EA046432B1 - VIRAL VECTORS ENCODING RECOMBINANT FVIII VARIANTS WITH INCREASED EXPRESSION FOR GENE THERAPY OF HEMOPHILIA A - Google Patents

Description

State of the art

Blood coagulation occurs through a complex and dynamic biological pathway of interdependent biochemical reactions called the coagulation cascade. Coagulation factor VIII (FVIII) is a key component of the cascade. Factor VIII enters the bleeding sites and forms a Khaza complex with activated Factor IX (FIXa) and Factor X (FX). The Khaza complex activates FX, which in turn activates the conversion of prothrombin to thrombin, which then activates other components in the coagulation cascade to create a stable thrombus (See Saenko et al., Trends Cardiovasc. Med., 9:185-192 (1999 ); Lenting et al., Blood, 92:3983-3996 (1998)).

Hemophilia A is a congenital X-linked blood clotting disorder characterized by a deficiency of factor VIII activity. Decreased factor VIII activity inhibits the positive feedback loop in the coagulation cascade. This results in incomplete coagulation, which manifests as cases of prolonged bleeding, extensive bruising, spontaneous oral and nasal bleeding, joint stiffness and chronic pain, and possibly internal bleeding and anemia in severe cases (Zhang et al., Clinic. Rev. Allerg. Immunol., 37:114-124 (2009).

Typically for hemophilia A, factor VIII replacement therapy is used, which consists of administering factor VIII protein (eg, plasma-derived or recombinant factor VIII) to a person with hemophilia A. Factor VIII is given prophylactically to prevent or reduce the incidence of bleeding events in response to an episode of acute bleeding. bleeding and/or perioperatively to ensure bleeding is controlled during surgery. However, there are several undesirable features of factor VIII replacement therapy.

First, factor VIII replacement therapy is used to treat or control hemophilia A, but does not cure the underlying factor VIII deficiency. Because of this, people with hemophilia A require factor VIII replacement therapy throughout their lives. Continuous treatment is expensive and requires a person to adhere to a strict treatment regimen, since failure to take just a few prophylactic doses can have serious consequences for people with severe hemophilia A.

Second, because factor VIII has a relatively short half-life in vivo, traditional prophylactic factor VIII replacement therapy requires administration every second or third day. This places a burden on the individual to adhere to therapy throughout their life. While third-generation long-acting factor VIII agents may reduce the frequency of administration, prophylactic FVIII replacement therapy with these agents still requires monthly, weekly, or more frequent dosing indefinitely. For example, prophylactic treatment with ELOCTATE™ [Antihemophilic Factor (Recombinant), Fc Fusion Protein] requires administration every three to five days (ELOCTATE™ Prescription Information, Biogen Idec Inc., (2015)). Moreover, the long-term effects of chemically modified biologics (eg, PEGylated polypeptides) are not yet fully understood.

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

Somatic gene therapy holds great promise in the treatment of hemophilia A because it addresses the underlying underexpression of factor VIII (eg, due to missense or nonsense mutations) rather than providing a one-time dose of factor VIII activity to an individual. Because of this difference in mechanism of action compared to factor VIII replacement therapy, a single administration of a factor VIII gene therapy vector can provide an individual with factor VIII for several years, reducing treatment costs and eliminating the need for continued patient compliance with therapy.

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Coagulation factor IX (FIX) gene therapy has been used effectively to treat people with hemophilia B, an associated blood clotting condition characterized by reduced factor IX activity (Manno C.S., et al., Nat Med., 12(3):342-47 (2006)). However, factor VIII gene therapy has 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). Essentially, the wild-type factor VIII coding sequence is 7053 base pairs, which is too large to be inserted into AAV gene therapy vectors. In addition, the observed expression of factor VIII B domain deleted (BDD-FVIII) recombinant variants was low. Thus, several groups have attempted to modify the codon usage of BDD-FVIII constructs with limited success.

Brief description of the invention

Accordingly, there is a need for factor VIII variants whose coding sequences are more efficiently packaged and transferred using gene therapy vectors. There is also a need for synthetic codon-altered nucleic acids that express factor VIII more efficiently. Such factor VIII variants and codon-altered nucleic acids may improve the treatment of factor VIII deficiencies (eg, hemophilia A). The above-mentioned disadvantages and other problems associated with the treatment of factor VIII deficiencies (eg, hemophilia A) are reduced or eliminated by the disclosed codon-altered variants of factor VIII.

In some embodiments, the invention provides nucleic acids encoding Factor VIII variants that have high sequence identity to the reported Factor VIII heavy chain codon altered sequences (e.g., CS01-HC-NA, CS04-HC-NA, or CS23- HC-NA) and light chain (CS01-LC-NA, CS04-LC-NA or CS23-LC-NA). In some embodiments, these nucleic acids further comprise a sequence encoding a linker sequence that replaces the native Factor VIII B domain (eg, linker sequences containing a furin cleavage site) between the factor VIII heavy and light chain encoding sequences.

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

In one embodiment of the polynucleotides described above, the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity with the sequence of BDLO04 (SEQ ID NO: 6).

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

In one embodiment of the polynucleotides described above, the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity with the sequence of BDLO01 (SEQ ID NO: 5).

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

In one embodiment of the polynucleotides described above, the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity with the sequence of BDLO23 (SEQ ID NO: 7).

In one embodiment of the polynucleotides described above, the first nucleotide sequence

- 2046432 The factor VIII polypeptide heavy chain encoding activity has at least 96% identity with the corresponding heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO: 3), CS01-HC-NA (SEQ ID NO: 24) or CS23-HC-NA (SEQ ID NO: 22)), and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide has at least 9-6% identity with the corresponding light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4), CS01-LC-NA (SEQ ID NO: 25) or CS23-LC-NA (SEQ ID NO: 23)).

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

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

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

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

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

In one embodiment of the polynucleotides described above, the first nucleotide sequence encoding the heavy chain of the factor VIII polypeptide is the sequence CS04-HC-NA (SEQ ID NO: 3), and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide is the sequence CS04-LC-NA (SEQ ID NO: 4).

In one embodiment of the polynucleotides described above, the first nucleotide sequence encoding the heavy chain of the factor VIII polypeptide is the sequence CS01-HC-NA (SEQ ID NO: 24), and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide is the sequence CS01-LC-NA (SEQ ID NO: 25).

In one embodiment of the polynucleotides described above, the first nucleotide sequence encoding the heavy chain of the factor VIII polypeptide is the sequence CS23-HC-NA (SEQ ID NO: 22), and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide is the sequence CS23-LC-NA (SEQ ID NO: 23).

In one aspect, the description provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to the sequence of CS04-FL-NA, wherein the polynucleotide encodes a factor VIII polypeptide.

In one aspect, the description provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to the sequence of CS01-FL-NA, wherein the polynucleotide encodes a factor VIII polypeptide.

In one aspect, the description provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to the sequence of CS23-FL-NA, wherein the polynucleotide encodes a factor VIII polypeptide.

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In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 96% identity with the corresponding full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1), CS01-FL-nA (SEQ ID NO: 13 ) or CS23FL-NA (SEQ ID NO: 20)).

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 97% identity with the corresponding full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1), CS01-FL-NA (SEQ ID NO: 13 ) or CS23FL-NA (SEQ ID NO: 20)).

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 98% identity with the corresponding full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1), CS01-FL-NA (SEQ ID NO: 13 ) or CS23FL-NA (SEQ ID NO: 20)).

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 99% identity with the corresponding full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1), CS01-FL-nA (SEQ ID NO: 13 ) or CS23FL-NA (SEQ ID NO: 20)).

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

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

In one embodiment of the polynucleotides described above, the nucleotide sequence is the sequence CS04-FL-NA (SEQ ID NO: 1).

In one embodiment of the polynucleotides described above, the nucleotide sequence is the sequence CS01-FL-NA (SEQ ID NO: 13).

In one embodiment of the polynucleotides described above, the nucleotide sequence is the sequence CS23-FL-NA (SEQ ID NO: 20).

In one embodiment of the polynucleotides described above, the polynucleotide encodes a factor VIII polypeptide comprising an amino acid sequence having at least 95% identity with the sequence CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotide encodes a factor VIII polypeptide comprising an amino acid sequence having at least 96% identity with the sequence CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotide encodes a factor VIII polypeptide comprising an amino acid sequence having at least 97% identity with the sequence of CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotide encodes a factor VIII polypeptide comprising an amino acid sequence having at least 98% identity with the sequence of CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotide encodes a factor VIII polypeptide comprising an amino acid sequence having at least 99% identity with the sequence of CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotide encodes a factor VIII polypeptide containing an amino acid sequence having at least 99.5% identity with the sequence CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotide encodes a factor VIII polypeptide containing an amino acid sequence having at least 99.9% identity with the sequence CS04-FL-AA (SEQ ID NO: 2).

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

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

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

In one aspect, this disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to the sequence of CS01-SC1-NA (SEQ ID NO: 26), wherein the polynucleotide encodes a single-chain factor VIII polypeptide.

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In one aspect, this disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to the sequence of CS01-SC2-NA (SEQ ID NO: 27), wherein the polynucleotide encodes a single-chain factor VIII polypeptide.

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

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

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 96% identity with the corresponding full-length polynucleotide sequence (e.g., CS04-SC1-NA (SEQ ID NO: 9), CS04-SC2-NA (SEQ ID no: 11 ), CS01SC1-NA (SEQ ID NO: 26), CS01-SC2-NA (SEQ ID NO: 27), CS23-SC1-NA (SEQ ID NO: 28) or CS23-SC2NA (SEQ ID NO: 29)) .

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 97% identity with the corresponding full-length polynucleotide sequence (e.g., CS04-SC1-NA (SEQ ID NO: 9), CS04-SC2-NA (SEQ ID no: 11 ), CS01SC1-NA (SEQ ID NO: 26), CS01-SC2-NA (SEQ ID NO: 27), CS23-SC1-NA (SEQ ID NO: 28) or CS23-SC2NA (SEQ ID NO: 29)) .

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 98% identity with the corresponding full-length polynucleotide sequence (e.g., CS04-SC1-NA (SEQ ID NO: 9), CS04-SC2-NA (SEQ ID nO: 11 ), CS01SC1-NA (SEQ ID NO: 26), CS01-SC2-NA (SEQ ID NO: 27), CS23-SC1-NA (SEQ ID NO: 28) or CS23-SC2NA (SEQ ID NO: 29)) .

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 99% identity with the corresponding full-length polynucleotide sequence (e.g., CS04-SC1-NA (SEQ ID NO: 9), CS04-SC2-NA (SEQ ID nO: 11 ), CS01SC1-NA (SEQ ID NO: 26), CS01-SC2-NA (SEQ ID NO: 27), CS23-SC1-NA (SEQ ID NO: 28) or CS23-SC2NA (SEQ ID NO: 29)) .

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

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

In one embodiment of the polynucleotides described above, the nucleotide sequence is the sequence CS04-SC1-NA (SEQ ID NO: 9).

In one embodiment of the polynucleotides described above, the nucleotide sequence is the sequence CS04-SC2-NA (SEQ ID NO: 11).

In one embodiment of the polynucleotides described above, the nucleotide sequence is the sequence CS01-SC1-NA (SEQ ID NO: 26).

In one embodiment of the polynucleotides described above, the nucleotide sequence is the sequence CS01-SC2-NA (SEQ ID NO: 27).

In one embodiment of the polynucleotides described above, the nucleotide sequence is the sequence CS23-SC1-NA (SEQ ID NO: 28).

In one embodiment of the polynucleotides described above, the nucleotide sequence is the sequence CS23-SC2-NA (SEQ ID NO: 29).

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 95% identity with a sequence selected from the group consisting of CS01-FL-NA, CS01-HC-NA, CS01-LC-NA, CS04-FL-NA , CS04-HC-NA, CS04-LC-NA, CS23-FLNA, CS23-HC-NA, CS23-LC-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1-NA, CS01-SC2 -NA, CS04-SC2NA and CS23-SC2-NA.

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 96% identity with a sequence selected from the group consisting of CS01-FL-NA, CS01-HC-NA, CS01-LC-NA, CS04-FL-NA , CS04-HC-NA, CS04-LC-NA, CS23-FLNA, CS23-HC-NA, CS23-LC-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1-NA, CS01-SC2 -NA, CS04-SC2NA and CS23-SC2-NA.

In one embodiment of the polynucleotides described above, the nucleotide sequence

- 5 046432 has at least 97% identity with a sequence selected from the group consisting of CS01-FL-NA, CS01-HC-NA, CS01-LC-NA, CS04-FL-NA, CS04-HC-NA, CS04-LC-NA, CS23-FLNA, CS23-HC-NA, CS23-LC-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1-NA, CS01-SC2-NA, CS04-SC2NA and CS23-SC2-NA.

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 98% identity with a sequence selected from the group consisting of CS01-FL-NA, CS01-HC-NA, CS01-LC-NA, CS04-FL-NA , CS04-HC-NA, CS04-LC-NA, CS23-FLNA, CS23-HC-NA, CS23-LC-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1-NA, CS01-SC2 -NA, CS04-SC2NA and CS23-SC2-NA.

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 99% identity with a sequence selected from the group consisting of CS01-FL-NA, CS01-HC-NA, CS01-LC-NA, CS04-FL-NA , CS04-HC-NA, CS04-LC-NA, CS23-FLNA, CS23-HC-NA, CS23-LC-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1-NA, CS01-SC2 -NA, CS04-SC2NA and CS23-SC2-NA.

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 99.5% identity with a sequence selected from the group consisting of CS01-FL-NA, CS01-HC-NA, CS01-LC-NA, CS04-FL-NA , CS04-HC-NA, CS04-LC-NA, CS23FL-NA, CS23-HC-NA, CS23-LC-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1-NA, CS01-SC2 -NA, CS04SC2-NA and CS23-SC2-NA.

In one embodiment of the polynucleotides described above, the nucleotide sequence has at least 99.5% identity with a sequence selected from the group consisting of CS01-FL-NA, CS01-HC-NA, CS01-LC-NA, CS04-FL-NA , CS04-HC-NA, CS04-LC-NA, CS23FL-NA, CS23-HC-NA, CS23-LC-NA, CS01-SC1-NA, CS04-SC1-NA, CS23-SC1-NA, CS01-SC2 -NA, CS04SC2-NA and CS23-SC2-NA.

In one embodiment of the polynucleotides described above, the nucleotide sequence is selected from the group consisting of cSo1-FL-NA, CS01-HC-NA, CS01-LC-NA, CS04-FL-NA, CS04HC-NA, CS04-LC-NA , CS23-FL-NA, CS23-HC-NA, CS23-LC-NA, CS01-SC1-NA, CS04-SC1-NA, CS23SC1-NA, CS01-SC2-NA, CS04-SC2-NA and CS23-SC2 -NA.

In one embodiment of the polynucleotides described above, the encoded factor VIII polypeptide comprises a glycosylatable polypeptide located between two consecutive amino acids.

In one embodiment of the polynucleotides described above, the polynucleotide also contains a promoter element operably linked to the polynucleotide encoding a factor VIII polypeptide.

In one embodiment of the polynucleotides described above, the polynucleotide also contains an enhancer element operably linked to a polynucleotide encoding a factor VIII polypeptide.

In one embodiment of the polynucleotides described above, the polynucleotide also contains a polyadenylation element operably linked to the polynucleotide encoding a factor VIII polypeptide.

In one embodiment of the polynucleotides described above, the polynucleotide also contains an intron operably linked to a nucleotide sequence encoding a factor VIII polypeptide.

In one embodiment of the polynucleotides described above, the intron is located between the promoter element and the translation initiation site (eg, the first encoding ATG) of the nucleotide sequence encoding the factor VIII polypeptide.

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

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

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

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

In another aspect, the disclosure provides a mammalian gene therapy vector as described above for the treatment of hemophilia A.

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

Brief description of graphic materials

In fig. 1 shows schematic illustrations of wild-type and ReFacto-type human factor VIII protein constructs.

In fig. 2A and 2B depict the codon-altered nucleotide sequence of CS04 (SEQ ID NO: 1) encoding a factor VIII variant in accordance with certain embodiments (CS04-FL-NA for the full-length coding sequence).

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In fig. 3 depicts the amino acid sequence of Factor VIII (SEQ ID NO: 2) encoded by the codon-altered nucleotide sequence CS04, in accordance with certain embodiments (CS04-FL-AA for the full-length amino acid sequence).

In fig. 4 depicts a portion of the codon-altered nucleotide sequence of CS04 (SEQ ID NO: 3) encoding the heavy chain of a factor VIII variant in accordance with certain embodiments (CS04-HC-NA).

In fig. 5 depicts a portion of the codon-altered nucleotide sequence of CS04 (SEQ ID NO: 4) encoding the light chain of a factor VIII variant in accordance with certain embodiments (CS04-LC-NA).

In fig. 6 depicts exemplary coding sequences (SEQ ID NOS: 5-7) for B domain replacement linkers in accordance with some embodiments. BDLO01 (SEQ ID NO: 5), BDLO04 (SEQ ID NO: 6) and BDLO23 (SEQ ID NO: 7) are the corresponding portions of the CS01, CS04 and CS23 nucleotide sequences with altered codons that encode the B domain replacement linker, respectively .

In fig. 7A-7C depict an AAV vector sequence (SEQ ID NO: 8) containing the codon-altered nucleotide sequence of CS04, in accordance with some embodiments (CS04-AV-NA).

In fig. 8A and 8B depict the nucleotide sequence of CS04Δ with altered codons (760-1667) (SPI; CS04Δ (741-1648), SPE) (SEQ ID NO: 9), encoding a single-chain variant of factor VIII in accordance with certain embodiments (CS04-SC1 -NA).

In fig. 9 shows the amino acid sequence of a factor VIII variant (SEQ ID NO: 10) encoded by CS0U (760-1667) (SPI; CSOU (741-1648), SPE), CS04Δ (760-1667) (SPI, CS04Δ (741-1648) , SPE) and CS23Δ (760-1667) (SPI; CS23Δ (741-1648), SPE) nucleotide sequences with altered codons in accordance with certain embodiments (CS01-SC1-AA CS04-SC1AA and CS23-SC1-AA, respectively) .

In fig. 10A and 10B depict the nucleotide sequence of CS04Δ with altered codons (772-1667) (SPI; CS04Δ (753-1648), SPE) (SEQ ID NO: 11), encoding a single-chain variant of factor VIII in accordance with certain embodiments (CS04-SC2 -NA).

In fig. 11 shows the amino acid sequence of factor VIII (SEQ ID NO: 12) encoded by CS0M (772-1667) (SPI; CS0M (753-1648), SPE), CS04Δ (772-1667) (SPI, CS04Δ (753-1648), SPE) and CS23Δ (772-1667) (SPI; CS23Δ (753-1648), SPE) codon-altered nucleotide sequences, in accordance with certain embodiments (CS01-SC2-AA CS04-SC2-AA and CS23-SC2-AA respectively).

In fig. 12A and 12B depict the codon-altered nucleotide sequence of CS01 (SEQ ID NO: 13) encoding a factor VIII variant in accordance with certain embodiments (CS01-FL-NA).

In fig. 13A and 13B depict the codon-altered nucleotide sequence of CS08 (SEQ ID NO: 14) encoding a factor VIII variant in accordance with certain embodiments (CS08-FL-NA).

In fig. 14A and 14B depict the codon-altered nucleotide sequence of CS10 (SEQ ID NO: 15) encoding a factor VIII variant in accordance with certain embodiments (CS10-FL-NA).

In fig. 15A and 15B depict the codon-altered nucleotide sequence of CS11 (SEQ ID NO: 16) encoding a factor VIII variant in accordance with certain embodiments (CS11-FL-NA).

In fig. 16A and 16B depict the coding sequence of wild-type ReFacto CS40 (SEQ ID NO: 17) according to certain embodiments (CS40-FL-NA).

In fig. 17A and 17B depict the codon-altered nucleotide sequence of CH25 (SEQ ID NO: 18) encoding a factor VIII variant in accordance with certain embodiments (CH25-FL-NA).

In fig. 18 depicts the amino acid sequence of human wild-type Factor VIII (SEQ ID NO: 19) according to certain embodiments (FVIII-FL-AA).

In fig. 19 shows a scheme for cloning constructs pCS40, pCS01, pCS04, pCS08, pCS10, pCS11 and pCh25 by inserting synthetic Refacto type BDD-FVIII DNA sequences into the pCh-BB01 vector backbone using AscI and NotI restriction sites.

In fig. 20 depicts the integrity of AAV vector genome preparations analyzed by agarose gel electrophoresis. Lane 1, DNA marker; track 2, VCS40; Lane 3, vCS01; track 4, VCS04. AAV vectors all have similar genome sizes, migrating approximately 5 kb (arrow, right side). The scale on the left side indicates the size of the DNA fragments in kilobases (kb).

In fig. 21 depicts protein analysis of AAV vector preparations by PAGE and silver staining. Lane 1, protein marker (M); track 2, VCS40; Lane 3, vCS01; and track 4, vCS04.

- 7 046432

All constructs have the same AAV8 capsids, consisting of VP1, VP2, and VP3 (arrows on the right). The scale on the left side indicates the size of the protein marker in kilodaltons (kDa).

In fig. 22A and 22B depict the codon-altered nucleotide sequence of CS23 (SEQ ID NO: 20) encoding a factor VIII variant in accordance with certain embodiments (CS23-FL-NA).

In fig. 23 depicts the amino acid sequence of Factor VIII (SEQ ID NO: 21) encoded by the codon-altered nucleotide sequence CS23, in accordance with certain embodiments (CS23-FL-AA).

In fig. 24 depicts a portion of the codon-altered nucleotide sequence of CS23 (SEQ ID NO: 22) encoding the heavy chain of a factor VIII variant in accordance with certain embodiments (CS23-HC-NA).

In fig. 25 depicts a codon-altered portion of the nucleotide sequence of CS23 (SEQ ID NO: 23) encoding a factor VIII variant light chain in accordance with certain embodiments (CS23-LC-NA).

In fig. 26 depicts a portion of the codon-altered nucleotide sequence of CS01 (SEQ ID NO: 24) encoding the heavy chain of a factor VIII variant in accordance with certain embodiments (CS01-HC-NA).

In fig. 27 depicts a portion of the codon-altered nucleotide sequence of CS01 (SEQ ID NO: 25) encoding the light chain of a factor VIII variant in accordance with certain embodiments (CS01-LC-NA).

In fig. 28A and 28B depict the nucleotide sequence of C.'S01A (760-1667) (SPI; C.'S01A (741-1648), SPE) with altered codons (SEQ ID NO: 26), encoding a single-chain variant of factor VIII in accordance with certain implementation options (CS01-SC1-NA).

In fig. 29A and 29B depict the nucleotide sequence of C.'S01A (772-1667) (SPI; C.'S01A (753-1648), SPE) with altered codons (SEQ ID NO: 27), encoding a single-chain variant of factor VIII in accordance with certain implementation options (CS01-SC2-NA).

In fig. 30A and 30B depict the nucleotide sequence of CS23Δ(760-1667) (SPI; CS23Δ(741-1648), SPE) with altered codons (SEQ ID NO: 28), encoding a single-chain variant of factor VIII in accordance with certain embodiments (CS23-SC1 -NA).

In fig. 31A and 31B depict the nucleotide sequence of CS23Δ with altered codons (772-1667) (SPI; CS23Δ (753-1648), SPE) (SEQ ID NO: 29), encoding a single-chain variant of factor VIII in accordance with certain embodiments (CS23-SC2 -NA).

Detailed description of the invention

Introduction.

AAV-based gene therapy holds great promise for the treatment of hemophilia. For hemophilia B, early clinical data are encouraging in that FIX levels of about 10% can be maintained in at least some patients for more than 1 year. However, for hemophilia A, achieving therapeutic expression levels of 5–10% using AAV vectors remains challenging for various reasons. First, the factor VIII coding sequence is too large for conventional AAV-based vectors.

Second, engineered factor VIII constructs with the B-domain deleted or truncated exhibit poor expression in vivo, even with codon optimization. Third, these factor VIII variants with the B domain deleted or truncated have a short half-life in vivo, exacerbating the consequences of poor expression. Fourth, even when expressed, FVIII is not efficiently secreted from cells, like other coagulation factors such as factor IX.

Moreover, these problems cannot be solved by simply administering higher doses of the gene therapy construct. According to current knowledge, the dose of AAV-based gene therapy vector should be increased above 2x 10 ¹² vg/kg body weight (vector genomes/kg). This is due to the fact that at such high doses, a T-cell immune response is activated, which destroys the transfected cells and, as a result, transgene expression is reduced or even lost. Therefore, strategies to improve FVIII expression are needed to make FVIII gene therapy a viable therapeutic option for patients with hemophilia A.

This disclosure relates to the discovery of factor VIII coding sequence variants with altered codons that address these and other problems associated with factor VIII gene therapy. For example, the polynucleotides described herein provide markedly improved expression in mammalian cells and exhibit improved virion packaging due to stabilized packaging interactions. In some embodiments, these advantages are realized by using coding sequences for factor VIII heavy and light chains with high sequence identity to codon altered constructs CS01, CS04, and CS23 (e.g., high sequence identity to one of CS01-HC, CS04- HC and CS23-HC heavy chain and high sequence identity compared to one

- 8 046432 from the light chain coding sequences CS01-LC, CS04-LC and CS23-LC).

In some embodiments, the factor VIII molecules encoded by the polynucleotides described herein have been truncated by truncation, deletion, or replacement of the wild-type B domain. As such, polynucleotides are better suited for expression of factor VIII using conventional gene therapy vectors, which do not efficiently express larger polypeptides such as wild-type factor VIII.

Advantageously, this document shows that the codon-altered coding sequences CS01, CS04 and CS23 encoding factor VIII provide superior expression of the B-domain deleted factor VIII construct in vivo. For example, in example 2 and in table. 4 shows that intravenous administration of AAV-based gene therapy vectors having CS01 (SEQ ID NO: 13), CS04 (SEQ ID NO: 1) and CS23 (SEQ ID NO: 20) provides 18-fold, 74-fold and 30-fold -fold increase in factor VIII expression compared to the corresponding CS40 construct, encoded by the wild-type polynucleotide sequence (SEQ ID NO: 17), in factor VIII knockout mice (Table 4).

In addition, this paper also shows that factor VIII variable coding sequences CS01 and CS04 with altered codons provide superior virion packaging and virus production. For example, Example 1 shows that AAV vector constructs containing constructs CS01 and CS04 provide 5-7 times greater viral yield compared to the corresponding construct CS40, encoded by the wild-type polynucleotide sequence, when isolated from the same amount of cell pellet.

Definitions.

As used herein, the following terms have the meanings ascribed to them unless otherwise noted.

As used herein, the terms Factor VIII and FVIII are used interchangeably and refer to any protein with Factor VIII activity (e.g., active FVIII, often referred to as FVIIIa) or a precursor (e.g., pro-protein or pre-pro-protein) of a protein with Factor VIII activity , in particular the cofactor activity of Factor IXa. 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 greater) of heavy and mild wild-type factor VIII polypeptide chains. In some embodiments, the B domain of the factor VIII polypeptide is removed, truncated, or replaced with a linker polypeptide to reduce the size of the polynucleotide encoding the factor VIII polypeptide. In an exemplary embodiment, amino acids 20-1457 of CS04-FL-AA constitute a Factor VIII polypeptide.

Non-limiting examples of wild-type Factor VIII polypeptides include human pre-proFactor VIII (e.g., under GenBank accession numbers AAA52485, CAA25619, AAA58466, AAA52484, AAA52420, AAV85964, BAF82636, BAG36452, CAI41660, CAI41666, CAI4 1672, CAI43241, САО03404, EAW72645, AAH22513, AAH64380, AAH98389, AAI11968, AAI11970 or AAB61261), the corresponding pro-Factor VIII and their natural variants; porcine pre-pro-Factor VIII (eg, according to UniProt accession numbers F1RZ36 or K7GSZ5), the corresponding pro-Factor VIII and natural variants thereof; mouse pre-pro-Factor VIII (eg, GenBank accession numbers AAA37385, CAM15581, CAM26492, or EDL29229), the corresponding pro-Factor VIII, and natural variants thereof; rat pre-pro-Factor VIII (eg, according to GenBank accession number AAQ21580), the corresponding pro-Factor VIII, and natural variants thereof; rat pre-pro Factor VIII; and other mammalian homologs of Factor VIII (eg, monkey, primate, hamster, guinea pig, etc.).

As used herein, the term Factor VIII polypeptide includes natural variants and artificial constructs with Factor IX cofactor activity. As used herein, the term factor VIII encompasses any naturally occurring variants, alternative sequences, isoforms, or mutant proteins that retain the basal cofactor activity of factor IX (e.g., at least 5%, 10%, 25%, 50%, 75%, or more corresponding to wild-type activity). Examples of factor VIII amino acid variations (relative to FVIII-FL-AA (SEQ ID NO: 19)) found in the human population include, but are not limited to,

S19R, R22T, Y24C, Y25C,

- 9 046432

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, A111T/V, 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, T2 94I, F295L, V297A, N299I, R301C/H/L, A303E/P, I307S, S308L, F312S, T314A/I, A315V, G323E, L326P, L327P/V, C329F, I331V, M339T, E340K, V345A/L, C348R/S/Y, Y365C, R391C/H/P, S392L/P, A394S, W401G, I405F/S, E409G, W412G/R, K427I, L431F/S, R437P/W, I438F, G439D/S/V, Y442C, K444R, Y450D/N, T454I, F455C, G466E, P470L/R/T, G474E/R/V, E475K, G477V, D478N, T479R, F484C, A488G, R490G, Y492C/H, Y492H, I494T, P496R, G498R, R503H, G513S/ V, I522Y, K529E, W532G, P540T, T541S, D544N, R546W, R550C/G/H, S553P, S554C/G, V556D, R560T, D561G/H/Y, I567T, P569R, S577F, V578A, D579A/H, N583 S, 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, I16 98T, Y1699C/F, E1701K, Q1705H, R1708C/H, T1714S, R1715G, A1720V, E1723K, D1727V, Y1728C, R1740G, K1751Q, F1762L, R1768H, G1769R, L1771P, L1775F/V, L1777P, G1779E/R, P1780L , I1782R, D1788H, M1791T, A1798P, S1799H, R1800C/G/H, P1801A, Y1802C, S1803Y, F1804S, L1808F, M1842I, P1844S, T1845P, E1848G, A1853T/V, S1858C, K1864E, D1865N/Y, H1867P/R, G1869D/V, G1872E, P1873R, L1875P, V1876L, C1877R/Y, L1882P, R1888I, E1894G, I1901F, E1904D/K, S1907C/R, W1908L, Y1909C, A1939T/V,

- 10 046432

N1941D/S, G1942A, M1945V, L1951F, R1960L/Q, L1963P, S1965I, M1966I/V, G1967D, S1968R, N1971T, H1973L, G1979V, H1980P/Y, F1982I, R1985Q, L199 4P, Y1998C, G2000A, T2004R, M2007I, G2013R, W2015C, R2016P/W, E2018G, G2022D, G2028R, S2030N, V2035A, Y2036C, N2038S, 2040Y, G2045E/V, I2051S, I2056N, A2058P, W2065R, P2067L, A 2070V, S2082N, S2088F, D2093G/Y, H2101D, T2105N, Q2106E/P/R, G2107S, R2109C, I2117F/S, Q2119R, F2120C/L, Y2124C, R2135P, S2138Y, T2141N, M2143V, F2145C, N2148S, N2157D, P2162L, R216 9C/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 contain polypeptides containing post-translational modifications.

Typically, factor VIII-encoding polynucleotides encode an inactive single-chain polypeptide (eg, a pre-pro protein) that is post-translationally processed to produce an active factor VIII protein (eg, FVIIIa). For example, according to FIG. 1, the human factor VIII pre-pro-protein is first cleaved to release the encoded signal peptide (not shown), generating the first single-chain pro-protein (shown as wild-type human FVIII). The pro protein is then cleaved between the B and A3 domains to produce a first polypeptide that contains the factor VIII heavy chain (eg, the A1 and A2 domains) and the B domain, and a second polypeptide that contains the factor VIII light chain (eg, contains the A3 domains , C1 and C3). The first polypeptide is further cleaved to remove the B domain and also to separate the A1 and A2 domains, which remain associated with the factor VIII light chain in the mature factor Villa protein. For a review of the maturation process of factor VIII, see Graw et al., Nat Rev Genet., 6 (6): 488-501 (2005), the contents of which are incorporated herein by reference in their entirety for all purposes.

However, in some embodiments, the factor VIII polypeptide is a single chain factor VIII polypeptide. Single-chain factor VIII polypeptides are designed to remove natural cleavage sites and optionally remove, shorten or replace the factor VIII B domain. 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 (1): 49-55 (1995)), the description of which is incorporated herein by reference in its entirety for all purposes.

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

As used herein, the term factor VIII light chain or simply light chain refers to the union of the A3, C1 and C2 domains of a factor VIII polypeptide. In an exemplary embodiment, amino acids 774-1457 of CS04-FL-AA (SEQ ID NO: 2) constitute the factor VIII light chain. In some embodiments, the factor VIII light chain does not contain the acidic peptide α3, which is released during in vivo maturation.

Typically, the factor VIII heavy and light chains are expressed as a single polypeptide chain, for example, together with an optional B domain or a B domain replacement linker. However, in some embodiments, factor VIII heavy chain and factor VIII light chain are expressed as separate polypeptide chains (eg, co-expressed) and are reduced to form factor VIII protein (eg, in vivo or in vitro).

As used herein, the terms B domain replacement linker and Factor VIII linker are used interchangeably and refer to shortened versions of wild-type Factor VIII B domain (e.g., amino acids 760-1667 FVIII-FL-AA (SEQ ID NO: 19)) or peptides , designed to replace the B domain of the factor VIII polypeptide. As used herein, the Factor VIII linker is located between the C-terminus of the Factor VIII heavy chain and the N-terminus of the Factor VIII light chain in a variant Factor VIII polypeptide in accordance with some embodiments. Non-limiting examples of B-domain replacement linkers are described in US Patent Nos. 4,868,112, 5,112,950,

- 11 046432

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; US Patent Application Publications No. 2013/024960, 2015/0071883 and 2015/0158930; and in PCT publications WO 2014/064277 and WO 2014/127215, the description of which is incorporated herein by reference in its entirety for all purposes.

Unless otherwise noted, Factor VIII amino acid numbering refers to the corresponding amino acid in the full-length wild-type human Factor VIII sequence (FVIII-FL-AA) shown as SEQ ID NO: 19 in FIG. 18. Thus, when referring to an amino acid substitution in a variant of the Factor VIII protein described herein, the number of amino acids specified refers to similar (e.g., structurally or functionally equivalent) and/or homologous (e.g., evolutionarily conserved in the primary amino acid sequence) full-length amino acid sequence of wild-type factor VIII. For example, amino acid substitution T2105N refers to a T-N substitution at position 2105 of the full-length wild-type human factor VIII sequence (FVIII-FL-AA, SEQ ID NO: 19) and a T to N substitution at position 1211 of the variant factor VIII protein encoded by CS04 (CS04- FL-AA, SEQ ID NO: 2).

As described herein, the factor VIII amino acid numbering system depends on whether the factor VIII signal peptide is included (eg, amino acids 1-19 of the full-length wild-type human factor VIII sequence). When a signal peptide is included, the numbering is designated as inclusive of the signal peptide or SPI. When no signal peptide is included, the numbering is designated as no signal peptide or SPE. For example, F328S in SPI numbering represents the same amino acid as F309S in SPE numbering. Unless otherwise indicated, all amino acid numbering refers to the corresponding amino acid in the full-length wild-type human factor VIII sequence (FVIII-FL-AA) shown as SEQ ID NO: 19 in FIG. 18.

As described herein, codon altered polynucleotides provide increased expression of a transgenic factor VIII in vivo (eg, when administered as part of a gene therapy vector) compared to the level of factor VIII expression provided by a naturally occurring factor VIII encoded construct (eg, a polynucleotide encoding the same human factor VIII construct using wild-type codons). As used herein, the term increased expression refers to the increased level of activity of a transgenic factor VIII in the blood of an animal that has been administered a codon-altered polynucleotide encoding factor VIII, compared to the level of activity in the blood of an animal of a transgenic factor VIII administered as a naturally encoded factor VIII construct. Activity levels can be measured using any factor VIII activity known in the art. A typical assay for determining factor VIII activity is the Technochrome FVIII assay (Technoclone, Vienna, Austria).

In some embodiments, increased expression refers to at least 25% greater transgenic factor VIII activity in the blood of an animal administered a codon-altered factor VIII polynucleotide compared to the level of factor VIII transgenic activity in the blood of an animal administered a naturally encoded polynucleotide factor VIII. In some embodiments, increased expression refers to at least 50% greater, at least 75% greater, at least 100% greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times larger, at least 20 times larger, at least 25 times larger, at least 30 times larger, at least 40 times larger, at least 50 times larger, at least 60 times larger greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, at least 100 times greater, at least 125 times greater, at least 150 times greater, at least 175 times greater, at least 200 times greater, at least 225 times greater, or at least 250 times greater transgenic factor VIII activity in the blood of an animal injected with a factor VIII polynucleotide with altered codons , compared with the level of transgenic factor VIII activity in the blood of an animal that was injected with a naturally encoded factor VIII polynucleotide.

As described herein, codon altered polynucleotides provide increased vector production compared to the level of vector production provided by a naturally encoded factor VIII construct (eg, a polynucleotide encoding the same human factor VIII construct using wild-type codons). As used herein, the term increased virus production refers to an increase in vector yield in cell culture (e.g., titer per liter of culture) inoculated with a codon-altered polynucleotide encoding factor VIII, compared to vector yield in cell culture inoculated with a naturally encoded factor VIII construct. . Vector yield data can be obtained using any vector titer assay known in the art. A typical assay for determining the yield of a vector (e.g., AAV vector) is qPCR using

- 12 046432 by inverted terminal repeats of AAV2 (Aurnhammer, Human Gene Therapy Methods: Part B 23:18-28 (2012)).

In some embodiments, increased virus production refers to at least 25% greater yield of the codon-mutated vector compared to the yield of a naturally encoded factor VIII construct in the same type of culture. In some embodiments, increased vector production refers to at least 50% more, at least 75% more, at least 100% more, at least 3 times more, at least 4 times more , at least 5 times larger, at least 6 times larger, at least 7 times larger, at least 8 times larger, at least 9 times larger, at least 10 times larger, according to at least 15 times greater or at least 20 times greater vector yield compared to the yield of a naturally encoded factor VIII construct in a culture of the same type.

As used herein, the term hemophilia refers to a group of disease conditions broadly characterized by decreased blood clotting or coagulation. Hemophilia can refer to hemophilia type A, type B, or type C, or a combination of all three types of diseases. Hemophilia type A (hemophilia A) is caused by a decrease or loss of factor VIII (FVIII) activity and is the best known of the hemophilia subtypes. Hemophilia type B (hemophilia B) results from loss or decreased function of clotting factor IX (FIX). Hemophilia type C (hemophilia C) results from the loss or decreased activity of coagulation factor XI (FXI). Hemophilia A and B are X-linked diseases, while hemophilia C is autosomal. Conventional treatments for hemophilia include both prophylactic and as-needed administration of coagulation factors such as FVIII, FIX, including Bebulin®-VH and FXI, as well as infusions of FEIBA-VH, desmopressin and plasma.

As used herein, the term FVIII gene therapy includes any therapeutic approach to provide a patient with a nucleic acid encoding factor VIII to alleviate, reduce, or prevent the recurrence of one or more symptoms (eg, clinical factors) associated with hemophilia. The term includes performing a procedure, maintaining a regimen, administering any compound, drug containing a nucleic acid encoding a factor VIII molecule, including any modified form of factor VIII (eg, a variant of factor VIII) to maintain or improve the health of a person with hemophilia. One skilled in the art will appreciate that either the course of FVIII therapy or the dose of the FVIII therapeutic agent may be modified, for example, based on the results obtained in accordance with the present invention.

As used herein, the term bypass therapy includes any therapeutic approach that provides non-factor VIII hemostatic agents, compounds, or coagulation factors to a patient to alleviate, reduce, or prevent the recurrence of one or more symptoms (eg, clinical factors) associated with hemophilia . Non-factor VIII compounds and coagulation factors include, but are not limited to, factor VIII bypass activity (FEIBA), recombinant activated factor VII (FVIIa), prothrombin complex concentrates and activated prothrombin complex concentrates. These non-factor VIII compounds and coagulation factors can be obtained recombinantly or from plasma. One skilled in the art will appreciate that either the course of bypass therapy or the dose of bypass therapy may be modified, for example, based on the results obtained in accordance with the present invention.

As used herein, the term combination therapy includes the administration of a nucleic acid encoding a factor VIII molecule and conventional therapeutic agents for the treatment of hemophilia A, includes any therapeutic approach that provides both a nucleic acid encoding a factor VIII molecule and a factor VIII molecule and/or hemostatic a non-factor VIII agent (eg, bypass therapeutic agent) to a patient to relieve, reduce, or prevent the recurrence of one or more symptoms (eg, clinical factors) associated with hemophilia. The term includes the performance of a procedure or regimen, the administration of any compound, drug, including a nucleic acid encoding a factor VIII molecule, including any modified form of factor VIII, that is useful for maintaining or improving the health of a person with hemophilia and including any of the agents that are described in this document.

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 the therapeutic effects for which it is administered. For example, a therapeutically effective amount of a drug useful for treating hemophilia may be an amount that is capable of preventing or ameliorating one or more symptoms associated with hemophilia. The exact dosage will depend on the purpose of treatment and will be determined by one skilled in the art using known methods (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols.

- 13 046432

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).

As used herein, the term gene refers to a segment of a DNA molecule that encodes a polypeptide chain (eg, a coding region). In some embodiments, the gene is located in regions immediately preceding, following and/or overlapping a coding region that is 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).

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

As used herein, the term promoter element refers to a nucleotide sequence that helps control the expression of a coding sequence. Typically, promoter elements are located at the 5' end of the initial translation site of a gene. However, in some embodiments, the promoter element may be located within an intron sequence or 3' end of the coding sequence. In some embodiments, the promoter useful for the gene therapy vector is derived from a native target protein gene (eg, the factor VIII promoter). In some embodiments, the promoter useful for the gene therapy vector is specific for expression in a particular cell or tissue of the target organism (eg, a liver-specific promoter). In other embodiments, one of a variety 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 β-actin promoter, and the methylCpG binding protein 2 (MeCP2) promoter. In some embodiments, the promoter is a constitutive promoter that provides substantially constant expression of the target protein. In other embodiments, the promoter is an inducible promoter that stimulates expression of a target protein in response to a particular stimulus (eg, exposure to a particular treatment or agent). For a review of the preparation of 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.

As used herein, the term vector refers to any carrier used to transfer a nucleic acid (eg, encoding a factor VIII gene therapy construct) into a host cell. In some embodiments, the vector contains a replicon that functions to replicate the carrier along with the target nucleic acid. Non-limiting examples of vectors useful for gene therapy include plasmids, phages, cosmids, artificial chromosomes and viruses that function as autonomous replication units in vivo. In some embodiments, the vector is a viral carrier for introducing a target nucleic acid (eg, 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 the virus's natural host, naturally occurring viruses are not known to cause any disease, and these viruses elicit little immune response.

As used herein, the term CpG island refers to a region within a polynucleotide that has a statistically increased density of CpG dinucleotides. As used herein, a region of a polynucleotide (eg, a codon-swapped polynucleotide encoding a factor VIII protein) is a CpG island if, among 200 bases: (i) the region has a GC content greater than 50%, and (ii) the ratio of observed CpG- dinucleotides to expected CpG dinucleotides is at least 0.6, as determined by the ratio:

A'ltpA'Ii:, 0.6.

For further information on methods for identifying CpG islands, see Gardiner-Garden M. et al., J Mol Biol., 196 (2): 261-82 (1987), the contents of which are expressly incorporated herein by reference in their entirety, for all purposes.

As used herein, the term nucleic acid refers to deoxyribonucleotides or ribonucleotides and their polymers in single- or double-stranded form and their complementary molecules. This term covers nucleic acids containing known nucleotide analogues or modified residues or linkages that are synthetic, naturally occurring and non-naturally occurring, that have similar binding properties as the reference

- 14 046432 nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogues include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides and peptide nucleic acids (PNAs).

The term amino acid refers to natural and non-natural amino acids, including amino acid analogs and amino acid mimetics, which function similarly to 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, such as hydroxyproline, carboxyglutamate, and O-phosphoserine. Naturally occurring amino acids may include, for example, D- and L-amino acids. Amino acids used herein may also include unnatural amino acids. Amino acid analogues refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, that is, any carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogues have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but their function is similar to that of a naturally occurring amino acid. Amino acids can be designated by their commonly used three-letter symbols, or by the single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Nucleotides may also be referred to by their common single-letter designations.

With regard to amino acid sequences, one skilled in the art will appreciate that individual substitutions, deletions or insertions in a nucleic acid or peptide sequence that alter, add or remove one amino acid or a small percentage of amino acids in the encoded sequence are conservatively modified variants where the change results in to the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables representing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecific homologs and alleles in this description.

Conservative amino acid substitutions that provide functionally similar amino acids are well known in the art. Depending on the functionality of a particular amino acid, for example for catalytic, structural or sterically important amino acids, different groups of amino acids can be considered conservative replacements for each other. In table 1 shows groups 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 effects/structural characteristics of the amino acid, and the effect of the amino acid on secondary structure.

Table 1

Groups of conservative amino acid substitutions based on the functionality of the residue in the protein

Important Feature Conservative Groups Charge/Polarity 1. H, R and K 2. D and E

- 15 046432

3. C, T, S, G, N, Q and Y 4. A, P, M, L, I, V, G and W Hydrophobicity 1. D, E, N, Q, R and K 2. C, S, T, P, G, H and Y 3. A, M, I, L, V, F and W Structural/Superficial Location 1. D, E, N, Q, H, R and K 2. C, S, T, P, A, G, W and Y 3. M, I, L, V and F Impact on Secondary Structure 1. A, E, Q, H, K, M, L and R 2. C, T, I, V, F, Y and W 3. S, G, P, D and N Evolutionary Conservation 1. D and E 2. H, K and R 3. N and Q 4. S and T 5. L, I and V 6. E, Y and W 7. A and G 8. M and S

The terms identical or percentage identity in the context of two or more nucleic acid or peptide sequences refer to two or more sequences or subsequences that are the same or have a certain percentage of amino acid residues or nucleotides that are the same (e.g., about 60% identity, preferably 65% , 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity in a specific area when compared and alignment to closely match the comparison site or highlighted region) when measured, for example, using the BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below, or by manual alignment and visual inspection.

As is known in the art, a variety of different programs can be used to determine the identity or similarity of a protein (or nucleic acid, as described below) sequence when compared to a known sequence. Sequence identity and/or similarity is determined using standard methods 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. USA, 85: 2444 (1988), through computer 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 default settings or by checking. Preferably, percent identity is calculated using FastDB based on the following parameters: mismatch penalty 1; omission penalty 1; omission penalty 0.33; and Box 30 fine, 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.

An example of a useful algorithm is PILEUP. PILEUP generates multiple sequence alignments from a group of related sequences using progressive pairwise alignments. It can also display a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method 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 include a default skip value of 3.00, a default skip length value of 0.10, and weighted trailing skips.

Another example of a useful algorithm is the BLAST algorithm, described in: Altschul et al., J. Mol. Biol. 215, 403-410, (1990); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); and Karlin et al., Proc. Natl. Acad. Sci. USA 90: 5873-5787 (1993), both incorporated by reference. A particularly useful BLAST program is the WU-BLAST-2 program from Altschul et al., Methods in Enzymology.

- 16 046432

266:460-480 (1996); http://blast.wustl/edu/blast/README.html]. WU-BLAST-2 uses several search parameters, most of which have default values. The adjustable parameters are set with the following values: overlap interval=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are determined by the program itself, depending on the composition of a specific sequence and the composition of a specific database with which the sequence of interest is searched; however, values can be adjusted to improve sensitivity.

An additional useful algorithm is gapped BLAST, as reported in Altschul et al., Nucl. Acids Res., 25: 3389-3402, incorporated by reference. Gapped BLAST uses BLOSUM-62 substitution scores; threshold parameter T is set to 9; two-hit method for running extensions without skipping; the estimate of the length k of gaps is 10+k; Xu is set to 16 and Xg is set to 40 for the database search stage and 67 for the output stage of the algorithms. Alignments with gaps are triggered using an estimate corresponding to ~22 bits.

Percentage amino acid sequence identity values are 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 that has more significant residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored). Similarly, percent nucleic acid sequence identity (%) to the coding sequence of the identified polypeptides is defined as the percentage of nucleotide residues in the candidate sequence that are identical to nucleotide residues in the cell cycle protein coding sequence. The preferred method uses the default BLASTN module of WU-BLAST-2 with the overlap interval and overlap fraction set to 1 and 0.125, respectively.

Alignment may involve introducing gaps in sequences to be aligned. Additionally, for sequences that contain either more or fewer amino acids than the protein encoded by the sequence depicted in FIG. 2 (SEQ ID NO: 1), it is understood that in one embodiment of the invention, the percentage of sequence identity will be determined based on the number of identical amino acids or nucleotides relative to the total number of amino acids or nucleotides. Thus, for example, the identity of sequences that are shorter than that shown in FIG. 2 (SEQ ID NO: 1), as discussed below, will be determined using the number of nucleotides in a shorter sequence in one embodiment of the invention. In percent identity calculations, relative weights are not assigned to different manifestations of sequence variation such as insertions, deletions, substitutions, etc.

In one embodiment of the invention, only identities are scored positively (+1), and all forms of sequence variation, including gaps, are assigned a value of 0, eliminating the need for weighted scales or parameters as described below for sequence similarity calculations. Sequence identity can 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 that has more residues in the aligned region.

The term allelic variants refers to polymorphic forms of a gene at a specific genetic locus, as well as cDNAs derived from the mRNA of gene transcripts and the polypeptides they encode. The term mammalian preferred codon refers to a subset of codons from the set of amino acid-encoding codons that are most commonly used in proteins expressed in mammalian cells, as shown in the following list: Gly (GGC, GGG); Glu (GAG); Asp (GAC); Val (GTG, GTC); Ala (GCC, GCT); Ser (AGC, TCC); Lys (AAG); Asn(AAC); Met (ATG); Ile (PBX); Thr(ACC); Trp(TGG); Cys (TGC); Tyr (TAT, TAC); Leu (CTG); Phe (TTC); Arg (CGC, AGG, AGA); Gln (CAG); His (CAC); and Pro (CCC).

As used herein, the term altered codon refers to a polynucleotide sequence encoding a polypeptide (eg, a variant VIII protein) wherein at least one codon of the naturally occurring polynucleotide encoding the polypeptide has been altered to improve the property of the polynucleotide sequence. In some embodiments, the improved property results in increased transcription of the mRNA encoding the polypeptide, increased stability of the mRNA (eg, increased half-life of the mRNA), increased translation of the polypeptide, and/or improved packaging of the polynucleotide within the vector. Non-limiting examples of changes that can be used to obtain improved properties include changing codon usage and/or distribution for certain amino acids, adjusting global and/or local GC content, removing sequences with high AT content, removing repetitive sequence elements, adjusting global and/or local CpG dinucleotide content, removal of critical regulatory elements (for example, TATA box and CCAAT box), removal of intron/exon splice sites, improvement of regulatory sequences

- 17 046432 activities (for example, the introduction of a Kozak consensus sequence) and the removal of sequence elements capable of forming secondary structure (for example, hairpins) in the transcribed mRNA.

As discussed herein, there are various nomenclatures to refer to the components described in this document. CS number (eg, CS04, CS01, CS23, etc.) refers to codon altered polynucleotides encoding FVIII polypeptides and/or encoded polypeptides, including variants. For example, CS01-FL refers to the Full Length polynucleotide sequence of CS01 with altered codons, or amino acid sequence (sometimes referred to herein as CS01-FL-AA for the Amino Acid sequence and CS01-FL-NA for the sequence nucleic acid sequence (Nucleic Acid sequence) encoded by the polynucleotide sequence CS01. Likewise, CS01-LC refers to either a codon altered nucleic acid sequence (CS01-LC-NA) encoding the light chain of a FVIII polypeptide, or an amino acid sequence (also sometimes referred to herein as CS01-LC-AA) of the FVIII light chain encoded by polynucleotide sequence CS01. Similarly, CS01-HC, CS01-HC-AA and CS01-HC-NA are the same for FVIII heavy chain. As will be appreciated by those skilled in the art, for constructs such as CS01, CS04, CS23, etc., which are modified only by altered codons (e.g., they do not contain additional amino acid substitutions compared to Refacto), the amino acid sequences will be identical , since amino acid sequences do not change during codon optimization. Thus, description sequence constructs include, but are not limited to, CS01-FL-NA, CS01-FL-AA, CS01-LC-NA, CS01-LC-AA, CS01-HC-AA, CS01-HC-NA, CS04 -FL-NA, CS04-FL-AA, CS04-LC-NA, CS04-LC-AA, CS04-HC-AA, CS04-HC-NA, CS23-FL-NA, CS23-FL-AA, CS23 -LC -NA, CS23-LC-AA, CS23-HC-AA and CS23-HC-NA.

Factor VIII variants with altered codons.

In some embodiments, this disclosure provides codon altered polynucleotides encoding variants of factor VIII. These codon-altered polynucleotides provide significantly improved factor VIII expression when incorporated into AAV-based gene therapy constructs. Codon-altered polynucleotides also demonstrate improved packaging of AAV virions compared to traditional codon-optimized constructs. As shown in example 2 and table. 4, Applicants achieve these advantages through the discovery of three codon altered polynucleotides (CS01-FL-NA, CS04-FL-NA and CS23-FL-NA) encoding a factor VIII polypeptide with human factor VIII heavy and light chains and a short 14 amino acid substituent B -linker domain (SQ linker) containing a furin cleavage site to facilitate maturation of active FVIIIa protein in vivo.

In one embodiment of the invention, the codon altered polynucleotide provided herein has nucleotide sequences with high identity to at least the sequences CS01, CS04 or CS23 (SEQ ID NOs 13, 1 and 20, respectively) encoding the factor VIII heavy chain and factor VIII light chain. As is known in the art, the Factor VIII B domain is unsuitable for in vivo activity. Thus, in some embodiments, the codon altered polynucleotides provided herein lack the Factor VIII B domain entirely. In some embodiments, the native Factor VIII B domain is replaced with a short amino acid linker containing a furin cleavage site, for example, an SQ linker consisting of amino acids 760-773 CS01, CS04 or CS23 (SEQ ID NOs 2, 2 and 21, respectively). The SQ linker also refers to the sequence BDLO04 (-AA for amino acid sequence and -NA for nucleotide sequence).

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 (eg, porcine factor VIII). In other embodiments, the factor VIII heavy and light chains are chimeric heavy and light chains (eg, a combination of a first human sequence and a second mammalian sequence). In other embodiments, the factor VIII heavy and light chains are a humanized version of the heavy and light chains of another mammal, for example, the heavy and light chain sequences of another mammal, in which human sequence residues are substituted at selected positions to reduce the immunogenicity of the resulting peptide when administered to a human .

The GC content of human genes varies widely: from less than 25% to more than 90%. However, in general, human genes with higher GC content are expressed at higher levels. For example, in Kudla et al. (PLoS Biol., 4 (6): 80 (2006)) it is shown that increasing the content of the GC gene increases the expression of the encoded polypeptide, primarily by increasing transcription and achieving a higher level of stability of the mRNA transcript. As a rule, what is desired

- 18 046432 the GC content in the codon optimized gene construct is equal to or greater than 60%. However, native AAV genomes have a GC content of about 56%.

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., about 56% GC) that is lower than the preferred CG content of polynucleotides that typically undergo codon optimization for expression in mammalian cells (eg, 60% GC or higher). As indicated in Example 1, CS04-FLNA(SEQ ID NO: 1), which has a GC content of approximately 56%, has improved virion packaging compared to similar codon-edited sequences with higher GC content.

Thus, in some embodiments, the total GC content of the codon-mutated polynucleotide encoding the factor VIII polypeptide is less than 60%. In some embodiments, the total GC content of the codon-mutated polynucleotide encoding the factor VIII polypeptide is less than 59%. In some embodiments, the total GC content of the codon-mutated polynucleotide encoding the factor VIII polypeptide is less than 58%. In some embodiments, the total GC content of the codon-mutated polynucleotide encoding the factor VIII polypeptide is less than 57%. In some embodiments, the total GC content of the codon-mutated polynucleotide encoding the factor VIII polypeptide is no more than 56%.

In some embodiments, the total GC content of the codon-altered polynucleotide encoding the Factor VIII polypeptide is between 54% and 59%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor VIII polypeptide is between 55% and 59%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the Factor VIII polypeptide is between 56% and 59%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor VIII polypeptide is between 54% and 58%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor VIII polypeptide is between 55% and 58%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor VIII polypeptide is between 56% and 58%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the Factor VIII polypeptide is between 54% and 57%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the Factor VIII polypeptide is between 55% and 57%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the Factor VIII polypeptide is between 56% and 57%. In some embodiments, the total GC content of the codon-mutated polynucleotide encoding the factor VIII polypeptide is between 54% and 56%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the Factor VIII polypeptide is between 55% and 56%.

In some embodiments, the total GC content of the codon-mutated polynucleotide encoding the factor VIII polypeptide is 56±0.5%. In some embodiments, the total GC content of the codon-mutated polynucleotide encoding the factor VIII polypeptide is 56±0.4%. In some embodiments, the total GC content of the codon-mutated polynucleotide encoding the factor VIII polypeptide is 56±0.3%. In some embodiments, the total GC content of the codon-mutated polynucleotide encoding the Factor VIII polypeptide is 56±0.2%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the Factor VIII polypeptide is 56±0.1%. In some embodiments, the total GC content of the codon-mutated polynucleotide encoding the Factor VIII polypeptide is 56%.

Linker replacing the B domain of Factor VIII.

In some embodiments, the association between the FVIII heavy chain and the light chain (eg, the B domain in wild-type Factor VIII) is further altered. Due to packaging capacity limitations of AAV, variants with the Bdomain deleted, truncated, and/or linker substituted should improve the efficacy of FVIII gene therapy designs. The most traditionally used B domain replacement linker is SQ FVIII, which retains only the 14 amino acids of the B domain as a linker sequence. Another porcine VIII variant (OBI-1, described in US Pat. No. 6,458,563) is well expressed in CHO cells and has a longer linker of 24 amino acids. In some embodiments, the factor VIII constructs encoded by the codon-altered polynucleotides described herein comprise a B domain SQ type linker sequence. In other embodiments of the invention

- 19 046432 factor VIII constructs encoded by codon altered polynucleotides described herein contain a B domain type 0BI-1 linker sequence.

In some embodiments, the encoded factor VIII polypeptides described herein comprise an SQ type B domain linker (SFSQNPPVLKRHQR; BDL-SQ-AA; SEQ ID NO: 30), including B domain amino acids 760-762/1657-1667 human wild-type factor VIII (FVIIIFL-AA, SEQ ID NO: 19) (Sandberg et al., Thromb. Haemost. 85:93 (2001)). In some embodiments, the SQ-type B domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the SQ-type V-domain linker has two amino acid substitutions relative to the corresponding wild-type sequence.

In some embodiments, the encoded Factor VIII polypeptides described herein comprise a Greengene-type B domain linker, including amino acids 760/15821667 of the human wild-type Factor VIII B domain (FVIII-FL-AA, SEQ ID NO: 19 ) (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, the encoded Factor VIII polypeptides described herein comprise an extended SQ-type B-domain linker, including human wild-type Factor VIII amino acids 760769/1657-1667 (FVIII-FL-AA, SEQ ID NO: 19) (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 domain linker has two amino acid substitutions relative to the corresponding wild-type sequence.

In some embodiments, the encoded factor VIII polypeptides described herein comprise a porcine OBI-1 type B domain linker, including the amino acids SFAQNSRPPSASAPKPPVLRRHQR (SEQ ID NO: 31) 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 OBI1 B-domain linker has two amino acid substitutions relative to the corresponding wild-type sequence.

In some embodiments, the encoded Factor VIII polypeptides described herein comprise a human OBI-1 type B domain linker, including amino acids 760772/1655-1667 of the human wild-type Factor VIII B domain (FVIII-FL-AA, SEQ ID NO: 19). In some embodiments, the human OBI-1 type B domain linker has one amino acid substitution 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, the encoded factor VIII polypeptides described herein comprise a type 08 B domain linker including the amino acids SFSQNSRHQAYRYRRG (SEQ ID NO: 32) from the porcine wild type 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 0BI-1 B domain linker has two amino acid substitutions relative to the corresponding wild-type sequence.

Removal of the B domain from factor VIII constructs does not appear to affect the activity of the activated enzyme (eg, FVIIIa), presumably because the B domain is removed during activation. However, the factor VIII B domain contains several residues that are post-translationally modified, for example, by N- or O-mediated 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 domain predicts that at least four of these sites are glycosylated in vivo . These modifications within the B domain are thought to contribute to the post-translational regulation and/or half-life of factor VIII in vivo.

Although the factor VIII B domain is absent from the mature factor Villa protein, glycosylation in the B domain of the factor VIII precursor molecule can increase the half-life of the protein before activation. Thus, in some embodiments, the polypeptide linker of the encoded factor VIII construct described herein contains one or more glycosylation sequences to enable glycosylation in vivo. In some embodiments, the polypeptide linker comprises at least one glycosylatable consensus sequence (e.g., an N- or O-mediated glycation consensus sequence

- 20 046432 cosylation). In some embodiments, the polypeptide linker comprises at least two consensus glycosylation sequences. In some embodiments, the polypeptide linker contains at least three consensus glycosylation sequences. In some embodiments, the polypeptide linker contains at least four consensus glycosylation sequences. In some embodiments, the polypeptide linker contains at least five consensus glycosylation sequences. In some embodiments, the polypeptide linker contains at least 6, 7, 8, 9, 10 or more consensus glycosylation sequences.

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

Codon-altered polynucleotides encoding a variant of Factor VIII with a cleavable linker.

CS04 polynucleotides with altered codons.

In one embodiment, the codon altered polynucleotides provided herein comprise a nucleotide sequence encoding a variant Factor VIII polypeptide with a linker that is cleaved in vivo. A factor VIII polypeptide contains a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The factor VIII polypeptide heavy chain is encoded by the first nucleotide sequence having high sequence identity with CS04-HC-NA (SEQ ID NO: 3), which is part of CS04-FL-NA (SEQ ID NO: 1) encoding the factor VIII heavy chain . The factor VIII polypeptide light chain is encoded by a second nucleotide sequence with high sequence identity to CS04-LC-NA (SEQ ID NO: 4), which is a portion of CS04-FL-NA (SEQ ID NO: 1) encoding the factor VIII light chain. The polypeptide linker contains a furin cleavage site that allows for in vivo maturation (eg, following in vivo expression or introduction of a precursor polypeptide).

In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity with CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity with CS04-HC-NA and CS04-LC-NA (SEQ ID N0 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity with CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity with CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity with CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity with CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity with CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS04-HC-NA and CS04-LC-NA (SEQ ID NOS 3 and 4), respectively.

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

- 21 046432 at least 96% identity with BDLO04 (SEQ ID NO: 6). In some embodiments, the third nucleotide sequence has at least 97% identity with BDLO04 (SEQ ID NO: 6). In some embodiments, the third nucleotide sequence has at least 98% identity with BDLO04 (SEQ ID NO: 6). In some embodiments, the third nucleotide sequence is identical to BDLO04 (SEQ ID NO: 6).

In some embodiments, the codon-altered polynucleotide has a nucleotide sequence with high sequence identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 95% identity with CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 96% identity with CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 97% identity with CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 98% identity with CS04FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 99% identity with CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 99.5% identity with CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 99.9% identity with CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence is identical to CS04-FL-NA (SEQ ID NO: 1).

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

CS01 codon altered polynucleotides In one embodiment, the codon altered polynucleotides provided herein comprise a nucleotide sequence encoding a variant factor VIII polypeptide with a linker that is cleaved in vivo. A factor VIII polypeptide contains a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The factor VIII polypeptide heavy chain is encoded by the first nucleotide sequence having high sequence identity with CS01-HC-NA (SEQ ID NO: 24), which is part of CS01-FL-NA (SEQ ID NO: 13) encoding the factor VIII heavy chain . The factor VIII polypeptide light chain is encoded by a second nucleotide sequence with high sequence identity to CS01-LC-NA (SEQ ID NO: 25), which is a portion of CS01-FL-NA (SEQ ID NO: 13) encoding the factor VIII light chain. The polypeptide linker contains a furin cleavage site that allows for in vivo maturation (eg, following in vivo expression or introduction of a precursor polypeptide).

In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS01-HC-NA and

- 22 046432

CS01-LC-NA (SEQ ID NO: 24 and 25), respectively.

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

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

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

CS23 polynucleotides with altered codons.

In one embodiment, the codon altered polynucleotides provided herein comprise a nucleotide sequence encoding a variant Factor VIII polypeptide with a linker that is cleaved in vivo. A factor VIII polypeptide contains a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The factor VIII polypeptide heavy chain is encoded by the first nucleotide sequence having high sequence identity with CS23-HC-NA (SEQ ID NO: 22), which is part of CS23-FL-NA (SEQ ID NO: 20) encoding the factor VIII heavy chain . The factor VIII polypeptide light chain is encoded by a second nucleotide sequence with high sequence identity to CS23-LC-NA (SEQ ID NO: 23), which is a portion of CS23-FL-NA (SEQ ID NO: 20) encoding the factor VIII light chain. The polypeptide linker contains a furin cleavage site that allows for in vivo maturation (eg, following in vivo expression or introduction of a precursor polypeptide).

In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity with CS23-HC-NA and CS23-LC-NA (SEQ ID NOs 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity with CS23-HC-NA and CS23-LC-NA (SEQ ID NOs 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity with CS23-HC-NA and CS23-LC-NA (SEQ ID NOs 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity with CS23-HC-NA and CS23-LC-NA (SEQ ID NOs 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences are

- 23 046432 at least 99% sequence identity with CS23-HC-NA and CS23-LC-NA (SEQ ID NOs 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity with CS23-HC-NA and CS23-LC-NA (SEQ ID NOs 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity with CS23-HC-NA and CS23-LC-NA (SEQ ID NOs 22 and 23), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS23-HC-NA and CS23-LC-NA (SEQ ID NOs: 22 and 23), respectively.

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

In some embodiments, the codon altered polynucleotide has a nucleotide sequence with high sequence identity to CS23-FL-NA (SEQ ID NO: 20). In some embodiments, the nucleotide sequence has at least 95% identity with CS23-FL-NA (SEQ ID NO: 20). In some embodiments, the nucleotide sequence has at least 96% identity with CS23-FL-NA (SEQ ID NO: 20). In some embodiments, the nucleotide sequence has at least 97% identity with CS23-FL-NA (SEQ ID NO: 20). In some embodiments, the nucleotide sequence has at least 98% identity with CS23-FL-NA (SEQ ID NO: 20). In some embodiments, the nucleotide sequence has at least 99% identity with CS23-FL-NA (SEQ ID NO: 20). In some embodiments, the nucleotide sequence has at least 99.5% identity with CS23-FL-NA (SEQ ID NO: 20). In some embodiments, the nucleotide sequence has at least 99.9% identity with CS23-FL-NA (SEQ ID NO: 20). In some embodiments, the nucleotide sequence is identical to CS23-FL-NA (SEQ ID NO: 20).

In some embodiments, the factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high identity to CS23-FL-AA (SEQ ID NO: 21). In some embodiments, the amino acid sequence has at least 97% identity with CS23-FL-AA (SEQ ID NO: 21). In some embodiments, the amino acid sequence has at least 98% identity with CS23-FL-AA (SEQ ID NO: 21). In some embodiments, the amino acid sequence has at least 99% identity with CS23-FL-AA (SEQ ID NO: 21). In some embodiments, the amino acid sequence has at least 99.5% identity with CS23-FL-AA (SEQ ID NO: 21). In some embodiments, the amino acid sequence has at least 99.9% identity with CS23FL-AA (SEQ ID NO: 21). In some embodiments, the amino acid sequence is identical to CS23-FL-AA (SEQ ID NO: 21).

Polynucleotides with altered codons encoding single-stranded Factor VIII protein.

Factor VIII constructs in which the furin cleavage site located at the C-terminus of the B domain is removed retain single-chain polypeptide activity despite the fact that normal maturation of the factor VIII molecule cannot occur (Leyte et al. (1991)). Similarly, a Factor VIII construct lacking the B domain with an attenuated furin site (containing the R1664H amino acid substitution) is more biologically active than the corresponding Factor VIII construct with a wild-type furin cleavage site (Siner et al. (2013)). Accordingly, in some embodiments, the codon altered polynucleotides provided herein comprise a nucleotide sequence encoding a single-strand variant of a Factor VIII polypeptide. A single-chain factor VIII polypeptide contains a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The polypeptide linker does not contain a furin cleavage site.

CS04 single-stranded polynucleotides with altered codons.

In one embodiment, the codon altered polynucleotides provided herein comprise a nucleotide sequence encoding a single-strand variant of a Factor VIII polypeptide. Factor VIII polypeptide contains factor VIII light chain,

- 24 046432 factor VIII heavy chain and, optionally, a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The factor VIII polypeptide heavy chain is encoded by the first nucleotide sequence having high sequence identity with CS04-HC-NA (SEQ ID NO: 3), which is part of CS04-FL-NA (SEQ ID NO: 1) encoding the factor VIII heavy chain . The factor VIII polypeptide light chain is encoded by a second nucleotide sequence with high sequence identity to CS04-LC-NA (SEQ ID NO: 4), which is a portion of CS04-FL-NA (SEQ ID NO: 1) encoding the factor VIII light chain. The optional polypeptide linker does not contain a furin cleavage site.

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

In some embodiments, the codon altered polynucleotide has a nucleotide sequence with high sequence identity to CS04-SC1-NA (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 95% identity with CS04-SC1-NA (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 96% identity with CS04-SC1NA (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 97% identity with CS04-SC1-NA (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 98% identity with CS04-SC1-NA (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 99% identity with CS04-SC1-NA (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 99.5% identity with CS04-SC1-NA (SEQ ID NO: 9). In some embodiments, the nucleotide sequence has at least 99.9% identity with CS04-SC1-NA (SEQ ID NO: 9). In some embodiments, the nucleotide sequence is identical to CS04-SC1-NA (SEQ ID NO: 9).

In some embodiments, the codon altered polynucleotide has a nucleotide sequence with high sequence identity to CS04-SC2-NA (SEQ ID NO: 11). In some embodiments, the nucleotide sequence has at least 95% identity with CS04-SC2-NA (SEQ ID NO: 11). In some embodiments, the nucleotide sequence has at least 96% identity with CS04-SC2NA (SEQ ID NO: 11). In some embodiments, the nucleotide sequence has at least 97% identity with CS04-SC2-NA (SEQ ID NO: 11). In some embodiments, the nucleotide sequence has at least 98% identity with CS04-SC2-NA (SEQ ID NO: 11). In some embodiments, the nucleotide sequence has at least 99% identity with CS04-SC2-NA (SEQ ID NO: 11). In some embodiments, the nucleotide sequence has at least 99.5% identity with CS04-SC2-NA (SEQ ID NO: 11). In some embodiments, the nucleotide sequence has at least 99.9% identity with CS04-SC2-NA (SEQ ID NO: 11). In some embodiments, the nucleotide sequence is identical to CS04SC2-NA (SEQ ID NO: 11).

In some embodiments, the single-strand variant of factor VIII encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to CS04-SC1-AA (SEQ ID NO: 10, human factor VIIIA (760-1667) (SPI; HsFVIIIA (741 -1648), SPE)). In some embodiments, the factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with

- 25 046432 high identity with CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 97% identity with CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 98% identity with CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 99% identity with CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 99.5% identity with CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 99.9% identity with CS04-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is identical to CS04-SC1-AA (SEQ ID NO: 10).

In some embodiments, the single-strand variant of factor VIII encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to CS04-SC2-AA (SEQ ID NO: 12, human factor FVIIIA (7721667) (SPI; HsFVIIIA (753-1648) ), SPE)). In some embodiments, the factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high identity to CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 97% identity with CS04SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 98% identity with CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99% identity with CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99.5% identity with CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99.9% identity with CS04-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is identical to CS04-SC2-AA (SEQ ID NO: 12).

CS01 single-stranded codon altered polynucleotides In one embodiment of the invention, the codon altered polynucleotides provided herein comprise a nucleotide sequence encoding a single-stranded variant of a factor VIII polypeptide. A factor VIII polypeptide comprises a factor VIII light chain, a factor VIII heavy chain, and optionally a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The factor VIII polypeptide heavy chain is encoded by the first nucleotide sequence having high sequence identity with CS01-HC-NA (SEQ ID NO: 24), which is part of CS01-FL-NA (SEQ ID NO: 13) encoding the factor VIII heavy chain . The factor VIII polypeptide light chain is encoded by a second nucleotide sequence with high sequence identity to CS01-LC-NA (SEQ ID NO: 25), which is a portion of CS01-FL-NA (SEQ ID NO: 13) encoding the factor VIII light chain. The optional polypeptide linker does not contain a furin cleavage site.

In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity with CS01-HC-NA and CS01-LC-NA (SEQ ID NOs 24 and 25), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS01-HC-NA and CS01-LC-NA (SEQ ID NOs: 24 and 25), respectively.

In some embodiments, the codon altered polynucleotide has a nucleotide sequence with high sequence identity to CS01-SC1-NA (SEQ ID NO: 26). In some embodiments, the nucleotide sequence has at least 95% identity with CS01-SC1-NA (SEQ ID NO: 26). In some embodiments, the nucleotide sequence has at least 96% identity with CS01-SC1NA (SEQ ID NO: 26). In some embodiments, the nucleotide sequence

- 26046432 has at least 97% identity with CS01-SC1-NA (SEQ ID NO: 26). In some embodiments, the nucleotide sequence has at least 98% identity with CS01-SC1-NA (SEQ ID NO: 26). In some embodiments, the nucleotide sequence has at least 99% identity with CS01-SC1-NA (SEQ ID NO: 26). In some embodiments, the nucleotide sequence has at least 99.5% identity with CS01-SC1-NA (SEQ ID NO: 26). In some embodiments, the nucleotide sequence has at least 99.9% identity with CS01-SC1-NA (SEQ ID NO: 26). In some embodiments, the nucleotide sequence is identical to CS01SC1-NA (SEQ ID NO: 26).

In some embodiments, the codon altered polynucleotide has a nucleotide sequence with high sequence identity to CS01-SC2-NA (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 95% identity with CS01-SC2-NA (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 96% identity with CS01-SC2NA (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 97% identity with CS01-SC2-NA (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 98% identity with CS01-SC2-NA (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 99% identity with CS01-SC2-NA (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 99.5% identity with CS01-SC2-NA (SEQ ID NO: 27). In some embodiments, the nucleotide sequence has at least 99.9% identity with CS01-SC2-NA (SEQ ID NO: 27). In some embodiments, the nucleotide sequence is identical to CS01SC2-NA (SEQ ID NO: 27).

In some embodiments, the single-strand variant of factor VIII encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to CS01-SC1-AA (SEQ ID NO: 10, human factor FVIIIA (7601667) (SPI; HsFVIIIA (741-1648) ), SPE)). In some embodiments, the factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high identity to CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 97% identity with CS01SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 98% identity with CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 99% identity with CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 99.5% identity with CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 99.9% identity with CS01-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is identical to CS01-SC1-AA (SEQ ID NO: 10).

In some embodiments, the single-chain variant of factor VIII encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to CS01-SC2-AA (SEQ ID NO: 12, human factor FVIIIA (7721667) (SPI; HsFVIIIA (753-1648) ), SPE)). In some embodiments, the factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high identity to CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 97% identity with CS01SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 98% identity with CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99% identity with CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99.5% identity with CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99.9% identity with CS01-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is identical to CS01-SC2-AA (SEQ ID NO: 12).

CS23 single-stranded codon altered polynucleotides In one embodiment of the invention, the codon altered polynucleotides provided herein comprise a nucleotide sequence encoding a single-stranded variant of a factor VIII polypeptide. A factor VIII polypeptide comprises a factor VIII light chain, a factor VIII heavy chain, and optionally a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The factor VIII polypeptide heavy chain is encoded by the first nucleotide sequence having high sequence identity with CS23-HC-NA (SEQ ID NO: 22), which is

- 27 046432 part of CS23-FL-NA (SEQ ID NO: 20), encoding the heavy chain of factor VIII. The factor VIII polypeptide light chain is encoded by a second nucleotide sequence with high sequence identity to CS23-LC-NA (SEQ ID NO: 23), which is a portion of CS23-FL-NA (SEQ ID NO: 20) encoding the factor VIII light chain. The optional polypeptide linker does not contain a furin cleavage site.

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

In some embodiments, the codon altered polynucleotide has a nucleotide sequence with high sequence identity to CS23-SC1-NA (SEQ ID NO: 28). In some embodiments, the nucleotide sequence has at least 95% identity with CS23-SC1-NA (SEQ ID NO: 28). In some embodiments, the nucleotide sequence has at least 96% identity with CS23-SC1NA (SEQ ID NO: 28). In some embodiments, the nucleotide sequence has at least 97% identity with CS23-SC1-NA (SEQ ID NO: 28). In some embodiments, the nucleotide sequence has at least 98% identity with CS23-SC1-NA (SEQ ID NO: 28). In some embodiments, the nucleotide sequence has at least 99% identity with CS23-SC1-NA (SEQ ID NO: 28). In some embodiments, the nucleotide sequence has at least 99.5% identity with CS23-SC1-NA (SEQ ID NO: 28). In some embodiments, the nucleotide sequence has at least 99.9% identity with CS23-SC1-NA (SEQ ID NO: 28). In some embodiments, the nucleotide sequence is identical to CS23SC1-NA (SEQ ID NO: 28).

In some embodiments, the codon altered polynucleotide has a nucleotide sequence with high sequence identity to CS23-SC2-NA (SEQ ID NO: 29). In some embodiments, the nucleotide sequence has at least 95% identity with CS23-SC2-NA (SEQ ID NO: 29). In some embodiments, the nucleotide sequence has at least 96% identity with CS23-SC2NA (SEQ ID NO: 29). In some embodiments, the nucleotide sequence has at least 97% identity with CS23-SC2-NA (SEQ ID NO: 29). In some embodiments, the nucleotide sequence has at least 98% identity with CS23-SC2-NA (SEQ ID NO: 29). In some embodiments, the nucleotide sequence has at least 99% identity with CS23-SC2-NA (SEQ ID NO: 29). In some embodiments, the nucleotide sequence has at least 99.5% identity with CS23-SC2-NA (SEQ ID NO: 29). In some embodiments, the nucleotide sequence has at least 99.9% identity with CS23-SC2-NA (SEQ ID NO: 29). In some embodiments, the nucleotide sequence is identical to CS23SC2-NA (SEQ ID NO: 29).

In some embodiments, the single-strand variant of factor VIII encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to CS23-SC1-AA (SEQ ID NO: 10, human factor FVIIIA (7601667) (SPI; HsFVIIIA (741-1648) ), SPE)). In some embodiments, the factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high identity to CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 97% identity with CS23SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence

- 28 046432 has at least 98% identity with CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 99% identity with CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 99.5% identity with CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence has at least 99.9% identity with CS23-SC1-AA (SEQ ID NO: 10). In some embodiments, the amino acid sequence is identical to CS23-SC1-AA (SEQ ID NO: 10).

In some embodiments, the single-strand variant of factor VIII encoded by the codon-altered polynucleotide has an amino acid sequence with high sequence identity to CS23-SC2-AA (SEQ ID NO: 12, human factor VIIIA (772-1667) (SPI; HsFVIIIA (753 -1648), SPE)). In some embodiments, the factor VIII variant encoded by the codon-altered polynucleotide has an amino acid sequence with high identity to CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 97% identity with CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 98% identity with CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99% identity with CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99.5% identity with CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence has at least 99.9% identity with CS23-SC2-AA (SEQ ID NO: 12). In some embodiments, the amino acid sequence is identical to CS23-SC2-AA (SEQ ID NO: 12).

Factor VIII expression vectors.

In some embodiments, the codon altered polynucleotides described herein are integrated into expression vectors.

Non-limiting examples of expression vectors include viral vectors (eg, vectors suitable for gene therapy), plasmid vectors, bacteriophage vectors, cosmids, phasmids, artificial chromosomes, and the like.

Non-limiting examples of viral vectors include a retrovirus, such as Moloney murine leukemia virus (MMLV), Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; viruses like SV40; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; and polio viruses.

In some embodiments, the codon altered polynucleotides described herein are integrated into gene therapy vectors. In some embodiments, the gene therapy vector is a retrovirus and, in particular, a replication-defective 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).

In one embodiment of the invention, 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. Ther., 20(4): 699-708 (2012), which are incorporated herein by reference in their entirety for all purposes). Details regarding the generation and use of rAAV vectors are described, for example, in US Patent Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference in its entirety for all purposes. In a specific embodiment of the invention, the AAV vector is an AAV-8 vector.

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, 272 (5259):263-267, 1996; Zufferey et al., Nat Biotechnol, 15(9):871- 875, 1997; Blomer et al., J Virol., 71(9):6641-6649, 1997; US Patent Nos. 6,013,516 and 5,994,136).

A wide variety of vectors can be used to express factor VIII polypeptide from a codon-altered polypeptide in cell culture, including eukaryotic and prokaryotic expression vectors. In some embodiments of the invention it is assumed that

- 29 046432 that the plasmid vector is used to express factor VIII polypeptide in cell culture. In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts. The vector may contain a replication site, as well as marking sequences that are capable of providing phenotypic selection in transformed cells. The plasmid will contain a codon-altered polynucleotide encoding a factor VIII polypeptide operably linked to one or more control sequences, such as a promoter.

Non-limiting examples of prokaryotic expression vectors include plasmids such as pRSET, pET, pBAD, etc., where the promoters used in the prokaryotic expression vectors contain lac, trc, trp, recA, araBAD, etc. Examples of eukaryotic expression vectors include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET using promoters such as AOX1, GAP, GAL1, AUG1, etc.; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc. . and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., 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 β-actin.

Examples

Example 1. Construction of an expression sequence with modified Factor VIII variant codons.

Two obstacles had to be overcome to create a factor VIII coding sequence that is effective for gene therapy for hemophilia A. First, due to the genome size limitations of conventional gene therapy delivery vectors (eg, AAV virions), the polypeptide coding for factor VIII must be significantly shortened. Second, the coding sequence needed to be modified to: (i) stabilize packaging interactions within the delivery vector, (ii) stabilize the intermediate mRNA, and (iii) improve mRNA transcription/translation stability.

To achieve the first goal, applicants began with the design of a B-domain deleted variant of Factor VIII, referred to herein as FVIII-BDD-SQ. In this design, the B domain is replaced with a 14 amino acid sequence called the SQ sequence. Recombinant FVIII-BDD-SQ is sold under the trade name REFACTO® and has been shown to be effective in the treatment of hemophilia A. However, the native coding sequence for FVIII-BDD-SQ, which contains the wild-type human nucleotide sequence for the factor VIII heavy and light chains, inefficiently expressed in gene therapy vectors.

To enhance the weak expression of native FVIII-BDD-SQ, the codon optimization algorithm described in Fath et al. (PLoS ONE, 6:e17596 (2011)), modified as described in Ward et al. (Blood, 117:798 (2011)) and McIntosh et al. (Blood, 121, 3335-3344 (2013)) was applied to the FVIIIBDD-SQ sequence to create the first intermediate coding sequence CS04a. However, the applicants acknowledged that the CS04a sequence generated using the modified algorithm could be improved by further modification of the sequence. Accordingly, applicants reintroduced CpG dinucleotides, reintroduced the CGC codon for arginine, changed the leucine and serine codon distributions, reintroduced highly conserved codon pairs, and removed the cryptic TATA box, CCAAT box, and splice site elements while avoiding CpG islands and local predominance of AT and GC-rich areas.

First, the modified algorithm systematically replaces codons containing CpG dinucleotides (eg, arginine codons) with non-CpG dinucleotide codons, and eliminates/avoids CpG dinucleotides generated by adjacent codons. This strict avoidance of CpG dinucleotides is usually done to prevent TLR-induced immunity after intramuscular injection of DNA vaccines. However, this limits the possibilities for codon optimization. For example, the modified algorithm eliminates the use of the full set of arginine CGX codons. It is particularly disruptive in encoding genes for expression in human cells, since CGC is the most commonly used arginine codon in highly expressed human genes. In addition, preventing neighboring codons from creating CpGs further limits optimization opportunities (eg, limiting the number of codon pairs that can be used together).

Since TLR-induced immunity is not expected to be a problem associated with hepatic AAV-based gene therapy, CpG-containing codons and adjacent CpG-forming codons were reintroduced into the intermediate coding sequence of CS04a, preferably into the factor VIII light chain coding sequence (eg at the 3' end of the FVIII-BDD-SQ coding sequence). This allowed for increased use of human preferred codons, especially for arginine. However, care has been taken to

- 30 046432 avoid the creation of CpG islands, which are regions of the coding sequence that have a high frequency of CpG sites. This is contrary to the teachings of Krinner et al. (Nucleic Acids Res., 42 (6): 3551-64 (2014)), which suggests that CpG domains downstream of transcription start sites contribute to high levels of gene expression.

Second, the modified algorithm exclusively uses codons such as CTG for leucine, GTG for valine, and CAG for glutamine. However, this violates the principles of codon balancing, for example, as proposed in Haas et al. (Current Biology, 6(3):315-24 (1996)). To account for overuse of preferred codons by the modified algorithm, alternative leucine codons were reintroduced if allowed by other rules applied to codon variation (e.g., CpG frequency and GC content).

Third, the modified algorithm replaces codon pairs no matter how conserved they are when certain conditions are met (for example, the presence of CG dinucleotides). To account for beneficial properties that may have been conserved by evolution, the most conserved codon pairs that were replaced by the algorithm and the most conserved preferred codon pairs, for example, as described in Tats et al. (BMC Genomics 9:463 (2008)) were analyzed and adjusted where allowed by other rules applying to codon changes (eg, CpG frequency and GC content).

Fourth, the serine codons used in the intermediate coding sequence were also re-modified. In particular, the serine codons AGC, TCC and TCT were introduced into the modified coding sequence at higher frequency to better match the natural human codon distribution (Haas et al., supra).

Fifth, TATA box elements, CCAAT box elements, and intron/exon spline sites were identified and removed from the modified coding sequence. When modifying the coding sequence, care was taken to avoid local over-increase in the occurrence of AT- or GC-rich regions.

Finally, in addition to optimizing codon usage in the coding sequence, the structural requirements for the AAV virion were considered by further modifying the intermediate coding sequence CS04a. AAV vectors (eg, the nucleic acid portion of the AAV virion) are packaged as single-stranded DNA molecules in their capsids (for review, see Daya and Berns, Clin. Microbiol Rev., 21(4):583-93 (2008)). Therefore, the GC content of the vector is likely to influence genome packaging and therefore vector yield during production. Like many algorithms, the modified algorithm used here produces an optimized gene sequence with at least 60% GC content (see Fath et al., PLoS One, 6 (3): e17596 (2011) (erratum in: PLoS One , (6) 3 (2011). However, the AAV8 capsid protein is encoded by a nucleotide sequence that has a lower GC content of about 56%. Thus, to better mimic the natural coding sequence of the AAV8 capsid protein, the GC content of the intermediate coding sequence CS04a was reduced to 56%.

The resulting CS04 coding sequence shown in FIG. 2, has a total GC content of 56%.

The content of CpG dinucleotide in the sequence is moderate. However, CpG dinucleotides are predominantly present in the downstream portion of the coding sequence, for example, the portion encoding the factor VIII light chain. The CS04 sequence has 79.77% nucleotide sequence identity with the corresponding wild-type factor VIII coding sequences (Genbank accession number M14113).

Several other codon optimized ReFacto constructs were prepared for comparison. CS01 was designed by applying the codon optimization algorithm of Fath et al., modified according to Ward et al., as done for CS04. However, unlike CS04, the CS01 construct does not contain any CpG islands. The CS08 ReFacto construct was subjected to codon optimization as described in Radcliff P.M. et al., Gene Therapy, 15: 289-97 (2008), the contents of which are incorporated herein by reference in their entirety for all purposes. The codon-optimized ReFacto construct based on CS10 codons was obtained from Eurofins Genomics (Ebersberg, Germany). The codon optimized CS11 ReFacto construct was obtained from Integrated DNA Technologies, Inc. (Coralville, USA). The codon optimized CH25 ReFacto construct was obtained from ThermoFischer Scientific's GeneArt services (Regensburg, Germany). The CS4 0 ReFacto construct consists of the wild-type factor VIII coding sequence. The algorithm used to construct CS23 is based on the JCAT tool (www.jcat.de), an online tool for codon optimization (Grote et al., 2005; Nucl. Acids Res. W526-31). The sequence was further modified to be more consistent with the use of albumin superfamily codons (Mirsafian et al., 2014: Sc. Word Journal 2014, ID 639682). Sequence identifiers common to each of the ReFacto coding sequences are shown in Table 1. 2 below.

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table 2

Percentage identity matrix for factor VIII constructs with altered codons CSO1 CS04 CS08 CS1O CS11 CS40 CH25 CS23 CSO1 100% CS04 93.0% 100% CS08 80.7% 82.2.% 100% CS1O 79.1% 79.4% 78.4% 100% CS11 78.3% 78.3% 78.1% 77.5% 100% CS40 79.6% 79.8% 76.7% 77.6% 75.4% 100% CH25 81.3% 85.1% 85.0% 79.9% 79.4% 75.8% 100% CS23 84.3% 89.2% 85.1% 80.3% 79.9 76.5% 93.2% 100%

The plasmids of each construct were constructed by cloning different synthetic DNA fragments into the same vector master plasmid (pCh-BB01). Synthesis of BDD-FVIII Refacto-type DNA fragments with flanking AscI and NotI restriction enzyme sites was performed using ThermoFischer Scientific (Regensburg, Germany). The vector backbone contains two flanking AAV2 inverted terminal repeats (ITRs) that contain a promoter/enhancer sequence derived from murine liver transthyretin, AscI and NotI enzyme restriction sites for insertion of the corresponding Refacto type BDD-FVIII, and a synthetic polyA site. After ligation of the prepared vector backbone and inserts with AscI and NotI sites, the resulting plasmids were amplified on a milligram scale. The sequences of the Refacto type BDD-FVIII constructs were confirmed by direct sequencing (Microsynth, Balgach, Switzerland). Cloning resulted in the creation of seven different plasmid constructs named pCS40, pCS0l, pCS04, pCS08, pCS10, pCS11, pCh25 and pCS23 (Fig. 19). The constructs have the same vector backbone and encode the same FVIII protein with the B domain missing (Refacto-type BDD-FVIII), but differ in their FVIII coding sequence.

AAV8 vectors were generated by three plasmid transfection methods as described in Grieger J.C., et al. (Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector, Mol Ther., Oct 6. (2015) doi: 10.1038/mt.2015.187. [Epub ahead of print]), whose the contents are incorporated herein by reference in their entirety for all purposes. Suspended HEK293 cells were used for plasmid transfection using the corresponding FVIII vector plasmid, helper plasmid pXX6-80 (carrying adenoviral helper genes) and packaging plasmid pGSK2/8 (containing the rep2 and cap8 genes). To isolate AAV8 constructs, cell pellets from one liter of culture were treated using iodixanol gradients as described in Grieger et al. (2015, Supra). The procedure resulted in vector drugs called vCS01, vCS04, vCS08, vCS10, vCS11 and vCH25. Vectors were quantified by qPCR using the generic qPCR procedure using AAV2 inverted terminal repeats (Aurnhammer, Human Gene Therapy Methods: Part B 23:18-28 (2012)). A control vector plasmid carrying AAV2 inverted terminal repeats was used to generate a standard curve. The resulting VCS04 design is shown as SEQ ID NO: 8 in FIG. 7A-7C.

The integrity of the vector genomes was analyzed by AAV agarose gel electrophoresis. Electrophoresis was performed as described in Fagone et al., Human Gene Therapy Methods 23:1-7 (2012). Briefly, AAV vector preparations were incubated at 75°C for 10 min in the presence of 0.5% SDS and then cooled to room temperature. Approximately 1.5E10 vector genomes (vg) were loaded onto a 1% 1xTAE agarose gel lane and electrophoresed for 60 min at an electric field strength of 7 V/cm of gel length. The gel was then stained with 2x GelRed solution (Biotium Cat #41003) and visualized with ChemiDocTMMP (Biorad). The results shown in FIG. 20 show that the viral vectors vCS0l, vCS04 and vCS40 have a genome of the same size, indicated by a distinct band in the 5 kb range (Fig. 20, lanes 2-4). Despite the vector size of about 5.2 kb, the genome appears as a uniform stripe, confirming the correct packaging of the somewhat oversized genome (relative to the wild-type AAV genome of 4.7 kb). All other vCS vectors exhibit the same genomic size (data not shown).

To confirm the expected pattern of capsid proteins, SDS PAGE followed by silver staining was performed with vectors vCS01, vCS04 and vCS40 (Fig. 21). As shown in the drawing, a further purification procedure resulted in a highly purified preparation exhibiting

- 32 046432 expected pattern of proteins VP1, VP2 and VP3 (Fig. 21, lanes 2-4). The same pattern was obtained for all other viral preparations (not shown). The SDS-PAGE procedure of AAV preparations was performed according to standard protocols. Each lane contained 1E10 vg of the corresponding viral construct and was separated on a 4-12% Bis-Tris gel (NuPAGE® Novex, Life Technologies) according to the manufacturer's instructions. Silver staining was performed using the SilverQuestTM kit (Novex, Life Technologies) according to the manufacturer's instructions.

Surprisingly, the AAV vectors vCS01 and VCS04 had denser virion packaging, as measured by higher yields in AAV virus production compared to the wild-type VCS40 coding construct and other codon-optimized constructs. As shown in table. 3, vectors vCS01 and vCS04 replicate significantly better than VCS40, resulting in a 5- to 7-fold increase in AAV yield.

Table 3

Yield per 1 liter of cell culture obtained using AAV vector constructs vCS01, vCS04 and vCD40 when purified from cell sediment

Design Vector concentration [vg/ml] X10E12 Output [vg/l] X10E12 Magnification factor relative to wt VCS4 0 2.0 11.0 - VCS01 9.2 51.4 4.7 VCS04 - Sample 1 17.6 79.2 7.2 VCS04 - Sample 2 15.9 58.8 5.4

Example 2. In Vivo Expression of Codon-Altered Factor VIII Expression Sequence Variants.

To test the biological activity of codon-altered factor VIII sequence variants, the ReFacto type FVIII constructs described in Example 1 were administered to factor VIII null mice. Briefly, assays were performed using C57B1/6 FVIII knockout mice (na) (68 animals per group) by tail vein injection of 4E12 vector genomes (vg) per kilogram of mouse body weight. Blood was collected 14 days after injection by retroorbital puncture, and plasma was prepared and frozen using standard procedures. Expression levels were measured on day 14 due to the minimal effect of inhibitory antibodies at this time, which is observed in some animals in this mouse model at later times. FVIII activity in mouse plasma was determined using the Technochrome FVIII assay with minor modifications as suggested by the manufacturer (Technoclone, Vienna, Austria). For analysis, plasma samples were appropriately diluted and mixed with analytical reagents containing thrombin, activated factor IX (FIXa), phospholipids, factor X and calcium. After activation of FVIII by thrombin, a complex is formed with FIXa, phospholipids and calcium. This complex activates FX resulting in the formation of active FX (FXa), which in turn cleaves para-nitroanilide (pNA) from the chromogenic substrate. The kinetics of pNA formation is measured at 405 nm. The rate is directly proportional to the concentration of FVIII in the sample. FVIII concentrations are read from the control curve and the results are reported as IU FVIII/milliliter.

The results presented in table. 4 below demonstrate that the codon-altered sequences designed using commercial algorithms (CS10, CS11 and CH25) provided only a small increase in BDD factor VIII (3- to 4-fold) compared to the wild-type BDD factor VIII construct (CS40). . Similarly, a codon-altered BDD factor VIII construct prepared as described in Radcliffe et al. (CS08) provided only a 3- to 4-fold increase in BDD-FVIII expression. This result is consistent with the results obtained in Radcliff et al. Surprisingly, constructs CS01, CS04, and CS23 provided significantly higher expression of BDD-FVIII in in vivo activity assays (18-, 74-, and 30-fold, respectively).

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Table 4

FVIII expression in plasma of FVIII knockout mice induced by different AAV vector constructs

Design Codon Algorithm Average FVIII Expression Level per Day 14 [IU/ml] Standard deviation Quantity mice Magnification factor relative to wt VCS40 Human, wild type 0.03 0.03 12 - vCSOl Way Applicants 0.55 0.28 22 18.3 VCS04 Way Applicants 2.21 1.20 55 73.7 VCS0 8 Radcliffe et al. 0.11 0.01 6 3.6 vCSIO Eurofins 0.09 0.01 7 3.0 vCSll IDT 0.08 0.02 8 2.7 vCH2 5 GeneArt 0.13 0.12 18 4.3 vCS23 Way Applicants 0.91 0.32 5 30.3

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

Claims (11)

1. A polynucleotide comprising a nucleotide sequence having at least 95% identity with SEQ ID NO: 13, wherein the polynucleotide encodes a factor VIII polypeptide. 2. The polynucleotide according to claim 1, wherein the nucleotide sequence has at least 99% identity with SEQ ID NO: 13. 3. The polynucleotide according to claim 1, wherein the nucleotide sequence is SEQ ID NO: 13. 4. A polynucleotide comprising a nucleotide sequence encoding a factor VIII polypeptide, wherein the factor VIII polypeptide comprises a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain, wherein the heavy chain of the factor VIII polypeptide is encoded by the first nucleotide sequence having at least 98% identity with SEQ ID NO: 24; the light chain of the factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 98% identity with SEQ ID NO: 25; and the polypeptide linker contains a furin cleavage site and is encoded by the third nucleotide sequence of SEQ ID NO: 5. 5. An adeno-associated virus (AAV) vector containing a polynucleotide according to any one of claims 1-4. 6. An adeno-associated virus (AAV) particle containing a polynucleotide according to any one of claims 1 to 4. 7. A host cell infected with an adeno-associated virus (AAV) particle containing a polynucleotide according to any one of claims 1 to 4. 8. A method for producing an adeno-associated virus (AAV) particle, comprising introducing a polynucleotide according to any one of claims 1-4 into a plasmid vector and transfecting a mammalian host cell with the plasmid vector, wherein the polynucleotide is competent for replication in the mammalian host cell. 9. A method for treating hemophilia A, including administering to the patient an adeno-associated virus (AAV) particle according to claim 6. - 34 046432 10. A method for transducing a host cell, comprising contacting the host cell with an adeno-associated virus (AAV) particle according to claim 6. 11. Use of an adeno-associated virus (AAV) particle according to claim 6 for the production of a medicinal product for the treatment of hemophilia A.