Viral vectors encoding recombinant fviii variants with increased expression for gene therapy of hemophilia a

Abstract

The present disclosure provides, among other aspects, codon-altered polynucleotides encoding Factor VIII variants for expression in mammalian cells. In some embodiments, the disclosure also provides mammalian gene therapy vectors and methods for treating hemophilia A.

Description

VIRAL VECTORS ENCODING RECOMBINANT FVIII VARIANTS WITH INCREASED EXPRESSION FOR GENE THERAPY OF HEMOPHILIA ACROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 511,979, filed July 5, 2023, the content of which is hereby incorporated by reference herein, in its entirety, for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on July 1, 2024, is named 008073-5248-WO_SequenceListing, and is 199,237 bytes in size.BACKGROUND OF THE DISCLOSURE

[0003] Blood coagulation proceeds through a complex and dynamic biological pathway of interdependent biochemical reactions, referred to as the coagulation cascade. Coagulation Factor VIII (FVIII) is a key component in the cascade. Factor VIII is recruited to bleeding sites, and forms a Xase complex with activated Factor IX (FIXa) and Factor X (FX). The Xase complex activates FX, which in turn activates prothrombin to thrombin, which then activates other components in the coagulation cascade to generate a stable clot.

[0004] Hemophilia A is a congenital X-linked bleeding disorder characterized by a deficiency in Factor VIII activity. Diminished Factor VIII activity inhibits a positive feedback loop in the coagulation cascade. This causes incomplete coagulation, which manifests as bleeding episodes with increased duration, extensive bruising, spontaneous oral and nasal bleeding, joint stiffness and chronic pain, and possibly internal bleeding and anemia in severe cases.

[0005] Conventionally, hemophilia A is treated by Factor VIII replacement therapy, which consists of administering Factor VIII protein (e.g., plasma-derived or recombinantly-produced Factor VIII) to an individual with hemophilia A. Factor VIII is administered prophylactically to prevent or reduce frequency of bleeding episodes, in response to an acute bleeding episode, and / orperioperatively to manage bleeding during surgery. However, there are several undesirable features of Factor VIII replacement therapy.

[0006] First, Factor VIII replacement therapy is used to treat or manage hemophilia A, but does not cure the underlying Factor VIII deficiency. Because of this, individuals with hemophilia A require Factor VIII replacement therapy for the duration of their lives. Continuous treatment is expensive and requires the individual to maintain strict compliance, as missing only 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 vivo, conventional prophylactic Factor VIII replacement therapy requires administration every second or third day. This places a burden on the individual to maintain compliance throughout their life. While third generation “long-acting” Factor VIII drugs may reduce the frequency of administration, prophylactic Factor FVIII replacement therapy with these drugs still requires monthly, weekly, or more frequent administration in perpetuity. For example, prophylactic treatment with ELOCTATE™ [Antihemophilic Factor (Recombinant), FL Fusion Protein] requires administration every three to five days (ELOCTATE™ Prescribing Information, Biogen Idee Inc., (2015)). Moreover, the long-term effects of chemically modified biologies (e.g., pegylated polypeptides) are not yet fully understood.

[0008] Third, between 15% and 30% of all individuals receiving Factor VIII replacement therapy form anti-Factor VIII inhibitor antibodies, rendering the therapy inefficient. Factor VIII bypass therapy (e.g., administration of plasma-derived or recombinantly-produced prothrombin complex concentrates) can be used to treat hemophilia in individuals that form inhibitor antibodies. However, Factor VIII bypass therapy is less effective than Factor VIII replacement therapy.

[0009] Somatic gene therapy holds great promise for the treatment of hemophilia A because it would remedy the underlying under-expression functional Factor VIII activity (e.g., due to missense or nonsense mutations), rather than provide a one-time dose of Factor VIII activity to the individual. Because of this difference in the mechanism of action, as compared to Factor VIII replacement therapy, one-time administration of a Factor VIII gene therapy vector may provide an individual with Factor VIII for several years, reducing the cost of treatment and eliminating the need for continued patient compliance.

[0010] Coagulation Factor IX (FIX) gene therapy has been used effectively to treat individuals with hemophilia B, a related blood coagulation condition characterized by diminished Factor IX activity. However, Factor VIII gene therapy presents several unique 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). The coding sequence of wild-type Factor VIII is 7053 base pairs, which is too large to be packaged in conventional AAV gene therapy vectors. Further, reported recombinant expression of B-domain deleted variants of Factor VIII (BDD-FVIII) has been poor. It is believed that several groups have attempted to alter the codon usage of BDD- FVIII constructs, with limited success.BRIEF SUMMARY OF DISCLOSURE

[0011] Accordingly, there is a need for Factor VIII variants whose coding sequences are more efficiently packaged into, and delivered via, gene therapy vectors. There is also a need for synthetic, codon-altered nucleic acids which express Factor VIII more efficiently. The Factor VIII variants with codon-altered nucleic acids disclosed herein provide significantly higher Factor VIII activity when expressed in cell culture and in vivo.BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 shows schematic illustrations of the wild-type human Factor VIII and a B-domain deleted human Factor VIII construct, in accordance with some embodiments of the disclosure.

[0013] Figure 2 shows the BDD-FVIII full length (FL) nucleic acid sequence (BDD-FVIII-FL- NA, SEQ ID NO: 1) encoding a Factor VIII protein.

[0014] Figure 3 shows partial nucleic acid sequences of BDD-FVIII-FL-NA encoding the FVIII heavy chain (SEQ ID NO:2), FVIII light chain (SEQ ID NO: 3), and a B domain linker (SEQ ID NO:4).

[0015] Figure 4 shows the BDD-FVIII full length (SEQ ID NO: 5), heavy chain (SEQ ID NO: 6), light chain (SEQ ID NO: 7), and B domain linker (SEQ ID NO: 8) amino acid sequences.

[0016] Figure 5 shows the codon-optimized CS01 full length (FL) nucleic acid sequence (SEQ ID NOV).

[0017] Figure 6 shows partial nucleic acid sequences of the codon-optimized CS01 construct encoding the FVIII heavy chain (SEQ ID NO: 10), FVIII light chain (SEQ ID NO: 11), and a B domain linker (SEQ ID NO:4).

[0018] Figure 7 shows the PS01 full length (SEQ ID NO: 12), heavy chain (SEQ ID NO: 13), light chain (SEQ ID NO: 14), and B domain linker (SEQ ID NO: 8) amino acid sequences.

[0019] Figure 8 shows the codon-optimized CS02 full length (FL) nucleic acid sequence (SEQ ID NO: 15).

[0020] Figure 9 shows partial nucleic acid sequences of the codon-optimized CS02 construct encoding the FVIII heavy chain (SEQ ID NO: 16), FVIII light chain (SEQ ID NO: 17), and a B domain linker (SEQ ID NO:4).

[0021] Figure 10 shows the PS02 full length (SEQ ID NO: 18), heavy chain (SEQ ID NO: 19), light chain (SEQ ID NO:20), and B domain linker (SEQ ID NO: 8) amino acid sequences.

[0022] Figure 11 shows the codon-optimized CS03 full length (FL) nucleic acid sequence (SEQ ID NO:21).

[0023] Figure 12 shows partial nucleic acid sequences of the codon-optimized CS03 construct encoding the FVIII heavy chain (SEQ ID NO:22), FVIII light chain (SEQ ID NO:23), and a B domain linker (SEQ ID NO:4).

[0024] Figure 13 shows the PS03 full length (SEQ ID NO:24), heavy chain (SEQ ID NO:25), light chain (SEQ ID NO:26), and B domain linker (SEQ ID NO: 8) amino acid sequences.

[0025] Figure 14 shows the codon-optimized CS04 full length (FL) nucleic acid sequence (SEQ ID NO:27).

[0026] Figure 15 shows partial nucleic acid sequences of the codon-optimized CS04 construct encoding the FVIII heavy chain (SEQ ID NO:28), FVIII light chain (SEQ ID NO:29), and a B domain linker (SEQ ID NO:4).

[0027] Figure 16 shows thePS04 full length (SEQ ID NO:30), heavy chain (SEQ ID NO:31), light chain (SEQ ID NO:32), and B domain linker (SEQ ID NO: 8) amino acid sequences.

[0028] Figure 17 shows the codon-optimized CS05 full length (FL) nucleic acid sequence (SEQ ID NO:33).

[0029] Figure 18 shows partial nucleic acid sequences of the codon-optimized CS05 construct encoding the FVIII heavy chain (SEQ ID NO:34), FVIII light chain (SEQ ID NO:35), and a B domain linker (SEQ ID NO:4).

[0030] Figure 19 shows the PS05 full length (SEQ ID NO: 36), heavy chain (SEQ ID NO: 37), light chain (SEQ ID NO:38), and B domain linker (SEQ ID NO: 8) amino acid sequences.

[0031] Figure 20 shows the codon-optimized CS06 full length (FL) nucleic acid sequence (SEQ ID NO:39).

[0032] Figure 21 shows partial nucleic acid sequences of the codon-optimized CS06 construct encoding the FVIII heavy chain (SEQ ID NO:40), FVIII light chain (SEQ ID NO:41), and a B domain linker (SEQ ID NO:42).

[0033] Figure 22 shows the codon-optimized CS07 full length (FL) nucleic acid sequence (SEQ ID NO:43).

[0034] Figure 23 shows partial nucleic acid sequences of the codon-optimized CS07 construct encoding the FVIII heavy chain (SEQ ID NO:44), FVIII light chain (SEQ ID NO:45), and a B domain linker (SEQ ID NO:46).

[0035] Figure 24 shows the codon-optimized CS08 full length (FL) nucleic acid sequence (SEQ ID NO:47).

[0036] Figure 25 shows partial nucleic acid sequences of the codon-optimized CS08 construct encoding the FVIII heavy chain (SEQ ID NO:48), FVIII light chain (SEQ ID NO:49), and a B domain linker (SEQ ID NO: 50).

[0037] Figure 26 shows the codon-optimized CS09 full length (FL) nucleic acid sequence (SEQ ID NO:51).

[0038] Figure 27 shows partial nucleic acid sequences of the codon-optimized CS09 construct encoding the FVIII heavy chain (SEQ ID NO: 52), FVIII light chain (SEQ ID NO: 53), and a B domain linker (SEQ ID NO: 54).

[0039] Figure 28 shows the codon-optimized CS10 full length (FL) nucleic acid sequence (SEQ ID NO:55).

[0040] Figure 29 shows partial nucleic acid sequences of the codon-optimized CS10 construct encoding the FVIII heavy chain (SEQ ID NO: 56), FVIII light chain (SEQ ID NO: 57), and a B domain linker (SEQ ID NO:58).

[0041] Figure 30 shows the codon-optimized CS11 full length (FL) nucleic acid sequence (SEQ ID NO: 59).

[0042] Figure 31 shows partial nucleic acid sequences of the codon-optimized CS05 construct encoding the FVIII heavy chain (SEQ ID NO:60), FVIII light chain (SEQ ID NO:61), and a B domain linker (SEQ ID NO:62).

[0043] Figure 32 shows the codon-optimized CS12 full length (FL) nucleic acid sequence (SEQ ID NO:63).

[0044] Figure 33 shows partial nucleic acid sequences of the codon-optimized CS12 construct encoding the FVIII heavy chain (SEQ ID NO:64), FVIII light chain (SEQ ID NO:65), and a B domain linker (SEQ ID NO:66).

[0045] Figure 34 shows the sequence for the CRM8 enhancer (SEQ ID NO:67), the hTTR promoter (SEQ ID NO: 68), the MVM intron (SEQ ID NO: 69), the WPRE mut6delATG (SEQ ID NO:70), and the bGH polyA (SEQ ID NO:71) elements of the pMY168 plasmid backbone.

[0046] Figure 35 shows schematics of 13 codon-optimized BDD Factor VIII constructs in the pMY168 cloning plasmid, in accordance with some embodiments of the disclosure.

[0047] Figures 36A to 36C show the in vitro evaluation data of 13 codon-optimized BDD Factor VIII constructs in 4 different cell lines. Figure 36A summarizes the FVIII antigen (Factor VIII Antigen Plus Kit, Affinity Biologicals), and the FVIII activity (BIOPHENTM FVIII: C kit, HYPHEN BioMed) in Hepal-6, HepG2, HEK293T, and Huh7 cells compared to the cell onlynegative control, the no DNA negative control, and the pBDDFVIII control vector expression (BDD-FVIII). Figure 36B shows the graphical representation of the FVIII antigen data and Figure 36C shows the FVIII activity data of the data in Figure 36 A.

[0048] Figure 37 shows the experimental conditions for in vivo expression assays for 13 BDD Factor VIII variant constructs, in accordance with some embodiments of the disclosure.

[0049] Figures 38A, and 38B, show in vivo FVIII antigen data (Figure 38A) measured in mouse plasma harvested 48h after hydrodynamic tail vein (HTV) injections in C57BL / 6 mice (Figure 38A), and the FVIII activity data (Figure 38B) from the same experiment.

[0050] Figures 39A-39D provide sequence alignments across PS01-FL, PS02-FL, PS03-FL, PS04-FL, PS05-FL and BDD-FVIII-FL-AA amino acid sequences.

[0051] Figure 40 provide the amino acid sequence of wild type full-length FVIII (FVIII-WT-FL- AA (SEQ ID NO: 72)).

[0052] Figure 41 provides a summary of FVIII nucleic acid constructs, in accordance with some embodiments of the disclosure.

[0053] Figure 42 shows in vivo FVIII antigen data measured in mouse plasma harvested 48h after hydrodynamic tail vein (HTV) injections in C57BL / 6 mice.DETAILED DESCRIPTION OF DISCLOSUREI. Introduction

[0054] AAV-based gene therapy holds great promise for the treatment of hemophiliacs. For hemophilia B, first clinical data are encouraging in that FIX levels of about 10% can be maintained in at least some patients for more than 1 year. For hemophilia A however, achieving therapeutic expression levels of 5-10% with AAV vectors remains challenging for various 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 suffer from poor expression in vivo, even when codon-optimized. Third, these B-domain deleted or truncated Factor VIII variant constructs have short half-lives in vivo, exacerbating the effects of poor expression. Fourth, even whenexpressed, FVIII is not efficiently secreted from cells, as are other coagulation factors, such as Factor IX.

[0055] Moreover, these challenges cannot be addressed by simply administering higher doses of the gene therapy construct. According to current knowledge, the vector dose of an AAV-based gene therapy vector should be increased above 2x1012vg / kg body weight. This is because at such high doses a T cell immune response is triggered, which destroys transduced cells and, as a consequence, transgene expression is reduced or even eliminated. Therefore, strategies to improve the expression of FVIII are needed to make FVIII gene therapy a viable therapeutic option for hemophilia A patients.

[0056] The present disclosure relates to the discovery of codon-altered Factor VIII variant coding sequences that solve these and other problems associated with Factor VIII gene therapy. For example, the polynucleotides disclosed herein provide markedly improved expression in mammalian cells, and display improved virion packaging due to stabilized packing interactions. In some implementations, these advantages are realized by using coding sequences for the heavy and light chains of Factor VIII with high sequence identity to the codon altered CS01, CS04, and CS23 constructs (e.g., with high sequence identity to one of the CS01-HC, CS04-HC, and CS23- HC heavy chain coding sequences and high sequence identity to one of the CS01-LC, CS04-LC, and CS23-LC light chain coding sequences).

[0057] In some implementations, the Factor VIII molecules encoded by the polynucleotides described herein have been shortened by truncating, deleting, or replacing the wild-type B-domain. As such, the polynucleotides are better suited for expressing Factor VIII via conventional gene therapy vectors, which inefficiently express larger polypeptides, such as the wild-type Factor VIII.

[0058] Advantageously, it is shown herein that the CS01, CS02, CS03, CS04, CS05, CS06, CS07, CS08, CS09, CS10, CS11, and CS12 codon-altered Factor VIII variant coding sequences provide superior expression of a B-domain deleted Factor VIII construct in vivo. For example, it is demonstrated in Example 5 that intravenous administration of AAV-based gene therapy vectors having the CS01 (SEQ ID NOV), CS02 (SEQ ID NO: 15), CS03 (SEQ ID NO:21), CS04 (SEQ ID NO:27), CS05 (SEQ ID NO:33), CS06 (SEQ ID NO:39), CS07 (SEQ ID NO:43), CS08 (SEQ IDNO:47), CS09 (SEQ ID N0:51), CS10 (SEQ ID NO:55), CS11 (SEQ ID NO:59), and CS12 (SEQ ID NO:63) coding sequence induced detectable Factor VIII expression.II. Definitions

[0059] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0060] As used herein, the terms “Factor VIII” and “FVHI” are used interchangeably, and refer to any protein with Factor VIII activity (e.g., active FVIII, often referred to as FVIIIa) or protein precursor (e.g., pro-protein or pre-pro-protein) of a protein with Factor VIII activity, particularly Factor IXa cofactor activity. In an exemplary embodiment, a Factor VIII polypeptide refers to a polypeptide that has sequences with high 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.

[0061] Non-limiting examples of wild type Factor VIII polypeptides include human pre-pro- Factor VIII (e.g., GenBank accession nos. AAA52485, CAA25619, AAA58466, AAA52484, AAA52420, AAV85964, BAF82636, BAG36452, CAI41660, CAI41666, CAI41672, CAI43241, CA003404, EAW72645, AAH22513, AAH64380, AAH98389, AAI11968, AAI11970, or AAB61261), corresponding pro-Factor VIII, and natural variants thereof; porcine pre-pro-Factor VIII (e.g., UniProt accession nos. F1RZ36 or K7GSZ5), corresponding pro-Factor VIII, and natural variants thereof; mouse pre-pro-Factor VIII (e.g., GenBank accession nos. AAA37385, CAM15581, CAM26492, or EDL29229), corresponding pro-Factor VIII, and natural variants thereof; rat pre-pro-Factor VIII (e.g., GenBank accession no. AAQ21580), corresponding pro- Factor VIII, and natural variants thereof; rat pre-pro-Factor VIII; and other mammalian Factor VIII homologues (e.g., monkey, ape, hamster, guinea pig, etc.).

[0062] As used herein, a Factor VIII polypeptide includes natural variants and artificial constructs with Factor IX cofactor activity. As used in the present disclosure, Factor VIII encompasses any natural variants, alternative sequences, isoforms, or mutant proteins that retain some basal FactorIX 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 variations (relative to FVIII-WT-FL-AA (SEQ ID NO: 72)) found in the human population include, without limitation, 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, M110V, A111T7V, H113R / Y, L117F / R, G121S, E129V, G130R, E132D, Y133C, D135G / Y, T137A / I, S138R, E141K, D145H, V147D, Y155H, 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, S308L, F312S, T314A / I, A315V, G323E, L326P, L327P / V, C329F, 133 IV, 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, D56 I G / 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, Ml 8421, 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, W2015C, R2016P / W, E2018G, G2022D, G2028R,S2030N, V2035A, Y2036C, N2038S, 2040Y, G2045E / V, I2051S, I2056N, A2058P, W2065R, P2067L, A2070V, S2082N, S2088F, D2093G / Y, H2101D, T2105N, Q2106E / P / R, G2107S, R2109C, 12117F / S, Q2119R, F2120C / L, Y2124C, R2135P, S2138Y, T2141N, M2143V, F2145C, N2148S, N2157D, P2162L, R2169C / H, P2172L / Q / R, T2173A / I, H2174D, R2178C / H / L, R2182C / H / P, M2183R / V, L2185S / W, S2192I, C2193G, P2196R, G2198V, E2200D, I2204T, I2209N, A2211P, A2220P, P2224L, 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.

[0063] Generally, polynucleotides encoding Factor VIII encode for an inactive single-chain polypeptide (e.g., a pre-pro-protein) that undergoes post-translational processing to form an active Factor VIII protein (e.g., FVIIIa). For example, referring to Figure 1, the wild type human Factor VIII pre-pro-protein is first cleaved to release the encoded signal peptide (not shown), forming a first single-chain pro-protein (shown as “human wild-type FVIII). The pro-protein is then cleaved between the B and A3 domains to form a first polypeptide that includes the Factor VIII heavy chain (e.g., the Al and A2 domains) and B-domain, and a second polypeptide that includes the Factor VIII light chain (e.g., including the A3, Cl, and C3 domains). The first polypeptide is further cleaved to remove the B-domain, and also to separate the Al and A2 domains, which remain associated with the Factor VIII light chain in the mature Factor Villa protein. For review of the Factor VIII maturation process, see Graw et al., Nat Rev Genet., 6(6):488-501 (2005), the content of which is incorporated herein by reference in its entirety for all purposes.

[0064] However, in some embodiments, the Factor VIII polypeptide is a single-chain Factor VIII polypeptide. Single-chain Factor VIII polypeptides are engineered to remove natural cleavage sites, and optionally remove, truncate, or replace the B-domain of Factor VIII. As such, they are not matured by cleavage (other than cleavage of an optional signal and / or leader peptide), and are active as a single chain. Non-limiting examples of single-chain Factor VIII polypeptides are described in Zollner et al. (Thromb Res, 134(1): 125-31 (2014)) and Donath et al. (Biochem J., 312(l):49-55 (1995)), the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

[0065] As used herein, the terms “Factor VIII heavy chain,” or simply “heavy chain,” refers to the aggregate of the Al and A2 domains of a Factor VIII polypeptide. In an exemplary embodiment, amino acids 20-759 of BDDF VIII-FL- AA (SEQ ID NO: 5) constitute a Factor VIII heavy chain.

[0066] As used herein, the term “Factor VIII light chain,” or simply “light chain,” refers to the aggregate of the A3, Cl, and C2 domains of a Factor VIII polypeptide. In an exemplary embodiment, amino acids 774-1457 of BDDFVIII-FL-AA (SEQ ID NO: 5) constitute a Factor VIII light chain. In some embodiments, a Factor VIII light chain excludes the acidic a3 peptide, which is released during maturation in vivo.

[0067] Generally, Factor VIII heavy and light chains are expressed as a single polypeptide chain, e.g., along with an optional B-domain or B-domain substituted linker. However, in some embodiments, a Factor VIII heavy chain and Factor VIII light chain are expressed as separate polypeptide chains (e.g., co-expressed), and reconstituted to form a Factor VIII protein (e.g., in vivo or in vitro).

[0068] As used herein, the terms “B-domain substituted linker” and “Factor VIII linker” are used interchangeably, and refer to truncated versions of a wild type Factor VIII B-domain (e.g., amino acids 760-1667 of FVIII-WT-FL-AA (SEQ ID NO:72)) or peptides 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 a Factor VIII heavy chain and the N-terminus of a Factor VIII light chain in a Factor VIII variant polypeptide in accordance with some embodiments. Non-limiting examples of B-domain substituted linkers are disclosed in U.S. Patent Nos. 10,189,888, 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, 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. WO 2014 / 064277 and WO 2014 / 127215, the disclosures of which are hereby incorporated by reference, in their entireties, for all purposes.

[0069] In some embodiments, the present disclosure provides a B-domain deleted Factor VIII variant construct, referred to herein as “BDDFVIII-FL” (SEQ ID NO: 5 in Figure 4). In this construct, the B-domain is replaced with a fourteen amino acid sequence referred to as the “SQ” sequence SFSQNPPVLKRHQR (BDDFVIII-BDL-AA (SEQ ID NO: 8)). Unless otherwisespecified herein, the numbering of Factor VIII amino acids refers to the corresponding amino acid in the B-domain deleted Factor VIII variant (BDDFVIII-FL-AA), presented as SEQ ID NO: 5. As such, when referring to an amino acid substitution in a Factor VIII variant protein disclosed herein, the recited amino acid number refers to the (e.g., structurally or functionally equivalent) and / or homologous (e.g., evolutionarily conserved in the primary amino acid sequence) amino acid in the BDDFVIII-FL-AA. For example, a V63Q amino acid substitution refers to a V to Q substitution at position 2105 of the B-domain truncated Factor VIII variant sequence (BDDFVIII-FL-AA; SEQ ID NO: 5).

[0070] As described herein, the Factor VIII amino acid numbering system is dependent on whether the Factor VIII signal peptide (e.g., amino acids 1-19 of the full-length, wild-type human Factor VIII sequence FVIII-WT-FL-AA (SEQ ID NO:72) and BDD-FVIII-AA (SEQ ID NO: 5)) is included. Where the signal peptide is included, the numbering is referred to as “signal peptide inclusive” or “SPI”. Where the signal peptide is not included, the numbering is referred to as “signal peptide exclusive” or “SPE.” For example, F328S is SPI numbering for the same amino acid as F309S, in SPE numbering.

[0071] As described herein, the codon-altered polynucleotides provide increased expression of transgenic Factor VIII in vivo (e.g., when administered as part of a gene therapy vehicle including but not limited to a viral vector such as an AAV vector or a lipid nanoparticle), as compared to the level of Factor VIII expression provided by a natively-coded Factor VIII construct (e.g., a polynucleotide encoding the same Factor VIII construct using the wild-type human codons). As used herein, the term “increased expression” refers to an increased level of transgenic Factor VIII activity in the blood of an animal administered the codon-altered polynucleotide encoding Factor VIII, as compared to the level of transgenic Factor VIII activity in the blood of an animal administered a natively-coded Factor VIII construct. The activity levels can be measured using any Factor VIII activity known in the art. An exemplary assay for determining Factor VIII activity is the Technochrome FVIII assay (Technoclone, Vienna, Austria).

[0072] In some embodiments, increased expression refers to at least 25% greater transgenic Factor VIII activity in the blood of an animal administered the codon-altered Factor VIII polynucleotide, as compared to the level of transgenic Factor VIII activity in the blood of an animal administered a natively coded Factor VIII polynucleotide. In some embodiments, increased expression refersto 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, at least 20-fold greater, at least 25-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, at least 125-fold greater, at least 150-fold greater, at least 175- fold greater, at least 200-fold greater, at least 225-fold greater, or at least 250-fold greater transgenic Factor VIII activity in the blood of an animal administered the codon-altered Factor VIII polynucleotide, as compared to the level of transgenic Factor VIII activity in the blood of an animal administered a natively coded Factor VIII polynucleotide.

[0073] As described herein, the codon-altered polynucleotides provide increased vector production, as compared to the level of vector production provided by a natively-coded Factor VIII construct (e.g., a polynucleotide encoding the same Factor VIII construct using the wild-type human codons). As used herein, the term “increased virus production” refers to an increased vector yield in cell culture (e.g., titer per liter culture) inoculated with the codon-altered polynucleotide encoding Factor VIII, as compared to the vector yield in cell culture inoculated with a natively- coded Factor VIII construct. The vector yields can be measured using any vector titer assay known in the art. An exemplary assay for determining vector yield (e.g., of an AAV vector) is qPCR targeting the AAV2 inverted terminal repeats (Aurnhammer, Human Gene Therapy Methods: Part B 23: 18-28 (2012)).

[0074] In some embodiments, increased virus production refers to at least 25% greater codon- altered vector yield, as compared to the yield of a natively-coded Factor VIII construct in the same type of culture. In some embodiments, increased vector production refers to 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 codon-altered vector yield, as compared to the yield of a natively-coded Factor VIII construct in the same type of culture.

[0075] As used herein, the term "hemophilia" refers to a group of disease states broadly characterized by reduced blood clotting or coagulation. Hemophilia may refer to Type A, TypeB, or Type C hemophilia, or to the composite of all three diseases types. Type A hemophilia (hemophilia A) is caused by a reduction or loss of Factor VIII (FVIII) activity and is the most prominent of the hemophilia subtypes. Type B hemophilia (hemophilia B) results from the loss or reduction of factor IX (FIX) clotting function. Type C hemophilia (hemophilia C) is a consequence of the loss or reduction in factor XI (FXI) clotting activity. Hemophilia A and B are X-linked diseases, while hemophilia C is autosomal. Conventional treatments for hemophilia include both 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.

[0076] As used herein, the term "FVIII gene therapy" includes any therapeutic approach of providing a nucleic acid encoding Factor VIII to a patient to relieve, diminish, or prevent the reoccurrence of one or more symptoms (e.g., clinical factors) associated with hemophilia. The term encompasses administering any compound, drug, procedure, or regimen comprising a nucleic acid encoding a Factor VIII molecule, including any modified form of Factor VIII (e.g., Factor VIII variant), for maintaining or improving the health of an individual with hemophilia. One skilled in the art will appreciate that either the course of FVIII therapy or the dose of a FVIII therapeutic agent can be changed, e.g., based upon the results obtained in accordance with the present disclosure.

[0077] As used herein, the term "bypass therapy" includes any therapeutic approach of providing non-Factor VIII hemostatic agents, compounds or coagulation factors to a patient to relieve, diminish, or prevent the reoccurrence of one or more symptoms (e.g., clinical factors) associated with hemophilia. Non-Factor VIII compounds and coagulation factors include, but are not limited to, Factor VIII Inhibitor Bypass Activity (FEIBA), recombinant activated factor VII (FVIIa), prothrombin complex concentrates, and activated prothrombin complex concentrates. These nonFactor VIII compounds and coagulation factors may be recombinant or plasma-derived. One skilled in the art will appreciate that either the course of bypass therapy or the dose of bypass therapy can be changed, e.g., based upon the results obtained in accordance with the present disclosure.

[0078] As used herein, a “combination therapy” including administration of a nucleic acid encoding a Factor VIII molecule and a conventional hemophilia A therapeutic agent includes any therapeutic approach of providing both a nucleic acid encoding a Factor VIII molecule and a FactorVIII molecule and / or non-Factor VIII hemostatic agent (e.g., bypass therapeutic agent) to a patient to relieve, diminish, or prevent the reoccurrence of one or more symptoms (e.g., clinical factors) associated with hemophilia. The term encompasses administering any compound, drug, procedure, or regimen including a nucleic acid encoding a Factor VIII molecule, including any modified form of Factor VIII, which is useful for maintaining or improving the health of an individual with hemophilia and includes any of the therapeutic agents described herein.

[0079] 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 therapeutic effects for which it is administered. For example, a therapeutically effective amount of a drug useful for treating hemophilia can be the amount that is capable of preventing or relieving one or more symptoms associated with hemophilia. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled 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).

[0080] As used herein, the term "gene" refers to the segment of a DNA molecule that codes for a polypeptide chain (e.g., the coding region). In some embodiments, a gene is positioned by regions immediately preceding, 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 intron).

[0081] As used herein, the term "regulatory elements" refers to nucleotide sequences, such as promoters, enhancers, terminators, polyadenylation sequences, introns, etc., that provide for the expression of a coding sequence in a cell.

[0082] As used herein, the term “promoter element” refers to a nucleotide sequence that assists with controlling expression of a coding sequence. Generally, promoter elements are located 5’ of the translation start site of a gene. However, in certain embodiments, a promoter element may be located within an intron sequence, or 3’ of the coding sequence. In some embodiments, a promoter useful for a gene therapy vector is derived from the native gene of the target protein (e.g., a FactorVIII promoter). In some embodiments, a promoter useful for a gene therapy vector is specific for expression in a particular cell or tissue of the target organism (e.g., a liver-specific promoter). In yet other embodiments, one of a plurality of well characterized promoter elements is used in a gene therapy vector described herein. Non-limiting examples of well-characterized promoter elements include the CMV early promoter, the 0-actin promoter, and the methyl CpG binding protein 2 (MeCP2) promoter. In some embodiments, the promoter is a constitutive promoter, which drives substantially constant expression of the target protein. In other embodiments, the promoter is an inducible promoter, which drives expression of the target protein in response to a particular stimulus (e.g., exposure to a particular treatment or agent). For a review of designing promoters 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.

[0083] As used herein, the term “AAV vector”, “AAV virion,” “AAV particle,” or “AAV gene therapy particle,” “AAV gene therapy vector,” or “rAAV gene therapy vector” refers to a complete virus unit, such as an AAV virus particle (comprising a linear, single-stranded AAV nucleic acid genome associated with at least one AAV capsid protein coat). In some embodiments, singlestranded AAV nucleic acid molecules of either complementary sense (e.g., “sense” or “anti-sense” strands) are packaged into any one AAV virion and both strands are equally infectious. Also included are infectious viral particles containing a heterologous polynucleotide of interest (e.g., FVIII polynucleotide or a biologically active portion thereof), which is flanked on both sides by AAV ITRs.

[0084] As used herein, the term “enhancer” refers to a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants (US Patent Application Publication Number 2009 / 0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie et al., (1989) Molecular Biology ofRNA ed. Cech (Liss, New York) 237-256 and Gallic et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey et al., (1990) EMBO J. 9:1685-96), Cis-regulatory modules (CRMs, see, e.g., Niu et al. Nucleic acids research 46.11 (2018): 5395-5409; Visel et al. Nature 461.7261 (2009):199) and the enhancers of U.S. Pat.No. 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments.

[0085] As used herein, the term “CRM8” refers to a cis-acting regulatory module derived from a polynucleotide sequence from the human Serpinal gene (see, e.g., Chuah et al., (2014) Molecular Therapy: 22(9): 1605-1613).

[0086] As used herein, the term “Woodchuck hepatitis virus posttranscriptional regulatory element” or “WPRE,” refers to a DNA sequence that, when transcribed, creates a tertiary structure capable of enhancing the expression of a gene. See, Lee Y et al, Exp. Physiol. 2005; 90(l):33-37 and Donello J et al, J. Virol. 1998; 72(6): 5085-5092.

[0087] As used herein, the term “intron” refers to a DNA molecule that may be isolated or identified from a gene and may be defined generally as a region spliced out during messenger RNA (mRNA) processing prior to translation. Alternately, an intron may be a synthetically produced or manipulated DNA element. An intron may contain enhancer elements that effect the transcription of operably linked genes. An intron may be used as a regulatory element for modulating expression of an operably linked transcribable DNA molecule. A construct may comprise an intron, and the intron may or may not be heterologous with respect to the transcribable DNA molecule. Examples of introns include the rice actin intron, the corn HSP70 intron, the P-globin intron, the SV40 intron, the minute virus of mice (MVM) intron, or other artificially synthesized introns. In some embodiments, the vector / polynucleotide of the present disclosure includes an MVM intron (see, e.g., Wu et al., Mol. Ther. 16(2):280-289, 2008).

[0088] As used herein, the term "vector" refers to any vehicle used to transfer a 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 units of replication in vivo. In some embodiments, a vector is a viral vehicle for introducing a target nucleic acid (e.g., a codon- altered polynucleotide encoding a Factor VIII variant). Many modified eukaryotic viruses useful for gene therapy are known in the art. For example, adeno-associated viruses (AAVs) are particularly well suited for use in human gene therapy because humans are a natural host for thevirus, the native viruses are not known to contribute to any diseases, and the viruses illicit a mild immune response.

[0089] As used herein, the term “CpG dinucleotide” describes a part of a DNA molecule in which two nucleotides containing the nucleobases cytosine and guanine are linked to each other. Typically, the CpG dinucleotide contains or consists of the unit deoxycytidine-phosphate- deoxyguanosine (in a 5’ to 3’ direction). In general, without being limited to this, the presence and level of the CpG-dinucleotide in a DNA molecule is organism- and region-dependent. CpG sites occur with high frequency in genomic regions called CpG islands (or CG islands) with the cytosine often being subject to methylation.

[0090] As used herein, the term “CpG island” refers to a region within a polynucleotide having a statistically elevated density of CpG dinucleotides. As used herein, a region of a polynucleotide (e.g., a polynucleotide encoding a codon-altered Factor VIII protein) is a CpG island if, over a 200-base pair window: (i) the region has GC content of greater than 50%, and (ii) the ratio of observed CpG dinucleotides per expected CpG dinucleotides is at least 0.6, as defined by the relationship: 0.6.For additional information on methods for identifying CpG islands, see Gardiner-Garden M. et al., J Mol Biol., 196(2):261-82 (1987), the content of which is expressly incorporated herein by reference, in its entirety, for all purposes.

[0091] As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O- methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0092] The term “amino acid” refers to naturally occurring and non-natural amino acids, including amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Naturally occurring amino acids can include, e.g., D- and L-amino acids. The amino acids used herein can also include non-natural amino acids. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., any carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, or methionine methyl sulfonium. 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. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function 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. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0093] The nucleotide sequences that encode the mutant Factor VIII constructs herein may be identical to the coding sequence provided herein or may be a different coding sequence, which sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the coding sequences provided herein. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each variation of a nucleic acid which encodes a same polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual gene therapy constructs.

[0094] As to amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid or peptide sequence that alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acidwith a chemically similar amino acid. Conservative substitution tables providing 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 disclosure.

[0095] Conservative amino acid substitutions providing functionally similar amino acids are well known in the art. Dependent on the functionality of the particular amino acid, e.g., catalytic, structural, or sterically important amino acids, different groupings of amino acid may be considered conservative substitutions for each other. Table 1 provides groupings of amino acids that are considered conservative substitutions based on the charge and polarity of the amino acid, the hydrophobicity of the amino acid, the surface exposure / structural nature of the amino acid, and the secondary structure propensity of the amino acid.Table 1. Groupings of conservative amino acid substitutions based on the functionality of the residue in the protein.

[0096] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection.

[0097] As is known in the art, a number of different programs may be used to identify whether a protein (or nucleic acid as discussed below) has sequence identity or similarity to a known sequence. Sequence identity and / or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math., 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol., 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res., 12:387-395 (1984), preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon thefollowing 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,” Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc, all of which are incorporated by reference.

[0098] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pair wise alignments. It may also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5: 151-153 (1989), both incorporated by reference. Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0099] 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. U.S.A. 90:5873-5787 (1993), both incorporated by reference. A particularly useful BLAST program is the WU-BLAST-2 program which was obtained 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 with the following values: overlap span =1, overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

[0100] An additional useful algorithm is gapped BLAST, as reported by Altschul et al., Nucl. Acids Res., 25:3389-3402, incorporated by reference. Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions; charges gap lengths of k a cost of 10+k; Xu set to 16, and Xg set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to ~22 bits.

[0101] A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “longer” sequence in the aligned region. The “longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored). In a similar manner, “percent (%) nucleic acid sequence identity” with respect to the coding sequence of the polypeptides identified is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the cell cycle protein. A preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

[0102] The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the protein encoded by the sequences provided herein in the figures, 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 in relation to the total number of amino acids or nucleotides. Thus, for example, sequence identity of sequences shorter than that shown in the figures, as discussed below, will be determined using the number of nucleotides in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.

[0103] In one embodiment, only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of “0”, which obviates the need for a weighted scale or parameters as described below for 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 of the “shorter” sequence in the aligned region and multiplying by 100. The “longer” sequence is the one having the most actual residues in the aligned region.

[0104] The term “allelic variants” refers to polymorphic forms of a gene at a particular genetic locus, as well as cDNAs derived from mRNA transcripts of the genes, and the polypeptides encoded by them. The term “preferred mammalian codon” refers a subset of codons from among the set of codons encoding an amino acid that are most frequently used in proteins expressed in mammalian cells as chosen from the following list: Gly (GGC, GGG); Glu (GAG); Asp (GAC);Vai (GTG, GTC); Ala (GCC, GCT); Ser (AGC, TCC); Lys (AAG); Asn (AAC); Met (ATG); He (ATC); Thr (ACC); Trp (TGG); Cys (TGC); Tyr (TAT, TAC); Leu (CTG); Phe (TTC); Arg (CGC, AGG, AGA); Gin (CAG); His (CAC); and Pro (CCC).

[0105] As used herein, the term codon-altered refers to a polynucleotide sequence encoding a polypeptide (e.g., a Factor VIII variant protein), where at least one codon of the native polynucleotide encoding the polypeptide has been changed to improve a property of the polynucleotide sequence. In some embodiments, the improved property promotes increased transcription of mRNA coding for the polypeptide, increased stability of the mRNA (e.g., improved mRNA half-life), increased translation of the polypeptide, and / or increased packaging of the polynucleotide within the vector. Non-limiting examples of alterations that can be used to achieve the improved properties include changing the usage and / or distribution of codons for particular amino acids, adjusting global and / or local GC content, removing AT-rich sequences, removing repeated sequence elements, adjusting global and / or local CpG dinucleotide content, removing cryptic regulatory elements (e.g., TATA box and CCAAT box elements), removing of intron / exon splice sites, improving regulatory sequences (e.g., introduction of a Kozak consensus sequence), and removing sequence elements capable of forming secondary structure (e.g., stemloops) in the transcribed mRNA.

[0106] As discussed herein, there are various nomenclatures to refer to components of the disclosure herein. “CS-number” (e.g. “CS01”, “CS02”, etc.) refers to codon altered polynucleotides encoding FVIII polypeptides including variants. For example, “CS01-FL” refers to the Full Length codon altered CS01 polynucleotide sequence (also referred to herein as “CS01- FL-NA” for the Nucleic Acid sequence). Similarly, “CS01-LC” and “CS01-LC-NA” refer to the codon altered nucleic acid sequence encoding the light chain of a FVIII polypeptide. Likewise, “CS01-HC” and “CS01-HC-NA" refer to the CS01 polynucleotide sequence encoding the FVIII heavy chain. As will be appreciated by those in the art, constructs CS04, CS06, CS07, CS08, CS09, CS10, CS11, and CS12 encode for the same BDD-FVIII variant. Thus, nucleic acid constructs encoding the FVIII polypeptide variants of the disclosure include, but are not limited to, CS01-FL, CS02-FL, CS03-FL, CS04-FL, CS05-FL, CS06-FL, CS07-FL, CS08-FL, CS09-FL, CS10-FL, CS11-FL, CS12-FL, CS01-HC, CS02-HC, CS03-HC, CS04-HC, CS05-HC, CS06-HC, CS07-HC, CS08-HC, CS09-HC, CS10-HC, CS11-HC, CS12-HC, CS01-LC, CS02-LC, CS03-LC,CS04-LC, CS05-LC, CS06-LC, CS07-LC, CS08-LC, CS09-LC, CS10-LC, CS11-LC, and CS12- LC.

[0107] ‘PS-number” (e.g. “PS01”, “PS02”, etc.) refers to FVIII polypeptides including variants encoded by the codon altered polynucleotides as described by the CS-numbered sequences. For example, PS01-FL refers to the Full Length polypeptide sequence of a BDD- FVIII-AA (i.e., FVIII polypeptide with the B-domain replaced with an SQ linker) shown in Figure 7 as SEQ ID NO: 12. Similarly, “PS01-LC” refers to the light chain of a BDD-FVIII polypeptide shown in Figure 7 as SEQ ID NO: 14. Likewise, PS01-HC refers to a BDD-FVIII polypeptide shown in Figure 7 as SEQ ID NO: 13. Thus, the FVIII polypeptide variants of the disclosure include, but are not limited to, PS01-FL, PS02-FL, PS03-FL, PS04-FL, PS05-FL, PS01- HC, PS02-HC, PS03-HC, PS04-HC, PS05-HC, PS01-LC, PS02-LC, PS03-LC, PS04-LC, and PS05-LC.

[0108] The disclosure also includes additional new Factor VIII variants, as described below, with the appropriate nomenclature.III. Codon-Altered Factor VIII Variants

[0109] In some embodiments, the present disclosure provides codon-altered polynucleotides encoding Factor VIII variants. These codon-altered polynucleotides provide markedly improved expression of Factor VIII when administered in a gene therapy vehicle such as an AAV-based gene therapy construct or a lipid nanoparticle. The codon-altered polynucleotides also demonstrate improved AAV-virion packaging, as compared to conventionally codon-optimized constructs. As demonstrated in Examples 1-3, Applicants have achieve these advantages through the discovery of twelve codon-altered polynucleotides (CS01- FL, CS02-FL, CS03-FL, CS04-FL, CS05-FL, CS06-FL, CS07-FL, CS08-FL, CS09-FL, CS10-FL, CS11-FL, CS12-FL), encoding variant Factor VIII polypeptides with human Factor VIII heavy and light chains, and a short, 14 amino acid, B-domain substituted linker (the “SQ” linker) containing a furin cleavage site to facilitate maturation of an active FVIIIa protein in vivo. As demonstrated in Examples 4 and 5, the codon-alteration and incorporation of various amino acid mutations into the encoded Factor VIII molecule increased in vitro and in vivo expression of Factor VIII antigen and activity.

[0110] In one embodiment, a codon-altered polynucleotide provided herein has nucleotide sequences with high sequence identity to at least the sequences within CS01, CS02, CS03, CS04, CS05, CS06, CS07, CS08, CS09, CS10, CS11, or CS12 encoding the Factor VIII heavy chain and Factor VIII light chains. As known in the art, the B-domain of Factor VIII is dispensable for activity in vivo. Thus, in some embodiments, the codon-altered polynucleotides provided herein completely lack a 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., the “SQ” linker consisting of ammo acids 760-773 of the PS01-FL, PS02-FL, PS03-FL, PS04-FL, and PS05-FL constructs. The “SQ” linker is also referred to as BDDFVIII-BDL-AA or BDDFVIII-BDL-NA, (-AA for the amino acid sequence and -NA for the nucleotide sequence). In some embodiments, the present disclosure provides a variety of codon-optimized polynucleotide sequences encoding the SQ linker having the same amino acid sequence of BDDFVIII-BDL-AA, including but not limited to CS01-BDL, CS02-BDL, CS03-BDL, CS04-BDL, CS05-BDL, CS06-BDL, CS07-BDL, CS08-BDL, CS09-BDL, CS10-BDL, CS11-BDL, and CS12-BDL.

[0111] In one embodiment, the Factor VIII heavy and light chains encoded by the codon- altered polynucleotide are human Factor VIII heavy and light chains, respectively. In other embodiments, the Factor VIII heavy and light chains encoded by the codon-altered polynucleotide are heavy and light chain sequences from another mammal (e.g., porcine Factor VIII). In yet other embodiments, the Factor VIII heavy and light chains are chimeric heavy and light chains (e.g., a combination of human and a second mammalian sequence). In yet other embodiments, the Factor VIII heavy and light chains are humanized version of the heavy and light chains from another mammal, e.g., heavy and light chain sequences from another mammal in which human residues are substituted at select positions to reduce the immunogenicity of the resulting peptide when administered to a human.

[0112] The GC content of human genes varies widely, from less than 25% to greater than 90%. However, in general, human genes with higher GC contents are expressed at higher levels. For example, Kudla et al. (PLoS Biol., 4(6): 80 (2006)) demonstrate that increasing a gene’s GC content increases expression of the encoded polypeptide, primarily by increasing transcription and effecting a higher steady state level of the mRNA transcript. Generally, the desired GC content ofa codon-optimized gene construct is equal or greater than 60%. However, native AAV genomes have GC contents of around 56%.

[0113] Accordingly, in some embodiments, the codon-altered polynucleotides provided herein have a CG content that more closely matches the GC content of native AAV virions (e.g., around 56% GC), which is lower than the preferred CG contents of polynucleotides that are conventionally codon-optimized for expression in mammalian cells (e.g., at or above 60% GC). The codon-optimized BDDFVIII-NA sequence (SEQ ID NO: 1), which has a GC content of about 56%, has improved virion packaging as compared to similarly codon-altered coding sequences with higher GC content.

[0114] Thus, in some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is less than 60%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is less than 59%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is less than 58%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is less than 57%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is no more than 56%.

[0115] In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is from 54% to 59%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is from 55% to 59%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is from 56% to 59%. In some embodiments, the overall GC content of a codon- altered polynucleotide encoding a Factor VIII polypeptide is from 54% to 58%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is from 55% to 58%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is from 56% to 58%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is from 54% to 57%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is from 55% to 57%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide isfrom 56% to 57%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is from 54% to 56%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is from 55% to 56%.

[0116] In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is 56±0.5%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is 56±0.4%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is 56±0.3%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is 56±0.2%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is 56±0.1%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is 56%.

[0117] In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is less than 56%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is from 50% to 56%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is from 51% to 56%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is from 52% to 56%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is from 53% to 56%.

[0118] In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is of 53±3%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is of 53±2%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is of 53±1%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is of 53±0.5%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is of 53±0.2%. In some embodiments, theoverall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide provided herein is of 53±0.1%. In some embodiments, the overall GC content of a codon-altered polynucleotide encoding a Factor VIII polypeptide is 53%.A. Factor VIII Amino Acid Substitutions

[0119] To further increase the efficiency of expression of the Factor VIII constructs described herein, amino acid substitutions known to improve secretion, increase specific activity, and / or enhanced the stability of Factor VIII are further incorporated, in some implementations. A number of potential variants were identified that increase the plasma levels of FVIII activity at a given vector dose. These variants include those with a more efficient signal peptide, amino acid substitutions that prevent BiP interactions (i.e., interactions with the binding immunoglobulin protein (BiP), which are a reflection of misfolded proteins present in the ER lumen), amino acid substitutions resembling more efficiently secreted Factor VIII orthologs (e. g. , porcine Factor VIII), single-chain Factor VIII variants, and amino acid substitutions that stabilize Factor VIII and / or reduce subunit dissociation.

[0120] In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 1 to 30 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 5 to 29 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 10 to 28 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 15 to 27 acid substitutions relative to BDD-FVIII- AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 20 to 26 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 acid substitutions relative to BDD- FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 23 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5).

[0121] In some embodiments, the FVIII polypeptide contains 22 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 23 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIIIpolypeptide contains 24 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 25 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 26 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5). In some embodiments, the FVIII polypeptide contains 27 acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5).

[0122] In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, L171, H212, S260, L318, M320, S332, F387, 1390, V393, 1499, V534, M558, M681, S700, K732, Y738, L749, L750, K752, 1775, E800, E809, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, L171, H212, S260, E306, M320, S332, F387, 1390, V393, 1499, V534, M558, M681, S700, K732, L749, L750, 1775, E809, T1148, L1248, LI 377, and VI 439. In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, L171, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, K732, Y738, L749, L750, K752, 1775, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, L171, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, S700, K732, L749, L750, 1775, E800, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, L171, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, S700, K732, Y738, L749, L750, K752, 1775, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, L171, H212, S260, E306, M320, S332, F387, 1390, V393, V534, M558, M681, K732, Y738, L749, L750, 1775, K1093, T1148, L1248, LI 377, and V1439.

[0123] In some embodiments, the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, L171, H212, S260, L318, M320, S332, F387, 1390, V393, 1499, V534, M558, M681, S700, K732, Y738, L749, L750, K752, 1775, E800, E809, K1093, T1148, L1248, L1377, and VI 439. In some embodiments, the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, L171, H212, S260, E306, M320, S332, F387, 1390, V393, 1499, V534, M558, M681, S700, K732, L749, L750, 1775, E809, T1148, L1248, L1377, and V1439. In some embodiments, the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD- FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, LI 71, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, K732, Y738, L749, L750, K752, 1775, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, L171, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, S700, K732, L749, L750, 1775, E800, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, LI 71, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, S700, K732, Y738, L749, L750, K752, 1775, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, L171, H212, S260, E306, M320, S332, F387, 1390, V393, V534, M558, M681, K732, Y738, L749, L750, 1775, K1093, T1148, L1248, L1377, and V1439.

[0124] In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E809G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F,L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E800V, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F3871, 1390 A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, L750D, I775G, K1093A, T1148N, L1248D, L1377D, and V1439E.

[0125] In some embodiments, the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including but not limited to V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E809G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including but not limited to V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including but not limited to V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E800V, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including but not limited to V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F3871, 1390 A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptideof the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including but not limited to V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, L750D, I775G, K1093A, T1148N, L1248D, L1377D, and V1439E.

[0126] In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, L171, H212, S260, L318, M320, S332, F387, 1390, V393, 1499, V534, M558, M681, S700, K732, Y738, L749, L750, and K752. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, L171, H212, S260, E306, M320, S332, F387, 1390, V393, 1499, V534, M558, M681, S700, K732, L749, and L750. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, LI 71, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, K732, Y738, L749, L750, and K752. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, LI 71, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, S700, K732, L749, and L750. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, L171, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, S700, K732, Y738, L749, L750, and K752. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: V63, L171, H212, S260, E306, M320, S332, F387, 1390, V393, V534, M558, M681, K732, Y738, L749, and L750.

[0127] In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, L171, H212, S260, E306, M320, S332, F387, 1390,V393, 1499, V534, M558, M681, S700, K732, L749, and L750. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, L171, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, K732, Y738, L749, L750, and K752. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, L171, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, S700, K732, L749, and L750. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, L171, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, S700, K732, L749, L750, 1775, E800, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, L171, H212, S260, E306, L318, M320, S332, F387, 1390, V393, V534, M558, M681, S700, K732, Y738, L749, L750, and K752. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: V63, L171, H212, S260, E306, M320, S332, F387, 1390, V393, V534, M558, M681, K732, Y738, L749, and L750.

[0128] In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, and L750D. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, and K752E. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, and L750D. In some embodiments,the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting ofV63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, and K752E. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, and L750D.

[0129] In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including not limited to V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, and L750D. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including not limited to V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, and K752E. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, and L750D. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including not limited to V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, and K752E. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including not limited to V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, and L750D.

[0130] In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: 1775, E800, E809, K1093, T1148, L1248,L1377, and V1439. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: 1775, E809, T1148, L1248, L1377, and VI 439. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: 1775, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: 1775, E800, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions selected from the group consisting of: 1775, K1093, T1148, L1248, L1377, and V1439.

[0131] In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: 1775, E800, E809, K1093, T1148, L1248, L1377, and VI 439. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: 1775, E809, T1148, L1248, L1377, and V1439. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: 1775, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD- FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: 1775, E800, K1093, T1148, L1248, L1377, and V1439. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) at positions including but not limited to: 1775, K1093, T1148, L1248, L1377, and V1439.

[0132] In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of I775G, E809G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure containsone or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of I775G, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of K1093G; T1148N; L1248D; L1377D; and V1439E. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.

[0133] In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including not limited to I775G, E809G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including not limited to I775G, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) selected from the group consisting of T1148N; L1248D; L1377D; and V1439E. In some embodiments, the light chains of the FVIII polypeptide of the present disclosure contains amino acid substitutions relative to BDD-FVIII-AA (SEQ ID NO: 5) including not limited to I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.

[0134] In some embodiments, the FVIII polypeptide contains a heavy chain comprising one or more amino acid substitutions selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F3871, 1390 A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, and L750D and a light chain comprising one or more amino acid substitutions selected from the group consisting of I775G, E809G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide contains a heavy chain comprising one or more amino acid substitutions selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, and K752E and a light chain comprising one or more amino acid substitutions selected from the group consisting of I775G, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the heavy chains of the FVIII polypeptide of the presentdisclosure contains one or more amino acid substitutions selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, and L750D and a light chain comprising one or more amino acid substitutions selected from the group consisting of KI 093 G; T1148N; L1248D; L1377D; and V1439E. In some embodiments, the FVIII polypeptide contains a heavy chain comprising one or more amino acid substitutions selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, and K752E and a light chain comprising one or more amino acid substitutions selected from the group consisting of I775G, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide contains a heavy chain comprising one or more amino acid substitutions selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, and L750D and a light chain comprising one or more amino acid substitutions selected from the group consisting of I775G, K1093A, T1148N, L1248D, L1377D, and V1439E.

[0135] In some embodiments, the FVIII polypeptide contains a heavy chain comprising amino acid substitutions including not limited to V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, and L750D and a light chain comprising amino acid substitutions including not limited to I775G, E809G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide contains a heavy chain comprising amino acid substitutions including not limited to V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, and K752E and a light chain comprising amino acid substitutions including not limited to I775G, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the heavy chains of the FVIII polypeptide of the present disclosure contains one or more amino acid substitutions selected from the group consisting of V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, and L750D and a light chain comprising amino acid substitutions including not limited to K1093G; T1148N; L1248D; L1377D; and V1439E. In some embodiments, the FVIII polypeptide contains a heavy chain comprising amino acid substitutions including not limited to V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I,M558W, M681N, S700E, K732W, Y738E, L749F, L750D, and K752E and a light chain comprising amino acid substitutions including not limited to I775G, K1093G, T1148N, L1248D, L1377D, and V1439E. In some embodiments, the FVIII polypeptide contains a heavy chain comprising amino acid substitutions including not limited to V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, and L750D and a light chain comprising amino acid substitutions including not limited to I775G, K1093A, T1148N, L1248D, L1377D, and V1439E.

[0136] In some embodiments, the FVIII polypeptide comprises an amino acid sequence of PS01-FL (SEQ ID NO: 12). In some embodiments, the FVIII polypeptide comprises an amino acid sequence of PS02-FL (SEQ ID NO: 18). In some embodiments, the FVIII polypeptide comprises an amino acid sequence of PS03-FL (SEQ ID NO: 24). In some embodiments, the FVIII polypeptide comprises an amino acid sequence of PS04-FL (SEQ ID NO: 30). In some embodiments, the FVIII polypeptide comprises an amino acid sequence of PS05-FL (SEQ ID NO: 36).

[0137] In some embodiments, the FVIII polypeptide comprises a heavy chain comprising an amino acid sequence of PS01-HC (SEQ ID NO: 13). In some embodiments, the FVIII polypeptide comprises a heavy chain comprising an amino acid sequence of PS02-HC (SEQ ID NO: 19). In some embodiments, the FVIII polypeptide comprises a heavy chain comprising an amino acid sequence of PS03-HC (SEQ ID NO: 25). In some embodiments, the FVIII polypeptide comprises a heavy chain comprising an amino acid sequence of PS04-HC (SEQ ID NO: 31). In some embodiments, the FVIII polypeptide comprises a heavy chain comprising an amino acid sequence of PS05-HC (SEQ ID NO: 37).

[0138] In some embodiments, the FVIII polypeptide comprises a light chain comprising an amino acid sequence of PS01-LC (SEQ ID NO: 14). In some embodiments, the FVIII polypeptide comprises a light chain comprising an amino acid sequence of PS02-LC (SEQ ID NO: 20). In some embodiments, the FVIII polypeptide comprises a light chain comprising an amino acid sequence of PS03-LC (SEQ ID NO: 26). In some embodiments, the FVIII polypeptide comprises a light chain comprising an amino acid sequence of PS04-LC (SEQ ID NO: 32). In some embodiments, the FVIII polypeptide comprises a light chain comprising an amino acid sequence of PS05-LC (SEQ ID NO: 38).

[0139] In some embodiments, the FVIII polypeptide comprises a heavy chain comprising an amino acid sequence of PS01-HC (SEQ ID NO: 13) and a light chain comprising an amino acid sequence of PS01-LC (SEQ ID NO: 14). In some embodiments, the FVIII polypeptide comprises a heavy chain comprising an amino acid sequence of PS02-HC (SEQ ID NO: 19) and a light chain comprising an amino acid sequence of PS02-LC (SEQ ID NO: 20). In some embodiments, the FVIII polypeptide comprises a heavy chain comprising an amino acid sequence of PS 03 -HC (SEQ ID NO: 25) and a light chain comprising an amino acid sequence of PS03-LC (SEQ ID NO: 26). In some embodiments, the FVIII polypeptide comprises a heavy chain comprising an amino acid sequence of PS04-HC (SEQ ID NO: 31) and a light chain comprising an amino acid sequence of PS04-LC (SEQ ID NO: 32). In some embodiments, the FVIII polypeptide comprises a heavy chain comprising an amino acid sequence of PS05-HC (SEQ ID NO: 37) and a light chain comprising an amino acid sequence of PS05-LC (SEQ ID NO: 38).

[0140] Other examples of known Factor VIII variants include mutation of residues Al 08, R121, and L2302 (SPE), located at the interface between the Al and C2 domains, which increase the stability of Factor VIII. For example, the Al 081 amino acid substitution introduces a hydrophobic residue that better fills the inter-domain space, stabilizing the interaction. Likewise, an R121C / L2302C (SPE) double amino acid substitution introduces a disulfide bond spanning the A1-C2 domains, further stabilizing the interaction. Taken together, all three amino acid substitutions increase the thermal stability of Factor VIII by 3 to 4-fold. For review, see Wakabayashi et al., J Biol Chem. 286(29):25748-55 (2011) and Wakabayashi et al., Thromb Haemost. 10(3):492-95 (2012). Accordingly, in some embodiments, the encoded Factor VIII polypeptide includes A108I and / or R121C / L2302C amino acid substitutions.

[0141] Mutation of El 13 (SPE), located within the calcium binding domain of Factor VIII, increases the specific FVIII clotting activity. For example, E113A appears to increase FXase formation through increased FVIII affinity for Factor IXa. Specifically, the E113A amino acid substitution increases specific FVIII clotting activity two-fold and increases affinity for Factor IXa by four-fold (Biochemistry, 41:8485 (2002); J. Biol. Chem., 279:12677 (2004); and Biochemistry, 44: 10298 (2005)). Accordingly, in some embodiments, the encoded Factor VIII polypeptides include an El 13A amino acid substitution.

[0142] Substitution of one or more amino acid residues surrounding the Factor VIII APC cleavage site (residues 331-341 (SPE)) reduce Factor Villa inactivation by activated protein C, without affecting FVIII activity. For example, PQL333-335VDQ (SPE) amino acid substitutions reduce Factor VIII inactivation by 16-fold. Likewise, MKN336-339GNQ amino acid substitutions reduce Factor VIII inactivation by 9-fold. When combined, the two triple amino acid substitutions (e.g., PQLRMKN333-339VDQRGNQ) reduce Factor VIII inactivation by 100-fold (J. Biol. Chem., 282:20264 (2007). Accordingly, in some embodiments, the encoded Factor VIII polypeptide include PQL333-335VDQ and / or MKN337-339GNQ (SPE) amino acid substitutions.

[0143] Mutations within the A2 domain interface also increase Factor VIII stability. Specifically, mutating charged residues in the A1-A2 and A2-A3 domain interfaces increases stability and retention of the A2 subunit in Factor Villa. For example, mutation of D519, E665, and El 984 (SPE) to V or A yields up to 2-fold increased stability in Factor VIII and up to 5-fold stability in Factor Villa. Specifically, D519A / E665V amino acid substitutions provide a 3-fold increase in stability; D519V / E665V amino acid substitutions provide a 2-fold increase in stability, an 8-fold decrease in A2 dissociation, and a 2-4-fold increase in thrombin generation potential; D519V / E1984A amino acid substitutions provide a 2-fold increase in stability; and D519V / E665V / E1984A amino acid substitution provide a 2-fold increase in stability (Blood 112:2761-69 (2008); J. Thromb. Haemost., 7:438-44 (2009)). Accordingly, in some embodiments, the encoded Factor VIII polypeptides include one or more of D519A / V, E665A / V, and E1984A / V amino acid substitutions.

[0144] Mutations within an 11 amino acid hydrophobic P-sheet in the Al domain, which interacts with BiP, increase secretion of Factor VIII. For example, an F328S (SPI, F309S SPE) amino acid substitution within the pocket increased Factor VIII secretion 3-fold. See, for example, U.S. Patent No. 10,189,888, which is hereby disclosed herein by reference in its entirety.

[0145] The set of ammo acid substitutions I105V / A127S / G151K / M166T / L171P (SPI numbering; (SPE numbering is V86I / S108A / K132G / T147M / P152L, respectively) was based on the discovery that substitution of porcine amino acids 82-176 for the corresponding human amino acids in a B-domain deleted gene therapy construct increased Factor VIII activity when expressed in HEK293 cells. Back-mutation of single porcine amino acids into the human BDD-FVIII construct identified five amino acids within the Al domain that contribute to this phenomenon:I105V, A127S, G151K, M166T, and L171P (SPI). Introduction of the combination of these mutations into the human construct recapitulated the improved activity of the larger porcine substitution. See, for example, U.S. Patent No. 10,189,888, which is hereby disclosed herein by reference in its entirety.

[0146] Deletion of amino acids AIEPRSF755-761 and the insertion of amino acids TTYVNRSL (SEQ ID NO: 73) after N754, relative to FVIII-WT-FL-AA (SEQ ID NO:72) (e g., AIEPRSF755-761 TTYVNRSL) ("TTYVNRSL" disclosed as SEQ ID NO: 73) introduces an additional glycosylation site close to the interface. Residues ALEPR755-759, relative to SEQ ID NO: 72, fall within the end of the heavy chain, while residues S760 and F761 fall within the B- domain. In some embodiments, where the FVIII B-domain is deleted, truncated, or replaced, residues S760 and F761 may not be present in the underlying amino acid sequence being mutated. See, for example, U.S. Patent No. 10,189,888, which is hereby disclosed herein by reference in its entirety.

[0147] Elimination of the C1899-C1903 disulfide bond in Factor VIII also increased secretion. Moreover, the increases in Factor VIII secretion are additive for the combination of F328S (SPI, F309S SPE, full-length FVIII) and C1918G / C1922G amino acid substitutions (Miao et al., Blood, 103:3412-19 (2004); Selvaraj et al., J. Thromb. Haemost., 10:107-15 (2012)). Accordingly, in some embodiments, the encoded Factor VIII polypeptides include m4 mutations, which is the F328S (SPI, F309S SPE) and C1918G / C1922G (SPI) amino acid substitutions.

[0148] As above, elimination of the C1899-C1903 disulfide bond in Factor VIII also increased secretion. C1918G / C1922G (SPI) amino acid substitutions, contained within the light chain, are referred to herein as the m5 mutation set.

[0149] B. Factor VIII B-domain Substituted Linkers

[0150] In some embodiments, the linkage between the FVIII heavy chain and the light chain (e.g., the B-domain in wild-type Factor VIII) is further altered. Due to size constraints of AAV packaging capacity, B-domain deleted, truncated, and or linker substituted variants should improve the efficacy of the FVIII gene therapy construct. The most conventionally used B-domainsubstituted linker is that of SQ FVIII, which retains only 14 amino acids of the B domain as linker sequence. Another variant of porcine VIII (“OBI-1,” described in U.S. Patent No. 6,458,563) is well expressed in CHO cells and has a slightly longer linker of 24 amino acids. In some embodiments, the Factor VIII constructs encoded by the codon-altered polynucleotides described herein include an SQ-type B-domain linker sequence. In other embodiments, the Factor VIII constructs encoded by the codon-altered polynucleotides described herein include an OBI- 1 -type B-domain linker sequence.

[0151] In some embodiments, the encoded Factor VIII polypeptides described herein include an SQ-type B-domain linker, including amino acids 760-762 / 1657-1667 of the wild-type human Factor VIII B-domain (FVIII-WT-FL-AA (SEQ ID NO: 72)) (Sandberg et al. Thromb. Haemost. 85:93 (2001)). In some embodiments, the SQ-type B-domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the SQ-type B-domain linker has two amino acid substitutions relative to the corresponding wild-type sequence. In some embodiments, a glycosylation peptide is inserted into the SQ-type B-domain linker.

[0152] In some embodiments, the encoded Factor VIII polypeptides described herein include a Greengene-type B-domain linker, including amino acids 760 / 1582-1667 of the wild-type human Factor VIII B-domain (FVIII-WT-FL-AA (SEQ ID NO:72)) (Oh et al., Biotechnol. Prog., 17: 1999 (2001)). In some embodiments, the Greengene-type B-domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the Greengene-type B-domain linker has two amino acid substitutions relative to the corresponding wild-type sequence. In some embodiments, a glycosylation peptide is inserted into the Greengenetype B-domain linker. .

[0153] In some embodiments, the encoded Factor VIII polypeptides described herein include an extended SQ-type B-domain linker (SFSQNPPVLKRHQR; BDL-SQ-AA; SEQ ID NO: 8), including amino acids 760-769 / 1657-1667 of the wild-type human Factor VIII B-domain (FVIII-WT-FL-AA (SEQ ID NO:72)) (Thim et al., Haemophilia, 16:349 (2010)). In some embodiments, the extended SQ-type B-domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the extended SQ-type B-domainlinker has two amino acid substitutions relative to the corresponding wild-type sequence. In some embodiments, a glycosylation peptide is inserted into the extended SQ-type B-domain linker. .

[0154] In some embodiments, the encoded Factor VIII polypeptides described herein include a porcine OBI- 1 -type B-domain linker, including the amino acids SFAQNSRPPSASAPKPPVLRRHQR (SEQ ID NO: 74) from the wild-type porcine Factor VIII B-domain (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 relative to the corresponding wild-type sequence. In some embodiments, the porcine OBI- 1 -type B-domain linker has two amino acid substitutions relative to the corresponding wild-type sequence. In some embodiments, a glycosylation peptide is inserted into the porcine OBI- 1 -type B-domain linker. .

[0155] In some embodiments, the encoded Factor VIII polypeptides described herein include a human OBI-l-type B-domain linker, including amino acids 760-772 / 1655-1667 of the wild-type human Factor VIII B-domain (FVIII-WT-FL-AA (SEQ ID NO: 72)). In some embodiments, the human OBI- 1 -type B-domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the human OBI- 1 -type B-domain linker has two amino acid substitutions relative to the corresponding wild-type sequence. In some embodiments, a glycosylation peptide is inserted into the human OBI- 1 -type B-domain linker. .

[0156] In some embodiments, the encoded Factor VIII polypeptides described herein include an 08-type B-domain linker, including the amino acids SFSQNSRHQAYRYRRG (SEQ ID NO:75 from the wild-type porcine Factor VIII B-domain (Toschi et al., Curr. Opin. Mol. Ther. 12:517 (2010)). In some embodiments, the porcine OBI-l-type B-domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the porcine OBI-l-type B-domain linker has two amino acid substitutions relative to the corresponding wild-type sequence. In some embodiments, a glycosylation peptide is inserted into the porcine OBI- 1 -type B-domain linker. .

[0157] Removal of the B-domain from Factor VIII constructs does not appear to affect the activity of the activated enzyme (e.g., FVIIIa), presumably because the B-domain is removed during activation. However, the B-domain of Factor VIII contains several residues that are post- translationally modified, e.g., by N- or O-linked glycosylation. In silico analysis (Prediction of N-glycosylation sites in human proteins, R. Gupta, E. Jung and S. Brunak, in preparation (2004)) of the wild-type Factor VIII B-domain predicts that at least four of these sites are glycosylated in vivo. It is thought that these modifications within the B-domain contribute to the post-translational regulation and / or half-life of Factor VIII in vivo.

[0158] While the Factor VIII B-domain is absent in mature Factor Villa protein, glycosylation within the B-domain of the precursor Factor VIII molecule may increase 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 glycosylation in vivo. In some embodiments, the polypeptide linker includes at least one consensus glycosylation sequence (e.g., an N- or O-linked glycosylation consensus sequence). In some embodiments, the polypeptide linker includes at least two consensus glycosylation sequences. In some embodiments, the polypeptide linker includes at least three consensus glycosylation sequences. In some embodiments, the polypeptide linker includes at least four consensus glycosylation sequences. In some embodiments, the polypeptide linker includes at least five consensus glycosylation sequences. In some embodiments, the polypeptide linker includes at least 6, 7, 8, 9, 10, or more consensus glycosylation sequences.

[0159] In some embodiments, the polypeptide linker contains at least one N-linked glycosylation sequence N-X-S / 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 N-X- S / 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 N-X-S / 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 N-X-S / 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 N-X- S / 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 N-X-S / T, where X is any amino acid other than P, S, or T.

[0160] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to BDDFVIII-BDL-AA (SEQ ID NO: 8). In some embodiments, the third nucleotide sequence has at least 95% identityto SEQ ID NO: 8. In some embodiments, the third nucleotide sequence has at least 96% identity to SEQ ID NO: 8. In some embodiments, the third nucleotide sequence has at least 97% identity to SEQ ID NO: 8. In some embodiments, the third nucleotide sequence has at least 98% identity to SEQ ID NO: 8. In some embodiments, the third nucleotide sequence has at least 99% identity to SEQ ID NO: 8. In some embodiments, the third nucleotide sequence has at least 99.5% identity to SEQ ID NO: 8. In some embodiments, the third nucleotide sequence has at least 99.9% identity to SEQ ID NO: 8. In some embodiments, the third nucleotide sequence is identical to SEQ IDNO: 8.

[0161] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS06-BDL (SEQ ID NO: 42). In some embodiments, the third nucleotide sequence has at least 95% identity to CS06-BDL (SEQ ID NO: 42). In some embodiments, the third nucleotide sequence has at least 96% identity to CS06-BDL (SEQ ID NO: 42). In some embodiments, the third nucleotide sequence has at least 97% identity to CS06-BDL (SEQ ID NO: 42). In some embodiments, the third nucleotide sequence has at least 98% identity to CS06-BDL (SEQ ID NO: 42). In some embodiments, the third nucleotide sequence has at least 99% identity to CS06-BDL (SEQ ID NO: 42). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS06-BDL (SEQ ID NO: 42). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS06- BDL (SEQ ID NO: 42). In some embodiments, the third nucleotide sequence is identical to CS06- BDL (SEQ ID NO: 42).

[0162] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS07-BDL (SEQ ID NO: 46). In some embodiments, the third nucleotide sequence has at least 95% identity to CS07-BDL (SEQ ID NO: 46). In some embodiments, the third nucleotide sequence has at least 96% identity to CS07-BDL (SEQ ID NO: 46). In some embodiments, the third nucleotide sequence has at least 97% identity to CS07-BDL (SEQ ID NO: 46). In some embodiments, the third nucleotide sequence has at least 98% identity to CS07-BDL (SEQ ID NO: 46). In some embodiments, the third nucleotide sequence has at least 99% identity to CS07-BDL (SEQ ID NO: 46). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS07-BDL (SEQ ID NO: 46). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS07-BDL (SEQ ID NO: 46). In some embodiments, the third nucleotide sequence is identical to CS07- BDL (SEQ ID NO: 46).

[0163] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS08-BDL (SEQ ID NO: 50). In some embodiments, the third nucleotide sequence has at least 95% identity to CS08-BDL (SEQ ID NO: 50). In some embodiments, the third nucleotide sequence has at least 96% identity to CS08-BDL (SEQ ID NO: 50). In some embodiments, the third nucleotide sequence has at least 97% identity to CS08-BDL (SEQ ID NO: 50). In some embodiments, the third nucleotide sequence has at least 98% identity to CS08-BDL (SEQ ID NO: 50). In some embodiments, the third nucleotide sequence has at least 99% identity to CS08-BDL (SEQ ID NO: 50). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS08-BDL (SEQ ID NO: 50). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS08- BDL (SEQ ID NO: 50). In some embodiments, the third nucleotide sequence is identical to CS08- BDL (SEQ ID NO: 50).

[0164] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS09-BDL (SEQ ID NO: 54). In some embodiments, the third nucleotide sequence has at least 95% identity to CS09-BDL (SEQ ID NO: 54). In some embodiments, the third nucleotide sequence has at least 96% identity to CS09-BDL (SEQ ID NO: 54). In some embodiments, the third nucleotide sequence has at least 97% identity to CS09-BDL (SEQ ID NO: 54). In some embodiments, the third nucleotide sequence has at least 98% identity to CS09-BDL (SEQ ID NO: 54). In some embodiments, the third nucleotide sequence has at least 99% identity to CS09-BDL (SEQ ID NO: 54). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS09-BDL (SEQ ID NO: 54). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS09- BDL (SEQ ID NO: 54). In some embodiments, the third nucleotide sequence is identical to CS09- BDL (SEQ ID NO: 54).

[0165] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS10-BDL (SEQ ID NO: 58). In some embodiments, the third nucleotide sequence has at least 95% identity to CS10-BDL (SEQ ID NO: 58). In some embodiments, the third nucleotide sequence has at least 96% identityto CS10-BDL (SEQ ID NO: 58). In some embodiments, the third nucleotide sequence has at least 97% identity to CS10-BDL (SEQ ID NO: 58). In some embodiments, the third nucleotide sequence has at least 98% identity to CS10-BDL (SEQ ID NO: 58). In some embodiments, the third nucleotide sequence has at least 99% identity to CS10-BDL (SEQ ID NO: 58). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS10-BDL (SEQ ID NO: 58). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS10- BDL (SEQ ID NO: 58). In some embodiments, the third nucleotide sequence is identical to CS10- BDL (SEQ ID NO: 58).

[0166] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS11-BDL (SEQ ID NO: 62). In some embodiments, the third nucleotide sequence has at least 95% identity to CS11-BDL (SEQ ID NO: 62). In some embodiments, the third nucleotide sequence has at least 96% identity to CS11-BDL (SEQ ID NO: 62). In some embodiments, the third nucleotide sequence has at least 97% identity to CS11-BDL (SEQ ID NO: 62). In some embodiments, the third nucleotide sequence has at least 98% identity to CS11-BDL (SEQ ID NO: 62). In some embodiments, the third nucleotide sequence has at least 99% identity to CS11-BDL (SEQ ID NO: 62). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS11-BDL (SEQ ID NO: 62). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS11- BDL (SEQ ID NO: 62). In some embodiments, the third nucleotide sequence is identical to CS11- BDL (SEQ ID NO: 62).

[0167] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS12-BDL (SEQ ID NO: 66). In some embodiments, the third nucleotide sequence has at least 95% identity to CS12-BDL (SEQ ID NO: 66). In some embodiments, the third nucleotide sequence has at least 96% identity to CS12-BDL (SEQ ID NO: 66). In some embodiments, the third nucleotide sequence has at least 97% identity to CS12-BDL (SEQ ID NO: 66). In some embodiments, the third nucleotide sequence has at least 98% identity to CS12-BDL (SEQ ID NO: 66). In some embodiments, the third nucleotide sequence has at least 99% identity to CS12-BDL (SEQ ID NO: 66). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS12-BDL (SEQ ID NO: 66). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS12-BDL (SEQ ID NO: 66). In some embodiments, the third nucleotide sequence is identical to CS12- BDL (SEQ ID NO: 66).C. Codon-altered Polynucleotides Encoding a Factor VIII VariantCS01 Codon Altered Polynucleotides

[0168] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain, and a polypeptide linker joining 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 high sequence identity to CS01-HC (SEQ ID NO: 10), which is the portion of CS01-FL (SEQ ID NO: 9) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS01-LC (SEQ ID NO: 11), which is the portion of CS01-FL (SEQ ID NO: 9) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0169] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS01-HC and CS01-LC (SEQ ID NOs: 10 and 11), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS01-HC and CS01-LC (SEQ ID NOs: 10 and 11), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS01-HC and CS01-LC (SEQ ID NOs: 10 and 11), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS01-HC and CS01-LC (SEQ ID NOs: 10 and 11), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS01-HC and CS01-LC (SEQ ID NOs: 10 and 11), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS01 -HC and CS01 -LC (SEQ ID NOs: 10 and 11), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS01-HC and CS01-LC (SEQ ID NOs: 10 and 11), respectively. In some embodiments, the first and secondnucleotide sequences are identical to CS01-HC and CS01-LC (SEQ ID NOs: 10 and 11), respectively.

[0170] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS01 -BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS01-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS01-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS01-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS01-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99% identity to CS01-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS01-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS01- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS01- BDL (SEQ ID NO: 4).

[0171] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS01-FL (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 95% identity to CS01-FL (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 96% identity to CS01-FL (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 97% identity to CS01-FL (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 98% identity to CS01-FL (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 99% identity to CS01-FL (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS01-FL (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS01- FL (SEQ ID NO: 9). In some embodiments, the nucleotide sequence is identical to CS01-FL (SEQ ID NO: 9).

[0172] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS01-FL (SEQ ID NO: 9). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS01-FL (SEQ ID NO: 12). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all 24 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E809G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO: 5).

[0173] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS01-FL (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 97% identity to PS01-FL (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 98% identity to PS01- FL (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99% identity to PS01-FL (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99.5% identity to PS01-FL (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99.9% identity to PS01-FL (SEQ ID NO: 12). In some embodiments, the amino acid sequence is identical to PS01-FL (SEQ ID NO: 12).

[0174] In some embodiments, the Factor VIII variant encoded by the CS01 polynucleotide, having high sequence homology to PS01-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F3871, 1390 A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E809G, T1148N, L1248D, L1377D, and V1439E.

[0175] In one embodiment, the Factor VIII variant encoded by the CS01 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, 1390 A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E809G, T1148N, L1248D, L1377D, and V1439E.CS02 Codon Altered Polynucleotides

[0176] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain,and a polypeptide linker joining 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 high sequence identity to CS02-HC (SEQ ID NO: 16), which is the portion of CS02-FL (SEQ ID NO: 15) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS02-LC (SEQ ID NO: 17), which is the portion of CS02-FL (SEQ ID NO: 15) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0177] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS02-HC and CS02-LC (SEQ ID NOs: 16 and 17), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS02-HC and CS02-LC (SEQ ID NOs: 16 and 17), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS02-HC and CS02-LC (SEQ ID NOs: 16 and 17), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS02-HC and CS02-LC (SEQ ID NOs: 16 and 17), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS02-HC and CS02-LC (SEQ ID NOs: 16 and 17), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS02-HC and CS02-LC (SEQ ID NOs: 16 and 17), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS02-HC and CS02-LC (SEQ ID NOs: 16 and 17), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS02-HC and CS02-LC (SEQ ID NOs: 16 and 17), respectively.

[0178] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS02-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS02-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS02-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS02-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS02-BDL (SEQ ID NO: 4). In some embodiments, the thirdnucleotide sequence has at least 99% identity to CS02-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS02-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS02- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS02- BDL (SEQ ID NO: 4).

[0179] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS02-FL (SEQ ID NO: 15). In some embodiments, the nucleotide sequence has at least 95% identity to CS02-FL (SEQ ID NO: 15). In some embodiments, the nucleotide sequence has at least 96% identity to CS02-FL (SEQ ID NO: 15). In some embodiments, the nucleotide sequence has at least 97% identity to CS02-FL (SEQ ID NO: 15). In some embodiments, the nucleotide sequence has at least 98% identity to CS02-FL (SEQ ID NO: 15). In some embodiments, the nucleotide sequence has at least 99% identity to CS02-FL (SEQ ID NO: 15). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS02- FL (SEQ ID NO: 15). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS02-FL (SEQ ID NO: 15). In some embodiments, the nucleotide sequence is identical to CS02-FL (SEQ ID NO: 15).

[0180] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS02-FL (SEQ ID NO: 15). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS02-FL (SEQ ID NO: 18). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all 25 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO: 5).

[0181] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS02-FL (SEQ ID NO:18). In some embodiments, the amino acid sequence has at least 97% identity to PS02-FL (SEQ ID NO: 18). In some embodiments, the amino acid sequence has at least 98% identity to PS02- FL (SEQ ID NO: 18). In some embodiments, the amino acid sequence has at least 99% identity to PS02-FL (SEQ ID NO: 18). In some embodiments, the amino acid sequence has at least 99.5% identity to PS02-FL (SEQ ID NO: 18). In some embodiments, the amino acid sequence has at least 99.9% identity to PS02-FL (SEQ ID NO: 18). In some embodiments, the amino acid sequence is identical to PS02-FL (SEQ ID NO: 18).

[0182] In some embodiments, the Factor VIII variant encoded by the CS02 polynucleotide, having high sequence homology to PS02-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.

[0183] In one embodiment, the Factor VIII variant encoded by the CS02 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.CS03 Codon Altered Polynucleotides

[0184] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain, and a polypeptide linker joining 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 high sequence identity to CS03-HC (SEQ ID NO: 22), which is the portion of CS03-FL (SEQ ID NO: 21) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS03-LC (SEQ ID NO: 23), which is the portion of CS03-FL (SEQ ID NO: 21) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0185] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS03-HC and CS03-LC (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS03-HC and CS03-LC (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS03-HC and CS03-LC (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS03-HC and CS03-LC (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS03-HC and CS03-LC (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS03-HC and CS03-LC (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS03-HC and CS03-LC (SEQ ID NOs: 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS03-HC and CS03-LC (SEQ ID NOs: 22 and 23), respectively.

[0186] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS03-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS03-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS03-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS03-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS03-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99% identity to CS03-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS03-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS03- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS03- BDL (SEQ ID NO: 4).

[0187] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS03-FL (SEQ ID NO: 21). In some embodiments, the nucleotide sequence has at least 95% identity to CS03-FL (SEQ ID NO: 21). In some embodiments, thenucleotide sequence has at least 96% identity to CS03-FL (SEQ ID NO: 21). In some embodiments, the nucleotide sequence has at least 97% identity to CS03-FL (SEQ ID NO: 21). In some embodiments, the nucleotide sequence has at least 98% identity to CS03-FL (SEQ ID NO: 21). In some embodiments, the nucleotide sequence has at least 99% identity to CS03-FL (SEQ ID NO: 21). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS03- FL (SEQ ID NO: 21). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS03-FL (SEQ ID NO: 21). In some embodiments, the nucleotide sequence is identical to CS03-FL (SEQ ID NO: 21).

[0188] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS03-FL (SEQ ID NO: 21). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS03-FL (SEQ ID NO:24). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all 25 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F3871, 1390 A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E800V, K1093G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO: 5).

[0189] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS03-FL (SEQ ID NO: 24). In some embodiments, the amino acid sequence has at least 97% identity to PS03-FL (SEQ ID NO: 24). In some embodiments, the amino acid sequence has at least 98% identity to PS03- FL (SEQ ID NO: 24). In some embodiments, the amino acid sequence has at least 99% identity to PS03-FL (SEQ ID NO: 24). In some embodiments, the amino acid sequence has at least 99.5% identity to PS03-FL (SEQ ID NO: 24). In some embodiments, the amino acid sequence has at least 99.9% identity to PS03-FL (SEQ ID NO: 24). In some embodiments, the amino acid sequence is identical to PS03-FL (SEQ ID NO: 24).

[0190] In some embodiments, the Factor VIII variant encoded by the CS03 polynucleotide, having high sequence homology to PS03-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E800V, K1093G, T1148N, L1248D, L1377D, and V1439E.

[0191] In one embodiment, the Factor VIII variant encoded by the CS03 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E800V, K1093G, T1148N, L1248D, L1377D, and V1439E.CS04 Codon Altered Polynucleotides

[0192] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain, and a polypeptide linker joining 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 high sequence identity to CS04-HC (SEQ ID NO: 28), which is the portion of CS04-FL (SEQ ID NO: 27) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS04-LC (SEQ ID NO: 29), which is the portion of CS04-FL (SEQ ID NO: 27) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0193] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS04-HC and CS04-LC (SEQ ID NOs: 28 and 29), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS04-HC and CS04-LC (SEQ ID NOs: 28 and 29), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS04-HC and CS04-LC (SEQ ID NOs: 28 and 29), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS04-HC and CS04-LC (SEQ ID NOs: 28 and29), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS04-HC and CS04-LC (SEQ ID NOs: 28 and 29), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS04-HC and CS04-LC (SEQ ID NOs: 28 and 29), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS04-HC and CS04-LC (SEQ ID NOs: 28 and 29), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS04-HC and CS04-LC (SEQ ID NOs: 28 and 29), respectively.

[0194] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS04-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS04-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS04-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS04-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS04-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99% identity to CS04-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS04-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS04- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS04- BDL (SEQ ID NO: 4).

[0195] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS04-FL (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 95% identity to CS04-FL (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 96% identity to CS04-FL (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 97% identity to CS04-FL (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 98% identity to CS04-FL (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 99% identity to CS04-FL (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS04- FL (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 99.9% identityto CS04-FL (SEQ ID NO: 27). In some embodiments, the nucleotide sequence is identical to CS04-FL (SEQ ID NO: 27).

[0196] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS04-FL (SEQ ID NO: 27). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or all 26 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO:5).

[0197] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 97% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 98% identity to PS04- FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.5% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.9% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence is identical to PS04-FL (SEQ ID NO: 30).

[0198] In some embodiments, the Factor VIII variant encoded by the CS04 polynucleotide, having high sequence homology to PS04-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and VI 439E.

[0199] In one embodiment, the Factor VIII variant encoded by the CS04 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.CS05 Codon Altered Polynucleotides

[0200] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain, and a polypeptide linker joining 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 high sequence identity to CS05-HC (SEQ ID NO: 34), which is the portion of CS05-FL (SEQ ID NO: 33) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS05-LC (SEQ ID NO: 35), which is the portion of CS05-FL (SEQ ID NO: 33) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0201] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS05-HC and CS05-LC (SEQ ID NOs: 34 and 35), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS05-HC and CS05-LC (SEQ ID NOs: 34 and 35), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS05-HC and CS05-LC (SEQ ID NOs: 34 and 35), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS05-HC and CS05-LC (SEQ ID NOs: 34 and 35), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS05-HC and CS05-LC (SEQ ID NOs: 34 and 35), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS05-HC and CS05-LC (SEQ ID NOs: 34 and 35), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS05-HC and CS05-LC (SEQ ID NOs: 34 and 35), respectively. In some embodiments, the first and secondnucleotide sequences are identical to CS05-HC and CS05-LC (SEQ ID NOs: 34 and 35), respectively.

[0202] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS05-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS05-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS05-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS05-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS05-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99% identity to CS05-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS05-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS05- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS05- BDL (SEQ ID NO: 4).

[0203] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS05-FL (SEQ ID NO: 33). In some embodiments, the nucleotide sequence has at least 95% identity to CS05-FL (SEQ ID NO: 33). In some embodiments, the nucleotide sequence has at least 96% identity to CS05-FL (SEQ ID NO: 33). In some embodiments, the nucleotide sequence has at least 97% identity to CS05-FL (SEQ ID NO: 33). In some embodiments, the nucleotide sequence has at least 98% identity to CS05-FL (SEQ ID NO: 33). In some embodiments, the nucleotide sequence has at least 99% identity to CS05-FL (SEQ ID NO: 33). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS05- FL (SEQ ID NO: 33). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS05-FL (SEQ ID NO: 33). In some embodiments, the nucleotide sequence is identical to CS05-FL (SEQ ID NO: 33).

[0204] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS05-FL (SEQ ID NO: 33). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS05-FL (SEQ ID NO: 36). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or all 23 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, L750D, I775G, K1093A, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO: 5).

[0205] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS05-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 97% identity to PS05-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 98% identity to PS05- FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99% identity to PS05-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.5% identity to PS05-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.9% identity to PS05-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence is identical to PS05-FL (SEQ ID NO: 30).

[0206] In some embodiments, the Factor VIII variant encoded by the CS05 polynucleotide, having high sequence homology to PS05-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, L750D, I775G, K1093A, T1148N, L1248D, L1377D, and V1439E.

[0207] In one embodiment, the Factor VIII variant encoded by the CS05 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, L750D, I775G, K1093A, T1148N, L1248D, L1377D, and V1439E.CS06 Codon Altered Polynucleotides

[0208] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain,and a polypeptide linker joining 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 high sequence identity to CS06-HC (SEQ ID NO: 40), which is the portion of CS06-FL (SEQ ID NO: 39) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS06-LC (SEQ ID NO: 41), which is the portion of CS06-FL (SEQ ID NO: 39) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0209] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS06-HC and CS06-LC (SEQ ID NOs: 40 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS06-HC and CS06-LC (SEQ ID NOs: 40 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS06-HC and CS06-LC (SEQ ID NOs: 40 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS06-HC and CS06-LC (SEQ ID NOs: 40 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS06-HC and CS06-LC (SEQ ID NOs: 40 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS06-HC and CS06-LC (SEQ ID NOs: 40 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS06-HC and CS06-LC (SEQ ID NOs: 40 and 41), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS06-HC and CS06-LC (SEQ ID NOs: 40 and 41), respectively.

[0210] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS06-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS06-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS06-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS06-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS06-BDL (SEQ ID NO: 4). In some embodiments, the thirdnucleotide sequence has at least 99% identity to CS06-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS06-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS06- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS06- BDL (SEQ ID NO: 4).

[0211] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS06-FL (SEQ ID NO: 39). In some embodiments, the nucleotide sequence has at least 95% identity to CS06-FL (SEQ ID NO: 39). In some embodiments, the nucleotide sequence has at least 96% identity to CS06-FL (SEQ ID NO: 39). In some embodiments, the nucleotide sequence has at least 97% identity to CS06-FL (SEQ ID NO: 39). In some embodiments, the nucleotide sequence has at least 98% identity to CS06-FL (SEQ ID NO: 39). In some embodiments, the nucleotide sequence has at least 99% identity to CS06-FL (SEQ ID NO: 39). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS06- FL (SEQ ID NO: 39). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS06-FL (SEQ ID NO: 39). In some embodiments, the nucleotide sequence is identical to CS06-FL (SEQ ID NO: 39).

[0212] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS06-FL (SEQ ID NO: 39). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or all 26 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO:5).

[0213] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS04-FL (SEQ ID NO:30). In some embodiments, the amino acid sequence has at least 97% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 98% identity to PS04- FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.5% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.9% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence is identical to PS04-FL (SEQ ID NO: 30).

[0214] In some embodiments, the Factor VIII variant encoded by the CS06 polynucleotide, having high sequence homology to PS04-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and VI 439E.

[0215] In one embodiment, the Factor VIII variant encoded by the CS06 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.CS07 Codon Altered Polynucleotides

[0216] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain, and a polypeptide linker joining 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 high sequence identity to CS07-HC (SEQ ID NO: 44), which is the portion of CS07-FL (SEQ ID NO: 43) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS07-LC (SEQ ID NO: 45), which is the portion of CS07-FL (SEQ ID NO: 43) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0217] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS07-HC and CS07-LC (SEQ ID NOs: 44 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS07-HC and CS07-LC (SEQ ID NOs: 44 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS07-HC and CS07-LC (SEQ ID NOs: 44 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS07-HC and CS07-LC (SEQ ID NOs: 44 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS07-HC and CS07-LC (SEQ ID NOs: 44 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS07-HC and CS07-LC (SEQ ID NOs: 44 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS07-HC and CS07-LC (SEQ ID NOs: 44 and 45), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS07-HC and CS07-LC (SEQ ID NOs: 44 and 45), respectively.

[0218] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS07-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS07-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS07-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS07-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS07-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99% identity to CS07-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS07-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS07- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS07- BDL (SEQ ID NO: 4).

[0219] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS07-FL (SEQ ID NO: 43). In some embodiments, the nucleotide sequence has at least 95% identity to CS07-FL (SEQ ID NO: 43). In some embodiments, thenucleotide sequence has at least 96% identity to CS07-FL (SEQ ID NO: 43). In some embodiments, the nucleotide sequence has at least 97% identity to CS07-FL (SEQ ID NO: 43). In some embodiments, the nucleotide sequence has at least 98% identity to CS07-FL (SEQ ID NO: 43). In some embodiments, the nucleotide sequence has at least 99% identity to CS07-FL (SEQ ID NO: 43). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS07- FL (SEQ ID NO: 43). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS07-FL (SEQ ID NO: 43). In some embodiments, the nucleotide sequence is identical to CS07-FL (SEQ ID NO: 43).

[0220] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS07-FL (SEQ ID NO: 43). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or all 26 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO:5).

[0221] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 97% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 98% identity to PS04- FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.5% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.9% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence is identical to PS04-FL (SEQ ID NO: 30).

[0222] In some embodiments, the Factor VIII variant encoded by the CS07 polynucleotide, having high sequence homology to PS04-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and VI 439E.

[0223] In one embodiment, the Factor VIII variant encoded by the CS07 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.CS08 Codon Altered Polynucleotides

[0224] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain, and a polypeptide linker joining 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 high sequence identity to CS08-HC (SEQ ID NO: 48), which is the portion of CS08-FL (SEQ ID NO: 47) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS08-LC (SEQ ID NO: 49), which is the portion of CS08-FL (SEQ ID NO: 47) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0225] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS08-HC and CS08-LC (SEQ ID NOs: 48 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS08-HC and CS08-LC (SEQ ID NOs: 48 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS08-HC and CS08-LC (SEQ ID NOs: 48 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS08-HC and CS08-LC (SEQ ID NOs: 48 and49), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS08-HC and CS08-LC (SEQ ID NOs: 48 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS08-HC and CS08-LC (SEQ ID NOs: 48 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS08-HC and CS08-LC (SEQ ID NOs: 48 and 49), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS08-HC and CS08-LC (SEQ ID NOs: 48 and 49), 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 CS08-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS08-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS08-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS08-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS08-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99% identity to CS08-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS08-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS08- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS08- BDL (SEQ ID NO: 4).

[0227] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS08-FL (SEQ ID NO: 47). In some embodiments, the nucleotide sequence has at least 95% identity to CS08-FL (SEQ ID NO: 47). In some embodiments, the nucleotide sequence has at least 96% identity to CS08-FL (SEQ ID NO: 47). In some embodiments, the nucleotide sequence has at least 97% identity to CS08-FL (SEQ ID NO: 47). In some embodiments, the nucleotide sequence has at least 98% identity to CS08-FL (SEQ ID NO: 47). In some embodiments, the nucleotide sequence has at least 99% identity to CS08-FL (SEQ ID NO: 47). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS08- FL (SEQ ID NO: 47). In some embodiments, the nucleotide sequence has at least 99.9% identityto CS08-FL (SEQ ID NO: 47). In some embodiments, the nucleotide sequence is identical to CS08-FL (SEQ ID NO: 47).

[0228] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS08-FL (SEQ ID NO: 47). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or all 26 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO:5).

[0229] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 97% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 98% identity to PS04- FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.5% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.9% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence is identical to PS04-FL (SEQ ID NO: 30).

[0230] In some embodiments, the Factor VIII variant encoded by the CS08 polynucleotide, having high sequence homology to PS04-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and VI 439E.

[0231] In one embodiment, the Factor VIII variant encoded by the CS08 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.CS09 Codon Altered Polynucleotides

[0232] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain, and a polypeptide linker joining 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 high sequence identity to CS09-HC (SEQ ID NO: 52), which is the portion of CS09-FL (SEQ ID NO: 51) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS09-LC (SEQ ID NO: 53), which is the portion of CS09-FL (SEQ ID NO: 51) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0233] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS09-HC and CS09-LC (SEQ ID NOs: 52 and 53), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS09-HC and CS09-LC (SEQ ID NOs: 52 and 53), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS09-HC and CS09-LC (SEQ ID NOs: 52 and 53), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS09-HC and CS09-LC (SEQ ID NOs: 52 and 53), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS09-HC and CS09-LC (SEQ ID NOs: 52 and 53), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS09-HC and CS09-LC (SEQ ID NOs: 52 and 53), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS09-HC and CS09-LC (SEQ ID NOs: 52 and 53), respectively. In some embodiments, the first and secondnucleotide sequences are identical to CS09-HC and CS09-LC (SEQ ID NOs: 52 and 53), respectively.

[0234] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS09-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS09-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS09-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS09-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS09-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99% identity to CS09-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS09-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS09- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS09- BDL (SEQ ID NO: 4).

[0235] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS09-FL (SEQ ID NO: 51). In some embodiments, the nucleotide sequence has at least 95% identity to CS09-FL (SEQ ID NO: 51). In some embodiments, the nucleotide sequence has at least 96% identity to CS09-FL (SEQ ID NO: 51). In some embodiments, the nucleotide sequence has at least 97% identity to CS09-FL (SEQ ID NO: 51). In some embodiments, the nucleotide sequence has at least 98% identity to CS09-FL (SEQ ID NO: 51). In some embodiments, the nucleotide sequence has at least 99% identity to CS09-FL (SEQ ID NO: 51). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS09- FL (SEQ ID NO: 51). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS09-FL (SEQ ID NO: 51). In some embodiments, the nucleotide sequence is identical to CS09-FL (SEQ ID NO: 51).

[0236] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS09-FL (SEQ ID NO: 51). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or all 26 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO:5).

[0237] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 97% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 98% identity to PS04- FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.5% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.9% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence is identical to PS04-FL (SEQ ID NO: 30).

[0238] In some embodiments, the Factor VIII variant encoded by the CS09 polynucleotide, having high sequence homology to PS04-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and VI 439E.

[0239] In one embodiment, the Factor VIII variant encoded by the CS09 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.CS10 Codon Altered Polynucleotides

[0240] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable invivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain, and a polypeptide linker joining 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 high sequence identity to CS10-HC (SEQ ID NO: 56), which is the portion of CS10-FL (SEQ ID NO: 55) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS10-LC (SEQ ID NO: 57), which is the portion of CS10-FL (SEQ ID NO: 55) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0241] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS10-HC and CS10-LC (SEQ ID NOs: 56 and 57), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS10-HC and CS10-LC (SEQ ID NOs: 56 and 57), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS10-HC and CS10-LC (SEQ ID NOs: 56 and 57), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS10-HC and CS10-LC (SEQ ID NOs: 56 and 57), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS10-HC and CS10-LC (SEQ ID NOs: 56 and 57), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS 10-HC and CS 10-LC (SEQ ID NOs: 56 and 57), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS10-HC and CS10-LC (SEQ ID NOs: 56 and 57), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS10-HC and CS10-LC (SEQ ID NOs: 56 and 57), respectively.

[0242] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS10-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS10-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS10-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS10-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequencehas at least 98% identity to CS10-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99% identity to CS10-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS10-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS10- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS10- BDL (SEQ ID NO: 4).

[0243] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS10-FL (SEQ ID NO: 55). In some embodiments, the nucleotide sequence has at least 95% identity to CS10-FL (SEQ ID NO: 55). In some embodiments, the nucleotide sequence has at least 96% identity to CS10-FL (SEQ ID NO: 55). In some embodiments, the nucleotide sequence has at least 97% identity to CS10-FL (SEQ ID NO: 55). In some embodiments, the nucleotide sequence has at least 98% identity to CS10-FL (SEQ ID NO: 55). In some embodiments, the nucleotide sequence has at least 99% identity to CS10-FL (SEQ ID NO: 55). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS10- FL (SEQ ID NO: 55). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS10-FL (SEQ ID NO: 55). In some embodiments, the nucleotide sequence is identical to CS10-FL (SEQ ID NO: 55).

[0244] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS10-FL (SEQ ID NO: 55). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or all 26 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO:5).

[0245] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 97% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 98% identity to PS04- FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.5% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.9% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence is identical to PS04-FL (SEQ ID NO: 30).

[0246] In some embodiments, the Factor VIII variant encoded by the CS 10 polynucleotide, having high sequence homology to PS04-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and VI 439E.

[0247] In one embodiment, the Factor VIII variant encoded by the CS10 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.CS11 Codon Altered Polynucleotides

[0248] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain, and a polypeptide linker joining 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 high sequence identity to CS11-HC (SEQ ID NO: 60), which is the portion of CS11-FL (SEQ ID NO: 59) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS11-LC (SEQ ID NO: 61), which is the portion of CS11-FL (SEQ ID NO: 59) encoding for a Factor VIIIlight chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0249] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS11-HC and CS11-LC (SEQ ID NOs: 60 and 61), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS11-HC and CS11-LC (SEQ ID NOs: 60 and 61), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS11-HC and CS11-LC (SEQ ID NOs: 60 and 61), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS11-HC and CS11-LC (SEQ ID NOs: 60 and 61), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS11-HC and CS11-LC (SEQ ID NOs: 60 and 61), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS 11 -HC and CS 11 -LC (SEQ ID NOs: 60 and 61 ), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS11-HC and CS11-LC (SEQ ID NOs: 60 and 61), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS11-HC and CS11-LC (SEQ ID NOs: 60 and 61), respectively.

[0250] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS 11 -BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS11-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS11-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS11-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS11-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99% identity to CS11-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS11-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS11- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS11- BDL (SEQ ID NO: 4).

[0251] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS11-FL (SEQ ID NO: 59). In some embodiments, the nucleotide sequence has at least 95% identity to CS11-FL (SEQ ID NO: 59). In some embodiments, the nucleotide sequence has at least 96% identity to CS11-FL (SEQ ID NO: 59). In some embodiments, the nucleotide sequence has at least 97% identity to CS11-FL (SEQ ID NO: 59). In some embodiments, the nucleotide sequence has at least 98% identity to CS11-FL (SEQ ID NO: 59). In some embodiments, the nucleotide sequence has at least 99% identity to CS11-FL (SEQ ID NO: 59). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS11- FL (SEQ ID NO: 59). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS11-FL (SEQ ID NO: 59). In some embodiments, the nucleotide sequence is identical to CS11-FL (SEQ ID NO: 59).

[0252] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS11-FL (SEQ ID NO: 59). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or all 26 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO:5).

[0253] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 97% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 98% identity to PS04- FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.5% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has atleast 99.9% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence is identical to PS04-FL (SEQ ID NO: 30).

[0254] In some embodiments, the Factor VIII variant encoded by the CS11 polynucleotide, having high sequence homology to PS04-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and VI 439E.

[0255] In one embodiment, the Factor VIII variant encoded by the CS11 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.CS12 Codon Altered Polynucleotides

[0256] In one embodiment, the codon-altered polynucleotides provided herein include a nucleotide sequence encoding a Factor VIII variant polypeptide with a linker that is cleavable in vivo. The Factor VIII polypeptide includes a Factor VIII light chain, a Factor VIII heavy chain, and a polypeptide linker joining 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 high sequence identity to CS12-HC (SEQ ID NO: 64), which is the portion of CS12-FL (SEQ ID NO: 63) encoding for a Factor VIII heavy chain. The light chain of the Factor VIII polypeptide is encoded by a second nucleotide sequence with high sequence identity to CS12-LC (SEQ ID NO: 65), which is the portion of CS12-FL (SEQ ID NO: 63) encoding for a Factor VIII light chain. The polypeptide linker includes a furin cleavage site, which allows for maturation in vivo (e.g., after expression in vivo or administration of the precursor polypeptide).

[0257] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS12-HC and CS12-LC (SEQ ID NOs: 64 and 65), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS12-HC and CS12-LC (SEQ ID NOs: 64 and 65), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS12-HC and CS12-LC(SEQ ID NOs: 64 and 65), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS12-HC and CS12-LC (SEQ ID NOs: 64 and 65), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS12-HC and CS12-LC (SEQ ID NOs: 64 and 65), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS12-HC and CS12-LC (SEQ ID NOs: 64 and 65), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS12-HC and CS12-LC (SEQ ID NOs: 64 and 65), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS12-HC and CS12-LC (SEQ ID NOs: 64 and 65), respectively.

[0258] In some embodiments, the polypeptide linker of the Factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to CS12-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 95% identity to CS12-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 96% identity to CS12-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 97% identity to CS 12-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 98% identity to CS 12-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99% identity to CS12-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.5% identity to CS 12-BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence has at least 99.9% identity to CS 12- BDL (SEQ ID NO: 4). In some embodiments, the third nucleotide sequence is identical to CS12- BDL (SEQ ID NO: 4).

[0259] In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS12-FL (SEQ ID NO: 63). In some embodiments, the nucleotide sequence has at least 95% identity to CS12-FL (SEQ ID NO: 63). In some embodiments, the nucleotide sequence has at least 96% identity to CS12-FL (SEQ ID NO: 63). In some embodiments, the nucleotide sequence has at least 97% identity to CS12-FL (SEQ ID NO: 63). In some embodiments, the nucleotide sequence has at least 98% identity to CS12-FL (SEQ ID NO: 63). In some embodiments, the nucleotide sequence has at least 99% identity to CS12-FL (SEQ ID NO: 63). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS12-FL (SEQ ID NO: 63). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS12-FL (SEQ ID NO: 63). In some embodiments, the nucleotide sequence is identical to CS12-FL (SEQ ID NO: 63).

[0260] In some embodiments, the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to CS12-FL (SEQ ID NO: 63). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the nucleotide sequence encodes for a BDD-FVIII variant having at least 1, at least 2, at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or all 26 of the ammo acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E (numbered relative to the BDDFVIII-FL-AA sequence of SEQ ID NO:5).

[0261] In some embodiments, the Factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 97% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 98% identity to PS04- FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.5% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence has at least 99.9% identity to PS04-FL (SEQ ID NO: 30). In some embodiments, the amino acid sequence is identical to PS04-FL (SEQ ID NO: 30).

[0262] In some embodiments, the Factor VIII variant encoded by the CS12 polynucleotide, having high sequence homology to PS04-FL (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity), comprises one or more amino acid substitutions selected from V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and VI 439E.

[0263] In one embodiment, the Factor VIII variant encoded by the CS12 polynucleotide comprises amino acid substitutions V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E.Example Embodiments

[0264] In a first aspect, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a heavy chain, a light chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N- terminus of the light chain, wherein the nucleic acid sequence has at least 99% identity to a nucleic acid sequence selected from the group consisting of: CS01 -FL, CS02-FL, CS03-FL, CS04-FL, and CS05-FL.

[0265] In a second aspect, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a heavy chain, a light chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N- terminus of the light chain, wherein: the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS01-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS01 -LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS02-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS02-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS03-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS03-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS04-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS04-LC; or the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS05-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS05-LC.

[0266] In some embodiments of the first or second aspect, the Factor VIII polypeptide comprises: V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E809G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E800V, K1093G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); or V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F3871, 1390 A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, L750D, I775G, K1093A, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5).

[0267] In a third aspect, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a Factor VIII polypeptide, wherein the nucleic acid sequence has at least 95% identity to a nucleic acid sequence selected from the group consisting of: CS01-FL, wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, 1390 A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E809G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); CS02-FL, wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, 1390 A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); CS03-FL, wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E800V, K1093G, T1148N, L1248D, L1377D, and V1439E ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); CS04-FL, wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G,V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and CS05-FL, wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, L750D, I775G, K1093A, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5).

[0268] In a fourth aspect, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a heavy chain, a light chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N- terminus of the light chain, wherein: the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS01-HC, wherein the heavy chain comprises V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F3871, 1390 A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, and L750D amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS01-LC, wherein the light chain comprises I775G, E809G, T1148N, L1248D, L1377D, and V1439E ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS02-HC, wherein the heavy chain comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, and K752E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS02-LC, wherein the light chain comprises I775G, K1093G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS03-HC, wherein the heavy chain comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, and L750D amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS03-LC, wherein the light chain comprises I775G; E800V; K1093G; T1148N; L1248D; L1377D; and V1439E ammo acid substitutionrelative to BDDFVIII-FL-AA (SEQ ID NO: 5); the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS04-HC, wherein the heavy chain comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, and K752E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS04-LC, wherein the light chain comprises I775G, K1093G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); or the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS05-HC, wherein the heavy chain comprises V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, and L750D amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS05-LC, wherein the light chain comprises I775G, KI 093 A, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5).

[0269] In some embodiments of the aspects disclosed above, the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having at least 99% identity to PS01-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS01-LC; the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having at least 99% identity to PS02-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS02- LC; the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having at least 99% identity to PS03-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS03-LC; the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having at least 99% identity to PS04-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS04-LC; or the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having at least 99% identity to PS05-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS05-LC.

[0270] In some embodiments of the aspects disclosed above, the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence of PS01-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence of PS01-LC; the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence of PS02-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence of PS02-LC; the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence of PS03-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence of PS03-LC; the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence of PS04-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence of PS04- LC; or the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence of PS05- HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence ofPS05-LC.

[0271] In some embodiments of the aspects disclosed above, the polypeptide linker comprises a furin cleavage site.

[0272] In some embodiments of the aspects disclosed above, the polypeptide linker comprises an amino acid sequence that is BDDFVIII BDL AA.

[0273] In some embodiments of the aspects disclosed above, the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to a sequence selected from the group consisting of: BDDFVIII-BDL-NA; CS06-BDL; CS07-BDL; CS08-BDL; CS09-BDL; CS10-BDL; CS11-BDL; and CS12-BDL.

[0274] In some embodiments of the aspects disclosed above, the third nucleotide sequence has at least 97.5% identity to a sequence selected from the group consisting of: BDDFVIII-BDL- NA; CS06-BDL; CS07-BDL; CS08-BDL; CS09-BDL; CS10-BDL; CS11-BDL; and CS12-BDL.

[0275] In some embodiments of the aspects disclosed above, the third nucleotide sequence is selected from the group consisting of: BDDFVIII-BDL-NA; CS06-BDL; CS07-BDL; CS08- BDL; CS09-BDL; CS10-BDL; CS11-BDL; and CS12-BDL.

[0276] In some embodiments of the aspects disclosed above, the polynucleotide encodes a polypeptide comprising a sequence having at least 95% identity to an amino acid sequence selected from the group consisting of: PS01-FL; PS02-FL; PS03-FL; PS04-FL; and PS05-FL.

[0277] In some embodiments of the aspects disclosed above, the polynucleotide encodes a polypeptide comprising a sequence having at least 99% identity an amino acid sequence selected from the group consisting of: PS01-FL (SEQ ID NO: 12); PS02-FL (SEQ ID NO: 18); PS03-FL (SEQ ID NO: 24); PS04-FL (SEQ ID NO: 30); and PS05-FL (SEQ ID NO: 36).

[0278] In some embodiments of the aspects disclosed above, the polynucleotide encodes a polypeptide comprising an amino acid sequence selected from the group consisting of: PS01-FL (SEQ ID NO: 12); PS02-FL (SEQ ID NO: 18); PS03-FL (SEQ ID NO: 24); PS04-FL (SEQ ID NO: 30); and PS05-FL (SEQ ID NO: 36).

[0279] In some embodiments of the aspects disclosed above, the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS01-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS01-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS02- HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS02-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS03-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS03-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS04-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS04-LC; or the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS05-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS05-LC.

[0280] In some embodiments of the aspects disclosed above, the nucleic acid sequence is selected from the group consisting of: CS01-FL; CS02-FL; CS03-FL; CS04-FL; and CS05-FL.

[0281] In a fifth aspect, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a heavy chain, a light chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N-terminus of the light chain, wherein the nucleic acid sequence has at least 95% identity to a nucleic acid sequence selected from the group consisting of: CS06-FL; CS07-FL; CS08-FL; CS09-FL; CS10-FL; CS11-FL; and CS12-FL.

[0282] In some embodiments of the fifth aspect, the polynucleotide comprises a sequence having at least 99% identity to a nucleic acid sequence selected from the group consisting of: CS06-FL (SEQ ID NO: 39); CS07-FL (SEQ ID NO: 43); CS08-FL (SEQ ID NO: 47); CS09-FL (SEQ ID NO: 51); CS10-FL (SEQ ID NO: 55); CS11-FL (SEQ ID NO: 59); and CS12-FL (SEQ ID NO: 63).

[0283] In a sixth aspect, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a heavy chain, a light chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N- terminus of the light chain, wherein: the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS06-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS06-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS07-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS07-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS08-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS08-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS09-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS09-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS10- HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS10-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS11-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS11-LC; or the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS12-HC; and wherein the light chainof the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS12-LC.

[0284] In some embodiments of the sixth aspect, the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS06-HC (SEQ ID NO: 40); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS06-LC (SEQ ID NO: 41); the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS07-HC (SEQ ID NO: 44); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS07-LC (SEQ ID NO: 45); the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS08-HC (SEQ ID NO: 48); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS08-LC (SEQ ID NO: 49); the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS09-HC (SEQ ID NO: 52); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS09-LC (SEQ ID NO: 53); the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS10-HC (SEQ ID NO: 56); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS10-LC (SEQ ID NO: 57); the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS11- HC (SEQ ID NO: 60); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS11-LC (SEQ ID NO: 61); or the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS12-HC (SEQ ID NO: 64); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS12-LC (SEQ ID NO: 65).

[0285] In some embodiments of the sixth aspect, the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N,L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5).

[0286] In some embodiments of the sixth aspect, the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having at least 99% identity to PS04-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS04-LC.

[0287] In some embodiments of the sixth aspect, the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence of PS04-HC; and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence of PS04-LC.

[0288] In some embodiments of the sixth aspect, the polypeptide linker comprises a furin cleavage site.

[0289] In some embodiments of the sixth aspect, the polypeptide linker comprises an amino acid sequence that is BDDFVIII BDL AA.

[0290] In some embodiments of the sixth aspect, the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to a sequence selected from the group consisting of: BDDFVIII-BDL-NA; CS06-BDL; CS07-BDL; CS08-BDL; CS09-BDL; CS10- BDL; CS11-BDL; and CS12-BDL.

[0291] In some embodiments of the sixth aspect, the third nucleotide sequence has at least 97.5% identity to a sequence selected from the group consisting of: BDDFVIII-BDL-NA; CS06- BDL; CS07-BDL; CS08-BDL; CS09-BDL; CS10-BDL; CS11-BDL; and CS12-BDL.

[0292] In some embodiments of the sixth aspect, the third nucleotide sequence is selected from the group consisting of: BDDFVIII-BDL-NA; CS06-BDL; CS07-BDL; CS08-BDL; CS09- BDL; CS10-BDL; CS11-BDL; and CS12-BDL.

[0293] In some embodiments of the sixth aspect, the nucleic acid sequence encodes a polypeptide having an amino acid sequence of PS04-FL.

[0294] In some embodiments of the sixth aspect, the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS06-HC; and wherein the light chain ofthe Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS06-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS07-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS07-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS08-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS08-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS09-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS09-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS10-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS 10-LC; the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS11-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS11-LC; or the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS12-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS12-LC.

[0295] In some embodiments of the sixth aspect, the nucleic acid sequence is selected from the group consisting of: CS06-FL; CS07-FL; CS08-FL; CS09-FL; CS10-FL; CS11-FL; and CS12- FL.

[0296] In some embodiments of the aspects described above, the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the promoter is an hTTR promoter located 5' of the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the hTTR promoter has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 68. In some embodiments, the hTTR promoter has a sequence of SEQ ID NO: 68.

[0297] In some embodiments of the aspects described above, the polynucleotide further comprises a liver-specific regulatory element operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the liver-specific regulatory element is an enhancer located 5' of the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the enhancer comprises at least 1 copy of CRM8 (lxCRM8), 2 copies of CRM8 (2xCRM8), or at least 3 copies of CRM8 (3xCRM8). In some embodiments, the enhanceris 3xCRM8. In some embodiments, the CRM8 enhancer has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 67. In some embodiments, the CRM8 enhancer has a sequence of SEQ ID NO: 67.

[0298] In some embodiments, the promoter and the liver-specific regulatory element are directly attached.

[0299] In some embodiments, the polynucleotide further comprises an intron operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the intron comprises an MVM intron located 5' of the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the MVM intron has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69. In some embodiments, said MVM intron has a sequence of SEQ ID NO: 69. In some embodiments, said intron is positioned between the promoter and the translation initiation site of the polynucleotide encoding the Factor FVIII polypeptide.

[0300] In some embodiments of the aspects described above, the polynucleotide further comprises a post-translational regulatory element operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the post-translational regulatory element is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) located 3' of the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the WPRE has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 70.

[0301] In some embodiments of the aspects described above, the polynucleotide further comprises a terminator operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the terminator is a polyadenylation element located 3' of the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the polyadenylation element is selected from the group consisting of SV40-polyA, hGH-polyA, BGH- polyA, and rbGlob-polyA. In some embodiments, the polyadenylation element is BGH-polyA having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 71.

[0302] In some embodiments of the aspects described above, the polynucleotide comprises from the 5'-terminus to the 3'-terminus, a 3xCRM8 enhancer, an hTTR promoter, an MVM intron, a polynucleotide encoding the Factor FVIII polypeptide, a WPRE, and a BGH-polyA terminator.

[0303] D. Factor VIII Expression Vectors

[0304] In some embodiments, the codon-altered polynucleotides described herein are integrated into expression vectors. Non-limiting examples of expression vectors include viral vectors (e.g., vectors suitable for gene therapy), non- viral vectors, liposomes, other lipid containing complexes including lipid nanoparticles (LNPs), other macromolecular complexes, inorganic nanoparticles, synthetic modified mRNA, unmodified mRNA, small molecules, non- biologically active molecules (e.g., gold particles), polymerized molecules (e.g., dendrimers), naked DNA, phages, transposons, episomes. plasmid vectors, bacteriophage vectors, cosmids, phagemids, artificial chromosomes, and the like.

[0305] Non-limiting examples of viral vectors include: retrovirus, e.g., Moloney murine leukemia virus (MMLV), Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; and polio viruses.

[0306] In some embodiments, the codon-altered polynucleotides described herein are integrated into a gene therapy vector. In some embodiments, the gene therapy vector is a retrovirus, and particularly a replication-deficient retrovirus. Protocols for the production of replication-deficient retroviruses are known in the art. For review, 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, N.J. (1991).

[0307] In one embodiment, the gene therapy vector is an adeno-associated virus (AAV) based gene therapy vector. AAV systems have been described previously and are generally well 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. Then, 20(4):699-708 (2012), each incorporated herein by reference in their entireties for all purposes). Details concerning the generation and use of rAAV vectors are described, for example, in U. S. Patent Nos.5,139,941 and 4,797,368, each incorporated herein by reference in their entireties for all purposes. In a particular embodiment, the AAV vector is an AAV-8 vector.

[0308] In some embodiments, the codon-altered polynucleotides described herein are integrated into a retroviral expression vector. These systems have been described previously, and are generally well 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 a specific embodiment, the retroviral vector is a lentiviral vector (see, for example, Naldini et al., Science, 212(5259)' '.263-26rl , 1996; Zufferey et al.,NatBiotechnol, 15(9): 871 -875, 1997; Blomer etal., J Virol., 71(9): 6641-6649, 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136).

[0309] A wide variety of vectors can be used for the expression of a Factor VIII polypeptide from a codon-altered polypeptide in cell culture, including eukaryotic and prokaryotic expression vectors. In certain embodiments, a plasmid vector is contemplated for use in expressing a Factor VIII polypeptide in cell culture. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector can carry a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. The plasmid will include the codon-altered polynucleotide encoding the Factor VIII polypeptide, operably linked to one or more control sequences, for example, a promoter.

[0310] Non-limiting examples of vectors for prokaryotic expression include plasmids such as pRSET, pET, pBAD, etc., wherein the promoters used in prokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET, using promoters such as A0X1, GAP, GALI, AUG1, etc; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., using promoters such as PH, plO, MT, Ac5, OpIE2, gp64, polh, etc., and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc / RSV, pcDNA3, pBPV, etc., and vectors derived from viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses, retroviruses, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and P-actin.

[0311] In some embodiments, the expression vector provided herein further comprises an hTTR promoter operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the hTTR promoter has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 68. In some embodiments, the hTTR promoter sequence is identical to SEQ ID NO: 68.

[0312] In some embodiments, the expression vector further comprises a liver-specific regulatory element operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the liver-specific regulatory element is an enhancer located 5’ of the nucleic acid sequence encoding the Factor VIII polypeptide.

[0313] In some embodiments, the enhancer comprises at least 1 copy of CRM8 (lxCRM8),2 copies of CRM8 (2xCRM8), 3 copies of CRM8 (3xCRM8), 4 copies or more of CRM8 (4xCRM8). In some embodiments, the enhancer is 3xCRM8. In some embodiments, the 3xCRM8 enhancer has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 67. In some embodiments, the 3xCRM8 enhancer sequence is identical SEQ ID NO: 67. In some embodiments, the promoter and the liver-specific regulatory element are directly attached.

[0314] In some embodiments, the expression vector further comprises an intron operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the intron comprises an MVM intron located 5’ of the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the MVM intron has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69. In some embodiments, said MVM intron has a sequence of SEQ ID NO: 69. In some embodiments, said intron is positioned between the promoter and the translation initiation site of the polynucleotide encoding the Factor FVIII polypeptide.

[0315] In some embodiments, the expression vector further comprises a post-translational regulatory element operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the post-translational regulatory element is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) located 3’ of the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the WPRE has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 70. In some embodiments, thepolynucleotide further comprises a terminator operably linked to the polynucleotide encoding the Factor VIII polypeptide.

[0316] In some embodiments, the terminator is a polyadenylation element located 3 ’ of the polynucleotide encoding the Factor VIII polypeptide. In some embodiments, the polyadenylation element is selected from the group consisting of SV40-polyA, hGH-polyA, BGH-polyA, and rbGlob-polyA. In some embodiments, the polyadenylation element is BGH-polyA having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 71.

[0317] In some embodiments, the expression vector comprises from 5’ to 3’ a 3xCRM8 enhancer, an hTTR promoter, an MVM intron, a polynucleotide encoding the Factor FVIII polypeptide, a WPRE, and a BGH-polyA terminator.IV. Gene Therapy Vehicle

[0318] In one aspect, the present disclosure provides a gene therapy vehicle. Non-limiting examples of gene therapy vehicles include: adeno-associated virus (AAV), adenovirus (AV), lentivirus (LV), non-viral vectors, liposomes, other lipid containing complexes including lipid nanoparticles (LNPs), other macromolecular complexes, inorganic nanoparticles, synthetic modified mRNA, unmodified mRNA, small molecules, non-biologically active molecules (e.g., gold particles), polymerized molecules (e.g., dendrimers), naked DNA, phages, transposons, episomes. plasmid vectors, bacteriophage vectors, cosmids, phagemids, artificial chromosomes, and the like. It is not intended that the present disclosure be limited to any specific gene therapy vector, as any vehicle suitable for a given setting finds use. The gene therapy vector may be designed to deliver genes to specific cells, tissues, species or host, or may find more general applicability.

[0319] In some embodiments, the disclosure provides non-viral delivery vehicles for delivering the variant Factor VIII polynucleotides described herein. In some embodiments, the non-vinal delivery vehicle is a particle-based delivery vehicle.

[0320] In some embodiments, the particle is a polymer-based particle. A polymer-based particle may be made of a biodegradable polymer or a non-biodegradable polymer. Non-limiting examples of non-biodegradable polymers include polyethylenimine (PEI), Poly(amidoamine)(PAMAM), amine-terminated PAMAM, and poly(vinylimidazole) (PVI). Non-limiting examples of biodegradable polymers include chitosan (CS), poly(P-amino ester)s (PBAEs), and polylactide (PLA).

[0321] In some embodiments, the particle is a liposome or lipid nanoparticle (LNP). Nonlimiting examples of lipids useful for forming a liposome or LNP include cationic lipids, such as N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N- dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and1.2-Dioleyloxy-3 -trimethylaminopropane chloride salt), N-(l-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1.2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N,N-dimethylaminopropane (y- DLenDMA), 1 ,2-Dilinoleylcarbamoyloxy-3 -dimethylaminopropane (DLin-C-DAP), 1,2- Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilinoleyoxy-3- morpholinopropane (DLin-MA), 1 ,2-Dilinoleoy 1-3 -dimethylaminopropane (DLinDAP), 1,2- Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-Linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. Cl), l,2-Dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2- Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-l,2- propanediol (DLinAP), 3-(N,N-Dioleylamino)-l,2-propanediol (DOAP), l,2-Dilinoleyloxo-3-(2- N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl- [l,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31 -tetraen- 19-yl4-(dimethylamino)butanoate (MC3), 1 , 1 '-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dihnoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K- DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N- dimethylpropan-1 -amine (MC3 Ether), 4-((6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31-tetraen-19- yloxy)-N,N-dimethylbutan-l -amine (MC4 Ether), or any combination thereof. Other cationiclipids include, but are not limited to, N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P — (N — (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(l-(2,3- dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dileoyl-sn-3- phosphoethanolamine (DOPE), l,2-dioleoyl-3 -dimethylammonium propane (DODAP), N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), and 2,2- Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (XTC). Additionally, commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL), and Lipofectamine (comprising DOSPA and DOPE, available from GIBCO / BRL).

[0322] Other suitable cationic lipids are disclosed in International Publication Nos. WO 09 / 086558, WO 09 / 127060, WO 10 / 048536, WO 10 / 054406, WO 10 / 088537, WO 10 / 129709, and WO 2011 / 153493; U.S. Patent Publication Nos. 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803; U.S. Pat. No. 8,158,601; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are herein incorporated by reference.

[0323] In some embodiments, the particle is a peptide-based particle. A peptide-based particle may be made of peptide nucleic acids (PNAs), polypeptides, functional peptides, and combination thereof. In some embodiments, the peptides are from about 2 to about 50 amino acids in length and are used to form nanoscale structures that interact with a polynucleotide, e.g., a variant BDD-FVIII polynucleotide described herein, to form a complex that facilitates delivery.

[0324] In some embodiments, the particle is formed from an inorganic material. Nonlimiting examples of inorganic materials that can be used to form a nanoparticle include mesoporous silica, gold, magnetic ferric particles, carbon nanotubes, graphine, and quantum dots.

[0325] Non-limiting examples of non-vial delivery vehicles for polynucleotide delivery are described, for example, in Ramamoorth M and Narvekar A., Non viral vectors in gene therapy- an overview, J Clin Diagn Res., 9(l):GE01-6 (2015), Zu, H. and Gao, D., Non-viral Vectors in Gene Therapy: Recent Development, Challenges, and Prospects, AAPS J, 23(78) (2021), and Kanvinde S, Kulkarni T, Deodhar S, Bhattacharya D, and Dasgupta A., Non-Viral Vectors forDelivery of Nucleic Acid Therapies for Cancer, BioTech, 11(1):6 (2022), the contents of which are disclosed herein by reference, in their entireties, for all purposes.A. AAV vector

[0326] In one aspect, the present disclosure provides an adenoviral-based vector comprising the polynucleotide encoding Factor VIII variants as described herein. AAV vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0327] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and w2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lackof ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. Reference is made to US 2003 / 0087817, International Patent Application No. WO 2016 / 205764, International Patent Application No. WO 2018 / 071868, U.S. Patent Publication No. 2018 / 0127780, and International Publication No. WO 2020 / 236982, the disclosures of each of which are incorporated herein by reference.

[0328] In some embodiments, the base editor constructs (including, the split-constructs) may be engineered for delivery in one or more AAV vectors. An AAV as related to any of the methods and compositions provided herein may be of any serotype including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2 / 1, 2 / 5, 2 / 8, 2 / 9, 3 / 1, 3 / 5, 3 / 8, or 3 / 9). In other embodiments, the AAV may be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, or AAV12 vector or particle. In some embodiments, the AAV vector of the present disclosure is AAV-8.

[0329] Methods of making or packaging AAV vectors are known in the art and reagents are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Nos. US 2007 / 0015238 and US 2012 / 0322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid comprising a gene of interest may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP2 region as described herein), and transfected into a recombinant cells such that the AAV vector can be packaged and subsequently purified.

[0330] In some embodiments, the AAV vector comprises a nucleic acid vector, which may comprise at a minimum: (a) the polynucleotide encoding the FVIII polypeptide described herein, and (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the polynucleotide.

[0331] In some embodiments, the AAV vector provided herein has capsid proteins that have amino acids of different serotypes outside of the VP1 region. In some embodiments, the serotype of the backbone of the VP1 protein is different from the serotype of the ITRs and / or theRep gene. In some embodiments, the serotype of the backbone of the VP1 capsid protein of a particle is the same as the serotype of the ITRs. In some embodiments, the serotype of the backbone of the VP1 capsid protein of a particle is the same as the serotype of the Rep gene. In some embodiments, capsid proteins of the AAV vector comprise amino acid mutations that result in improved transduction efficiency.

[0332] In some embodiments, the AAV vectors provided herein are single-stranded (i.e., ssAAV vectors) or self-complementary (i.e., scAAV vectors). In some embodiments, the AAV vectors are single-stranded ssAAV vectors. In some embodiments, the AAV vectors are self- complementary scAAV vectors.

[0333] In some embodiments, the AAV vector further comprises one or more regulatory elements that facilitate expression of the FVIII polypeptide in a host upon transfection. As will be appreciated by a skilled person in the art, non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails.

[0334] In some embodiments, the AAV vector further comprises an hTTR promoter operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the hTTR promoter has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 68. In some embodiments, the hTTR promoter sequence is identical to SEQ ID NO: 68.

[0335] In some embodiments, the AAV vector further comprises a liver-specific regulatory element operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the liver-specific regulatory element is an enhancer located 5’ of the nucleic acid sequence encoding the Factor VIII polypeptide.

[0336] In some embodiments, the enhancer comprises at least 1 copy of CRM8 (lxCRM8), 2 copies of CRM8 (2xCRM8), 3 copies of CRM8 (3xCRM8), 4 copies or more of CRM8 (4xCRM8). In some embodiments, the enhancer is 3xCRM8. In some embodiments, the 3xCRM8 enhancer has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 67. In some embodiments, the 3xCRM8 enhancer sequence is identical SEQ ID NO: 67. In some embodiments, the promoter and the liver-specific regulatory element are directly attached.

[0337] In some embodiments, the AAV vector further comprises an intron operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the intron comprises an MVM intron located 5’ of the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the MVM intron has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69. In some embodiments, said MVM intron has a sequence of SEQ ID NO: 69. In some embodiments, said intron is positioned between the promoter and the translation initiation site of the polynucleotide encoding the Factor FVIII polypeptide.

[0338] In some embodiments, the AAV vector further comprises a post-translational regulatory element operably linked to the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the post-translational regulatory element is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) located 3’ of the nucleic acid sequence encoding the Factor VIII polypeptide. In some embodiments, the WPRE has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 70. In some embodiments, the polynucleotide further comprises a terminator operably linked to the polynucleotide encoding the Factor VIII polypeptide.

[0339] In some embodiments, the terminator is a polyadenylation element located 3 ’ of the polynucleotide encoding the Factor VIII polypeptide. In some embodiments, the polyadenylation element is selected from the group consisting of SV40-polyA, hGH-polyA, BGH-polyA, and rbGlob-polyA. In some embodiments, the polyadenylation element is BGH-polyA having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 71.

[0340] In some embodiments, the AAV vector comprises from 5’ to 3’ a 3xCRM8 enhancer, an hTTR promoter, an MVM intron, a polynucleotide encoding the Factor FVIII polypeptide, a WPRE, and a BGH-polyA terminator.B. Lipid Nanoparticles

[0341] Lipid nanoparticles (LNP) have a structure that includes a single monolayer or bilayer of lipids that encapsulates a compound in a solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous phase or other liquid phase in its interior, but rather the lipids from the bilayer or monolayer shell are directly complexed to the internal compound therebyencapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles having a relatively uniform dispersion of shape and size. While sources vary on what size qualifies a lipid particle as being a nanoparticle, there is some overlap in agreement that a lipid nanoparticle can have a diameter in the range of from 10 nm to 1000 nm. However, more commonly they are considered to be smaller than 120 nm or even 100 nm.

[0342] For lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to include an ionizable cationic lipid which can complex to and associate with the negatively charged backbone of the nucleic acid core. Ionizable cationic lipids with apparent pKa values below about 7 have the benefit of providing a cationic lipid for complexing with the nucleic acid's negatively charged backbone and loading into the lipid nanoparticle at pH values below the pKa of the ionizable lipid where it is positively charged. Then, at physiological pH values, the lipid nanoparticle can adopt a relatively neutral exterior allowing for a significant increase in the circulation half-lives of the particles following i.v. administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutics, and low levels of cytotoxicity and immunogenicity.

[0343] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids were widely studied as synthetic materials for delivery of nucleic acid medicines. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed by cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and characterized by broad size distributions ranging from the submicron scale to a few microns. Lipoplexes, such as the Lipofectamine© reagent, have found considerable utility for in vitro transfection. However, these first-generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (imparted by the cationic lipid) result in rapid plasma clearance, hemolytic and other toxicities, as well as immune system activation.

[0344] The polynucleotide disclosed herein or a pharmaceutically acceptable salt thereof can be incorporated into a lipid nanoparticle.

[0345] In some embodiments, the present disclosure provides lipid nanoparticles comprising the FVHLencoding polynucleotide encapsulated within the lipid nanoparticle.

[0346] In some embodiments, the polynucleotide is fully encapsulated within the lipid portion of the lipid nanoparticle such that the polynucleotide in the lipid nanoparticle is resistant in aqueous solution to nuclease degradation. In other embodiments, the lipid nanoparticles described herein are substantially non-toxic to mammals such as humans.

[0347] In some embodiments, the lipid nanoparticles comprise a polynucleotide provided herein, a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates). The lipid nanoparticles can also include cholesterol.

[0348] In the nucleic acid-lipid nanoparticles, the polynucleotide may be fully encapsulated within the lipid portion of the nanoparticle, thereby protecting the nucleic acid from nuclease degradation. In preferred embodiments, a lipid nanoparticle comprising an polynucleotide is fully encapsulated within the lipid portion of the lipid nanoparticle, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the polynucleotide in the lipid nanoparticle is not substantially degraded after exposure of the particle to a nuclease at 37° C for at least 20, 30, 45, or 60 minutes. In certain other instances, the polynucleotide in the lipid nanoparticle is not substantially degraded after incubation of the nanoparticle in serum at 37° C. for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the polynucleotide is complexed with the lipid portion of the nanoparticle.

[0349] In some embodiments, full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid. Encapsulation is determined by adding the dye to a lipid nanoparticle, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane- impermeable dye.

[0350] In some embodiments, the lipid nanoparticles comprise the polynucleotide that is fully encapsulated within the lipid portion of the nanoparticle, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction thereof or range therein) of the particles have the polynucleotide encapsulated therein. The amount may be any value or subvalue within the recited ranges, including endpoints.

[0351] Depending on the intended use of the lipid nanoparticle, the proportions of the components can be varied, and the delivery efficiency of a particular nanoparticle can be measured using assays known in the art.

[0352] In some embodiments, a lipid nanoparticle comprises: (a) the polynucleotide of the present disclosure, (b) a cationic lipid, (c) an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG-modified lipid), (d) optionally a non-cationic lipid (such as a neutral lipid), and (e) optionally, a sterol.

[0353] Suitable cationic lipids are disclosed in International Publication Nos. WO 09 / 086558, WO 09 / 127060, WO 10 / 048536, WO 10 / 054406, WO 10 / 088537, WO 10 / 129709, and WO 2011 / 153493; U.S. Patent Publication Nos. 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803; U.S. Pat. No. 8,158,601; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are herein incorporated by reference.

[0354] In one aspect, the disclosure provides a method for treating hemophilia A comprising administering, to a patient in need thereof, the gene therapy vehicle as described herein.

[0355] In one aspect, the disclosure provides a gene therapy vehicle as described herein for treating hemophilia A.

[0356] In one aspect, the disclosure provides for use of a gene therapy vehicle as described herein in treating hemophilia A.

[0357] In one aspect, the disclosure provides for use of a gene therapy vehicle as described herein for the manufacture of a medicament for treating hemophilia A.

[0358] In one aspect, the disclosure provides a method for treating hemophilia A comprising administering, to a patient in need thereof, the pharmaceutical composition as described herein.

[0359] In one aspect, the disclosure provides a pharmaceutical composition as described herein for treating hemophilia A.

[0360] In one aspect, the disclosure provides for use of a pharmaceutical composition as described herein in treating hemophilia A.

[0361] In one aspect, the disclosure provides for use of pharmaceutical composition as described herein for the manufacture of a medicament for treating hemophilia A.V. ExamplesExample 1 — Construction of a Codon-Altered Factor VIII Variant Expression Sequence

[0362] Two hurdles had to be overcome to create a Factor VIII coding sequence that is effective for gene therapy intended to treat hemophilia A. First, because of the genomic size limitations of conventional gene therapy delivery vectors (e.g., AAV virions), the encoded Factor VIII polypeptide had to be shortened considerably. Second, the coding sequence had to be altered to: (i) stabilize packaging interactions within the delivery vector, (ii) stabilize the mRNA intermediary, and (iii) improve the robustness of transcription / translation of the mRNA.

[0363] To achieve the first objective, Applicants started with a human wild type nucleic acid sequence encoding a B-domain deleted Factor VIII variant construct, referred to herein as “BDDFVIII-FL-AA” (SEQ ID NO: 5). In this construct, the B-domain is replaced with a fourteen amino acid sequence referred to as the “SQ” sequence (FIG. 1). To address the poor expression of the native BDD-FVIII, the codon optimization algorithm described in Fath et al. (PLoS ONE,6:el7596 (2011)), modified as described in Ward et al. (Blood, 117:798 (2011)) and in McIntosh et al. (Blood, 121, 3335-3344 (2013)), was applied to the BDDFVIII-FL sequence to create an intermediate coding sequence. However, the intermediate sequence was then manually modified to further improve expression.

[0364] The resulting BDDFVIII-FL-NA coding sequence (SEQ ID NO: 1), shown in Figure 2, has an overall GC content of 56%. The CpG-dinucleotide content of the sequence is moderate. However, CpG dinucleotides are predominantly present in the downstream portion of the coding sequence, e.g., the portion coding for the Factor VIII light chain. The CS04 sequence has 79.77% nucleotide sequence identity to the corresponding coding sequences in wild-type Factor VIII (Genbank accession Ml 4113).Example 2 — Production of Codon Optimized FVIII variants from the BDD-FVIII sequence

[0365] To further increase the FVIII activity of the codon-optimized BDD-FVIII construct, the BDDFVIII-FL-NA coding sequence (SEQ ID NO: 1) was altered to introduce codons for five sets of amino acid substitutions, resulting in the CS01 (SEQ ID NOV), CS02 (SEQ ID NO: 15), CS03 (SEQ ID NO:21), CS04 (SEQ ID NO:27) and CS05 (SEQ ID NO:33) constructs, encoding the PS01 (SEQ ID NO: 12), PS02 (SEQ ID NO: 18), PS03 (SEQ ID NO:24), PS04 (SEQ ID NO:30), and PS05 (SEQ ID NO:36) variant BDD-FVIII polypeptides, respectively. The sequence identities shared between each of the coding sequences is shown in Table 2, below.Table 2 - Percent identity matrix for codon-altered Factor VIII constructs.

[0366] Plasmids containing each construct were constructed by cloning different synthetic DNA fragments into the same vector backbone plasmid (pMY168). DNA synthesis of the BDD- FVIII fragments with flanking Asci and Notl enzyme restriction sites was done by ThermoFischer Scientific (Regensburg, Germany). The vector backbone contains two flanking AAV2-derived inverted terminal repeats (ITRs) that encompass a human transthyretin (hTTR) promoter / 3XCRM8 enhancer sequence derived from the liver-specific murine transthyretin gene, Asci and Notl enzyme restriction sites for insertion of the respective BDD-FVIII coding sequence, a MVM intron, a WHV Posttranscriptional Regulatory Element (WPRE) containing a mut6delATG mutation, and a synthetic polyA site. After ligation of the prepared vector backbone and inserts via the Asci and Notl sites, the resulting plasmids were amplified in milligram scale. The coding sequence for the BDD-FVIII constructs were verified by direct sequencing and restriction digestion (Azenta Life Sciences, Burlington, MA, USA). The cloning resulted in 9 different plasmid constructs named pBDDFVIII, pCSOl, pCS02, pCS03, pCS04, and pCS05 (FIG. 35). The constructs have the same vector backbone and encode the corresponding BDDFVIII-AA, PS01, PS02, PS03, PS04, and PS05 BDD-FVIII constructs, with the BDDFVIII-NA, CS01, CS02, CS03, CS04, and CS05 sequences, respectively.Example 3 — De Novo Optimization of CS04 Variant Using COOL Algorithm

[0367] To address the poor expression of the native BDD-VIII, the codon optimization algorithm described in Chin JX etal., Bioinformatics, 30(15):2210-12 (2014), the content of whichis hereby incorporated by reference, in its entirety, for all purposes, was applied with a human liver tissue-specific codon usage bias to generate seven different codon-optimized sequences encoding the PS04-FL BDD-Factor VIII polypeptide: CS06 (SEQ ID NO: 39), CS07 (SEQ ID NO: 43), CS08 (SEQ ID NO:47), CS09 (SEQ ID NO:51), CS10 (SEQ ID NO:55), CS11 (SEQ ID NO:59), and CS12 (SEQ ID NO: 63), using different parameters for each construct. Evolution design criteria and statistics for the constructs are shown in Table 3, below. The CS04 construct which encodes for the same PS04 variant BDD-FVIII construct, as described in Example 2, is included for reference.Table 3 - Design criteria and statistics for the evolved codon-altered Factor VIII constructs.

[0368] Briefly, human liver tissue-specific codon usage was determined by first identifying those genes that are most highly expressed in primary human liver cells or human liver tissue, e.g., the top 1-10% highest expressed genes. Three sources of information were used to compile this codon-bias. First, expression data was compiled using RNA-seq on thirty-two different healthy human tissue types from 122 individuals. Second, microarray expression data was obtained from assorted liver tissue samples. Third, proteome data was obtained by from pooled healthy tissue from ten individuals, using liquid chromatography-mass spectrometry (LC- MS). For more information on the data sources, see, Uhlen M. et al., Science, 347(6220): 1260419 (2015); Yu Y. et al., Genomics. 96(5):281-89 (2010); and Chinese Human Liver ProteomeProfiling Consortium, First insight into the human liver proteome from PROTEOMESKY - LIVERHu 1.0, a publicly available database, J Proteome Res., 9(l):79-94 (2010), the contents of which are expressly incorporated herein by reference, in their entireties, for all purposes.

[0369] The human liver tissue-specific codon bias generated above was used for a multiobjective genetic algorithm designed to optimize codon context fitness (e.g., the context of consecutive codon pairs) and individual codon fitness, with an emphasis on one or the other. See, Chin JX et al., Bioinformatics, 30(15):2210-12 (2014). The design of a first set of constructs (CS06-CS11) were constrained to a GC content of 53±2% during the evolution process. In addition, the evolution was constrained to avoid introduction of novel mRNA splice sites, TATA boxes, CC(T / C)CAATCT boxes, and large nucleotide repeats (e.g., greater than 20 bp). After evolution, the number of CpG sites was further reduced manually, e.g., to less than 10, as summarized in Table 3. One additional construct (CS12) was generated using a similar strategy, except that their GC contents were not constrained, in an attempt to not reduce overall sequence fitness. The CpG content of CS12 was not further reduced, for the same reason.

[0370] The sequence similarity between each of these BDD-FVIII coding sequences, and the CS04 construct (SEQ ID NO:27), is shown in Table 4, below.Table 4 - Percent identity matrix for codon-altered Factor VIII constructs.

[0371] Plasmids of each construct were constructed by cloning different synthetic DNA fragments into the same vector backbone plasmid (pMY168). DNA synthesis of the sequences encoding the BDD-FVIII constructs, with flanking Asci and Notl enzyme restriction sites, was performed by ThermoFischer Scientific (Regensburg, Germany). The vector backbone contains two flanking AAV2-derived inverted terminal repeats (ITRs) that encompass a human transthyretin (hTTR) promoter (SEQ ID NO:68) and 3XCRM8 enhancer (SEQ ID NO:67) sequence derived from the liver-specific murine transthyretin gene, Asci and Notl enzyme restriction sites for insertion of the respective BDD-FVIII coding sequence, a MVM intron (SEQ ID NO:69), a WHV Posttranscriptional Regulatory Element (WPRE) containing a mut6delATG mutation (SEQ ID NO:70), and a synthetic polyA site (SEQ ID NO:71) (Figure 34). After ligation of the prepared vector backbone and inserts via the Asci and Notl sites, the resulting plasmids were amplified in milligram scale. The BDD-FVIII coding sequences of the constructs were verified by direct sequencing and restriction digestion (Azenta Life Sciences, Burlington, MA, USA). The cloning resulted in 7 different plasmid constructs named pCS06, pCS07, pCS08, pCS09, pCSIO, pCSl l, and pCS12 (Figure 35). The constructs have the same vector backbone and encode the same B-domain deleted FVIII protein (PCS04 DNA sequence source), but differ in their FVIII coding sequence.Example 4 —In vitro Evaluation Codon-Altered Factor VIII Variant Expression Plasmids

[0372] To evaluate the expression and activity of the codon-altered Factor VIII variants, the 13 plasmids constructed as described in Examples 2 and 3 (Figure 35) were transfected into 4 different cell types, and expression levels were measured using the Factor VIII Antigen Plus Kit from Affinity Biologicals (F8PLUS-AG). In parallel, Factor VIII activity was measured using the BIOPHENTM FVIII: C kit, from HYPHEN BioMed (221046-RUO). The data are summarized in Figures 36A-36B.

[0373] Briefly, HEK293T, Huh7, HepG2 and Hepal-6 cells maintained in DMEM media supplemented with 10% fetal bovine serum (FBS) were seeded in 6 cm plate at a density of 2million / plate. At 80% cell confluence, cells were transfected with Factor VIII plasmids using Lipofectamine 3000. Then OPTIMEM media was replaced with 2 mL of expression media (DMEM / F12 supplemented with ITS liquid media (Sigma I3146-5mL), L-glutamine (2 mM), albumax (1.5 mg / mL) and CaCh (2 mM)). After 48 hours the media was collected and frozen. For negative control, cells only (no Lipofectamine and no DNA) or cells transfected with Lipofectamine 3000 alone (no DNA) were used.

[0374] Factor VIII antigen was detected in all samples at various degrees across all four cell lines. Likewise, Factor VIII activity was detected in all samples across all four cell lines (Figure 36A, 36B).Example 5 — In Vivo Expression of Codon-Altered Factor VIII Variant Expression Sequences

[0375] To test the biological potency of the codon-altered Factor VIII variant sequences, plasmids containing the codon-optimized sequences encoding BDD -FVIII constructs (pCSOl- pCS08 and pCS10-pCS12 as described in Examples 1, 2, and 3), as well as a codon-optimized construct (pBDDFVIII) encoding a BDD-FVIII (SQ) construct without any amino acid substitutions that otherwise shares the codon-optimization schema of CS01-CS05 were administered to C57BL / 6 mice. Briefly, the assays were performed in C57BL / 6 mice (with 5 animals per group, except for the positive control where 12 animals were used) by tail vein injection of 25 ug of each plasmid, in a volume of 80 mL per kilogram body weight of mouse (Figure 37A). Blood was drawn 48 hours after injection by retroorbital puncture and plasma was prepared and frozen using standard procedures. FVIII antigen and activity levels at 48 hours were chosen because there is minimal influence of inhibitory antibodies at this time, which are seen in some animals of this mouse model at later times. FVIII antigen was measured using the Factor VIII Antigen Plus Kit from Affinity Biologicals (F8PLUS-AG). In parallel, FVIII activity in the mouse plasma was determined using the BIOPHENTM FVIII: C kit, from HYPHEN BioMed (221046-RUO).

[0376] The results, presented in Figures 38A-38B, demonstrate that the codon-altered sequences designed using the algorithms detailed in Examples 1, 2, and 3, (CS01 through CS08, CS10 through CS12) were all detected several-fold more as compared to the untreated mice negative control.Example 6 — In Vivo Expression of Codon-Altered Factor VIII Variant Expression Sequences

[0377] To test the biological potency of the codon-altered Factor VIII variant sequences, plasmids containing the codon-optimized sequences encoding BDD-FVIII constructs (pCSOl- pCS08 and pCS10-pCS12 as described in Examples 1, 2, and 3), as well as a non-codon optimized construct (CS13) encoding the PS04-FL BDD-FVIII construct and a codon-optimized construct (pBDDFVIII) encoding a BDD-FVIII (SQ) construct without any amino acid substitutions that otherwise shares the codon-optimization schema of CS01-CS05, were administered to C57BL / 6 mice. Briefly, the assays were performed in C57BL / 6 mice (with 5 animals per group, except for the positive control where 12 animals were used) by tail vein injection of 25 ug of each plasmid, in a volume of 80 mL per kilogram body weight of mouse. Blood was drawn 48 hours after injection by retroorbital puncture and plasma was prepared and frozen using standard procedures. FVIII antigen and activity levels at 48 hours were chosen because there is minimal influence of inhibitory antibodies at this time, which are seen in some animals of this mouse model at later times. FVIII antigen was measured using the Factor VIII Antigen Plus Kit from Affinity Biologicals (F8PLUS-AG).

[0378] The results, presented in Figure 42, demonstrate that administration of codon- altered sequences (CS01-CS08 and CS10-CS12) resulted in significantly greater FVIII antigen in the blood of animals administered the non-codon-optimized BDD-FVIII construct (CS13).

[0379] 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 persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A gene therapy vehicle comprising a polynucleotide encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a heavy chain, a light chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N-terminus of the light chain, wherein the polynucleotide comprises a sequence having at least 99% identity to a nucleic acid sequence selected from the group consisting of: a) CS01-FL (SEQ ID NO: 9); b) CS02-FL (SEQ ID NO: 15); c) CS03-FL (SEQ ID NO: 21); d) CS04-FL (SEQ ID NO: 27); and e) CS05-FL (SEQ ID NO: 33).

2. A gene therapy vehicle comprising a polynucleotide encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a heavy chain, a light chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N-terminus of the light chain, wherein: a) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS01-HC (SEQ ID NO: 10); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS01-LC (SEQ ID NO: 11); b) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS02-HC (SEQ ID NO: 16); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS02-LC (SEQ ID NO: 17); c) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS03-HC (SEQ ID NO: 22); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS03-LC (SEQ ID NO: 23); d) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS04-HC (SEQ ID NO: 28); and wherein the light chain of the Factor FVIII polypeptide is encoded by a secondnucleotide sequence having at least 99% identity to CS04-LC (SEQ ID NO: 29); or e) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS05-HC (SEQ ID NO: 34); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS05-LC (SEQ ID NO: 35).

3. The gene therapy vehicle of claim 1 or 2, wherein the Factor VIII polypeptide comprises: a) V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E809G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); b) V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); c) V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E800V, K1093G, T1148N, L1248D, L1377D, and V1439E ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); d) V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); or e) V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, L750D, I775G, K1093A, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5).

4. A gene therapy vehicle comprising a polynucleotide encoding a Factor VIII polypeptide, wherein the polynucleotide comprises a sequence having at least 95% identity to a nucleic acid sequence selected from the group consisting of: a) CS01-FL (SEQ ID NOV), wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F3871, 1390 A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E809G,T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); b) CS02-FL (SEQ ID NO: 15), wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); c) CS03-FL (SEQ ID NO:21), wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, L750D, I775G, E800V, K1093G, T1148N, L1248D, L1377D, and V1439E ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); d) CS04-FL (SEQ ID NO:27), wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and e) CS05-FL (SEQ ID NO: 33), wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, L750D, I775G, K1093A, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII- FL-AA (SEQ ID NO: 5).

5. A gene therapy vehicle comprising a polynucleotide encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a heavy chain, a light chain, and apolypeptide linker joining the C-terminus of the heavy chain to the N-terminus of the light chain, wherein: a) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS01-HC, wherein the heavy chain comprises V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, I499L, V534I, M558W, M681G, S700E, K732W, L749F, and L750D amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS01-LC, wherein the light chain comprises I775G, E809G, T1148N, L1248D, L1377D, and V1439E ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); b) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS02-HC, wherein the heavy chain comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, K732W, Y738E, L749F, L750D, and K752E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS02-LC, wherein the light chain comprises I775G, K1093G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); c) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS03-HC, wherein the heavy chain comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, S700E, K732W, L749F, and L750D amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS03-LC, wherein the light chain comprises I775G; E800V; K1093G; T1148N; L1248D; L1377D; and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5);d) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS04-HC, wherein the heavy chain comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, and K752E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS04-LC, wherein the light chain comprises I775G, K1093G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); or e) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS05-HC, wherein the heavy chain comprises V63Q, L171P, H212A, S260T, E306G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, and L750D ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS05-LC, wherein the light chain comprises I775G, K1093A, T1148N, L1248D, L1377D, and V1439E ammo acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5).

6. The gene therapy vehicle of any one of claims 1 to 5, wherein: a) the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having 99% sequence identity to PS01-HC (SEQ ID NO: 13); and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS01-LC (SEQ ID NO: 14); b) the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having 99% sequence identity to PS02-HC (SEQ ID NO: 19); and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having 99% sequence identity to PS02-LC (SEQ ID NO: 20); c) the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having at least 99% identity to PS03-HC (SEQ ID NO: 25); and wherein the lightchain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS03-LC (SEQ ID NO: 26); d) the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having at least 99% identity to PS04-HC (SEQ ID NO: 31); and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS04-LC (SEQ ID NO: 27); or e) the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having at least 99% identity to PS05-HC (SEQ ID NO: 37); and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS05-LC (SEQ ID NO: 33).

7. The gene therapy vehicle of any one of claims 1 to 5, the Factor VIII polypeptide comprising a heavy chain, a light chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N-terminus of the light chain, wherein: f) the heavy chain of the Factor VIII polypeptide comprises the amino acid sequence of PS01-HC (SEQ ID NO: 13); and wherein the light chain of the Factor FVIII polypeptide comprises the amino acid sequence of PS01-LC (SEQ ID NO: 14); g) the heavy chain of the Factor VIII polypeptide comprises the amino acid sequence of PS02-HC (SEQ ID NO: 19); and wherein the light chain of the Factor FVIII polypeptide comprises the amino acid sequence of PS02-LC (SEQ ID NO: 20); h) the heavy chain of the Factor VIII polypeptide comprises the amino acid sequence of PS03-HC (SEQ ID NO: 25); and wherein the light chain of the Factor FVIII polypeptide comprises the amino acid sequence of PS03-LC (SEQ ID NO: 26); i) the heavy chain of the Factor VIII polypeptide comprises the amino acid sequence of PS04-HC (SEQ ID NO: 31); and wherein the light chain of the Factor FVIII polypeptide comprises the amino acid sequence of PS04-LC (SEQ ID NO: 27); or j) the heavy chain of the Factor VIII polypeptide comprises the amino acid sequence of PS05-HC (SEQ ID NO: 37); and wherein the light chain of the Factor FVIII polypeptide comprises the amino acid sequence of PS05-LC (SEQ ID NO: 33).

8. The gene therapy vehicle of any one of claims 1 to 7, wherein the polypeptide linker comprises a furin cleavage site.

9. The gene therapy vehicle of any one of claims 1 to 7, wherein the polypeptide linker comprises the amino acid sequence of BDDFVIII BDL AA (SEQ ID NO: 8).

10. The gene therapy vehicle of any one of claims 1 to 9, 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: a) BDDFVIII-BDL-NA (SEQ ID NO:4); b) CS06-BDL (SEQ ID NO: 42); c) CS07-BDL (SEQ ID NO: 46); d) CS08-BDL (SEQ ID NO: 50); e) CS09-BDL (SEQ ID NO: 54); f) CS10-BDL (SEQ ID NO: 58); g) CS11-BDL (SEQ ID NO: 62); and h) CS12-BDL (SEQ ID NO: 66).

11. The gene therapy vehicle of claim 10, wherein the third nucleotide sequence has at least 97.5% identity to a sequence selected from the group consisting of: a) BDDFVIII-BDL-NA; b) CS06-BDL; c) CS07-BDL; d) CS08-BDL; e) CS09-BDL; f) CS10-BDL; g) CS11-BDL; and h) CS12-BDL.

12. The gene therapy vehicle of claim 10, wherein the third nucleotide sequence is selected from the group consisting of: a) BDDFVIII-BDL-NA;b) CS06-BDL; c) CS07-BDL; d) CS08-BDL; e) CS09-BDL; f) CS10-BDL; g) CSl l-BDL; and h) CS12-BDL.

13. The gene therapy vehicle of any one of claims 1 to 12, wherein the polynucleotide encodes a polypeptide comprising a sequence having at least 95% identity to an amino acid sequence selected from the group consisting of: a) PS01-FL (SEQ ID NO: 12); b) PS02-FL (SEQ ID NO: 18); c) PS03-FL (SEQ ID NO: 24); d) PS04-FL (SEQ ID NO: 30); and e) PS05-FL (SEQ ID NO: 36).

14. The gene therapy vehicle of any one of claims 1 to 12, wherein the polynucleotide encodes a polypeptide comprising a sequence having at least 99% identity an amino acid sequence selected from the group consisting of: a) PS01-FL (SEQ ID NO: 12); b) PS02-FL (SEQ ID NO: 18); c) PS03-FL (SEQ ID NO: 24); d) PS04-FL (SEQ ID NO: 30); and e) PS05-FL (SEQ ID NO: 36).

15. The gene therapy vehicle of any one of claims 1 to 12, wherein the polynucleotide encodes a polypeptide comprising an amino acid sequence selected from the group consisting of: a) PS01-FL (SEQ ID NO: 12); b) PS02-FL (SEQ ID NO: 18); c) PS03-FL (SEQ ID NO: 24); d) PS04-FL (SEQ ID NO: 30); ande) PS05-FL (SEQ ID NO: 36).

16. The gene therapy vehicle of any one of claims 1 to 15, wherein: a) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS01-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS01-LC; b) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS02-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS02-LC; c) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS03-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS03-LC; d) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS04-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS04-LC; or e) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS05-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS05-LC.

17. The gene therapy vehicle of any one of claims 1 to 16, wherein the polynucleotide comprises a sequence selected from the group consisting of: a) CS01-FL; b) CS02-FL; c) CS03-FL; d) CS04-FL; and e) CS05-FL.

18. A gene therapy vehicle comprising a polynucleotide encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a heavy chain, a light chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N-terminus of the light chain, wherein the polynucleotide comprises a sequence having at least 95% identity to a nucleic acid sequence selected from the group consisting of:a) CS06-FL (SEQ ID NO: 39); b) CS07-FL (SEQ ID NO: 43); c) CS08-FL (SEQ ID NO: 47); d) CS09-FL (SEQ ID NO: 51); e) CS10-FL (SEQ ID NO: 55); f) CS11-FL (SEQ ID NO: 59); and g) CS12-FL (SEQ ID NO: 63).

19. The gene therapy vehicle of claim 18, wherein the polynucleotide comprises a sequence having at least 99% identity to a nucleic acid sequence selected from the group consisting of: a) CS06-FL (SEQ ID NO: 39); b) CS07-FL (SEQ ID NO: 43); c) CS08-FL (SEQ ID NO: 47); d) CS09-FL (SEQ ID NO: 51); e) CS10-FL (SEQ ID NO: 55); f) CS11-FL (SEQ ID NO: 59); and g) CS12-FL (SEQ ID NO: 63).

20. A gene therapy vehicle comprising a polynucleotide encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a heavy chain, a light chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N-terminus of the light chain, wherein: a) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS06-HC (SEQ ID NO: 40); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS06-LC (SEQ ID NO: 41); b) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS07-HC (SEQ ID NO: 44); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS07-LC (SEQ ID NO: 45);c) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS08-HC (SEQ ID NO: 48); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS08-LC (SEQ ID NO: 49); d) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS09-HC (SEQ ID NO: 52); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS09-LC (SEQ ID NO: 53); e) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS10-HC (SEQ ID NO: 56); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS10-LC (SEQ ID NO: 57); f) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS11-HC (SEQ ID NO: 60); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS11-LC (SEQ ID NO: 61); or g) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS12-HC (SEQ ID NO: 64); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS12-LC (SEQ ID NO: 65).

21. The gene therapy vehicle of claim 20, wherein: a) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS06-HC (SEQ ID NO: 40); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS06-LC (SEQ ID NO: 41); b) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS07-HC (SEQ ID NO: 44); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS07-LC (SEQ ID NO: 45);c) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS08-HC (SEQ ID NO: 48); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS08-LC (SEQ ID NO: 49); d) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS09-HC (SEQ ID NO: 52); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS09-LC (SEQ ID NO: 53); e) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS10-HC (SEQ ID NO: 56); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS10-LC (SEQ ID NO: 57); f) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS11-HC (SEQ ID NO: 60); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS11-LC (SEQ ID NO: 61); or g) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity to CS12-HC (SEQ ID NO: 64); and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to CS12-LC (SEQ ID NO: 65).

22. The gene therapy vehicle of any one of claims 18 to 21 , wherein the Factor VIII polypeptide comprises V63Q, L171P, H212A, S260T, E306G, L318G, M320R, S332P, F387I, I390A, V393G, V534I, M558W, M681N, S700E, K732W, Y738E, L749F, L750D, K752E, I775G, K1093G, T1148N, L1248D, L1377D, and V1439E amino acid substitution relative to BDDFVIII-FL-AA (SEQ ID NO: 5).

23. The gene therapy vehicle of any one of claims 18 to 22, wherein the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence having at least 99% identity toPS04-HC (SEQ ID NO: 31); and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence having at least 99% identity to PS04-LC (SEQ ID NO: 32).

24. The gene therapy vehicle of any one of claims 18 to 22, wherein the heavy chain of the Factor VIII polypeptide comprises an amino acid sequence of PS04-HC (SEQ ID NO: 31); and wherein the light chain of the Factor FVIII polypeptide comprises an amino acid sequence of PS04-LC (SEQ ID NO: 32).

25. The gene therapy vehicle of any one of claims 18 to 24, wherein the polypeptide linker comprises a furin cleavage site.

26. The gene therapy vehicle of any one of claims 18 to 25, wherein the polypeptide linker comprises an amino acid sequence that is BDDFVIII BDL AA (SEQ ID NO: 8).

27. The gene therapy vehicle of any one of claims 18 to 26, 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: a) BDDFVIII-BDL-NA; b) CS06-BDL; c) CS07-BDL; d) CS08-BDL; e) CS09-BDL; f) CS10-BDL; g) CSl l-BDL; and h) CS12-BDL.

28. The gene therapy vehicle of claim 27, wherein the third nucleotide sequence has at least 97.5% identity to a sequence selected from the group consisting of: a) BDDFVIII-BDL-NA; b) CS06-BDL; c) CS07-BDL; d) CS08-BDL;e) CS09-BDL; f) CS10-BDL; g) CSl l-BDL; and h) CS12-BDL.

29. The gene therapy vehicle of claim 27, wherein the third nucleotide sequence is selected from the group consisting of: a) BDDFVIII-BDL-NA; b) CS06-BDL; c) CS07-BDL; d) CS08-BDL; e) CS09-BDL; f) CS10-BDL; g) CSl l-BDL; and h) CS12-BDL.

30. The gene therapy vehicle of any one of claims 18 to 29, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of PS04-FL.

31. The gene therapy vehicle of any one of claims 18 to 30, wherein: a) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS06-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS06-LC; b) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS07-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS07-LC; c) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS08-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS08-LC; d) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS09-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS09-LC;e) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS10-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS10-LC; f) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS11-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS11-LC; or g) the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence of CS12-HC; and wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence of CS12-LC.

32. The gene therapy vehicle of any one of claims 18 to 31 , wherein the polynucleotide comprises a sequence selected from the group consisting of: a) CS06-FL; b) CS07-FL; c) CS08-FL; d) CS09-FL; e) CS10-FL; f) CS 11 -FL; and g) CS12-FL.

33. The gene therapy vehicle of any one of claims 1 to 32, wherein the polynucleotide further comprises a promoter operably linked to the polynucleotide encoding the Factor VIII polypeptide.

34. The gene therapy vehicle of claim 33, wherein the promoter is an hTTR promoter located 5’ of the polynucleotide encoding the Factor VIII polypeptide.

35. The gene therapy vehicle of claim 34, wherein the hTTR promoter has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 68.

36. The gene therapy vehicle of claim 34 or 35, wherein the hTTR promoter has a sequence of SEQ ID NO: 68.

37. The gene therapy vehicle of any one of claims 1 to 36, wherein the polynucleotide further comprises a liver-specific regulatory element operably linked to the polynucleotide encoding the Factor VIII polypeptide.

38. The gene therapy vehicle of claim 37, wherein the liver-specific regulatory element is an enhancer located 5’ of the polynucleotide encoding the Factor VIII polypeptide.

39. The gene therapy vehicle of claim 38, wherein the enhancer comprises at least 1 copy of CRM8 (lxCRM8), at least 2 copies of CRM8 (2xCRM8), or at least 3 copies of CRM8 (3xCRM8).

40. The gene therapy vehicle of claim 39, wherein the enhancer is 3xCRM8.

41. The gene therapy vehicle of claim 40, wherein the CRM8 enhancer has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 67.

42. The gene therapy vehicle of claim 39 or 40, wherein the CRM8 enhancer has a sequence of SEQ ID NO: 67.

43. The gene therapy vehicle of any one of claims 37 to 42, wherein the promoter and the liver-specific regulatory element are directly attached.

44. The gene therapy vehicle of any one of claims 1 to 43, wherein the polynucleotide further comprises an intron operably linked to the polynucleotide encoding the Factor VIII polypeptide.

45. The gene therapy vehicle of claim 44, wherein the intron comprises an MVM intron located 5’ of the polynucleotide encoding the Factor VIII polypeptide.

46. The gene therapy vehicle of claim 45, wherein the MVM intron has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69.

47. The gene therapy vehicle of claim 44 or 45, wherein said MVM intron has a sequence of SEQ ID NO: 69.

48. The gene therapy vehicle of any one of claims 44 to 47, wherein said intron is positioned between the promoter and the translation initiation site of the polynucleotide encoding the Factor FVIII polypeptide.

49. The gene therapy vehicle of any one of claims 1 to 48, wherein the polynucleotide further comprises a post-translational regulatory element operably linked to the polynucleotide encoding the Factor VIII polypeptide.

50. The gene therapy vehicle of claim 49, wherein the post-translational regulatory element is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) located 3’ of the polynucleotide encoding the Factor VIII polypeptide.

51. The gene therapy vehicle of claim 50, wherein the WPRE has at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 70.

52. The gene therapy vehicle of any one of claims 1 to 51, wherein the polynucleotide further comprises a terminator operably linked to the polynucleotide encoding the Factor VIII polypeptide.

53. The gene therapy vehicle of claim 52, wherein the terminator is a polyadenylation element located 3’ of the polynucleotide encoding the Factor VIII polypeptide.

54. The gene therapy vehicle of claim 53, wherein the polyadenylation element is selected from the group consisting of SV40-polyA, hGH-polyA, BGH-polyA, and rbGlob- polyA.

55. The gene therapy vehicle of claim 53 or 54, wherein the polyadenylation element is BGH-polyA having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 71.

56. The gene therapy vehicle of any one of claims 1 to 55, wherein the polynucleotide comprises from the 5 ’-terminus to the 3 ’-terminus, a 3xCRM8 enhancer, an hTTR promoter, an MVM intron, a polynucleotide encoding the Factor FVIII polypeptide, a WPRE, and a BGH- polyA terminator.

57. The gene therapy vehicle of any one of claims 1 to 56, wherein the gene therapy vehicle is an adeno-associated virus (AAV) vector.

58. The gene therapy vehicle of claim 57, wherein the AAV vector is a serotype 8 adeno-associated virus (AAV-8) vector.

59. The gene therapy vehicle of claim 57 or 58, wherein the polynucleotide is a single-stranded polynucleotide.

60. The gene therapy vehicle of any one of claims 1 to 56, wherein the gene therapy vehicle comprises a lipid nanoparticle (LNP) encapsulating the polynucleotide encoding the Factor VIII polypeptide.

61. The gene therapy vehicle of any one of claims 1 to 56, wherein the gene therapy vehicle comprises a liposome or a multilamellar vesicle encapsulating the polynucleotide encoding the Factor VIII polypeptide..

62. An expression vector comprising a variant BDD-FVIII polynucleotide sequence described herein.

63. A pharmaceutical composition comprising a gene therapy vehicle according to any one of claims 1-61.

64. A method for treating hemophilia A comprising administering, to a patient in need thereof, the gene therapy vehicle according to any one of claims 1 to 61.

65. The gene therapy vehicle according to any one of claims 1 to 61 for treating hemophilia A.

66. Use of the gene therapy vehicle according to any one of claims 1 to 61 in treating hemophilia A.

67. Use of the gene therapy vehicle according to any one of claims 1 to 61 for the manufacture of a medicament for treating hemophilia A.

68. A method for treating hemophilia A comprising administering, to a patient in need thereof, the pharmaceutical composition according to claim 63.

69. The pharmaceutical composition according to claim 63 for treating hemophilia A.

70. Use of the pharmaceutical composition according to claim 63 in treating hemophilia A.

71. Use of pharmaceutical composition according to claim 63 for the manufacture of a medicament for treating hemophilia A.

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