High-expression viral vectors encoding recombinant FVIII mutants for gene therapy of hemophilia A

Abstract

To provide codon-altered polynucleotides encoding Factor VIII variants for expression in mammalian cells.SOLUTION: A polynucleotide comprises a nucleotide sequence having at least 95% identity to CS01-FL-NA relating to a predetermined sequence, wherein the polynucleotide encodes a Factor VIII polypeptide.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 255,317, filed November 13, 2015, the entire disclosure of which is incorporated herein by reference for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of this ASCII file, created on November 9, 2016, has the filename 008073_5107_WO_Sequence_Listing.txt and is 353,479 bytes in size. [Background technology]

[0003] Background Art of the Disclosure Blood coagulation proceeds through a complex and dynamic biological pathway, the coagulation cascade, in which multiple biochemical reactions are interdependent. Coagulation factor VIII (FVIII) is a key component of the cascade. Factor VIII is recruited to the site of bleeding and forms an Xase (tenase) complex with activated factor IX (FIXa) and factor x (FX). The Xase complex activates FX, which activates prothrombin to thrombin, which then activates other components of the coagulation cascade to form a stable clot (reviewed in Saenko et al., Trends Cardiovasc. Med., 9:185-192 (1999) (Non-Patent Document 1); Lenting et al., Blood, 92:3983-3996 (1998) (Non-Patent Document 2)).

[0004] Hemophilia A is a congenital, X-linked bleeding disorder characterized by a deficiency in factor VIII activity. Decreased factor VIII activity inhibits a positive feedback loop within the coagulation cascade, resulting in defective clotting and manifesting as bleeding episodes with prolonged bleeding times, widespread ecchymosis, spontaneous bleeding from the mouth and nose, joint stiffness, and chronic pain, and in severe cases, possible internal bleeding and anemia (Zhang et al., Clinic. Rev. Allerg. Immunol., 37:114-124 (2009)).

[0005] Traditionally, hemophilia A is treated with factor VIII replacement therapy, which consists of administering factor VIII protein (e.g., plasma-derived or recombinantly produced factor VIII) to individuals with hemophilia A. Factor VIII is administered prophylactically to prevent or reduce the frequency of bleeding episodes, in response to acute bleeding episodes, and / or perioperatively to control bleeding during surgery. However, factor VIII replacement therapy has several undesirable characteristics.

[0006] First, although factor VIII replacement therapy is used to treat or manage hemophilia A, it does not cure the underlying factor VIII deficiency. As a result, individuals with hemophilia A must receive factor VIII replacement therapy throughout their lives. Continuing treatment is expensive, and patients must maintain strict compliance, as missing even a few prophylactic doses can have serious consequences for individuals with severe hemophilia A.

[0007] Second, because factor VIII has a relatively short half-life in the body, conventional prophylactic factor VIII replacement therapy requires dosing every two or three days, which places a burden on individuals who maintain lifelong compliance. While third-generation "long-acting" factor VIII drugs can reduce dosing frequency, prophylactic factor VIII replacement therapy with these drugs still requires monthly, weekly, or more frequent dosing indefinitely. For example, prophylactic treatment with ELOCTATE™ [Antihemophilic Factor (Recombinant), Fc Fusion Protein] requires dosing every three to five days (ELOCTATE™ Prescribing Information, Biogen Idec Inc. (2015)). Furthermore, the long-term effects of chemically modified biologics (e.g., PEGylated polypeptides) remain poorly understood.

[0008] Third, 15% to 30% of all individuals who receive factor VIII replacement therapy develop anti-factor VIII inhibitor antibodies, rendering the therapy ineffective. Factor VIII bypass therapy (e.g., administration of plasma-derived or recombinantly produced prothrombin complex concentrates) can be used to treat hemophilia in individuals who develop inhibitor antibodies. However, factor VIII bypass therapy is less effective than factor VIII replacement therapy (Mannucci PM, J Thromb Haemost., 1(7):1349-55 (2003)) and may be associated with an increased risk of cardiovascular complications (Luu and Ewenstein, Haemophilia, 10 Suppl. 2:10-16 (2004)).

[0009] Somatic cell gene therapy holds great promise for the treatment of hemophilia A because it appears to treat the underlying low expression of factor VIII activity function (e.g., due to missense or nonsense mutations) rather than providing an individual with a single dose of factor VIII activity. Because of this difference in mechanism of action, compared with factor VIII replacement therapy, a single administration of a factor VIII gene therapy vector could provide an individual with factor VIII for several years, reducing treatment costs and eliminating the need for ongoing patient compliance.

[0010] Coagulation factor IX (FIX) gene therapy has been used effectively to treat individuals with hemophilia B, a related blood clotting condition characterized by reduced factor IX activity (Manno CS, et al., Nat Med., 12(3):342-47 (2006)). However, factor VIII gene therapy presents several inherent challenges. For example, the full-length wild-type factor VIII polypeptide (2351 amino acids; UniProt accession number P00451) is five times larger than the full-length wild-type factor IX polypeptide (461 amino acids; UniProt accession number P00740). Thus, the coding sequence for wild-type factor VIII is 7053 base pairs, too large to be packaged into conventional AAV gene therapy vectors. Furthermore, reported recombinant expression of a B-domain deleted variant of factor VIII (BDD-FVIII) has been poor. Therefore, several groups have attempted to modify the codon usage of the BDD-FVIII construct, but have not had much success. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Saenko et al.,Trends Cardiovasc.Med.,9:185-192(1999) [Non-patent document 2] Lenting et al.,Blood,92:3983-3996(1998) [Non-patent document 3] Zhang et al.,Clinic.Rev.Allerg.Immunol.,37:114-124(2009) [Non-patent document 4] ELOCTATE™ Prescribing Information, Biogen Idec Inc. (2015) [Non-patent document 5] Mannucci PM, J Thromb Haemost.,1(7):1349-55(2003) [Non-patent document 6] Luu and Ewenstein, Haemophilia, 10 Suppl.2:10-16(2004) [Non-Patent Document 7] Manno CS, et al., Nat Med., 12(3):342-47(2006) Summary of the Invention

[0012] Disclosure Overview Thus, there is a need for factor VIII variants whose coding sequences can be more efficiently packaged into and delivered via gene therapy vectors. There is also a need for codon-modified synthetic nucleic acids that more efficiently express factor VIII. Such factor VIII variants and codon-modified nucleic acids enable improved treatment of factor VIII deficiency (e.g., hemophilia A). The disclosed codon-modified factor VIII variants reduce or eliminate the above-mentioned deficiencies and other problems associated with treating factor VIII deficiency (e.g., hemophilia A).

[0013] According to some embodiments, the present disclosure provides nucleic acids encoding Factor VIII variants with high sequence identity to the disclosed codon-modified sequences of the Factor VIII heavy chain (e.g., CS01-HC-NA, CS04-HC-NA, or CS23-HC-NA) and light chain (e.g., CS01-LC-NA, CS04-LC-NA, or CS23-LC-NA). In some embodiments, these nucleic acids further include sequences encoding a linker sequence (e.g., a linker sequence comprising a furin cleavage site) that replaces the native Factor VIII B domain between the sequences encoding the Factor VIII heavy and light chains.

[0014] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide. The Factor VIII polypeptide includes a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS04-HC-NA (SEQ ID NO: 3). The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS04-LC-NA (SEQ ID NO: 4). The polypeptide linker comprises a furin cleavage site.

[0015] In one embodiment of the above polynucleotide, the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to BDLO04 (SEQ ID NO: 6).

[0016] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide. The Factor VIII polypeptide includes a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS01-HC-NA (SEQ ID NO: 24). The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS01-LC-NA (SEQ ID NO: 25). The polypeptide linker comprises a furin cleavage site.

[0017] In one embodiment of the above polynucleotide, the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to BDLO01 (SEQ ID NO:5).

[0018] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide. The Factor VIII polypeptide includes a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS23-HC-NA (SEQ ID NO: 22). The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS23-LC-NA (SEQ ID NO: 23). The polypeptide linker comprises a furin cleavage site.

[0019] In one embodiment of the above polynucleotide, the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to BDLO23 (SEQ ID NO: 7).

[0020] In one embodiment of the above polynucleotides, the first nucleotide sequence encoding the heavy chain of the Factor VIII polypeptide has at least 96% identity to the respective heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO: 3), CS01-HC-NA (SEQ ID NO: 24), or CS23-HC-NA (SEQ ID NO: 22)), and the second nucleotide sequence encoding the light chain of the Factor VIII polypeptide has at least 96% identity to the respective light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4), CS01-LC-NA (SEQ ID NO: 25), or CS23-LC-NA (SEQ ID NO: 23)).

[0021] In one embodiment of the above polynucleotides, the first nucleotide sequence encoding the heavy chain of the Factor VIII polypeptide has at least 97% identity to the respective heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO: 3), CS01-HC-NA (SEQ ID NO: 24), or CS23-HC-NA (SEQ ID NO: 22)), and the second nucleotide sequence encoding the light chain of the Factor VIII polypeptide has at least 97% identity to the respective light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4), CS01-LC-NA (SEQ ID NO: 25), or CS23-LC-NA (SEQ ID NO: 23)).

[0022] In one embodiment of the above polynucleotides, the first nucleotide sequence encoding the heavy chain of the Factor VIII polypeptide has at least 98% identity to the respective heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO: 3), CS01-HC-NA (SEQ ID NO: 24), or CS23-HC-NA (SEQ ID NO: 22)), and the second nucleotide sequence encoding the light chain of the Factor VIII polypeptide has at least 98% identity to the respective light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4), CS01-LC-NA (SEQ ID NO: 25), or CS23-LC-NA (SEQ ID NO: 23)).

[0023] In one embodiment of the above polynucleotides, the first nucleotide sequence encoding the heavy chain of the Factor VIII polypeptide has at least 99% identity to the respective heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO: 3), CS01-HC-NA (SEQ ID NO: 24), or CS23-HC-NA (SEQ ID NO: 22)), and the second nucleotide sequence encoding the light chain of the Factor VIII polypeptide has at least 99% identity to the respective light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4), CS01-LC-NA (SEQ ID NO: 25), or CS23-LC-NA (SEQ ID NO: 23)).

[0024] In one embodiment of the above polynucleotides, the first nucleotide sequence encoding the heavy chain of the Factor VIII polypeptide has at least 99.5% identity to the respective heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO: 3), CS01-HC-NA (SEQ ID NO: 24), or CS23-HC-NA (SEQ ID NO: 22)), and the second nucleotide sequence encoding the light chain of the Factor VIII polypeptide has at least 99.5% identity to the respective light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4), CS01-LC-NA (SEQ ID NO: 25), or CS23-LC-NA (SEQ ID NO: 23)).

[0025] In one embodiment of the above polynucleotides, the first nucleotide sequence encoding the heavy chain of the Factor VIII polypeptide has at least 99.9% identity to the respective heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO: 3), CS01-HC-NA (SEQ ID NO: 24), or CS23-HC-NA (SEQ ID NO: 22)), and the second nucleotide sequence encoding the light chain of the Factor VIII polypeptide has at least 99.9% identity to the respective light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4), CS01-LC-NA (SEQ ID NO: 25), or CS23-LC-NA (SEQ ID NO: 23)).

[0026] In one embodiment of the above polynucleotide, the first nucleotide sequence encoding the heavy chain of the Factor VIII polypeptide is CS04-HC-NA (SEQ ID NO: 3), and the second nucleotide sequence encoding the light chain of the Factor VIII polypeptide is CS04-LC-NA (SEQ ID NO: 4).

[0027] In one embodiment of the above polynucleotide, the first nucleotide sequence encoding the heavy chain of the Factor VIII polypeptide is CS01-HC-NA (SEQ ID NO: 24), and the second nucleotide sequence encoding the light chain of the Factor VIII polypeptide is CS01-LC-NA (SEQ ID NO: 25).

[0028] In one embodiment of the above polynucleotide, the first nucleotide sequence encoding the heavy chain of the Factor VIII polypeptide is CS23-HC-NA (SEQ ID NO: 22), and the second nucleotide sequence encoding the light chain of the Factor VIII polypeptide is CS23-LC-NA (SEQ ID NO: 23).

[0029] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to CS04-FL-NA, wherein such polynucleotide encodes a Factor VIII polypeptide.

[0030] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to CS01-FL-NA, wherein such polynucleotide encodes a Factor VIII polypeptide.

[0031] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to CS23-FL-NA, wherein such polynucleotide encodes a Factor VIII polypeptide.

[0032] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 96% identity to the respective full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1), CS01-FL-NA (SEQ ID NO: 13), or CS23-FL-NA (SEQ ID NO: 20)).

[0033] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 97% identity to the respective full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1), CS01-FL-NA (SEQ ID NO: 13), or CS23-FL-NA (SEQ ID NO: 20)).

[0034] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 98% identity to the respective full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1), CS01-FL-NA (SEQ ID NO: 13), or CS23-FL-NA (SEQ ID NO: 20)).

[0035] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 99% identity to the respective full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1), CS01-FL-NA (SEQ ID NO: 13), or CS23-FL-NA (SEQ ID NO: 20)).

[0036] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 99.5% identity to the respective full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1), CS01-FL-NA (SEQ ID NO: 13), or CS23-FL-NA (SEQ ID NO: 20)).

[0037] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 99.9% identity to the respective full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1), CS01-FL-NA (SEQ ID NO: 13), or CS23-FL-NA (SEQ ID NO: 20)).

[0038] In one embodiment of the above polynucleotide, the nucleotide sequence is CS04-FL-NA (SEQ ID NO: 1).

[0039] In one embodiment of the above polynucleotide, the nucleotide sequence is CS01-FL-NA (SEQ ID NO: 13).

[0040] In one embodiment of the above polynucleotide, the nucleotide sequence is CS23-FL-NA (SEQ ID NO: 20).

[0041] In one embodiment of the above polynucleotide, the polynucleotide encodes a Factor VIII polypeptide comprising an amino acid sequence having at least 95% identity to CS04-FL-AA (SEQ ID NO: 2).

[0042] In one embodiment of the above polynucleotide, the polynucleotide encodes a Factor VIII polypeptide comprising an amino acid sequence having at least 96% identity to CS04-FL-AA (SEQ ID NO: 2).

[0043] In one embodiment of the above polynucleotide, the polynucleotide encodes a Factor VIII polypeptide comprising an amino acid sequence having at least 97% identity to CS04-FL-AA (SEQ ID NO: 2).

[0044] In one embodiment of the above polynucleotide, the polynucleotide encodes a Factor VIII polypeptide comprising an amino acid sequence having at least 98% identity to CS04-FL-AA (SEQ ID NO: 2).

[0045] In one embodiment of the above polynucleotide, the polynucleotide encodes a Factor VIII polypeptide comprising an amino acid sequence having at least 99% identity to CS04-FL-AA (SEQ ID NO: 2).

[0046] In one embodiment of the above polynucleotide, the polynucleotide encodes a Factor VIII polypeptide comprising an amino acid sequence having at least 99.5% identity to CS04-FL-AA (SEQ ID NO: 2).

[0047] In one embodiment of the above polynucleotide, the polynucleotide encodes a Factor VIII polypeptide comprising an amino acid sequence having at least 99.9% identity to CS04-FL-AA (SEQ ID NO: 2).

[0048] In one embodiment of the above polynucleotide, the polynucleotide encodes a Factor VIII polypeptide comprising the amino acid sequence of CS04-FL-AA (SEQ ID NO: 2).

[0049] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to CS04-SC1-NA (SEQ ID NO: 9), wherein such polynucleotide encodes a Factor VIII single-chain polypeptide.

[0050] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to CS04-SC2-NA (SEQ ID NO: 11), wherein such polynucleotide encodes a Factor VIII single-chain polypeptide.

[0051] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to CSO1-SC1-NA (SEQ ID NO: 26), wherein such polynucleotide encodes a Factor VIII single-chain polypeptide.

[0052] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to CSO1-SC2-NA (SEQ ID NO: 27), wherein such polynucleotide encodes a Factor VIII single-chain polypeptide.

[0053] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to CS23-SC1-NA (SEQ ID NO: 28), wherein such polynucleotide encodes a Factor VIII single-chain polypeptide.

[0054] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to CS23-SC2-NA (SEQ ID NO: 29), wherein such polynucleotide encodes a Factor VIII single-chain polypeptide.

[0055] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 96% identity to the respective full-length polynucleotide sequence (e.g., CS04-SC1-NA (SEQ ID NO: 9), CS04-SC2-NA (SEQ ID NO: 11), CS01-SC1-NA (SEQ ID NO: 26), CS01-SC2-NA (SEQ ID NO: 27), CS23-SC1-NA (SEQ ID NO: 28), or CS23-SC2-NA (SEQ ID NO: 29)).

[0056] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 97% identity to the respective full-length polynucleotide sequence (e.g., CS04-SC1-NA (SEQ ID NO: 9), CS04-SC2-NA (SEQ ID NO: 11), CS01-SC1-NA (SEQ ID NO: 26), CS01-SC2-NA (SEQ ID NO: 27), CS23-SC1-NA (SEQ ID NO: 28), or CS23-SC2-NA (SEQ ID NO: 29)).

[0057] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 98% identity to the respective full-length polynucleotide sequence (e.g., CS04-SC1-NA (SEQ ID NO: 9), CS04-SC2-NA (SEQ ID NO: 11), CS01-SC1-NA (SEQ ID NO: 26), CS01-SC2-NA (SEQ ID NO: 27), CS23-SC1-NA (SEQ ID NO: 28), or CS23-SC2-NA (SEQ ID NO: 29)).

[0058] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 99% identity to the respective full-length polynucleotide sequence (e.g., CS04-SC1-NA (SEQ ID NO: 9), CS04-SC2-NA (SEQ ID NO: 11), CS01-SC1-NA (SEQ ID NO: 26), CS01-SC2-NA (SEQ ID NO: 27), CS23-SC1-NA (SEQ ID NO: 28), or CS23-SC2-NA (SEQ ID NO: 29)).

[0059] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 99.5% identity to the respective full-length polynucleotide sequence (e.g., CS04-SC1-NA (SEQ ID NO: 9), CS04-SC2-NA (SEQ ID NO: 11), CS01-SC1-NA (SEQ ID NO: 26), CS01-SC2-NA (SEQ ID NO: 27), CS23-SC1-NA (SEQ ID NO: 28), or CS23-SC2-NA (SEQ ID NO: 29)).

[0060] In one embodiment of the above polynucleotides, the nucleotide sequence has at least 99.9% identity to the respective full-length polynucleotide sequence (e.g., CS04-SC1-NA (SEQ ID NO: 9), CS04-SC2-NA (SEQ ID NO: 11), CS01-SC1-NA (SEQ ID NO: 26), CS01-SC2-NA (SEQ ID NO: 27), CS23-SC1-NA (SEQ ID NO: 28), or CS23-SC2-NA (SEQ ID NO: 29)).

[0061] In one embodiment of the above polynucleotide, the nucleotide sequence is CS04-SC1-NA (SEQ ID NO: 9).

[0062] In one embodiment of the above polynucleotide, the nucleotide sequence is CS04-SC2-NA (SEQ ID NO: 11).

[0063] In one embodiment of the above polynucleotide, the nucleotide sequence is CS01-SC1-NA (SEQ ID NO: 26).

[0064] In one embodiment of the above polynucleotide, the nucleotide sequence is CS01-SC2-NA (SEQ ID NO: 27).

[0065] In one embodiment of the above polynucleotide, the nucleotide sequence is CS23-SC1-NA (SEQ ID NO: 28).

[0066] In one embodiment of the above polynucleotide, the nucleotide sequence is CS23-SC2-NA (SEQ ID NO: 29).

[0067] Specifications for a range of cylinder heads are CS01-FL-NA, CS01-HC-NA, and CS01-LC-NA CS04-FL-NA、CS04-HC-NA、CS04-LC-NA、CS23-FL-NA、CS23-HC-NA、CS23-L C-NA, CS01m1-FL-NA, CS01m2-FL-NA, CS01m3-FL-NA, CS01m4-FL-NA, CS01m12-FL-NA, CS01m13-FL-NA, CS01m23-FL-NA, CS01m24-FL-NA, CS01m24-FL-NA NA、CS01m123-FL-NA、CS01m234-FL-NA、CS04m1-FL-NA、CS04m2-FL-NA、CS04m3-FL-NA、CS04m4-FL-NA、CS04m12-FL-NA、CS04m13-FL-NA、CS04m23-FL -NA、CS04m24-FL-NA、CS04m34-FL-NA、CS04m123-FL-NA、CS04m234-FL-NA、CS23m1-FL-NA、CS23m2-FL-NA、CS23m3-FL-NA、CS23m4-FL-NA、CS23m12-FL -NA, CS23m13-FL-NA, CS23m23-FL-NA, CS23m24-FL-NA, CS23m34-FL-NA, CS23m123-FL-NA, CS23m234-FL-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1- NA、CS01m1-SC1-NA、CS01m2-SC1-NA、CS01m3-SC1-NA、CS01m4-SC1-NA、CS0 1m12-SC1-NA、CS01m13-SC1-NA、CS01m23-SC1-NA、CS01m24-SC1-NA、CS01m 34-SC1-NA、CS01m123-SC1-NA、CS01m234-SC1-NA、CS04m1-SC1-NA、CS04m2-SC1-NA、CS04m3-SC1-NA、CS04m4-SC1-NA、CS04m12-SC1-NA、CS04m13-SC 1-NA、CS04m23-SC1-NA、CS04m24-SC1-NA、CS04m34-SC1-NA、CS04m123-SC1 -NA、CS04m234-SC1-NA、CS23m1-SC1-NA、CS23m2-SC1-NA、CS23m3-SC1-NA、CS23m4-SC1-NA, CS23m12-SC1-NA, CS23m13-SC1-NA, CS23m23-SC1-NA, CS23m24-SC1-NA, CS23m34-SC1 -NA, CS23m123-SC1-NA, CS23m234-SC1-NA, CS01-SC2-NA, CS04-SC2-NA, CS23-SC2-NA, CS01m1-SC2-NA , CS01m2-SC2-NA, CS01m3-SC2-NA, CS01m4-SC2-NA, CS01m12-SC2-NA, CS01m13-SC2-NA, CS01m23-SC2- NA, CS01m24-SC2-NA, CS01m34-SC2-NA, CS01m123-SC2-NA, CS01m234-SC2-NA, CS04m1-SC2-NA, CS04m2- SC2-NA, CS04m3-SC2-NA, CS04m4-SC2-NA, CS04m12-SC2-NA, CS04m13-SC2-NA, CS04m23-SC2-NA, CS04m 24-SC2-NA, CS04m34-SC2-NA, CS04m123-SC2-NA, CS04m234-SC2-NA, CS23m1-SC2-NA, CS23m2-SC2-NA, It has at least 95% identity to a sequence selected from the group consisting of CS23m3-SC2-NA, CS23m4-SC2-NA, CS23m12-SC2-NA, CS23m13-SC2-NA, CS23m23-SC2-NA, CS23m24-SC2-NA, CS23m34-SC2-NA, CS23m123-SC2-NA, and CS23m234-SC2-NA.

[0068] Specifications for a range of cylinder heads are CS01-FL-NA, CS01-HC-NA, and CS01-LC-NA CS04-FL-NA、CS04-HC-NA、CS04-LC-NA、CS23-FL-NA、CS23-HC-NA、CS23-L C-NA, CS01m1-FL-NA, CS01m2-FL-NA, CS01m3-FL-NA, CS01m4-FL-NA, CS01m12-FL-NA, CS01m13-FL-NA, CS01m23-FL-NA, CS01m24-FL-NA, CS01m24-FL-NA NA、CS01m123-FL-NA、CS01m234-FL-NA、CS04m1-FL-NA、CS04m2-FL-NA、CS04m3-FL-NA、CS04m4-FL-NA、CS04m12-FL-NA、CS04m13-FL-NA、CS04m23-FL -NA、CS04m24-FL-NA、CS04m34-FL-NA、CS04m123-FL-NA、CS04m234-FL-NA、CS23m1-FL-NA、CS23m2-FL-NA、CS23m3-FL-NA、CS23m4-FL-NA、CS23m12-FL -NA, CS23m13-FL-NA, CS23m23-FL-NA, CS23m24-FL-NA, CS23m34-FL-NA, CS23m123-FL-NA, CS23m234-FL-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1- NA、CS01m1-SC1-NA、CS01m2-SC1-NA、CS01m3-SC1-NA、CS01m4-SC1-NA、CS0 1m12-SC1-NA、CS01m13-SC1-NA、CS01m23-SC1-NA、CS01m24-SC1-NA、CS01m 34-SC1-NA、CS01m123-SC1-NA、CS01m234-SC1-NA、CS04m1-SC1-NA、CS04m2-SC1-NA、CS04m3-SC1-NA、CS04m4-SC1-NA、CS04m12-SC1-NA、CS04m13-SC 1-NA、CS04m23-SC1-NA、CS04m24-SC1-NA、CS04m34-SC1-NA、CS04m123-SC1 -NA、CS04m234-SC1-NA、CS23m1-SC1-NA、CS23m2-SC1-NA、CS23m3-SC1-NA、CS23m4-SC1-NA, CS23m12-SC1-NA, CS23m13-SC1-NA, CS23m23-SC1-NA, CS23m24-SC1-NA, CS23m34-SC1 -NA, CS23m123-SC1-NA, CS23m234-SC1-NA, CS01-SC2-NA, CS04-SC2-NA, CS23-SC2-NA, CS01m1-SC2-NA , CS01m2-SC2-NA, CS01m3-SC2-NA, CS01m4-SC2-NA, CS01m12-SC2-NA, CS01m13-SC2-NA, CS01m23-SC2- NA, CS01m24-SC2-NA, CS01m34-SC2-NA, CS01m123-SC2-NA, CS01m234-SC2-NA, CS04m1-SC2-NA, CS04m2- SC2-NA, CS04m3-SC2-NA, CS04m4-SC2-NA, CS04m12-SC2-NA, CS04m13-SC2-NA, CS04m23-SC2-NA, CS04m 24-SC2-NA, CS04m34-SC2-NA, CS04m123-SC2-NA, CS04m234-SC2-NA, CS23m1-SC2-NA, CS23m2-SC2-NA, It has at least 96% identity to a sequence selected from the group consisting of CS23m3-SC2-NA, CS23m4-SC2-NA, CS23m12-SC2-NA, CS23m13-SC2-NA, CS23m23-SC2-NA, CS23m24-SC2-NA, CS23m34-SC2-NA, CS23m123-SC2-NA, and CS23m234-SC2-NA.

[0069] Specifications for a range of cylinder heads are CS01-FL-NA, CS01-HC-NA, and CS01-LC-NA CS04-FL-NA、CS04-HC-NA、CS04-LC-NA、CS23-FL-NA、CS23-HC-NA、CS23-L C-NA, CS01m1-FL-NA, CS01m2-FL-NA, CS01m3-FL-NA, CS01m4-FL-NA, CS01m12-FL-NA, CS01m13-FL-NA, CS01m23-FL-NA, CS01m24-FL-NA, CS01m24-FL-NA NA、CS01m123-FL-NA、CS01m234-FL-NA、CS04m1-FL-NA、CS04m2-FL-NA、CS04m3-FL-NA、CS04m4-FL-NA、CS04m12-FL-NA、CS04m13-FL-NA、CS04m23-FL -NA、CS04m24-FL-NA、CS04m34-FL-NA、CS04m123-FL-NA、CS04m234-FL-NA、CS23m1-FL-NA、CS23m2-FL-NA、CS23m3-FL-NA、CS23m4-FL-NA、CS23m12-FL -NA, CS23m13-FL-NA, CS23m23-FL-NA, CS23m24-FL-NA, CS23m34-FL-NA, CS23m123-FL-NA, CS23m234-FL-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1- NA、CS01m1-SC1-NA、CS01m2-SC1-NA、CS01m3-SC1-NA、CS01m4-SC1-NA、CS0 1m12-SC1-NA、CS01m13-SC1-NA、CS01m23-SC1-NA、CS01m24-SC1-NA、CS01m 34-SC1-NA、CS01m123-SC1-NA、CS01m234-SC1-NA、CS04m1-SC1-NA、CS04m2-SC1-NA、CS04m3-SC1-NA、CS04m4-SC1-NA、CS04m12-SC1-NA、CS04m13-SC 1-NA、CS04m23-SC1-NA、CS04m24-SC1-NA、CS04m34-SC1-NA、CS04m123-SC1 -NA、CS04m234-SC1-NA、CS23m1-SC1-NA、CS23m2-SC1-NA、CS23m3-SC1-NA、CS23m4-SC1-NA, CS23m12-SC1-NA, CS23m13-SC1-NA, CS23m23-SC1-NA, CS23m24-SC1-NA, CS23m34-SC1 -NA, CS23m123-SC1-NA, CS23m234-SC1-NA, CS01-SC2-NA, CS04-SC2-NA, CS23-SC2-NA, CS01m1-SC2-NA , CS01m2-SC2-NA, CS01m3-SC2-NA, CS01m4-SC2-NA, CS01m12-SC2-NA, CS01m13-SC2-NA, CS01m23-SC2- NA, CS01m24-SC2-NA, CS01m34-SC2-NA, CS01m123-SC2-NA, CS01m234-SC2-NA, CS04m1-SC2-NA, CS04m2- SC2-NA, CS04m3-SC2-NA, CS04m4-SC2-NA, CS04m12-SC2-NA, CS04m13-SC2-NA, CS04m23-SC2-NA, CS04m 24-SC2-NA, CS04m34-SC2-NA, CS04m123-SC2-NA, CS04m234-SC2-NA, CS23m1-SC2-NA, CS23m2-SC2-NA, It has at least 97% identity to a sequence selected from the group consisting of CS23m3-SC2-NA, CS23m4-SC2-NA, CS23m12-SC2-NA, CS23m13-SC2-NA, CS23m23-SC2-NA, CS23m24-SC2-NA, CS23m34-SC2-NA, CS23m123-SC2-NA, and CS23m234-SC2-NA.

[0070] Specifications for a range of cylinder heads are CS01-FL-NA, CS01-HC-NA, and CS01-LC-NA CS04-FL-NA、CS04-HC-NA、CS04-LC-NA、CS23-FL-NA、CS23-HC-NA、CS23-L C-NA, CS01m1-FL-NA, CS01m2-FL-NA, CS01m3-FL-NA, CS01m4-FL-NA, CS01m12-FL-NA, CS01m13-FL-NA, CS01m23-FL-NA, CS01m24-FL-NA, CS01m24-FL-NA NA、CS01m123-FL-NA、CS01m234-FL-NA、CS04m1-FL-NA、CS04m2-FL-NA、CS04m3-FL-NA、CS04m4-FL-NA、CS04m12-FL-NA、CS04m13-FL-NA、CS04m23-FL -NA、CS04m24-FL-NA、CS04m34-FL-NA、CS04m123-FL-NA、CS04m234-FL-NA、CS23m1-FL-NA、CS23m2-FL-NA、CS23m3-FL-NA、CS23m4-FL-NA、CS23m12-FL -NA, CS23m13-FL-NA, CS23m23-FL-NA, CS23m24-FL-NA, CS23m34-FL-NA, CS23m123-FL-NA, CS23m234-FL-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1- NA、CS01m1-SC1-NA、CS01m2-SC1-NA、CS01m3-SC1-NA、CS01m4-SC1-NA、CS0 1m12-SC1-NA、CS01m13-SC1-NA、CS01m23-SC1-NA、CS01m24-SC1-NA、CS01m 34-SC1-NA、CS01m123-SC1-NA、CS01m234-SC1-NA、CS04m1-SC1-NA、CS04m2-SC1-NA、CS04m3-SC1-NA、CS04m4-SC1-NA、CS04m12-SC1-NA、CS04m13-SC 1-NA、CS04m23-SC1-NA、CS04m24-SC1-NA、CS04m34-SC1-NA、CS04m123-SC1 -NA、CS04m234-SC1-NA、CS23m1-SC1-NA、CS23m2-SC1-NA、CS23m3-SC1-NA、CS23m4-SC1-NA, CS23m12-SC1-NA, CS23m13-SC1-NA, CS23m23-SC1-NA, CS23m24-SC1-NA, CS23m34-SC1 -NA, CS23m123-SC1-NA, CS23m234-SC1-NA, CS01-SC2-NA, CS04-SC2-NA, CS23-SC2-NA, CS01m1-SC2-NA , CS01m2-SC2-NA, CS01m3-SC2-NA, CS01m4-SC2-NA, CS01m12-SC2-NA, CS01m13-SC2-NA, CS01m23-SC2- NA, CS01m24-SC2-NA, CS01m34-SC2-NA, CS01m123-SC2-NA, CS01m234-SC2-NA, CS04m1-SC2-NA, CS04m2- SC2-NA, CS04m3-SC2-NA, CS04m4-SC2-NA, CS04m12-SC2-NA, CS04m13-SC2-NA, CS04m23-SC2-NA, CS04m 24-SC2-NA, CS04m34-SC2-NA, CS04m123-SC2-NA, CS04m234-SC2-NA, CS23m1-SC2-NA, CS23m2-SC2-NA, It has at least 98% identity to a sequence selected from the group consisting of CS23m3-SC2-NA, CS23m4-SC2-NA, CS23m12-SC2-NA, CS23m13-SC2-NA, CS23m23-SC2-NA, CS23m24-SC2-NA, CS23m34-SC2-NA, CS23m123-SC2-NA, and CS23m234-SC2-NA.

[0071] Specifications for a range of cylinder heads are CS01-FL-NA, CS01-HC-NA, and CS01-LC-NA CS04-FL-NA、CS04-HC-NA、CS04-LC-NA、CS23-FL-NA、CS23-HC-NA、CS23-L C-NA, CS01m1-FL-NA, CS01m2-FL-NA, CS01m3-FL-NA, CS01m4-FL-NA, CS01m12-FL-NA, CS01m13-FL-NA, CS01m23-FL-NA, CS01m24-FL-NA, CS01m24-FL-NA NA、CS01m123-FL-NA、CS01m234-FL-NA、CS04m1-FL-NA、CS04m2-FL-NA、CS04m3-FL-NA、CS04m4-FL-NA、CS04m12-FL-NA、CS04m13-FL-NA、CS04m23-FL -NA、CS04m24-FL-NA、CS04m34-FL-NA、CS04m123-FL-NA、CS04m234-FL-NA、CS23m1-FL-NA、CS23m2-FL-NA、CS23m3-FL-NA、CS23m4-FL-NA、CS23m12-FL -NA, CS23m13-FL-NA, CS23m23-FL-NA, CS23m24-FL-NA, CS23m34-FL-NA, CS23m123-FL-NA, CS23m234-FL-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1- NA、CS01m1-SC1-NA、CS01m2-SC1-NA、CS01m3-SC1-NA、CS01m4-SC1-NA、CS0 1m12-SC1-NA、CS01m13-SC1-NA、CS01m23-SC1-NA、CS01m24-SC1-NA、CS01m 34-SC1-NA、CS01m123-SC1-NA、CS01m234-SC1-NA、CS04m1-SC1-NA、CS04m2-SC1-NA、CS04m3-SC1-NA、CS04m4-SC1-NA、CS04m12-SC1-NA、CS04m13-SC 1-NA、CS04m23-SC1-NA、CS04m24-SC1-NA、CS04m34-SC1-NA、CS04m123-SC1 -NA、CS04m234-SC1-NA、CS23m1-SC1-NA、CS23m2-SC1-NA、CS23m3-SC1-NA、CS23m4-SC1-NA, CS23m12-SC1-NA, CS23m13-SC1-NA, CS23m23-SC1-NA, CS23m24-SC1-NA, CS23m34-SC1 -NA, CS23m123-SC1-NA, CS23m234-SC1-NA, CS01-SC2-NA, CS04-SC2-NA, CS23-SC2-NA, CS01m1-SC2-NA , CS01m2-SC2-NA, CS01m3-SC2-NA, CS01m4-SC2-NA, CS01m12-SC2-NA, CS01m13-SC2-NA, CS01m23-SC2- NA, CS01m24-SC2-NA, CS01m34-SC2-NA, CS01m123-SC2-NA, CS01m234-SC2-NA, CS04m1-SC2-NA, CS04m2- SC2-NA, CS04m3-SC2-NA, CS04m4-SC2-NA, CS04m12-SC2-NA, CS04m13-SC2-NA, CS04m23-SC2-NA, CS04m 24-SC2-NA, CS04m34-SC2-NA, CS04m123-SC2-NA, CS04m234-SC2-NA, CS23m1-SC2-NA, CS23m2-SC2-NA, It has at least 99% identity to a sequence selected from the group consisting of CS23m3-SC2-NA, CS23m4-SC2-NA, CS23m12-SC2-NA, CS23m13-SC2-NA, CS23m23-SC2-NA, CS23m24-SC2-NA, CS23m34-SC2-NA, CS23m123-SC2-NA, and CS23m234-SC2-NA.

[0072] Specifications for a range of cylinder heads are CS01-FL-NA, CS01-HC-NA, and CS01-LC-NA CS04-FL-NA、CS04-HC-NA、CS04-LC-NA、CS23-FL-NA、CS23-HC-NA、CS23-L C-NA, CS01m1-FL-NA, CS01m2-FL-NA, CS01m3-FL-NA, CS01m4-FL-NA, CS01m12-FL-NA, CS01m13-FL-NA, CS01m23-FL-NA, CS01m24-FL-NA, CS01m24-FL-NA NA、CS01m123-FL-NA、CS01m234-FL-NA、CS04m1-FL-NA、CS04m2-FL-NA、CS04m3-FL-NA、CS04m4-FL-NA、CS04m12-FL-NA、CS04m13-FL-NA、CS04m23-FL -NA、CS04m24-FL-NA、CS04m34-FL-NA、CS04m123-FL-NA、CS04m234-FL-NA、CS23m1-FL-NA、CS23m2-FL-NA、CS23m3-FL-NA、CS23m4-FL-NA、CS23m12-FL -NA, CS23m13-FL-NA, CS23m23-FL-NA, CS23m24-FL-NA, CS23m34-FL-NA, CS23m123-FL-NA, CS23m234-FL-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1- NA、CS01m1-SC1-NA、CS01m2-SC1-NA、CS01m3-SC1-NA、CS01m4-SC1-NA、CS0 1m12-SC1-NA、CS01m13-SC1-NA、CS01m23-SC1-NA、CS01m24-SC1-NA、CS01m 34-SC1-NA、CS01m123-SC1-NA、CS01m234-SC1-NA、CS04m1-SC1-NA、CS04m2-SC1-NA、CS04m3-SC1-NA、CS04m4-SC1-NA、CS04m12-SC1-NA、CS04m13-SC 1-NA、CS04m23-SC1-NA、CS04m24-SC1-NA、CS04m34-SC1-NA、CS04m123-SC1 -NA、CS04m234-SC1-NA、CS23m1-SC1-NA、CS23m2-SC1-NA、CS23m3-SC1-NA、CS23m4-SC1-NA, CS23m12-SC1-NA, CS23m13-SC1-NA, CS23m23-SC1-NA, CS23m24-SC1-NA, CS23m34-SC1 -NA, CS23m123-SC1-NA, CS23m234-SC1-NA, CS01-SC2-NA, CS04-SC2-NA, CS23-SC2-NA, CS01m1-SC2-NA, CS01m2-SC2-NA, CS01m3-SC2-NA, CS01m4-SC2-NA, CS01m12-SC2-NA, CS01m13-SC2-NA, CS01m23-SC2-N A, CS01m24-SC2-NA, CS01m34-SC2-NA, CS01m123-SC2-NA, CS01m234-SC2-NA, CS04m1-SC2-NA, CS04m2-S C2-NA, CS04m3-SC2-NA, CS04m4-SC2-NA, CS04m12-SC2-NA, CS04m13-SC2-NA, CS04m23-SC2-NA, CS04m2 4-SC2-NA, CS04m34-SC2-NA, CS04m123-SC2-NA, CS04m234-SC2-NA, CS23m1-SC2-NA, CS23m2-SC2-NA, CS CS23m3-SC2-NA, CS23m4-SC2-NA, CS23m12-SC2-NA, CS23m13-SC2-NA, CS23m23-SC2-NA, CS23m24-SC2-NA, CS23m34-SC2-NA, CS23m123-SC2-NA, and CS23m234-SC2-NA.

[0073] Specifications for a range of cylinder heads are CS01-FL-NA, CS01-HC-NA, and CS01-LC-NA CS04-FL-NA、CS04-HC-NA、CS04-LC-NA、CS23-FL-NA、CS23-HC-NA、CS23-L C-NA, CS01m1-FL-NA, CS01m2-FL-NA, CS01m3-FL-NA, CS01m4-FL-NA, CS01m12-FL-NA, CS01m13-FL-NA, CS01m23-FL-NA, CS01m24-FL-NA, CS01m24-FL-NA NA、CS01m123-FL-NA、CS01m234-FL-NA、CS04m1-FL-NA、CS04m2-FL-NA、CS04m3-FL-NA、CS04m4-FL-NA、CS04m12-FL-NA、CS04m13-FL-NA、CS04m23-FL -NA、CS04m24-FL-NA、CS04m34-FL-NA、CS04m123-FL-NA、CS04m234-FL-NA、CS23m1-FL-NA、CS23m2-FL-NA、CS23m3-FL-NA、CS23m4-FL-NA、CS23m12-FL -NA, CS23m13-FL-NA, CS23m23-FL-NA, CS23m24-FL-NA, CS23m34-FL-NA, CS23m123-FL-NA, CS23m234-FL-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1- NA、CS01m1-SC1-NA、CS01m2-SC1-NA、CS01m3-SC1-NA、CS01m4-SC1-NA、CS0 1m12-SC1-NA、CS01m13-SC1-NA、CS01m23-SC1-NA、CS01m24-SC1-NA、CS01m 34-SC1-NA、CS01m123-SC1-NA、CS01m234-SC1-NA、CS04m1-SC1-NA、CS04m2-SC1-NA、CS04m3-SC1-NA、CS04m4-SC1-NA、CS04m12-SC1-NA、CS04m13-SC 1-NA、CS04m23-SC1-NA、CS04m24-SC1-NA、CS04m34-SC1-NA、CS04m123-SC1 -NA、CS04m234-SC1-NA、CS23m1-SC1-NA、CS23m2-SC1-NA、CS23m3-SC1-NA、CS23m4-SC1-NA, CS23m12-SC1-NA, CS23m13-SC1-NA, CS23m23-SC1-NA, CS23m24-SC1-NA, CS23m34-SC1 -NA, CS23m123-SC1-NA, CS23m234-SC1-NA, CS01-SC2-NA, CS04-SC2-NA, CS23-SC2-NA, CS01m1-SC2-NA, CS01m2-SC2-NA, CS01m3-SC2-NA, CS01m4-SC2-NA, CS01m12-SC2-NA, CS01m13-SC2-NA, CS01m23-SC2-N A, CS01m24-SC2-NA, CS01m34-SC2-NA, CS01m123-SC2-NA, CS01m234-SC2-NA, CS04m1-SC2-NA, CS04m2-S C2-NA, CS04m3-SC2-NA, CS04m4-SC2-NA, CS04m12-SC2-NA, CS04m13-SC2-NA, CS04m23-SC2-NA, CS04m2 4-SC2-NA, CS04m34-SC2-NA, CS04m123-SC2-NA, CS04m234-SC2-NA, CS23m1-SC2-NA, CS23m2-SC2-NA, CS CS23m3-SC2-NA, CS23m4-SC2-NA, CS23m12-SC2-NA, CS23m13-SC2-NA, CS23m23-SC2-NA, CS23m24-SC2-NA, CS23m34-SC2-NA, CS23m123-SC2-NA, and CS23m234-SC2-NA.

[0074] Specifications for a range of cylinder heads are CS01-FL-NA, CS01-HC-NA, and CS01-LC-NA CS04-FL-NA、CS04-HC-NA、CS04-LC-NA、CS23-FL-NA、CS23-HC-NA、CS23-L C-NA, CS01m1-FL-NA, CS01m2-FL-NA, CS01m3-FL-NA, CS01m4-FL-NA, CS01m12-FL-NA, CS01m13-FL-NA, CS01m23-FL-NA, CS01m24-FL-NA, CS01m24-FL-NA NA、CS01m123-FL-NA、CS01m234-FL-NA、CS04m1-FL-NA、CS04m2-FL-NA、CS04m3-FL-NA、CS04m4-FL-NA、CS04m12-FL-NA、CS04m13-FL-NA、CS04m23-FL -NA、CS04m24-FL-NA、CS04m34-FL-NA、CS04m123-FL-NA、CS04m234-FL-NA、CS23m1-FL-NA、CS23m2-FL-NA、CS23m3-FL-NA、CS23m4-FL-NA、CS23m12-FL -NA, CS23m13-FL-NA, CS23m23-FL-NA, CS23m24-FL-NA, CS23m34-FL-NA, CS23m123-FL-NA, CS23m234-FL-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1- NA、CS01m1-SC1-NA、CS01m2-SC1-NA、CS01m3-SC1-NA、CS01m4-SC1-NA、CS0 1m12-SC1-NA、CS01m13-SC1-NA、CS01m23-SC1-NA、CS01m24-SC1-NA、CS01m 34-SC1-NA、CS01m123-SC1-NA、CS01m234-SC1-NA、CS04m1-SC1-NA、CS04m2-SC1-NA、CS04m3-SC1-NA、CS04m4-SC1-NA、CS04m12-SC1-NA、CS04m13-SC 1-NA、CS04m23-SC1-NA、CS04m24-SC1-NA、CS04m34-SC1-NA、CS04m123-SC1 -NA、CS04m234-SC1-NA、CS23m1-SC1-NA、CS23m2-SC1-NA、CS23m3-SC1-NA、CS23m4-SC1-NA, CS23m12-SC1-NA, CS23m13-SC1-NA, CS23m23-SC1-NA, CS23m24-SC1-NA, CS23m34- SC1-NA, CS23m123-SC1-NA, CS23m234-SC1-NA, CS01-SC2-NA, CS04-SC2-NA, CS23-SC2-NA, CS01m1-S C2-NA, CS01m2-SC2-NA, CS01m3-SC2-NA, CS01m4-SC2-NA, CS01m12-SC2-NA, CS01m13-SC2-NA, CS01 m23-SC2-NA, CS01m24-SC2-NA, CS01m34-SC2-NA, CS01m123-SC2-NA, CS01m234-SC2-NA, CS04m1-SC2 -NA, CS04m2-SC2-NA, CS04m3-SC2-NA, CS04m4-SC2-NA, CS04m12-SC2-NA, CS04m13-SC2-NA, CS04m2 3-SC2-NA, CS04m24-SC2-NA, CS04m34-SC2-NA, CS04m123-SC2-NA, CS04m234-SC2-NA, CS23m1-SC2-N A, CS23m2-SC2-NA, CS23m3-SC2-NA, CS23m4-SC2-NA, CS23m12-SC2-NA, CS23m13-SC2-NA, CS23m23-SC2-NA, CS23m24-SC2-NA, CS23m34-SC2-NA, CS23m123-SC2-NA, and CS23m234-SC2-NA.

[0075] In one embodiment of the above polynucleotide, the encoded Factor VIII polypeptide comprises a glycosylated polypeptide located between two consecutive amino acids.

[0076] In one embodiment of the above polynucleotide, the encoded polypeptide linker includes a glycosylated peptide having an amino acid sequence having at least 92% identity to a glycosylated peptide selected from the group consisting of NG1-AA, NG4-AA, NG5-AA, NG6-AA, NG7-AA, NG9-AA, NG10-AA, NG16-AA, NG17-AA, NG18-AA, NG19-AA, NG20-AA, NG21-AA, and NGV-AA.

[0077] In one embodiment of the above polynucleotide, the encoded polypeptide linker comprises a glycosylated peptide having an amino acid sequence selected from the group consisting of NG1-AA, NG4-AA, NG5-AA, NG6-AA, NG7-AA, NG9-AA, NG10-AA, NG16-AA, NG17-AA, NG18-AA, NG19-AA, NG20-AA, NG21-AA, and NGV-AA.

[0078] In one embodiment of the above polynucleotide, the glycosylated peptide is encoded by a polynucleotide having a nucleotide sequence having at least 95% identity to a sequence selected from the group consisting of NG1-NA, NG4-NA, NG5-NA, NG6-NA, NG7-NA, NG9-NA, NG10-NA, NG16-NA, NG17-NA, NG18-NA, NG19-NA, NG20-NA, NG21-NA, and NGV-NA.

[0079] In one embodiment of the above polynucleotide, the glycosylated peptide is encoded by a polynucleotide having a nucleotide sequence selected from one of NG1-NA, NG4-NA, NG5-NA, NG6-NA, NG7-NA, NG9-NA, NG10-NA, NG16-NA, NG17-NA, NG18-NA, NG19-NA, NG20-NA, NG21-NA, and NGV-NA.

[0080] In one embodiment of the above polynucleotide, the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to a sequence selected from the group consisting of BDLNG1-NA, BDLNG3-NA, BDLNG5-NA, BDLNG6-NA, BDLNG9-NA, BDLNG10-NA, BDLNG16-NA, BDLNG17-NA, BDLNG18-NA, BDLNG19-NA, BDLNG20-NA, and BDLNG21-NA.

[0081] In one embodiment of the above polynucleotide, the encoded Factor VIII polypeptide includes the amino acid substitution F328S (in SPI; F309S in SPE) relative to FVIII-FL-AA (SEQ ID NO: 19).

[0082] In one embodiment of the above polynucleotide, the encoded Factor VIII polypeptide includes the amino acid substitutions I105V, A127S, G151K, M166T, and L171P (in the case of SPI; I86V, A108S, G132K, M147T, and L152P, respectively, in the case of SPE) relative to FVIII-FL-AA (SEQ ID NO: 19).

[0083] In one embodiment of the above polynucleotide, the encoded Factor VIII polypeptide includes (a) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), and (b) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19). In some embodiments (e.g., when the encoded FVIII molecule includes a portion of the N-terminal region of the wild-type B domain), the encoded Factor VIII polypeptide also includes a deletion of amino acids SF760-761 relative to FVIII-FL-AA (SEQ ID NO: 19).

[0084] In one embodiment of the above polynucleotide, the encoded Factor VIII polypeptide includes (a) the amino acid substitution F328S (in SPI; F309S in SPE) relative to FVIII-FL-AA (SEQ ID NO: 19), and (b) the amino acid substitutions C1918G and C1922G (in SPI; C1899G and C1903, respectively, in SPE) relative to FVIII-FL-AA (SEQ ID NO: 19).

[0085] In one embodiment of the above polynucleotide, the encoded Factor VIII polypeptide includes (a) the amino acid substitution F328S (in SPI; F309S in SPE) relative to FVIII-FL-AA (SEQ ID NO: 19), and (b) the amino acid substitutions I105V, A127S, G151K, M166T, and L171P (in SPI; I86V, A108S, G132K, M147T, and L152P, respectively, in SPE) relative to FVIII-FL-AA (SEQ ID NO: 19).

[0086] In one embodiment of the above polynucleotide, the encoded Factor VIII polypeptide includes (a) the amino acid substitution F328S relative to FVIII-FL-AA (SEQ ID NO: 19), (b) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), and (c) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 relative to FVIII-FL-AA (SEQ ID NO: 19). In some embodiments (e.g., when the encoded FVIII molecule includes a portion of the N-terminal region of the wild-type B domain), the encoded Factor VIII polypeptide also includes a deletion of amino acids SF760-761 relative to FVIII-FL-AA (SEQ ID NO: 19).

[0087] In one embodiment of the above polynucleotide, the encoded Factor VIII polypeptide includes (a) amino acid substitutions I105V, A127S, G151K, M166T, and L171P relative to FVIII-FL-AA (SEQ ID NO: 19), (b) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), and (c) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 relative to FVIII-FL-AA (SEQ ID NO: 19). In some embodiments (e.g., when the encoded FVIII molecule includes a portion of the N-terminal region of the wild-type B domain), the encoded Factor VIII polypeptide also includes a deletion of amino acids SF760-761 relative to FVIII-FL-AA (SEQ ID NO: 19).

[0088] In one embodiment of the above polynucleotide, the encoded Factor VIII polypeptide includes (a) the amino acid substitution F328S relative to FVIII-FL-AA (SEQ ID NO: 19), (b) the amino acid substitutions C1918G and C1922G relative to FVIII-FL-AA (SEQ ID NO: 19), and (c) the amino acid substitutions I105V, A127S, G151K, M166T, and L171P relative to FVIII-FL-AA (SEQ ID NO: 19).

[0089] In one embodiment of the polynucleotide, the encoded Factor VIII polypeptide includes (a) the amino acid substitution F328S relative to FVIII-FL-AA (SEQ ID NO: 19), (b) the amino acid substitutions C1918G and C1922G relative to FVIII-FL-AA (SEQ ID NO: 19), (c) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), and (d) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 relative to FVIII-FL-AA (SEQ ID NO: 19). In some embodiments (e.g., when the encoded FVIII molecule includes a portion of the N-terminal region of the wild-type B domain), the encoded Factor VIII polypeptide also includes a deletion of amino acids SF760-761 relative to FVIII-FL-AA (SEQ ID NO: 19).

[0090] In one embodiment of the above polynucleotide, the encoded Factor VIII polypeptide includes (a) amino acid substitutions I105V, A127S, G151K, M166T, and L171P relative to FVIII-FL-AA (SEQ ID NO: 19), (b) amino acid substitution F328S relative to FVIII-FL-AA (SEQ ID NO: 19), (c) amino acid substitutions C1918G and C1922G relative to FVIII-FL-AA (SEQ ID NO: 19), (d) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), and (e) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 relative to FVIII-FL-AA (SEQ ID NO: 19). In some embodiments (e.g., when the encoded FVIII molecule includes a portion of the N-terminal region of the wild-type B domain), the encoded factor VIII polypeptide also includes a deletion of amino acids SF760-761 relative to FVIII-FL-AA (SEQ ID NO: 19).

[0091] In one embodiment of the above polynucleotide, the polynucleotide also includes a promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide.

[0092] In one embodiment of the above polynucleotide, the polynucleotide also comprises an enhancer element operably linked to the polynucleotide encoding the Factor VIII polypeptide.

[0093] In one embodiment of the above polynucleotide, the polynucleotide also includes a polyadenylation element operably linked to the polynucleotide encoding the Factor VIII polypeptide.

[0094] In one embodiment of the above polynucleotide, the polynucleotide also includes an intron operably linked to the nucleotide sequence encoding the Factor VIII polypeptide.

[0095] In one embodiment of the above polynucleotide, an intron is located between the promoter element and the translation start site (eg, the first coding ATG) of the nucleotide sequence encoding the Factor VIII polypeptide.

[0096] In another aspect, the disclosure provides a mammalian gene therapy vector comprising a polynucleotide as described above.

[0097] In one embodiment of the above mammalian gene therapy vector, the mammalian gene therapy vector is an adeno-associated virus (AAV) vector.

[0098] In one embodiment of the above mammalian gene therapy vector, the AAV vector is an AAV-8 vector.

[0099] In another aspect, the disclosure provides a method of treating hemophilia A comprising administering to a patient in need thereof a mammalian gene therapy vector as described above.

[0100] In another aspect, the disclosure provides a mammalian gene therapy vector as described above for treating hemophilia A.

[0101] In another aspect, the disclosure provides the use of a mammalian gene therapy vector as described above for the manufacture of a medicament for the treatment of hemophilia A.

[0102] In another aspect, the present disclosure provides a Factor VIII polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the Factor VIII polypeptide has a sequence at least 95% identical to the sequence CSO1-HC-AAm23. The light chain of the Factor VIII polypeptide has a sequence at least 95% identical to the sequence CSO1-LC-AAm23. The polypeptide linker of the Factor VIII polypeptide comprises a furin cleavage site. The Factor VIII polypeptide comprises (i) amino acid substitutions I105V, A127S, G151K, M166T, and L171P, (ii) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), and (iii) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19).

[0103] In another aspect, the present disclosure provides a Factor VIII polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the Factor VIII polypeptide has a sequence at least 95% identical to the sequence CSO1-HC-AAm123. The light chain of the Factor VIII polypeptide has a sequence at least 95% identical to the sequence CSO1-LC-AAm123. The polypeptide linker of the Factor VIII polypeptide comprises a furin cleavage site. The Factor VIII polypeptide comprises (i) amino acid substitutions I105V, A127S, G151K, M166T, and L171P, (ii) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), (iii) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19), and iv) an amino acid substitution F328S.

[0104] In another aspect, the present disclosure provides a Factor VIII polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the Factor VIII polypeptide has a sequence at least 95% identical to the sequence CS01-HC-AAm234. The light chain of the Factor VIII polypeptide has a sequence at least 95% identical to the sequence CS01-LC-AAm234. The polypeptide linker of the Factor VIII polypeptide comprises a furin cleavage site. The Factor VIII polypeptide includes (i) amino acid substitutions I105V, A127S, G151K, M166T, and L171P, (ii) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), (iii) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19), and iv) amino acid substitutions F328S / C1918G / C1922G.

[0105] In one embodiment of the described Factor VIII polypeptide, the heavy chain of the Factor VIII polypeptide has a sequence at least 96% identical to the respective heavy chain sequence (e.g., CS01-HC-AAm23, CS01-HC-AAm123, or CS01-HC-AAm234), and the light chain of the Factor VIII polypeptide has a sequence at least 96% identical to the respective light chain sequence (e.g., CS01-LC-AAm23, CS01-LC-AAm123, or CS01-LC-AAm234).

[0106] In one embodiment of the described Factor VIII polypeptide, the heavy chain of the Factor VIII polypeptide has a sequence at least 97% identical to the respective heavy chain sequence (e.g., CS01-HC-AAm23, CS01-HC-AAm123, or CS01-HC-AAm234), and the light chain of the Factor VIII polypeptide has a sequence at least 97% identical to the respective light chain sequence (e.g., CS01-LC-AAm23, CS01-LC-AAm123, or CS01-LC-AAm234).

[0107] In one embodiment of the described Factor VIII polypeptide, the heavy chain of the Factor VIII polypeptide has a sequence at least 98% identical to the respective heavy chain sequence (e.g., CS01-HC-AAm23, CS01-HC-AAm123, or CS01-HC-AAm234), and the light chain of the Factor VIII polypeptide has a sequence at least 98% identical to the respective light chain sequence (e.g., CS01-LC-AAm23, CS01-LC-AAm123, or CS01-LC-AAm234).

[0108] In one embodiment of the described Factor VIII polypeptide, the heavy chain of the Factor VIII polypeptide has a sequence at least 99% identical to the respective heavy chain sequence (e.g., CS01-HC-AAm23, CS01-HC-AAm123, or CS01-HC-AAm234), and the light chain of the Factor VIII polypeptide has a sequence at least 99% identical to the respective light chain sequence (e.g., CS01-LC-AAm23, CS01-LC-AAm123, or CS01-LC-AAm234).

[0109] In one embodiment of the described Factor VIII polypeptide, the heavy chain of the Factor VIII polypeptide has a sequence at least 99.5% identical to the respective heavy chain sequence (e.g., CS01-HC-AAm23, CS01-HC-AAm123, or CS01-HC-AAm234), and the light chain of the Factor VIII polypeptide has a sequence at least 99.5% identical to the respective light chain sequence (e.g., CS01-LC-AAm23, CS01-LC-AAm123, or CS01-LC-AAm234).

[0110] In one embodiment of the described Factor VIII polypeptide, the heavy chain of the Factor VIII polypeptide has a sequence identical to the respective heavy chain sequence (e.g., CS01-HC-AAm23, CS01-HC-AAm123, or CS01-HC-AAm234), and the light chain of the Factor VIII polypeptide has a sequence identical to the respective light chain sequence (e.g., CS01-LC-AAm23, CS01-LC-AAm123, or CS01-LC-AAm234).

[0111] In one embodiment of the above-mentioned Factor VIII polypeptide, the polypeptide linker has at least 95% identity to BDL-SQ-AA (SEQ ID NO: 30).

[0112] In one embodiment of the above-mentioned Factor VIII polypeptide, the polypeptide linker has the amino acid sequence BDL-SQ-AA (SEQ ID NO: 30).

[0113] In one embodiment of the above-described Factor VIII polypeptide, the polypeptide linker comprises a glycosylated peptide having an amino acid sequence having at least 92% identity to a glycosylated peptide selected from the group consisting of NG1-AA, NG4-AA, NG5-AA, NG6-AA, NG7-AA, NG9-AA, NG10-AA, NG16-AA, NG17-AA, NG18-AA, NG19-AA, NG20-AA, NG21-AA, and NGV-AA.

[0114] In one embodiment of the above-mentioned Factor VIII polypeptide, the polypeptide linker comprises a glycosylated peptide selected from the group consisting of NG1-AA, NG4-AA, NG5-AA, NG6-AA, NG7-AA, NG9-AA, NG10-AA, NG16-AA, NG17-AA, NG18-AA, NG19-AA, NG20-AA, NG21-AA, and NGV-AA.

[0115] In one embodiment of the above-mentioned Factor VIII polypeptide, the polypeptide linker has an amino acid sequence having at least 95% identity to a sequence selected from the group consisting of BDLNG1-AA, BDLNG3-AA, BDLNG5-AA, BDLNG6-AA, BDLNG9-AA, BDLNG10-AA, BDLNG16-AA, BDLNG17-AA, BDLNG18-AA, BDLNG19-AA, BDLNG20-AA and BDLNG21-AA.

[0116] In one embodiment of the above-mentioned Factor VIII polypeptide, the polypeptide linker has an amino acid sequence selected from the group consisting of BDLNG1-AA, BDLNG3-AA, BDLNG5-AA, BDLNG6-AA, BDLNG9-AA, BDLNG10-NA, BDLNG16-AA, BDLNG17-AA, BDLNG18-AA, BDLNG19-AA, BDLNG20-AA and BDLNG21-AA.

[0117] In another aspect, the disclosure provides a Factor VIII polypeptide having an amino acid sequence at least 95% identical to CS40-FL-AAm23 (SEQ ID NO: 104), including (i) amino acid substitutions I105V, A127S, G151K, M166T, and L171P, (ii) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), and (iii) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19).

[0118] In another aspect, the present disclosure provides a Factor VIII polypeptide having an amino acid sequence at least 95% identical to CS40-FL-AAm123, including (i) amino acid substitutions I105V, A127S, G151K, M166T, and L171P, (ii) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), (iii) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19), and iv) an amino acid substitution F328S.

[0119] In another aspect, the present disclosure provides a Factor VIII polypeptide having an amino acid sequence at least 95% identical to CS40-FL-AAm234, including (i) amino acid substitutions I105V, A127S, G151K, M166T, and L171P, (ii) a deletion of amino acids AIEPR755-759 relative to FVIII-FL-AA (SEQ ID NO: 19), (iii) an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19), and iv) amino acid substitutions F328S / C1918G / C1922G.

[0120] In one embodiment of the described Factor VIII polypeptides, the Factor VIII polypeptide has a sequence that is at least 96% identical to the respective full-length sequence (e.g., CS40-FL-AAm23 (SEQ ID NO: 104), CS40-FL-AAm123, or CS40-FL-AAm234).

[0121] In one embodiment of the described Factor VIII polypeptides, the Factor VIII polypeptide has a sequence that is at least 97% identical to the respective full-length sequence (e.g., CS40-FL-AAm23 (SEQ ID NO: 104), CS40-FL-AAm123, or CS40-FL-AAm234).

[0122] In one embodiment of the described Factor VIII polypeptides, the Factor VIII polypeptide has a sequence that is at least 98% identical to the respective full-length sequence (e.g., CS40-FL-AAm23 (SEQ ID NO: 104), CS40-FL-AAm123, or CS40-FL-AAm234).

[0123] In one embodiment of the described Factor VIII polypeptides, the Factor VIII polypeptide has a sequence that is at least 99% identical to the respective full-length sequence (e.g., CS40-FL-AAm23 (SEQ ID NO: 104), CS40-FL-AAm123, or CS40-FL-AAm234).

[0124] In one embodiment of the described Factor VIII polypeptides, the Factor VIII polypeptide has a sequence that is at least 99.5% identical to the respective full-length sequence (e.g., CS40-FL-AAm23 (SEQ ID NO: 104), CS40-FL-AAm123, or CS40-FL-AAm234).

[0125] In one embodiment of the described Factor VIII polypeptides, the Factor VIII polypeptide has a sequence identical to the respective full-length sequence (e.g., CS40-FL-AAm23 (SEQ ID NO: 104), CS40-FL-AAm123, or CS40-FL-AAm234). [The present invention 1001] A polynucleotide comprising a nucleotide sequence having at least 95% identity to CS01-FL-NA (SEQ ID NO: 13), wherein the polynucleotide encodes a Factor VIII polypeptide. [The present invention 1002] 1001. The polynucleotide of the present invention, wherein said nucleotide sequence has at least 99% identity to CS01-FL-NA (SEQ ID NO: 13). [The present invention 1003] 1001. The polynucleotide of the present invention, wherein the nucleotide sequence is CS01-FL-NA (SEQ ID NO: 13). [The present invention 1004] 1. A polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide, wherein the Factor VIII polypeptide comprises a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CSO1-HC-NA (SEQ ID NO: 24); the light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS01-LC-NA (SEQ ID NO: 25); and The polynucleotide, wherein the polypeptide linker comprises a furin cleavage site. [The present invention 1005] 1004. The polynucleotide of claim 10, wherein said polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to BDLO01 (SEQ ID NO: 5). [The present invention 1006] the first nucleotide sequence has at least 99% identity to CSO1-HC-NA (SEQ ID NO: 24); and The polynucleotide of claim 1004 or 1005, wherein said second nucleotide sequence has at least 99% identity to CS01-LC-NA (SEQ ID NO: 25). [The present invention 1007] the first nucleotide sequence is CSO1-HC-NA (SEQ ID NO: 24), and The polynucleotide of claim 1004 or 1005, wherein the second nucleotide sequence is CS01-LC-NA (SEQ ID NO: 25). [The present invention 1008] A polynucleotide comprising a nucleotide sequence having at least 95% identity to CS04-FL-NA (SEQ ID NO: 1), wherein the polynucleotide encodes a Factor VIII polypeptide. [The present invention 1009] 1004. The polynucleotide of the present invention, wherein said nucleotide sequence has at least 99% identity to CS04-FL-NA (SEQ ID NO: 1). [The present invention 1010] The polynucleotide of the present invention 1004, wherein the nucleotide sequence is CS04-FL-NA (SEQ ID NO: 1). [The present invention 1011] 1. A polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide, said Factor VIII polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS04-HC-NA (SEQ ID NO: 3); the light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS04-LC-NA (SEQ ID NO: 4); and The polynucleotide, wherein the polypeptide linker comprises a furin cleavage site. [The present invention 1012] The polynucleotide of claim 1011, wherein said polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to BDLO04 (SEQ ID NO: 6). [The present invention 1013] the first nucleotide sequence has at least 99% identity to CS04-HC-NA (SEQ ID NO: 3); and The polynucleotide of claim 1011 or 1012, wherein the second nucleotide sequence has at least 99% identity to CS04-LC-NA (SEQ ID NO: 4). [The present invention 1014] the first nucleotide sequence is CS04-HC-NA (SEQ ID NO: 3), and The polynucleotide of claim 1011 or 1012, wherein the second nucleotide sequence is CS04-LC-NA (SEQ ID NO: 4). [The present invention 1015] A polynucleotide comprising a nucleotide sequence having at least 95% identity to CS23-FL-NA, wherein the polynucleotide encodes a Factor VIII polypeptide. [The present invention 1016] 1015. The polynucleotide of the present invention, wherein the nucleotide sequence has at least 99% identity to CS23-FL-NA (SEQ ID NO: 20). [The present invention 1017] The polynucleotide of the present invention 1015, wherein the nucleotide sequence is CS23-FL-NA (SEQ ID NO: 20). [The present invention 1018] 1. A polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide, said Factor VIII polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS23-HC-NA (SEQ ID NO: 22); the light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS23-LC-NA (SEQ ID NO: 23); and The polynucleotide, wherein the polypeptide linker comprises a furin cleavage site. [The present invention 1019] The polynucleotide of claim 1018, wherein said polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to BDLO23 (SEQ ID NO: 7). [The present invention 1020] the first nucleotide sequence has at least 99% identity to CS23-HC-NA (SEQ ID NO: 22); and The polynucleotide of claim 1018 or 1019, wherein said second nucleotide sequence has at least 99% identity to CS23-LC-NA (SEQ ID NO: 23). [The present invention 1021] the first nucleotide sequence is CS23-HC-NA (SEQ ID NO: 22), and The polynucleotide of claim 1018 or 1019, wherein the second nucleotide sequence is CS23-LC-NA (SEQ ID NO: 23). [The present invention 1022] The polynucleotide of any of claims 1004, 1011, and 1018, wherein the encoded polypeptide linker comprises a glycosylated peptide having an amino acid sequence having at least 92% identity to a glycosylated peptide selected from the group consisting of NG1-AA, NG4-AA, NG5-AA, NG6-AA, NG7-AA, NG9-AA, NG10-AA, NG16-AA, NG17-AA, NG18-AA, NG19-AA, NG20-AA, NG21-AA, and NGV-AA. [The present invention 1023] The polynucleotide of any of claims 1004, 1011, and 1018, wherein the encoded polypeptide linker comprises a glycosylated peptide selected from the group consisting of NG1-AA, NG4-AA, NG5-AA, NG6-AA, NG7-AA, NG9-AA, NG10-AA, NG16-AA, NG17-AA, NG18-AA, NG19-AA, NG20-AA, NG21-AA, and NGV-AA. [The present invention 1024] The polynucleotide of the present invention 1022 or 1023, wherein the glycosylated peptide is encoded by a polynucleotide having a nucleotide sequence having at least 95% identity to a sequence selected from the group consisting of NG1-NA, NG4-NA, NG5-NA, NG6-NA, NG7-NA, NG9-NA, NG10-NA, NG16-NA, NG17-NA, NG18-NA, NG19-NA, NG20-NA, NG21-NA and NGV-NA. [The present invention 1025] The polynucleotide of the present invention 1022 or 1023, wherein the glycosylated peptide is encoded by a polynucleotide having a nucleotide sequence selected from one of NG1-NA, NG4-NA, NG5-NA, NG6-NA, NG7-NA, NG9-NA, NG10-NA, NG16-NA, NG17-NA, NG18-NA, NG19-NA, NG20-NA, NG21-NA and NGV-NA. [The present invention 1026] The polynucleotide of any of claims 1004, 1011, and 1018, wherein the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to a sequence selected from the group consisting of BDLNG1-NA, BDLNG3-NA, BDLNG5-NA, BDLNG6-NA, BDLNG9-NA, BDLNG10-NA, BDLNG16-NA, BDLNG17-NA, BDLNG18-NA, BDLNG19-NA, BDLNG20-NA, and BDLNG21-NA. [The present invention 1027] The polynucleotide of any of claims 1001 to 1021, which encodes a Factor VIII polypeptide comprising an amino acid sequence having at least 95% identity to CS04-FL-AA (SEQ ID NO: 2). [The present invention 1028] Any of the polynucleotides of 1001 to 1021 of the present invention, which encode a Factor VIII polypeptide comprising the amino acid sequence of CS04-FL-AA (SEQ ID NO: 2). [The present invention 1029] A polynucleotide comprising a nucleotide sequence having at least 95% identity to CS01-SC1-NA (SEQ ID NO: 26), wherein the polynucleotide encodes a Factor VIII single-chain polypeptide. [The present invention 1030] 1029. The polynucleotide of the present invention, wherein said nucleotide sequence has at least 99% identity to CS01-SC1-NA (SEQ ID NO: 26). [The present invention 1031] The polynucleotide of the present invention 1029, wherein the nucleotide sequence is CS01-SC1-NA (SEQ ID NO: 26). [The present invention 1032] A polynucleotide comprising a nucleotide sequence having at least 95% identity to CS04-SC1-NA (SEQ ID NO: 9), wherein the polynucleotide encodes a Factor VIII single-chain polypeptide. [The present invention 1033] 1029. The polynucleotide of the present invention, wherein said nucleotide sequence has at least 99% identity to CS04-SC1-NA (SEQ ID NO: 9). [The present invention 1034] The polynucleotide of the present invention 1029, wherein the nucleotide sequence is CS04-SC1-NA (SEQ ID NO: 9). [This invention 1035] A polynucleotide comprising a nucleotide sequence having at least 95% identity to CS23-SC1-NA (SEQ ID NO: 28), wherein the polynucleotide encodes a Factor VIII single-chain polypeptide. [The present invention 1036] 1029. The polynucleotide of the present invention, wherein the nucleotide sequence has at least 99% identity to CS23-SC1-NA (SEQ ID NO: 28). [This invention 1037] The polynucleotide of the present invention 1029, wherein the nucleotide sequence is CS23-SC1-NA (SEQ ID NO: 28). [The present invention 1038] The polynucleotide of any one of claims 1029 to 1037, which encodes a Factor VIII single-chain polypeptide comprising an amino acid sequence having at least 95% identity to CS01-SC1-AA (SEQ ID NO: 10). [This invention 1039] The polynucleotide of any one of 1029 to 1037 of the present invention, which encodes a Factor VIII single-chain polypeptide comprising the amino acid sequence of CS01-SC1-AA (SEQ ID NO: 10). [The present invention 1040] A polynucleotide comprising a nucleotide sequence having at least 95% identity to CS01-SC2-NA (SEQ ID NO: 27), wherein the polynucleotide encodes a Factor VIII single-chain polypeptide. [This invention 1041] 1040. The polynucleotide of the present invention, wherein the nucleotide sequence has at least 99% identity to CS01-SC2-NA (SEQ ID NO: 27). [The present invention 1042] The polynucleotide of the present invention 1040, wherein the nucleotide sequence is CS01-SC2-NA (SEQ ID NO: 27). [This invention 1043] A polynucleotide comprising a nucleotide sequence having at least 95% identity to CS04-SC2-NA (SEQ ID NO: 11), wherein the polynucleotide encodes a Factor VIII single-chain polypeptide. [This invention 1044] The polynucleotide of the present invention 1043, wherein the nucleotide sequence has at least 99% identity to CS04-SC2-NA (SEQ ID NO: 11). [This invention 1045] The polynucleotide of the present invention 1043, wherein the nucleotide sequence is CS04-SC2-NA (SEQ ID NO: 11). [The present invention 1046] A polynucleotide comprising a nucleotide sequence having at least 95% identity to CS23-SC2-NA (SEQ ID NO: 29), wherein the polynucleotide encodes a Factor VIII single-chain polypeptide. [This invention 1047] The polynucleotide of the present invention 1043, wherein the nucleotide sequence has at least 99% identity to CS23-SC2-NA (SEQ ID NO: 29). [This invention 1048] The polynucleotide of the present invention 1043, wherein the nucleotide sequence is CS23-SC2-NA (SEQ ID NO: 29). [This invention 1049] Any of the polynucleotides of 1040 to 1048 of the present invention, which encode a Factor VIII single-chain polypeptide comprising an amino acid sequence having at least 95% identity to CS01-SC2-AA (SEQ ID NO: 12). [The present invention 1050] Any of the polynucleotides of 1040 to 1048 of the present invention, which encode a Factor VIII single-chain polypeptide comprising the amino acid sequence of CS01-SC2-AA (SEQ ID NO: 12). [This invention 1051] A polynucleotide comprising a sequence having at least 95% identity to a sequence selected from the group consisting of CS01-HC-NA, CS01-LC-NA, CS04-HC-NA, CS04-LC-NA, CS23-HC-NA, and CS23-LC-NA. [This invention 1052] A polynucleotide comprising a sequence having at least 99% identity to a sequence selected from the group consisting of CS01-HC-NA, CS01-LC-NA, CS04-HC-NA, CS04-LC-NA, CS23-HC-NA, and CS23-LC-NA. [This invention 1053] A polynucleotide comprising a sequence selected from the group consisting of CS01-HC-NA, CS01-LC-NA, CS04-HC-NA, CS04-LC-NA, CS23-HC-NA, and CS23-LC-NA. [This invention 1054] The polynucleotide of any of claims 1051 to 1053, which encodes a Factor VIII polypeptide comprising an amino acid sequence having at least 95% identity to CS01-FL-AA (SEQ ID NO: 2). [This invention 1055] The polynucleotide of any one of 1051 to 1053 of the present invention, which encodes a Factor VIII polypeptide comprising the amino acid sequence of CS01-FL-AA (SEQ ID NO: 2). [This invention 1056] The polynucleotide of any of claims 1001 to 1055, wherein the encoded Factor VIII polypeptide comprises the amino acid substitution F328S relative to FVIII-FL-AA (SEQ ID NO: 19). [This invention 1057] The polynucleotide of any of claims 1001 to 1055, wherein the encoded Factor VIII polypeptide comprises the amino acid substitutions I105V, A127S, G151K, M166T, and L171P relative to FVIII-FL-AA (SEQ ID NO: 19). [This invention 1058] the encoded Factor VIII polypeptide is (a) deletion of amino acids AIEPRSF755 to 761 with respect to FVIII-FL-AA (SEQ ID NO: 19); and (b) Insertion of the amino acid TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19) The polynucleotide of any one of 1001 to 1055 of the present invention, comprising: [This invention 1059] the encoded Factor VIII polypeptide is (a) the amino acid substitution F328S relative to FVIII-FL-AA (SEQ ID NO: 19), and (b) Amino acid substitutions C1918G and C1922G relative to FVIII-FL-AA (SEQ ID NO: 19) The polynucleotide of any one of 1001 to 1055 of the present invention, comprising: [The present invention 1060] 1056. The polynucleotide of any of claims 1001 to 1055, wherein the encoded Factor VIII polypeptide comprises the amino acid substitutions I105V, A127S, G151K, M166T, L171P, and F328S relative to FVIII-FL-AA (SEQ ID NO: 19). [The present invention 1061] the encoded Factor VIII polypeptide is (a) the amino acid substitution F328S relative to FVIII-FL-AA (SEQ ID NO: 19); (b) deletion of amino acids AIEPRSF755 to 761 with respect to FVIII-FL-AA (SEQ ID NO: 19); and (c) Insertion of the amino acid TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19) The polynucleotide of any one of 1001 to 1055 of the present invention, comprising: [The present invention 1062] the encoded Factor VIII polypeptide is (a) amino acid substitutions I105V, A127S, G151K, M166T, and L171P relative to FVIII-FL-AA (SEQ ID NO: 19); (b) deletion of amino acids AIEPRSF755 to 761 with respect to FVIII-FL-AA (SEQ ID NO: 19); and (c) Insertion of the amino acid TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19) The polynucleotide of any one of 1001 to 1055 of the present invention, comprising: [The present invention 1063] the encoded Factor VIII polypeptide is (a) the amino acid substitution F328S relative to FVIII-FL-AA (SEQ ID NO: 19); (b) the amino acid substitutions C1918G and C1922G relative to FVIII-FL-AA (SEQ ID NO: 19), and (c) Amino acid substitutions I105V, A127S, G151K, M166T, and L171P relative to FVIII-FL-AA (SEQ ID NO: 19) The polynucleotide of any one of 1001 to 1055 of the present invention, comprising: [The present invention 1064] the encoded Factor VIII polypeptide is (a) the amino acid substitution F328S relative to FVIII-FL-AA (SEQ ID NO: 19); (b) amino acid substitutions C1918G and C1922G relative to FVIII-FL-AA (SEQ ID NO: 19); (c) deletion of amino acids AIEPRSF755 to 761 with respect to FVIII-FL-AA (SEQ ID NO: 19); and (d) Insertion of the amino acid TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19) The polynucleotide of any one of 1001 to 1055 of the present invention, comprising: [This invention 1065] the encoded Factor VIII polypeptide is (a) amino acid substitutions I105V, A127S, G151K, M166T, and L171P relative to FVIII-FL-AA (SEQ ID NO: 19); (b) the amino acid substitution F328S relative to FVIII-FL-AA (SEQ ID NO: 19); (c) amino acid substitutions C1918G and C1922G relative to FVIII-FL-AA (SEQ ID NO: 19); (d) deletion of amino acids AIEPRSF755 to 761 with respect to FVIII-FL-AA (SEQ ID NO: 19); and (e) Insertion of the amino acid TTYVNRSL (SEQ ID NO: 33) after N754 in FVIII-FL-AA (SEQ ID NO: 19) The polynucleotide of any one of 1001 to 1055 of the present invention, comprising: [The present invention 1066] The polynucleotide of any of claims 1001 to 1065, further comprising a promoter element operably linked to said polynucleotide encoding said Factor VIII polypeptide. [This invention 1067] 1067. The polynucleotide of claim 1066, wherein said promoter element is a liver-specific promoter sequence upstream of said nucleotide sequence encoding said Factor VIII polypeptide. [The present invention 1068] 1068. The polynucleotide of claim 1067, further comprising an intron sequence located between said liver-specific promoter sequence and said nucleotide sequence encoding said Factor VIII polypeptide. [This invention 1069] An adeno-associated virus (AAV) vector comprising any one of the polynucleotides of the present inventions 1001 to 1068. [The present invention 1070] An adeno-associated virus (AAV) particle comprising any one of the polynucleotides of the present inventions 1001 to 1068. [This invention 1071] A host cell infected with an adeno-associated virus (AAV) particle containing any one of the polynucleotides of the present inventions 1001 to 1068. [This invention 1072] A method for producing adeno-associated virus (AAV) particles, comprising the step of introducing any one of the polynucleotides of the present invention 1001 to 1068 into a mammalian host cell, wherein the polynucleotide is capable of replication in the mammalian host cell. [This invention 1073] A method for treating hemophilia A, comprising administering to a patient in need thereof an adeno-associated virus (AAV) particle of the present invention. [This invention 1074] A method for transducing a host cell, comprising contacting said host cell with an adeno-associated virus (AAV) particle of the present invention. [Brief explanation of the drawings]

[0126] [Figure 1] Schematic diagram of wild-type and ReFacto human Factor VIII protein constructs. [Figure 2A] 1 shows the CS04 codon-modified nucleotide sequence (SEQ ID NO: 1) encoding a Factor VIII variant according to some embodiments ("CS04-FL-NA" for full-length coding sequence). [Figure 2B] 1 shows the CS04 codon-modified nucleotide sequence (SEQ ID NO: 1) encoding a Factor VIII variant according to some embodiments ("CS04-FL-NA" for full-length coding sequence). [Figure 3] 1 shows the Factor VIII variant amino acid sequence (SEQ ID NO: 2) encoded by the CS04 codon-modified nucleotide sequence according to some embodiments ("CS04-FL-AA" for full-length amino acid sequence). [Figure 4] 1 shows a portion of the CS04 codon-modified nucleotide sequence (SEQ ID NO: 3) encoding the heavy chain of a Factor VIII variant according to some embodiments ("CS04-HC-NA"). [Figure 5]1 shows a portion of the CS04 codon-modified nucleotide sequence (SEQ ID NO: 4) encoding the light chain of a Factor VIII variant according to some embodiments ("CS04-LC-NA"). [Figure 6] Exemplary coding sequences for B domain replacement linkers according to some embodiments are shown (SEQ ID NOS: 5-7 and 36-48, respectively, in order of appearance). BDLO01 (SEQ ID NO: 5), BDLO04 (SEQ ID NO: 6), and BDLO23 (SEQ ID NO: 7) are portions of codon-modified nucleotide sequences CS01, CS04, and CS23, respectively, that encode the B domain replacement linkers. [Figure 7A] 1 shows an AAV vector sequence (SEQ ID NO: 8) including the CS04 codon-modified nucleotide sequence according to some embodiments ("CS04-AV-NA"). [Figure 7B] 1 shows an AAV vector sequence (SEQ ID NO: 8) including the CS04 codon-modified nucleotide sequence according to some embodiments ("CS04-AV-NA"). [Figure 7C] 1 shows an AAV vector sequence (SEQ ID NO: 8) including the CS04 codon-modified nucleotide sequence according to some embodiments ("CS04-AV-NA"). [Figure 8A] 1 shows the CS01m1 codon-altered nucleotide sequence (SEQ ID NO: 49) encoding a Factor VIII variant having the amino acid substitution F328S according to some embodiments ("CS01m1-FL-NA"). [Figure 8B] 1 shows the CS01m1 codon-altered nucleotide sequence (SEQ ID NO: 49) encoding a Factor VIII variant having the amino acid substitution F328S according to some embodiments ("CS01m1-FL-NA"). [Figure 9A] 1 shows the codon-modified nucleotide sequence (SEQ ID NO: 9) of CS04Δ(760-1667) (for SPI; CS04Δ(741-1648) for SPE) encoding a single-chain factor VIII variant according to some embodiments ("CS04-SC1-NA"). [Figure 9B]1 shows the codon-modified nucleotide sequence (SEQ ID NO: 9) of CS04Δ(760-1667) (for SPI; CS04Δ(741-1648) for SPE) encoding a single-chain factor VIII variant according to some embodiments ("CS04-SC1-NA"). [Figure 10] 1 shows the factor VIII variant amino acid sequences (SEQ ID NO: 10) encoded by the codon-altered nucleotide sequences of CS01Δ(760-1667) (for SPI; CS01Δ(741-1648) for SPE), CS04Δ(760-1667) (for SPI; CS04Δ(741-1648) for SPE), and CS23Δ(760-1667) (for SPI; CS23Δ(741-1648) for SPE) according to some embodiments ("CS01-SC1-AA," "CS04-SC1-AA," and "CS23-SC1-AA," respectively). [Figure 11A] 1 shows the codon-modified nucleotide sequence (SEQ ID NO: 11) of CS04Δ(772-1667) (for SPI; CS04Δ(753-1648) for SPE) encoding a single-chain factor VIII variant according to some embodiments ("CS04-SC2-NA"). [Figure 11B] 1 shows the codon-modified nucleotide sequence (SEQ ID NO: 11) of CS04Δ(772-1667) (for SPI; CS04Δ(753-1648) for SPE) encoding a single-chain factor VIII variant according to some embodiments ("CS04-SC2-NA"). [Figure 12] 1 shows the factor VIII variant amino acid sequences (SEQ ID NO: 12) encoded by the codon-altered nucleotide sequences of CS01Δ(772-1667) (for SPI; CS01Δ(753-1648) for SPE), CS04Δ(772-1667) (for SPI; CS04Δ(753-1648) for SPE), and CS23Δ(772-1667) (for SPI; CS23Δ(753-1648) for SPE) according to some embodiments ("CS01-SC2-AA," "CS04-SC2-AA," and "CS23-SC2-AA," respectively). [Figure 13A]

[0013] Figures 13A and 13B show the amino acid and nucleotide sequences of exemplary glycosylated peptides inserted into B domain replacement linkers according to some embodiments. "NG1" or "NG1-AA" is the code for the amino acid sequence shown on the top line. "NG1-NA" is the code for the nucleic acid sequence shown on the bottom line of each set. Figures 13A and 13B disclose the amino acid sequences as SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, and the nucleotide sequences as SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, all in their respective order of appearance. [Figure 13B]

[0013] Figures 13A and 13B show the amino acid and nucleotide sequences of exemplary glycosylated peptides inserted into B domain replacement linkers according to some embodiments. "NG1" or "NG1-AA" is the code for the amino acid sequence shown on the top line. "NG1-NA" is the code for the nucleic acid sequence shown on the bottom line of each set. Figures 13A and 13B disclose the amino acid sequences as SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, and the nucleotide sequences as SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, all in their respective order of appearance. [Figure 14] 1 shows the results of in silico prediction of in vivo N-glycosylation of wild-type factor VIII B domain, which discloses SEQ ID NOS: 76 and 76 to 82 in order of appearance. [Figure 15] 1 shows the results of in silico prediction of in vivo N-glycosylation of the V3 peptide linker, which are shown in SEQ ID NOS: 83 and 83-89, respectively. [Figure 16A] 1 shows the CS01 codon-modified nucleotide sequence (SEQ ID NO: 13) encoding a Factor VIII variant according to some embodiments ("CS01-FL-NA"). [Figure 16B] 1 shows the CS01 codon-modified nucleotide sequence (SEQ ID NO: 13) encoding a Factor VIII variant according to some embodiments ("CS01-FL-NA"). [Figure 17A] 1 shows the CS08 codon-modified nucleotide sequence (SEQ ID NO: 14) encoding a Factor VIII variant according to some embodiments ("CS08-FL-NA"). [Figure 17B] 1 shows the CS08 codon-modified nucleotide sequence (SEQ ID NO: 14) encoding a Factor VIII variant according to some embodiments ("CS08-FL-NA"). [Figure 18A] 1 shows the CS10 codon-modified nucleotide sequence (SEQ ID NO: 15) encoding a Factor VIII variant according to some embodiments ("CS10-FL-NA"). [Figure 18B] 1 shows the CS10 codon-modified nucleotide sequence (SEQ ID NO: 15) encoding a Factor VIII variant according to some embodiments ("CS10-FL-NA"). [Figure 19A] 1 shows the CS11 codon-modified nucleotide sequence (SEQ ID NO: 16) encoding a Factor VIII variant according to some embodiments ("CS11-FL-NA"). [Figure 19B] 1 shows the CS11 codon-modified nucleotide sequence (SEQ ID NO: 16) encoding a Factor VIII variant according to some embodiments ("CS11-FL-NA"). [Figure 20A] 1 shows the CS40 wild-type ReFacto coding sequence (SEQ ID NO: 17) according to some embodiments ("CS40-FL-NA"). [Figure 20B] 1 shows the CS40 wild-type ReFacto coding sequence (SEQ ID NO: 17) according to some embodiments ("CS40-FL-NA"). [Figure 21A] 1 shows a CH25 codon-altered nucleotide sequence (SEQ ID NO: 18) encoding a Factor VIII variant according to some embodiments ("CH25-FL-NA"). [Figure 21B] 1 shows a CH25 codon-altered nucleotide sequence (SEQ ID NO: 18) encoding a Factor VIII variant according to some embodiments ("CH25-FL-NA"). [Figure 22]1 shows the wild-type human Factor VIII amino acid sequence (SEQ ID NO: 19) according to some embodiments ("FVIII-FL-AA"). [Figure 23] Figure 1 shows the cloning scheme for constructs pCS40, pCS01, pCS04, pCS08, pCS10, pCS11, and pCh25 by inserting the DNA sequence of Refacto-synthesized BDD-FVIII into the vector backbone pCh-BB01 via both the AscI and NotI restriction sites. [Figure 24] Figure 1 shows the integrity of AAV vector genome preparations analyzed by agarose gel electrophoresis. Lane 1 is a DNA marker, lane 2 is vCS40, lane 3 is vCS01, and lane 4 is vCS04. All AAV vectors have genomes of the same size, migrating approximately 5 kb (arrows on the right). The scale on the left indicates the size of the DNA fragments in kilobases (kb). [Figure 25] Protein analysis of AAV vector preparations by PAGE and silver staining is shown. Lane 1 is a protein marker (M), lane 2 is vCS40, lane 3 is vCS01, and lane 4 is vCS04. All constructs have the same AAV8 capsid consisting of VP1, VP2, and VP3 (arrows on the right). The scale on the left indicates the size of the protein markers in kilodaltons (kDa). [Figure 26A] 1 shows the CS23 codon-modified nucleotide sequence (SEQ ID NO: 20) encoding a Factor VIII variant according to some embodiments ("CS23-FL-NA"). [Figure 26B] 1 shows the CS23 codon-modified nucleotide sequence (SEQ ID NO: 20) encoding a Factor VIII variant according to some embodiments ("CS23-FL-NA"). [Figure 27] 1 shows the Factor VIII variant amino acid sequence (SEQ ID NO: 21) encoded by the CS23 codon-modified nucleotide sequence according to some embodiments ("CS23-FL-AA"). [Figure 28]1 shows a portion of the CS23 codon-modified nucleotide sequence (SEQ ID NO: 22) encoding the heavy chain of a Factor VIII variant according to some embodiments ("CS23-HC-NA"). [Figure 29] 1 shows a portion of the CS23 codon-modified nucleotide sequence (SEQ ID NO: 23) encoding the light chain of a Factor VIII variant according to some embodiments ("CS23-LC-NA"). [Figure 30A] 1 shows the CS01m13 codon-altered nucleotide sequence (SEQ ID NO: 90) encoding a Factor VIII variant having m1(F328S) and m3 amino acid substitutions according to some embodiments ("CS01-FL-NA-m13"). [Figure 30B] 1 shows the CS01m13 codon-altered nucleotide sequence (SEQ ID NO: 90) encoding a Factor VIII variant having m1(F328S) and m3 amino acid substitutions according to some embodiments ("CS01-FL-NA-m13"). [Figure 31A] 1 shows the CS01m23 codon-altered nucleotide sequence (SEQ ID NO:91) encoding a Factor VIII variant having the m2 and m3 mutation sets according to some embodiments ("CS01-FL-NA-m23"). [Figure 31B] 1 shows the CS01m23 codon-altered nucleotide sequence (SEQ ID NO:91) encoding a Factor VIII variant having the m2 and m3 mutation sets according to some embodiments ("CS01-FL-NA-m23"). [Figure 32A] 1 shows the CS01m3 codon-altered nucleotide sequence (SEQ ID NO: 92) encoding a Factor VIII variant having an amino acid substitution of m3 according to some embodiments ("CS01-FL-NA-m3"). [Figure 32B] 1 shows the CS01m3 codon-altered nucleotide sequence (SEQ ID NO: 92) encoding a Factor VIII variant having an amino acid substitution of m3 according to some embodiments ("CS01-FL-NA-m3"). [Figure 33A]1 shows the CS01m2 codon-altered nucleotide sequence (SEQ ID NO: 93) encoding a Factor VIII variant having amino acid substitutions of the m2 mutation set (I105V / A127S / G151K / M166T / L171P (SPI)) according to some embodiments ("CS01-FL-NA-m2"). [Figure 33B] 1 shows the CS01m2 codon-altered nucleotide sequence (SEQ ID NO: 93) encoding a Factor VIII variant having amino acid substitutions of the m2 mutation set (I105V / A127S / G151K / M166T / L171P (SPI)) according to some embodiments ("CS01-FL-NA-m2"). [Figure 34A] 1 shows the CS04m2 codon-altered nucleotide sequence (SEQ ID NO: 94) encoding a Factor VIII variant having amino acid substitutions of the m2 mutant (I105V / A127S / G151K / M166T / L171P (SPI)) according to some embodiments ("CS01-FL-NA-m2"). [Figure 34B] 1 shows the CS04m2 codon-altered nucleotide sequence (SEQ ID NO: 94) encoding a Factor VIII variant having amino acid substitutions of the m2 mutant (I105V / A127S / G151K / M166T / L171P (SPI)) according to some embodiments ("CS01-FL-NA-m2"). [Figure 35A] 1 shows the CS04m3 codon-altered nucleotide sequence (SEQ ID NO: 95) encoding a Factor VIII variant having amino acid substitutions of m3 according to some embodiments ("CS04-FL-NA-m3"). [Figure 35B] 1 shows the CS04m3 codon-altered nucleotide sequence (SEQ ID NO: 95) encoding a Factor VIII variant having amino acid substitutions of m3 according to some embodiments ("CS04-FL-NA-m3"). [Figure 36A] 1 shows the CS04m23 codon-altered nucleotide sequence (SEQ ID NO: 96) encoding a Factor VIII variant having the m2 mutation set (I105V / A127S / G151K / M166T / L171P (SPI)) and the m3 amino acid substitutions according to some embodiments ("CS04-FL-NA-m23"). [Figure 36B] 1 shows the CS04m23 codon-altered nucleotide sequence (SEQ ID NO: 96) encoding a Factor VIII variant having the m2 mutation set (I105V / A127S / G151K / M166T / L171P (SPI)) and the m3 amino acid substitutions according to some embodiments ("CS04-FL-NA-m23"). [Figure 37A] 1 shows the CS04m1 codon-altered nucleotide sequence (SEQ ID NO:97) encoding a Factor VIII variant having the m1 (F328S) amino acid substitution according to some embodiments ("CS04-FL-NA-m1"). [Figure 37B] 1 shows the CS04m1 codon-altered nucleotide sequence (SEQ ID NO:97) encoding a Factor VIII variant having the m1 (F328S) amino acid substitution according to some embodiments ("CS04-FL-NA-m1"). [Figure 38A] 1 shows the CS04m13 codon-altered nucleotide sequence (SEQ ID NO: 98) encoding a Factor VIII variant having m1 and m3 amino acid substitutions according to some embodiments ("CS04-FL-NA-m13"). [Figure 38B] 1 shows the CS04m13 codon-altered nucleotide sequence (SEQ ID NO: 98) encoding a Factor VIII variant having m1 and m3 amino acid substitutions according to some embodiments ("CS04-FL-NA-m13"). [Figure 39A] 1 shows the CS23m13 codon-altered nucleotide sequence (SEQ ID NO: 99) encoding a Factor VIII variant having m1 and m3 amino acid substitutions according to some embodiments ("CS23m13-FL-NA"). [Figure 39B] 1 shows the CS23m13 codon-altered nucleotide sequence (SEQ ID NO: 99) encoding a Factor VIII variant having m1 and m3 amino acid substitutions according to some embodiments ("CS23m13-FL-NA"). [Figure 40A] 1 shows the CS23m3 codon-altered nucleotide sequence (SEQ ID NO: 100) encoding a Factor VIII variant having an amino acid substitution of m3 according to some embodiments ("CS23-FL-NA-m3"). [Figure 40B] 1 shows the CS23m3 codon-altered nucleotide sequence (SEQ ID NO: 100) encoding a Factor VIII variant having an amino acid substitution of m3 according to some embodiments ("CS23-FL-NA-m3"). [Figure 41A] 1 shows the CS23m2 codon-modified nucleotide sequence (SEQ ID NO: 101) encoding a Factor VIII variant having the m2 mutation set (amino acid substitutions I105V / A127S / G151K / M166T / L171P) according to some embodiments ("CS23-FL-NA-m2"). [Figure 41B] 1 shows the CS23m2 codon-modified nucleotide sequence (SEQ ID NO: 101) encoding a Factor VIII variant having the m2 mutation set (amino acid substitutions I105V / A127S / G151K / M166T / L171P) according to some embodiments ("CS23-FL-NA-m2"). [Figure 42A] 1 shows the CS23m1 codon-altered nucleotide sequence (SEQ ID NO: 102) encoding a Factor VIII variant having the m1 (F328S) amino acid substitution according to some embodiments ("CS23-FL-NA-m1"). [Figure 42B] 1 shows the CS23m1 codon-altered nucleotide sequence (SEQ ID NO: 102) encoding a Factor VIII variant having the m1 (F328S) amino acid substitution according to some embodiments ("CS23-FL-NA-m1"). [Figure 43A] 1 shows the CS23m23 codon-altered nucleotide sequence (SEQ ID NO: 103) encoding a Factor VIII variant having the m2 mutation set (I105V / A127S / G151K / M166T / L171P) and m3 amino acid substitutions according to some embodiments ("CS23-FL-NA-m23"). [Figure 43B] 1 shows the CS23m23 codon-altered nucleotide sequence (SEQ ID NO: 103) encoding a Factor VIII variant having the m2 mutation set (I105V / A127S / G151K / M166T / L171P) and m3 amino acid substitutions according to some embodiments ("CS23-FL-NA-m23"). [Figure 44]Cloning of pCS constructs is shown, which is performed by inserting Refacto-synthesized BDD-FVIII with different mutations (see inset) into the vector backbone pCh-BB01 via both AscI and NotI restriction sites. [Figure 45] Protein analysis of AAV vector preparations by PAGE and silver staining is shown. Lane 1 is a protein marker (M), lane 2 is vCS01, lane 3 is vCS17, lane 4 is vCS19, lane 5 is vCS20, lane 6 is vCS40, lane 7 is vCS04, lane 8 is vCS17, and lane 9 is vCS24 construct. All constructs have the same AAV8 capsid consisting of VP1, VP2, and VP3 (arrows on the right). The scale on the left indicates the size of the protein markers in kilodaltons (kDa). [Figure 46] The integrity of AAV vector genome preparations analyzed by agarose gel electrophoresis is shown. Lane 1 is a DNA marker (M), lane 2 is vCS04, lane 3 is vCS17, lane 4 is vCS20, lane 5 is vCS24, lane 6 is vCS16, and lane 7 is vCS40 construct. The vector amount is 1.5E10 vg per lane. AAV vectors have genomes of identical size and migrate approximately 5 kb (arrows on the right). The scale on the left indicates the size of the DNA fragments in kilobases (kb). [Figure 47] 1 shows a portion of the CS01 codon-modified nucleotide sequence (SEQ ID NO: 24) encoding the heavy chain of a Factor VIII variant according to some embodiments ("CS01-HC-NA"). [Figure 48] 1 shows a portion of the CS01 codon-modified nucleotide sequence (SEQ ID NO: 25) encoding the light chain of a Factor VIII variant according to some embodiments ("CS01-LC-NA"). [Figure 49A] 1 shows the codon-modified nucleotide sequence (SEQ ID NO: 26) encoding the CS01Δ(760-1667) (for SPI; CS01Δ(741-1648) for SPE) single-chain factor VIII variant according to some embodiments ("CS01-SC1-NA"). [Figure 49B]1 shows the codon-modified nucleotide sequence (SEQ ID NO: 26) encoding the CS01Δ(760-1667) (for SPI; CS01Δ(741-1648) for SPE) single-chain factor VIII variant according to some embodiments ("CS01-SC1-NA"). [Figure 50A] 1 shows the codon-modified nucleotide sequence (SEQ ID NO: 27) encoding the single-chain factor VIII variant CS01Δ(772-1667) (for SPI; CS01Δ(753-1648) for SPE) according to some embodiments ("CS01-SC2-NA"). [Figure 50B] 1 shows the codon-modified nucleotide sequence (SEQ ID NO: 27) encoding the single-chain factor VIII variant CS01Δ(772-1667) (for SPI; CS01Δ(753-1648) for SPE) according to some embodiments ("CS01-SC2-NA"). [Figure 51A] 1 shows the codon-modified nucleotide sequence (SEQ ID NO: 28) of CS23Δ(760-1667) (for SPI; CS23Δ(741-1648) for SPE) encoding a single-chain factor VIII variant according to some embodiments ("CS23-SC1-NA"). [Figure 51B] 1 shows the codon-modified nucleotide sequence (SEQ ID NO: 28) of CS23Δ(760-1667) (for SPI; CS23Δ(741-1648) for SPE) encoding a single-chain factor VIII variant according to some embodiments ("CS23-SC1-NA"). [Figure 52A] 1 shows the codon-modified nucleotide sequence (SEQ ID NO: 29) of CS23Δ(772-1667) (for SPI; CS23Δ(753-1648) for SPE) encoding a single-chain factor VIII variant according to some embodiments ("CS23-SC2-NA"). [Figure 52B] 1 shows the codon-modified nucleotide sequence (SEQ ID NO: 29) of CS23Δ(772-1667) (for SPI; CS23Δ(753-1648) for SPE) encoding a single-chain factor VIII variant according to some embodiments ("CS23-SC2-NA"). [Figure 53]1 shows the Factor VIII variant amino acid sequence (SEQ ID NO: 104) encoded by the CS01m23 codon-altered nucleotide sequence according to some embodiments ("CS01m23-FL-AA"). [Figure 54] 1 shows the Factor VIII variant amino acid sequence (SEQ ID NO: 105) encoded by the CS04m3 codon-altered nucleotide sequence according to some embodiments ("CS01m23-FL-AA"). [Figure 55] 1 shows the Factor VIII variant amino acid sequence (SEQ ID NO: 106) encoded by the CS01m12 codon-altered nucleotide sequence according to some embodiments ("CS01m12-FL-AA"). [Figure 56] 1 shows the Factor VIII variant amino acid sequence (SEQ ID NO: 107) encoded by the CS04m12 codon-altered nucleotide sequence according to some embodiments ("CS04m12-FL-AA"). [Figure 57A] 1 shows the CS01m12 codon-altered nucleotide sequence (SEQ ID NO: 108) encoding a Factor VIII variant having m1(F328S) and m2 amino acid substitutions according to some embodiments ("CS01-FL-NAm12"). [Figure 57B] 1 shows the CS01m12 codon-altered nucleotide sequence (SEQ ID NO: 108) encoding a Factor VIII variant having m1(F328S) and m2 amino acid substitutions according to some embodiments ("CS01-FL-NAm12"). [Figure 58A] 1 shows the CS04m12 codon-altered nucleotide sequence (SEQ ID NO: 109) encoding a Factor VIII variant having m1 (F328S) and m2 amino acid substitutions according to some embodiments ("CS04-FL-NAm12"). [Figure 58B] 1 shows the CS04m12 codon-altered nucleotide sequence (SEQ ID NO: 109) encoding a Factor VIII variant having m1 (F328S) and m2 amino acid substitutions according to some embodiments ("CS04-FL-NAm12"). DETAILED DESCRIPTION OF THE INVENTION

[0127] Detailed Description of Disclosure I. Introduction AAV-based gene therapy holds promise for the treatment of hemophilia patients. In hemophilia B, initial clinical data are promising, demonstrating that FIX levels of approximately 10% can be maintained for at least a year in at least some patients. However, in hemophilia A, achieving therapeutic expression levels of 5–10% with AAV vectors remains challenging for a variety of reasons. First, the factor VIII coding sequence is too large for conventional AAV-based vectors. Second, engineered B-domain deleted or truncated factor VIII constructs, even when codon-optimized, suffer from low in vivo expression. Third, these B-domain deleted or truncated factor VIII mutant constructs have a short in vivo half-life, further exacerbating the effects of low expression. Fourth, even when expressed, FVIII is not secreted from cells as efficiently as other clotting factors, such as factor IX.

[0128] Furthermore, these issues cannot be addressed by simply administering high doses of gene therapy constructs. Current knowledge suggests that AAV-based gene therapy vectors should be administered at a vector dose of 2x10 / kg body weight. 12 vg. Such high doses induce a T-cell immune response that destroys the transduced cells, resulting in reduced or even lost transgene expression. Therefore, strategies to improve FVIII expression are needed to make FVIII gene therapy a viable treatment option for patients with hemophilia A.

[0129] The present disclosure relates to the discovery of codon-modified factor VIII variant coding sequences that solve these and other problems associated with factor VIII gene therapy. For example, the polynucleotides disclosed herein exhibit significantly improved expression in mammalian cells and improved virion packaging due to stabilized packing interactions. In some embodiments, these advantages are achieved through the use of factor VIII heavy and light chain coding sequences with high sequence identity to the codon-modified constructs CS01, CS04, and CS23 (e.g., high sequence identity to one of the heavy chain coding sequences CS01-HC, CS04-HC, and CS23-HC and high sequence identity to one of the light chain coding sequences CS01-LC, CS04-LC, and CS23-LC).

[0130] In some embodiments, the Factor VIII molecules encoded by the polynucleotides described herein are truncated by truncation, deletion, or substitution of the wild-type B domain, making such polynucleotides more suitable for expression of Factor VIII via conventional gene therapy vectors, which are less efficient at expressing large polypeptides such as wild-type Factor VIII.

[0131] Advantageously, the codon-modified factor VIII variant coding sequences CS01, CS04, and CS23 are shown herein to provide superior expression of B-domain deleted factor VIII constructs in vivo. For example, it is demonstrated in Examples 2 and 4 that intravenous administration of AAV-based gene therapy vectors containing the coding sequences CS01 (SEQ ID NO: 13), CS04 (SEQ ID NO: 1), and CS23 (SEQ ID NO: 20) in factor VIII knockout mice results in 18-fold, 74-fold, and 30-fold increased factor VIII expression compared to the corresponding CS40 construct encoded by the wild-type polynucleotide sequence (SEQ ID NO: 17) (Tables 4 and 7).

[0132] Furthermore, the codon-modified Factor VIII variant coding sequences CS01 and CS04 are shown herein to provide superior virion packaging and virus production. For example, it is demonstrated in Example 1 that, when isolated from the same amount of cell pellet, AAV vector constructs containing constructs CS01 and CS04 produce 5-7 times greater virus yields than the corresponding CS40 construct encoded by the wild-type polynucleotide sequence.

[0133] Advantageously, Applicants have also discovered that the improved Factor VIII activity resulting from the codon-modified sequences CS01, CS04, and CS23 can be further enhanced by introducing mutations into the underlying Factor VIII polypeptide sequence. For example, as demonstrated in Example 4, the mutations F328S, X5, and X1, when expressed in vivo in the codon-modified background of CS01 or CS04, either alone or in combination with each other, further enhanced FVIII activity by 2-7 fold compared to the wild-type codon-modified construct (Table 7). Even more strikingly, these codon-modified sequences encoding mutant Factor VIII variants exhibited up to a 246-fold enhancement compared to the corresponding CS40 construct encoded by the wild-type polynucleotide sequence (Table 7).

[0134] II. Definition As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0135] As used herein, the terms "factor VIII" and "FVIII" are used interchangeably and refer to any protein with factor VIII activity (e.g., active FVIII, often referred to as FVIIIa) or a protein precursor (e.g., a proprotein or preproprotein) of a protein with factor VIII activity, particularly factor IXa cofactor activity. In an exemplary embodiment, a factor VIII polypeptide refers to a polypeptide having a high degree of sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more) to the heavy and light chains of a wild-type factor VIII polypeptide. In some embodiments, the B domain of a factor VIII polypeptide is deleted, truncated, or replaced with a linker polypeptide to reduce the size of the polynucleotide encoding the factor VIII polypeptide. In an exemplary embodiment, amino acids 20-1457 of SEQ ID NO: 2 constitute a factor VIII polypeptide.

[0136] Non-limiting examples of wild-type factor VIII polypeptides include human preprofactor VIII (e.g., GenBank Accession Nos. AAA52485, CAA25619, AAA58466, AAA52484, AAA52420, AAV85964, BAF82636, BAG36452, CAI41660, CAI41666, CAI41672, CAI43241, CAO03404, EAW72645, AAH22513, AAH64380, AAH98389, AAI11968, AAI11970, or AAB61261), the corresponding profactor VIII, and natural variants thereof; porcine preprofactor VIII (e.g., mouse preprofactor VIII (e.g., UniProt accession number F1RZ36 or K7GSZ5), the corresponding profactor VIII, and natural variants thereof; mouse preprofactor VIII (e.g., GenBank accession number AAA37385, CAM15581, CAM26492, or EDL29229), the corresponding profactor VIII, and natural variants thereof; rat preprofactor VIII (e.g., GenBank accession number AAQ21580), the corresponding profactor VIII, and natural variants thereof; rat preprofactor VIII; and other mammalian factor VIII homologs (e.g., monkeys, apes, hamsters, guinea pigs, etc.).

[0137] As used herein, Factor VIII polypeptides include natural variants and artificial constructs with Factor IX cofactor activity. As used in this disclosure, Factor VIII encompasses any natural variant, alternative sequence, isoform, or mutein that retains some basal Factor IX cofactor activity (e.g., at least 5%, 10%, 25%, 50%, 75%, or more of the corresponding wild-type activity). Examples of Factor VIII amino acid mutations (relative to FVIII-FL-AA (SEQ ID NO: 19)) found in the human population include, but are not limited to, S19R, R22T, Y24C, Y25C, L26P / R, E30V, W33G, Y35C / H, G41C, R48C / K, K67E / N, L69P, E72K, D75E / V / Y, P83R, G89D / V, G92A / V, A97P, E98K, V99D, D101G / H / V, V104D, K108T, M 110V, A111T / V, H113R / Y, L117F / R, G121S, E129V, G130R, E132D, Y133C, D135G / Y, T137A / I, S138R, E141K, D145H, V147D, Y1 55H, V159A, N163K, G164D / V, P165S, C172W, S176P, S179P, V181E / M, K185T, D186G / N / Y, S189L, L191F, G193R, L195P, C198G , S202N / R, F214V, L217H, A219D / T, V220G, D222V, E223K, G224W, T252I, V253F, N254I, G255V, L261P, P262L, G263S, G266F , C267Y, W274C, H275L, G278R, G280D, E284K, V285G, E291G / K, T294I, F295L, V297A, N299I, R301C / H / L, A303E / P, I307S, S3 08L, F312S, T314A / I, A315V, G323E, L326P, L327P / V, C329F, I331V, M339T, E340K, V345A / L, C348R / S / Y, Y365C, R391C / H / P , S392L / P, A394S, W401G, I405F / S, E409G, W412G / R, K427I, L431F / S, R437P / W, I438F, G439D / S / V, Y442C, K444R, Y450D / N,T454I、F455C、G466E、P470L / R / T、G474E / R / V、E475K、G477V、D478N、T479R、F484C、A488G、R490G、Y492C / H、Y492H、I494T、P496R、G498R、R503H、G513S / V、I522Y、K529E、W532G、P540T、T541S、D544N、R546W、R550C / G / H、S553P、S554C / G、V556D、R560T、D561G / H / Y、I567T、P569R、S577F、V578A、D579A / H、N583S、Q584H / K / R、I585R / T、M586V、D588G / Y、L594Q、S596P、N601D / K、R602G、S603I / R、W604C、Y605H / S、N609I、R612C、N631K / S、M633I、S635N、N637D / I / S、Y639C、L644V、L650F、V653A / M、L659P、A663V、Q664P、F677L、M681I、V682F、Y683C / N、T686R、F698L、M699T / V、M701I、G705V、G710W、N713I、R717L / W、G720D / S、M721I / L、A723T、L725Q、V727F、E739K、Y742C、R795G、P947R、V1012L、E1057K、H1066Y、D1260E、K1289Q、Q1336K、N1460K、L1481P、A1610S、I1698T、Y1699C / F、E1701K、Q1705H、R1708C / H、T1714S、R1715G、A1720V、E1723K、D1727V、Y1728C、R1740G、K1751Q、F1762L、R1768H、G1769R、L1771P、L1775F / V、L1777P、G1779E / R、P1780L、I1782R、D1788H、M1791T、A1798P、S1799H、R1800C / G / H、P1801A、Y1802C、S1803Y、F1804S、L1808F、M1842I、P1844S、T1845P、E1848G、A1853T / V、S1858C、K1864E、D1865N / Y、H1867P / R、G1869D / V、G1872E、P1873R、L1875P、V1876L、C1877R / Y、L1882P、R1888I、E1894G、I1901F、E1904D / K, S1907C / R, W1908L, Y1909C, A1939T / V, N1941D / S, G1942A, M1945V, L1951F, R1960L / Q, L1963P, S1965I, M1966I / V , G1967D, S1968R, N1971T, H1973L, G1979V, H1980P / Y, F1982I, R1985Q, L1994P, Y1998C, G2000A, T2004R, M2007I, G2013R, W2 015C, R2016P / W, E2018G, G2022D, G2028R, S2030N, V2035A, Y2036C, N2038S, 2040Y, G2045E / V, I2051S, I2056N, A2058P, W20 65R, P2067L, A2070V, S2082N, S2088F, D2093G / Y, H2101D, T2105N, Q2106E / P / R, G2107S, R2109C, I2117F / S, Q2119R, F2120C / L, Y2124C, R2135P, S2138Y, T2141N, M2143V, F2145C, N2148S, N2157D, P2162L, R2169C / H, P2172L / Q / R, T2173A / I, H2174D, R 2178C / H / L, R2182C / H / P, M2183R / V, L2185S / W, S2192I, C2193G, P2196R, G2198V, E2200D, I2204T, I2209N, A2211P, A2220P, P Factor VIII proteins include polypeptides containing post-translational modifications, such as 2224L, R2228G / L / P / Q, L2229F, V2242M, W2248C / S, V2251A / E, M2257V, T2264A, Q2265R, F2279C / I, I2281T, D2286G, W2290L, G2304V, D2307A, P2319L / S, R2323C / G / H / L, R2326G / L / P / Q, Q2330P, W2332R, I2336F, R2339T, G2344C / D / S, and C2345S / Y. Factor VIII proteins also include polypeptides containing post-translational modifications.

[0138] Generally, a polynucleotide encoding factor VIII encodes an inactive single-chain polypeptide (e.g., a preproprotein) that is post-translationally processed to form an active factor VIII protein (e.g., FVIIIa). For example, with reference to FIG. 1, wild-type human factor VIII preproprotein is first cleaved to release the encoded signal peptide (not shown) and form a first single-chain proprotein (designated "human wild-type FVIII"). The proprotein is then cleaved between the B and A3 domains to form a first polypeptide containing the factor VIII heavy chain (e.g., the A1 and A2 domains) and the B domain, and a second polypeptide containing the factor VIII light chain (e.g., the A3, C1, and C3 domains). The first polypeptide is further cleaved to remove the B domain and separate the A1 and A2 domains, which remain associated with the factor VIII light chain in the mature factor VIIIa protein. For a review of the maturation process of Factor VIII, see Graw et al., Nat Rev Genet., 6(6):488-501 (2005), the contents of which are incorporated by reference in their entirety for all purposes.

[0139] However, in some embodiments, the Factor VIII polypeptide is a Factor VIII single-chain polypeptide. Factor VIII single-chain polypeptides are engineered to remove the native cleavage site and, optionally, to remove, truncate, or replace the Factor VIII B domain. Thus, they are active as single chains, rather than in the mature state due to cleavage (other than cleavage of the optional signal peptide and / or leader peptide). Non-limiting examples of Factor VIII single-chain polypeptides are described in Zollner et al. (Thromb Res, 134(1):125-31 (2014)) and Donath et al. (Biochem J., 312(1):49-55 (1995)), the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0140] As used herein, the term "Factor VIII heavy chain" or simply "heavy chain" refers to the assembly of the A1 and A2 domains of a Factor VIII polypeptide. In an exemplary embodiment, amino acids 20-759 of CS04-FL-AA (SEQ ID NO: 2) constitute the Factor VIII heavy chain.

[0141] As used herein, the term "factor VIII light chain" or simply "light chain" refers to the assembly of the A3, C1, and C2 domains of a factor VIII polypeptide. In an exemplary embodiment, amino acids 774-1457 of CS04-FL-AA (SEQ ID NO: 2) constitute the factor VIII light chain. In some embodiments, the acidic a3 peptide, which is released during maturation in vivo, is excluded from the factor VIII light chain.

[0142] Generally, the Factor VIII heavy and light chains are expressed as a single polypeptide chain, e.g., with an optional B domain or B domain-substituted linker, although in some embodiments, the Factor VIII heavy chain and the Factor VIII light chain are expressed (e.g., co-expressed) as separate polypeptide chains and reconstituted to form the Factor VIII protein (e.g., in vivo or in vitro).

[0143] As used herein, the terms "B domain replacement linker" and "Factor VIII linker" are used interchangeably and refer to a truncated version of the wild-type Factor VIII B domain (e.g., amino acids 760-1667 of FVIII-FL-AA (SEQ ID NO: 19)) or a peptide engineered to replace the B domain of a Factor VIII polypeptide. As used herein, a Factor VIII linker is positioned between the C-terminus of the Factor VIII heavy chain and the N-terminus of the Factor VIII light chain of a Factor VIII variant polypeptide according to some embodiments. Non-limiting examples of B domain replacement linkers are described in U.S. Patent Nos. 4,868,112, 5,112,950, 5,171,844, 5,543,502, 5,595,886, 5,610,278, 5,789,203, 5,972,885, 6,048,720, 6,060,447, 6,114,148, 6,228,620, 6,316,226, and 6,346,513. , 6,458,563, 6,924,365, 7,041,635, and 7,943,374; U.S. Patent Application Publication Nos. 2013 / 024960, 2015 / 0071883, and 2015 / 0158930; and PCT Publication Nos. WO2014 / 064277 and WO2014 / 127215, the disclosures of which are incorporated by reference in their entireties herein for all purposes.

[0144] Unless otherwise specified herein, factor VIII amino acid numbering refers to the corresponding amino acid in the full-length wild-type human factor VIII sequence (FVIII-FL-AA) presented in Figure 22 as SEQ ID NO: 19. Thus, when referring to an amino acid substitution in a factor VIII variant protein disclosed herein, the recited amino acid number refers to the similar (e.g., structurally or functionally equivalent) and / or homologous (e.g., evolutionarily conserved in the primary amino acid sequence) amino acid in the full-length wild-type factor VIII sequence. For example, the amino acid substitution T2105N refers to a T to N substitution at position 2105 of the full-length wild-type human factor VIII sequence (FVIII-FL-AA; SEQ ID NO: 19), a T to N substitution at position 1211 of the factor VIII variant protein encoded by CS04 (CS04-FL-AA; SEQ ID NO: 2), and a T to N substitution at position 1212 of the factor VIII variant encoded by CS04m3 (CS04m3-FL-AA; SEQ ID NO: 105).

[0145] As used herein, the Factor VIII amino acid numbering system varies depending on whether the Factor VIII signal peptide (e.g., amino acids 1-19 of the full-length wild-type human Factor VIII sequence) is included. If the signal peptide is included, the numbering is designated "signal peptide included" or "SPI." If the signal peptide is not included, the numbering is designated "signal peptide excluded" or "SPE." For example, F328S is the SPI numbering representation of the same amino acid as F309S in SPE numbering. Unless otherwise specified, all amino acid numbering refers to the corresponding amino acid in the full-length wild-type human Factor VIII sequence (FVIII-FL-AA) presented in Figure 22 as SEQ ID NO: 19.

[0146] As described herein, the codon-modified polynucleotide increases the in vivo expression of transgenic factor VIII (e.g., when administered as part of a gene therapy vector) compared to the factor VIII expression level obtained with a naturally encoded factor VIII construct (e.g., a polynucleotide encoding the same factor VIII construct using wild-type human codons). As used herein, the term "increased expression" refers to an increase in the transgenic factor VIII blood activity level of an animal administered the codon-modified polynucleotide encoding factor VIII compared to the transgenic factor VIII blood activity level of an animal administered the naturally encoded factor VIII construct. Activity levels can be measured using any factor VIII activity assay known in the art. An exemplary assay for determining factor VIII activity is the Technochrome FVIII assay (Technoclone, Vienna, Austria).

[0147] In some embodiments, increased expression refers to at least a 25% increase in transgenic Factor VIII blood activity in animals administered the codon-modified Factor VIII polynucleotide compared to the transgenic Factor VIII blood activity level in animals administered the naturally encoded Factor VIII polynucleotide. In some embodiments, increased expression refers to at least 50% higher, at least 75% higher, at least 100% higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 15-fold higher, at least 20-fold higher, at least 25-fold higher, at least 30-fold higher, at least 40-fold higher, at least 50-fold higher, at least 60-fold higher, at least 70-fold higher, at least 80-fold higher, at least 90-fold higher, at least 100-fold higher, at least 125-fold higher, at least 150-fold higher, at least 175-fold higher, at least 200-fold higher, at least 225-fold higher, or at least 250-fold higher transgenic Factor VIII blood activity in an animal administered the codon-modified Factor VIII polynucleotide compared to the transgenic Factor VIII blood activity level in an animal administered the naturally encoded Factor VIII polynucleotide.

[0148] As described herein, the codon-modified polynucleotide increases vector production compared to the vector production level obtained with a naturally encoded Factor VIII construct (e.g., a polynucleotide encoding the same Factor VIII construct using wild-type human codons). As used herein, the term "increased virus production" refers to an increase in vector yield (e.g., titer per liter of culture medium) in a cell culture inoculated with a codon-modified polynucleotide encoding Factor VIII compared to the vector yield (e.g., titer per liter of culture medium) in a cell culture inoculated with a naturally encoded Factor VIII construct. Vector yield can be measured using any vector titer assay known in the art. An exemplary assay for determining vector yield (e.g., AAV vector yield) is qPCR targeting the AAV2 terminal inverted repeat (Aurnhammer, Human Gene Therapy Methods: Part B 23:18-28 (2012)).

[0149] In some embodiments, increased virus production refers to a codon-modified vector yield that is at least 25% greater than the naturally encoded Factor VIII construct yield in homogeneous culture. In some embodiments, increased vector production refers to a codon-modified vector yield that is at least 50% greater, at least 75% greater, at least 100% greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 15-fold greater, or at least 20-fold greater than the naturally encoded Factor VIII construct yield in homogeneous culture.

[0150] As used herein, the term "hemophilia" refers to a group of disease states broadly characterized by reduced blood clotting or coagulation. Hemophilia can refer to hemophilia type A, type B, or type C, or a combination of all three disease types. Hemophilia type A (hemophilia A) is caused by reduced or deficient factor VIII (FVIII) activity and is the most prominent hemophilia subtype. Hemophilia type B (hemophilia B) results from reduced or deficient clotting function of factor IX (FIX). Hemophilia type C (hemophilia C) results from reduced or deficient clotting activity of factor XI (FXI). Hemophilia A and B are X-linked disorders, while hemophilia C is an autosomal disorder. Conventional hemophilia treatments include prophylactic and on-demand administration of clotting factors such as FVIII, FIX (including Bebulin®-VH), and FXI, as well as FEIBA-VH, desmopressin, and plasma infusions.

[0151] As used herein, the term "FVIII gene therapy" includes any treatment that provides a patient with a nucleic acid encoding factor VIII to alleviate, reduce, or prevent the recurrence of one or more symptoms (e.g., clinical factors) associated with hemophilia. The term encompasses the administration of any compound, drug, treatment, or regimen, including a nucleic acid encoding a factor VIII molecule, including any modified form of factor VIII (e.g., a factor VIII variant), to maintain or improve the health of an individual with hemophilia. Those skilled in the art will appreciate that the duration of FVIII therapy, the dosage of the FVIII therapeutic agent, can both be modified, for example, based on the results obtained in accordance with the present disclosure.

[0152] As used herein, the term "bypass therapy" includes any treatment that provides a patient with a non-factor VIII hemostatic agent, compound, or clotting factor to alleviate, reduce, or prevent the recurrence of one or more symptoms (e.g., clinical factors) associated with hemophilia. Non-factor VIII compounds and clotting factors include, but are not limited to, factor VIII inhibitor bypassing activity (FEIBA), recombinant activated factor VII (FVIIa), prothrombin complex concentrate, and activated prothrombin complex concentrate. These non-factor VIII compounds and clotting factors may be recombinant or plasma-derived. One skilled in the art will appreciate that the duration of bypass therapy, the dose of bypass therapy, and both can be modified based on, for example, the results obtained in accordance with the present disclosure.

[0153] As used herein, "combination therapy," which includes the administration of a nucleic acid encoding a Factor VIII molecule and a conventional hemophilia A therapeutic agent, includes any therapy that provides a patient with both a nucleic acid encoding a Factor VIII molecule and a Factor VIII molecule and / or a non-Factor VIII hemostatic agent (e.g., a bypassing therapy) to alleviate, reduce, or prevent the recurrence of one or more symptoms (e.g., clinical factors) associated with hemophilia. Such term encompasses the administration of any compound, drug, treatment, or regimen that is useful for maintaining or improving the health of an individual with hemophilia and includes a nucleic acid encoding a Factor VIII molecule, including any modified form of Factor VIII, including any of the therapeutic agents described herein.

[0154] The terms "therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" or "effective or sufficient amount or dose" refer to a dose that produces a therapeutic effect in the recipient. For example, a therapeutically effective amount of a drug useful for treating hemophilia would be an amount that is capable of preventing or alleviating one or more symptoms associated with hemophilia. The exact dose will vary depending on the therapeutic purpose and can be ascertained by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0155] As used herein, the term "gene" refers to a segment of a DNA molecule that encodes a polypeptide chain (e.g., a coding region). In some embodiments, a gene refers to the regions immediately preceding, immediately following, and / or intervening the coding region that are involved in producing the polypeptide chain (e.g., regulatory elements such as a promoter, enhancer, polyadenylation sequence, 5' untranslated region, 3' untranslated region, or introns).

[0156] As used herein, the term "regulatory element" refers to nucleotide sequences such as promoters, enhancers, terminators, polyadenylation sequences, introns, etc. that direct the expression of a coding sequence in a cell.

[0157] As used herein, the term "promoter element" refers to a nucleotide sequence that helps control the expression of a coding sequence. Generally, promoter elements are located 5' to the translation start site of a gene. However, in certain embodiments, promoter elements can be located within intron sequences or at the 3' end of a coding sequence. In some embodiments, promoters useful in gene therapy vectors are derived from the native gene of the target protein (e.g., the Factor VIII promoter). In some embodiments, promoters useful in gene therapy vectors are specifically expressed in particular cells or tissues of the target organism (e.g., a liver-specific promoter). In yet other embodiments, one of several well-characterized promoter elements is used in the gene therapy vectors described herein. Non-limiting examples of well-characterized promoter elements include the CMV early promoter, the β-actin promoter, and the methylated CpG-binding protein 2 (MeCP2) promoter. In some embodiments, the promoter is a constitutive promoter, which induces substantially constant expression of the target protein. In other embodiments, the promoter is an inducible promoter, which induces expression of the target protein in response to a particular stimulus (e.g., exposure to a particular treatment or drug). For a review of promoter design for AAV-mediated gene therapy, see Gray et al. (Human Gene Therapy 22:1143-53 (2011)), the contents of which are expressly incorporated by reference in their entirety for all purposes.

[0158] As used herein, the term "vector" refers to any vehicle used to introduce nucleic acid (e.g., encoding a Factor VIII gene therapy construct) into a host cell. In some embodiments, a vector includes a replicon, which functions to replicate the vehicle along with the target nucleic acid. Non-limiting examples of vectors useful for gene therapy include plasmids, phages, cosmids, artificial chromosomes, and viruses, which function as autonomous replication units in vivo. In some embodiments, a vector is a viral vehicle for introducing a target nucleic acid (e.g., a codon-modified polynucleotide encoding a Factor VIII variant). Numerous modified eukaryotic viruses useful for gene therapy are known in the art. For example, adeno-associated virus (AAV) is particularly suitable for use in human gene therapy because humans are the natural host for the virus, the wild-type form of this virus is not known to contribute to disease, and the virus elicits a mild immune response.

[0159] As used herein, the term "CpG island" refers to a region within a polynucleotide that has a statistically high density of CpG dinucleotides. As used herein, a region of a polynucleotide (e.g., a polynucleotide encoding a codon-modified Factor VIII protein) is a CpG island if, over a 200 base pair region, (i) the GC content of the region is greater than 50%, and (ii) the ratio of observed to expected CpG dinucleotides is at least 0.6, as defined by the following relationship: TIFF0007631425000001.tif8128For further information regarding methods for identifying CpG islands, see Gardiner-Garden M. et al., J Mol Biol., 196(2):261-82 (1987), the contents of which are expressly incorporated by reference in their entirety into this specification for all purposes.

[0160] As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form, and their complementary strands. The term encompasses synthetic, naturally occurring, and non-naturally occurring nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties and are metabolized in a manner similar to the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0161] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, including amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring amino acids can include, for example, D-amino acids and L-amino acids. As used herein, amino acids can also include non-naturally occurring amino acids. An amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., any carbon atom bonded to a hydrogen atom, a carboxyl group, an amino group, or an R group, such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0162] As used herein, the nucleotide sequence encoding a mutant Factor VIII construct may be the same as or a different coding sequence from the coding sequences provided herein, which, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as the coding sequences provided herein. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, each variation of a nucleic acid that encodes the same polypeptide is included in each described sequence for the expression product, but not for the actual gene therapy construct.

[0163] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid or peptide sequences that alter, add, or delete a single amino acid or a small number of amino acids in the encoded sequence are "conservatively modified variants," in which such alterations replace an amino acid with a chemically similar amino acid. Conservative substitution tables listing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present disclosure.

[0164] Conservative amino acid substitutions that provide functionally similar amino acids are well known in the art. Depending on the functionality of a particular amino acid, for example, catalytic amino acids, structural amino acids, or sterically important amino acids, different classes of amino acids may be considered as conservative substitutions for each other. Table 1 lists amino acid classes that are considered as conservative substitutions based on the charge and polarity of the amino acid, the hydrophobicity of the amino acid, the surface exposure / structural properties of the amino acid, and the tendency of the amino acid to form a secondary structure.

[0165] Table 1. Classification of conservative amino acid substitutions based on the functionality of protein residues. TIFF0007631425000002.tif203164

[0166] The terms "identical" or "percent identity" in the context of two or more nucleic acid or peptide sequences means that two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides when measured using the BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below, or when manually aligned and visually inspected (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region).

[0167] As is known in the art, a variety of programs may be used to determine whether a protein (or nucleic acid, as discussed below) has sequence identity or sequence similarity to a known sequence. Sequence identity and / or similarity may be determined using standard techniques known in the art, including the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math., 2:482 (1981), preferably using default settings; the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol., 48:443 (1970); the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988); computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, all included in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Drive, Madison, Wis.)); or the Best Fit algorithm described in Devereux et al., Nucl. Acid Res., 12:387-395 (1984). Methods for calculating percent identity include, but are not limited to, the Fit sequence program, or by inspection. Preferably, percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecules Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc., all of which are incorporated by reference.

[0168] One useful algorithm is PILEUP, which creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also generate a tree plot showing the clustering relationships used to create the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987), which is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989), both of which are incorporated by reference. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0169] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993), all of which are incorporated by reference. A particularly useful BLAST program is the WU-BLAST-2 program, available from Altschul et al., Methods in Enzymology, 266:460-480 (1996); http: / / blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values ​​determined by the program itself depending on the composition of the particular sequence and the particular database in which the sequence of interest is searched, but each value may be adjusted to increase sensitivity.

[0170] Another useful algorithm is gapped BLAST, reported by Altschul et al., Nucl. Acids Res., 25:3389-3402, which is incorporated by reference. Gapped BLAST uses BLOSUM-62 substitution scores, the threshold T parameter is set to 9, ungapped extensions are generated using the two-hit method, the gap length k is multiplied by the cost 10 + k, Xu is set to 16, and Xg is set to 40 in the database search phase and 67 in the output phase of the algorithm. Gapped alignments are generated with scores equivalent to approximately 22 bits.

[0171] The percent amino acid sequence identity is calculated by dividing the number of matching identical residues by the total number of residues in the "longer" sequence in the aligned region. The "longer" sequence is the sequence with the most actual residues in the aligned region (ignoring gaps introduced by WU-Blast-2 to maximize the alignment score). In a similar manner, "percent nucleic acid sequence identity" with respect to the coding sequence of an identified polypeptide is defined as the percentage of nucleotide residues in the candidate sequence that are identical to those in the coding sequence of the cell cycle protein. A preferred method utilizes the BLASTN module of WU-BLAST-2, set to default parameters with an overlap span and overlap fraction of 1 and 0.125, respectively.

[0172] Alignment may include the introduction of gaps into the sequences being aligned. Furthermore, for sequences containing more or fewer amino acids than the protein encoded by the sequence of Figure 2 (SEQ ID NO: 1), it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids or nucleotides relative to the total number of amino acids or nucleotides. Thus, for example, as discussed below, the sequence identity of sequences shorter than the sequence shown in Figure 2 (SEQ ID NO: 1) will in one embodiment be determined using the number of nucleotides in such shorter sequence. The percent identity calculation does not assign relative weight to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.

[0173] In one embodiment, only identities are given a positive score (+1), and all forms of sequence variation, including gaps, are assigned a value of "0," thereby eliminating the need for weighting scales or parameters, as described below, in sequence similarity calculations. Percent sequence identity may be calculated, for example, by dividing the number of matching identical residues by the total number of residues in the "shorter" sequence in the aligned region, and multiplying by 100. The "longer" sequence is the sequence with the most actual residues in the aligned region.

[0174] The term "allelic variant" refers to polymorphic forms of a gene at a particular locus, as well as to the cDNAs derived from mRNA transcripts of the gene and the polypeptides encoded thereby. The term "preferred mammalian codons" refers to a subset of codons within the set of codons encoding certain amino acids that are most frequently used in proteins expressed in mammalian cells, selected from the following list: Gly (GGC, GGG); Glu (GAG); Asp (GAC); Val (GTG, GTC); Ala (GCC, GCT); Ser (AGC, TCC); Lys (AAG); Asn (AAC); Met (ATG); Ile (ATC); Thr (ACC); Trp (TGG); Cys (TGC); Tyr (TAT, TAC); Leu (CTG); Phe (TTC); Arg (CGC, AGG, AGA); Gln (CAG); His (CAC); and Pro (CCC).

[0175] As used herein, the term "codon modified" refers to a polynucleotide sequence encoding a polypeptide (e.g., a Factor VIII mutant protein) in which at least one codon of a wild-type polynucleotide encoding such polypeptide has been altered to improve a property of the polynucleotide sequence. In some embodiments, the improved property promotes enhanced transcription of the mRNA encoding the polypeptide, increased stability of the mRNA (e.g., improved mRNA half-life), enhanced translation of the polypeptide, and / or increased packaging of the polynucleotide in a vector. Non-limiting examples of modifications that can be used to achieve improved properties include altering codon usage and / or distribution for particular amino acids, adjusting global and / or local GC content, removing AT-rich sequences, removing repetitive sequence elements, adjusting global and / or local CpG dinucleotide content, removing potential regulatory elements (e.g., TATA box and CCAAT box elements), removing intron / exon splice sites, improving regulatory sequences (e.g., introducing Kozak consensus sequences), and removing sequence elements that can form secondary structures (e.g., stem-loops) within the transcribed mRNA.

[0176] In the discussion herein, various nomenclatures refer to components of the present disclosure. "CS-number" (e.g., "CS04," "CS01," "CS23," etc.) refers to a codon-modified polynucleotide encoding a FVIII polypeptide and / or the encoded polypeptide, including variants thereof. For example, CS01-FL refers to the full-length codon-modified CS01 polynucleotide sequence or amino acid sequence encoded by the CS01 polynucleotide sequence (sometimes referred to herein as "CS01-FL-AA" for the amino acid sequence and "CS01-FL-NA" for the nucleic acid sequence). Similarly, "CS01-LC" refers to the codon-modified nucleic acid sequence encoding the light chain of a FVIII polypeptide ("CS01-LC-NA"), or the amino acid sequence of the FVIII light chain encoded by the CS01 polynucleotide sequence (sometimes referred to herein as "CS01-LC-AA"). Similarly, CS01-HC, CS01-HC-AA, and CS01-HC-NA are identical for the FVIII heavy chain. As will be appreciated by those skilled in the art, constructs such as CS01, CS04, CS23, etc., in which only codons have been modified (e.g., they contain no additional amino acid substitutions when compared to Refacto) will result in identical amino acid sequences, as the amino acid sequence has not been modified by codon optimization. Thus, disclosed sequence constructs include, but are not limited to, CS01-FL-NA, CS01-FL-AA, CS01-LC-NA, CS01-LC-AA, CS01-HC-AA, CS01-HC-NA, CS04-FL-NA, CS04-FL-AA, CS04-LC-NA, CS04-LC-AA, CS04-HC-AA, CS04-HC-NA, CS23-FL-NA, CS23-FL-AA, CS23-LC-NA, CS23-LC-AA, CS23-HC-AA, and CS23-HC-NA.

[0177] This nomenclature also applies to the glycosylated peptides shown in Figure 13, whereby "NGA1-AA" refers to the amino acid sequence and NGA1-NA refers to the nucleic acid sequence.

[0178] The present disclosure also includes additional appropriately named novel Factor VIII variants, described below.

[0179] III. Codon-modified Factor VIII variants In some embodiments, the present disclosure provides codon-modified polynucleotides encoding factor VIII variants. These codon-modified polynucleotides significantly improve factor VIII expression when administered in AAV-based gene therapy constructs. The codon-modified polynucleotides also exhibit improved AAV-virion packaging compared to conventional codon-optimized constructs. As demonstrated in Examples 2 and 4, Applicants achieve these advantages through the discovery of three codon-modified polynucleotides (CS01-FL-NA, CS04-FL-NA, and CS23-FL-NA) encoding factor VIII polypeptides containing human wild-type factor VIII heavy and light chains and a short 14-amino acid B-domain replacement linker ("SQ" linker) containing a furin cleavage site that promotes maturation of active FVIIIa protein in vivo. As further demonstrated in Example 4, incorporation of various combinations of the F328S, X5, and X1 amino acid mutations into the encoded factor VIII molecule further enhanced in vivo expression of factor VIII activity.

[0180] In one embodiment, the codon-modified polynucleotides provided herein have a nucleotide sequence that is highly identical to at least the sequences in CS01, CS04, or CS23 (SEQ ID NOS: 13, 1, and 20, respectively) encoding the factor VIII heavy chain and the factor VIII light chain. As is known in the art, the B domain of factor VIII is not required for activity in vivo. Thus, in some embodiments, the codon-modified polynucleotides provided herein completely lack the factor VIII B domain. In some embodiments, the native factor VIII B domain is replaced with a short amino acid linker containing a furin cleavage site, e.g., an "SQ" linker consisting of amino acids 760-773 of the construct CS01, CS04, or CS23 (SEQ ID NOS: 2, 2, and 21, respectively). The "SQ" linker is also referred to as BDLO04 (in Figure 6, the amino acid sequence is -AA and the nucleotide sequence is -NA).

[0181] In one embodiment, the factor VIII heavy and light chains encoded by the codon-modified polynucleotide are human factor VIII heavy and light chains, respectively. In other embodiments, the factor VIII heavy and light chains encoded by the codon-modified polynucleotide are heavy and light chain sequences derived from another mammal (e.g., porcine factor VIII). In yet another embodiment, the factor VIII heavy and light chains are chimeric heavy and light chains (e.g., combined sequences of a human and a second mammal). In yet another embodiment, the factor VIII heavy and light chains are humanized versions of the heavy and light chains of another mammal, e.g., heavy and light chain sequences derived from another mammal in which human residues have been substituted at selected positions to reduce immunogenicity of the resulting peptide when administered to humans.

[0182] The GC content of human genes varies widely, ranging from less than 25% to more than 90%. However, human genes with a high GC content are generally expressed at higher levels. For example, Kudla et al. (PLoS Biol., 4(6):80(2006)) demonstrated that increasing the GC content of a gene increases the expression of the encoded polypeptide, primarily by increasing transcription and increasing the steady-state mRNA transcript abundance. Generally, the desired GC content of a codon-optimized gene construct is 60% or higher. However, the wild-type AAV genome has a GC content of approximately 56%.

[0183] Thus, in some embodiments, the codon-modified polynucleotides provided herein have a CG content that is more consistent with the GC content of wild-type AAV virions (e.g., about 56% GC), which is lower than the preferred CG content of polynucleotides that have been conventionally codon-optimized for expression in mammalian cells (e.g., 60% GC or higher). As outlined in Example 1, CS04-FL-NA (SEQ ID NO: 1), which has a GC content of about 56%, exhibits improved virion packaging compared to a similarly codon-modified coding sequence with a high GC content.

[0184] Thus, in some embodiments, the overall GC content of a codon-modified polynucleotide encoding a Factor VIII polypeptide is less than 60%. In some embodiments, the overall GC content of a codon-modified polynucleotide encoding a Factor VIII polypeptide is less than 59%. In some embodiments, the overall GC content of a codon-modified polynucleotide encoding a Factor VIII polypeptide is less than 58%. In some embodiments, the overall GC content of a codon-modified polynucleotide encoding a Factor VIII polypeptide is less than 57%. In some embodiments, the overall GC content of a codon-modified polynucleotide encoding a Factor VIII polypeptide is 56% or less.

[0185] In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 54% and 59%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 55% and 59%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 56% and 59%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 54% and 58%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 55% and 58%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 56% and 58%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 54% and 57%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 55% and 57%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 56% and 57%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 54% and 56%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is between 55% and 56%.

[0186] In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is 56±0.5%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is 56±0.4%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is 56±0.3%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is 56±0.2%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is 56±0.1%. In some embodiments, the overall GC content of the codon-modified polynucleotide encoding a Factor VIII polypeptide is 56%.

[0187] A. Factor VIII Amino Acid Substitutions To further enhance the efficiency of AAV vector expression of the Factor VIII constructs described herein, some embodiments further incorporate amino acid substitutions known to improve secretion, increase specific activity, and / or enhance Factor VIII stability. A number of potential variants have been identified that increase plasma levels of FVIII activity at a given vector dose. These variants include those with increased signal peptide efficiency, amino acid substitutions that prevent BiP interaction, amino acid substitutions that resemble more efficiently secreted Factor VIII orthologs (e.g., porcine Factor VIII), single-chain Factor VIII variants, and variants with amino acid substitutions that stabilize Factor VIII and / or reduce subunit dissociation.

[0188] Mutations of residues A108, R121, and L2302 (SPE), located at the interface between the A1 and C2 domains, enhance the stability of factor VIII. For example, the amino acid substitution A108I introduces a hydrophobic residue that better fills the interdomain space and stabilizes the interaction. Similarly, the double amino acid substitution R121C / L2302C (SPE) introduces a disulfide bond that extends from the A1 to C2 domains and further stabilizes the interaction. Collectively, all three amino acid substitutions increase the thermal stability of factor VIII by 3-4 fold. For reviews, see Wakabayashi et al., J Biol Chem. 286(29):25748-55 (2011) and Wakabayashi et al., Thromb Haemost. 10(3):492-95 (2012). Thus, in some embodiments, the encoded Factor VIII polypeptide includes the amino acid substitutions A108I and / or R121C / L2302C.

[0189] Mutation of E113 (SPE), located within the calcium-binding domain of factor VIII, enhances specific FVIII clotting activity. For example, E113A appears to enhance the formation of FXase by increasing the affinity of FVIII for factor IXa. Specifically, the amino acid substitution E113A increases specific FVIII clotting activity by two-fold and affinity for factor IXa by four-fold (Biochemistry, 41:8485 (2002); J. Biol. Chem., 279:12677 (2004); and Biochemistry, 44:10298 (2005)). Thus, in some embodiments, the encoded factor VIII polypeptide includes the amino acid substitution E113A.

[0190] Substitution of one or more amino acid residues (residues 331-341 (SPE)) surrounding the factor VIII APC cleavage site inhibits inactivation of factor VIIIa by activated protein C without affecting FVIII activity. For example, the amino acid substitution PQL333-335VDQ (SPE) reduces factor VIII inactivation by 16-fold. Similarly, the amino acid substitution MKN336-339GNQ reduces factor VIII inactivation by 9-fold. Combining two triple amino acid substitutions (e.g., PQLRMKN333-339VDQRGNQ) (SEQ ID NOs: 34 and 35, respectively) reduces factor VIII inactivation by 100-fold (J. Biol. Chem., 282:20264 (2007)). Thus, in some embodiments, the encoded factor VIII polypeptide includes the amino acid substitutions PQL333-335VDQ and / or MKN337-339GNQ (SPE).

[0191] Mutations within the A2 domain interface also enhance the stability of factor VIII. Specifically, mutations of charged residues at the interface between the A1-A2 and A2-A3 domains enhance the stability and retention of the A2 subunit of factor VIIIa. For example, mutations of D519, E665, and E1984 to V or A can increase the stability of factor VIII by up to two-fold and that of factor VIIIa by up to five-fold. Specifically, the amino acid substitution D519A / E665V results in a three-fold increase in stability, the amino acid substitution D519V / E665V results in a two-fold increase in stability, an eight-fold decrease in A2 dissociation, and a two- to four-fold increase in thrombin generation capacity, the amino acid substitution D519V / E1984A results in a two-fold increase in stability, and the amino acid substitution D519V / E665V / E1984A results in a two-fold increase in stability (Blood 112:2761-69 (2008); J. Thromb. Haemost., 7:438-44 (2009)). Thus, in some embodiments, the encoded factor VIII polypeptide includes one or more of the amino acid substitutions D519A / V, E665A / V, and E1984A / V.

[0192] Of particular importance to this disclosure is the large number of specific mutations that can be included individually or in combination with other mutations described herein. These mutations are designated herein as codes, i.e., "m1" refers to a single amino acid change, "m2" is a set of five amino acid mutations, "m3" is a combination of a seven amino acid deletion and a six amino acid insertion extending to the junction between the polypeptide linker and the heavy chain, "m4" is a combination of an m1 single mutation and an m5 double mutation, and "m5" is a set of two cysteine ​​deletions. These mutations are described below. These mutations, alone or in combination with other mutations, can be included in any individual construct and are designated accordingly. For example, "m23" is a combination of the m2 and m3 variants for a particular scaffold as outlined herein, and accordingly, "CS01m23-FL-NA" or "CS01-FL-NAm23" refers to a codon-modified polynucleotide CS01 sequence containing nucleotides encoding the m2 and m3 mutations, and "CS01m23-FL-AA" or "CS01-FL-AAm23" refers to such an amino acid sequence. Because CS01 is codon-modified but does not alter the Refacto amino acid sequence, the above are considered mutations at the amino acid level when compared to the Refacto amino acid sequence CS01-FL-AA (SEQ ID NO: 2).

[0193] In many embodiments, the disclosed polypeptides are made with an "m1" mutation. Mutations within the 11-amino acid hydrophobic β-sheet of the A1 domain, which interacts with BiP, increase factor VIII secretion. For example, the amino acid substitution F328S (in the case of SPI; F309S in the case of SPE) within such a pocket increases factor VIII secretion by three-fold. The F328S mutation is referred to herein as an "m1" mutation and is located within the heavy chain. Again, as described herein, variants may be numbered as "Signal Peptide Inclusive" or "SPI," which includes the signal peptide, or "Signal Peptide Exclusive" or "SPE," which starts from the final processed protein sequence. Thus, the mutation F328S using SPI numbering is identical to the mutation F309 with SPE. Generally, SPI numbering is used herein, but as will be understood by those skilled in the art, either numbering system results in the same mutation(s).

[0194] Thus, the present disclosure includes polypeptides containing m1 mutations such as CS01-FL-AAm1, CS01-HC-AAm1, CS04-FL-AAm1, CS04-HC-AAm1 CS23-FL-AAm1, CS23-HC-AAm1, CS40-FL-AAm1 and CS40-HC-AAm1 (all of which encode the same corresponding protein sequence).

[0195] Additionally, the present disclosure includes not only polypeptide sequences containing the m1 mutation, but also codon-modified polynucleotide sequences encoding proteins having the m1 mutation, such as CS01-FL-NAm1, CS01-HC-NAm1, CS04-FL-NAm1, CS04-HC-NAm1, CS23-FL-NAm1, CS23-HC-NA-m1, CS40-FL-NAm1, and CS40-HC-NAm1.

[0196] In many embodiments, the disclosed polypeptides are engineered to contain the "m2" mutation set, i.e., the mutations I105V / A127S / G151K / M166T / L171P (SPI numbering; V86I / S108A / K132G / T147M / P152L in SPE numbering, respectively). The m2 mutation set is based on the fact that substitution of porcine amino acids 82-176 for the corresponding human amino acids in a B-domain deleted gene therapy construct enhances Factor VIII activity when expressed in HEK293 cells (W. Xiao, ibid.). Backmutation of single porcine amino acids into a human BDD-FVIII construct identified five amino acids in the A1 domain that contribute to this phenomenon: I105V, A127S, G151K, M166T, and L171P (SPI). Combinations of these mutations When introduced into a human construct, the improved activity of the larger porcine substitutions was reproduced. (Ibid.). Thus, in some embodiments, the encoded Factor VIII polypeptide contains one or more amino acid substitutions selected from I105V, A127S, G151K, M166T, and L171P, with the full set of five amino acids, m2, finding particular application in many embodiments. As with the m1 mutations, the m2 variants are in the heavy chain, and therefore the present disclosure includes polypeptides containing m2 mutations such as CS01-FL-AAm2, CS01-HC-AAm2, CS04-FL-AAm2, CS04-HC-AAm2, CS23-FL-AAm2, CS23-HC-AAm2, CS40-FL-AAm2, and CS40-HC-AAm2, all of which encode the same corresponding protein sequence.

[0197] Additionally, the present disclosure includes not only polypeptide sequences containing the m2 mutation, but also codon-modified polynucleotide sequences encoding proteins having the m2 mutation, such as CS01-FL-NAm2, CS01-HC-NAm2, CS04-FL-NAm2, CS04-HC-NAm2, CS23-FL-NAm2, CS23-HC-NA-m2, CS40-FL-NAm2, and CS40-HC-NAm2.

[0198] In further embodiments, the polypeptides and polynucleotides of the present disclosure include the m3 mutation. m3 is a substitution of six amino acids spanning the HC-to-B domain interface for seven amino acids, thereby introducing an additional glycosylation site near the interface. Thus, in some embodiments, m3 is a deletion of amino acids AIEPRSF755-761 and an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 (e.g., AIEPRSF755-761TTYVNRSL) relative to FVIII-FL-AA (SEQ ID NO: 19) ("TTYVNRSL" is disclosed as SEQ ID NO: 33). Residues AIEPR755-759 relative to SEQ ID NO: 19 are within the heavy chain terminus, and residues S760 and F761 are within the B domain. In some embodiments, when the B domain of FVIII is deleted, truncated, or substituted, residues S760 and F761 may not be present in the underlying amino acid sequence being mutated. Thus, in some embodiments, m3 is a deletion of amino acids AIEPR755-759 and an insertion of amino acids TTYVNRSL (SEQ ID NO: 33) after N754 relative to FVIII-FL-AA (SEQ ID NO: 19) (e.g., AIEPR755-759TTYVNRSL ("TTYVNRSL" is disclosed as SEQ ID NO: 33).

[0199] Because the m3 variants are at the junction between the heavy chain and B domains, the present disclosure includes polypeptides containing m3 variants such as CS01-FL-AAm3, CS01-HC-AAm3, CS04-FL-AAm3, CS04-HC-AAm3, CS23-FL-AAm3, CS23-HC-AAm3, CS40-FL-AAm3 and CS40-HC-AAm3 (all of which encode the same corresponding protein sequence).

[0200] Additionally, the present disclosure includes not only polypeptide sequences containing the m3 mutation, but also codon-modified polynucleotide sequences encoding proteins having the m3 mutation, such as CS01-FL-NAm3, CS01-HC-NAm3, CS04-FL-NAm3, CS04-HC-NAm3, CS23-FL-NAm3, CS23-HC-NA-m3, CS40-FL-NAm3, and CS40-HC-NAm3.

[0201] In further embodiments, the polypeptides and polynucleotides of the present disclosure include the m4 mutation. Removal of the C1899-C1903 disulfide bond of Factor VIII also increased secretion. Furthermore, increased Factor VIII secretion is additive when F328S (in SPI, F309S in SPE) is combined with the amino acid substitutions C1918G / C1922G (Miao et al., Blood, 103:3412-19 (2004); Selvaraj et al., J. Thromb. Haemost., 10:107-15 (2012)). Thus, in some embodiments, the encoded Factor VIII polypeptide includes the m4 mutation, F328S (in SPI, F309S in SPE) and the amino acid substitutions C1918G / C1922G (SPI). The F328S mutation is in the heavy chain and the two cysteine ​​mutations are in the light chain, so the polypeptide sequences containing the m4 mutations are CS01-FL-AAm4, CS01-HC-AAm4, CS01-LC-AAm4, CS04-FL-AAm4, CS04-HC-AAm4, CS04-LC-AAm4, CS23-FL-AAm4, CS23-HC-AAm4 and CS23-LC-AAm4.

[0202] Additionally, the present disclosure includes not only polypeptide sequences containing the m4 mutation, but also codon-modified polynucleotide sequences encoding proteins having the m4 mutation, such as CS01-FL-NAm4, CS01-HC-NAm4, CS01-LC-NAm4, CS04-FL-NAm4, CS04-HC-NAm4, CS04-LC-NAm4, CS23-FL-NAm4, CS23-HC-NAm4, CS23-LC-NAm4, CS40-FL-NA-m4, CS40-HC-NA-m4, and CS40-LC-NA-m4.

[0203] In further embodiments, the polypeptides and polynucleotides of the present disclosure comprise the m5 mutation. As noted above, removal of the C1899-C1903 disulfide bond in Factor VIII also increased secretion. The amino acid substitution C1918G / C1922G (SPI) contained within the light chain is referred to herein as the m5 mutation set.

[0204] Because the m5 variants are in the light chain, the present disclosure includes polypeptides containing m5 variants such as CS01-FL-AAm5, CS01-LC-AAm5, CS04-FL-AAm5, CS04-LC-AAm5, CS23-FL-AAm5, CS23-LC-AAm5, CS40-FL-AAm5 and CS40-LC-AAm5 (all of which encode the same corresponding protein sequence).

[0205] Additionally, the present disclosure includes not only polypeptide sequences containing the m5 mutation, but also codon-modified polynucleotide sequences encoding proteins having the m5 mutation, such as CS01-FL-NAm5, CS01-LC-NAm5, CS04-FL-NAm5, CS04-LC-NAm5, CS23-FL-NA-m5, CS23-LC-NA-m5, CS40-FL-NA-m5, and CS40-LC-NA-m5.

[0206] In addition to specific constructs (both amino acid and nucleic acid) containing m1, m2, m3, m4, and m5 individually, combinations of mutation sets can be made as outlined herein. As described herein, these are designated "m12" for the combination of m1 and m2 set, or "m123" for the combination of m1, m2, and m3 set. Accordingly, the present disclosure includes double combinations such as m12, m13, m14, m15, m23, m24, m25, m34, m35, and m45. Also included are triple combinations m123, m124, m125, m234, m235, and m345. Furthermore, the quadruple combinations m1234, m1235, m1345, and m12345 are included. Of particular interest in some embodiments are the mutation sets m1, m2, m3, and m4, m23, m123, and m234.

[0207] B. Factor VIII B Domain Replacement Linker In some embodiments, the linkage between the heavy and light chains of FVIII (e.g., the B domain of wild-type factor VIII) is further modified. Because of the limited size of AAV packaging, mutants with deleted, truncated, and / or substituted B domains are required to improve the efficiency of FVIII gene therapy constructs. The most commonly used B domain replacement linker to date is the SQ FVIII linker, which retains only 14 amino acids of the B domain as a linker sequence. Another variant of porcine factor VIII ("OBI-1," described in U.S. Pat. No. 6,458,563) expresses well in CHO cells and has a slightly longer linker of 24 amino acids. In some embodiments, factor VIII constructs encoded by the codon-modified polynucleotides described herein comprise an SQ-type B domain linker sequence. In other embodiments, factor VIII constructs encoded by the codon-modified polynucleotides described herein comprise an OBI-1-type B domain linker sequence.

[0208] In some embodiments, the encoded factor VIII polypeptides described herein include an SQ-type B-domain linker comprising amino acids 760-762 / 1657-1667 of the wild-type human factor VIII B-domain (FVIII-FL-AA; SEQ ID NO: 19) (Sandberg et al. Thromb. Haemost. 85:93 (2001)). In some embodiments, the SQ-type B-domain linker has one amino acid substitution compared to the corresponding wild-type sequence. In some embodiments, the SQ-type B-domain linker has two amino acid substitutions compared to the corresponding wild-type sequence. In some embodiments, a glycosylated peptide is inserted into the SQ-type B-domain linker. In some embodiments, the glycosylated peptide is selected from the peptides shown in FIG. 13 (SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance).

[0209] In some embodiments, the encoded factor VIII polypeptide described herein includes a Greengene-type B-domain linker (Oh et al., Biotechnol. Prog., 17:1999 (2001)) comprising amino acids 760 / 1582-1667 of the wild-type human factor VIII B-domain (FVIII-FL-AA; SEQ ID NO: 19). In some embodiments, the Greengene-type B-domain linker has one amino acid substitution compared to the corresponding wild-type sequence. In some embodiments, the Greengene-type B-domain linker has two amino acid substitutions compared to the corresponding wild-type sequence. In some embodiments, a glycosylated peptide is inserted into the Greengene-type B-domain linker. In some embodiments, the glycosylated peptide is selected from the peptides shown in FIG. 13 (SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance).

[0210] In some embodiments, the encoded factor VIII polypeptides described herein include an extended SQ B-domain linker comprising amino acids 760-769 / 1657-1667 of the wild-type human factor VIII B-domain (FVIII-FL-AA; SEQ ID NO: 19). TIFF0007631425000003.tif4128 is included (Thim et al., Haemophilia, 16:349 (2010)). In some embodiments, the extended SQ-type B-domain linker has one amino acid substitution compared to the corresponding wild-type sequence. In some embodiments, the extended SQ-type B-domain linker has two amino acid substitutions compared to the corresponding wild-type sequence. In some embodiments, a glycosylated peptide is inserted into the extended SQ-type B-domain linker. In some embodiments, the glycosylated peptide is selected from the peptides shown in FIG. 13 (SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance).

[0211] In some embodiments, the encoded Factor VIII polypeptides described herein contain the amino acid sequence from the wild-type porcine Factor VIII B domain. porcine OBI-1-type B domain linkers (Toschi et al., Curr. Opin. Mol. Ther. 12:517 (2010)), including TIFF0007631425000004.tif4128. In some embodiments, the porcine OBI-1-type B domain linker has one amino acid substitution compared to the corresponding wild-type sequence. In some embodiments, the porcine OBI-1-type B domain linker has two amino acid substitutions compared to the corresponding wild-type sequence. In some embodiments, a glycosylated peptide is inserted into the porcine OBI-1-type B domain linker. In some embodiments, the glycosylated peptide is selected from the peptides shown in FIG. 13 (SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance).

[0212] In some embodiments, the encoded Factor VIII polypeptide described herein includes a human OBI-1-type B domain linker comprising amino acids 760-772 / 1655-1667 of the wild-type human Factor VIII B domain (FVIII-FL-AA; SEQ ID NO: 19). In some embodiments, the human OBI-1-type B domain linker has one amino acid substitution compared to the corresponding wild-type sequence. In some embodiments, the human OBI-1-type B domain linker has two amino acid substitutions compared to the corresponding wild-type sequence. In some embodiments, a glycosylated peptide is inserted into the human OBI-1-type B domain linker. In some embodiments, the glycosylated peptide is selected from the peptides shown in FIG. 13 (SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance).

[0213] In some embodiments, the encoded Factor VIII polypeptides described herein contain the amino acid sequence from the wild-type porcine Factor VIII B domain. TIFF0007631425000005.tif5128, including O8-type B domain linkers (Toschi et al., Curr. Opin. Mol. Ther. 12:517 (2010)). In some embodiments, the porcine OBI-1-type B domain linker has one amino acid substitution compared to the corresponding wild-type sequence. In some embodiments, the porcine OBI-1-type B domain linker has two amino acid substitutions compared to the corresponding wild-type sequence. In some embodiments, a glycosylated peptide is inserted into the porcine OBI-1-type B domain linker. In some embodiments, the glycosylated peptide is selected from the peptides shown in FIG. 13 (SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance).

[0214] Removal of the B domain from factor VIII constructs does not appear to affect the activity of activating enzymes (e.g., FVIIIa), likely because the B domain is removed during activation. However, the B domain of factor VIII contains several residues that are subject to post-translational modification, for example, by N-linked or O-linked glycosylation. In silico analysis of wild-type factor VIII B domain (Prediction of N-glycosylation sites in human proteins, R. Gupta, E. Jung, and S. Brunak, in preparation (2004)) predicts that at least four of these sites are glycosylated in vivo (Figure 14). These modifications within the B domain are thought to contribute to post-translational regulation and / or half-life of factor VIII in vivo.

[0215] Although the mature factor VIIIa protein does not have a factor VIII B domain, glycosylation within the B domain of the precursor factor VIII molecule can extend the circulating half-life of the protein prior to activation. Thus, in some embodiments, the polypeptide linker of the encoded factor VIII constructs described herein includes one or more glycosylation sequences to allow for in vivo glycosylation. In some embodiments, the polypeptide linker includes at least one glycosylation consensus sequence (e.g., an N-linked or O-linked glycosylation consensus sequence). In some embodiments, the polypeptide linker includes at least two glycosylation consensus sequences. In some embodiments, the polypeptide linker includes at least three glycosylation consensus sequences. In some embodiments, the polypeptide linker includes at least four glycosylation consensus sequences. In some embodiments, the polypeptide linker includes at least five glycosylation consensus sequences. In some embodiments, the polypeptide linker includes at least 6, 7, 8, 9, 10, or more glycosylation consensus sequences.

[0216] In some embodiments, the polypeptide linker contains at least one N-linked glycosylation sequence NXS / T, where X is any amino acid other than P, S, or T. In some embodiments, the polypeptide linker contains at least two N-linked glycosylation sequences NXS / T, where X is any amino acid other than P, S, or T. In some embodiments, the polypeptide linker contains at least three N-linked glycosylation sequences NXS / T, where X is any amino acid other than P, S, or T. In some embodiments, the polypeptide linker contains at least four N-linked glycosylation sequences NXS / T, where X is any amino acid other than P, S, or T. In some embodiments, the polypeptide linker contains at least five N-linked glycosylation sequences NXS / T, where X is any amino acid other than P, S, or T. In some embodiments, the polypeptide linker contains at least 6, 7, 8, 9, 10, or more N-linked glycosylation sequences NXS / T, where X is any amino acid other than P, S, or T.

[0217] In some embodiments, the polypeptide linker includes a glycosylated peptide having high sequence identity to any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, in order of appearance, respectively, as shown in Figures 13A-13B. In some embodiments, the glycosylated polypeptide has at least 92% identity to any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, in order of appearance, respectively, as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide has no more than two amino acid substitutions to any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, in order of appearance, respectively, as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide has no more than one amino acid substitution with respect to any of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide has an amino acid sequence selected from any of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B.

[0218] In some embodiments, the glycosylated peptide is encoded by a polynucleotide sequence having at least 92% identity to any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B, and at least 90% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide is encoded by a polynucleotide sequence having at least 92% identity to any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B, and at least 95% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide is encoded by a polynucleotide sequence having at least 92% identity to any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B, and at least 98% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance as shown in Figures 13A-13B.

[0219] In some embodiments, the glycosylated peptide has no more than two amino acid substitutions with any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B, and is encoded by a polynucleotide sequence having at least 90% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide has no more than two amino acid substitutions with any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B, and is encoded by a polynucleotide sequence having at least 95% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide has no more than two amino acid substitutions with any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B, and is encoded by a polynucleotide sequence having at least 98% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance as shown in Figures 13A-13B.

[0220] In some embodiments, the glycosylated peptide has no more than one amino acid substitution with any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B, and is encoded by a polynucleotide sequence having at least 90% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide has no more than one amino acid substitution with any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B, and is encoded by a polynucleotide sequence having at least 95% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide has no more than one amino acid substitution with any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B, and is encoded by a polynucleotide sequence having at least 98% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance as shown in Figures 13A-13B.

[0221] In some embodiments, the glycosylated peptide has a sequence selected from any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, in order of appearance, respectively, as shown in Figures 13A-13B, and is encoded by a polynucleotide sequence having at least 90% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, in order of appearance, respectively, as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide has a sequence selected from any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, in order of appearance, respectively, as shown in Figures 13A-13B, and is encoded by a polynucleotide sequence having at least 95% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, in order of appearance, respectively, as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide has a sequence selected from any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, in order of appearance, respectively, as shown in Figures 13A-13B, and is encoded by a polynucleotide sequence having at least 98% identity to a corresponding nucleotide sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, in order of appearance, respectively, as shown in Figures 13A-13B.

[0222] In some embodiments, the Factor VIII polypeptides encoded by the codon-modified polynucleotides described herein have a B-domain replacement linker, wherein the glycosylated peptide is inserted into the SQ linker sequence (CS04-FL-AA; amino acids 760-773 of SEQ ID NO: 2). In certain embodiments, the glycosylated peptide is selected from any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, in order of appearance, respectively, as shown in Figures 13A-13B; a glycosylated peptide having at least 92% identity to any one of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, in order of appearance, respectively, as shown in Figures 13A-13B; 13A-13B, respectively, and glycosylated peptides having no more than one amino acid substitution with respect to any one of SEQ ID NOS: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, respectively, in order of appearance as shown in Figures 13A-13B. In some embodiments, the glycosylated peptide is inserted into the SQ peptide between residues N768 and P769 (CS04-FL-AA; for SEQ ID NO: 2).

[0223] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence that has high sequence identity to any one of the sequences shown in Figure 6 (SEQ ID NOS: 5-7 and 36-48, respectively, in order of appearance). In some embodiments, the third nucleotide sequence has at least 95% identity to any one of the sequences shown in Figure 13 (SEQ ID NOS: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance). In some embodiments, the third nucleotide sequence has at least 96% identity to any one of the sequences shown in Figure 13 (SEQ ID NOS: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance). In some embodiments, the third nucleotide sequence has at least 97% identity to any one of the sequences shown in Figure 13 (SEQ ID NOS: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance). In some embodiments, the third nucleotide sequence has at least 98% identity to any one of the sequences shown in Figure 13 (SEQ ID NOS: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance). In some embodiments, the third nucleotide sequence has at least 99% identity to any one of the sequences shown in Figure 13 (SEQ ID NOS: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance). In some embodiments, the third nucleotide sequence has at least 99.5% identity to any one of the sequences shown in Figure 13 (SEQ ID NOS: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance). In some embodiments, the third nucleotide sequence has at least 99.9% identity to any one of the sequences shown in Figure 13 (SEQ ID NOS: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance).In some embodiments, the third nucleotide sequence is identical to any one of the sequences shown in Figure 13 (SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, respectively, in order of appearance).

[0224] C. Codon-modified polynucleotides encoding factor VIII variants with cleavable linkers Codon-modified polynucleotide CS04 In one embodiment, the codon-modified polynucleotide provided herein includes a nucleotide sequence encoding a factor VIII variant polypeptide having an in vivo cleavable linker. The factor VIII polypeptide includes a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence that is part of CS04-FL-NA (SEQ ID NO: 1) and has high sequence identity to CS04-HC-NA (SEQ ID NO: 3), which encodes the factor VIII heavy chain. The light chain of the factor VIII polypeptide is encoded by a second nucleotide sequence that is part of CS04-FL-NA (SEQ ID NO: 1) and has high sequence identity to CS04-LC-NA (SEQ ID NO: 4), which encodes the factor VIII light chain. The polypeptide linker includes a furin cleavage site that allows for in vivo maturation (e.g., after in vivo expression or administration of a precursor polypeptide).

[0225] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively.

[0226] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to BDLO04 (SEQ ID NO:6), which encodes a 14 amino acid linker corresponding to amino acids 760-773 of CS04-FL-AA (SEQ ID NO:2). In some embodiments, the third nucleotide sequence has at least 95% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence has at least 96% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence has at least 97% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence has at least 98% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence is identical to BDLO04 (SEQ ID NO:6).

[0227] In some embodiments, the codon-modified polynucleotide has a nucleotide sequence with high sequence identity to CS04-FL-NA (SEQ ID NO:1). In some embodiments, the nucleotide sequence is at least 95% identical to CS04-FL-NA (SEQ ID NO:1). In some embodiments, the nucleotide sequence is at least 96% identical to CS04-FL-NA (SEQ ID NO:1). In some embodiments, the nucleotide sequence is at least 97% identical to CS04-FL-NA (SEQ ID NO:1). In some embodiments, the nucleotide sequence is at least 98% identical to CS04-FL-NA (SEQ ID NO:1). In some embodiments, the nucleotide sequence is at least 99% identical to CS04-FL-NA (SEQ ID NO:1). In some embodiments, the nucleotide sequence is at least 99.5% identical to CS04-FL-NA (SEQ ID NO:1). In some embodiments, the nucleotide sequence is at least 99.9% identical to CS04-FL-NA (SEQ ID NO:1). In some embodiments, the nucleotide sequence is identical to CS04-FL-NA (SEQ ID NO: 1).

[0228] In some embodiments, the factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence with high sequence identity to CS04-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is at least 97% identical to CS04-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is at least 98% identical to CS04-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is at least 99% identical to CS04-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is at least 99.5% identical to CS04-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is at least 99.9% identical to CS04-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is identical to CS04-FL-AA (SEQ ID NO:2).

[0229] In some embodiments, the factor VIII variants encoded by the CS04 polynucleotides having high sequence homology (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity) to CS04-FL-AA contain one or more amino acid substitutions selected from m1, m2, m3, m4, and m5.

[0230] In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m1. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m2. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m3. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m4. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m5.

[0231] In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m12. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m13. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m23. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m24. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m25. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m34. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m35.

[0232] In one embodiment, the Factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m123. In one embodiment, the Factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m234. In one embodiment, the Factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m125.

[0233] Codon-modified polynucleotide CS01 In one embodiment, the codon-modified polynucleotide provided herein includes a nucleotide sequence encoding a factor VIII variant polypeptide having an in vivo cleavable linker. The factor VIII polypeptide includes a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence that is part of CS01-FL-NA (SEQ ID NO: 13) and has high sequence identity to CS01-HC-NA (SEQ ID NO: 24), which encodes the factor VIII heavy chain. The light chain of the factor VIII polypeptide is encoded by a second nucleotide sequence that is part of CS01-FL-NA (SEQ ID NO: 13) and has high sequence identity to CS01-LC-NA (SEQ ID NO: 25), which encodes the factor VIII light chain. The polypeptide linker includes a furin cleavage site that allows for in vivo maturation (e.g., after in vivo expression or administration of the precursor polypeptide).

[0234] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences are identical to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively.

[0235] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to BDLO04 (SEQ ID NO:6), which encodes a 14 amino acid linker corresponding to amino acids 760-773 of CS01-FL-AA (SEQ ID NO:2). In some embodiments, the third nucleotide sequence has at least 95% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence has at least 96% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence has at least 97% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence has at least 98% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence is identical to BDLO04 (SEQ ID NO:6).

[0236] In some embodiments, the codon-modified polynucleotide has a nucleotide sequence that has high sequence identity to CS01-FL-NA (SEQ ID NO:13). In some embodiments, the nucleotide sequence is at least 95% identical to CS01-FL-NA (SEQ ID NO:13). In some embodiments, the nucleotide sequence is at least 96% identical to CS01-FL-NA (SEQ ID NO:13). In some embodiments, the nucleotide sequence is at least 97% identical to CS01-FL-NA (SEQ ID NO:13). In some embodiments, the nucleotide sequence is at least 98% identical to CS01-FL-NA (SEQ ID NO:13). In some embodiments, the nucleotide sequence is at least 99% identical to CS01-FL-NA (SEQ ID NO:13). In some embodiments, the nucleotide sequence is at least 99.5% identical to CS01-FL-NA (SEQ ID NO:13). In some embodiments, the nucleotide sequence is at least 99.9% identical to CS01-FL-NA (SEQ ID NO:13). In some embodiments, the nucleotide sequence is identical to CS01-FL-NA (SEQ ID NO: 13).

[0237] In some embodiments, the factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence with high sequence identity to CS01-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is at least 97% identical to CS01-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is at least 98% identical to CS01-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is at least 99% identical to CS01-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is at least 99.5% identical to CS01-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is at least 99.9% identical to CS01-FL-AA (SEQ ID NO:2). In some embodiments, the amino acid sequence is identical to CS01-FL-AA (SEQ ID NO:2).

[0238] In some embodiments, the factor VIII variant encoded by the CS01 polynucleotide having high sequence homology (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity) to CS01-FL-AA comprises one or more amino acid substitutions selected from m1, m2, m3, m4, and m5.

[0239] In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m1. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m2. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m3. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m4. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m5.

[0240] In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m12. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m13. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m23. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m24. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m25. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m34. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m35.

[0241] In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m123. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m234. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m125.

[0242] Codon-modified polynucleotide CS23 In one embodiment, the codon-modified polynucleotide provided herein includes a nucleotide sequence encoding a factor VIII variant polypeptide having an in vivo cleavable linker. The factor VIII polypeptide includes a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence that is part of CS23-FL-NA (SEQ ID NO: 20) and has high sequence identity to CS23-HC-NA (SEQ ID NO: 22), which encodes the factor VIII heavy chain. The light chain of the factor VIII polypeptide is encoded by a second nucleotide sequence that is part of CS23-FL-NA (SEQ ID NO: 20) and has high sequence identity to CS23-LC-NA (SEQ ID NO: 23), which encodes the factor VIII light chain. The polypeptide linker includes a furin cleavage site that allows in vivo maturation (e.g., after in vivo expression or administration of a precursor polypeptide).

[0243] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively.

[0244] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to BDLO04 (SEQ ID NO:6), which encodes a 14 amino acid linker corresponding to amino acids 760-773 of CS23-FL-AA (SEQ ID NO:21). In some embodiments, the third nucleotide sequence has at least 95% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence has at least 96% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence has at least 97% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence has at least 98% identity to BDLO04 (SEQ ID NO:6). In some embodiments, the third nucleotide sequence is identical to BDLO04 (SEQ ID NO:6).

[0245] In some embodiments, the codon-modified polynucleotide has a nucleotide sequence that is highly identical to CS23-FL-NA (SEQ ID NO:20). In some embodiments, the nucleotide sequence is at least 95% identical to CS23-FL-NA (SEQ ID NO:20). In some embodiments, the nucleotide sequence is at least 96% identical to CS23-FL-NA (SEQ ID NO:20). In some embodiments, the nucleotide sequence is at least 97% identical to CS23-FL-NA (SEQ ID NO:20). In some embodiments, the nucleotide sequence is at least 98% identical to CS23-FL-NA (SEQ ID NO:20). In some embodiments, the nucleotide sequence is at least 99% identical to CS23-FL-NA (SEQ ID NO:20). In some embodiments, the nucleotide sequence is at least 99.5% identical to CS23-FL-NA (SEQ ID NO:20). In some embodiments, the nucleotide sequence is at least 99.9% identical to CS23-FL-NA (SEQ ID NO:20). In some embodiments, the nucleotide sequence is identical to CS23-FL-NA (SEQ ID NO: 20).

[0246] In some embodiments, the factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence with high sequence identity to CS23-FL-AA (SEQ ID NO:21). In some embodiments, the amino acid sequence is at least 97% identical to CS23-FL-AA (SEQ ID NO:21). In some embodiments, the amino acid sequence is at least 98% identical to CS23-FL-AA (SEQ ID NO:21). In some embodiments, the amino acid sequence is at least 99% identical to CS23-FL-AA (SEQ ID NO:21). In some embodiments, the amino acid sequence is at least 99.5% identical to CS23-FL-AA (SEQ ID NO:21). In some embodiments, the amino acid sequence is at least 99.9% identical to CS23-FL-AA (SEQ ID NO:21). In some embodiments, the amino acid sequence is identical to CS23-FL-AA (SEQ ID NO:21).

[0247] In some embodiments, the factor VIII variant encoded by the CS23 polynucleotide having high sequence homology (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity) to CS23-FL-AA comprises one or more amino acid substitutions selected from m1, m2, m3, m4, and m5.

[0248] In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m1. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m2. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m3. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m4. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m5.

[0249] In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m12. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m13. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m23. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m24. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m25. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m34. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m35.

[0250] In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m123. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m234. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m125.

[0251] D. Codon-modified polynucleotides encoding factor VIII single-chain proteins Factor VIII constructs in which the furin cleavage site located at the C-terminus of the B domain has been removed retain activity as single-chain polypeptides, despite the inability of the factor VIII molecule to undergo normal maturation (Leyte et al. (1991)). Similarly, B-domain-deleted factor VIII constructs with a weak furin site (containing the amino acid substitution R1664H) are more biologically active than corresponding factor VIII constructs with a wild-type furin cleavage site (Siner et al. (2013)). Thus, in some embodiments, the codon-modified polynucleotides provided herein include a nucleotide sequence encoding a factor VIII mutant single-chain polypeptide. The factor VIII single-chain polypeptide includes a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The polypeptide linker does not include a furin cleavage site.

[0252] Codon-modified single-stranded polynucleotide CS04 In one embodiment, the codon-modified polynucleotide provided herein comprises a nucleotide sequence encoding a variant single-chain factor VIII polypeptide. The factor VIII polypeptide comprises a factor VIII light chain, a factor VIII heavy chain, and an optional polypeptide linker linking the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence that is part of CS04-FL-NA (SEQ ID NO: 1) and has high sequence identity to CS04-HC-NA (SEQ ID NO: 3), which encodes the factor VIII heavy chain. The light chain of the factor VIII polypeptide is encoded by a second nucleotide sequence that is part of CS04-FL-NA (SEQ ID NO: 1) and has high sequence identity to CS04-LC-NA (SEQ ID NO: 4), which encodes the factor VIII light chain. The optional polypeptide linker does not include a furin cleavage site.

[0253] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs: 3 and 4), respectively.

[0254] In some embodiments, the codon-modified polynucleotide has a nucleotide sequence with high sequence identity to CS04-SC1-NA (SEQ ID NO:9). In some embodiments, the nucleotide sequence is at least 95% identical to CS04-SC1-NA (SEQ ID NO:9). In some embodiments, the nucleotide sequence is at least 96% identical to CS04-SC1-NA (SEQ ID NO:9). In some embodiments, the nucleotide sequence is at least 97% identical to CS04-SC1-NA (SEQ ID NO:9). In some embodiments, the nucleotide sequence is at least 98% identical to CS04-SC1-NA (SEQ ID NO:9). In some embodiments, the nucleotide sequence is at least 99% identical to CS04-SC1-NA (SEQ ID NO:9). In some embodiments, the nucleotide sequence is at least 99.5% identical to CS04-SC1-NA (SEQ ID NO:9). In some embodiments, the nucleotide sequence is at least 99.9% identical to CS04-SC1-NA (SEQ ID NO:9). In some embodiments, the nucleotide sequence is identical to CS04-SC1-NA (SEQ ID NO: 9).

[0255] In some embodiments, the codon-modified polynucleotide has a nucleotide sequence that is highly identical to CS04-SC2-NA (SEQ ID NO:11). In some embodiments, the nucleotide sequence is at least 95% identical to CS04-SC2-NA (SEQ ID NO:11). In some embodiments, the nucleotide sequence is at least 96% identical to CS04-SC2-NA (SEQ ID NO:11). In some embodiments, the nucleotide sequence is at least 97% identical to CS04-SC2-NA (SEQ ID NO:11). In some embodiments, the nucleotide sequence is at least 98% identical to CS04-SC2-NA (SEQ ID NO:11). In some embodiments, the nucleotide sequence is at least 99% identical to CS04-SC2-NA (SEQ ID NO:11). In some embodiments, the nucleotide sequence is at least 99.5% identical to CS04-SC2-NA (SEQ ID NO:11). In some embodiments, the nucleotide sequence is at least 99.9% identical to CS04-SC2-NA (SEQ ID NO:11). In some embodiments, the nucleotide sequence is identical to CS04-SC2-NA (SEQ ID NO: 11).

[0256] In some embodiments, the single-chain factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS04-SC1-AA (SEQ ID NO: 10; human factor VIIIΔ(760-1667) (for SPI; HsFVIIIΔ(741-1648) for SPE)). In some embodiments, the factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 97% identical to CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 98% identical to CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 99% identical to CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 99.5% identical to CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 99.9% identical to CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is identical to CS04-SC1-AA (SEQ ID NO: 10).

[0257] In some embodiments, the factor VIII variant encoded by the CS04-SC1 polynucleotide having high sequence homology (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity) to CS04-SC1-AA comprises one or more amino acid substitutions selected from m1, m2, m3, m4, and m5.

[0258] In some embodiments, the single-chain factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS04-SC2-AA (SEQ ID NO: 12; human factor VIIIΔ(772-1667) (for SPI; HsFVIIIΔ(753-1648) for SPE)). In some embodiments, the factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 97% identical to CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 98% identical to CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 99% identical to CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 99.5% identical to CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 99.9% identical to CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is identical to CS04-SC2-AA (SEQ ID NO: 12).

[0259] In some embodiments, the single-chain factor VIII variant encoded by the CS04-SC2 polynucleotide having high sequence homology (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity) to CS04-SC2-AA comprises one or more amino acid substitutions selected from m1, m2, m3, m4, and m5.

[0260] In one embodiment, the single-chain factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m1. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m2. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m3. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m4. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m5.

[0261] In one embodiment, the single-chain factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m12. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m13. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m23. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m24. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m25. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m34. In one embodiment, the factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m35.

[0262] In one embodiment, the single-chain Factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m123. In one embodiment, the Factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m234. In one embodiment, the Factor VIII variant encoded by the CS04 polynucleotide comprises the amino acid substitution m125.

[0263] Codon-modified single-stranded polynucleotide CS01 In one embodiment, the codon-modified polynucleotide provided herein comprises a nucleotide sequence encoding a variant single-chain factor VIII polypeptide. The factor VIII polypeptide comprises a factor VIII light chain, a factor VIII heavy chain, and an optional polypeptide linker linking the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence that is part of CS01-FL-NA (SEQ ID NO: 13) and has high sequence identity to CS01-HC-NA (SEQ ID NO: 24), which encodes the factor VIII heavy chain. The light chain of the factor VIII polypeptide is encoded by a second nucleotide sequence that is part of CS01-FL-NA (SEQ ID NO: 13) and has high sequence identity to CS01-LC-NA (SEQ ID NO: 25), which encodes the factor VIII light chain. The optional polypeptide linker does not include a furin cleavage site.

[0264] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences are identical to CSO1-HC-NA and CSO1-LC-NA (SEQ ID NOs: 24 and 25), respectively.

[0265] In some embodiments, the codon-modified polynucleotide has a nucleotide sequence that is highly identical to CSO1-SC1-NA (SEQ ID NO:26). In some embodiments, the nucleotide sequence is at least 95% identical to CSO1-SC1-NA (SEQ ID NO:26). In some embodiments, the nucleotide sequence is at least 96% identical to CSO1-SC1-NA (SEQ ID NO:26). In some embodiments, the nucleotide sequence is at least 97% identical to CSO1-SC1-NA (SEQ ID NO:26). In some embodiments, the nucleotide sequence is at least 98% identical to CSO1-SC1-NA (SEQ ID NO:26). In some embodiments, the nucleotide sequence is at least 99% identical to CSO1-SC1-NA (SEQ ID NO:26). In some embodiments, the nucleotide sequence is at least 99.5% identical to CSO1-SC1-NA (SEQ ID NO:26). In some embodiments, the nucleotide sequence is at least 99.9% identical to CSO1-SC1-NA (SEQ ID NO:26). In some embodiments, the nucleotide sequence is identical to CS01-SC1-NA (SEQ ID NO: 26).

[0266] In some embodiments, the codon-modified polynucleotide has a nucleotide sequence that is highly identical to CSO1-SC2-NA (SEQ ID NO:27). In some embodiments, the nucleotide sequence is at least 95% identical to CSO1-SC2-NA (SEQ ID NO:27). In some embodiments, the nucleotide sequence is at least 96% identical to CSO1-SC2-NA (SEQ ID NO:27). In some embodiments, the nucleotide sequence is at least 97% identical to CSO1-SC2-NA (SEQ ID NO:27). In some embodiments, the nucleotide sequence is at least 98% identical to CSO1-SC2-NA (SEQ ID NO:27). In some embodiments, the nucleotide sequence is at least 99% identical to CSO1-SC2-NA (SEQ ID NO:27). In some embodiments, the nucleotide sequence is at least 99.5% identical to CSO1-SC2-NA (SEQ ID NO:27). In some embodiments, the nucleotide sequence is at least 99.9% identical to CSO1-SC2-NA (SEQ ID NO:27). In some embodiments, the nucleotide sequence is identical to CS01-SC2-NA (SEQ ID NO: 27).

[0267] In some embodiments, the single-chain factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS01-SC1-AA (SEQ ID NO: 10; human factor VIIIΔ(760-1667) (for SPI; HsFVIIIΔ(741-1648) for SPE)). In some embodiments, the factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 97% identical to CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 98% identical to CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 99% identical to CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 99.5% identical to CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 99.9% identical to CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is identical to CS01-SC1-AA (SEQ ID NO: 10).

[0268] In some embodiments, the factor VIII variant encoded by the CS01-SC1 polynucleotide having high sequence homology (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity) to CS01-SC1-AA comprises one or more amino acid substitutions selected from m1, m2, m3, m4, and m5.

[0269] In some embodiments, the single-chain factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS01-SC2-AA (SEQ ID NO: 12; human factor VIIIΔ(772-1667) (for SPI; HsFVIIIΔ(753-1648) for SPE)). In some embodiments, the factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 97% identical to CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 98% identical to CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 99% identical to CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 99.5% identical to CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 99.9% identical to CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is identical to CS01-SC2-AA (SEQ ID NO: 12).

[0270] In some embodiments, the single-chain factor VIII variant encoded by the CS01-SC2 polynucleotide having high sequence homology (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity) to CS01-SC2-AA comprises one or more amino acid substitutions selected from m1, m2, m3, m4, and m5.

[0271] In one embodiment, the single-chain factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m1. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m2. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m3. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m4. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m5.

[0272] In one embodiment, the single-chain factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m12. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m13. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m23. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m24. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m25. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m34. In one embodiment, the factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m35.

[0273] In one embodiment, the single-chain Factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m123. In one embodiment, the Factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m234. In one embodiment, the Factor VIII variant encoded by the CS01 polynucleotide comprises the amino acid substitution m125.

[0274] Codon-modified single-stranded polynucleotide CS23 In one embodiment, the codon-modified polynucleotide provided herein comprises a nucleotide sequence encoding a variant single-chain factor VIII polypeptide. The factor VIII polypeptide comprises a factor VIII light chain, a factor VIII heavy chain, and an optional polypeptide linker linking the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence that is part of CS23-FL-NA (SEQ ID NO: 20) and has high sequence identity to CS23-HC-NA (SEQ ID NO: 22), which encodes the factor VIII heavy chain. The light chain of the factor VIII polypeptide is encoded by a second nucleotide sequence that is part of CS23-FL-NA (SEQ ID NO: 20) and has high sequence identity to CS23-LC-NA (SEQ ID NO: 23), which encodes the factor VIII light chain. The optional polypeptide linker does not include a furin cleavage site.

[0275] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively.

[0276] In some embodiments, the codon-modified polynucleotide has a nucleotide sequence that has high sequence identity to CS23-SC1-NA (SEQ ID NO:28). In some embodiments, the nucleotide sequence is at least 95% identical to CS23-SC1-NA (SEQ ID NO:28). In some embodiments, the nucleotide sequence is at least 96% identical to CS23-SC1-NA (SEQ ID NO:28). In some embodiments, the nucleotide sequence is at least 97% identical to CS23-SC1-NA (SEQ ID NO:28). In some embodiments, the nucleotide sequence is at least 98% identical to CS23-SC1-NA (SEQ ID NO:28). In some embodiments, the nucleotide sequence is at least 99% identical to CS23-SC1-NA (SEQ ID NO:28). In some embodiments, the nucleotide sequence is at least 99.5% identical to CS23-SC1-NA (SEQ ID NO:28). In some embodiments, the nucleotide sequence is at least 99.9% identical to CS23-SC1-NA (SEQ ID NO:28). In some embodiments, the nucleotide sequence is identical to CS23-SC1-NA (SEQ ID NO: 28).

[0277] In some embodiments, the codon-modified polynucleotide has a nucleotide sequence that is highly identical to CS23-SC2-NA (SEQ ID NO:29). In some embodiments, the nucleotide sequence is at least 95% identical to CS23-SC2-NA (SEQ ID NO:29). In some embodiments, the nucleotide sequence is at least 96% identical to CS23-SC2-NA (SEQ ID NO:29). In some embodiments, the nucleotide sequence is at least 97% identical to CS23-SC2-NA (SEQ ID NO:29). In some embodiments, the nucleotide sequence is at least 98% identical to CS23-SC2-NA (SEQ ID NO:29). In some embodiments, the nucleotide sequence is at least 99% identical to CS23-SC2-NA (SEQ ID NO:29). In some embodiments, the nucleotide sequence is at least 99.5% identical to CS23-SC2-NA (SEQ ID NO:29). In some embodiments, the nucleotide sequence is at least 99.9% identical to CS23-SC2-NA (SEQ ID NO:29). In some embodiments, the nucleotide sequence is identical to CS23-SC2-NA (SEQ ID NO: 29).

[0278] In some embodiments, the single-chain factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS23-SC1-AA (SEQ ID NO: 10; human factor VIIIΔ(760-1667) (for SPI; CS04Δ(741-1648) for SPE)). In some embodiments, the factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 97% identical to CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 98% identical to CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 99% identical to CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is at least 99.5% identical to CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 99.9% identity to CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is identical to CS23-SC1-AA (SEQ ID NO: 10).

[0279] In some embodiments, the factor VIII variant encoded by the CS23-SC1 polynucleotide having high sequence homology (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity) to CS23-SC1-AA comprises one or more amino acid substitutions selected from m1, m2, m3, m4, and m5.

[0280] In some embodiments, the single-chain factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS23-SC2-AA (SEQ ID NO: 12; human factor VIIIΔ(772-1667) (for SPI; HsFVIIIΔ(753-1648) for SPE)). In some embodiments, the factor VIII variant encoded by the codon-modified polynucleotide has an amino acid sequence highly identical to CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 97% identical to CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 98% identical to CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 99% identical to CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 99.5% identical to CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is at least 99.9% identical to CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is identical to CS23-SC2-AA (SEQ ID NO: 12).

[0281] In some embodiments, the single-chain factor VIII variant encoded by the CS23-SC2 polynucleotide having high sequence homology (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity) to CS23-SC2-AA comprises one or more amino acid substitutions selected from m1, m2, m3, m4, and m5.

[0282] In one embodiment, the single-chain factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m1. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m2. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m3. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m4. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m5.

[0283] In one embodiment, the single-chain factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m12. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m13. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m23. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m24. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m25. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m34. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m35.

[0284] In one embodiment, the single-chain factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m123. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m234. In one embodiment, the factor VIII variant encoded by the CS23 polynucleotide comprises the amino acid substitution m125.

[0285] E. Factor VIII Expression Vector In some embodiments, the codon-modified polynucleotides described herein are incorporated into expression vectors, non-limiting examples of which include viral vectors (e.g., vectors suitable for gene therapy), plasmid vectors, bacteriophage vectors, cosmids, phagemids, artificial chromosomes, and the like.

[0286] Non-limiting examples of viral vectors include retroviruses, such as Moloney murine leukemia virus (MMLV), Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40 virus; polyoma virus; Epstein-Barr virus; papilloma virus; herpes virus; vaccinia virus; and polio virus.

[0287] In some embodiments, the codon-modified polynucleotides described herein are incorporated into gene therapy vectors. In some embodiments, the gene therapy vector is a retrovirus, specifically a replication-defective retrovirus. Protocols for generating replication-defective retroviruses are known in the art. For general reviews, see Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E.J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0288] In one embodiment, the gene therapy vector is an adeno-associated virus (AAV)-based gene therapy vector. The AAV system has been previously described and is generally known in the art (Kelleher and Vos, Biotechniques, 17(6):1110-17(1994); Cotten et al., Proc Natl Acad Sci USA, 89(13):6094-98(1992); Curiel, Nat Immun, 13(2-3):141-64(1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129(1992); and Asokan A, et al., Mol. Ther., 20(4):699-708(2012), each of which is incorporated herein by reference in its entirety for all purposes). Details regarding the production and use of rAAV vectors are described, for example, in U.S. Patent Nos. 5,139,941 and 4,797,368, each of which is incorporated by reference in its entirety for all purposes. In certain embodiments, the AAV vector is an AAV-8 vector.

[0289] In some embodiments, the codon-modified polynucleotides described herein are incorporated into retroviral expression vectors. These systems have been previously described and are generally known in the art (Mann et al., Cell, 33:153-159, 1983; Nicolas and Rubinstein, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986). In certain embodiments, the retroviral vector is a lentiviral vector (see, e.g., Naldini et al., Science, 272(5259):263-267, 1996; Zufferey et al., Nat Biotechnol, 15(9):871-875, 1997; Blomer et al., J Virol., 71(9):6641-6649, 1997; U.S. Patent Nos. 6,013,516 and 5,994,136).

[0290] A wide variety of vectors can be used to express Factor VIII polypeptides from codon-modified polypeptides in cell culture, including eukaryotic and prokaryotic expression vectors. In some embodiments, plasmid vectors are contemplated for use in expressing Factor VIII polypeptides in cell culture. Generally, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with the host. The vector can carry a replication site and marking sequences that can enable phenotypic selection in transformed cells. The plasmid will include a codon-modified polynucleotide encoding a Factor VIII polypeptide operably linked to one or more control sequences, e.g., a promoter.

[0291] Non-limiting examples of prokaryotic expression vectors include plasmids such as pRSET, pET, pBAD, etc., where promoters for use in prokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc. Examples of eukaryotic expression vectors include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET, which use promoters such as AOX1, GAP, GAL1, AUG1, etc.; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., which use promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc.; and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc / RSV, pcDNA3, pBPV, etc., which use promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin, as well as vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpes virus, retrovirus, etc. [Example]

[0292] IV. Working Examples Example 1 - Construction of codon-modified Factor VIII variant expression sequences To generate an effective factor VIII coding sequence for gene therapy of hemophilia A, two obstacles had to be overcome. First, the limited genome size of conventional delivery vectors (e.g., AAV virions) for gene therapy required that the encoded factor VIII polypeptide be significantly shortened. Second, the coding sequence had to be modified to (i) stabilize packaging interactions within the delivery vector, (ii) improve mRNA-mediated stabilization, and (iii) improve the robustness of mRNA transcription / translation.

[0293] To achieve the first goal, Applicants started with a B-domain deleted factor VIII variant construct, referred to herein as "FVIII-BDD-SQ." In this construct, the B domain is replaced with a 14 amino acid sequence, referred to as the "SQ" sequence. Recombinant FVIII-BDD-SQ is commercially available under the trade name REFACTO® and has been shown to be effective in managing hemophilia A. However, the wild-type coding sequence of FVIII-BDD-SQ, which contains the human wild-type nucleic acid sequences for the heavy and light chains of factor VIII, is not efficiently expressed in gene therapy vectors.

[0294] To address the poor expression of wild-type FVIII-BDD-SQ, the codon optimization algorithm described by Fath et al. (PLoS ONE, 6:e17596 (2011)), with modifications as described by Ward et al. (Blood, 117:798 (2011)) and McIntosh et al. (Blood, 121, 3335-3344 (2013)), was applied to the FVIII-BDD-SQ sequence to generate the first intermediate coding sequence, CS04a. However, Applicants recognized that the CS04a sequence generated using the modified algorithm could be improved by further sequence modifications. Therefore, Applicants reintroduced CpG dinucleotides, reintroduced CGC codons for arginine, altered the codon distribution of leucine and serine, reintroduced highly conserved codon pairs, and removed potential TATA box, CCAAT box, and splice site elements, while avoiding local over-representation of CpG islands and AT-rich and GC-rich stretches.

[0295] First, the modified algorithm systematically replaces codons containing CpG dinucleotides (e.g., arginine codons) with non-CpG dinucleotide codons and eliminates / avoids CpG dinucleotides created by adjacent codons. Such strict CpG dinucleotide avoidance is typically performed to prevent TLR-induced immunity after intramuscular injection of DNA vaccines. However, doing so limits the potential for codon optimization. For example, the modified algorithm excludes the use of the entire set of arginine codons, CGX. This is particularly destructive for gene coding when expressed in human cells, because CGC is the most frequently used codon for arginine in highly expressed human genes. Furthermore, avoiding the creation of CpGs by adjacent codons further limits the potential for optimization (e.g., by limiting the number of codon pairs that can be used together).

[0296] Because TLR-induced immunity is not expected to be a problem in liver-directed gene therapy using AAV, we selectively reintroduced CpG-containing codons and adjacent codons that create CpGs into the intermediate coding sequence CS04a in the sequence encoding the factor VIII light chain (e.g., at the 3' end of the FVIII-BDD-SQ coding sequence). This allowed for a higher frequency of preferred human codons, particularly arginine codons. However, we took care to avoid the creation of CpG islands, which are regions of the coding sequence that frequently contain CpG sites. This contradicts the teachings of Krinner et al. (Nucleic Acids Res., 42(6):3551-64(2014)), which suggests that CpG domains downstream of the transcription start site promote high levels of gene expression.

[0297] Second, the correction algorithm only applies certain codons, such as CTG for leucine, GTG for valine, and CAG for glutamine. However, this violates the principle of balanced codon usage, as proposed, for example, by Haas et al. (Current Biology, 6(3):315-24(1996)). To address the overuse of preferred codons by the correction algorithm, alternative codons for leucine were reintroduced when allowed by other rules applied to codon modification (e.g., CpG frequency and GC content).

[0298] Third, the modified algorithm replaces codon pairs regardless of their degree of conservation in nature if certain criteria (e.g., the presence of a CG dinucleotide) are met. To take into account potentially evolutionarily conserved beneficial properties, the most conserved codon pairs replaced by the algorithm and the most conserved preferred codon pairs, such as those described by Tats et al. (BMC Genomics 9:463 (2008)), were analyzed and adjusted if permitted by other rules applied to codon modification (e.g., CpG frequency and GC content).

[0299] Fourth, the serine codons used in the intermediate coding sequence were also re-engineered: specifically, the serine codons AGC, TCC, and TCT were introduced at high frequency into the modified coding sequence to better match overall human codon usage (Haas et al., supra).

[0300] Fifth, TATA box and CCAAT box elements, as well as intron / exon splice sites, were screened and removed from the modified coding sequences, taking care to avoid local over-representation of AT-rich or GC-rich stretches.

[0301] Finally, in addition to optimizing codon usage within the coding sequence, we considered the structural requirements of the underlying AAV virion for further refining the intermediate coding sequence CS04a. AAV vectors (e.g., the nucleic acid portion of the AAV virion) are packaged as single-stranded DNA molecules within the capsid (for a review, see Daya and Berns, Clin. Microbiol. Rev., 21(4):583-93 (2008)). Therefore, the GC content of the vector can affect genome packaging and, ultimately, vector yield during production. Like many algorithms, the modified algorithm used here generates optimized gene sequences with a GC content of at least 60% (Fath et al., PLoS One, 6(3):e17596 (2011) (see errata in PLoS One, (6)3 (2011))). However, AAV8 capsid proteins are encoded by nucleotide sequences with a low GC content of approximately 56%. Therefore, the GC content of the intermediate coding sequence CS04a was reduced to 56% to better mimic the wild-type AAV8 capsid protein coding sequence.

[0302] The resulting CS04 coding sequence, shown in Figure 2, has an overall GC content of 56%. The CpG-dinucleotide content of this sequence is moderate, although CpG dinucleotides are predominantly present in downstream portions of the coding sequence, e.g., in the portion encoding the light chain of factor VIII. The CS04 sequence shares 79.77% nucleotide sequence identity with the corresponding coding sequence of wild-type factor VIII (Genbank accession M14113).

[0303] For comparison, several other codon-optimized ReFacto constructs were prepared. CS01 was constructed by applying the codon optimization algorithm of Fath et al., as modified by Ward et al., as was done for CS04. However, unlike CS04, the CS01 construct does not contain a CpG island. ReFacto construct CS08 was codon-optimized as described in Radcliff PM et al., Gene Therapy, 15:289-97 (2008), the contents of which are expressly incorporated herein by reference in their entirety for all purposes. Codon-optimized ReFacto construct CS10 was obtained from Eurofins Genomics (Ebersberg, Germany). Codon-optimized ReFacto construct CS11 was obtained from Integrated DNA Technologies, Inc. (Coralville, USA). The codon-optimized ReFacto construct CH25 was obtained from ThermoFischer Scientific's GeneArt services (Regensburg, Germany). ReFacto construct CS40 consists of the wild-type factor VIII coding sequence. The algorithm used to construct CS23 was based on the JCAT tool (www.jcat.de), an online tool for codon optimization (Grote et al., 2005; Nucl. Acids Res. W526-31). The sequence was further modified to better reflect the codon usage of the albumin superfamily (Mirsafian et al. 2014: Sc. Word Journal 2014, ID 639682). The sequence identity shared between each ReFacto coding sequence is shown in Table 2 below.

[0304] Table 2. Percent identity matrix of codon-modified Factor VIII constructs TIFF0007631425000006.tif65149

[0305] Each construct was constructed by cloning different synthetic DNA fragments into the same vector backbone plasmid (pCh-BB01). DNA synthesis of the Refacto BDD-FVIII fragments, flanked by AscI and NotI restriction sites, was performed by ThermoFischer Scientific (Regensburg, Germany). The vector backbone contained two flanking AAV2-derived inverted terminal repeats (ITRs), which encompassed the promoter / enhancer sequence from the liver-specific mouse transthyretin gene, AscI and NotI restriction sites for insertion of the respective Refacto BDD-FVIII, and a synthetic poly(A) site. After ligation of the prepared vector backbone and insertion via both the AscI and NotI sites, the resulting plasmids were amplified on a milligram scale. The Refacto BDD-FVIII sequences of the constructs were verified by direct sequencing (Microsynth, Balgach, Switzerland). Cloning yielded seven different plasmid constructs, designated pCS40, pCS01, pCS04, pCS08, pCS10, pCS11, and pCh25 (Figure 23), each of which has the same vector backbone and encodes the same B-domain deleted FVIII protein (Refacto type BDD-FVIII), but differs in its FVIII coding sequence.

[0306] AAV8-based vectors were prepared using a three-plasmid transfection method as described by Grieger JC, et al. (Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector, Mol Ther., Oct 6, 2015, doi:10.1038 / mt.2015.187. [Epub ahead of print]), the entire disclosure of which is expressly incorporated herein by reference for all purposes. HEK293 suspension cells were used for plasmid transfection using the corresponding FVIII vector plasmid, helper plasmid pXX6-80 (carrying adenovirus helper genes), and packaging plasmid pGSK2 / 8 (contributing the rep2 and cap8 genes). To isolate AAV8 constructs, cell pellets from a 1-liter culture were processed using an iodixanol gradient as described by Grieger et al. (2015, supra). This procedure yielded vector preparations designated vCS01, vCS04, vCS08, vCS10, vCS11, and vCH25. Vectors were quantified by qPCR using a universal qPCR procedure targeting the AAV2 inverted terminal repeats (Aurnhammer, Human Gene Therapy Methods: Part B 23:18-28 (2012)). A control vector plasmid carrying the AAV2 inverted terminal repeats was used to generate a standard curve. The resulting vCS04 construct is presented in Figures 7A-7C as SEQ ID NO:8.

[0307] The integrity of the vector genome was analyzed by AAV agarose gel electrophoresis. Electrophoresis was performed as described in Fagone et al., Human Gene Therapy Methods 23:1-7 (2012). Briefly, AAV vector preparations were incubated at 75°C for 10 minutes in the presence of 0.5% SDS and then cooled to room temperature. Approximately 1.5E10 vector genomes (vg) were loaded per lane of a 1% 1xTAE agarose gel and electrophoresed at 7 V / cm for 60 minutes. The gel was then stained in 2x diluted GelRed (Biotium Cat. No. 41003) solution and imaged with a ChemiDoc™ MP (Biorad). The results, shown in Figure 24, show that the viral vectors vCS01, vCS04, and vCS40 exhibit distinct bands in the 5 kb range (Figure 24, lanes 2-4), indicating that their genome sizes are similar. The genome appears as a uniform band despite the vector size of approximately 5.2 kb, confirming the proper packaging of a slightly larger genome (compared to the 4.7 kb AAV wild-type genome), a size consistent with all other vCS vector preparations (data not shown).

[0308] To confirm the predicted capsid protein patterns, vectors vCS01, vCS04, and vCS40 were subjected to SDS-PAGE followed by silver staining (Figure 25). As shown, downstream purification yielded highly purified material that displayed the predicted protein patterns for VP1, VP2, and VP3 (Figure 25, lanes 2-4). The same patterns were observed for all other viral preparations (not shown). SDS-PAGE of AAV preparations was performed according to standard procedures. 1E10 vg of each viral construct was loaded per lane and separated on a 4-12% Bis-Tris (NuPAGE®, Novex, Life Technologies) gel according to the manufacturer's instructions. Silver staining was performed using the SilverQuest™ kit (Novex, Life Technologies) according to the manufacturer's instructions.

[0309] Surprisingly, AAV vectors vCS01 and vCS04 demonstrated higher AAV virus yields and virion packaging than the wild-type coding construct vCS40 and other codon-optimized constructs. As shown in Table 3, vectors vCS01 and vCS04 replicated substantially better than vCS40, producing AAV titers that were 5-7 times higher.

[0310] Table 3. Yields per liter of cell culture obtained using AAV vector constructs vCS01, vCS04, and vCD40, purified from cell pellets. TIFF0007631425000007.tif51170

[0311] Example 2 - In vivo expression of codon-modified Factor VIII variant expression sequences To test the biological potency of the codon-modified factor VIII variant sequences, the ReFacto FVIII construct described in Example 1 was administered to factor VIII-deficient mice. Briefly, evaluation was performed in C57Bl / 6 FVIII knockout (ko) mice (6–8 mice per group) by tail vein injection of 4E12 vector genomes (vg) per kilogram of mouse body weight. Blood was collected by retroorbital puncture 14 days after injection, and plasma was prepared and frozen using standard procedures. The expression level at day 14 was chosen because the influence of inhibitory antibodies observed in some animals in this mouse model is minimal at this time. FVIII activity in mouse plasma was measured using the Technochrome FVIII assay, performed with only minor modifications according to the manufacturer's recommendations (Technoclone, Vienna, Austria). For the assay, plasma samples were appropriately diluted and mixed with assay reagents containing thrombin, activated factor IX (FIXa), phospholipids, factor X, and calcium. Following FVIII activation by thrombin, a complex of FIXa, phospholipids, and calcium is formed. This complex activates FX to activated FXa, which cleaves the chromogenic substrate para-nitroanilide (pNA). The kinetics of pNA formation is measured at 405 nm. The rate is directly proportional to the FVIII concentration in the sample. FVIII concentrations are read from a standard curve and results are given in FVIII IU / ml.

[0312] The results, presented in Table 4 below, show that the codon-modified sequences (CS10, CS11, and CH25) designed using a commercial algorithm only slightly increased BDD-FVIII expression (3-4 fold) compared to the wild-type BDD-FVIII construct (CS40). Similarly, the codon-modified BDD-FVIII construct (CS08) prepared as described by Radcliffe et al. only increased BDD-FVIII expression 3-4 fold. This result is consistent with that reported by Radcliffe et al. Surprisingly, constructs CS01, CS04, and CS23 produced much higher BDD-FVIII expression in in vivo biological potency assays (18-fold, 74-fold, and 30-fold increases, respectively).

[0313] Table 4. Plasma FVIII expression in FVIII knockout mice induced by various AAV vector constructs. TIFF0007631425000008.tif86170

[0314] Example 3 - Design of glycosylated peptides for B domain replacement linkers Other researchers have shown that inserting a small peptide containing six putative N-linked glycosylation sites from the wild-type factor VIII B domain (the "V3 peptide") into a B-domain deletion gene therapy construct increased factor VIII levels in mouse plasma (McIntosh et al., Blood 121(17):3335-44 (2013)). However, to maintain the small size of the B-domain replacement linker, these glycosylation sites were removed from their context in the wild-type B domain. In silico predictions of the V3 peptide-containing linker (Gupta et al., supra) suggest that only two of these glycosylation sites within the V3 peptide will be modified in vivo (Figure 15).

[0315] Therefore, Applicants sought to identify alternatively glycosylated peptides that would likely support high levels of in vivo glycosylation and showed higher concordance with wild-type glycosylation than the V3 peptide. Applicants designed and tested several alternatively glycosylated peptides in silico. Some of these peptides, shown in Figures 13A-13B, were predicted to have in vivo glycosylation comparable to or greater than that of the V3 peptide when placed between amino acids N768 and P769 of the B-domain replacement linker in SEQ ID NO: 2. The in silico prediction results are shown in Table 5 below. Table 5 also reports the results of expression experiments performed on several constructs encoding ReFacto-type Factor VIII proteins with glycosylated peptides incorporated into the B-domain replacement linker in the codon-optimized CSO1 background.

[0316] Table 5. N-glycosylation predictions for B domain-substituted linker peptides and performance of AAV vector constructs in vivo. TIFF0007631425000009.tif103163

[0317] AAV vectors containing NG variants were constructed as described in Example 1 and tested in FVIII knockout mice as described in Example 2. All viral vectors listed in Table 5 (except for the control vector vCS40) are based on the algorithm used for vCS01. A parallel set of constructs using the vCS04 algorithm was also prepared (vNG / CS04 series) and tested in mouse models. Results were compared to the expression levels achieved with the wild-type construct vCS40. The expression level at day 28 was chosen in this example because the expression levels of most constructs reached their highest levels at this time point. Three AAV vectors, including vNG4 / CS01, vNG10 / CS01, and vNG16 / CS01, achieved over 40-fold higher FVIII expression levels (Table 5). The corresponding constructs vNG4 / CS04, vNG10 / CS04, and vNG16 / CS04 are expected to show even higher expression because they are based on the superior vCS04 algorithm.

[0318] Surprisingly, the vNG / CS01 series of AAV vectors demonstrated higher measured yields of AAV virus production and higher virion packaging compared to the vCS40 wild-type coding construct. As shown in Table 6, the vNG / CS01 vectors replicated substantially better than vCS40, producing approximately three-fold higher AAV titers.

[0319] Table 6. Yields per liter of cell culture obtained using AAV vector constructs purified from cell pellets. TIFF0007631425000010.tif103133

[0320] Example 4 - Construction of mutant BDD-FVIII constructs A number of different mutant Refacto BDD-FVIII constructs carrying amino acid mutations within the factor VIII heavy chain and / or B-domain replacement linkers were cloned and screened. The corresponding vectors, referred to herein as the "vCS" series of vectors, encode BDD-FVIII variants in the codon-modified CS01, CS04, and CS23 backgrounds. The methods used to construct the CS01 and CS04 backgrounds are described in Example 1. The method used to construct CS23 was based on the JCAT tool (www.jcat.de), an online tool for codon optimization (Grote et al., 2005; Nucl. Acids Res. W526-31). The sequence was further modified to better reflect the codon usage of the albumin superfamily (Mirsafian et al., Sc. Word Journal, ID 639682 (2014)), the contents of which are expressly incorporated herein by reference in their entirety for all purposes.

[0321] The FVIII sequences of the vCS series constructs contained a combination of three mutations. The first amino acid change introduced into the FVIII sequence was an X1 mutation (TTYVNRSL (SEQ ID NO: 33); X. Xiao), which introduces an additional glycosylation site near the B domain replacement linker. The X1 mutation is also referred to herein as the "m3" mutation. The second amino acid change made to the FVIII sequence included the F328S (in SPI, F309S in SPE), an amino acid change known to improve FVIII secretion (Swaaroop, J. Biol. Chem., 272:24121-24 (1997)). This mutation is also referred to herein as the "m1" mutation. The third change was the so-called X5 mutation, which is a combination of five amino acid changes in the heavy chain A1 domain that improves the specific activity and secretion of BDD-FVIII (Cao et al., 2014; ASGCT Abstract #460; mutation details will be disclosed in the oral presentation). The X5 mutation is also referred to herein as the "m2" mutation. Next, a combination of X1 with F328S (for SPI, F309S for SPE) was made, followed by a combination of X1 with X5, also referred to as "X6," and then another combination of X5 with F328S (for SPI, F309S for SPE) (Table 7).

[0322] Gene synthesis and cloning of vector plasmids. Plasmids were constructed by cloning different synthetic DNA fragments into the same vector backbone plasmid (pCh-BB01). DNA synthesis of the Refacto BDD-FVIII fragments, flanked by AscI and NotI restriction sites, was performed by ThermoFischer Scientific (Regensburg, Germany). The vector backbone contained two flanking AAV2-derived inverted terminal repeats (ITRs), which encompassed the promoter / enhancer sequence from the liver-specific mouse transthyretin gene, AscI and NotI restriction sites for insertion of the respective Refacto BDD-FVIII fragments, and a synthetic poly(A) site. After ligation of the prepared vector backbone and insertion via both the AscI and NotI sites, the resulting plasmids were amplified on a milligram scale. The Refacto BDD-FVIII sequences of the constructs were verified by direct sequencing (Microsynth, Balgach, Switzerland). Cloning yielded different plasmid constructs, as shown in Figure 44.

[0323] Small-scale vector preparation and quantification by quantitative PCR (qPCR). AAV8-based vectors were prepared by a three-plasmid transfection method essentially as described by Grieger et al. (2015, supra). HEK293 suspension cells were used for plasmid transfection using the corresponding FVIII vector plasmid, the helper plasmid pXX6X80 (carrying the adenovirus helper genes), and the packaging plasmid pGSK2 / 8 (contributing the rep2 and cap8 genes). For downstream processing, cell pellets from 1-liter cultures were processed using an iodixanol gradient as described above. This procedure yielded the vector preparations summarized in Table 8. Vectors were quantified by qPCR using a universal qPCR procedure targeting the AAV2 terminal inverted repeats (Aurnhammer, Human Gene Therapy Methods: Part B 23:18-28 (2012)). Accurately quantified vector plasmids carrying the AAV2 terminal inverted repeats were used to generate a standard curve.

[0324] AAV vector characterization. The integrity of the vector genome was analyzed by AAV agarose gel electrophoresis. Electrophoresis was performed as described by Fagone et al. (Human Gene Therapy Methods, 23:1-7 (2012)). AAV vector preparations were incubated at 75°C for 10 minutes in the presence of 0.5% SDS and then cooled to room temperature. Approximately 1.5E10 vector genomes (vg) were loaded per lane of a 1% 1xTAE agarose gel and electrophoresed at 7V / cm gel length for 60 minutes. The gel was then stained in 2x diluted GelRed (Biotium Cat. No. 41003) solution and imaged with a ChemiDoc™ MP (Biorad). The vector selection results are shown in Figure 45. The viral vectors vCS04 (control), vCS17, vCS20, vCS24, vCS16, and vCS40 (control) all showed distinct bands in the 5 kb range (Figure 45, lanes 2-7; right arrows), indicating identical genome sizes. The genomes were uniformly banded despite the vector size of approximately 5.2 kb, confirming the proper packaging of the slightly larger genome (compared to the 4.7 kb of the AAV wild-type genome).

[0325] To confirm vector purity and the expected capsid protein patterns, these vectors were subjected to SDS-PAGE followed by silver staining, as shown in Figure 46. As shown, downstream purification yielded highly purified material that displayed the expected protein patterns for VP1, VP2, and VP3 (Figure 46, lanes 2-9; right arrows). SDS-PAGE of AAV preparations was performed according to standard procedures. 1E10 vg per lane was separated on a 4-12% Bis-Tris (NuPAGE®, Novex, Life Technologies) gel according to the manufacturer's instructions. Silver staining was performed using the SilverQuest™ kit (Novex, Life Technologies) according to the manufacturer's instructions.

[0326] In vivo biological titer screening of vectors. Screening of various Refacto-type BDD-FVIII constructs was performed in mice. Assays were performed in C57Bl / 6 FVIII knockout (ko) mice (6–8 mice per group) by tail vein injection of 4E12 vector genomes (vg) per kilogram of mouse body weight. Blood was collected by retroorbital puncture 14 days after injection, and plasma was prepared and frozen using standard procedures. FVIII activity in mouse plasma was measured using a Technoclone chromogenic method (Technochrome FVIII, Technoclone, Vienna, Austria) with minor modifications. Briefly, plasma samples were appropriately diluted and mixed with an assay reagent containing thrombin, activated factor IX (FIXa), phospholipids, factor X, and calcium. Following FVIII activation by thrombin, a complex of FIXa, phospholipids, and calcium is formed. This complex activates FX to form activated FXa, which cleaves the chromogenic substrate para-nitroanilide (pNA). The kinetics of pNA production is measured at 405 nm. The rate is directly proportional to the FVIII concentration in the sample. The FVIII concentration is read from a standard curve and the results are given in FVIII IU / ml.

[0327] The results of the mouse biological potency studies (FVIII expression data in mouse plasma on day 14, expressed in international units per milliliter [IU / ml], and fold expression compared to the wild-type control vCS40) are shown in Table 7. The AAV vectors vCS19, vCS26, and vCS32 all contain an X1 glycosylation site in the codon-modified backgrounds CS01, CS04, and CS23, respectively. As can be seen in Table 7, surprisingly, high levels of expression were obtained compared to the wild-type construct vCS40 (set at a level of 1). For example, vCS26 was expressed at a 202-fold higher level than the wild-type vector vCS40. Another control construct, vCH111, an X1-series vector containing the X1 mutation in a Geneart codon background, showed a smaller increase in expression (12-fold).

[0328] Vectors vCS16, vCS28, and vCS34 all contain the F328S (F309S for SPE) mutation, which enhances secretion, in the codon-modified backgrounds CS01, CS04, and CS23, respectively. As seen in Table 7, high expression levels (45-93-fold higher than the wild-type control vCS40) were obtained with vCS16 and vCS28.

[0329] Vectors vCS20, vCS24, and vCS33 contain the X5 mutation in the codon-modified backgrounds CS01, CS04, and CS23, respectively. Within the X5 series, the best-performing variant was vCS20, achieving levels of over 3 units / ml after day 14 and a 121-fold increase over the wild-type control, vCS40.

[0330] Vectors vCS17, vCS29, and vCS31 contain a combination of the X1 mutation and the F328S (for SPI, F309S for SPE) mutation in the codon-modified backgrounds CS01, CS04, and CS23, respectively (Table 6). Constructs vCS17 and vCS29 achieved very high expression levels (115- to 246-fold increase over the control vCS40) during mouse testing. Remarkably, in the FVIII KO mouse model used, most mice treated with the vCS17 construct did not develop neutralizing antibodies over time, as indicated by increased FVIII levels at subsequent time points (e.g., days 28 and 42; data not shown). This was an unexpected finding, as some other constructs exhibited decreased expression levels over time due to the generation of neutralizing antibodies. When the secretion-enhancing mutation F328S (in the case of SPI, F309S in the case of SPE) was combined with X1 in the CS01 background, the induction of immunogenicity was low.

[0331] Vectors vCS18, vCS27, and vCS35 contain a combination of the X1 and X5 mutations in the codon-modified backgrounds CS01, CS04, and CS23, respectively. The combination of these two mutations was also highly effective. For example, vCS18 achieved a 145-fold increase over the control vCS40 (Table 7).

[0332] Vectors vCS48 and vCS49 contain a combination of the X5 mutation and the F328S (in SPI, F309S in SPE) mutation in the codon-modified backgrounds CS01 and CS04, respectively. The combination of these two mutations was also highly effective. One of the highest gains of all mutants, a 239-fold gain over the control vCS40, was achieved with vCS49, confirming the special value of the combination including the F328S (in SPI, F309S in SPE) mutation.

[0333] An even more surprising finding was that the mutant AAV vectors grew substantially better than construct vCS40, which contains the wild-type BDD-FVIII codon. Sequence optimization resulted in several-fold higher vector yields. For some of the best-expressing constructs (e.g., vCS29, vCS17, vCS20, and vCS26), the yield increase due to the codon modifications and / or mutated sequences was approximately 3- to 5-fold higher than the wild-type vector (Table 8).

[0334] Table 7 shows the BDD-FVIII expression in plasma of FVIII knockout mice induced by various AAV vector constructs. The constructs have the same vector backbone but encode different types of mutant FVIII, including different codon-optimized backgrounds. The expression level at day 14 was chosen because the influence of inhibitory antibodies, which are commonly seen in some animals in this mouse model at later time points, is minimal at this time point. nd: not detectable.

[0335] Table 7. In vivo biological potency data of vCS constructs TIFF0007631425000011.tif169169

[0336] Table 8. Yields per liter of cell culture (packaging efficiency) obtained with different AAV vector constructs. Vectors were purified from cell pellets. nd = not detected. TIFF0007631425000012.tif120151

[0337] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0338] Sequence information SEQUENCE LISTING <110> TAKEDA PHARMACEUTICAL COMPANY LIMITED <120> VIRAL VECTORS ENCODING RECOMBINANT FVIII VARIANTS WITH INCREASED EXPRESSION FOR GENE THERAPY OF HEMOPHILIA A <150> US 62 / 255,317 <151> 2015-11-13 <160> 124 <170> PatentIn version 3.5 <210> 1 <211> 4374 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 1 atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgcc 60 accaggagat actacctggg ggctgtggag ctttcttggg actacatgca gtctgacctg 120 ggggagctgc ctgtggatgc caggttccca cccagagtgc ccaaatcctt cccattcaac 180 acctctgtgg tctacaagaa gaccctcttt gtggagttca ctgaccacct gttcaacatt 240 gccaaaccca ggccaccctg gatgggactc ctgggaccca ccattcaggc tgaggtgtat 300 gacactgtgg tcatcaccct caagaacatg gcctcccacc ctgtgagcct gcatgctgtg 360 ggggtcagct actggaaggc ctctgagggg gctggattg atgaccagac ctcccagagg 420 gagaaggagg atgacaaagt gttccctggg ggcagccaca cctatgtgtg gcaggtcctc 480 aaggaagaatg gccccatggc ctctgaccca ctctgcctga cctactccta cctttctcat 540 gtggacctgg tcaaggacct caactctgga ctgattgggg ccctgctggt gtgcagggag 600 ggctccctgg ccaaaagaga gacccagacc ctgcacaagt tcattctcct gtttgctgtc 660 tttgatgagg gcaagagctg gcactctgaa accaagaact ccctgatgca ggacagggat 720 gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaacaggagc 780 ctgcctggac tcattggctg ccacaggaaa tctgtctact ggcatgtgat tggcatgggg 840 acaacccctg aggtgcactc cattttcctg gagggccaca ccttcctggt caggaaccac 900 agacaggcca gcctggagat cagccccatc accttcctca ctgcccagac cctgctgatg 960 gacctcggac agttcctgct gttctgccac atcagctccc accagcatga tggcatggag 1020 gcctatgtca aggtggacag ctgccctgag gagccacagc tcaggatgaa gaacaatgag 1080 gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt ccgctttgat 1140 gatgacaaca gcccatcctt cattcagatc aggtctgtgg ccaagaaaca ccccaagacc 1200 tgggtgcact acattgctgc tgaggaggag gactgggact atgccccact ggtcctggcc 1260 cctgatgaca ggagctacaa gagccagtac ctcaacaatg gcccacagag gattggacgc 1320 aagtacaaga aagtcaggtt catggcctac actgatgaaa ccttcaagac cagggaggcc 1380 attcagcatg agtctggcat cctgggccca ctcctgtatg gggaggtggg ggacaccctg 1440 ctcatcatct tcaagaacca ggcctccagg ccctacaaca tctacccaca tggcatcact 1500 gatgtcaggc ccctgtacag ccgcaggctg ccaaaggggg tgaaacacct caaggacttc 1560 cccattctgc ctggggagat cttcaagtac aagtggactg tcactgtgga ggatggacca 1620 accaaatctg accccaggtg cctcaccaga tactactcca gctttgtgaa catggagagg 1680 gacctggcct ctggcctgat tggcccactg ctcatctgct acaaggagtc tgtggaccag 1740 aggggaaacc agatcatgtc tgacaagagg aatgtgattc tgttctctgt ctttgatgag 1800 aacaggagct ggtacctgac tgagaacatt cagcgcttcc tgcccaaccc tgctggggtg 1860 cagctggagg accctgagtt ccaggccagc aacatcatgc actccatcaa tggctatgtg 1920 tttgacagcc tccagctttc tgtctgcctg catgaggtgg cctactggta cattctttct 1980 attggggccc agactgactt cctttctgtc ttcttctctg gctacacctt caaacacaag 2040 atggtgtatg aggacaccct gaccctcttc ccattctctg gggagactgt gttcatgagc 2100 atggagaacc ctggcctgtg gattctggga tgccacaact ctgacttccg caacaggggc 2160 atgactgccc tgctcaaagt ctcctcctgt gaagaaca ctggggacta ctatgaggac 2220 agctatgagg acatctctgc ctacctgctc agcaagaaca atgccattga gcccaggagc 2280 ttcagccaga atccacctgt cctgaaacgc caccagaggg agatcaccag gaccaccctc 2340 cagtctgacc aggagagat tgactatgat gacaccatt ctgtggagat gaagaagag 2400 gactttgaca tctatgacga ggacgagaac cagagcccaa ggagcttcca gaagagacc 2460 aggcactact tcattgctgc tgtggagcgc ctgtgggact atggcatgag ctccagcccc 2520 catgtcctca ggaacagggc ccagtctggc tctgtgccac agttcaagaa agtggtcttc 2580 caagagttca ctgatggcag cttcacccag cccctgtaca gagggagct gaatgagcac 2640 ctgggactcc tgggcccata catcagggct gaggtggagg acaacatcat ggtgaccttc 2700 cgcaaccagg cctccaggcc ctacagcttc tacagctccc tcatcagcta tgaggaggac 2760 cagaggcagg gggctgagcc acgcaagaac tttgtgaaac ccaatgaaac caagacctac 2820 ttctggaaag tccagcacca catggccccc accaaggatg agtttgactg caaggcctgg 2880 gcctacttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggcccactc 2940 ctggtctgcc acaccaacac cctgaaccct gcccatggaa ggcaagtgac tgtgcaggag 3000 tttgccctct tcttcaccat ctttgatgaa accaagagct ggtacttcac tgagaacatg 3060 gagcgcaact gcagggcccc atgcaacatt cagatggagg accccacctt caaagagaac 3120 taccgcttcc atgccatcaa tggctacatc atggacaccc tgcctgggct tgtcatggcc 3180 caggaccaga ggatcaggtg gtacctgctt tctatgggct ccaatgagaa cattcactcc 3240 atccacttct ctgggcatgt cttcactgtg cgcaagaagg aggagtacaa gatggccctg 3300 tacaacctct accctggggt ctttgagact gtggagatgc tgccctccaa agctggcatc 3360 tggagggtgg agtgcctcat tggggagcac ctgcatgctg gcatgagcac cctgttcctg 3420 gtctacagca acaagtgcca gacccccctg ggaatggcct ctggccacat cagggacttc 3480 cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctccactac 3540 tctggatcca tcaatgcctg gagcaccaag gagccattca gctggatcaa agtggacctg 3600 ctggccccca tgatcatcca tggcatcaag acccaggggg ccaggcagaa gttctccagc 3660 ctgtacatca gccagttcat catcatgtac agcctggatg gcaagaaatg gcagacctac 3720 agaggcaact ccactggaac actcatggtc ttctttggca atgtggacag ctctggcatc 3780 aagcacaaca tcttcaaccc cccaatcatc gccagataca tcaggctgca ccccacccac 3840 tacagcatcc gcagcaccct caggatggag ctgatgggct gtgacctgaa ctcctgcagc 3900 atgcccctgg gcatggagag caaggccatt tctgatgccc agatcactgc ctccagctac 3960 ttcaccaaca tgtttgccac ctggagccca agcaaggcca ggctgcacct ccagggaagg 4020 agcaatgcct ggaggcccca ggtcaacaac ccaaaggagt ggctgcaggt ggacttccag 4080 aagaccatga aggtcactgg ggtgaccacc cagggggtca agagcctgct caccagcatg 4140 tatgtgaagg agttcctgat cagctccagc caggatggcc accagtggac cctcttcttc 4200 cagaatggca aggtcaaggt gttccagggc aaccaggaca gcttcacccc tgtggtgaac 4260 agcctggacc cccccctcct gaccagatac ctgaggattc acccccagag ctgggtccac 4320 cagattgccc tgaggatgga ggtcctggga tgtgaggccc aggacctgta ctga 4374 <210> 2 <211> 1457 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 1 5 10 15 Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45 Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile 65 70 75 80 Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95 Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110 His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125 Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140 Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu 145 150 155 160 Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175 Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190 Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205 Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220 Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235 240 Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255 Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270 Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285 Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300 Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met 305 310 315 320 Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335 Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350 Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365 Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380 Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765 Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 770 775 780 Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu 785 790 795 800 Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 805 810 815 Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 820 825 830 Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845 Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860 Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His 865 870 875 880 Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885 890 895 Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 900 905 910 Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 915 920 925 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940 Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 945 950 955 960 Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 965 970 975 Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990 Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000 1005 Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1010 1015 1020 Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1025 1030 1035 Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1040 1045 1050 Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1055 1060 1065 Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1070 1075 1080 Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1085 1090 1095 Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1100 1105 1110 Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1115 1120 1125 Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1130 1135 1140 Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1145 1150 1155 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1160 1165 1170 Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1175 1180 1185 Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1190 1195 1200 Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1205 1210 1215 Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1220 1225 1230 Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240 1245 Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1250 1255 1260 Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1265 1270 1275 Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1280 1285 1290 Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1295 1300 1305 Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1310 1315 1320 Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1325 1330 1335 Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1340 1345 1350 Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1355 1360 1365 Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1370 1375 1380 Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1385 1390 1395 Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 1400 1405 1410 Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1415 1420 1425 Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1430 1435 1440 Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1445 1450 1455 <210> 3 <211> 2220 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 3 gccaccagga gatactacct gggggctgtg gagctttctt gggactacat gcagtctgac 60 ctgggggagc tgcctgtgga tgccaggttc ccacccagag tgcccaaatc cttcccattc 120 aacacctctg tggtctacaa gaagaccctc tttgtggagt tcactgacca cctgttcaac 180 attgccaaac ccaggccacc ctggatggga ctcctgggac ccaccattca ggctgaggtg 240 tatgacactg tggtcatcac cctcaagaac atggcctccc accctgtgag cctgcatgct 300 gtgggggtca gctactggaa ggcctctgag ggggctgagt atgatgacca gacctcccag 360 agggagaagg aggatgacaa agtgttccct gggggcagcc acacctatgt gtggcaggtc 420 ctcaaggaga atggccccat ggcctctgac ccactctgcc tgacctactc ctacctttct 480 catgtggacc tggtcaagga cctcaactct ggactgattg gggccctgct ggtgtgcagg 540 gagggctccc tggccaaaga gaagacccag accctgcaca agttcattct cctgtttgct 600 gtctttgatg agggcaagag ctggcactct gaaaccaaga actccctgat gcaggacagg 660 gatgctgcct ctgccagggc ctggcccaag atgcacactg tgaatggcta tgtgaacagg 720 agcctgcctg gactcattgg ctgccacagg aaatctgtct actggcatgt gattggcatg 780 gggacaaccc ctgaggtgca ctccattttc ctggagggcc acaccttcct ggtcaggaac 840 cacagacagg ccagcctgga gatcagcccc atcaccttcc tcactgccca gaccctgctg 900 atggacctcg gacagttcct gctgttctgc cacatcagct cccaccagca tgatggcatg 960 gaggcctatg tcaaggtgga cagctgccct gaggagccac agctcaggat gaagaacaat 1020 gaggaggctg aggactatga tgatgacctg actgactctg agatggatgt ggtccgcttt 1080 gatgatgaca acagcccatc cttcattcag atcaggtctg tggccaagaa acaccccaag 1140 acctgggtgc actacattgc tgctgaggag gaggactggg actatgcccc actggtcctg 1200 gcccctgatg acaggagcta caagagccag tacctcaaca atggcccaca gaggattgga 1260 cgcaagtaca agaaagtcag gttcatggcc tacactgatg aaaccttcaa gaccagggag 1320 gccattcagc atgagtctgg catcctgggc ccactcctgt atggggaggt gggggacacc 1380 ctgctcatca tcttcaagaa ccaggcctcc aggccctaca acatctaccc acatggcatc 1440 actgatgtca ggcccctgta cagccgcagg ctgccaaagg gggtgaaaca cctcaaggac 1500 ttccccattc tgcctgggga gatcttcaag tacaagtgga ctgtcactgt ggaggatgga 1560 ccaaccaaat ctgaccccag gtgcctcacc agatactact ccagctttgt gaacatggag 1620 agggacctgg cctctggcct gattggccca ctgctcatct gctacaagga gtctgtggac 1680 cagaggggaa accagatcat gtctgacaag aggaatgtga ttctgttctc tgtctttgat 1740 gagaacagga gctggtacct gactgagaac attcagcgct tcctgcccaa ccctgctggg 1800 gtgcagctgg aggaccctga gttccaggcc agcaacatca tgcactccat caatggctat 1860 gtgtttgaca gcctccagct ttctgtctgc ctgcatgagg tggcctactg gtacattctt 1920 tctattgggg cccagactga cttcctttct gtcttcttct ctggctacac cttcaaacac 1980 aagatggtgt atgaggacac cctgaccctc ttcccattct ctggggagac tgtgttcatg 2040 agcatggaga accctggcct gtggattctg ggatgccaca actctgactt ccgcaacagg 2100 ggcatgactg ccctgctcaa agtctcctcc tgtgacaaga acactgggga ctactatgag 2160 gacagctatg aggacatctc tgcctacctg ctcagcaaga acaatgccat tgagcccagg 2220 <210> 4 <211> 2052 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 4 gagatcacca ggaccaccct ccagtctgac caggaggaga ttgactatga tgacaccatt 60 tctgtggaga tgaagaaaga ggactttgac atctatgacg aggacgagaa ccagagccca 120 aggagcttcc agaagaagac caggcactac ttcattgctg ctgtggagcg cctgtgggac 180 tatggcatga gctccagccc ccatgtcctc aggaacaggg cccagtctgg ctctgtgcca 240 cagttcaaga aagtggtctt ccaagagttc actgatggca gcttcaccca gccctgtac 300 agaggggagc tgaatgagca cctgggactc ctgggcccat acatcagggc tgaggtggag 360 gacaacatca tggtgacctt ccgcaaccag gcctccaggc cctacagctt ctacagctcc 420 ctcatcagct atgaggagga ccagaggcag ggggctgagc cacgcaagaa ctttgtgaaa 480 cccaatgaaa ccaagaccta cttctggaaa gtccagcacc acatggcccc caccaaggat 540 gagtttgact gcaaggcctg ggcctacttc tctgatgtgg acctggagaa ggatgtgcac 600 tctggcctga ttggcccact cctggtctgc cacaccaaca ccctgaaccc tgcccatgga 660 aggcaagtga ctgtgcagga gtttgccctc ttcttcacca tctttgatga aaccaagagc 720 tggtacttca ctgagaacat ggagcgcaac tgcagggccc catgcaacat tcagatggag 780 gaccccacct tcaaagagaa ctaccgcttc catgccatca atggctacat catggacacc 840 ctgcctgggc ttgtcatggc ccaggaccag aggatcaggt ggtacctgct ttctatgggc 900 tccaatgaga acattcactc catccacttc tctgggcatg tcttcactgt gcgcaagaag 960 gaggagtaca agatggccct gtacaacctc taccctgggg tctttgagac tgtggagatg 1020 ctgccctcca aagctggcat ctggagggtg gagtgcctca ttggggagca cctgcatgct 1080 ggcatgagca ccctgttcct ggtctacagc aacaagtgcc agacccccct gggaatggcc 1140 tctggccaca tcagggactt ccagatcact gcctctggcc agtatggcca gtgggccccc 1200 aagctggcca ggctccacta ctctggatcc atcaatgcct ggagcaccaa ggagccattc 1260 agctggatca aagtggacct gctggccccc atgatcatcc atggcatcaa gacccagggg 1320 gccaggcaga agttctccag cctgtacatc agccagttca tcatcatgta cagcctggat 1380 ggcaagaaat ggcagaccta cagaggcaac tccactggaa cactcatggt cttctttggc 1440 aatgtggaca gctctggcat caagcacaac atcttcaacc ccccaatcat cgccagatac 1500 atcaggctgc accccaccca ctacagcatc cgcagcaccc tcaggatgga gctgatgggc 1560 tgtgacctga actcctgcag catgcccctg ggcatggaga gcaaggccat ttctgatgcc 1620 cagatcactg cctccagcta cttcaccaac atgtttgcca cctggagccc aagcaaggcc 1680 aggctgcacc tccagggaag gagcaatgcc tggaggcccc aggtcaacaa cccaaaggag 1740 tggctgcagg tggacttcca gaagaccatg aaggtcactg gggtgaccac ccagggggtc 1800 aagagcctgc tcaccagcat gtatgtgaag gagttcctga tcagctccag ccaggatggc 1860 caccagtgga ccctcttctt ccagaatggc aaggtcaagg tgttccaggg caaccaggac 1920 agcttcaccc ctgtggtgaa cagcctggac ccccccctcc tgaccagata cctgaggatt 1980 cacccccaga gctgggtcca ccagattgcc ctgaggatgg aggtcctggg atgtgaggcc 2040 caggacctgt ac 2052 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 5 agcttctctc agaatccacc tgtcctgaag agacaccaga ga 42 <210> 6 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 6 agcttcagcc agaatccacc tgtcctgaaa cgccaccaga gg 42 <210> 7 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 7 agcttcagcc agaacccccc cgtgctgaag aggcaccaga gg 42 <210> 8 <211> 7827 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 8 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cctcgagatt taaatgacgt 420 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc 480 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcaga gagggagtgg 540 ccaactccat cactaggggt tcctgagttt aaacttcgtc gacgattcga gcttgggctg 600 caggtcgagg gcactgggag gatgttgagt aagatggaaa actactgatg acccttgcag 660 agacagagta ttaggacatg tttgaacagg ggccgggcga tcagcaggta gctctagagg 720 atccccgtct gtctgcacat ttcgtagagc gagtgttccg atactctaat ctccctaggc 780 aaggttcata tttgtgtagg ttacttattc tccttttgtt gactaagtca ataatcagaa 840 tcagcaggtt tggagtcagc ttggcaggga tcagcagcct gggttggaag gagggggtat 900 aaaagcccct tcaccaggag aagccgtcac acagactagg cgcgccaccg ccaccatgca 960 gattgagctg agcacctgct tcttcctgtg cctgctgagg ttctgcttct ctgccaccag 1020 gagatactac ctgggggctg tggagctttc ttgggactac atgcagtctg acctggggga 1080 gctgcctgtg gatgccaggt tcccacccag agtgcccaaa tccttcccat tcaacacctc 1140 tgtggtctac aagaagaccc tctttgtgga gttcactgac cacctgttca acattgccaa 1200 acccaggcca ccctggatgg gactcctggg acccaccatt caggctgagg tgtatgacac 1260 tgtggtcatc accctcaaga acatggcctc ccaccctgtg agcctgcatg ctgtgggggt 1320 cagctactgg aaggcctctg agggggctga gtatgatgac cagacctccc agagggagaa 1380 ggaggatgac aaagtgttcc ctgggggcag ccacacctat gtgtggcagg tcctcaagga 1440 gaatggcccc atggcctctg acccactctg cctgacctac tcctaccttt ctcatgtgga 1500 cctggtcaag gacctcaact ctggactgat tggggccctg ctggtgtgca gggagggctc 1560 cctggccaaa gagaaccc agaccctgca caagttcatt ctcctgtttg ctgtctttga 1620 tgagggcaag agctggcact ctgaaaccaa gaactccctg atgcaggaca gggatgctgc 1680 ctctgccagg gcctggccca agatgcacac tgtgaatggc tatgtgaaca ggagcctgcc 1740 tggactcatt ggctgccaca ggaaatctgt ctactggcat gtgattggca tggggacaac 1800 ccctgaggtg cactccattt tcctggaggg ccacaccttc ctggtcagga accacagaca 1860 ggccagcctg gagatcagcc ccatcacctt cctcactgcc cagaccctgc tgatggacct 1920 cggacagttc ctgctgttct gccacatcag ctcccaccag catgatggca tggaggccta tgtcaaggtg gacagctgcc ctgaggagcc acagctcagg atgaagaaca atgaggaggc tgaggactat gatgacc tgactgactc tgagatgat gtggtccgct ttgatgatga caacagccca tccttcattc agatcaggtc tgtggccaag aaacacccca agacctgggt gcactacatt gctgctgagg aggaggactg ggactatgcc ccactggtcc tggcccctga 2220 tgacaggagc tacaagagcc agtacctcaa caatggccca cagaggattg gacgcaagta caagaagtc aggttcatgg cctacactga tgaaaccttc aagaccaggg aggccattca gcatgagtct ggcatcctgg gcccactcct gtatggggag gtggggaca ccctgctcat catcttcaag aaccaggcct ccaggcccta caacatctac ccacatggca tcactgatgt caggcccctg tacagccgca ggctgccaaa ggggggtgaaa cacctcaagg acttccccat tctgcctggg gagatcttca agtacaagtg gactgtcact gtgaggatg gaccaaccaa atctgacccc aggtgcctca ccagatacta ctccagcttt gtgaacatgg agagggacct 2640 ggcctctggc ctgattggcc cactgctcat ctgctacaag gagtctgtgg accagagggg 2700 aaaccagatc atgtctgaca agaggaatgt gattctgttc tctgtctttg atgagaacag 2760 gagctggtac ctgactgaga acattcagcg cttcctgccc aaccctgctg gggtgcagct 2820 ggaggaccct gagttccagg ccagcaacat catgcactcc atcaatggct atgtgtttga 2880 cagcctccag ctttctgtct gcctgcatga ggtggcctac tggtacattc tttctattgg 2940 ggcccagact gacttccttt ctgtcttctt ctctggctac accttcaaac acaagatggt 3000 gtatgaggac accctgaccc tcttcccatt ctctggggag actgtgttca tgagcatgga 3060 gaaccctggc ctgtggattc tgggatgcca caactctgac ttccgcaaca ggggcatgac 3120 tgccctgctc aaagtctcct cctgtgacaa gaacactggg gactactatg aggacagcta 3180 tgaggacatc tctgcctacc tgctcagcaa gaacaatgcc attgagccca ggagcttcag 3240 ccagaatcca cctgtcctga aacgccacca gagggagatc accaggacca ccctccagtc 3300 tgaccaggag gagattgact atgatgacac catttctgtg gagatgaaga aagaggactt 3360 tgacatctat gacgaggacg agaaccagag cccaaggagc ttccagaaga agaccaggca 3420 ctacttcatt gctgctgtgg agcgcctgtg ggactatggc atgagctcca gcccccatgt 3480 cctcaggaac agggcccagt ctggctctgt gccacagttc aagaaagtgg tcttccaaga 3540 gttcactgat ggcagcttca cccagcccct gtacagaggg gagctgaatg agcacctggg 3600 actcctgggc ccatacatca gggctgaggt ggaggacaac atcatggtga ccttccgcaa 3660 ccaggcctcc aggccctaca gcttctacag ctccctcatc agctatgagg aggaccagag 3720 gcagggggct gagccacgca agaactttgt gaaacccaat gaaaccaaga cctacttctg 3780 gaaagtccag caccacatgg cccccaccaa ggatgagttt gactgcaagg cctgggccta 3840 cttctctgat gtggacctgg agaaggatgt gcactctggc ctgattggcc cactcctggt 3900 ctgccacacc aacaccctga accctgccca tggaaggcaa gtgactgtgc aggagtttgc 3960 cctcttcttc accatctttg atgaaaccaa gagctggtac ttcactgaga acatggagcg 4020 caactgcagg gccccatgca acattcagat ggaggacccc accttcaaag agaactaccg 4080 cttccatgcc atcaatggct acatcatgga caccctgcct gggcttgtca tggcccagga 4140 ccagaggatc aggtggtacc tgctttctat gggctccaat gagaacattc actccatcca 4200 cttctctggg catgtcttca ctgtgcgcaa gaaggaggag tacaagatgg ccctgtacaa 4260 cctctaccct ggggtctttg agactgtgga gatgctgccc tccaaagctg gcatctggag 4320 ggtggagtgc ctcattgggg agcacctgca tgctggcatg agcaccctgt tcctggtcta 4380 cagcaacaag tgccagaccc ccctgggaat ggcctctggc cacatcaggg acttccagat 4440 cactgcctct ggccagtatg gccagtgggc ccccaagctg gccaggctcc actactctgg 4500 atccatcaat gcctggagca ccaaggagcc attcagctgg atcaaagtgg acctgctggc 4560 ccccatgatc atccatggca tcaagaccca gggggccagg cagaagttct ccagcctgta 4620 catcagccag ttcatcatca tgtacagcct ggatggcaag aaatggcaga cctacagagg 4680 caactccact ggaacactca tggtcttctt tggcaatgtg gacagctctg gcatcaagca 4740 caacatcttc aaccccccaa tcatcgccag atacatcagg ctgcacccca cccactacag 4800 catccgcagc accctcagga tggagctgat gggctgtgac ctgaactcct gcagcatgcc 4860 cctgggcatg gagagcaagg ccatttctga tgcccagatc actgcctcca gctacttcac 4920 caacatgttt gccacctgga gcccaagcaa ggccaggctg cacctccagg gaaggagcaa 4980 tgcctggagg ccccaggtca acaacccaaa ggagtggctg caggtggact tccagaagac 5040 catgaaggtc actggggtga ccacccaggg ggtcaagagc ctgctcacca gcatgtatgt 5100 gaaggagttc ctgatcagct ccagccagga tggccaccag tggaccctct tcttccagaa 5160 tggcaaggtc aaggtgttcc agggcaacca ggacagcttc acccctgtgg tgaacagcct 5220 ggaccccccc ctcctgacca gatacctgag gattcacccc cagagctggg tccaccagat 5280 tgccctgagg atggaggtcc tgggatgtga ggcccaggac ctgtactgat gacgagcggc 5340 cgctcttagt agcagtatcg ataataaaag atctttatttt tcattagatc tgtgtgttgg 5400 ttttttgtgt gttaattaag ctcgcgaagg aacccctagt gatggagttg gccactccct 5460 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct 5520 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aagacgattt 5580 aaatgacaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 5640 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 5700 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 5760 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 5820 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 5880 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 5940 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 6000 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 6060 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 6120 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 6180 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 6240 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 6300 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 6360 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 6420 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 6480 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 6540 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 6600 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 6660 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 6720 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 6780 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 6840 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 6900 taccgcgaga cccacgctca ccggctccag attackcagc ataaaccag cccaccggaa 6960 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct atttattgtt 7020 gccgggaagc tagatagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 7080 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 7140 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaagcggtt agctccttcg 7200 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 7260 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 7320 actcaaccaa gtcattctga gatagtgta tgcggcgacc gagttgctt tgcccggcgt 7380 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaac 7440 gttcttcggg gcgaaaactc tcaggatct taccgctgtt gagatccagt tcgatgtaac 7500 ccactcgtgc acccaactga tcttcagcat cttttactt caccagcgtt tctgggtgag 7560 caaaaacagg aaggcaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 7620 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 7680 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 7740 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 7800 ataggcgtat cacgaggccc tttcgtc 7827 <210> 9 <211> 4332 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 9 atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgcc 60 accaggagat actacctggg ggctgtggag ctttcttggg actacatgca gtctgacctg 120 ggggagctgc ctgtggatgc caggttccca cccagagtgc ccaaatcctt cccattcaac 180 acctctgtgg tctacaagaa gaccctcttt gtggagttca ctgaccacct gttcaacatt 240 gccaaaccca ggccaccctg gatgggactc ctgggaccca ccattcaggc tgaggtgtat 300 gacactgtgg tcatcaccct caagaacatg gcctcccacc ctgtgagcct gcatgctgtg 360 ggggtcagct actggaaggc ctctgagggg gctggattg atgaccagac ctcccagagg 420 gagaaggagg atgacaaagt gttccctggg ggcagccaca cctatgtgtg gcaggtcctc 480 aaggaagaatg gccccatggc ctctgaccca ctctgcctga cctactccta cctttctcat 540 gtggacctgg tcaaggacct caactctgga ctgattgggg ccctgctggt gtgcagggag 600 ggctccctgg ccaaaagaga gacccagacc ctgcacaagt tcattctcct gtttgctgtc 660 tttgatgagg gcaagagctg gcactctgaa accaagaact ccctgatgca ggacagggat 720 gctgcccttg ccagggcctg gcccaagatg caacactgtga atggctatgt gaacaggagc 780 ctgcctggac tcattggctg ccacaggaaa tctgtctact ggcatgtgat tggcatgggg 840 acaacccctg aggtgcactc cattttcctg gagggccaca ccttcctggt caggaaccac 900 agacaggcca gcctggagat cagccccatc accttctcca ctgcccagac cctgctgatg 960 gacctcggac agttcctgct gttctgccac atcagctccc accagcatga tggcatggag 1020 gcctatgtca aggtggacag ctgccctgag gagccacagc tcaggatgaa gaacaatgag 1080 gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt ccgctttgat 1140 gatgacaaca gcccatcctt cattcagatc aggtctgtgg ccaagaaaca ccccaagacc 1200 tgggtgcact acattgctgc tgaggaggag gactgggact atgccccact ggtcctggcc 1260 cctgatgaca ggagctacaa gagccagtac ctcaacaatg gcccacagag gattggacgc 1320 aagtacaaga aagtcaggtt catggcctac actgatgaaa ccttcaagac cagggaggcc 1380 attcagcatg agtctggcat cctgggccca ctcctgtatg gggaggtggg ggacaccctg 1440 ctcatcatct tcaagaacca ggcctccagg ccctacaaca tctacccaca tggcatcact 1500 gatgtcaggc ccctgtacag ccgcaggctg ccaaaggggg tgaaacacct caaggacttc 1560 cccattctgc ctggggagat cttcaagtac aagtggactg tcactgtgga ggatggacca 1620 accaaatctg accccaggtg cctcaccaga tactactcca gctttgtgaa catggagagg 1680 gacctggcct ctggcctgat tggcccactg ctcatctgct acaaggagtc tgtggaccag 1740 aggggaaacc agatcatgtc tgacaagagg aatgtgattc tgttctctgt ctttgatgag 1800 aacaggagct ggtacctgac tgagaacatt cagcgcttcc tgcccaaccc tgctggggtg 1860 cagctggagg accctgagtt ccaggccagc aacatcatgc actccatcaa tggctatgtg 1920 tttgacagcc tccagctttc tgtctgcctg catgaggtgg cctactggta cattctttct 1980 attggggccc agactgactt cctttctgtc ttcttctctg gctacacctt caaacacaag 2040 atggtgtatg aggacaccct gaccctcttc ccattctctg gggagactgt gttcatgagc 2100 atggagaacc ctggcctgtg gattctggga tgccacaact ctgacttccg caacaggggc 2160 atgactgccc tgctcaaagt ctcctcctgt gacaagaaca ctggggacta ctatgaggac 2220 agctatgagg acatctctgc ctacctgctc agcaagaaca atgccattga gcccagggag 2280 atcaccagga ccaccctcca gtctgaccag gaggagattg actatgatga caccatttct 2340 gtggagatga agaaagagga ctttgacatc tatgacgagg acgagaacca gagcccaagg 2400 agcttccaga agaagaccag gcactacttc attgctgctg tggagcgcct gtgggactat 2460 ggcatgagct ccagccccca tgtcctcagg aacagggccc agtctggctc tgtgccacag 2520 ttcaagaaag tggtcttcca agagttcact gatggcagct tcacccagcc cctgtacaga 2580 ggggagctga atgagcacct gggactcctg ggcccataca tcagggctga ggtggaggac 2640 aacatcatgg tgaccttccg caaccaggcc tccaggccct acagcttcta cagctccctc 2700 atcagctatg aggaggacca gaggcagggg gctgagccac gcaagaactt tgtgaaaccc 2760 aatgaaacca agacctactt ctggaaagtc cagcaccaca tggcccccac caaggatgag 2820 tttgactgca aggcctgggc ctacttctct gatgtggacc tggagaagga tgtgcactct 2880 ggcctgattg gcccactcct ggtctgccac accaacaccc tgaaccctgc ccatggaagg 2940 caagtgactg tgcaggagtt tgccctcttc ttcaccatct ttgatgaaac caagagctgg 3000 tacttcactg agaacatgga gcgcaactgc agggccccat gcaacattca gatggaggac 3060 cccaccttca aagagaacta ccgcttccat gccatcaatg gctacatcat ggacaccctg 3120 cctgggcttg tcatggccca ggaccagagg atcaggtggt acctgctttc tatgggctcc 3180 aatgagaaca ttcactccat ccacttctct gggcatgtct tcactgtgcg caagaaggag 3240 gagtacaaga tggccctgta caacctctac cctggggtct ttgagactgt ggagatgctg 3300 ccctccaaag ctggcatctg gagggtggag tgcctcattg gggagcacct gcatgctggc 3360 atgagcaccc tgttcctggt ctacagcaac aagtgccaga cccccctggg aatggcctct 3420 ggccacatca gggacttcca gatcactgcc tctggccagt atggccagtg ggcccccaag 3480 ctggccaggc tccactactc tggatccatc aatgcctgga gcaccaagga gccattcagc 3540 tggatcaaag tggacctgct ggcccccatg atcatccatg gcatcaagac ccagggggcc 3600 aggcagaagt tctccagcct gtacatcagc cagttcatca tcatgtacag cctggatggc 3660 aagaaatggc agacctacag aggcaactcc actggaacac tcatggtctt ctttggcaat 3720 gtggacagct ctggcatcaa gcacaacatc ttcaaccccc caatcatcgc cagatacatc 3780 aggctgcacc ccacccacta cagcatccgc agcaccctca ggatggagct gatgggctgt 3840 gacctgaact cctgcagcat gcccctgggc atggagagca aggccatttc tgatgcccag 3900 atcactgcct ccagctactt caccaacatg tttgccacct ggagcccaag caaggccagg 3960 ctgcacctcc agggaaggag caatgcctgg aggccccagg tcaacaaccc aaaggagtgg 4020 ctgcaggtgg acttccagaa gaccatgaag gtcactgggg tgaccaccca gggggtcaag 4080 agcctgctca ccagcatgta tgtgaaggag ttcctgatca gctccagcca ggatggccac 4140 cagtggaccc tcttcttcca gaatggcaag gtcaaggtgt tccagggcaa ccaggacagc 4200 ttcacccctg tggtgaacag cctggacccc cccctcctga cagatacct gaggattcac 4260 ccccagagct gggtccacca gattgccctg aggatggagg tcctgggatg tgaggcccag 4320 gacctgtact ga 4332 <210> 10 <211> 1443 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 With Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 1 5 10 15 Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45 Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile 65 70 75 80 Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95 Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110 His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125 Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140 Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu 145 150 155 160 Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175 Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190 Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205 Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220 Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235 240 Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255 Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270 Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285 Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300 Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met 305 310 315 320 Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335 Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350 Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr ...

Claims

1. A polynucleotide comprising a nucleotide sequence having at least 95% identity to CS04-FL-NA (SEQ ID NO: 1), The polynucleotide encoding a Factor VIII polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain.

2. The polynucleotide of claim 1, wherein the nucleotide sequence has at least 96% identity to CS04-FL-NA (SEQ ID NO:1).

3. The polynucleotide of claim 1 or 2, wherein the nucleotide sequence has at least 97% identity to CS04-FL-NA (SEQ ID NO: 1).

4. The polynucleotide of any one of claims 1 to 3, wherein the nucleotide sequence has at least 98% identity to CS04-FL-NA (SEQ ID NO: 1).

5. The polynucleotide of any one of claims 1 to 4, wherein the nucleotide sequence has at least 99% identity to CS04-FL-NA (SEQ ID NO: 1).

6. The polynucleotide of any one of claims 1 to 5, wherein the Factor VIII polypeptide has at least 97% identity to CS04-FL-AA (SEQ ID NO:2).

7. The polynucleotide of any one of claims 1 to 6, wherein the Factor VIII polypeptide has at least 98% identity to CS04-FL-AA (SEQ ID NO:2).

8. 8. The polynucleotide of any one of claims 1 to 7, wherein the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS04-HC-NA (SEQ ID NO:3).

9. 8. The polynucleotide of any one of claims 1 to 7, wherein the light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS04-LC-NA (SEQ ID NO: 4).

10. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS04-HC-NA (SEQ ID NO:3); and 8. The polynucleotide of any one of claims 1 to 7, wherein the light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS04-LC-NA (SEQ ID NO: 4).

11. The polynucleotide of claim 8 or 10, wherein the first nucleotide sequence has at least 96% identity to CS04-HC-NA (SEQ ID NO: 3).

12. The polynucleotide of claim 8 or 10, wherein the first nucleotide sequence has at least 97% identity to CS04-HC-NA (SEQ ID NO: 3).

13. The polynucleotide of claim 8 or 10, wherein the first nucleotide sequence has at least 98% identity to CS04-HC-NA (SEQ ID NO: 3).

14. The polynucleotide of claim 9 or 10, wherein the second nucleotide sequence has at least 96% identity to CS04-LC-NA (SEQ ID NO: 4).

15. The polynucleotide of claim 9 or 10, wherein the second nucleotide sequence has at least 97% identity to CS04-LC-NA (SEQ ID NO: 4).

16. The polynucleotide of claim 9 or 10, wherein the second nucleotide sequence has at least 98% identity to CS04-LC-NA (SEQ ID NO: 4).

17. The polynucleotide of any one of claims 1 to 16, wherein the polypeptide linker comprises a furin cleavage site.

18. An adeno-associated virus (AAV) vector comprising the polynucleotide of any one of claims 1 to 17.

19. An adeno-associated virus (AAV) particle comprising a polynucleotide according to any one of claims 1 to 17.

20. A host cell infected with an adeno-associated virus (AAV) particle comprising a polynucleotide according to any one of claims 1 to 17.

21. Introducing a polynucleotide according to any one of claims 1 to 17 into a mammalian host cell.

1. An in vitro method for producing an adeno-associated virus (AAV) particle, comprising:

22. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 17 for use in the treatment of hemophilia.

23. 20. Use of a polynucleotide according to any one of claims 1 to 17 in the manufacture of a medicament for the treatment of hemophilia.

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