, particularly a human cell. Such nucleic acid molecules may include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded.

[0060] The FVIII variants of the current disclosure may be prepared in a variety of ways. The protein may be purified from appropriate sources. For example, the sources can be transformed bacterial or animal (e.g., mammalian or human) cultured cells or tissues which can express FVIII variants. Purification methods can include, for example, immunoaffinity purification. In some embodiments, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such as pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocyte lysates. In vitro transcription and translation systems are commercially available, e.g., from Promega or Life Technologies.

[0061] In some embodiments, larger quantities of FVIII variant may be produced by an expression in a suitable prokaryotic or eukaryotic expression system. In some embodiments, part or all of a DNA molecule encoding the FVIII variant may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli, or a mammalian cell, such as CHO or HeLa cells. In some embodiments, tagged fusion proteins comprising the FVIII variant can be generated. Such variant-tagged fusion proteins can be encoded by part or all of a DNA molecule, ligated in the correct codon reading frame to a nucleotide sequence encoding a portion or all of a desired polypeptide tag which is inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli, or a eukaryotic cell, such as, yeast and mammalian cells. Vectors may comprise the regulatory elements necessary for expression of the DNA in the particular host cell permitting expression of the DNA in the particular host cell. Example regulatory elements required for expression include, but are not limited to, promoter sequences, transcription initiation sequences, and enhancer sequences.

[0062] FVIII variant proteins, produced by gene expression in a recombinant prokaryotic or eukaryotic system (e.g., human cells) may be purified according to known methods. In someWSGR Docket No. 57837-747.601 embodiments, an expression / secretion system (e.g., a commercially available one) can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be purified from the surrounding medium according to the manufacturer’s recommendations. In some embodiments, when expression / secretion vectors are not used, the recombinant protein can be purified by affinity separation, such as, immunological interaction with antibodies that bind specifically to the recombinant protein or nickel columns for isolation of recombinant proteins tagged with 6-8 histidine residues at their N-terminus or C- terminus. Tags used for such purposes may comprise the FLAG epitope, GST or the hemagglutinin epitope. The isolated FVIII variants can be analyzed accordingly to standard procedures, including, for example, amino acid sequence analysis or molecular weight analysis, etc.

[0063] Herein, novel FVIII variants are provided. These FVIII variants comprise at least one amino acid mutation at position 570, 571, 572, and / or 707 of mature human FVIII or its variants. For simplicity, the variants are generally described throughout the disclosure in the context of full length mature human FVIII. The mutation position is relative to a corresponding position of the amino acid sequence of SEQ ID NO: 2 or a variant thereof. However, the disclosure contemplates and encompasses mature human FVIII variants (or engineered FVIII variants), including, for example, Factor FVIIIa, F309 variant (including F309S variant), K659 variant (including K659M variant), K1813 variant (including K1813A variant), P290 variant (including P290T variant), R336 variant (including R336Q variant), R562 variant (including R562Q variant), A3-SP / DE, FVIII-AF, FVIILCC, FVIII-ET3, VIII-HR, VIII-IR8, FVIII-N8, FVIILOL, FVIII-Q, FVIII-QQ, FVIII-RH, FVIILSC, FVIII-SQ, FVIII- V3, FVIII- VV, FVIILX5, and FVIII prepeptide molecules having the same amino acid substitutions and / or linkers as described in FVIII. In a particular embodiment, the FVIII variants of the current disclosure are expressed as a single chain molecule or at least almost exclusively as a single chain molecule.

[0064] The FVIII variants of the current disclosure can be from any mammalian species. In some embodiments, the FVIII variant is human. SEQ ID NO: 2 provides an example of the amino acid sequence of human FVIII. SEQ ID NO: 1 provides the 19 amino acid signal peptide at FVIII N-terminus. Nucleic acid molecules which encode Factor FVIII variants can be readily determined from the corresponding amino acid sequences.

[0065] In some embodiments, the FVIII variants are B-domain deleted (BDD) FVIII molecules comprising a linker. B-domain corresponds to amino acids 741-1648 of SEQ ID NO: 2. (Full-length mature Human FVIII). In some embodiments, the linker comprises a sequence set forth in Table 1. In some embodiments, the linker comprises a sequence set forth in Table 2. In some embodiments, the linker comprises a sequence set forth in Table 3. In some embodiments, the linker consists of a sequence set forth in Table 2 or Table 3. 18. In some embodiments, theWSGR Docket No. 57837-747.601B-domain is replaced with an amino acid sequence up to about 50, up to about 45, up to about 40, up to about 35, up to about 30, up to about 25, up to about 20, up to about 15, up to about 10, or up to about 5 amino acids in length. In some embodiments, the B-domain is replaced with an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity) with any one of SEQ ID NOs: 4-20, particularly at least 90%, 95%, 97%, 99%, or 100% homology (identity).

[0066] In accordance with one aspect of the current disclosure, the Factor VIII variants comprise at least one amino acid mutation at position 570, 571, 572, and / or 707. In some embodiments, the Factor VIII variant comprises a single mutation selected from the group consisting of mutation at position 570, 571, 572, and 707. In some embodiments, the Factor VIII variant comprises two mutations selected from the group consisting of mutation at position 570, 571, 572, and 707. In some embodiments, the Factor VIII variant comprises three mutations selected from the group consisting of mutation at position 570, 571, 572, and 707. In some embodiments, the FVIII variants disclosed herein possess higher specific activity than wild type full length human FVIII or existing engineered human FVIII variants. In some embodiments, the FVIII variants disclosed herein possess higher specific activity than wild type full length human FVIII.

[0067] In some embodiments, the Factor VIII variants comprise a mutation at position 570. In some embodiments, the Factor VIII variants comprise a mutation at position 570 and comprises a sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the Factor VIII variants comprise a non-Lys (K) amino acid at position 570. In some embodiments, the Lys at position 570 is substituted with Ala (A), Asp (D), Glu (E), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the Lys at position 570 is substituted with Arg (R), Ser (S), Thr (T), or Vai (V). In some embodiments, the Lys at position 570 is substituted with Ala (A), Ser (S), Thr (T), or Vai (V). In some embodiments, the Lys at position 570 is substituted with Ser (S) or Vai (V). In some embodiments, the Lys at position 570 is substituted with Ala (A), Gin (Q), Trp (W), or Tyr (Y). In some embodiments, the Lys at position 570 is substituted with Asp (D), Glu (E), His (H), He (I), Leu (L), Met (M), or Asn (N). In some embodiments, the Lys at position 570 is substituted with Ala (A). In some embodiments, the Lys at position 570 is substituted with Asp (D). In some embodiments, the Lys at position 570 is substituted with Glu (E). In some embodiments, the Lys at position 570 is substituted with His (H). In some embodiments, the Lys at position 570 is substituted with lie (I). In some embodiments, the Lys at position 570 is substituted with Leu (L). In some embodiments, the Lys at position 570 is substituted with MetWSGR Docket No. 57837-747.601(M). In some embodiments, the Lys at position 570 is substituted with Asn (N). In some embodiments, the Lys at position 570 is substituted with Gin (Q). In some embodiments, the Lys at position 570 is substituted with Arg (R). In some embodiments, the Lys at position 570 is substituted with Ser (S). In some embodiments, the Lys at position 570 is substituted with Thr (T). In some embodiments, the Lys at position 570 is substituted with Vai (V). In some embodiments, the Lys at position 570 is substituted with Trp (W). In some embodiments, the Lys at position 570 is substituted with Tyr (Y).

[0068] In some embodiments, the Factor VIII variants comprise a mutation at position 571. In some embodiments, the Factor VIII variants comprise a mutation at position 571 and comprises a sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the Factor VIII variants comprise a non- Arg (R) amino acid at position 571. In some embodiments, the Arg at position 571 is substituted with Ala (A), Cys (C), Asp (D), Glu (E), Phe (F), Gly (G), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the Arg at position 571 is substituted with Ala (A), Cys (C), Phe (F), He (I), Met (M), Ser (S), Thr (T), or Vai (V). In some embodiments, the Arg at position 571 is substituted with Phe (F), Met (M), Ser (S), Thr (T), or Vai (V). In some embodiments, the Arg at position 571 is substituted with Ser (S) or Thr (T). In some embodiments, the Arg at position 571 is substituted with Glu (E), His (H), Leu (L), Gin (Q), Trp (W), or Tyr (Y). In some embodiments, the Arg at position 571 is substituted with Asp (D) or Gly (G). In some embodiments, the Arg at position 571 is substituted with Ala (A). In some embodiments, the Arg at position 571 is substituted with Cys (C). In some embodiments, the Arg at position 571 is substituted with Asp (D). In some embodiments, the Arg at position 571 is substituted with Glu (E). In some embodiments, the Arg at position 571 is substituted with Phe (F). In some embodiments, the Arg at position 571 is substituted with Gly (G). In some embodiments, the Arg at position 571 is substituted with His (H). In some embodiments, the Arg at position 571 is substituted with He (I). In some embodiments, the Arg at position 571 is substituted with Leu (L). In some embodiments, the Arg at position 571 is substituted with Met (M). In some embodiments, the Arg at position 571 is substituted with Asn (N). In some embodiments, the Arg at position 571 is substituted with Gin (Q). In some embodiments, the Arg at position 571 is substituted with Ser (S). In some embodiments, the Arg at position 571 is substituted with Thr (T). In some embodiments, the Arg at position 571 is substituted with Vai (V). In some embodiments, the Arg at position 571 is substituted with Trp (W). In some embodiments, the Arg at position 571 is substituted with Tyr (Y).

[0069] In some embodiments, the Factor VIII variants comprise a mutation at position 572.WSGR Docket No. 57837-747.601In some embodiments, the Factor VIII variants comprise a mutation at position 572 and comprises a sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the Factor VIII variants comprise a non-Asn (N) amino acid at position 572. In some embodiments, the Asn at position 572 is substituted with is substituted with Ala (A), Cys (C), Glu (E), Phe (F), Gly (G), He (I), Leu (L), Met (M), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the Asn at position 572 is substituted with Ala (A), Cys (C), Glu (E), Phe (F), Gly (G), He (I), Leu (L), Met (M), or Ser (S). In some embodiments, the Asn at position 572 is substituted with Ala (A), Glu (E), Phe (F), or Ser (S). In some embodiments, the Asn at position 572 is substituted with Ala (A), Glu (E), or Phe (F). In some embodiments, the Asn at position 572 is substituted with Cys (C), Gly (G), He (I), Leu (L), or Met (M). In some embodiments, the Asn at position 572 is substituted with Gin (Q), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the Asn at position 572 is substituted with is substituted with Ala (A). In some embodiments, the Asn at position 572 is substituted with is substituted with Cys (C). In some embodiments, the Asn at position 572 is substituted with is substituted with Glu (E). In some embodiments, the Asn at position 572 is substituted with is substituted with Phe (F). In some embodiments, the Asn at position 572 is substituted with is substituted with Gly (G). In some embodiments, the Asn at position 572 is substituted with is substituted with He (I). In some embodiments, the Asn at position 572 is substituted with is substituted with Leu (L). In some embodiments, the Asn at position 572 is substituted with is substituted with Met (M). In some embodiments, the Asn at position 572 is substituted with is substituted with Gin (Q). In some embodiments, the Asn at position 572 is substituted with is substituted with Ser (S). In some embodiments, the Asn at position 572 is substituted with is substituted with Thr (T). In some embodiments, the Asn at position 572 is substituted with is substituted with Vai (V). In some embodiments, the Asn at position 572 is substituted with is substituted with Trp (W). In some embodiments, the Asn at position 572 is substituted with is substituted with Tyr (Y).

[0070] In some embodiments, the Factor VIII variants comprise a mutation at position 707. In some embodiments, the Factor VIII variants comprise a mutation at position 707 and comprises a sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the Factor VIII variants comprise a non-Lys (K) amino acid at position 707. In some embodiments, the Lys at position 707 is substituted with Ala (A), Asp (D), Glu (E), Phe (F), Gly (G), His (H), lie (I), Leu (L), Met (M), Asn (N), Pro (P), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y). In some embodiments, the Lys at position 707 is substituted with Gly (G), Met (M), Asn (N), Pro (P), Ser (S), Trp (W), or Tyr (Y). In some embodiments, the Lys at position 707 isWSGR Docket No. 57837-747.601 substituted with Met (M), Asn (N), Ser (S), Trp (W), or Tyr (Y). In some embodiments, the Lys at position 707 is substituted with Asn (N). In some embodiments, the Lys at position 707 is substituted with Ala (A), His (H), Leu (L), or Gin (Q). In some embodiments, the Lys at position 707 is substituted with Asp (D), Glu (E), Phe (F), He (I), Thr (T), or Vai (V). In some embodiments, the Lys at position 707 is substituted with Ala (A). In some embodiments, the Lys at position 707 is substituted with Asp (D). In some embodiments, the Lys at position 707 is substituted with Glu (E). In some embodiments, the Lys at position 707 is substituted with Phe (F). In some embodiments, the Lys at position 707 is substituted with Gly (G). In some embodiments, the Lys at position 707 is substituted with His (H). In some embodiments, the Lys at position 707 is substituted with He (I). In some embodiments, the Lys at position 707 is substituted with Leu (L). In some embodiments, the Lys at position 707 is substituted with Met (M). In some embodiments, the Lys at position 707 is substituted with Asn (N). In some embodiments, the Lys at position 707 is substituted with Pro (P). In some embodiments, the Lys at position 707 is substituted with Gin (Q). In some embodiments, the Lys at position 707 is substituted with Ser (S). In some embodiments, the Lys at position 707 is substituted with Thr (T). In some embodiments, the Lys at position 707 is substituted with Vai (V). In some embodiments, the Lys at position 707 is substituted with Trp (W). In some embodiments, the Lys at position 707 is substituted with Tyr (Y).

[0071] In some embodiments, the FVIII variants of the present disclosure may comprise at least one mutation at positions 570, 571, 572, and / or 707 as described herein with or without a linker in place of the B-domain as described herein. In other words, the present disclosure encompasses (i) FVIII variants with at least a mutation at positions 570, 571, 572, and / or 707 as described herein, and the FVIII variants comprise a full B domain, and (ii) FVIII variants comprising at least a mutation at positions 570, 571, 572, and / or 707 as described herein and a linker in place of the B-domain. In some embodiments, the FVIII variants of the present disclosure may comprise a mutation at position 570 as described herein with a linker in place of the B-domain as described herein. In some embodiments, the FVIII variants of the present disclosure may comprise a mutation at position 571 as described herein with a linker in place of the B-domain as described herein. In some embodiments, the FVIII variants of the present disclosure may comprise a mutation at position 572 as described herein with a linker in place of the B-domain as described herein. In some embodiments, the FVIII variants of the present disclosure may comprise a mutation at position 707 as described herein with a linker in place of the B-domain as described herein.

[0072] In some embodiments, the FVIII variants of the present disclosure may comprise two mutations at positions 570, 571, 572, and / or 707 as described herein with or without a linker inWSGR Docket No. 57837-747.601 place of the B-domain as described herein. In other words, the present disclosure encompasses (i) FVIII variants with two mutations at positions 570, 571, 572, and / or 707 as described herein, and the FVIII variants comprise a full B domain, and (ii) FVIII variants comprising two mutations at position 570, 571, 572, and / or 707 as described herein and a linker in place of the B-domain. In some embodiments, the FVIII variants of the present disclosure may comprise two mutations at positions selected from the group consisting of positions 570, 571, 572, and 707 as described herein with a linker in place of the B-domain as described herein.

[0073] In some embodiments, the FVIII variants of the present disclosure may comprise three mutations at positions 570, 571, 572, and / or 707 as described herein with or without a linker in place of the B-domain as described herein. In other words, the present disclosure encompasses (i) FVIII variants with three mutations at positions 570, 571, 572, and / or 707 as described herein, and the FVIII variants comprise a full B domain, and (ii) FVIII variants comprising three mutations at position 570, 571, 572, and / or 707 as described herein and a linker in place of the B-domain. In some embodiments, the FVIII variants of the present disclosure may comprise three mutations at positions selected from the group consisting of positions 570, 571, 572, and 707 as described herein with a linker in place of the B-domain as described herein.

[0074] In some embodiments, the FVIII variants of the present disclosure may comprise mutations at positions 570, 571, 572, and 707 as described herein with or without a linker in place of the B-domain as described herein. In other words, the present disclosure encompasses (i) FVIII variants with mutations at positions 570, 571, 572, and 707 as described herein, and the FVIII variants comprise a full B domain, and (ii) FVIII variants comprising four mutations at position 570, 571, 572, and 707 as described herein and a linker in place of the B-domain.

[0075] As used herein, the phrase “comprise at least one mutation at positions 570, 571, 572, or 707 includes (1) one mutation at positions 570, 571, 572, or 707; (2) two mutations at positions 570, 571, 572, or 707; (3) three mutations at positions 570, 571, 572, or 707; or (4) four mutations at positions 570, 571, 572, and 707.

[0076] In some embodiments, the FVIII variants of the present disclosure may comprise at least one mutation at positions 570, 571, 572, and / or 707 as described herein, and at least an additional mutation at positions 290, 309, 336, 562, 659, or 1813, with or without a linker in place of the B-domain as described herein. In other words, the present disclosure encompasses (i) FVIII variants with at least a mutation at positions 570, 571, 572, and / or 707 as described herein, at least an additional mutation at positions 290, 309, 336, 562, 659, or 1813, and the FVIII variants comprise a full B domain, and (ii) FVIII variants comprising at least a mutation at positions 570, 571, 572, and / or 707 as described herein, at least an additional mutation atWSGR Docket No. 57837-747.601 positions 290, 309, 336, 562, 659, or 1813, and the FVIII variants comprise a linker in place of the B-domain.

[0077] In some embodiments, the FVIII variants of the present disclosure may comprise at least one mutation at positions 570, 571, 572, and / or 707 as described herein on a FVIII variant (e g., A3-SP / DE, FVIII-AF, FVIII-CC, FVIII-ET3, VIII-HR, VIII-IR8, FVIII-N8, FVIII-OL, FVIII-Q, FVIII-QQ, FVIII-RH, FVIII-SC, FVIII- SQ, FVIII-V3, FVIII-VV, or FVIII-X5) with or without a linker in place of the B-domain as described herein. In some embodiment, the FVIII variants of the present disclosure may comprise at least one mutation at positions 570, 571, 572, and / or 707 as described herein on FVIII-SQ. In some embodiment, the FVIII variants of the present disclosure may comprise at least one mutation at positions 570, 571, 572, and / or 707 as described herein on FVIII-X5.

[0078] In some embodiments, the FVIII variants of the present disclosure may comprise at least one mutation at positions 570, 571, 572, and / or 707 as described herein and at least one mutation up to all mutations from other FVIII variants (e.g., A3-SP / DE, FVIII-AF, FVIII-CC, FVIII-ET3, VIII-HR, VIII-IR8, FVIII-N8, FVIII-OL, FVIII-Q, FVIII-QQ, FVIII-RH, FVIII-SC, FVIII-SQ, FVIII-V3, FVIII-VV, or FVIII-X5) with or without a linker in place of the B-domain as described herein.

[0079] In some embodiments, the FVIII variants disclosed above comprise a linker peptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity) with any one of SEQ ID NOs: 4-20, particularly at least 90%, 95%, 97%, 99%, or 100% homology (identity) with any one of SEQ ID NOs: 4-20 (Tables 2 and 3).

[0080] As stated hereinabove, the FVIII variant of the present disclosure may be human, human and animal hybrid, or animal. In some embodiments, the FVIII variant of the present disclosure has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity) with SEQ ID NO: 2 or SEQ ID NO: 3 (or an activated FVIII fragment thereof), particularly at least 90%, 95%, 97%, 99%, or 100% homology (identity) with SEQ ID NO: 2 or SEQ ID NO: 3 (or an activated FVIII fragment thereof). In some 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: 2 (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: 2 (or an activated FVIII fragment thereof). The homology (identity) percentages above exclude the substitutions at position 570, 571, 572, and / or 707.

[0081] The FVIII variants of the present disclosure may also be post-translationallyWSGR Docket No. 57837-747.601 modified. The FVIII variants may be post-translationally modified in a cell (particularly a human cell) or in vitro.

[0082] In some embodiments, the FVIII variants of the present disclosure have increased expression compared to wild-type FVIII or FVIII-SQ. In a particular embodiment, the FVIII variants of the present disclosure have increased FVIII activity or increased specific activity compared to wild-type FVIII or FVIII-SQ. In some embodiments, expression is assessed using ELISA.

[0083] Nucleic acid molecules encoding the above FVIII variants are also encompassed by the present disclosure. Nucleic acid molecules encoding the variants may be prepared by known methods. The nucleic acid molecules may be maintained in any vector, particularly an expression vector.

[0084] Provided herein are methods and compositions for assessing activity of FVIII and FVIII-SQ variants of the present disclosure. In some embodiments, a one stage clotting assay is used to assess activity. In some embodiments, a one stage clotting assay is used to assess specific activity. In some embodiments, activity is assessed at 1, 2, 3, 4, 5, or 6 weeks following the start of expression in an organism or cell. In some embodiments, activity is assessed at 1, 2, 3, 4, 5, or 6 weeks following injection of a plasmid encoding a construct of the present disclosure into an organism. In some embodiments, an FVIII-SQ variant comprising a substitution mutation shows higher activity compared to an FVIII-SQ variant lacking said mutation. In some embodiments, an FVIII-SQ variant comprising a substitution mutation of Arg at position 571 with Ser shows higher activity and / or higher specific activity compared to an FVIII-SQ variant lacking said mutation.Use of FVIII Variants

[0085] FVIII variant proteins and the corresponding nucleic acid molecules encoding the same may be used, for example, as therapeutic and / or prophylactic agents which modulate the blood coagulation cascade. The FVIII variant proteins and the corresponding nucleic acid molecules encoding the same may be administered in a therapeutically effective amount to modulate (e.g., increase) hemostasis and / or form a clot and / or stop or inhibit bleeding or aberrant bleeding. The disclosed FVIII variants possess superior properties and can provide effective hemostasis. In some embodiments, FVIII variants or the corresponding nucleic acid molecules may be administered to a patient via infusion in a biologically compatible carrier, e.g., via intravenous injection. In some embodiments, the FVIII variants or the corresponding nucleic acid molecules may be encapsulated into liposomes, lipid nanoparticles, or mixed with other phospholipids or micelles to increase stability of the molecule. FVIII variants or theWSGR Docket No. 57837-747.601 corresponding nucleic acid molecules may be administered alone or in combination with other agents to modulate hemostasis. In come embodiments, the other agents comprise emicizumab (Hemlibra®), FVIII Fc-fusion protein (rFVIII-Fc; Eloctate®), or PEGylated, full-length, recombinant FVIII peptides, or nucleic acid molecules encoding thereof.

[0086] In some embodiments, the FVIII variants disclosed herein are used in a protein therapy. As used herein, the term “protein therapy” generally refers to a medical treatment where a specific protein is administered to a subject to replace a deficient, defective or absent protein in their body, essentially aiming to repair or restore normal function of the normal protein by introducing another functional protein or a protein variant (either naturally occurring or genetically engineered) within the body. Protein therapy is often achieved through the use of recombinant DNA technology to produce the desired protein in a lab, then introduce the desired protein into the body of the subject, thereby supplementing a missing or malfunctioning protein in the subject. This is a common therapy treat various diseases including genetic disorders, where a specific protein deficiency is present in the subject. Desired proteins can be administered through injections, infusions, or other targeted delivery methods depending on the desired site of action.

[0087] In some embodiments, the FVIII variants or the corresponding nucleic acid molecules disclosed herein are used in a gene therapy. As used herein, the term “gene therapy” generally refers to a medical procedure that treats or prevents disease by altering a person’s genes. This treatment option may improve quality of life for a subject who has genetic defects that lead to a deficient, defective or absent protein in their body. Gene therapy may work by: replacing a faulty gene by introducing a healthy copy of a gene to replace a disease-causing defective gene; or inactivating a faulty gene by turning off a gene that is not functioning properly; or introducing new genes, by adding a new gene to help treat a disease; or genome editing by making precise changes to the subject's DNA and restore a protein’s or cell’s function. For example, one can edit a patient’s gene to express a FVIII variant disclosed herein for treating hemophilia A. Gene therapy can be performed in a few different ways: by gene transfer, also known as gene addition, which involves adding a new gene to affected cells; or by ex vivo treatment, wherein cells are collected from the body, treated outside of the body, and then returned to the body of the subject. The procedure for gene therapy varies depending on the disease and the type of therapy used. A vector, or carrier, is used to introduce the new therapeutic genes into the body. Vectors can be made from a reprogrammed virus, stem cells, or fatty particles called liposomes.

[0088] In some embodiments, the FVIII variants or the corresponding nucleic acid molecules disclosed herein are used in a cell therapy. As used herein, the term “cell therapy”WSGR Docket No. 57837-747.601 generally refers to a treatment that involves introducing cells into a patient's body to treat or prevent disease. Cell therapy can be used to replace or repair damaged cells and tissue, or to restore or modulate the function of a subject’s cells. The type of therapeutic cells used may depend on the disease being treated and the desired effect. Some common cells used in cell therapy include hematopoietic stem cells, skeletal muscle stem cells, lymphocytes, dendritic cells, and pancreatic islet cells. Therapeutic cells can be used in their original form, or they can be modified in a laboratory to achieve specific characteristics. For example, in CAR T-cell therapy, a patient’s T-cells are modified to produce special receptors that help them attack cancer cells. Cell therapy can be classified as autologous or allogeneic. Autologous means that cells are isolated from the patient, modified, and then reinfused into the patient. Allogeneic means that cells are derived from a donor, and may or may not be modified. Cell therapy can be used to treat a wide range of conditions, including cancers, autoimmune disease, urinary problems, infectious disease, cardiovascular disease, and neurological disorders. For example, one can engineer hematopoietic stem cells so that the cells or their derivatives can produce FVIII variants disclosed herein to treat hemophilia A.

[0089] In some embodiments, the FVIII variants are introduced into a subject’s body in the forms of protein, peptide, RNA, or DNA, or via gene editing techniques to modify a gene in a subject’s body to produce a gene encoding for a FVIII variant disclosed herein.

[0090] In some embodiments, a therapeutically effective amount of the composition can be determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present disclosure. As disclosed above, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., treating, modulating, curing, preventing and / or ameliorating hemophilia A). For example, a therapeutically effective amount can be an amount sufficient to achieve a desired therapeutic and / or prophylactic effect. In some cases, the amount of a therapeutic agent (e.g., rAAV comprising a DNA sequence encoding a FVIII protein) administered to a subject in need thereof depends upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject. In some cases, both objective and subjective assays can optionally be employed to identify optimal dosage ranges.

[0091] A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein or therapeutic agent or therapeutic composition, a therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) can vary, for example, depending on route of administration, on combination with other pharmaceutical agents. In some cases, the specific therapeutically effective amount (and / or unit dose) for any particular patient can depend upon aWSGR Docket No. 57837-747.601 variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific protein employed; the duration of the treatment; etc.

[0092] In some embodiments, a therapeutically effective dose of the provided composition, when administered regularly, results in increased expression of hepatic FVIII protein as compared to baseline levels before treatment. In some embodiments, administering the provided composition results in the expression of a FVIII protein level at or above about 100 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, about 500 ng / ml, about 600 ng / ml, about 700 ng / ml, about 800 ng / ml, about 900 ng / ml, about 1,000 ng / ml, about 1,200 ng / ml or about 1,400 ng / ml of total protein in serum .

[0093] In some embodiments, administering provided compositions results in increased serum FVIII protein levels. In some embodiments, administering provided compositions results in increased serum FVIII protein levels by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to baseline FVIII protein level before treatment. Baseline FVIII protein level in serum is measured immediately before or at a commonly selected time before the treatment.

[0094] In some embodiments, the composition comprising the FVIII variant may comprise a physiologically acceptable matrix and may be formulated as a pharmaceutical preparation. The pharmaceutical preparation may be formulated using substantially known methods. For example, the FVIII variants can be mixed with a buffer containing salts, such as NaCl, CaCh, and amino acids, such as glycine and / or lysine. The buffer may be in a pH range from about 6 to about 8. The pharmaceutical preparation can be stored in in a finished solution or in a lyophilized form or a deep-frozen form. In some embodiments, the pharmaceutical preparation is stored in lyophilized form and is dissolved into a visually clear solution using an appropriate reconstitution solution. Alternatively, the pharmaceutical preparation can also be made available as a liquid preparation or as a liquid that is deep-frozen. The pharmaceutical preparation is stable, i.e., it can be allowed to stand in dissolved form for a prolonged time prior to its application to a subject. In some embodiments, the pharmaceutical preparation is a one- component preparation. In some embodiments, the pharmaceutical preparation is a multicomponent preparation with other components.

[0095] The pharmaceutical preparation may contain, as an example, dosages of from about 1 to about 1000 pg / kg, from about 10 to about 500 pg / kg, from about 10 to about 250 pg / kg, or from about 10 to about 100 pg / kg. In some embodiments, the pharmaceutical protein preparationWSGR Docket No. 57837-747.601 may comprise a dosage of from about 30 to about 100 lU / kg (e.g., as a single daily injection or up to 3 times or more / day). Patients may be treated immediately upon presentation at the clinic with a bleed or prior to the delivery of cut / wound causing a bleed. In some embodiments, patients may receive a bolus infusion every one to three, eight, or twelve hours or, if sufficient improvement is observed, a once daily infusion of the FVIII variant.

[0096] Nucleic acid molecules encoding FVIII variants may be used for a variety of purposes. In some embodiments, a nucleic acid delivery vehicle (e.g., an expression vector such as a viral vector) for modulating blood coagulation is provided wherein the expression vector comprises a nucleic acid sequence coding for a FVIII variant as described herein. Administration of the FVIII variant-encoding expression vectors to a patient may result in the expression of the FVIII variant which is configured to alter the coagulation cascade. In some embodiments, such a nucleic acid molecule may encode a FVIII variant polypeptide as described herein whose expression increases hemostasis. In a particular embodiment, the nucleic acid sequence encodes a human FVIII variant.

[0097] Expression vectors comprising nucleic acid sequences encoding a FVIII variant may be administered alone, or in combination with other therapeutic agents useful for modulating hemostasis. In some embodiments, the expression vectors or combination of therapeutic agents may be administered to the patient alone or in a pharmaceutically acceptable or biologically compatible composition.

[0098] In some embodiments, the expression vector comprising nucleic acid sequences encoding a FVIII variant is a viral vector. Viral vectors include, but are not limited to, adenoviral vectors (with or without tissue specific promoters / enhancers), adeno-associated virus (AAV) vectors of any serotype (e.g., AAV1 to AAV12, particularly AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, and AAV9), hybrid AAV vectors (e.g., AAV-DJ), and other AAV variants, lentivirus vectors and pseudo-typed lentivirus vectors (e.g., Ebola virus, vesicular stomatitis virus (VSV), and feline immunodeficiency virus (FIV)), herpes simplex virus vectors, vaccinia virus vectors, and retroviral vectors. In some embodiments, the vector is an AAV vector. In some embodiments, the vector is a lentiviral vector. These vectors can transduce, including but not limited to, hematopoietic stem cells, immune cells, skeletal muscle, fat tissue, central nervous system and / or liver cells; and have been used as carriers for many clinical trials in progress.

[0099] In some embodiments, engineered CD34 stem cells can be used to express the FVIII variants disclosed herein to treat hemophilia A. In some embodiments, lentivirus vector- mediated gene therapy can be used to introduce FVIII variant expression in human platelets by transducing CD34+ cells from human cord blood. This therapy may improve hemostasis in aWSGR Docket No. 57837-747.601 subject with hemophilia A.

[0100] In some embodiments, fetal-derived term placental cells (PLCs) can be used as cellular delivery vehicles for the FVIII variants disclosed herein. PLCs can be genetically modified to produce and secrete therapeutic levels of the FVIII variants disclosed herein. In some embodiments, PLCs can be transduced with a lentiviral vector to increase FVIII variants secretion. In some embodiments, the FVIII transgene for PLCs may be a myeloid codon- optimized, bioengineered FVIII variants.

[0101] In some embodiments, particle-mediated transfection, electroporation, and hydrodynamics-based transfection may be used as physical methods that introduce the nucleic acid materials encoding the FVIII variants disclosed herein into hematopoietic progenitors, which are then introduced into a subject’s body.

[0102] Non-viral vectors such as using transfection, electroporation can be relied upon as well to introduce the FVIII variants into the subject’s body.

[0103] Included in the present disclosure is a method for modulating hemostasis comprising providing cells of a subject with a nucleic acid delivery vehicle encoding a FVIII variant and allowing the cells to grow under conditions wherein the FVIII variant is expressed. The FVIII variants and FVIII variant expressing nucleic acid vectors as disclosed herein may be used in the treatment of disorders associated with aberrant blood coagulation. The expression vectors may be incorporated into pharmaceutical compositions that may be delivered to a subject, so as to allow the production of a biologically active protein (e.g., a FVIII variant) or by inducing expression of the FVIII variant in vivo by gene- and or cell-based therapies or by ex vivo modification / transduction of the patient's or donor's cells. The compositions may be administered alone or in combination with other agents, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents (e.g., co-factors) which influence hemostasis.

[0104] In some embodiments, the compositions (e.g., pharmaceutical compositions) may comprise a pharmaceutically acceptable carrier. Such carriers include a pharmaceutical agent that may be administered without undue toxicity. Pharmaceutically acceptable carriers may include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and theWSGR Docket No. 57837-747.601 like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., 18th Edition, Easton, Pa.

[1990] ).

[0105] 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 HC1, 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. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.

[0106] Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0107] The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding, free base forms. In other embodiments, the preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0. l%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. Other formulations are possible.

[0108] Pharmaceutical compositions may include compositions wherein the active ingredients are contained in an effective amount to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the capability of a skilled medical practitioner using the techniques and guidance provided in the present disclosure. Therapeutic doses will depend on, among other factors, the age and general condition of the subject, the severity of the aberrant blood coagulation phenotype, and the strength of the control sequences regulating the expression levels of the variant polypeptide.WSGR Docket No. 57837-747.601

[0109] As used herein, “Pharmaceutically acceptable” generally indicates 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.

[0110] The FVIII variants, alone or in combination with other agents, may be directly infused into a patient in an appropriate biological / pharmaceutical carrier as described hereinabove. Expression vectors of the present disclosure comprising nucleic acid sequences encoding variant or functional fragments thereof, may be administered to a patient by a variety of means (see below) to achieve and maintain a prophylactically and / or therapeutically effective level of the variant polypeptide. In some embodiments, the compositions may be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intraarterially, orally, intrahepatically or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications. A clinician specializing in the treatment of patients with blood coagulation disorders may determine the optimal route for administration of the adenoviral vectors comprising variant nucleic acid sequences based on a number of criteria, including, but not limited to: the condition of the patient and the purpose of the treatment (e.g., enhanced or reduced blood coagulation).EXAMPLES[oni] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.EXAMPLE 1. Some FVIII variants show enhanced specific activity compared to WT human FVIII in transient transfection in hepatocyte-like cells.

[0112] Wild-type FVIII and amino acid substitution variants thereof were transiently expressed in hepatocyte-like cells. FIG. 1 shows screening results of transient transfections for some FVIII variants disclosed herein. Each of these variants shown has a single amino acid change relative to the wildtype (WT) FVIII-SQ. FVIII variants tested are K570V variant, and R571S variant, K659M variant, and K707N variant. The antigen expression, activity and specific activity data were generated from plasmid transient transfections in hepatocyte-like cell lines (Huh7 cells and HepG2 cells). In these experiments, 24-well plates of each cell line wereWSGR Docket No. 57837-747.601 transfected with 500 ng of each DNA construct comprising either the WT FVIII-SQ or a FVIII variant. After 24 hours the media were changed to expression media that does not contain fetal bovine serum (FBS). Another 24 hours after the media was changed, the conditioned media were collected from each well. The conditioned media was then assayed for FVIII antigen by enzyme-linked immunosorbent assay (ELISA), and for FVIII activity with a one-stage activated partial thromboplastin time (aPTT) assay. FVIII antigen and activity levels were then normalized to wells transduced with FVIII-SQ (WT) to determine fold-changes in antigen and activity, and specific activity was calculated as the ratio of activity to antigen expression. The data for antigen expression, activity, and specific activity of some top variants relative to WT FVIII-SQ in transient transfections in hepatocyte-like cells are shown in FIG. 1.EXAMPLE 2. Site saturation mutagenesis at position K707 of FVIII to evaluate impact of each mutation on specific activity of FVIII.

[0113] FIG. 2 shows antigen, activity, and specific activity of various FVIII variants at site 707 of human FVIII relative to WT FVIII-SQ (K707) in transient transfections of Huh7 cells. FVIII variants tested are K707A, K707C, K707D, K707E, K707F, K707G, K707H, K707I, K707L, K707M, K707N, K707P, K707Q, K707R, K707S, K707T, K707V, K707W, and K707Y. In these experiments, FVIII antigen and activity levels were normalized to wells transduced with WT FVIII-SQ (K707) to determine fold-changes in antigen, activity, and specific activity. Of tested K707 mutations, K707N appeared to have the most significant change in FVIII antigen levels.EXAMPLE 3. Site saturation mutagenesis at position K570 of FVIII to evaluate impact of each mutation on specific activity of FVIII.

[0114] FIGS. 3A-3C shows antigen, activity, and specific activity of site saturation mutagenesis of K570 of human FVIII relative to WT FVIII-SQ (K570) in transient transfections of Huh7 cells. FVIII variants tested are K570A, K570C, K570D, K570E, K570F, K570G, K570H, K570I, K570L, K570M, K570N, K570P, K570Q, K570R, K570S, K570T, K570V, K570W, and K570Y. In these experiment, the assay was done using Huh7 cells transfected with 500 ng of each DNA construct in 24-well plates to determine FVIII antigen and activity levels which were then used to calculate specific activity relative to WT (FVIII-SQ). FIG. 3A shows FVIII variant antigen expression of various FVIII variants at site 570 of human FVIII relative to WT FVIII-SQ. FIG. 3B shows FVIII variant one-stage activity of various FVIII variants at site 570 of human FVIII relative to WT FVIII-SQ. FIG. 3C shows FVIII variant specific activity of various FVIII variants at site 570 of human FVIII relative to WT FVIII-SQ.EXAMPLE 4. Site saturation mutagenesis at position R571 of FVIII to evaluate impact of each mutation on specific activity of FVIII.WSGR Docket No. 57837-747.601

[0115] FIGS. 4A-4C shows antigen, activity, and specific activity of site saturation mutagenesis of R571 of human FVIII relative to WT FVIII-SQ (R571) in transient transfections of Huh7 cells. FVIII variants tested are R571 A, R571C, R571D, R571E, R571F, R571G, R571H, R571I, R571L, R571M, R571N, R571P, R571Q, R571R, R571S, R571T, R571V, R571W, and R571 Y. In these experiment, the assay was done using Huh7 cells transfected with 500 ng of each DNA construct in 24-well plates to determine FVIII antigen and activity levels which were then used to calculate specific activity relative to WT (FVIII-SQ). FIG. 4A shows FVIII variant antigen expression of various FVIII variants at site 571 of human FVIII relative to WT FVIII-SQ. FIG. 4B shows FVIII variant one-stage activity of various FVIII variants at site 571 of human FVIII relative to WT FVIII-SQ. FIG. 4C shows FVIII variant specific activity of various FVIII variants at site 571 of human FVIII relative to WT FVIII-SQ.EXAMPLE 5. Some FVIII variants show enhanced specific activity compared to WT human FVIII.

[0116] FIG. 5 shows specific activity of some FVIII variants with single amino acid mutations at different sites compared to WT FVIII-SQ. FVIII variants tested are K570A, K570S, K570T, K570V, R571F, R571M, R571S, R571T, R571V, and K659M. The assay was done using Huh7 cells transfected with 500 ng of each DNA construct in 24-well plates to determine FVIII antigen and activity levels which were then used to calculate specific activity relative to WT FVIII-SQ.EXAMPLE 6. Multiple mutations combined may further augment FVIII specific activity.

[0117] FIG. 6 shows that the specific activities of some FVIII variants containing multiple mutations are higher than those of the original variants. FVIII variants tested are K659M, X5, K570V, R571S, K570V / K659M, R571S / K659M, R571S / X5. The assay was done using Huh7 cells transfected with 500 ng of each DNA construct in 24-well plates to determine FVIII antigen and activity levels which were then used to calculate specific activity relative to WT FVIII-SQ.EXAMPLE 7. Site saturation mutagenesis at position of N572 to evaluate impact of each mutation on specific activity of FVIII.

[0118] FIG. 7 shows specific activity of site saturation mutagenesis of N572 of human FVIII relative to WT FVIII-SQ (N572) in transient transfections of Huh7 cells. FVIII variants tested are N572A, N572C, N572D, N572E, N572F, N572G, N572I, N572K, N572L, N572M, N572Q, N572R, N572S, N572T, N572V, N572W, and N572Y. The assay was done using Huh7 cells transfected with 500 ng of each DNA construct in 24-well plates to determine FVIII antigen and activity levels which were then used to calculate specific activity relative to WT (FVIII-SQ).WSGR Docket No. 57837-747.601EXAMPLE 8. Combo FVIII variants

[0119] FIG. 8 shows antigen, activity, and specific activity of combinations of various amino acid substitutions on FVIII variants relative to WT FVIII-SQ in transient transfections of Huh7 cells. The assay was done using Huh7 cells transfected with 500 ng of each DNA construct in 24-well plates to determine FVIII antigen and activity levels which were then used to calculate specific activity relative to WT. The data were normalized to WT (FVIII-SQ). The data show a combination of different amino acid mutations may further enhance FVIII antigen concentration and / or specific activity relative to each single amino acid mutation.EXAMPLE 9. In vivo validation of mutant R571S in F8 KO hemophilia A mice

[0120] Expression cassettes containing FVIII-SQ and FVIII-SQ with R571 S mutation were separately packaged into AAV8 capsids and dosed in F8 knockout hemophilia A mice at 2.5xl012vg / kg. An FVIII ELISA was used to measure plasma human FVIII antigen level at week 4 post dosing. One stage clotting assay was used to measure FVIII activity.

[0121] As shown in FIG. 9A, FVIII-SQ with R571S mutation showed similar expression compared to FVIII-SQ without the mutation at 4 weeks after injection. FIG. 9B shows that FVIII-SQ with R571S mutation showed higher clotting assay activity and higher specific activity (FIG. 9C) when compared with FVIII-SQ. The specific activity of R571S was about 4 times that of SQ.NUMBERED EMBODIMENTS

[0122] Embodiment 1. A Factor VIII (FVIII) variant comprising at least one amino acid substitution mutation of (i) Lys at position 570, (ii) Arg at position 571, (iii) Asn at position 572, and / or (iv) Lys at position 707 of mature human FVIII or its variants.

[0123] Embodiment 2. The FVIII variant of embodiment 1, wherein the amino acid Lys (K) at position 570 is substituted with amino acid Ala (A), Asp (D), Glu (E), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

[0124] Embodiment 3. The FVIII variant of embodiment 1, wherein the Lys at position 570 is substituted with Arg (R), Ser (S), Thr (T), or Vai (V).

[0125] Embodiment 4. The FVIII variant of embodiment 1, wherein the Lys at position 570 is substituted with Ala (A), Ser (S), Thr (T), or Vai (V).

[0126] Embodiment 5. The FVIII variant of embodiment 1, wherein the Lys at position 570 is substituted with Ser (S) or Vai (V).

[0127] Embodiment 6. The FVIII variant of embodiment 1, wherein the Lys at position 570 is substituted with Arg.

[0128] Embodiment 7. The FVIII variant of embodiment 1, wherein the Lys at position 570 is substituted with Ala.WSGR Docket No. 57837-747.601

[0129] Embodiment 8. The FVIII variant of embodiment 1, wherein the Lys at position 570 is substituted with Ser.

[0130] Embodiment 9. The FVIII variant of embodiment 1, wherein the Lys at position 570 is substituted with Thr.

[0131] Embodiment 10. The FVIII variant of embodiment 1, wherein the Lys at position 570 is substituted with Vai.

[0132] Embodiment 11. The FVIII variant of embodiment 1, wherein the amino acid Arg(R) at position 571 is substituted with Ala (A), Cys (C) Asp (D), Glu (E), Phe (F), Gly (G), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

[0133] Embodiment 12. The FVIII variant of embodiment 1, wherein the Arg at position 571 is substituted with Ala (A), Cys (C), Phe (F), He (I), Met (M), Ser (S), Thr (T), or Vai (V).

[0134] Embodiment 13. The FVIII variant of embodiment 1, wherein the Arg at position 571 is substituted with Phe (F), Met (M), Ser (S), Thr (T), or Vai (V).

[0135] Embodiment 14. The FVIII variant of embodiment 1, wherein the Arg at position 571 is substituted with Ser (S) or Thr (T).

[0136] Embodiment 15. The FVIII variant of embodiment 1, wherein the Arg at position 571 is substituted with Phe.

[0137] Embodiment 16. The FVIII variant of embodiment 1, wherein the Arg at position 571 is substituted with Met.

[0138] Embodiment 17. The FVIII variant of embodiment 1, wherein the Arg at position 571 is substituted with Ser.

[0139] Embodiment 18. The FVIII variant of embodiment 1, wherein the Arg at position 571 is substituted with Thr.

[0140] Embodiment 19. The FVIII variant of embodiment 1, wherein the Arg at position 571 is substituted with Vai.

[0141] Embodiment 20. The FVIII variant of embodiment 1, wherein the amino acid Asn(N) at position 572 is substituted with Ala (A), Cys (C), Glu (E), Phe (F), Gly (G), lie (I), Leu (L), Met (M), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

[0142] Embodiment 21. The FVIII variant of embodiment 1, wherein the Asn at position572 is substituted with Ala (A), Cys (C), Glu (E), Phe (F), Gly (G), He (I), Leu (L), Met (M), or Ser (S).

[0143] Embodiment 22. The FVIII variant of embodiment 1, wherein the Asn at position 572 is substituted with Ala (A), Glu (E), Phe (F), or Ser (S).

[0144] Embodiment 23. The FVIII variant of embodiment 1, wherein the Asn at position 572 is substituted with Ala.WSGR Docket No. 57837-747.601

[0145] Embodiment 24. The FVIII variant of embodiment 1, wherein the Asn at position 572 is substituted with Glu.

[0146] Embodiment 25. The FVIII variant of embodiment 1, wherein the Asn at position 572 is substituted with Phe.

[0147] Embodiment 26. The FVIII variant of embodiment 1, wherein the Asn at position 572 is substituted with Ser.

[0148] Embodiment 27. The FVIII variant of embodiment 1, wherein the amino acid Lys(K) at position 707 is substituted with Ala (A), Asp (D), Glu (E), Phe (F), Gly (G), His (H), He (I), Leu (L), Met (M), Asn (N), Pro (P), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

[0149] Embodiment 28. The FVIII variant of embodiment 1, wherein the Lys at position 707 is substituted with Gly (G), Met (M), Asn (N), Pro (P), Ser (S), Trp (W), or Tyr (Y).

[0150] Embodiment 29. The FVIII variant of embodiment 1, wherein the Lys at position 707 is substituted with Met (M), Asn (N), Ser (S), Trp (W), or Tyr (Y).

[0151] Embodiment 30. The FVIII variant of embodiment 1, wherein the Lys at position 707 is substituted with Asn.

[0152] Embodiment 31. The FVIII variant of embodiment 1, wherein the Lys at position 707 is substituted with Gly.

[0153] Embodiment 32. The FVIII variant of embodiment 1, wherein the Lys at position 707 is substituted with Met.

[0154] Embodiment 33. The FVIII variant of embodiment 1, wherein the Lys at position 707 is substituted with Ser.

[0155] Embodiment 34. The FVIII variant of embodiment 1, wherein the Lys at position 707 is substituted with Trp.

[0156] Embodiment 35. The FVIII variant of embodiment 1, wherein the Lys at position 707 is substituted with Tyr.

[0157] Embodiment 36. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, and (ii) the substitution mutation of Arg at position 571 of mature human FVIII or its variants.

[0158] Embodiment 37. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570 with Vai; and (2) the substitution mutation of Arg at position 571 with Ser.

[0159] Embodiment 38. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, and (ii) the substitution mutation of Asn at position 572 of mature human FVIII or its variants.

[0160] Embodiment 39. The FVIII variant of embodiment 1, wherein the FVIII variantWSGR Docket No. 57837-747.601 comprises (i) the substitution mutation of Lys at position 570, and (ii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

[0161] Embodiment 40. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Arg at position 571, and (ii) the substitution mutation of Asn at position 572 of mature human FVIII or its variants.

[0162] Embodiment 41. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Arg at position 571, and (ii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

[0163] Embodiment 42. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Asn at position 572, and (ii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

[0164] Embodiment 43. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Arg at position 571, and (iii) the substitution mutation of Asn at position 572 of mature human FVIII or its variants.

[0165] Embodiment 44. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Arg at position 571, and (iii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

[0166] Embodiment 45. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Asn at position 572, and (iii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

[0167] Embodiment 46. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Arg at position 571, (ii) the substitution mutation of Asn at position 572, and (iii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

[0168] Embodiment 47. The FVIII variant of embodiment 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Arg at position 571, (iii) the substitution mutation of Asn at position 572, and (iv) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

[0169] Embodiment 48. The FVIII variant of any one of embodiments 36-39, 43-46, or 47, wherein the Lys at position 570 is substituted with Ala (A), Asp (D), Glu (E), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

[0170] Embodiment 49. The FVIII variant of embodiment 48, wherein the Lys at positionWSGR Docket No. 57837-747.601570 is substituted with Arg.

[0171] Embodiment 50. The FVIII variant of embodiment 48, wherein the Lys at position 570 is substituted with Ala.

[0172] Embodiment 51. The FVIII variant of embodiment 48, wherein the Lys at position 570 is substituted with Ser.

[0173] Embodiment 52. The FVIII variant of embodiment 48, wherein the Lys at position 570 is substituted with Thr.

[0174] Embodiment 53. The FVIII variant of embodiment 48, wherein the Lys at position570 is substituted with Vai.

[0175] Embodiment 54. The FVIII variant of any one of embodiments 36, 37, 40, 41, 42, 44, 46, or 47-53, wherein the Arg at position 571 is substituted with Ala (A), Cys (C) Asp (D), Glu (E), Phe (F), Gly (G), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

[0176] Embodiment 55. The FVIII variant of embodiment 54, wherein the Arg at position571 is substituted with Phe.

[0177] Embodiment 56. The FVIII variant of embodiment 54, wherein the Arg at position 571 is substituted with Met.

[0178] Embodiment 57. The FVIII variant of embodiment 54, wherein the Arg at position 571 is substituted with Ser.

[0179] Embodiment 58. The FVIII variant of embodiment 54, wherein the Arg at position 571 is substituted with Thr.

[0180] Embodiment 59. The FVIII variant of embodiment 54, wherein the Arg at position571 is substituted with Vai.

[0181] Embodiment 60. The FVIII variant of any one of embodiments 38, 40, 42, 43, or 45- 59, wherein the Asn at position 572 is substituted with Ala (A), Cys (C), Glu (E), Phe (F), Gly (G), He (I), Leu (L), Met (M), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

[0182] Embodiment 61. The FVIII variant of embodiment 60, wherein the Asn at position572 is substituted with Ala.

[0183] Embodiment 62. The FVIII variant of embodiment 60, wherein the Asn at position 572 is substituted with Glu.

[0184] Embodiment 63. The FVIII variant of embodiment 60, wherein the Asn at position 572 is substituted with Phe.

[0185] Embodiment 64. The FVIII variant of embodiment 60, wherein the Asn at position 572 is substituted with Ser.

[0186] Embodiment 65. The FVIII variant of any one of embodiments 38, 41, 42, or 44-64,WSGR Docket No. 57837-747.601 wherein the Lys at position 707 is substituted with Ala (A), Asp (D), Glu (E), Phe (F), Gly (G), His (H), He (I), Leu (L), Met (M), Asn (N), Pro (P), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

[0187] Embodiment 66. The FVIII variant of embodiment 65, wherein the Lys at position 707 is substituted with Asn.

[0188] Embodiment 67. The FVIII variant of embodiment 65, wherein the Lys at position 707 is substituted with Gly.

[0189] Embodiment 68. The FVIII variant of embodiment 65, wherein the Lys at position 707 is substituted with Met.

[0190] Embodiment 69. The FVIII variant of embodiment 65, wherein the Lys at position 707 is substituted with Ser.

[0191] Embodiment 70. The FVIII variant of embodiment 65, wherein the Lys at position 707 is substituted with Trp.

[0192] Embodiment 71. The FVIII variant of embodiment 65, wherein the Lys at position 707 is substituted with Tyr.

[0193] Embodiment 72. The FVIII variant of embodiment 65, wherein the Arg at position 571 is substituted with Ser, and wherein the Lys at position 707 is substituted with Asn.

[0194] Embodiment 73. The FVIII variant of embodiment 65, wherein the Lys at position 570 is substituted with Vai, and wherein the Lys at position 707 is substituted with Asn.

[0195] Embodiment 74. The FVIII variant of embodiment 65, wherein the Lys at position 570 is substituted with Vai, wherein the Arg at position 571 is substituted with Ser, and wherein the Lys at position 707 is substituted with Asn.

[0196] Embodiment 75. The FVIII variant of any one of embodiments 1-74, further comprising at least one substitution mutation of: (i) Pro at position 290 is substituted with Thr; (ii) Phe at position 309 is substituted with Ser; (iii) Arg at position 336 is substituted with Gin; (iv) Arg at position 562 is substituted with Gin; (v) Lys at position 659 is substituted with Met; (vi) Lys at position 659 is substituted with Vai; or (vii) Lys at position 1813 is substituted with Ala; (viii) wherein the positions are based on the full-length mature human FVIII.

[0197] Embodiment 76. The FVIII variant of any one of embodiments 1-74, further comprising two substitution mutations selected from the group consisting of: (i) Pro at position 290 is substituted with Thr; (ii) Phe at position 309 is substituted with Ser; (iii) Arg at position 336 is substituted with Gin; (iv) Arg at position 562 is substituted with Gin; (v) Lys at position 659 is substituted with Met; (vi) Lys at position 659 is substituted with Vai; or (vii) Lys at position 1813 is substituted with Ala; wherein the positions are based on the full-length mature human FVIII.WSGR Docket No. 57837-747.601

[0198] Embodiment 77. The FVIII variant of any one of embodiments 1-74, further comprising three substitution mutations selected from the group consisting of: (i) Pro at position 290 is substituted with Thr; (ii) Phe at position 309 is substituted with Ser; (iii) Arg at position 336 is substituted with Gin; (iv) Arg at position 562 is substituted with Gin; (v) Lys at position 659 is substituted with Met; (vi) Lys at position 659 is substituted with Vai; or (vii) Lys at position 1813 is substituted with Ala; wherein the positions are based on the full-length mature human FVIII.

[0199] Embodiment 78. The FVIII variant of any one of embodiments 1-74, further comprising at least one substitution mutation of Lys at position 659 being substituted with Met, wherein the position is based on the full-length mature human FVIII.

[0200] Embodiment 79. The FVIII variant of embodiment 78, wherein Lys at position 570 is substituted with Vai, and wherein Lys at position 659 is substituted with Met, wherein the positions are based on the full-length mature human FVIII.

[0201] Embodiment 80. The FVIII variant of embodiment 78, wherein Arg at position 571 is substituted with Ser, and wherein Lys at position 659 is substituted with Met, and wherein the positions are based on the full-length mature human FVIII.

[0202] Embodiment 81. The FVIII variant of embodiment 78, wherein Asn at position 572 is substituted with at least one selected from the group consisting of Ala, Glu, Phe, and Ser, and wherein Lys at position 659 is substituted with Met, and wherein the positions are based on the full-length mature human FVIII.

[0203] Embodiment 82. The FVIII variant of embodiment 78, wherein Lys at position 707 is substituted with Asn, and wherein Lys at position 659 is substituted with Met, and wherein the positions are based on the full-length mature human FVIII.

[0204] Embodiment 83. The FVIII variant of embodiment 78, wherein Lys at position 570 is substituted with Vai, wherein Lys at position 707 is substituted with Asn, and wherein Lys at position 659 is substituted with Met, wherein the positions are based on the full-length ma-ture human FVIII.

[0205] Embodiment 84. The FVIII variant of embodiment 78, wherein Arg at position 571 is substituted with Ser, wherein Lys at position 707 is substituted with Asn, and wherein Lys at position 659 is substituted with Met, and wherein the positions are based on the full-length mature human FVIII.

[0206] Embodiment 85. The FVIII variant of any one of embodiments 1-84, wherein the FVIII variant is based on mature human FVIII.

[0207] Embodiment 86. The FVIII variant of any one of embodiments 1-84, wherein the FVIII variant is based on any of the following engineered FVIII proteins: A3-SP / DE, FVIII-AF,WSGR Docket No. 57837-747.601FVIII-CC, FVIII-ET3, VIII-HR, VIII-IR8, FVIII-N8, FVIII-OL, FVIILQ, FVIII-QQ, FVIII-RH, FVIII-SC, FVIII-SQ, FVIII-V3, FVIII- VV, FVIII-X5, or any combination thereof.

[0208] Embodiment 87. The FVIII variant of embodiment 86, wherein the FVIII variant is based on FVIII-X5.

[0209] Embodiment 88. The FVIII variant of embodiment 87, wherein Arg at position 571 is substituted with Ser, and wherein the FVIII variant is based on FVIII-X5.

[0210] Embodiment 89. The FVIII variant of embodiment 87, wherein Lys at position 570 is substituted with Vai, and wherein the FVIII variant is based on FVIII-X5.

[0211] Embodiment 90. The FVIII variant of embodiment 87, wherein Lys at position 570 is substituted with Vai, wherein Arg at position 571 is substituted with Ser, and wherein the FVIII variant is based on FVIII-X5.

[0212] Embodiment 91. The FVIII variant of embodiment 87, wherein Lys at position 570 is substituted with Vai, wherein Lys at position 659 is substituted with Met, and wherein the FVIII variant is based on FVIII-X5.

[0213] Embodiment 92. The FVIII variant of embodiment 87, wherein Arg at position 571 is substituted with Ser, wherein Lys at position 659 is substituted with Met, and wherein the FVIII variant is based on FVIII-X5.

[0214] Embodiment 93. The FVIII variant of embodiment 86, wherein the FVIII variant is based on FVIII-SQ.

[0215] Embodiment 94. The FVIII variant of any one of embodiments 1-93, wherein the FVIII variant comprises a linker sequence having at least 90% identity with any one of SEQ ID NOs: 4-20, wherein B-domain is replaced with the linker sequence.

[0216] Embodiment 95. The FVIII variant of any one of embodiments 1-93, wherein the FVIII variant comprises a linker sequence of any one of SEQ ID NOs: 4-20, wherein B-domain is re-placed with the linker sequence.

[0217] Embodiment 96. The FVIII variant of any one of embodiment 94 or embodiment 95, wherein the B do-main consists of amino acids 741-1648 of wild type FVIII (SEQ ID NO: 2).

[0218] Embodiment 97. A composition comprising at least one FVIII variant of any one of embodiments 1-96 and at least one pharmaceutically acceptable carrier.

[0219] Embodiment 98. A method for treatment of a hemostasis-related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of the FVIII variant of any one of embodiments 1-96 in a pharmaceutically acceptable carrier, where-in the FVIII variant is in a form of polypeptide, DNA or RNA.

[0220] Embodiment 99. The method of embodiment 98, wherein the FVIII variant is a component of or a product of lentivirus-based vector, adenovirus-based vector, another viralWSGR Docket No. 57837-747.601 vector, or non-viral vector.

[0221] Embodiment 100. The method of embodiment 98, wherein a nucleic acid encoding for the FVIII variant is transfected into a cell via electroporation.

[0222] Embodiment 101. The method of embodiment 98, wherein the FVIII variant is expressed by an engineered CD34 stem cell.

[0223] Embodiment 102. The method of any one of embodiments 98-101, wherein the hemostasis related disorder is hemophilia A.

[0224] Embodiment 103. A polynucleotide comprising a nucleic acid encoding the FVIII variant of any one of embodiments 1- 96.

[0225] Embodiment 104. The polynucleotide of embodiment 103, wherein the polynucleotide is a recombinant nucleotide.

[0226] Embodiment 105. The polynucleotide of embodiment 103, wherein the polynucleotide is a synthetic polynucleotide.

[0227] Embodiment 106. The polynucleotide of any one of embodiments 103-105, wherein said polynucleotide is codon optimized.

[0228] Embodiment 107. The polynucleotide of embodiment 106, wherein the nucleic acid encoding the human Factor VIII variant comprises a reduced number of CpG dinucleotides as compared to a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain.

[0229] Embodiment 108. The polynucleotide of any one of embodiments 103-107, wherein the nucleic acid encoding the human Factor VIII variant has a reduced immunogenicity compared to a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain.

[0230] Embodiment 109. The polynucleotide of any one of embodiments 103-108, wherein the polynucleotide comprises a promoter operably connected to the nucleic acid.

[0231] Embodiment 110. The polynucleotide of embodiment 109, wherein the promoter is a liver-specific promoter.

[0232] Embodiment 111. A recombinant adeno-associated virus (rAAV) particle, comprising a polynucleotide of any one of embodiments 103-110, wherein the rAAV particle comprises an AAV capsid protein selected from the group consisting of AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAVHSC, AAVDJ, LK03, NP59, KPI, or a variant thereof.

[0233] Embodiment 112. The rAAV particle of embodiment 111, wherein the rAAV particle is a single-stranded AAV (ssAAV).

[0234] Embodiment 113. The rAAV particle of embodiment 111, wherein the AAV capsid protein is AAV5 or AAV8.WSGR Docket No. 57837-747.601

[0235] Embodiment 114. A recombinant adeno-associated virus (rAAV) particle, prepared by transfecting the polynucleotide of any one of embodiments 103-110 into a host cell.

[0236] Embodiment 115. The rAAV particle of embodiment 114, wherein the host cell is an insect cell, a human cell, or an animal cell.

[0237] Embodiment 116. The rAAV particle of embodiment 115, wherein the insect cell is a Drosophila S2 cell or a Sf9 cell.

[0238] Embodiment 117. The rAAV particle of embodiment 115, wherein the animal cell is a fibroblasts, a Chinese hamster ovary (CHO) cell, a COS cell, a murine myeloma (NS0) cell, a HeLa cell, or a Baby Hamster Kidney (BEK) cell.

[0239] Embodiment 118. The rAAV particle of embodiment 115, wherein the human cell is a human embryonic kidney 293 (HEK293) cell, a HEK293 derivative (such as 293 T), a human fibrosarcoma (HT-1080) cell, a differentiated hepatocyte-derived carcinoma (Huh-7) cell, or a PER.C6 cell.

[0240] Embodiment 119. A method for treatment of a hemostasis related disorder in a patient in need thereof comprising administering a therapeutically effective amount of the polynucleotide of any one of embodiments 103-110 in a pharmaceutically acceptable carrier.

[0241] Embodiment 120. The method of embodiment 119, wherein the hemostasis related disorder is hemophilia A.

[0242] Embodiment 121. A method for treatment of a hemostasis related disorder in a patient in need thereof comprising administering a therapeutically effective amount of the rAAV particle of any one of embodiments 111-118 in a pharmaceutically acceptable carrier.

[0243] Embodiment 122. The method of embodiment 111, wherein the hemostasis related disorder is hemophilia A.

[0244] Embodiment 123. A protein comprising an activated form of the FVIII variant of any one of embodiments 1-96.

[0245] Embodiment 124. A Factor VIII-SQ (FVIII-SQ) variant comprising at least one amino acid substitution mutation, wherein the at least one amino acid substitution mutation comprises substitution of Arg at position 571 with Ser, wherein the FVIII-SQ variant comprising the substitution mutation of Arg at position 571 with Ser has higher activity compared with a FVIII-SQ variant lacking a substitution mutation of Arg at position 571 with Ser.

[0246] Embodiment 125. The FVIII-SQ variant of embodiment 124, wherein a one stage clotting assay is used to measure activity.

[0247] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will nowWSGR Docket No. 57837-747.601 occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Docket No. 57837-747.601CLAIMSWHAT IS CLAIMED IS:

1. A Factor VIII (FVIII) variant comprising at least one amino acid substitution mutation of (i) Lys at position 570, (ii) Arg at position 571, (iii) Asn at position 572, and / or (iv) Lys at position 707 of mature human FVIII or its variants.

2. The FVIII variant of claim 1, wherein the amino acid Lys (K) at position 570 is substituted with amino acid Ala (A), Asp (D), Glu (E), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

3. The FVIII variant of claim 1, wherein the amino acid Arg (R) at position 571 is substituted with Ala (A), Cys (C) Asp (D), Glu (E), Phe (F), Gly (G), His (H), He (I), Leu (L), Met (M), Asn (N), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

4. The FVIII variant of claim 1, wherein the amino acid Asn (N) at position 572 is substituted with Ala (A), Cys (C), Glu (E), Phe (F), Gly (G), lie (I), Leu (L), Met (M), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

5. The FVIII variant of claim 1, wherein the amino acid Lys (K) at position 707 is substituted with Ala (A), Asp (D), Glu (E), Phe (F), Gly (G), His (H), lie (I), Leu (L), Met (M), Asn (N), Pro (P), Gin (Q), Ser (S), Thr (T), Vai (V), Trp (W), or Tyr (Y).

6. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, and (ii) the substitution mutation of Arg at position 571 of mature human FVIII or its variants.

7. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570 with Vai; and (2) the substitution mutation of Arg at position 571 with Ser.

8. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, and (ii) the substitution mutation of Asn at position 572 of mature human FVIII or its variants.

9. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, and (ii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

10. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Arg at position 571, and (ii) the substitution mutation of Asn at position 572 of mature human FVIII or its variants.WSGR Docket No. 57837-747.60111. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Arg at position 571, and (ii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

12. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Asn at position 572, and (ii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

13. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Arg at position 571, and (iii) the substitution mutation of Asn at position 572 of mature human FVIII or its variants.

14. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Arg at position 571, and (iii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

15. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Asn at position 572, and (iii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

16. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Arg at position 571, (ii) the substitution mutation of Asn at position 572, and (iii) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

17. The FVIII variant of claim 1, wherein the FVIII variant comprises (i) the substitution mutation of Lys at position 570, (ii) the substitution mutation of Arg at position 571, (iii) the substitution mutation of Asn at position 572, and (iv) the substitution mutation of Lys at position 707 of mature human FVIII or its variants.

18. The FVIII variant of any one of claims 1-17, further comprising at least one substitution mutation of:(i) Pro at position 290 is substituted with Thr;(ii) Phe at position 309 is substituted with Ser;(iii) Arg at position 336 is substituted with Gin;(iv) Arg at position 562 is substituted with Gin;(v) Lys at position 659 is substituted with Met;(vi) Lys at position 659 is substituted with Vai; or(vii) Lys at position 1813 is substituted with Ala; wherein the positions are based on the full-length mature human FVIII.

19. The FVIII variant of any one of claims 1-17, further comprising two substitution mutations selected from the group consisting of:(i) Pro at position 290 is substituted with Thr;WSGR Docket No. 57837-747.601(ii) Phe at position 309 is substituted with Ser;(iii) Arg at position 336 is substituted with Gin;(iv) Arg at position 562 is substituted with Gin;(v) Lys at position 659 is substituted with Met;(vi) Lys at position 659 is substituted with Vai; or(vii) Lys at position 1813 is substituted with Ala; wherein the positions are based on the full-length mature human FVIII.

20. The FVIII variant of any one of claims 1-17, further comprising three substitution mutations selected from the group consisting of:(i) Pro at position 290 is substituted with Thr;(ii) Phe at position 309 is substituted with Ser;(iii) Arg at position 336 is substituted with Gin;(iv) Arg at position 562 is substituted with Gin;(v) Lys at position 659 is substituted with Met;(vi) Lys at position 659 is substituted with Vai; or(vii) Lys at position 1813 is substituted with Ala; wherein the positions are based on the full-length mature human FVIII.

21. The FVIII variant of any one of claims 1-20, wherein the FVIII variant is based on any of the following engineered FVIII proteins: A3-SP / DE, FVIII-AF, FVIII-CC, FVIII-ET3, VIII- HR, VIII-IR8, FVIII-N8, FVIII-OL, FVIII-Q, FVIII-QQ, FVIII-RH, FVIII-SC, FVIII-SQ, FVIII- V3, FVIII- VV, FVIII-X5, or any combination thereof.

22. The FVIII variant of any one of claims 1-21, wherein the FVIII variant comprises a linker sequence having at least 90% sequence identity with any one of SEQ ID NOs: 4-20, wherein a B-domain is replaced with the linker sequence.

23. The FVIII variant of any one of claim 22, wherein the B domain consists of amino acids 741-1648 of wild type FVIII (SEQ ID NO: 2).

24. A composition comprising at least one FVIII variant of any one of claims 1-23 and at least one pharmaceutically acceptable carrier.

25. A method for treatment of a hemostasis-related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of the FVIII variant of any one of claims 1-23 in a pharmaceutically acceptable carrier, wherein the FVIII variant is in a form of polypeptide, DNA or RNA.

26. The method of claim 25, wherein the FVIII variant is a component of or a product of lentivirus-based vector, adenovirus-based vector, another viral vector, or non-viral vector.WSGR Docket No. 57837-747.60127. The method of claim 25, wherein a nucleic acid encoding for the FVIII variant is transfected into a cell via electroporation.

28. The method of claim 25, wherein the FVIII variant is expressed by an engineered CD34 stem cell.

29. The method of any one of claims 25-28, wherein the hemostasis related disorder is hemophilia A.

30. A polynucleotide comprising a nucleic acid encoding the FVIII variant of any one of claims 1-23.

31. The polynucleotide of claim 30, wherein the polynucleotide is a recombinant nucleotide.

32. The polynucleotide of claim 30, wherein the polynucleotide is a synthetic polynucleotide.

33. The polynucleotide of any one of claims 30-32, wherein the polynucleotide is codon optimized.

34. The polynucleotide of claim 33, wherein the nucleic acid encoding the human Factor VIII variant comprises a reduced number of CpG dinucleotides as compared to a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain.

35. The polynucleotide of any one of claims 30-34, wherein the nucleic acid encoding the human Factor VIII variant has a reduced immunogenicity compared to a wild-type nucleic acid that encodes a wild-type Factor VIII peptide without a B domain.

36. The polynucleotide of any one of claims 30-35, wherein the polynucleotide comprises a promoter operably connected to the nucleic acid.

37. The polynucleotide of claim 36, wherein the promoter is a liver-specific promoter.

38. A recombinant adeno-associated virus (rAAV) particle comprising a polynucleotide of any one of claims 30-37, wherein the rAAV particle comprises an AAV capsid protein selected from the group consisting of AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAVHSC, AAVDJ, LK03, NP59, KPI, or a variant thereof.

39. The rAAV particle of claim 38, wherein the rAAV particle is a single-stranded AAV (ssAAV).

40. The rAAV particle of claim 38, wherein the AAV capsid protein is AAV5 or AAV8.

41. A recombinant adeno-associated virus (rAAV) particle prepared by transfecting the polynucleotide of any one of claims 30-37 into a host cell.

42. The rAAV particle of claim 41, wherein the host cell is an insect cell, a human cell, or an animal cell.

43. The rAAV particle of claim 42, wherein the insect cell is a Drosophila S2 cell or a Sf9 cell.WSGR Docket No. 57837-747.60144. The rAAV particle of claim 42, wherein the animal cell is a fibroblasts, a Chinese hamster ovary (CHO) cell, a COS cell, a murine myeloma (NSO) cell, a HeLa cell, or a Baby Hamster Kidney (BHK) cell.

45. The rAAV particle of claim 42, wherein the human cell is a human embryonic kidney 293 (HEK293) cell, a HEK293 derivative (such as 293T), a human fibrosarcoma (HT-1080) cell, a differentiated hepatocyte-derived carcinoma (Huh-7) cell, or a PER.C6 cell.

46. A method for treatment of a hemostasis related disorder in a patient in need thereof comprising administering a therapeutically effective amount of the polynucleotide of any one of claims 30-37 in a pharmaceutically acceptable carrier.

47. The method of claim 46, wherein the hemostasis related disorder is hemophilia A.

48. A method for treatment of a hemostasis related disorder in a patient in need thereof comprising administering a therapeutically effective amount of the rAAV particle of any one of claims 38-45 in a pharmaceutically acceptable carrier.

49. The method of claim 48, wherein the hemostasis related disorder is hemophilia A.

50. A protein comprising an activated form of the FVIII variant of any one of claims 1-23.

51. A Factor VIII-SQ (FVIII-SQ) variant comprising at least one amino acid substitution mutation, wherein the at least one amino acid substitution mutation comprises substitution of Arg at position 571 with Ser, wherein the FVIII-SQ variant comprising the substitution mutation of Arg at position 571 with Ser has higher activity compared with a FVIII-SQ variant lacking a substitution mutation of Arg at position 571 with Ser.

52. The FVIII-SQ variant of claim 124, wherein a one stage clotting assay is used to measure activity.

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