RU2773956C2 - Viral vectors encoding recombinant fix variants with increased expression for gene therapy of hemophilia b - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to polynucleotide encoding factor IX protein; it can be used in medicine. Polynucleotide contains a nucleic acid sequence that is at least 99% identical to a nucleic acid sequence SEQ ID NO: 17.

EFFECT: obtained polynucleotide can be used in the composition of a vector for effective gene therapy of hemophilia B in mammals.

26 cl, 25 dwg, 7 tbl, 6 ex

Description

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/509,616, filed May 22, 2017, which is hereby incorporated by reference in its entirety for all purposes.

LINK TO "SEQUENCE LIST", TABLE OR COMPUTER PROGRAM, APPENDIX WITH THE LIST IS PROVIDED ON THE CD

[0002] This disclosure incorporates by reference herein a copy of the text of the Sequence Listing created on May 21, 2018 and titled 008073_5117_WO_Sequence_Listing.txt, which is 73 kilobytes in size.

BACKGROUND OF THE INVENTION

[0003] Blood coagulation occurs through a complex and dynamic biological pathway of interdependent biochemical reactions called the coagulation cascade. Coagulation factor VIII (FVIII) and factor IX (FIX) are key components in the cascade. Factor VIII is recruited to bleeding sites and forms a Xase complex with activated factor IX and factor X (FX). The Xase 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 clot (reviewed by Saenko et al ., Trends Cardiovasc. Med ., 9:185-192 (1999); Lenting et al ., Blood , 92:3983-3996 (1998)).

[0004] Hemophilia B is a congenital X-linked bleeding disorder characterized by a deficiency in factor IX activity. Generally, decreased factor VIII/factor IX activity inhibits the positive feedback loop in the coagulation cascade. This results in incomplete coagulation, which manifests as bleeding episodes of prolonged duration, extensive bruising, spontaneous oral and epistaxis 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)).

[0005] Hemophilia B is usually treated with factor IX replacement therapy, which consists of administering a factor IX protein (e.g., factor IX derived from plasma or recombinantly produced) to a person with hemophilia B. Factor IX is administered prophylactically to prevent or reduce the frequency of bleeding episodes, in response to an acute bleeding episode, and/or perioperatively to control bleeding during surgery. However, there are several undesirable features of factor IX replacement therapy.

[0006] First, factor IX replacement therapy is used to treat or control hemophilia B, but does not treat the underlying factor IX deficiency. Because of this, people with hemophilia B need factor IX replacement therapy throughout their lives. Continuous treatment is costly and requires the individual to maintain a strict regimen, as skipping even just a few prophylactic doses can have serious consequences for individuals with severe hemophilia B.

[0007] Second, because conventional factor IX products have a relatively short in vivo half-life of about 24 hours, prophylactic factor IX replacement therapy requires administration two or three times a week. Compliance with such a regimen throughout life can be burdensome for an individual. Third-generation long-acting factor IX products may reduce the frequency of administration, but prophylactic factor FIX replacement therapy with these drugs still requires monthly, weekly, or more frequent use at all times. For example, prophylactic treatment with beta-pegol Nonacog [pegylated recombinant factor IX] (Novo Nordisk, pending US and EF regulatory approval) still requires weekly dosing (Collins PW, et al., Blood, 124(26):3880-86 (2014)). Moreover, the long-term effects of chemically modified biologics (eg, pegylated polypeptides) are not yet fully understood.

[0008] Third, up to 5% of patients with severe hemophilia B develop antibodies that inhibit factor IX in response to factor IX replacement therapy, rendering the therapy ineffective (Osooli and Berntorp, J. Intern. Med., 277(1 ):1-15 (2015)). Unlike factor VIII bypass therapy, which can be used to treat patients with hemophilia A who develop antibodies that inhibit factor VIII, factor IX bypass therapy does not exist for the treatment of hemophilia B.

[0009] Fourth, factor IX replacement therapy is expensive, ranging from US$1,000 to US$3,000 per dose, depending on the weight of the patient (available online from the Hemophilia Federation of America). Thus, at twice-weekly dosing, factor IX replacement therapy can cost up to $300,000 per year.

[0010] Gene therapy has great promise in the treatment of hemophilia B because it will eliminate the underexpression of the functional activity of factor IX (for example, due to a missense or nonsense mutation) that causes conditions, and will not provide the individual with a single dose of activity factor IX. Because of the difference in mechanism of factor IX delivery compared to factor IX replacement therapy, a single administration of a factor IX gene therapy vector can provide an individual with sufficient levels of factor IX for several years, if not longer. This reduces the cost of treatment and eliminates the need for constant patient compliance.

[0011] A proof of concept for factor IX gene therapy in hemophilia B has been presented. See, for example, Manno C.S., et al., Nat Med., 12(3):342-47 (2006). However, questions remain as to whether therapeutically effective amounts of factor IX can be expressed for sufficient periods of time. See, for example, Giangrande, Semin Thromb Hemost. 42(5):513-17 (2016).

[0012] Several attempts have been made to construct a codon-optimized factor IX. For example, WO 2006/036502 describes a codon-optimized factor IX gene therapy AAV vector with the HCR-1 ApoE enhancer and the alpha-1 antitrypsin (AAT) promoter. Similarly, WO 2014/064277 and WO 2016/146757 disclose codon-optimized factor VIII and factor IX AAV gene therapy vectors that include one or more copies of the liver-specific regulatory element SERPIN. Finally, WO 2016/210170 describes codon-optimized factor IX gene therapy AAV vectors with the HCR-1 ApoE enhancer and the alpha-1 antitrypsin (AAT) promoter.

SUMMARY OF THE DISCLOSURE

[0013] Accordingly, there is a need for improved factor IX gene therapy constructs. For example, there is a need for synthetic codon-altered nucleic acids encoding factor IX that are more efficiently packaged and delivered by gene therapy vectors. There is also a need for synthetic codon-altered nucleic acids that express factor IX more efficiently. There is also a need for codon-altered nucleic acids encoding factor IX polypeptides with improved folding properties, improved secretion from expressing cells, and/or increased activity compared to wild-type factor IX. Such factor IX-encoding codon-altered nucleic acids allow for improved treatment of factor IX deficiency (eg, hemophilia B). The above deficiencies and other problems associated with the treatment of factor IX deficiency (eg, hemophilia B) are reduced or abolished by the disclosed codon-altered nucleic acids encoding factor IX proteins.

[0014] In one aspect, nucleic acid compositions (eg, codon-altered polynucleotides) encoding factor IX and factor IX variants are described. Nucleic acid compositions include polynucleotides of high sequence identity with sequences CS02, CS03, CS04, CS05 and CS06 encoding Factor IX as described herein. The nucleic acid compositions described herein provide increased factor IX expression relative to wild-type factor IX coding sequences. Nucleic acid compositions also allow for increased production of AAV-based gene therapy virions. In some embodiments, the nucleic acid compositions described herein have reduced GC content and/or contain fewer CpG dinucleotides compared to factor IX-encoding wild-type sequences.

[0015] In some embodiments, the nucleic acid composition comprises a polynucleotide encoding factor IX that has a nucleotide sequence with at least 95% sequence identity (e.g., at least 95% sequence identity, 96%, 97%, 98%, 99 % or 100%) relative to the disclosed sequence selected from CS02-FL-NA (SEQ ID NO: 5), CS02-MP-NA (SEQ ID NO: 13), CS03-FL-NA (SEQ ID NO: 6), CS03-MP-NA (SEQ ID NO: 14), CS04-FL-NA (SEQ ID NO: 7), CS04-MP-NA (SEQ ID NO: 15), CS05-FL-NA (SEQ ID NO: 8 ), CS05 -MP-NA (SEQ ID NO: 16), CS06-FL-NA (SEQ ID NO: 9) and CS06-MP-NA (SEQ ID NO: 17).

[0016] In some embodiments, the nucleic acid composition comprises a polynucleotide encoding factor IX that has a nucleotide sequence with at least 95% sequence identity (e.g., at least 95% sequence identity, 96%, 97%, 98%, 99% or 100%) relative to the disclosed factor IX light chain encoding sequence (e.g., CS02-LC-NA (SEQ ID NO: 42), CS03-LC-NA (SEQ ID NO: 44), CS04-LC-NA ( SEQ ID NO: 46), CS05-LC-NA (SEQ ID NO: 48) or CS06-LC-NA (SEQ ID NO: 50)), and the disclosed factor IX heavy chain encoding sequence (e.g., CS02-HC- NA (SEQ ID NO: 41), CS03-HC-NA (SEQ ID NO: 43), CS04-HC-NA (SEQ ID NO: 45), CS05-HC-NA (SEQ ID NO: 47) or CS06- HC-HA (SEQ ID NO: 49)).

[0017] In some embodiments, the nucleic acid composition comprises a polynucleotide encoding a Factor IX polypeptide having a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the light chain to the N-terminus of the heavy chain (e.g., an activation peptide). The factor IX polypeptide light chain is encoded by a first nucleotide sequence with high sequence identity to one of CS02-LC-NA (SEQ ID NO: 42), CS03-LC-NA (SEQ ID NO: 44), CS04-LC-NA (SEQ ID NO: 46), CS05-LC-NA (SEQ ID NO: 48) and CS06-LC-NA (SEQ ID NO: 50). The factor IX polypeptide heavy chain is encoded by a second nucleotide sequence with high sequence identity to one of CS02-HC-NA (SEQ ID NO: 41), CS03-HC-NA (SEQ ID NO: 43), CS04-HC-NA (SEQ ID NO: 45), CS05-HC-NA (SEQ ID NO: 47) and CS06-HC-NA (SEQ ID NO: 49). The polypeptide linker contains a protease cleavage site (eg, two factor XIa cleavage sites).

[0018] In some embodiments of the polynucleotides described above, the polypeptide linker has an amino acid sequence with high sequence identity to wild-type factor IX activation peptide FIX-AP-AA (SEQ ID NO: 56; amino acids 192-226 of FIX-FL-AA (SEQ ID NO: 2)). In some embodiments, the polypeptide linker is encoded by a third nucleic acid sequence having high sequence identity with one of CS02-AP-NA (SEQ ID NO: 57), CS03-AP-NA (SEQ ID NO: 58), CS04-AP-NA (SEQ ID NO: 59), CS05-AP-NA (SEQ ID NO: 60) and CS06-AP-NA (SEQ ID NO: 61).

[0019] In some embodiments, the codon-altered polynucleotides described herein encode a factor IX prepropolypeptide, for example, wherein the encoded factor IX protein contains a signal peptide and a propeptide. In some embodiments, the signal peptide, the propeptide, or both the signal peptide and the propeptide are encoded by a codon-altered sequence. In some embodiments, the signal peptide, the propeptide, or both the signal peptide and the propeptide are encoded by the wild-type sequence, while the portion of the nucleic acid encoding the mature single-stranded factor IX polypeptide (e.g., FIX-MP-AA (SEQ ID NO: 10); amino acids 47- 461 of FIX-FL-AA (SEQ ID NO: 2)) are codon-altered.

[0020] In some embodiments, the codon-altered polynucleotides described herein encode a variant factor IX polypeptide, e.g., having one or more amino acid substitutions relative to the wild-type factor IX amino acid sequence (e.g., FIX-FL-AA (SEQ ID NO : 2) or FIX-MP-AA (SEQ ID NO: 10)). In some embodiments, the factor IX variant is a hyperactive factor IX variant with increased factor IX activity relative to wild-type factor IX. In a specific embodiment, the encoded Factor IX polypeptide has the amino acid substitution R384L, ie. variant "Padua" (relative to the prepropolypipeptide sequence of factor IX FIX-FL-AA (SEQ ID NO: 2), amino acid substitution R338L relative to the mature single-stranded sequence of factor IX FIX-MP-AA (SEQ ID NO: 10)).

[0021] In one aspect, methods for treating hemophilia B are described. The methods include administering to a patient in need thereof a nucleic acid composition (e.g., a codon-altered factor IX polynucleotide construct) described herein (e.g., a polynucleotide having high sequence identity with factor IX coding sequence CS02, CS03, CS04, CS05 or CS06). In some embodiments, the factor IX polynucleotide construct is a mammalian gene therapy vector as described herein. In a specific embodiment, the factor IX polynucleotide construct is an adeno-associated virus (AAV) vector. In some embodiments, the gene therapy vector includes one or more copies of a liver-specific regulatory control element (eg, 1 to 3 copies of the CRM8 regulatory control element).

[0022] In one aspect, methods for producing adeno-associated virus (AAV) particles are described. The method comprises introducing a nucleic acid composition (e.g., a codon-altered Factor IX polynucleotide construct) described herein (e.g., a polynucleotide having high sequence identity with a factor IX coding sequence of CS02, CS03, CS04, CS05, or CS06) into a host cell a mammal, in which the polynucleotide is capable of replication in a mammalian host cell.

BRIEF DESCRIPTION OF GRAPHICS

[0023] Figure 1 shows exemplary factor IX gene therapy constructs in accordance with some embodiments. Sequence elements for self-complementary (A, B) and single-stranded (C, D) vectors are shown without (A, C) and with (B, D) liver-specific cis-regulatory modules (CRM8).

[0024] In FIG. 2 shows the coding sequence for wild-type factor IX (SEQ ID NO: 1) for accession number CCDS14666.1 ("FIX-FL-NA").

[0025] FIG. 3 shows the amino acid sequences of two wild type factor IX prepropolypeptide isoforms expressed in humans. In FIG. 3A shows the wild-type amino acid sequence of the first longer Factor IX prepropolypeptide isoform (SEQ ID NO: 2) corresponding to UniProt Accession P00740 and NCBI Accession NP_000124.1 ("FIX-FL-AA"). In FIG. 3B shows the wild-type amino acid sequence for the second shorter factor IX prepropolypeptide isoform (SEQ ID NO: 3) corresponding to NCBI accession number NP_001300842.1 ("FIX2-FL-AA").

[0026] FIG. 4 shows the amino acid sequence of the Padova variant (R384L) factor IX (SEQ ID NO: 4; "FIXp-FL-AA").

[0027] In FIG. 5 shows the codon-altered CS02 nucleotide sequence (SEQ ID NO: 5) encoding the factor IX variant with the amino acid substitution R384L (CS02-FL-NA), according to some embodiments.

[0028] FIG. 6 shows the codon-altered nucleotide sequence of CS03 (SEQ ID NO: 6) encoding the factor IX variant with the amino acid substitution R384L (CS03-FL-NA), according to some embodiments.

[0029] FIG. 7 shows the codon-altered nucleotide sequence of CS04 (SEQ ID NO: 7) encoding the factor IX variant with the amino acid substitution R384L (CS04-FL-NA), according to some embodiments.

[0030] FIG. 8 shows the codon-altered nucleotide sequence of CS05 (SEQ ID NO: 8) encoding the factor IX variant with the amino acid substitution R384L (CS05-FL-NA), according to some embodiments.

[0031] In FIG. 9 shows the codon-altered nucleotide sequence of CS06 (SEQ ID NO: 9) encoding the factor IX variant with the amino acid substitution R384L (CS06-FL-NA), according to some embodiments.

[0032] FIG. 10 illustrates FIX antigen levels in wild-type mice injected with a CS02 gene therapy construct having 0, 1, 2, or 3 copies of the liver-specific cis-regulatory control element CRM8 at a dose of 2×10E11 µg/kg body weight.

[0033] FIG. 11 shows the amino acid sequences of two single-stranded wild-type factor IX protein isoforms (eg, lacking signal and propeptides) expressed in humans. In FIG. 11A shows the wild type amino acid sequence of the first longer factor IX prepropolypeptide isoform (SEQ ID NO: 10) corresponding to UniProt accession number P00740 and NCBI accession number NP_000124.1 ("FIX-MA-AA"). In FIG. 11B shows the wild-type amino acid sequence for the second shorter factor IX prepropolypeptide isoform (SEQ ID NO: 11) corresponding to NCBI accession number NP_001300842.1 ("FIX2-MA-AA").

[0034] FIG. 12 shows the single strand amino acid sequence of factor IX, Padua variant (R338L) (SEQ ID NO: 12; "FIXp-MP-AA").

[0035] FIG. 13 shows the codon-altered nucleotide sequence of CS02 (SEQ ID NO: 13) encoding a single chain variant of factor IX with the amino acid substitution R338L (CS02-MP-NA), according to some embodiments.

[0036] FIG. 14 shows the codon-altered nucleotide sequence of CS03 (SEQ ID NO: 14) encoding a single chain variant of factor IX with the amino acid substitution R338L (CS03-MP-NA), according to some embodiments.

[0037] FIG. 15 shows the codon-altered nucleotide sequence of CS04 (SEQ ID NO: 15) encoding a single-stranded factor IX variant with the amino acid substitution R338L (CS04-MP-NA), according to some embodiments.

[0038] FIG. 16 shows the codon-altered nucleotide sequence of CS05 (SEQ ID NO: 16) encoding a single-stranded factor IX variant with the amino acid substitution R338L (CS05-MP-NA), according to some embodiments.

[0039] FIG. 17 shows the codon-altered nucleotide sequence of CS06 (SEQ ID NO: 17) encoding a single-stranded factor IX variant with the amino acid substitution R338L (CS06-MP-NA), according to some embodiments.

[0040] FIG. 18 shows nucleic acid (NA) sequences encoding a prepropeptide (PPP) of a number of constructs described herein, in accordance with some embodiments.

[0041] In FIG. 19 shows nucleic acid (NA) sequences encoding a signal peptide (SP) for a number of constructs described herein, in accordance with some embodiments.

[0042] FIG. 20 shows nucleic acid (NA) sequences encoding a propeptide (PP) for a number of constructs described herein, in accordance with some embodiments.

[0043] FIG. 21 shows the amino acid sequence (AA) of the FIX prepropeptide (PPP).

[0044] FIG. 22 shows the amino acid sequence (AA) of the FIX signal peptide (SP).

[0045] FIG. 23 shows the amino acid sequence (AA) of the FIX propeptide (PP).

[0046] FIG. 24 shows the nucleic acid sequence of the CRM8 sequence (SEQ ID NO: 39).

[0047] FIG. 25A and B show the nucleic acid sequence of construct CS06-CRM8.3-ssV (SEQ ID NO:40).

DETAILED DESCRIPTION OF THE DISCLOSURE

Introduction

[0048] AAV-based gene therapy holds great promise for the treatment of hemophilia. With regard to hemophilia B, early clinical data are encouraging, as FIX levels of around 10% can be maintained in at least some patients for more than 1 year. For example, initial human studies have shown that hepatic artery catherization with AVV-FIX constructs resulted in transient expression of factor IX in vivo . Kay M. et al., Nat Genet. 24(3):257-61 (2000). However, transduction resulted in moderate activation of the immune system against AAV-derived capsid antigens. Manno CS et al., Nat Med. 12(3):342-47 (2006) and Mingozzi F. et al., Nat Med. 13(4):419-22 (2007).

[0049] Non-viral vectors may be less immunogenic because they rely on the delivery of deproteinized DNA or DNA associated with non-antigenic carriers (eg, inert polymers, lipids, or nanoparticles). However, the cellular transfection rates of non-viral vectors are lower than those of viral delivery vectors. In addition, long-term expression from non-viral vectors is hindered by the presence of bacterial sequences used for large-scale production of constructs.

[0050] However, these problems cannot be solved by simply introducing higher doses of the construct during gene therapy. According to current knowledge, the vector dose of the AAV-based gene therapy vector should not exceed 2 × 10 ¹² μg/kg of body weight. This is due to the fact that at such high doses, the immune response of T cells is triggered, which destroys the transduced cells, as a result of which the expression of the transgene decreases or even stops. Therefore, strategies for improving FIX expression are needed to make FIX gene therapy a viable therapeutic option for patients with hemophilia B.

[0051] Thus, improved factor IX polypeptide constructs that support improved factor IX expression and activity will improve both therapeutic approaches. For example, virus delivery methods can be improved by reducing the initial dose of the construct, thereby reducing the stimulation of the subject's immune system. Methods based on the introduction of deproteinized DNA could be improved by supporting more factor IX activity with fewer copies of the therapeutic polynucleotide.

[0052] The present disclosure relates to the discovery of codon-altered factor IX variant coding sequences that address these and other problems associated with factor IX gene therapy. For example, the polynucleotides described herein provide markedly improved factor IX expression and activity in a mammalian host. In some embodiments, these advantages are realized by using factor IX encoding polynucleotides of high sequence identity with codon-altered constructs CS02, CS03, CS04, CS05, and CS06. In some embodiments, such sequences include significantly fewer CpG dinucleotides compared to wild-type constructs, as described more fully below.

[0053] Advantageously, the factor IX sequences described herein with codon-altered constructs CS02, CS03, CS04, CS05, and CS06 provide increased expression of factor IX in vivo compared to equivalent wild-type sequences. For example, Example 1 shows that self-complementary AAV vectors carrying the factor IX coding sequence (R384L) with codon-altered constructs CS02, CS03, CS04, CS05, and CS06 provide a 20- to 40-fold increase in factor IX in vivo relative to a self-complementary AAV vector, carrying the wild-type factor IX coding sequence. Similarly, there is a 2-4-fold increase in factor IX expression relative to that of a self-complementary AAV vector carrying the wild-type factor IX (R384L) coding sequence (Table 2).

[0054] Advantageously, the improved factor IX activity generated at the codon-altered sequences CS02, CS03, CS04, CS05, and CS06 can be further improved by introducing one or more copies of the liver-specific regulatory element upstream of the factor IX coding sequence. For example, as shown in Examples 2 and 3, incorporation of one or more liver-specific CRM8 regulatory control elements into a factor IX self-complementing AAV vector further increased factor IX expression 2-3-fold in a mouse model and 2-13-fold in human hepatocytes (Tables 3 and 4, respectively). Similarly, incorporation of one or more copies of the liver-specific regulatory control element CRM8 into a factor IX single chain AAV vector increased factor IX activity 2-fold in vivo (mouse model; Table 5) and up to 26-fold in human hepatocytes (Table 6).

[0055] Surprisingly, while self-complementary AAV vectors encoding a codon-altered factor IX polypeptide lacking liver-specific regulatory control elements CRM8 provided greater increases in factor IX expression than similar single-stranded AAV vectors (compare 6.2 -fold increase in FIX activity provided by CS06-CRM.0-scV with 3.9-fold increase in factor IX activity provided by CS06-CRM.0-ssV (SEQ ID NO: 40) in Table 6), AAV factor IX single chain vectors containing multiple copies of the CRM8 liver-specific regulatory control elements were significantly superior to similar self-complementary AAV vectors (compare the 12.8-fold increase in factor IX activity provided by CS02-CRM8.3-scV compared to CS02-CRM8.0-scV, in Table 4 with a 16.8-fold increase in factor IX activity provided by CS06-CRM8.3-ssV (SEQ ID NO: 40) compared to CS06-CRM8.0-scV, in Table 6).

Definitions

[0056] In the context of this document, the following terms have their assigned meanings, unless otherwise indicated.

[0057] As used herein, the terms "factor IX" and "FIX" (where "IX" refers to the Roman numerals for the number "nine") are used interchangeably and refer to any protein with factor IX activity (e.g., active FIX, often referred to as "FIXa") or a protein precursor (e.g., proprotein or preproprotein, often referred to as pFIX and ppFIX) with factor IX activity, especially factor X cleavage activity in the presence of factor VIII, e.g. as measured using the one-step factor IX clotting assay described in chapter 2.7.11 of the European Pharmacopoeia 9.0, the contents of which are included in this description by reference

[0058] Factor IX is translated as an inactive single chain polypeptide that includes a signal peptide, a propeptide, a light chain, an activation peptide and a heavy chain, often referred to as the factor IX prepropolypeptide. The factor IX prepropeptide undergoes post-translational processing to form an active factor IX protein (eg, FIXa). This treatment includes removal (eg, cleavage) of the signal peptide followed by removal (eg, cleavage) of the propeptide to form a single chain mature factor IX polypeptide containing the factor IX light chain and the factor IX heavy chain that is still inactive. The mature factor IX polypeptide is further cleaved to remove the activation peptide between the factor IX light chain and the factor IX heavy chain to form an active factor IX protein (eg, FIXa). The factor IX light chain and the factor IX heavy chain remain linked via a disulfide bond.

[0059] For example, the wild-type human Factor IX pre-proprotein is first cleaved to release the encoded signal peptide (FIX-FL-AA amino acids 1-28 (SEQ ID NO: 2)), forming the first single strand of the proprotein. This single chain propeptide is then cleaved to release the propeptide (amino acids 29-46 FIX-FL-AA (SEQ ID NO: 2)) to form a second single chain propeptide (e.g., FIX-MP-AA (SEQ ID NO: 10) designated "MP ”, denoting mature protein (mature protein)). The second single chain proprotein is then double cleaved between the FIX light chain and the FIX heavy chain by factor XIa to release the activation peptide (amino acids 192-226 FIX-FL-AA (SEQ ID NO: 2)). Thus, an active factor IXa protein is formed, consisting of separate light and heavy chains linked by a disulfide bond. For more information on the structure, function, and activation of factor IX, see, for example, Brandstetter H. et al. P.N.A.S. USA, 92(21):9796-800 (1995), Hopfner KP et al., Structure, 7(8):989-96 (1999), and Gailani D. et al., Thromb Res., 133 Suppl 1: S48-51 (2014), the contents of which are incorporated herein by reference in their entirety for any purpose.

[0060] As described herein, such an active Factor IXa protein may include one or more variants, with the R338L variant finding particular use in some embodiments. This is referred to as "FIXp-MP-AA" (SEQ ID NO: 12) and the nucleic acid sequence is referred to herein as "FIXp-MP-NA"; "FIXp" refers to the incorporation of the R338L Padua variant into the final protein. It should be noted that the codon-optimized sequences CS02-CS06 exemplified herein encode a FIXp protein, including the R338L variant. Thus, specifically included in the definition of FIX is FIXp.

[0061] As used herein, the terms "Factor IX polypeptide" and "FIX polypeptide" refer to a polypeptide having Factor IX serine protease activity under certain conditions, e.g., as measured using the factor IX one-step clotting assay described in Chapter 2.7.11 European Pharmacopoeia 9.0. Factor IX polypeptides include single chain precursor polypeptides (including factor IX prepropolypeptides, factor IX propeptides, and mature factor IX single chain polypeptides) that, when activated by the post-translational processing described above, become an active factor IX protein with factor IX serine protease activity, and also active factor IX proteins per se. Factor IX polypeptides, including the R338L variant, are specifically included within the definition of factor IX polypeptides. In a typical embodiment, a human factor IX polypeptide refers to a polypeptide that includes an amino acid sequence with high sequence identity (e.g., at least 85%, 90%, 95%, 99% or more) to a portion of a wild-type human factor IX polypeptide, which includes the light and heavy chains, FIX-MP-AA (SEQ ID NO: 10, shown in Fig. 11A) or to a portion of the human factor IX polypeptide, Padua variant, which includes the light and heavy chains, FIXp-MP-AA (SEQ ID NO: 12 shown in figure 12).

[0062] As used herein, "factor IX light chain" or simply "light chain" refers to a polypeptide in the activated factor IXa protein derived from the N-terminal portion of a single-chain factor IX polypeptide containing the Gla module, EGF-like domains 1 and 2 factors IX. In a typical embodiment, amino acids 47-191 of the human factor IX prepropolypeptide (FIX-FL-AA (SEQ ID NO: 2)) comprise the factor IX light chain. In the present description, the amino acid sequence of the wild-type factor IX light chain is referred to as FIX-LC-AA (SEQ ID NO: 62).

[0063] As used herein, "Factor IX heavy chain" or simply "heavy chain" refers to a polypeptide in the activated Factor IXa protein derived from the C-terminal portion of a Factor IX single chain polypeptide containing the Factor IX peptidase S1 domain. In one exemplary embodiment, amino acids 227-461 of the human factor IX prepropolypeptide (FIX-FL-AA (SEQ ID NO: 2)) comprise the factor IX heavy chain. In the present specification, the wild type factor IX heavy chain amino acid sequence is referred to as FIX-HC-AA (SEQ ID NO: 63) and FIXp-HC-AA (SEQ ID NO: 64) when the R338L variant is included.

[0064] Typically, the factor IX light and heavy chains are expressed as a single polypeptide chain, eg, together with an activation peptide. However, in some embodiments, the factor IX light chain and the factor VIII heavy chain are expressed as separate polypeptide chains (eg, co-expressed) and reconstituted to form the factor IX protein (eg, in vivo or in vitro ). In general, for the purposes of the present invention, even if two strands are expressed separately, they are generally on the same expression vector (eg, viral genome) and not on different expression vectors.

[0065] As used herein, "factor IX activation peptide" or simply "activation peptide" refers to a peptide cut from a single chain factor IX polypeptide upon activation of the factor IXa protein. In a typical embodiment, amino acids 192-226 of the human factor IX prepropolypeptide (FIX-FL-AA (SEQ ID NO: 2)) constitute a factor IX activation peptide. In the present specification, the amino acid sequence of the wild type factor IX activation peptide is referred to as FIX-AP-AA (SEQ ID NO: 56).

[0066] As used herein, the term "factor IX signal peptide" or simply "signal peptide" refers to a peptide cut from the N-terminus of a factor IX prepropolypeptide by a signal peptidase. The signal peptide directs the newly translated factor IX preprotein to the endoplasmic reticulum. In a typical embodiment, amino acids 1-28 of the human factor IX prepropolypeptide (FIX-FL-AA (SEQ ID NO: 2)) constitute the factor IX signal peptide. In the present specification, the amino acid sequence of the wild-type factor IX signal peptide is referred to as FIX-SP-AA (SEQ ID NO: 37). A number of signal peptides according to the invention are shown in Figures 19 and 22.

[0067] As used herein, the term "factor IX propeptide" or simply "propeptide" refers to a peptide cut with furin from the N-terminus of a factor IX propolypeptide (eg, after signal peptide cleavage). The propeptide includes a γ-carboxylation recognition site that recruits a carboxylase to the neighboring Gla module, thereby facilitating the carboxylation of glutamine residues. In a typical embodiment, amino acids 29-46 of the human factor IX prepropolypeptide (FIX-FL-AA (SEQ ID NO: 2)) constitute the factor IX propeptide. In the present specification, the amino acid sequence of the wild-type factor IX propeptide is referred to as FIX-PP-AA (SEQ ID NO: 38).

[0068] As used herein, the term "factor IX prepropeptide" or simply "prepropeptide" refers to an aggregate of a factor IX signal peptide and a propolypeptide. In a typical embodiment, amino acids 1-46 of the human factor IX prepropolypeptide (FIX-FL-AA (SEQ ID NO: 2)) constitute the factor IX prepropeptide. In the present specification, the amino acid sequence of the wild-type factor IX prepropeptide is referred to as FIX-PPP-AA (SEQ ID NO: 36) with the nucleic acid sequence shown in FIG. 18, referred to as FIX-PPP-NA (SEQ ID NO: 18) (with corresponding FIXp-PPP-AA and FIXp-PPP-NA when R338L variant is used).

[0069] Unless otherwise noted herein, factor IX amino acid numbering refers to the corresponding amino acid in the full-length wild-type human factor IX prepropolypeptide sequence (FIX-FL-AA) shown as SEQ ID NO:2 in Figure 3A. Thus, when referring to an amino acid substitution in a Factor IX polypeptide disclosed herein, the amino acid number given refers to a similar (e.g., structurally or functionally equivalent) and/or homologous (e.g., evolutionarily conserved in primary amino acid sequence) amino acid in the full-length prepropolypeptide sequence. wild-type factor IX. For example, the R384L amino acid substitution refers to an R to L change at position 384 of the full-length wild-type human factor IX pre-propeptide sequence (FIX-FL-AA (SEQ ID NO: 2)), an R to L change at position 338 of the mature wild-type factor IX single chain polypeptide type (FIX-MP-AA (SEQ ID NO: 10)), replacing R with L at position 346 of the full-length wild-type human factor IX prepropeptide isoform 2 sequence (FIX2-FL-AA (SEQ ID NO: 3)), replacing R with L at position 300 of the mature wild-type human factor IX prepropolypeptide isoform 2 sequence (FIX2-FL-AA (SEQ ID NO: 3)) and an R to L change at position 158 of the wild-type human factor IX heavy chain sequence (FIX-HC-AA (SEQ ID NO: 63)).Thus, all of these nomenclature describe the same "Padua" amino acid substitution in different factor IX constructs.

[0070] As described herein, the factor IX amino acid numbering system depends on whether a factor IX prepropeptide is included (eg, amino acids 1-46 of the full-length wild-type human factor IX sequence, inclusive of signal peptide and propeptide). When a prepropeptide is included, the numbering is referred to as pre-pro-peptide inclusive (prepropeptide inclusive) or PPI. If the prepropeptide is not included, the numbering is designated as pre-pro-peptide exclusive (the pre-peptide is excluded) or PPE. For example, R384L is the PPI numbering for the same amino acid substitution as R338L in the PPE numbering. Similarly, the amino acid numbering of factor IX also depends on the isoform of factor IX. For example, R384L is the isoform 1 numbering for the same amino acid substitution as R346L in the isoform 2 numbering. Unless otherwise noted, all amino acid numbering refers to the corresponding amino acid in the full-length wild-type human factor IX isoform 1 sequence (FIX-FL-AA) , shown as SEQ ID NO: 2 in FIG. 3A. This numbering is identical to FIXp-FL-AA (SEQ ID NO: 4) which has the same amino acid sequence, except for the R384L "Padua" mutation.

[0071] Non-limiting examples of wild-type factor IX polypeptides include human preprofactor IX (e.g., GenBank accession number NP_000124.1 (FIX-FL-AA (SEQ ID NO: 2)) and NP_001300842.1 (FIX2-FL-AA (SEQ ID NO: 3)), the corresponding single chain factor IX lacking the signal peptide (preproprotein amino acids 1-28) and/or propeptide (preproprotein amino acids 29-46) and natural variants thereof; porcine preprofactor IX (e.g. accession number UniProt P00741), corresponding single chain factor IX lacking a signal peptide and natural variants thereof; mouse preprofactor IX (e.g. UniProt accession number P16294), corresponding single chain factor IX lacking a signal peptide and natural variants thereof; rat preprofactor IX (e.g., UniProt accession number P16296), the corresponding single chain factor IX lacking the signal peptide, and natural variants thereof; and other mammalian factor VIII homologues (e.g., chimpan se, great apes, hamsters, guinea pigs, etc.).

[0072] As used herein, the factor IX polypeptide includes natural variants and artificial constructs with factor X cleavage activity in the presence of factor VIII. As used in the present disclosure, factor IX encompasses any natural variants, alternative sequences, isoforms, or mutant proteins that retain some basal factor IX cleavage activity (e.g., at least 5%, 10%, 25%, 50%, 75 % or more of the corresponding wild-type activity, in accordance with the results of a single-stage analysis of blood coagulation in accordance with chapter 2.7.11 of the European Pharmacopoeia 9.0, which is specifically included in this description by reference due to guidelines regarding the analysis of human blood coagulation factor IX See chapter 2.7.11 Examples of factor IX amino acid variations (related to FIX-FL-AA (SEQ ID NO: 2)) found in the human population include, without limitation, I17N, L20S, C28R, C28Y, V30I, R43L, R43Q ,R43W,K45N,R46S,R46T,N48I,S49P,L52S,E53A,E54D,E54G,F55C,G58A,G58E,G58R,E66V,E67K,F71S,E73K,E73V,R75Q,E79D,T84R,Y91C,D93G , C97S, P101R, C102R, C102R, G10 6D G106S C108S D110N I112S N113K Y115C C119F C119R E124K G125E G125R G125V C134Y I136T N138H G139D G139S C155F G160E R191C, R191H, R226G, R226Q, R226W, V227D, V227F, V228F, V228L, Q241H, Q241K, C252S, C252Y, G253E, G253R, A265T, C268W, A279T, N283D, E291V, R294G, R294Q, V296M, H302R, N306S, I316F, L318R, L321Q, N328K, N328Y, P333H, P333T, T342K, T342M, I344L, G351D, W356C, G357E, G357R, K362E, G363W, A366D, R379G, R379Q, C382Y, L392F, L383I, R384L, K387E, I390F, M394K, F395I, F395L, C396F, C396S, A397P, R404T, C407R, C407S, D410H, S411G, S411I, G412E, G413R, P414T, V419E, F424V, T426P, S430T, W431G, W431R, G432S, E433A, G433K, C435Y, A436V, G442E, G442R, I443T, R449Q, R449W, Y450C, W453R and I454T. As discussed in more detail below, this numbering refers to wild-type human FIX. Other amino acid variations identified in the human population are known and can be detected, for example, using the National Center for Biotechnology Information (NCBI) Variation Viewer, accession number GCF_000001405.25. Factor VIII proteins also include polypeptides containing post-translational modifications.

[0073] A particular use in the present disclosure is the FIX protein, which includes the so-called "Padua" mutation, a substitution of arginine for leucine at position 338 of the mature factor IX single chain protein (R338L), position 384 of the factor IX prepropolypeptide (R384L). This mutation confers hyperfunctional activity on the FIX protein. For example, the Padua protein (eg, factor IX containing the R338L mutation) has been shown to be 5-10 times more active than wild-type factor IX in vivo. US patent No. 6,531,298; Simioni P. et al., N Engl J Med. 361(17): 1671-75 (2009), incorporated herein by reference in its entirety. Accordingly, the specification discloses amino acid and nucleic acid constructs that encode the Padua-FIX protein, sometimes referred to herein as FIXp or pFIX.

[0074] As used herein, the terms “factor IX polynucleotide” and “FIX polynucleotide” refer to a polynucleotide encoding a factor IX polypeptide having factor IX serine protease activity under specified conditions, e.g., as measured by a one-step factor IX fold assay, described in chapter 2.7.11 of the European Pharmacopoeia 9.0. Factor IX polynucleotides include polynucleotides encoding single-stranded factor IX precursor polypeptides, including factor IX pre-polypropeptides, factor IX propeptides, and mature single-stranded factor IX polypeptides, which, when activated by the post-translational processing described above, become an active factor IX protein with activity serine protease factor IX. Specifically included within the definition of factor IX polynucleotides are polynucleotides encoding a factor IX polypeptide that includes the R338L variant. In an exemplary embodiment, a human factor IX polynucleotide refers to a polynucleotide encoding a polypeptide that includes an amino acid sequence with high sequence identity (e.g., at least 85%, 90%, 95%, 99% or more) to a portion of the human factor IX polypeptide wild-type, which includes light and heavy chains, FIX-MP-AA (SEQ ID NO: 10, shown in Fig. 11A) or to a portion of the human factor IX polypeptide, Padua variant, which includes light and heavy chains, FIXp -MP- AA (SEQ ID NO: 12 shown in Figure 12).

[0075] As described herein, Factor IX polynucleotides may include regulatory elements such as promoters, enhancers, terminators, polyadenylation sequences, and introns, as well as virus packaging elements such as inverted terminal repeats (ITRs) and/or other elements which support the replication of the polynucleotide in a non-viral host cell, for example, a replicon supporting the expansion of the polynucleotide, for example in a bacterial, yeast or mammalian host cell.

[0076] Particularly useful in the present invention are codon-altered Factor IX polynucleotides. As described herein, codon-altered FIX polynucleotides provide increased expression of a factor IX transgenic in vivo compared to the level of factor IX expression provided by a natively encoded factor IX construct (e.g., a polynucleotide encoding the same factor IX amino acid sequence using wild-type human codons). As used herein, the term "overexpression" refers to an increased level of factor IX transgenic protein in the blood of an animal administered a codon-altered factor IX-encoding polynucleotide compared to the level of factor IX transgenic protein in the blood of an animal administered a natively encoded factor construct. IX. Increased protein expression results in increased factor IX activity; thus, increased expression leads to increased activity.

[0077] In some embodiments, increased expression refers to at least 25% more factor IX transgenic polypeptide in the blood of an animal that has been administered a codon-altered Factor IX polynucleotide compared to the level of factor IX transgenic polypeptide in the blood of an animal that has been administered natively encoded factor IX polynucleotide. For the purposes of the present invention, overexpression refers to an effect caused by a change in codon sequence, and not an overactivity caused by a basic amino acid substitution, such as a "Padua" mutation. That is, the expression level obtained from the codon-optimized sequence encoding the Padua factor IX polynucleotide is compared with the expression level obtained using the natively encoded Padua protein. In some embodiments, increased expression refers to a level of a factor IX transgenic polypeptide in the blood of an animal administered a codon-altered factor IX polynucleotide that is at least 50% higher, at least 75% higher, at least 100% higher , at least 3 times higher, at least 4 times higher, at least 5 times higher, at least 6 times higher, at least 7 times higher, at least 8 times higher, by at least 9 times higher, at least 10 times higher, at least 15 times higher, at least 20 times higher, at least 25 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 90 times higher, at least 100 times higher times higher, at least 125 times higher, at least 150 times higher, at least 175 times higher, at least 200 times higher, at least 225-fold higher or at least 250-fold higher than the level of transgenic factor IX polypeptide in the blood of an animal administered with a natively encoded factor IX polynucleotide. The levels of the factor IX polypeptide in the blood of an animal can be measured, for example, using an ELISA assay specific for the factor IX polypeptide.

[0078] By “factor IX activity” or “factor IX serine protease activity”, as used herein, is meant the ability to cleave a factor X polypeptide in the presence of a factor VIIIa cofactor, e.g. factor X to factor Xa. Activity levels can be measured using any factor IX activity known in the art; suitable assays are described herein; an exemplary assay for determining factor IX activity is the one-step factor IX clotting assay described in EP 9.0 chapter 2.7.11, which is used in the examples herein. In some embodiments, human plasma deficient in FIX activity is used as a control in a one-step clotting assay to determine factor IX specificity.

[0079] Since some factor IX variants have increased specific activity compared to wild-type factor IX in vivo , for example, the human Padua variant factor IX serine protease activity is 5-10 times higher than the activity of the native-coded human factor IX factor IX, in some In embodiments, the therapeutic potential of a factor IX polynucleotide composition is assessed by an increase in factor IX activity in the blood of an animal to which a factor IX polynucleotide is administered, eg, instead of or in addition to increased factor IX expression. In some embodiments, as used herein, increased factor IX activity refers to a greater increase in factor IX activity in the blood of an animal administered a codon-altered factor IX polynucleotide relative to baseline factor IX activity in the animal's blood prior to administration of the codon-altered factor IX polynucleotide. IX versus an increase in factor IX activity in the blood of an animal administered with the natively encoded factor IX polynucleotide relative to baseline factor IX activity in the blood of the animal prior to administration of the natively encoded factor IX polynucleotide. In some embodiments, increased factor IX activity refers to at least a 25% greater increase in factor IX activity in the blood of an animal administered a codon-altered factor IX polynucleotide relative to the baseline level of factor IX activity in the animal's blood prior to administration of the codon-altered factor IX polynucleotide. IX versus an increase in the level of factor IX activity in the blood of an animal administered the natively encoded factor IX polynucleotide relative to the animal's baseline factor IX activity prior to administration of the natively encoded factor IX polynucleotide. In some embodiments, increased factor IX activity refers to an increase in factor IX activity in the blood of an animal administered a codon-altered factor IX polynucleotide that is at least 50% higher, at least 75% higher, at least 100% higher, at least 3 times higher, at least 4 times higher, at least 5 times higher, at least 6 times higher, at least 7 times higher, at least 8 times higher, at least 9 times higher, at least 10 times higher, at least 15 times higher, at least 20 times higher, at least 25 times higher, at least 30 times higher, at least at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 90 times higher, at least 100 times higher, at least 125 times higher, at least 150 times higher, at least 175 times higher, at least 200 times higher, at least 225 times higher or at least 250 times higher than the initial level of factor IX activity in the blood of the animal before the introduction of the codon-altered factor IX polynucleotide compared with the increase in the level of factor IX activity in the blood of the animal that was injected with the natively encoded factor polynucleotide IX relative to the animal's baseline factor IX activity prior to administration of the natively encoded factor IX polynucleotide. Activity is measured using the factor IX one-step clotting assay described in EP 9.0 chapter 2.7.11 as described herein.

[0080] As used herein, the term "hemophilia" refers to a group of disease states 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 most prominent of the subtypes of hemophilia. Hemophilia type B (hemophilia B) is the result of a loss or decrease in the clotting function of factor IX (FIX). Hemophilia type C (hemophilia C) is the result of a loss or decrease in factor XI (FXI) clotting activity. Hemophilia A and B are X-linked diseases, while hemophilia C is an autosomal disease. Conventional treatments for hemophilia include both prophylactic and on-demand administration of clotting factors such as FVIII, FIX, including Bebulin®-VH and FXI, also FEIBA-VH, desmopressin, and plasma infusions.

[0081] As used herein, the term "factor IX gene therapy" or "FIX gene therapy" includes any therapeutic approach to providing a factor IX nucleic acid to a patient to alleviate, reduce, or prevent the recurrence of one or more symptoms (e.g., clinical factors) associated with factor IX deficiency (eg hemophilia B). The term includes the administration of any compound, drug, or procedure or regimen comprising a nucleic acid encoding a factor IX molecule, including any modified form of factor IX (e.g., factor VIII variant R384L), to maintain or improve the health of a factor IX deficient person (e.g., hemophilia B). One skilled in the art will appreciate that either the FIX gene therapy course or the dose of the FIX gene therapy therapeutic agent may be modified, for example, based on the results obtained in accordance with the present invention.

[0082] 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 in the treatment of hemophilia may be an amount that is capable of preventing or alleviating one or more of the symptoms associated with hemophilia.

[0083] In some embodiments, a therapeutically effective treatment results in a reduction in the frequency and/or severity of bleeding events in a subject.

[0084] As used herein, the term "gene" refers to a segment of a DNA molecule encoding a polypeptide chain (eg, a coding region). In some embodiments, the gene is located in the immediately preceding, following, and/or intermediate regions of the coding region that are involved in the production of the polypeptide chain (e.g., regulatory elements such as a promoter, enhancer, polyadenylation sequence, 5' untranslated region, 3' untranslated region, or intron).

[0085] As used herein, the term "regulatory elements" refers to nucleotide sequences, such as promoters, enhancers, terminators, polyadenylation sequences, introns, etc., that allow expression of a coding sequence in a cell.

[0086] As used herein, the term "promoter element" refers to a nucleotide sequence that helps control the expression of a coding sequence. Generally, promoter elements are located at the 5' end of the translation start site of a gene. However, in some embodiments, the promoter element may be located within the intron sequence or at the 3' end of the coding sequence. In some embodiments, the promoter usable for the gene therapy vector is derived from a native target protein gene (eg, a factor VIII promoter). In some embodiments, the promoter used 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 yet other embodiments, the gene therapy vector described herein uses one of a variety of well-characterized promoter elements. Non-limiting examples of well-characterized promoter elements include the CMV early promoter, the β-actin promoter, and the methyl CpG binding protein 2 (MeCP2) promoter. In some embodiments, the promoter is a constitutive promoter that drives substantially constant expression of the target protein. In other embodiments, the promoter is an inducible promoter that stimulates expression of the target protein in response to a specific stimulus (eg, exposure to a particular treatment or agent). For a review of promoter development 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.

[0087] As used herein, the term "CRM8" refers to a cis-acting regulatory module derived from the SERPINA1 gene (NCBI accession number NM_000295.4) that enhances the expression of an operably linked gene, e.g., a factor IX polypeptide coding sequence, in a liver-specific having high sequence identity with SEQ ID NO: 39. As used herein, a CRM8 element refers to one copy of a regulatory element that, in some embodiments, is included in one or more copies in a Factor IX polynucleotide, e.g., 1, 2, 3 or more copies. For more information on CRM elements such as CRM8, see Chuah MK et al., Mol Ther., 22 (9): 1605-13 (2014), which is incorporated herein by reference.

[0088] As used herein, the term "MVM intron" refers to an intron sequence derived from a mouse small virus having high sequence identity with SEQ ID NO: 53. For more information on the MVM intron itself, see Haut and Pintel, J Virol . 72 (3): 1834-43 (1998) and on the use of the MVM intron in AAV gene therapy vectors, see Wu Z et al., Mol Ther., 16 (2): 280-9 (2008), both of which are included to this document by reference.

[0089] As used herein, the term "operably linked" refers to a relationship between a first reference nucleotide sequence (e.g., a gene) and a second nucleotide sequence (e.g., a regulatory control element) that allows the second nucleotide sequence to influence one or more properties associated with the first reference nucleotide sequence (eg, transcription rate). In the context of the present disclosure, a regulatory control element is operably linked to a factor IX transgene when the regulatory element is located within a gene therapy vector such that it has an effect (eg, a promoter or tissue-specific effect) on the transcription of the factor IX transgene.

[0090] As used herein, the term "vector" refers to any nucleic acid construct used to transfer a factor IX nucleic acid into a host cell. In some embodiments, the vector includes a replicon that functions to replicate the nucleic acid construct. 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 vector for introducing a factor IX nucleic acid into a host cell. Many modified eukaryotic viruses used 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 natural carriers of the virus, native viruses are known to not contribute to any disease and the viruses elicit a weak immune response.

[0091] As used herein, the term "factor IX viral vector" refers to a recombinant virus containing a factor IX polynucleotide encoding a factor IX polypeptide that is sufficient to express the factor IX polypeptide when introduced into a suitable host animal (e.g., human). Specifically included within the definition of a factor IX viral vector are recombinant viruses in which a codon-altered factor IX polynucleotide encoding a factor IX polypeptide has been inserted into the viral genome. Also specifically included within the definition of a factor IX viral vector are recombinant viruses in which the native viral genome is replaced with a factor IX polynucleotide encoding a factor IX polypeptide. Included within the definition of a factor IX viral vector are recombinant viruses containing a factor IX polynucleotide encoding the Padua variant of factor IX.

[0092] As used herein, the term "factor IX viral particle" refers to a viral particle encapsulating a factor IX polynucleotide encoding a factor IX polypeptide that is specific for the expression of a factor IX polypeptide when introduced into a suitable host animal (e.g., human). Specifically included within the definition of factor IX virus particles are recombinant viral particles encapsulating a genome into which a codon-altered factor IX polynucleotide encoding a factor IX polypeptide has been inserted. Also specifically included within the definition of factor IX virus particles are recombinant virus particles encapsulating a factor IX polynucleotide encoding a factor IX polypeptide that replaces the native genome of the virus. Included in the definition of factor IX viral particles are recombinant viral particles encapsulating a factor IX polynucleotide encoding the factor IX "Padua" variant

[0093] By "AAV" or "adeno-associated virus", as used herein, is meant a dependdoparvovirus from the genus Parvoviridae . As used herein, the term "AAV" may refer to a virus derived from a naturally occurring "wild-type" AAV genome into which a factor IX polynucleotide has been inserted, a recombinant virus derived from a recombinant factor IX polynucleotide packaged in a capsid using capsid proteins encoded by a naturally occurring AAV gene, or by a recombinant virus derived from a recombinant factor IX polynucleotide packaged in a capsid using capsid proteins encoded by a non-natural capsid gene. The definition of AAV includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8) and AAV type 9 (AAV9) encapsulating a factor IX polynucleotide, and viruses formed by one or more AAV capsid protein variants encapsulating a factor IX polynucleotide.

[0094] By "AAV8", "AAV-8", or "AAV serotype 8", as used herein, is meant a virus formed by the AAV8 viral capsid protein that encapsulates a factor IX polynucleotide.

[0095] As used herein, the term "CpG" refers to a cytosine-guanine dinucleotide along one strand of DNA, where "p" represents a phosphate bond between them.

[0096] As used herein, the term "CpG island" refers to a region within a polynucleotide having a statistically increased density of CpG dinucleotides. As used herein, a region of a polynucleotide (e.g., a polynucleotide encoding a codon-altered Factor IX protein) is a CpG island if, when considering 200 base pairs: (i) the region has a GC content greater than 50%, and (ii) the ratio of observed dinucleotides CpG to expected CpG dinucleotides is at least 0.6 as defined by the ratio:

≥ 0,6.

≥ 0.6.

For more information on methods for identifying CpG islets, see Gardiner-Garden M. et al., J Mol Biol., 196 (2): 261-82 (1987), the contents of which are incorporated herein in their entirety for all purposes. through a link.

[0097] As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their polymers in single or double stranded form and their complements. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or units that are synthetic, naturally occurring and not naturally occurring; which have similar binding properties as the nucleic acid in question and are metabolized similarly to the reference nucleotides. Examples of such analogs include, without limitation, thiophosphates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptidonucleic acids (PNAs). However, particularly applicable embodiments herein for use in gene therapy in patients use phosphodiester linkages.

[0098] By "nucleic acid compositions" as used herein is meant any molecule or composition of molecules that includes a factor IX polynucleotide encoding a factor IX peptide. The definition of nucleic acid formulations includes factor IX polynucleotides, aqueous solutions of factor IX polynucleotides, viral particles encapsulating a factor IX polynucleotide, and aqueous formulations of viral particles encapsulating a factor IX polynucleotide. The nucleic acid composition as described herein comprises a codon-altered FIX gene encoding a FIX polypeptide.

[0099] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, including amino acid analogs and amino acid mimics, that 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, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring amino acids may include, for example, D- and L-amino acids. With respect to amino acid sequences, one of ordinary skill in the art will appreciate that individual substitutions, deletions, or additions to 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 a "conservatively modified variant" in which the change results in 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, interspecies homologues, and alleles of this disclosure.

[00100] 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, such as a catalytic, structural or sterically important amino acid, different groups of amino acids can be considered conservative substitutions for each other. Table 1 presents 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 attack/structural nature of the amino acid, and the propensity of the amino acid to secondary structure.

Table 1 Groupings of conservative amino acid substitutions are 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
3. C, T, S, G, N, Q and Y
4. A, P, M, L, I, V, F 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/surface impact 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 Secondary Structure Predisposition 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. F, Y and W
7. A and G
8. M and C

[00101] The terms "identical" or percent "identity" in the context of two or more nucleic acids or peptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same amino acid residues or nucleotides (i.e., about 60 % identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher domain identity when compared and aligned for maximum match in the comparison window or selection) measured using the BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below, or by manual alignment and visual inspection.

[00102] As is known in the art, a number of different programs can be used to identify whether a protein (or nucleic acid, as discussed below) has sequence identity or similarity to a known sequence. Sequence identity and/or similarity is determined using standard methods known in the art, including, but not limited to, the local sequence identification algorithm of Smith & Waterman, Adv. Appl. Math., 2:482 (1981), Sequence Identity Alignment Algorithm Needleman & Wunsch, J. Mol. Biol. 48:443 (1970). similarity search according to Pearson & Lipman, Proc. Natl. Acad. sci. U.S.A., 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, WI), 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; gap penalty (penalty for a gap in the sequence) - 1; gap size penalty - 0.33 and joining penalty - 30, "Current Methods in Sequence Comparison and Analysis,", Macromolecule Sequencing and Synthesis, Selected Methods and Applications, page 127 -149 (1988), Alan R. Liss,, with Inc., each of which is incorporated herein by reference.

[00103] An example of a suitable algorithm is PILEUP. The PILEUP algorithm creates a multiple sequence alignment 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. The PILEUP algorithm uses a simplification of the progressive alignment method by 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 are incorporated by reference. Applicable PILEUP parameters include the following: default gap weight (default deletion penalty) - 3.00, default gap length weight (default continuation penalty) - 0.10 and weighted end gaps (evaluated end deletions).

[00104] Another example of a useful algorithm is the BLAST algorithm described in: Altschul et al., J. Mol. Biol. 215, 403-410, (1990); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); and Karlin et al., Proc. Natl. Acad. sci. U.S.A. 90:5873-5787 (1993), both incorporated by reference. A particularly useful BLAST program is the WU-BLAST-2 program obtained from Altschul et al., Methods in Enzymology, 266:460-480 (1996); http://blast.wustl/edu/blast/README.html]. The WU-BLAST-2 program uses several search options, most of which are configured with default values. Adjustable parameters are set to the following values: overlap interval=1, overlap fraction=0.125, word threshold (T)=11. in which the search for the sequence under study is carried out; however, the values can be adjusted to improve sensitivity.

[00105] An additional useful algorithm is gapped BLAST as reported by Altschul et al., Nucl. Acids Res., 25:3389-3402, incorporated by reference. The Gapped BLAST algorithm uses BLOSUM-62 replacement points; parameter threshold T (threshold word length) is set to 9; two-hit method to trigger ungapped extensions (two-hit method of triggering a continuation without a deletion) changes the value of gap lengths (continuation of a deletion) k with a cost (price) of 10+k; Xu is set to 16 and Xg is set to 40 for the database lookup step and up to 67 for the output stage of the algorithms. Gap-containing alignments are triggered by an evaluation corresponding to ~22 bits.

[00106] The percent amino acid sequence identity is determined by the number of identical residues that match, divided by the total number of "longer" sequence residues in the aligned region. The "longer" sequence is the sequence that has the most valid residuals in the aligned region (the gaps introduced by WU-Blast-2 to maximize the alignment score are ignored). Similarly, "percent nucleic acid sequence identity" (%) in relation to the coding sequence of certain polypeptides is defined as the percentage of nucleotide residues in a candidate sequence that are identical to nucleotide residues in the coding sequence of a cell cycle protein. The preferred method uses the default BLASTN module of WU-BLAST-2 with the overlap interval and overlap ratio set to 1 and 0.125, respectively.

[00107] The alignment may include introducing alignment gaps into the sequences. In addition, for sequences that contain more or less amino acids than the protein encoded by the wild-type factor IX sequence in FIG. 3A (SEQ ID NO: 2), it is to be understood that in one embodiment, percent 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, sequence identities shorter than those shown in FIG. 3A (SEQ ID NO: 2), as discussed below, will be determined using the number of nucleotides in the shorter sequence in one embodiment. In percent identity calculations, various manifestations of sequence variation, such as insertions, deletions, substitutions, etc., are not assigned a relative weight.

[00108] In one embodiment, only identifiers are evaluated positively (+1), and all forms of resequencing, including gaps, are assigned the value "0", which eliminates the need for weighted scale or parameters, as described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of identical residues that match by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the sequence having the most valid residues in the aligned region.

[00109] The term "allelic variants" refers to polymorphic forms of a gene at a particular genetic locus, as well as cDNAs derived from gene mRNA transcripts and the polypeptides they encode. The term "preferred mammalian codon" refers to a subset of codons from a set of codons encoding an amino acid that are most commonly used in proteins expressed in mammalian cells selected from the following list: Gly (GGC, GGG); Glu (GAG); Asp (GAC); Val (GTG, GTC); Ala (GCC, GCT); Ser (AGC, TCC); Lys (AAG); Asn(AAC); Met (ATG); Ile (ATC); Thr (ACC); Trp(TGG); Cys (TGC); Tyr (TAT, TAC); Leu (CTG); Phe(TTC); Arg (CGC, AGG, AGA); Gln(CAG); His(CAC); and Pro (CCC).

[00110] As used herein, the term "codon-altered" refers to a polynucleotide sequence encoding a polypeptide (e.g., factor IX protein) in which at least one codon of the native polynucleotide encoding the polypeptide has been altered to improve a property of the polynucleotide sequence. In some embodiments, the improved property promotes increased transcription of the mRNA encoding the polypeptide, increased mRNA stability (eg, improved mRNA half-life), increased translation of the polypeptide, and/or increased packaging of the polynucleotide within the vector. Non-limiting examples of changes that can be used to achieve improved properties include changing codon usage and/or allocation of specific amino acids, adjusting global and/or local GC content, removing AT-rich sequences, removing repetitive sequence elements, adjusting global and/or or local content of CpG dinucleotides, removal of cryptic regulatory elements (eg, TATA-box and CCAAT-box elements), removal of intron/exon splicing sites, enhancement of regulatory sequences (eg, introduction of the Kozak consensus sequence), and removal of sequence elements capable of forming a secondary structure (eg, stem loops) in the transcribed mRNA.

[00111] As discussed herein, there are various nomenclature for referring to the components of the disclosure in this document. "CS number" (e.g., "CS02", "CS03", "CS04", "CS05", "CS06", etc.) refers to codon-altered polynucleotides encoding FIX polypeptides and/or encoded polypeptides, including variants . For example, CS02-FL refers to the codon-altered polynucleotide sequence or full length amino acid sequence CS02 ( F ull L ength) (sometimes referred to herein as CS02-FL-AA from A mino A cid (amino acid) and CS02-FL-HC (SEQ ID NO: 5) from N ucleic Acid (nucleic acid)), encoded by the polynucleotide sequence CS02. Similarly, "CS02-LC" refers to either a codon-altered nucleic acid sequence ("CS02-LC-NA" (SEQ ID NO: 42)) encoding the light chain of a FIX polypeptide or an amino acid sequence (also sometimes referred to herein as "CS02-LC-AA") of the FIX light chain encoded by the CS02 polynucleotide sequence. Similarly, CS02-HC, CS02-HC-AA and CS02-HC-NA (SEQ ID NO: 41) are the same for FIX heavy chain. Those skilled in the art will appreciate that for constructs such as CS02, CS03, CS04, CS05, CS06, etc., which are only codon-changed (e.g., they do not contain additional amino acid substitutions compared to factor IX, variant Padua) the amino acid sequences will be identical since the amino acid sequences are not changed by codon optimization. Thus, disclosure sequence constructs include, but are not limited to, CS02-FL-NA (SEQ ID NO:5), CS02-FL-AA, CS02-LC-NA (SEQ ID NO:42), CS02-LC -AA, CS02-HC-AA, CS02-HC-NA (SEQ ID NO:41), CS03-FL-NA (SEQ ID NO:6), CS03-FL-AA, CS03-LC-NA (SEQ ID NO: :44), CS03-LC-AA, CS03-HC-AA, CS03-HC-NA (SEQ ID NO:43), CS04-FL-NA (SEQ ID NO:7), CS04-FL-AA, CS04- LC-NA (SEQ ID NO:46), CS04-LC-AA, CS04-HC-AA, CS04-HC-NA, CS05-FL-NA (SEQ ID NO:8), CS05-FL-AA, CS05- LC-NA (SEQ ID NO:48), CS05-LC-AA, CS05-HC-AA, CS05-HC-NA (SEQ ID NO:47), CS06-FL-NA (SEQ ID NO:9), CS06 -FL-AA, CS06-LC-NA (SEQ ID NO:50), CS06-LC-AA, CS06-HC-AA and CS06-HC-NA (SEQ ID NO:49). It should be noted that all "CS" constructs provided herein encode or contain the amino acid sequence of FIXp, although the definition of CS constructs includes those that encode or contain the amino acid sequence of human wild-type FIX.

[00112] As used herein, the term "liver-specific expression" refers to preferential or preferential in vivo expression of a particular gene (eg, a codon-altered factor IX transgenic gene) in liver tissue over other tissues. In some embodiments, liver-specific expression means that at least 50% of all expression of a particular gene occurs in the subject's liver tissues. In other embodiments, liver-specific expression means that at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of all expression of a particular gene occurs in the liver tissues of the subject. Accordingly, a liver-specific regulatory element is a regulatory element that stimulates liver-specific gene expression in liver tissue.

[00113] As used herein, the terms "less than X" and "less than X%" refer to the range from 0 to X, excluding the value of X, for example, from 0% to X%, excluding X%. The terms used in this document are used interchangeably with a range starting at 0 or 0% but not including X or X%.

[00114] As used herein, the terms "not more than X" or "not more than X%" refer to the range from 0 to X, including the value of X, for example, from 0% to X%, including X%. The terms used herein are used interchangeably with a range starting at 0 or 0% and including X or X%.

[00115] As used herein, the terms "greater than X" or "greater than X%" refer to a range from X to the upper limit, excluding the value of X, for example, from X% to 100%, excluding X%. The terms used herein are used interchangeably with a range starting at, but not including, X or X% up to an upper limit that is 100% in the context of a percentage.

[00116] As used herein, the terms "at least X" or "at least X%" refer to the range from X to the upper limit, including the value of X, for example, from X% to 100%, including X%. The terms used herein are used interchangeably with a range starting at and including X or X% up to an upper limit that is 100% in the context of a percentage.

[00117] As used herein, the terms "between X and Y", "between X% and Y%", "from X to Y" and "from X% to Y%" refer to the range from X to Y, including the values of X and Y, for example, from X% to Y%, including X% and Y%. The terms used herein are used interchangeably with a range starting with X or X% and up to and including Y or Y%.

Codon-altered factor IX polynucleotide

[00118] In some embodiments, the present disclosure relates to codon-altered nucleic acid compositions encoding factor IX or a factor IX variant (wherein FIXp finds use in specific embodiments). Such codon-altered polynucleotides provide markedly improved expression of factor IX when introduced into AAV-based gene therapy constructs. The codon-altered polynucleotides also show improved packaging of the AAV virion compared to conventional codon-optimized constructs. As shown in Example 1, Applicants achieved these benefits through the discovery of several codon-altered polynucleotides (e.g., CS02-FL-NA, CS03-FL-NA, CS04-FL-NA, CS05-FL-NA and CS06-FL-NA ( SEQ ID NOs: 5-9 respectively)) encoding factor IXp polypeptides with an overactive amino acid substitution R338L (based on the mature factor IX single chain polypeptide sequence; R384L based on the factor IX pre-proprotein sequence). As shown in Examples 2 and 3, incorporation of one or more liver-specific regulatory controls (eg, CRM8) into gene therapy vectors encoding a factor IX molecule further increased factor IX expression and factor IX activity in vivo and in vitro .

[00119] Wild type Factor IX is encoded by a 28 amino acid signal peptide (FIX-SP-AA (SEQ ID NO: 37)) and an 18 amino acid propeptide (FIX-PP-AA (SEQ ID NO: 38)), which are cleaved from encoded polypeptide prior to activation of factor IXa. Those skilled in the art will appreciate that signal peptides and/or propeptides can be mutated, replaced with signal peptides and/or propeptides from other genes or other organisms, or removed entirely without affecting the sequence of the mature remaining polypeptide. after the signal and propeptide are removed by cellular processing.

[00120] Accordingly, in some embodiments, the codon-altered polynucleotide (e.g., nucleic acid composition) provided herein has a nucleotide sequence with high sequence identity to CS02-FL-NA, CS03-FL-NA, CS04-FL- NA, CS05-FL-NA, or CS06-FL-NA (SEQ ID NOs: 5-9, respectively) encoding a mature factor IX single chain polypeptide, i.e., a factor IX light chain, an activation peptide, and a heavy chain (e.g., amino acids 47 -461 full-length polypeptide encoded by the factor IX wild-type gene, FIX-FL-AA (SEQ ID NO: 2)).

[00121] In addition, as known in the art, wild-type human factor IX has a 34 amino acid activation peptide located between the factor IX light chain and the heavy chain, which is excised from the factor IX single chain polypeptide when the protein is activated. Because the activation peptide is removed from the active factor IX polypeptide, the peptide itself is not necessarily necessary for maximum factor IX activity. Accordingly, the Factor IX polypeptides encoded by the codon-altered polynucleotides disclosed herein are not required to have high sequence identity with the wild-type human activation peptide sequence (FIX-AP-AA (SEQ ID NO: 56)). However, the encoded activation peptide must be excised upon activation of the factor IX polypeptide. For example, in some embodiments, the encoded activation peptide should include factor XI cleavage sites at its N- and C-terminus that are recognized and cleaved by human factor IX in vivo .

[00122] Accordingly, in some embodiments, the codon-altered polynucleotide (e.g., nucleic acid composition) provided herein encodes a factor IX single-stranded polypeptide with high sequence identity to the human wild type FIX light chain sequence (FIX-LC-AA (SEQ ID NO: 62)) and human wild-type FIX heavy chain sequences (FIX-HC-AA (SEQ ID NO: 63)), and further encode a polypeptide linker connecting the C-terminus of the light chain to the N-terminus of the heavy chain (eg, an activation peptide) with two factor XI cleavage sites.

[00123] In some embodiments, the factor IX light and heavy chains encoded by the codon-altered polynucleotide are human factor IX light and heavy chains, respectively, including the FIXp heavy chain. In other embodiments, the factor IX light and heavy chains encoded by the codon-altered polynucleotide are heavy and light chain sequences from another mammal (eg, porcine factor IX). In yet other embodiments, the factor IX light and heavy chains are chimeric light and heavy chains (eg, a combination of a human and second mammalian sequence). In other embodiments, the factor IX light and heavy chains are a humanized version of the light and heavy chains from another mammal, for example. light and heavy chain sequences from another mammal in which human residues are substituted at selected positions to reduce the immunogenicity of the resulting peptide when administered to a human.

[00124] The content of GC in human genes varies widely: from less than 25% to more than 90%. However, in general, higher GC human genes are expressed at higher levels. For example, Kudla et al. (PLoS Biol., 4 (6): 80 (2006)) demonstrate that increasing the content of the GC gene increases the expression of the encoded polypeptide, mainly by increasing transcription and influencing a higher steady state level of the mRNA transcript. Typically, the desired GC content in a codon-optimized gene construct is considered to be equal to or greater than 60%. For example, the factor IX gene in the scAAV8.FIXR338L gene therapy vector was specifically codon-changed using the GeneOptimizer software (Geneart) to increase the GC content of the wild-type coding sequence from 41% GC to 61% GC. See Wu Z. et al., Mol Ther 16:280-89 (2008) and Monahan P.E. et al., Hum Gene Ther., 26(2):69-81 (2015). However, native AAV genomes have a GC content of about 56%.

[00125] Accordingly, in some embodiments, the codon-altered polynucleotides (e.g., nucleic acid compositions) provided herein have a CG content that more closely matches the GC content of native AAV virions (e.g., approximately 56% GC), such that lower than the preferred GC content of polynucleotides that are typically codon-optimized for expression in mammalian cells (eg, 60% GC or higher). For example, CS02-FL-NA (SEQ ID NO: 5) has a GC content of about 54%, CS03-FL-NA (SEQ ID NO: 6) has a GC content of about 55%, CS04-FL-NA (SEQ ID NO: 7) has a GC content of about 54.5%, CS05-FL-NA (SEQ ID NO: 8) has a GC content of about 56.6% and CS06-FL-NA (SEQ ID NO: 9) has a GC content of about 55% . These constructs should provide improved virion packaging compared to similarly codon-altered sequences with higher GC content.

[00126] Thus, in some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide (e.g., a polynucleotide having a high sequence identity with one of the Factor IX coding sequences CS02-CS06) is less than 60%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is less than 59%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding a Factor IX polypeptide is less than 58%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding a factor IX polypeptide is less than 57%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is no more than 56%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is no more than 55%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is no more than 54%.

[00127] In some embodiments, the total GC content of a codon-altered polynucleotide encoding a factor IX polypeptide is between 53% and 59%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 54% and 59%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 55% and 59%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a factor IX polypeptide is between 56% and 59%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 53% and 58%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 54% and 58%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 55% and 58%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 56% and 58%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a factor IX polypeptide is between 53% and 57%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a factor IX polypeptide is between 54% and 57%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 55% and 57%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a factor IX polypeptide is between 56% and 57%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 53% and 56%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 54% and 56%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 55% and 56%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a factor IX polypeptide is between 53% and 55%. In some embodiments, the total GC content of a codon-altered polynucleotide encoding a Factor IX polypeptide is between 54% and 55%.

[00128] In some embodiments, the total GC content of the codon-altered polynucleotide encoding a factor IX polypeptide is 54 ± 0.5%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 54 ± 0.4%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 54 ± 0.3%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the Factor IX polypeptide is 54 ± 0.2%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 54 ± 0.1%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 54%.

[00129] In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 55 ± 0.5%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 55 ± 0.4%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 55 ± 0.3%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 55 ± 0.2%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 55 ± 0.1%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the Factor IX polypeptide is 55%.

[00130] In some embodiments, the total GC content of the codon-altered polynucleotide encoding a Factor IX polypeptide is 56 ± 0.5%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding a factor IX polypeptide is 56 ± 0.4%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 56 ± 0.3%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the Factor IX polypeptide is 56 ± 0.2%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding the factor IX polypeptide is 56 ± 0.1%. In some embodiments, the total GC content of the codon-altered polynucleotide encoding a factor IX polypeptide is 56%.

[00131] These CpG dinucleotides (ie, a cytosine nucleotide followed by a guanine nucleotide) are expected to induce immune responses via toll-like receptors in vivo . Some evidence suggests that CpG-depleted AAV vectors under certain circumstances avoid immune detection in mice (Faust et al., J. Clin. Invest. 2013; 123, 2994-3001). The wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO: 1)) contains 20 CpG dinucleotides.

[00132] Accordingly, in some embodiments, the nucleic acid compositions (eg, codon-altered polynucleotides) provided herein are codon-altered to reduce the number of CpG dinucleotides in the Factor IX coding sequence. For example, CS02-FL-NA (SEQ ID NO: 5) does not contain CpG dinucleotides, CS03-FL-NA (SEQ ID NO: 6) does not contain CpG dinucleotides, CS04-FL-NA (SEQ ID NO: 7) does not contain CpG dinucleotides, CS05-FL-NA (SEQ ID NO: 8) contains 11 CpG dinucleotides, and CS06-FL-NA (SEQ ID NO: 9) contains 3 CpG dinucleotides. These constructs should elicit lower immunogenic responses than the wild-type factor IX coding sequence and similarly codon-altered sequences with higher numbers of CpG dinucleotides.

[00133] Thus, in some embodiments, the sequence of a codon-altered polynucleotide encoding a Factor IX polypeptide (e.g., a polynucleotide having a high sequence identity with one of the Factor IX coding sequences CS02-CS06) has less than 20 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the factor IX polypeptide has less than 15 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the factor IX polypeptide has less than 12 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the factor IX polypeptide has less than 10 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the factor IX polypeptide has less than 5 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the Factor IX polypeptide has less than 3 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the factor IX polypeptide does not contain CpG dinucleotides.

[00134] In some embodiments, the sequence of the codon-altered polynucleotide encoding the factor IX polypeptide has more than 15 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the Factor IX polypeptide has no more than 12 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the factor IX polypeptide has no more than 10 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the Factor IX polypeptide has no more than 5 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the Factor IX polypeptide has no more than 3 CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the factor IX polypeptide does not contain CpG dinucleotides. In some embodiments, the sequence of the codon-altered polynucleotide encoding the Factor IX polypeptide is at most 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 CpG dinucleotides or no CpG dinucleotides.

Factor IX amino acid substitutions

[00135] To further enhance the expression efficiency of the AAV vector-based factor IX constructs described herein, amino acid substitutions known to improve secretion, increase specific activity, and/or enhance factor stability have been further inserted in accordance with some embodiments. IX. Several potential factor IX variants are known in the art to increase plasma levels of FIX activity. These variants include amino acid substitutions that increase factor IX catalytic activity (eg, hyperactive mutants), increase antithrombin III and/or heparin resistance, increase serum half-life, and result in altered post-translational modification patterns.

[00136] For example, mutation of the R338 (PPE) residue can increase factor IX clotting activity. For an overview, see US Pat. No. 6,531,298, the contents of which are incorporated by reference in their entirety for all purposes. As disclosed in US Pat. No. 6,531,298, the amino acid substitution of arginine for leucine at this position increases factor IX activity. This was later confirmed in vivo , where the R338L (PPE) mutation increased factor IX activity 5-10-fold in vivo . For a review, see Simioni P. et al., N Engl J Med. 361(17): 1671-75 (2009), incorporated herein by reference in its entirety. Accordingly, in some embodiments, the codon-altered polynucleotides described herein encode a factor IX polypeptide with an arginine 384 amino acid substitution (PPI); residue 338 (PPE). In a specific embodiment, the amino acid substitution is R384L (PPI). In other embodiments, an amino acid substitution is made at residue 384 (PPI)/338 (PPE) with a residue other than leucine. For example, the R384A amino acid substitution (PPI) has been reported to provide 2-6 times higher activity in mice. Schuettrumpf J et al., Blood, 105 (6): 2316-23 (2005), the contents of which are expressly incorporated herein by reference in their entirety for all purposes.

[00137] Similarly, mutation of residues Y305, K311, S365 and Y391 results in increased factor IX activity on the synthetic substrate. In particular, single mutations of K311M and K311T resulted in a 2.8-fold and 6.7-fold increase in activity on the synthetic cleavage substrate. Sichler K. et al., J Biol Chem. 278(6):4121-26 (2003) (using different residue numbering). In addition, the Y305F/K311T/Y391T triple mutant resulted in a 7000-fold increase in activity on the synthetic substrate. Id. Accordingly, in some embodiments, the codon-altered polynucleotides described herein encode a factor IX polypeptide with an amino acid substitution at one or more of tyrosine 305 (PPI), lysine 311 (PPI), and tyrosine 391 (PPI). In a specific embodiment, the amino acid substitution is K311M (PPI). In a specific embodiment, the amino acid substitution is K311T (PPI). In another specific embodiment, the amino acid substitution is Y305F/K311T/Y391T (PPI).

[00138] Other amino acid substitutions that provide improved properties are known in the art and can be included in the described codon-altered Factor IX polynucleotides. For example, see US Patent No. 8,778,870, the contents of which are expressly incorporated herein by reference in its entirety for all purposes.

Codon-altered polynucleotides encoding factor IX protein

Codon-altered CS02 polynucleotides

[00139] In one embodiment, the nucleic acid composition provided herein comprises a factor IX polynucleotide (e.g., a codon-optimized polynucleotide) encoding a single-stranded factor IX polypeptide, wherein the factor IX polynucleotide comprises a nucleotide sequence having high sequence identity with CS02- FL-HA (SEQ ID NO: 5). In some embodiments, a nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS02-FL-NA (SEQ ID NO: 5) has a reduced GC content compared to a wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)). In some embodiments, the nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS02-FL-NA (SEQ ID NO: 5) has a reduced number of CpG dinucleotides compared to the wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)).

[00140] In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 95% identity with CS02-FL-NA (SEQ ID NO: 5). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 96% identity with CS02-FL-NA (SEQ ID NO: 5). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 97% identity with CS02-FL-NA (SEQ ID NO: 5). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 98% identity with CS02-FL-NA (SEQ ID NO: 5). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99% identity with CS02-FL-NA (SEQ ID NO: 5). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.5% identity with CS02-FL-NA (SEQ ID NO: 5). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.9% identity with CS02-FL-NA (SEQ ID NO: 5). In another embodiment, the codon-altered polynucleotide sequence is CS02-FL-NA (SEQ ID NO: 5).

[00141] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of less than 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of less than 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS02-FL-NA (SEQ ID NO: 5) has a GC content of less than 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of less than 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of less than 56%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of less than 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS02-FL-NA (SEQ ID NO: 5) has a GC content of less than 54%.

[00142] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 50% to 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS02-FL-NA (SEQ ID NO: 5) has a GC content of 50% to 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 50% to 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 50% to 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS02-FL-NA (SEQ ID NO: 5) has a GC content of 50% to 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 50% to 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 50% to 54%.

[00143] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 53.8%±1.0. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 53.8%±0.8. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 53.8%±0.6. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 53.8%±0.5. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 53.8%±0.4. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 53.8%±0.3. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 53.8%±0.2. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has a GC content of 53.8%±0.1. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS02-FL-NA (SEQ ID NO: 5) has a GC content of 53.8%.

[00144] In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 15 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 12 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 10 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 9 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 8 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 7 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 6 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 5 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 4 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 3 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 2 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) has no more than 1 CpG dinucleotide. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-FL-NA (SEQ ID NO: 5) does not contain CpG dinucleotides.

[00145] In some embodiments, the encoded Factor IX polypeptide, e.g., the polypeptide encoded by the polynucleotide having high sequence homology to CS02-FL-NA (SEQ ID NO: 5), has high sequence identity to the FIX-FL-AA preproprotein sequence ( SEQ ID NO: 2) wild-type factor IX and/or Padua variant FIXp-FL-AA pre-proprotein sequence (hFIX (R384L)) (SEQ ID NO: 4). The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing the signal peptide and propeptide and by removing the activating polypeptide).

[00146] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded Factor IX polypeptide has at least 96% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the encoded factor IX polypeptide sequence is FIX-FL-AA (SEQ ID NO: 2).

[00147] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-FL-AA (SEQ ID NO: 4).

[00148] In one embodiment, the nucleic acid composition provided herein comprises a factor IX polynucleotide (e.g., a codon-altered polynucleotide) encoding a single-stranded factor IX polypeptide (e.g., having a serine protease activity), wherein the factor IX polynucleotide comprises the nucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13). In some embodiments, a nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS02-MP-NA (SEQ ID NO: 13) has a reduced GC content compared to a wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)). In some embodiments, the nucleotide sequence of a Factor IX polynucleotide having a high sequence identity to CS02-MP-NA (SEQ ID NO: 13) has a reduced number of CpG dinucleotides compared to the wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)).

[00149] In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 95% identity with CS02-MP-NA (SEQ ID NO: 13). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 96% identity with CS02-MP-NA (SEQ ID NO: 13). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 97% identity with CS02-MP-NA (SEQ ID NO: 13). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 98% identity with CS02-MP-NA (SEQ ID NO: 13). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99% identity with CS02-MP-NA (SEQ ID NO: 13). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.5% identity with CS02-MP-NA (SEQ ID NO: 13). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.9% identity with CS02-MP-NA (SEQ ID NO: 13). In another embodiment, the codon-altered polynucleotide sequence is CS02-MP-NA (SEQ ID NO: 13).

[00150] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of less than 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of less than 59%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of less than 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of less than 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of less than 56%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of less than 55%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of less than 54%.

[00151] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 50% to 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 50% to 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 50% to 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 50% to 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 50% to 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 50% to 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 50% to 54%.

[00152] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 53.8%±1.0. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 53.8%±0.8. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 53.8%±0.6. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 53.8%±0.5. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 53.8%±0.4. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 53.8%±0.3. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 53.8%±0.2. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 53.8%±0.1. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has a GC content of 53.8%.

[00153] In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 15 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 12 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 10 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 9 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 8 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 7 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 6 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 5 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 4 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 3 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 2 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) has no more than 1 CpG dinucleotide. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS02-MP-NA (SEQ ID NO: 13) does not contain CpG dinucleotides.

[00154] In some embodiments, the high sequence identity of the factor IX polynucleotide to CS02-MP-NA (SEQ ID NO: 13) further comprises a factor IX signal polynucleotide encoding a factor IX signal peptide having the amino acid sequence FIX-SP-AA (SEQ ID No: 37). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-SP-NA (SEQ ID NO: 25 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-SP-NA (SEQ ID NO: 26 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-SP-NA (SEQ ID NO: 27 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-SP-NA (SEQ ID NO: 28 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-SP-NA (SEQ ID NO: 29 ).

[00155] In some embodiments, the high identity of the factor IX polynucleotide to CS02-MP-NA (SEQ ID NO: 13) further comprises a factor IX propeptide polynucleotide encoding a factor IX propeptide having the amino acid sequence FIX-PP-AA (SEQ ID NO: 38). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-PP-NA (SEQ ID NO: 31 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-PP-NA (SEQ ID NO: 32 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-PP-NA (SEQ ID NO: 33 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-PP-NA (SEQ ID NO: 34 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-PP-NA (SEQ ID NO: 35 ).

[00156] In some embodiments, the high identity of the factor IX polynucleotide to CS02-MP-NA (SEQ ID NO: 13) further includes a factor IX prepropeptide polynucleotide encoding a factor IX prepropeptide having the amino acid sequence FIX-PPP-AA (SEQ ID NO: 36). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-PPP-NA (SEQ ID NO: 19 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-PPP-NA (SEQ ID NO: 20 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-PPP-NA (SEQ ID NO: 21 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-PPP-NA (SEQ ID NO: 22 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-PPP-NA (SEQ ID NO: 23 ).

[00157] In some embodiments, the encoded Factor IX polypeptide, e.g., the polypeptide encoded by the polynucleotide having high sequence homology to CS02-FL-NA (SEQ ID NO: 5), has high sequence identity with the mature sequence of the single-stranded FIX factor IX polypeptide -MP-AA (SEQ ID NO: 10) and/or the mature single strand sequence of the Padua variant (hFIX (R384L)) FIXp-MP-AA (SEQ ID NO: 12). The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing any signal peptide and propeptide and by removing the activating polypeptide).

[00158] In one embodiment, the encoded factor IX polypeptide sequence has at least 85% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the encoded factor IX polypeptide sequence is FIX-MP-AA (SEQ ID NO: 10).

[00159] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-MP-AA (SEQ ID NO: 12).

[00160] In one embodiment, the codon-altered polynucleotides provided herein encode a single-stranded Factor IX polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the light chain to the N-terminus of the heavy chain. The factor IX polypeptide light chain is encoded by a first nucleotide sequence having high sequence identity with CS02-LC-NA (SEQ ID NO: 42), which is part of CS02-FL-NA (SEQ ID NO: 5) encoding the factor IX light chain. The factor IX polypeptide heavy chain is encoded by a second nucleotide sequence having high sequence identity with CS02-HC-NA (SEQ ID NO: 41), which is part of CS02-FL-NA (SEQ ID NO: 5) encoding the factor IX heavy chain. The polypeptide linker includes Factor XI cleavage sites that allow in vivo maturation (eg, following expression of a factor IX single chain polypeptide precursor).

[00161] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity with CS02-LC-NA and CS02-HC-NA (SEQ ID NOS: 42 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity with CS02-LC-NA and CS02-HC-NA (SEQ ID NOS: 42 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity with CS02-LC-NA and CS02-HC-NA (SEQ ID NOs: 42 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity with CS02-LC-NA and CS02-HC-NA (SEQ ID NOS: 42 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity with CS02-LC-NA and CS02-HC-NA (SEQ ID NOS: 42 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity with CS02-LC-NA and CS02-HC-NA (SEQ ID NOs: 42 and 41), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity with CS02-LC-NA and CS02-HC-NA (SEQ ID NOS: 42 and 41), respectively. In some embodiments, the first and second nucleotide sequences are CS02-LC-NA and CS02-HC-NA (SEQ ID NOs: 42 and 41), respectively.

[00162] In some embodiments, the factor IX construct polypeptide linker is encoded by a third nucleotide sequence having high sequence identity with CS02-AP-NA (SEQ ID NO: 57), which is a codon-altered sequence encoding a wild-type factor IX activation polypeptide, for example, amino acids 192-226 of FIX-FL-AA (SEQ ID NO: 2). In some embodiments, the implementation of the third nucleotide sequence has at least 80% identity with CS02-AP-NA (SEQ ID NO: 57). In some embodiments, the implementation of the third nucleotide sequence has at least 90% identity with CS02-AP-NA (SEQ ID NO: 57). In some embodiments, the implementation of the third nucleotide sequence has at least 95% identity with CS02-AP-NA (SEQ ID NO: 57). In some embodiments, the implementation of the third nucleotide sequence has at least 96% identity with CS02-AP-NA (SEQ ID NO: 57). In some embodiments, the implementation of the third nucleotide sequence has at least 97% identity with CS02-AP-NA (SEQ ID NO: 57). In some embodiments, the implementation of the third nucleotide sequence has at least 98% identity with CS02-AP-NA (SEQ ID NO: 57). In some embodiments, the implementation of the third nucleotide sequence has at least 99% identity with CS02-AP-NA (SEQ ID NO: 57). In some embodiments, the third nucleotide sequence is CS02-AP-NA (SEQ ID NO: 57).

[00163] In some embodiments, the encoded factor IX polypeptide also includes a signal peptide (eg, a factor IX signal peptide) and/or a propeptide (eg, a factor IX propeptide). In some embodiments, the signal peptide is a wild-type factor IX signal peptide (FIX-SP-AA (SEQ ID NO: 37)). In some embodiments, the signal peptide is encoded by a codon-altered polynucleotide sequence having high sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%) with CS02-SP-NA (SEQ ID NO: 25). In some embodiments, the propeptide is a wild-type factor IX propeptide (FIX-PP-AA (SEQ ID NO: 38)). In some embodiments, the propeptide peptide is encoded by a codon-altered polynucleotide sequence having high sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%) with CS02-PP-NA (SEQ ID NO: 31).

[00164] In some embodiments, a factor IX encoded polypeptide, e.g., a polynucleotide encoded by a polynucleotide having high sequence homology to CS02-LC-NA (SEQ ID NO:42) and CS02-HC-NA (SEQ ID NO:41), has high sequence identity with FIX-MP-AA (SEQ ID NO: 10) and/or mature FIXp-MP-AA (SEQ ID NO: 12) Padua variant single chain polypeptide (hFIX (R384L)) . The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing any signal peptide and propeptide and by removing the activating polypeptide).

[00165] In one embodiment, the encoded factor IX polypeptide sequence has at least 85% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the encoded factor IX polypeptide sequence is FIX-MP-AA (SEQ ID NO: 10).

[00166] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-MP-AA (SEQ ID NO: 12).

[00167] In some embodiments, with reference to Figure 1, a nucleic acid composition is provided that includes a self-complementary polynucleotide of structure A, wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has identity to at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% with CS02-MP-NA (SEQ ID NO: 13). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS02-FL-NA (SEQ ID NO: 5).

[00168] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a structure B self-complementary polynucleotide, wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has identity to at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% with CS02-MP-NA (SEQ ID NO: 13). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS02-FL-NA (SEQ ID NO: 5).

[00169] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a polynucleotide of structure C (e.g., a single-stranded polynucleotide), wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has an identity of at least 95%, 96%, 97% , 98%, 99%, 99.5%, 99.9%, or 100% with CS02-MP-NA (SEQ ID NO: 13). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS02-FL-NA (SEQ ID NO: 5).

[00170] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a polynucleotide of structure D (e.g., a single-stranded polynucleotide), wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has an identity of at least 95%, 96%, 97% , 98%, 99%, 99.5%, 99.9%, or 100% with CS02-MP-NA (SEQ ID NO: 13). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS02-FL-NA (SEQ ID NO: 5).

Codon-altered CS03 polynucleotides

[00171] In one embodiment, the nucleic acid composition provided herein comprises a factor IX polynucleotide (e.g., a codon-altered polynucleotide) encoding a single-stranded factor IX polypeptide, wherein the factor IX polynucleotide comprises a nucleotide sequence having a high sequence identity of CS03-FL -NA (SEQ ID NO: 6). In some embodiments, a nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS03-FL-NA (SEQ ID NO: 6) has a reduced GC content compared to a wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)). In some embodiments, the nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS03-FL-NA (SEQ ID NO: 6) has a reduced number of CpG dinucleotides compared to the wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)).

[00172] In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 95% identity with CS03-FL-NA (SEQ ID NO: 6). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 96% identity with CS03-FL-NA (SEQ ID NO: 6). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 97% identity with CS03-FL-NA (SEQ ID NO: 6). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 98% identity with CS03-FL-NA (SEQ ID NO: 6). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99% identity with CS03-FL-NA (SEQ ID NO: 6). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.5% identity with CS03-FL-NA (SEQ ID NO: 6). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.9% identity with CS03-FL-NA (SEQ ID NO: 6). In another embodiment, the codon-altered polynucleotide sequence is CS03-FL-NA (SEQ ID NO: 6).

[00173] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of less than 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of less than 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS03-FL-NA (SEQ ID NO: 6) has a GC content of less than 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS03-FL-NA (SEQ ID NO: 6) has a GC content of less than 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of less than 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of less than 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of less than 54%.

[00174] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 50% to 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 50% to 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 50% to 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 50% to 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 50% to 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 50% to 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 50% to 54%.

[00175] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 53.8%±1.0. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 53.8%±0.8. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 53.8% ±0.6. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 53.8%±0.5. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 53.8%±0.4. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 53.8%±0.3. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 53.8%±0.2. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has a GC content of 53.8%±0.1. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS03-FL-NA (SEQ ID NO: 6) has a GC content of 53.8%.

[00176] In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 15 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 12 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 10 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 9 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 8 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 7 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 6 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 5 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 4 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 3 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 2 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) has no more than 1 CpG dinucleotide. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-FL-NA (SEQ ID NO: 6) does not contain CpG dinucleotides.

[00177] In some embodiments, the encoded Factor IX polypeptide, e.g., the polypeptide encoded by the polynucleotide having high sequence homology to CS03-FL-NA (SEQ ID NO: 6), has high sequence identity to the FIX-FL-AA preproprotein sequence ( SEQ ID NO: 2) wild type factor IX and/or Padua variant FIXp-FL-AA preproprotein sequence (hFIX (R384L)) (SEQ ID NO: 4). The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing the signal peptide and propeptide and by removing the activating polypeptide).

[00178] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded Factor IX polypeptide has at least 96% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the encoded factor IX polypeptide sequence is FIX-FL-AA (SEQ ID NO: 2).

[00179] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-FL-AA (SEQ ID NO: 4).

[00180] In one embodiment, the nucleic acid composition provided herein comprises a factor IX polynucleotide (e.g., a codon-altered polynucleotide) encoding a single-stranded factor IX polypeptide (e.g., having serine protease activity), wherein the factor IX polynucleotide comprises the nucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14). In some embodiments, a nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS03-MP-NA (SEQ ID NO: 14) has a reduced GC content compared to a wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)). In some embodiments, the nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS03-MP-NA (SEQ ID NO: 14) has a reduced number of CpG dinucleotides compared to the wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)).

[00181] In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 95% identity with CS03-MP-NA (SEQ ID NO: 14). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 96% identity with CS03-MP-NA (SEQ ID NO: 14). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 97% identity with CS03-MP-NA (SEQ ID NO: 14). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 98% identity with CS03-MP-NA (SEQ ID NO: 14). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99% identity with CS03-MP-NA (SEQ ID NO: 14). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.5% identity with CS03-MP-NA (SEQ ID NO: 14). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.9% identity with CS03-MP-NA (SEQ ID NO: 14). In another embodiment, the codon-altered polynucleotide sequence is CS03-MP-NA (SEQ ID NO: 14).

[00182] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of less than 60%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of less than 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of less than 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of less than 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of less than 56%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of less than 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of less than 54%.

[00183] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 50% to 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 50% to 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 50% to 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 50% to 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 50% to 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 50% to 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 50% to 54%.

[00184] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 53.8%±1.0. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 53.8%±0.8. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 53.8%±0.6. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 53.8%±0.5. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 53.8%±0.4. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 53.8%±0.3. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 53.8%±0.2. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 53.8%±0.1. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has a GC content of 53.8%.

[00185] In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 15 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 12 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 10 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 9 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 8 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 7 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 6 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 5 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 4 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 3 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 2 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) has no more than 1 CpG dinucleotide. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS03-MP-NA (SEQ ID NO: 14) does not contain CpG dinucleotides.

[00186] In some embodiments, the high sequence identity of the factor IX polynucleotide to CS03-MP-NA (SEQ ID NO: 14) further comprises a factor IX signal polynucleotide encoding a factor IX signal peptide having the amino acid sequence FIX-SP-AA (SEQ ID No: 37). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-SP-NA (SEQ ID NO: 25 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-SP-NA (SEQ ID NO: 26 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-SP-NA (SEQ ID NO: 27 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-SP-NA (SEQ ID NO: 28 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-SP-NA (SEQ ID NO: 29 ).

[00187] In some embodiments, the high identity of the factor IX polynucleotide to CS03-MP-NA (SEQ ID NO: 14) further comprises a factor IX propeptide polynucleotide encoding a factor IX propeptide having the amino acid sequence FIX-PP-AA (SEQ ID NO: 38). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-PP-NA (SEQ ID NO: 31 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-PP-NA (SEQ ID NO: 32 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-PP-NA (SEQ ID NO: 33 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-PP-NA (SEQ ID NO: 34 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-PP-NA (SEQ ID NO: 35 ).

[00188] In some embodiments, the high identity of the factor IX polynucleotide to CS03-MP-NA (SEQ ID NO: 14) further includes a factor IX prepropeptide polynucleotide encoding a factor IX prepropeptide having the amino acid sequence FIX-PPP-AA (SEQ ID NO: 36). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-PPP-NA (SEQ ID NO: 19 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-PPP-NA (SEQ ID NO: 20 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-PPP-NA (SEQ ID NO: 21 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-PPP-NA (SEQ ID NO: 22 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-PPP-NA (SEQ ID NO: 23 ).

[00189] In some embodiments, the encoded Factor IX polypeptide, e.g., the polypeptide encoded by the polynucleotide having high sequence homology to CS03-FL-NA (SEQ ID NO: 6), has high sequence identity with the mature sequence of the single-stranded FIX factor IX polypeptide -MP-AA (SEQ ID NO: 10) and/or the mature single strand sequence of the Padua variant (hFIX (R384L)) FIXp-MP-AA (SEQ ID NO: 12). The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing any signal peptide and propeptide and by removing the activating polypeptide).

[00190] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the encoded factor IX polypeptide sequence is FIX-MP-AA (SEQ ID NO: 10).

[00191] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-MP-AA (SEQ ID NO: 12).

[00192] In one embodiment, the codon-altered polynucleotides provided herein encode a single-stranded Factor IX polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the light chain to the N-terminus of the heavy chain. The factor IX polypeptide light chain is encoded by a first nucleotide sequence having high sequence identity with CS03-LC-NA (SEQ ID NO: 44), which is part of CS03-FL-NA (SEQ ID NO: 6) encoding the factor IX light chain. The factor IX polypeptide heavy chain is encoded by a second nucleotide sequence having high sequence identity with CS03-HC-NA (SEQ ID NO: 43), which is part of CS03-FL-NA (SEQ ID NO: 6) encoding the factor IX heavy chain. The polypeptide linker includes Factor XI cleavage sites that allow in vivo maturation (eg, following expression of a factor IX single chain polypeptide precursor).

[00193] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity with CS03-LC-NA (SEQ ID NO: 44) and CS03-HC-NA (SEQ ID NO: 43), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity with CS03-LC-NA (SEQ ID NO: 44) and CS03-HC-NA (SEQ ID NO: 43), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity with CS03-LC-NA (SEQ ID NO: 44) and CS03-HC-NA (SEQ ID NO: 43), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity with CS03-LC-NA (SEQ ID NO: 44) and CS03-HC-NA (SEQ ID NO: 43), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity with CS03-LC-NA (SEQ ID NO: 44) and CS03-HC-NA (SEQ ID NO: 43), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity with CS03-LC-NA (SEQ ID NO: 44) and CS03-HC-NA (SEQ ID NO: 43), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity with CS03-LC-NA (SEQ ID NO: 44) and CS03-HC-NA (SEQ ID NO: 43), respectively. In some embodiments, the first and second nucleotide sequences are CS03-LC-NA (SEQ ID NO: 44) and CS03-HC-NA (SEQ ID NO: 43), respectively.

[00194] In some embodiments, the factor IX construct polypeptide linker is encoded by a third nucleotide sequence having high sequence identity with CS03-AP-NA (SEQ ID NO: 58), which is a codon-altered sequence encoding a wild-type factor IX activation polypeptide, for example, amino acids 192-226 of FIX-FL-AA (SEQ ID NO: 2). In some embodiments, the implementation of the third nucleotide sequence has at least 80% identity with CS03-AP-NA (SEQ ID NO: 58). In some embodiments, the implementation of the third nucleotide sequence has at least 90% identity with CS03-AP-NA (SEQ ID NO: 58). In some embodiments, the implementation of the third nucleotide sequence has at least 95% identity with CS03-AP-NA (SEQ ID NO: 58). In some embodiments, the implementation of the third nucleotide sequence has at least 96% identity with CS03-AP-NA (SEQ ID NO: 58). In some embodiments, the implementation of the third nucleotide sequence has at least 97% identity with CS03-AP-NA (SEQ ID NO: 58). In some embodiments, the implementation of the third nucleotide sequence has at least 98% identity with CS03-AP-NA (SEQ ID NO: 58). In some embodiments, the implementation of the third nucleotide sequence has at least 99% identity with CS03-AP-NA (SEQ ID NO: 58). In some embodiments, the third nucleotide sequence is CS03-AP-NA (SEQ ID NO: 58).

[00195] In some embodiments, the encoded factor IX polypeptide also includes a signal peptide (eg, a factor IX signal peptide) and/or a propeptide (eg, a factor IX propeptide). In some embodiments, the signal peptide is a wild-type factor IX signal peptide (FIX-SP-AA (SEQ ID NO: 37)). In some embodiments, the signal peptide is encoded by a codon-altered polynucleotide sequence having high sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%) with CS03-SP-NA (SEQ ID NO: 26). In some embodiments, the propeptide is a wild-type factor IX propeptide (FIX-PP-AA (SEQ ID NO: 38)). In some embodiments, the propeptide peptide is encoded by a codon-altered polynucleotide sequence having high sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%) with CS03-PP-NA (SEQ ID NO: 32).

[00196] In some embodiments, a factor IX encoded polypeptide, e.g., a polynucleotide encoded by a polynucleotide having high sequence homology to CS03-LC-NA (SEQ ID NO:44) and CS03-HC-NA (SEQ ID NO:43), has high sequence identity with FIX-MP-AA (SEQ ID NO: 10) and/or mature FIXp-MP-AA (SEQ ID NO: 12) Padua variant single chain polypeptide (hFIX (R384L)) . The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing any signal peptide and propeptide and by removing the activating polypeptide).

[00197] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the encoded factor IX polypeptide sequence is FIX-MP-AA (SEQ ID NO: 10).

[00198] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-MP-AA (SEQ ID NO: 12).

[00199] In some embodiments, with reference to Figure 1, a nucleic acid composition is provided that includes a self-complementary polynucleotide of structure A, wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has identity to at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% with CS03-MP-NA (SEQ ID NO: 14). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS03-FL-NA (SEQ ID NO: 6).

[00200] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a structure B self-complementary polynucleotide, wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature Factor IX polypeptide that has identity to at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% with CS03-MP-NA (SEQ ID NO: 14). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS03-FL-NA (SEQ ID NO: 6).

[00201] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a polynucleotide of structure C (e.g., a single-stranded polynucleotide), wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has an identity of at least 95%, 96%, 97% , 98%, 99%, 99.5%, 99.9%, or 100% with CS03-MP-NA (SEQ ID NO: 14). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS03-FL-NA (SEQ ID NO: 6).

[00202] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a polynucleotide of structure D (e.g., a single-stranded polynucleotide), wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has an identity of at least 95%, 96%, 97% , 98%, 99%, 99.5%, 99.9%, or 100% with CS03-MP-NA (SEQ ID NO: 14). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS03-FL-NA (SEQ ID NO: 6).

Codon-altered CS04 polynucleotides

[00203] In one embodiment, the nucleic acid composition provided herein comprises a factor IX polynucleotide (e.g., a codon-altered polynucleotide) encoding a single-stranded factor IX polypeptide, wherein the factor IX polynucleotide comprises a nucleotide sequence having a high sequence identity of CS04-FL -NA (SEQ ID NO: 7). In some embodiments, a nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a reduced GC content compared to a wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)). In some embodiments, the nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a reduced number of CpG dinucleotides compared to the wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)).

[00204] In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 95% identity with CS04-FL-NA (SEQ ID NO: 7). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 96% identity with CS04-FL-NA (SEQ ID NO: 7). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 97% identity with CS04-FL-NA (SEQ ID NO: 7). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 98% identity with CS04-FL-NA (SEQ ID NO: 7). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99% identity with CS04-FL-NA (SEQ ID NO: 7). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.5% identity with CS04-FL-NA (SEQ ID NO: 7). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.9% identity with CS04-FL-NA (SEQ ID NO: 7). In another embodiment, the sequence of the codon-altered polynucleotide is CS04-FL-NA (SEQ ID NO: 7).

[00205] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of less than 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of less than 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a GC content of less than 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a GC content of less than 57%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of less than 56%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of less than 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a GC content of less than 54%.

[00206] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of 50% to 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a GC content of 50% to 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a GC content of 50% to 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a GC content of 50% to 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of 50% to 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a GC content of 50% to 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of 50% to 54%.

[00207] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of 53.8%±1.0. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of 53.8%±0.8. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of 53.8%±0.6. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of 53.8%±0.5. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of 53.8%±0.4. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a GC content of 53.8%±0.3. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of 53.8%±0.2. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has a GC content of 53.8%±0.1. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS04-FL-NA (SEQ ID NO: 7) has a GC content of 53.8%.

[00208] In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 15 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 12 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 10 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 9 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 8 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 7 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 6 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 5 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 4 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 3 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 2 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) has no more than 1 CpG dinucleotide. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-FL-NA (SEQ ID NO: 7) does not contain CpG dinucleotides.

[00209] In some embodiments, the encoded Factor IX polypeptide, e.g., the polypeptide encoded by the polynucleotide having high sequence homology to CS04-FL-NA (SEQ ID NO: 7), has high sequence identity to the FIX-FL-AA preproprotein sequence ( SEQ ID NO: 2) wild type factor IX and/or Padua variant FIXp-FL-AA preproprotein sequence (hFIX (R384L)) (SEQ ID NO: 4). The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing the signal peptide and propeptide and by removing the activating polypeptide).

[00210] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded Factor IX polypeptide has at least 96% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the encoded factor IX polypeptide sequence is FIX-FL-AA (SEQ ID NO: 2).

[00211] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-FL-AA (SEQ ID NO: 4).

[00212] In one embodiment, the nucleic acid composition provided herein comprises a factor IX polynucleotide (e.g., a codon-altered polynucleotide) encoding a single-stranded factor IX polypeptide (e.g., having serine protease activity), wherein the factor IX polynucleotide comprises the nucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15). In some embodiments, a nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS04-MP-NA (SEQ ID NO: 15) has a reduced GC content compared to a wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)). In some embodiments, the nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS04-MP-NA (SEQ ID NO: 15) has a reduced number of CpG dinucleotides compared to the wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)).

[00213] In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 95% identity with CS04-MP-NA (SEQ ID NO: 15). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 96% identity with CS04-MP-NA (SEQ ID NO: 15). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 97% identity with CS04-MP-NA (SEQ ID NO: 15). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 98% identity with CS04-MP-NA (SEQ ID NO: 15). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99% identity with CS04-MP-NA (SEQ ID NO: 15). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.5% identity with CS04-MP-NA (SEQ ID NO: 15). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.9% identity with CS04-MP-NA (SEQ ID NO: 15). In another embodiment, the codon-altered polynucleotide sequence is CS04-MP-NA (SEQ ID NO: 15).

[00214] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of less than 60%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of less than 59%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of less than 58%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of less than 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of less than 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of less than 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of less than 54%.

[00215] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 50% to 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 50% to 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 50% to 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 50% to 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 50% to 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 50% to 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 50% to 54%.

[00216] In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 53.8%±1.0. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 53.8%±0.8. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 53.8%±0.6. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 53.8%±0.5. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 53.8%±0.4. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 53.8%±0.3. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 53.8%±0.2. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 53.8%±0.1. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has a GC content of 53.8%.

[00217] In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 15 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 12 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 10 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 9 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 8 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 7 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 6 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 5 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 4 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 3 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 2 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) has no more than 1 CpG dinucleotide. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS04-MP-NA (SEQ ID NO: 15) does not contain CpG dinucleotides.

[00218] In some embodiments, the high sequence identity of the factor IX polynucleotide to CS04-MP-NA (SEQ ID NO: 15) further comprises a factor IX signal polynucleotide encoding a factor IX signal peptide having the amino acid sequence FIX-SP-AA (SEQ ID No: 37). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-SP-NA (SEQ ID NO: 25 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-SP-NA (SEQ ID NO: 26 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-SP-NA (SEQ ID NO: 27 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-SP-NA (SEQ ID NO: 28 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-SP-NA (SEQ ID NO: 29 ).

[00219] In some embodiments, the high identity of the factor IX polynucleotide to CS04-MP-NA (SEQ ID NO: 15) further comprises a factor IX propeptide polynucleotide encoding a factor IX propeptide having the amino acid sequence FIX-PP-AA (SEQ ID NO: 38). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-PP-NA (SEQ ID NO: 31 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-PP-NA (SEQ ID NO: 32 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-PP-NA (SEQ ID NO: 33 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-PP-NA (SEQ ID NO: 34 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-PP-NA (SEQ ID NO: 35 ).

[00220] In some embodiments, the high identity of the factor IX polynucleotide to CS04-MP-NA (SEQ ID NO: 15) further includes a factor IX prepropeptide polynucleotide encoding a factor IX prepropeptide having the amino acid sequence FIX-PPP-AA (SEQ ID NO: 36). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-PPP-NA (SEQ ID NO: 19 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-PPP-NA (SEQ ID NO: 20 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-PPP-NA (SEQ ID NO: 21 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-PPP-NA (SEQ ID NO: 22 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-PPP-NA (SEQ ID NO: 23 ).

[00221] In some embodiments, an encoded Factor IX polypeptide, e.g., a polypeptide encoded by a polynucleotide having high sequence homology to CS04-FL-NA (SEQ ID NO: 7), has high sequence identity with the mature sequence of a single-stranded FIX factor IX polypeptide -MP-AA (SEQ ID NO: 10) and/or the mature single strand sequence of the Padua variant (hFIX (R384L)) FIXp-MP-AA (SEQ ID NO: 12). The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing any signal peptide and propeptide and by removing the activating polypeptide).

[00222] In one embodiment, the encoded factor IX polypeptide sequence has at least 85% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the encoded factor IX polypeptide sequence is FIX-MP-AA (SEQ ID NO: 10).

[00223] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-MP-AA (SEQ ID NO: 12).

[00224] In one embodiment, the codon-altered polynucleotides provided herein encode a single-stranded Factor IX polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the light chain to the N-terminus of the heavy chain. The factor IX polypeptide light chain is encoded by a first nucleotide sequence having high sequence identity with CS04-LC-NA (SEQ ID NO: 46), which is part of CS04-FL-NA (SEQ ID NO: 7) encoding the factor IX light chain. The factor IX polypeptide heavy chain is encoded by a second nucleotide sequence having high sequence identity with CS04-HC-NA (SEQ ID NO: 45), which is part of CS04-FL-NA (SEQ ID NO: 7) encoding the factor IX heavy chain. The polypeptide linker includes Factor XI cleavage sites that allow in vivo maturation (eg, following expression of a factor IX single chain polypeptide precursor).

[00225] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity with CS04-LC-NA and CS04-HC-NA (SEQ ID NOS: 46 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity with CS04-LC-NA and CS04-HC-NA (SEQ ID NOS: 46 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity with CS04-LC-NA and CS04-HC-NA (SEQ ID NOS: 46 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity with CS04-LC-NA and CS04-HC-NA (SEQ ID NOS: 46 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity with CS04-LC-NA and CS04-HC-NA (SEQ ID NOS: 46 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity with CS04-LC-NA and CS04-HC-NA (SEQ ID NOS: 46 and 45), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity with CS04-LC-NA and CS04-HC-NA (SEQ ID NOS: 46 and 45), respectively. In some embodiments, the first and second nucleotide sequences are CS04-LC-NA and CS04-HC-NA (SEQ ID NOS: 46 and 45), respectively.

[00226] In some embodiments, the factor IX construct polypeptide linker is encoded by a third nucleotide sequence having high sequence identity with CS04-AP-NA (SEQ ID NO: 59), which is a codon-altered sequence encoding a wild type factor IX activation polypeptide, for example, amino acids 192-226 of FIX-FL-AA (SEQ ID NO: 2). In some embodiments, the implementation of the third nucleotide sequence has at least 80% identity with CS04-AP-NA (SEQ ID NO: 59). In some embodiments, the implementation of the third nucleotide sequence has at least 90% identity with CS04-AP-NA (SEQ ID NO: 59). In some embodiments, the implementation of the third nucleotide sequence has at least 95% identity with CS04-AP-NA (SEQ ID NO: 59). In some embodiments, the implementation of the third nucleotide sequence has at least 96% identity with CS04-AP-NA (SEQ ID NO: 59). In some embodiments, the implementation of the third nucleotide sequence has at least 97% identity with CS04-AP-NA (SEQ ID NO: 59). In some embodiments, the implementation of the third nucleotide sequence has at least 98% identity with CS04-AP-NA (SEQ ID NO: 59). In some embodiments, the implementation of the third nucleotide sequence has at least 99% identity with CS04-AP-NA (SEQ ID NO: 59). In some embodiments, the third nucleotide sequence is CS04-AP-NA (SEQ ID NO: 59).

[00227] In some embodiments, the encoded factor IX polypeptide also includes a signal peptide (eg, a factor IX signal peptide) and/or a propeptide (eg, a factor IX propeptide). In some embodiments, the signal peptide is a wild-type factor IX signal peptide (FIX-SP-AA (SEQ ID NO: 37)). In some embodiments, the signal peptide is encoded by a codon-altered polynucleotide sequence having high sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%) with CS04-SP-NA (SEQ ID NO: 27). In some embodiments, the propeptide is a wild-type factor IX propeptide (FIX-PP-AA (SEQ ID NO: 38)). In some embodiments, the propeptide peptide is encoded by a codon-altered polynucleotide sequence having high sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%) with CS04-PP-NA (SEQ ID NO: 33).

[00228] In some embodiments, a factor IX encoded polypeptide, e.g., a polynucleotide encoded by a polynucleotide having high sequence homology to CS04-LC-NA (SEQ ID NO:46) and CS04-HC-NA (SEQ ID NO:45), has high sequence identity with FIX-MP-AA (SEQ ID NO: 10) and/or mature FIXp-MP-AA (SEQ ID NO: 12) Padua variant single chain polypeptide (hFIX (R384L)) . The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing any signal peptide and propeptide and by removing the activating polypeptide).

[00229] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the encoded factor IX polypeptide sequence is FIX-MP-AA (SEQ ID NO: 10).

[00230] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-MP-AA (SEQ ID NO: 12).

[00231] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a self-complementary polynucleotide of structure A, wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has identity to at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% with CS04-MP-NA (SEQ ID NO: 15). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS04-FL-NA (SEQ ID NO: 7).

[00232] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a structure B self-complementary polynucleotide, wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature Factor IX polypeptide that has identity to at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% with CS04-MP-NA (SEQ ID NO: 15). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS04-FL-NA (SEQ ID NO: 7).

[00233] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a polynucleotide of structure C (e.g., a single-stranded polynucleotide), wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has an identity of at least 95%, 96%, 97% , 98%, 99%, 99.5%, 99.9% or 100% with CS04-MP-NA (SEQ ID NO: 15). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS04-FL-NA (SEQ ID NO: 7).

[00234] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a polynucleotide of structure D (e.g., a single-stranded polynucleotide), wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has an identity of at least 95%, 96%, 97% , 98%, 99%, 99.5%, 99.9%, or 100% with CS04-MP-NA (SEQ ID NO: 15). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS04-FL-NA (SEQ ID NO: 7).

Codon-altered CS05 polynucleotides

[00235] In one embodiment, the nucleic acid composition provided herein comprises a factor IX polynucleotide (e.g., a codon-altered polynucleotide) encoding a single-stranded factor IX polypeptide, wherein the factor IX polynucleotide comprises a nucleotide sequence having a high sequence identity of CS05-FL -NA (SEQ ID NO: 8). In some embodiments, a nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS05-FL-NA (SEQ ID NO: 8) has a reduced GC content compared to a wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)). In some embodiments, the nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS05-FL-NA (SEQ ID NO: 8) has a reduced number of CpG dinucleotides compared to the wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)).

[00236] In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 95% identity with CS05-FL-NA (SEQ ID NO: 8). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 96% identity with CS05-FL-NA (SEQ ID NO: 8). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 97% identity with CS05-FL-NA (SEQ ID NO: 8). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 98% identity with CS05-FL-NA (SEQ ID NO: 8). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99% identity with CS05-FL-NA (SEQ ID NO: 8). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.5% identity with CS05-FL-NA (SEQ ID NO: 8). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.9% identity with CS05-FL-NA (SEQ ID NO: 8). In another embodiment, the codon-altered polynucleotide sequence is CS05-FL-NA (SEQ ID NO: 8).

[00237] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of less than 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS05-FL-NA (SEQ ID NO: 8) has a GC content of less than 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of less than 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of less than 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of less than 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of less than 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of less than 54%.

[00238] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 50% to 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 50% to 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 50% to 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 50% to 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 50% to 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 50% to 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 50% to 54%.

[00239] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 53.8%±1.0. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 53.8%±0.8. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 53.8% ±0.6. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 53.8%±0.5. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 53.8%±0.4. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 53.8%±0.3. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 53.8%±0.2. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has a GC content of 53.8%±0.1. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS05-FL-NA (SEQ ID NO: 8) has a GC content of 53.8%.

[00240] In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 15 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 12 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 10 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 9 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 8 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 7 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 6 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 5 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 4 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 3 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 2 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) has no more than 1 CpG dinucleotide. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-FL-NA (SEQ ID NO: 8) does not contain CpG dinucleotides.

[00241] In some embodiments, the encoded factor IX polypeptide, e.g., the polypeptide encoded by the polynucleotide having high sequence homology to CS05-FL-NA (SEQ ID NO: 8), has high sequence identity to the FIX-FL-AA preproprotein sequence ( SEQ ID NO: 2) wild type factor IX and/or Padua variant FIXp-FL-AA preproprotein sequence (hFIX (R384L)) (SEQ ID NO: 4). The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing the signal peptide and propeptide and by removing the activating polypeptide).

[00242] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded Factor IX polypeptide has at least 96% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the encoded factor IX polypeptide sequence is FIX-FL-AA (SEQ ID NO: 2).

[00243] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-FL-AA (SEQ ID NO: 4).

[00244] In one embodiment, the nucleic acid composition provided herein comprises a factor IX polynucleotide (e.g., a codon-altered polynucleotide) encoding a single-stranded factor IX polypeptide (e.g., having a serine protease activity), wherein the factor IX polynucleotide comprises the nucleotide sequence having a high sequence identity with CS05-MP-NA (SEQ ID NO: 16). In some embodiments, a nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS05-MP-NA (SEQ ID NO: 16) has a reduced GC content compared to a wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)). In some embodiments, the nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS05-MP-NA (SEQ ID NO: 16) has a reduced number of CpG dinucleotides compared to the wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)).

[00245] In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 95% identity with CS05-MP-NA (SEQ ID NO: 16). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 96% identity with CS05-MP-NA (SEQ ID NO: 16). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 97% identity with CS05-MP-NA (SEQ ID NO: 16). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 98% identity with CS05-MP-NA (SEQ ID NO: 16). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99% identity with CS05-MP-NA (SEQ ID NO: 16). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.5% identity with CS05-MP-NA (SEQ ID NO: 16). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.9% identity with CS05-MP-NA (SEQ ID NO: 16). In another embodiment, the codon-altered polynucleotide sequence is CS05-MP-NA (SEQ ID NO: 16).

[00246] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of less than 60%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of less than 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of less than 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of less than 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of less than 56%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of less than 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of less than 54%.

[00247] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 50% to 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 50% to 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 50% to 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 50% to 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 50% to 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 50% to 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 50% to 54%.

[00248] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 53.8%±1.0. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 53.8%±0.8. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 53.8%±0.6. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 53.8%±0.5. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 53.8%±0.4. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 53.8%±0.3. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 53.8%±0.2. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 53.8%±0.1. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has a GC content of 53.8%.

[00249] In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 15 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 12 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 10 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 9 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 8 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 7 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 6 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 5 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 4 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 3 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 2 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) has no more than 1 CpG dinucleotide. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS05-MP-NA (SEQ ID NO: 16) does not contain CpG dinucleotides.

[00250] In some embodiments, the high sequence identity of the factor IX polynucleotide with CS05-MP-NA (SEQ ID NO: 16) further comprises a factor IX signal polynucleotide encoding a factor IX signal peptide having the amino acid sequence FIX-SP-AA (SEQ ID No: 37). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-SP-NA (SEQ ID NO: 25 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-SP-NA (SEQ ID NO: 26 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-SP-NA (SEQ ID NO: 27 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-SP-NA (SEQ ID NO: 28 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-SP-NA (SEQ ID NO: 29 ).

[00251] In some embodiments, the high identity of the factor IX polynucleotide to CS05-MP-NA (SEQ ID NO: 16) further comprises a factor IX propeptide polynucleotide encoding a factor IX propeptide having the amino acid sequence FIX-PP-AA (SEQ ID NO: 38). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-PP-NA (SEQ ID NO: 31 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-PP-NA (SEQ ID NO: 32 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-PP-NA (SEQ ID NO: 33 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-PP-NA (SEQ ID NO: 34 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-PP-NA (SEQ ID NO: 35 ).

[00252] In some embodiments, the high identity of the factor IX polynucleotide to CS05-MP-NA (SEQ ID NO: 16) further includes a factor IX prepropeptide polynucleotide encoding a factor IX prepropeptide having the amino acid sequence FIX-PPP-AA (SEQ ID NO: 36). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-PPP-NA (SEQ ID NO: 19 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-PPP-NA (SEQ ID NO: 20 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-PPP-NA (SEQ ID NO: 21 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-PPP-NA (SEQ ID NO: 22 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-PPP-NA (SEQ ID NO: 23 ).

[00253] In some embodiments, an encoded Factor IX polypeptide, e.g., a polypeptide encoded by a polynucleotide having high sequence homology to CS05-FL-NA (SEQ ID NO: 8), has high sequence identity with the mature sequence of a single-stranded FIX factor IX polypeptide -MP-AA (SEQ ID NO: 10) and/or the mature single strand sequence of the Padua variant (hFIX (R384L)) FIXp-MP-AA (SEQ ID NO: 12). The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing any signal peptide and propeptide and by removing the activating polypeptide).

[00254] In one embodiment, the encoded factor IX polypeptide sequence has at least 85% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the encoded factor IX polypeptide sequence is FIX-MP-AA (SEQ ID NO: 10).

[00255] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-MP-AA (SEQ ID NO: 12).

[00256] In one embodiment, the codon-altered polynucleotides provided herein encode a single-stranded Factor IX polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the light chain to the N-terminus of the heavy chain. The factor IX polypeptide light chain is encoded by a first nucleotide sequence having high sequence identity with CS05-LC-NA (SEQ ID NO: 48), which is part of CS05-FL-NA (SEQ ID NO: 8) encoding the factor IX light chain. The factor IX polypeptide heavy chain is encoded by a second nucleotide sequence having high sequence identity with CS05-HC-NA (SEQ ID NO: 47), which is part of CS05-FL-NA (SEQ ID NO: 8) encoding the factor IX heavy chain. The polypeptide linker includes Factor XI cleavage sites that allow in vivo maturation (eg, following expression of a factor IX single chain polypeptide precursor).

[00257] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity with CS05-LC-NA and CS05-HC-NA (SEQ ID NOS: 48 and 47), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity with CS05-LC-NA and CS05-HC-NA (SEQ ID NOS: 48 and 47), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity with CS05-LC-NA and CS05-HC-NA (SEQ ID NOS: 48 and 47), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity with CS05-LC-NA and CS05-HC-NA (SEQ ID NOS: 48 and 47), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity with CS05-LC-NA and CS05-HC-NA (SEQ ID NOS: 48 and 47), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity with CS05-LC-NA and CS05-HC-NA (SEQ ID NOS: 48 and 47), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity with CS05-LC-NA and CS05-HC-NA (SEQ ID NOS: 48 and 47), respectively. In some embodiments, the first and second nucleotide sequences are CS05-LC-NA and CS05-HC-NA (SEQ ID NOs: 48 and 47), respectively.

[00258] In some embodiments, the factor IX construct polypeptide linker is encoded by a third nucleotide sequence having high sequence identity with CS05-AP-NA (SEQ ID NO: 60), which is a codon-altered sequence encoding a wild type factor IX activation polypeptide, for example, amino acids 192-226 of FIX-FL-AA (SEQ ID NO: 2). In some embodiments, the implementation of the third nucleotide sequence has at least 80% identity with CS05-AP-NA (SEQ ID NO: 60). In some embodiments, the implementation of the third nucleotide sequence has at least 90% identity with CS05-AP-NA (SEQ ID NO: 60). In some embodiments, the implementation of the third nucleotide sequence has at least 95% identity with CS05-AP-NA (SEQ ID NO: 60). In some embodiments, the implementation of the third nucleotide sequence has at least 96% identity with CS05-AP-NA (SEQ ID NO: 60). In some embodiments, the implementation of the third nucleotide sequence has at least 97% identity with CS05-AP-NA (SEQ ID NO: 60). In some embodiments, the implementation of the third nucleotide sequence has at least 98% identity with CS05-AP-NA (SEQ ID NO: 60). In some embodiments, the implementation of the third nucleotide sequence has at least 99% identity with CS05-AP-NA (SEQ ID NO: 60). In some embodiments, the third nucleotide sequence is CS05-AP-NA (SEQ ID NO: 60).

[00259] In some embodiments, the encoded factor IX polypeptide also includes a signal peptide (eg, a factor IX signal peptide) and/or a propeptide (eg, a factor IX propeptide). In some embodiments, the signal peptide is a wild-type factor IX signal peptide (FIX-SP-AA (SEQ ID NO: 37)). In some embodiments, the signal peptide is encoded by a codon-altered polynucleotide sequence having high sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%) with CS05-SP-NA (SEQ ID NO: 28). In some embodiments, the propeptide is a wild-type factor IX propeptide (FIX-PP-AA (SEQ ID NO: 38)). In some embodiments, the propeptide peptide is encoded by a codon-altered polynucleotide sequence having high sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%) with CS05-PP-NA (SEQ ID NO: 34).

[00260] In some embodiments, a factor IX encoded polypeptide, e.g., a polynucleotide encoded by a polynucleotide having high sequence homology to CS05-LC-NA (SEQ ID NO:48) and CS05-HC-NA (SEQ ID NO:47), has high sequence identity with FIX-MP-AA (SEQ ID NO: 10) and/or mature FIXp-MP-AA (SEQ ID NO: 12) Padua variant single chain polypeptide (hFIX (R384L)) . The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing any signal peptide and propeptide and by removing the activating polypeptide).

[00261] In one embodiment, the encoded factor IX polypeptide sequence has at least 85% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the encoded factor IX polypeptide sequence is FIX-MP-AA (SEQ ID NO: 10).

[00262] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-MP-AA (SEQ ID NO: 12).

[00263] In some embodiments, with reference to Figure 1, a nucleic acid composition is provided that includes a self-complementary polynucleotide of structure A, wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature Factor IX polypeptide that has identity to at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% with CS03-MP-NA (SEQ ID NO: 14). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS05-FL-NA (SEQ ID NO: 8).

[00264] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a structure B self-complementary polynucleotide, wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature Factor IX polypeptide that has identity to at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% with CS05-MP-NA (SEQ ID NO: 16). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS05-FL-NA (SEQ ID NO: 8).

[00265] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a polynucleotide of structure C (e.g., a single-stranded polynucleotide), wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has an identity of at least 95%, 96%, 97% , 98%, 99%, 99.5%, 99.9%, or 100% with CS05-MP-NA (SEQ ID NO: 16). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS05-FL-NA (SEQ ID NO: 8).

[00266] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a polynucleotide of structure D (e.g., a single-stranded polynucleotide), wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has an identity of at least 95%, 96%, 97% , 98%, 99%, 99.5%, 99.9%, or 100% with CS05-MP-NA (SEQ ID NO: 16). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS05-FL-NA (SEQ ID NO: 8).

Codon-altered CS06 polynucleotides

[00267] In one embodiment, the nucleic acid composition provided herein comprises a factor IX polynucleotide (e.g., a codon-altered polynucleotide) encoding a single-stranded factor IX polypeptide, wherein the factor IX polynucleotide comprises a nucleotide sequence having a high sequence identity of CS06-FL -NA (SEQ ID NO: 9). In some embodiments, a nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a reduced GC content compared to a wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)). In some embodiments, the nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a reduced number of CpG dinucleotides compared to the wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)).

[00268] In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 95% identity with CS06-FL-NA (SEQ ID NO: 9). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 96% identity with CS06-FL-NA (SEQ ID NO: 9). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 97% identity with CS06-FL-NA (SEQ ID NO: 9). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 98% identity with CS06-FL-NA (SEQ ID NO: 9). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99% identity with CS06-FL-NA (SEQ ID NO: 9). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.5% identity with CS06-FL-NA (SEQ ID NO: 9). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.9% identity with CS06-FL-NA (SEQ ID NO: 9). In another embodiment, the codon-altered polynucleotide sequence is CS06-FL-NA (SEQ ID NO: 9).

[00269] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of less than 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a GC content of less than 59%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of less than 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a GC content of less than 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a GC content of less than 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a GC content of less than 55%. In some embodiments, a codon-altered polynucleotide sequence having a high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a GC content of less than 54%.

[00270] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 50% to 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a GC content of 50% to 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 50% to 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a GC content of 50% to 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 50% to 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 50% to 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 50% to 54%.

[00271] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 53.8%±1.0. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a GC content of 53.8%±0.8. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 53.8%±0.6. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 53.8%±0.5. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 53.8%±0.4. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 53.8%±0.3. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 53.8%±0.2. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has a GC content of 53.8%±0.1. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity to CS06-FL-NA (SEQ ID NO: 9) has a GC content of 53.8%.

[00272] In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 15 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 12 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 10 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 9 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 8 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 7 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 6 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 5 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 4 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 3 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 2 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) has no more than 1 CpG dinucleotide. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-FL-NA (SEQ ID NO: 9) does not contain CpG dinucleotides.

[00273] In some embodiments, the encoded Factor IX polypeptide, e.g., the polypeptide encoded by the polynucleotide having high sequence homology to CS06-FL-NA (SEQ ID NO: 9), has high sequence identity to the FIX-FL-AA preproprotein sequence ( SEQ ID NO: 2) wild-type factor IX and/or Padua variant FIXp-FL-AA pre-proprotein sequence (hFIX (R384L)) (SEQ ID NO: 4). The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing the signal peptide and propeptide and by removing the activating polypeptide).

[00274] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded Factor IX polypeptide has at least 96% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-FL-AA (SEQ ID NO: 2). In one embodiment, the encoded factor IX polypeptide sequence is FIX-FL-AA (SEQ ID NO: 2).

[00275] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-FL-AA (SEQ ID NO: 4) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-FL-AA (SEQ ID NO: 4).

[00276] In one embodiment, the nucleic acid composition provided herein comprises a factor IX polynucleotide (e.g., a codon-altered polynucleotide) encoding a single-stranded factor IX polypeptide (e.g., having a serine protease activity), wherein the factor IX polynucleotide comprises the nucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17). In some embodiments, a nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS06-MP-NA (SEQ ID NO: 17) has a reduced GC content compared to a wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)). In some embodiments, the nucleotide sequence of a factor IX polynucleotide having a high sequence identity to CS06-MP-NA (SEQ ID NO: 17) has a reduced number of CpG dinucleotides compared to the wild-type factor IX coding sequence (FIX-FL-NA (SEQ ID NO:1)).

[00277] In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 95% identity with CS06-MP-NA (SEQ ID NO: 17). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 96% identity with CS06-MP-NA (SEQ ID NO: 17). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 97% identity with CS06-MP-NA (SEQ ID NO: 17). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 98% identity with CS06-MP-NA (SEQ ID NO: 17). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99% identity with CS06-MP-NA (SEQ ID NO: 17). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.5% identity with CS06-MP-NA (SEQ ID NO: 17). In a specific embodiment, the sequence of the codon-altered polynucleotide has at least 99.9% identity with CS06-MP-NA (SEQ ID NO: 17). In another embodiment, the codon-altered polynucleotide sequence is CS06-MP-NA (SEQ ID NO: 17).

[00278] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of less than 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of less than 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of less than 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of less than 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of less than 56%. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of less than 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of less than 54%.

[00279] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 50% to 60%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 50% to 59%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 50% to 58%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 50% to 57%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 50% to 56%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 50% to 55%. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 50% to 54%.

[00280] In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 53.8%±1.0. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 53.8%±0.8. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 53.8%±0.6. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 53.8%±0.5. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 53.8%±0.4. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 53.8%±0.3. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 53.8%±0.2. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 53.8%±0.1. In some embodiments, a codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has a GC content of 53.8%.

[00281] In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 15 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 12 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 10 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 9 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 8 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 7 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 6 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 5 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 4 CpG dinucleotides. In some embodiments, the sequence of a codon-altered polynucleotide having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 3 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 2 CpG dinucleotides. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) has no more than 1 CpG dinucleotide. In some embodiments, the codon-altered polynucleotide sequence having high sequence identity with CS06-MP-NA (SEQ ID NO: 17) does not contain CpG dinucleotides.

[00282] In some embodiments, the high sequence identity of the factor IX polynucleotide to CS06-MP-NA (SEQ ID NO: 17) further comprises a factor IX signal polynucleotide encoding a factor IX signal peptide having the amino acid sequence FIX-SP-AA (SEQ ID No: 37). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-SP-NA (SEQ ID NO: 25 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-SP-NA (SEQ ID NO: 26 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-SP-NA (SEQ ID NO: 27 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-SP-NA (SEQ ID NO: 28 ). In some embodiments, the factor IX signal polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-SP-NA (SEQ ID NO: 29 ).

[00283] In some embodiments, the high identity of the factor IX polynucleotide to CS06-MP-NA (SEQ ID NO: 17) further comprises a factor IX propeptide polynucleotide encoding a factor IX propeptide having the amino acid sequence FIX-PP-AA (SEQ ID NO: 38). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-PP-NA (SEQ ID NO: 31 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-PP-NA (SEQ ID NO: 32 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-PP-NA (SEQ ID NO: 33 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-PP-NA (SEQ ID NO: 34 ). In some embodiments, the Factor IX propeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-PP-NA (SEQ ID NO: 35 ).

[00284] In some embodiments, the high identity of the factor IX polynucleotide to CS06-MP-NA (SEQ ID NO: 17) further includes a factor IX prepropeptide polynucleotide encoding a factor IX prepropeptide having the amino acid sequence FIX-PPP-AA (SEQ ID NO: 36). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS02-PPP-NA (SEQ ID NO: 19 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS03-PPP-NA (SEQ ID NO: 20 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS04-PPP-NA (SEQ ID NO: 21 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS05-PPP-NA (SEQ ID NO: 22 ). In some embodiments, the factor IX prepropeptide polynucleotide has a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CS06-PPP-NA (SEQ ID NO: 23 ).

[00285] In some embodiments, an encoded Factor IX polypeptide, e.g., a polypeptide encoded by a polynucleotide having high sequence homology to CS06-FL-NA (SEQ ID NO: 9), has high sequence identity with the mature sequence of a single-stranded FIX factor IX polypeptide -MP-AA (SEQ ID NO: 10) and/or the mature single strand sequence of the Padua variant (hFIX (R384L)) FIXp-MP-AA (SEQ ID NO: 12). The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing any signal peptide and propeptide and by removing the activating polypeptide).

[00286] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the encoded factor IX polypeptide sequence is FIX-MP-AA (SEQ ID NO: 10).

[00287] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single-chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-MP-AA (SEQ ID NO: 12).

[00288] In one embodiment, the codon-altered polynucleotides provided herein encode a single-stranded Factor IX polypeptide comprising a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the light chain to the N-terminus of the heavy chain. The factor IX polypeptide light chain is encoded by a first nucleotide sequence having high sequence identity with CS06-LC-NA (SEQ ID NO: 50), which is part of CS06-FL-NA (SEQ ID NO: 9) encoding the factor IX light chain. The factor IX polypeptide heavy chain is encoded by a second nucleotide sequence having high sequence identity with CS06-HC-NA (SEQ ID NO: 49), which is part of CS06-FL-NA (SEQ ID NO: 9) encoding the factor IX heavy chain. The polypeptide linker includes Factor XI cleavage sites that allow in vivo maturation (eg, following expression of a factor IX single chain polypeptide precursor).

[00289] In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity with CS06-LC-NA and CS06-HC-NA (SEQ ID NOS: 50 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity with CS06-LC-NA and CS06-HC-NA (SEQ ID NOS: 50 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity with CS06-LC-NA and CS06-HC-NA (SEQ ID NOS: 50 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity with CS06-LC-NA and CS06-HC-NA (SEQ ID NOS: 50 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity with CS06-LC-NA and CS06-HC-NA (SEQ ID NOS: 50 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity with CS06-LC-NA and CS06-HC-NA (SEQ ID NOS: 50 and 49), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity with CS06-LC-NA and CS06-HC-NA (SEQ ID NOS: 50 and 49), respectively. In some embodiments, the first and second nucleotide sequences are CS06-LC-NA and CS06-HC-NA (SEQ ID NOs: 50 and 49), respectively.

[00290] In some embodiments, the factor IX construct linker polypeptide is encoded by a third nucleotide sequence having high sequence identity with CS06-AP-NA (SEQ ID NO: 61), which is a codon-altered sequence encoding a wild type factor IX activation polypeptide, for example, amino acids 192-226 of FIX-FL-AA (SEQ ID NO: 2). In some embodiments, the implementation of the third nucleotide sequence has at least 80% identity with CS06-AP-NA (SEQ ID NO: 61). In some embodiments, the implementation of the third nucleotide sequence has at least 90% identity with CS06-AP-NA (SEQ ID NO: 61). In some embodiments, the implementation of the third nucleotide sequence has at least 95% identity with CS06-AP-NA (SEQ ID NO: 61). In some embodiments, the implementation of the third nucleotide sequence has at least 96% identity with CS06-AP-NA (SEQ ID NO: 61). In some embodiments, the implementation of the third nucleotide sequence has at least 97% identity with CS06-AP-NA (SEQ ID NO: 61). In some embodiments, the implementation of the third nucleotide sequence has at least 98% identity with CS06-AP-NA (SEQ ID NO: 61). In some embodiments, the implementation of the third nucleotide sequence has at least 99% identity with CS06-AP-NA (SEQ ID NO: 61). In some embodiments, the third nucleotide sequence is CS06-AP-NA (SEQ ID NO: 61).

[00291] In some embodiments, the encoded factor IX polypeptide also includes a signal peptide (eg, a factor IX signal peptide) and/or a propeptide (eg, a factor IX propeptide). In some embodiments, the signal peptide is a wild-type factor IX signal peptide (FIX-SP-AA (SEQ ID NO: 37)). In some embodiments, the signal peptide is encoded by a codon-altered polynucleotide sequence having high sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%) with CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the propeptide is a wild-type factor IX propeptide (FIX-PP-AA (SEQ ID NO: 38)). In some embodiments, the propeptide peptide is encoded by a codon-altered polynucleotide sequence having high sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%) with CS06-PP-NA (SEQ ID NO: 35).

[00292] In some embodiments, a factor IX encoded polypeptide, e.g., a polynucleotide encoded by a polynucleotide having high sequence homology to CS06-LC-NA (SEQ ID NO:50) and CS06-HC-NA (SEQ ID NO: 49), has high sequence identity with FIX-MP-AA (SEQ ID NO: 10) and/or mature FIXp-MP-AA (SEQ ID NO: 12) Padua variant single chain polypeptide (hFIX (R384L)) . The encoded factor IX polypeptide must retain the ability to be activated in a functional factor IXa protein (eg, by removing any signal peptide and propeptide and by removing the activating polypeptide).

[00293] In one embodiment, the encoded factor IX polypeptide sequence has at least 85% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIX-MP-AA (SEQ ID NO: 10). In one embodiment, the encoded factor IX polypeptide sequence is FIX-MP-AA (SEQ ID NO: 10).

[00294] In one embodiment, the sequence of the encoded factor IX polypeptide has at least 85% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide (e.g., position 338 of the mature single chain factor IX polypeptide FIXp-MP-AA (SEQ ID NO: 12)). In one embodiment, the sequence of the encoded factor IX polypeptide has at least 90% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 95% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 96% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 97% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 98% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.5% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the sequence of the encoded factor IX polypeptide has at least 99.9% identity with FIXp-MP-AA (SEQ ID NO: 12) and includes leucine at position 384 of the prepropolypeptide. In one embodiment, the encoded factor IX polypeptide sequence is FIXp-MP-AA (SEQ ID NO: 12).

[00295] In some embodiments, with reference to Figure 1, a nucleic acid composition is provided that includes a self-complementary polynucleotide of structure A, wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has identity to at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% with CS06-MP-NA (SEQ ID NO: 17). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS06-FL-NA (SEQ ID NO: 9).

[00296] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a structure B self-complementary polynucleotide, wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature Factor IX polypeptide that has identity to at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% with CS06-MP-NA (SEQ ID NO: 17). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS06-FL-NA (SEQ ID NO: 9).

[00297] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a polynucleotide of structure C (e.g., a single-stranded polynucleotide), wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has an identity of at least 95%, 96%, 97% , 98%, 99%, 99.5%, 99.9%, or 100% with CS06-MP-NA (SEQ ID NO: 17). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS06-FL-NA (SEQ ID NO: 9).

[00298] In some embodiments, with reference to FIG. 1, a nucleic acid composition is provided that includes a polynucleotide of structure D (e.g., a single-stranded polynucleotide), wherein the FIX coding sequence portion of the polynucleotide comprises a nucleic acid sequence encoding a mature factor IX polypeptide that has an identity of at least 95%, 96%, 97% , 98%, 99%, 99.5%, 99.9%, or 100% with CS06-MP-NA (SEQ ID NO: 17). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one of FIX-SP-NA (SEQ ID NO: 24), CS02-SP-NA (SEQ ID NO: 25), CS03-SP-NA (SEQ ID NO: 26), CS04-SP-NA (SEQ ID NO: 27 ), CS05-SP-NA (SEQ ID NO: 28) and CS06-SP-NA (SEQ ID NO: 29). In some embodiments, the coding sequence portion of the FIX polynucleotide also includes a nucleic acid sequence encoding a factor IX propeptide (optionally in combination with a factor IX signal peptide nucleic acid sequence as described above) that has an identity of at least 90%, 95%, 96 %, 97%, 98%, 99% or 100% and one of FIX-PP-NA (SEQ ID NO: 30), CS02-PP-NA (SEQ ID NO: 31), CS03-PP-NA (SEQ ID NO: 32), CS04-PP-NA (SEQ ID NO: 33), CS05-PP-NA (SEQ ID NO: 34) and CS06-PP-NA (SEQ ID NO: 35). In some embodiments, the FIX polynucleotide coding sequence portion comprises a nucleic acid sequence encoding a factor IX prepropolypeptide that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity, or 100% with CS06-FL-NA (SEQ ID NO: 9).

Propeptides and codon-altered factor IX signal

[00299] In one aspect, the invention discloses codon-altered polynucleotides encoding factor IX signal peptides, factor IX propeptides, and both (eg, factor IX prepropeptides). Such codon-altered polynucleotides improve Factor IX expression and can be placed, for example, upstream of a codon-altered or otherwise altered polynucleotide encoding a single-stranded Factor IX polypeptide (e.g., Factor IX light chain, activation peptide, and heavy chain). Typically, the encoded peptides are wild type factor IX signal peptides (e.g. FIX-SP-AA (SEQ ID NO: 37)), propeptides (e.g. FIX-PP-AA (SEQ ID NO: 38) and prepropeptides (FIX -PPP-AA (SEQ ID NO: 36)).

[00300] In some embodiments, the codon-altered polynucleotides encoding factor IX signal peptides, propeptides, and prepropeptides have a high sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or 100%) with one of CS02-SP-NA (SEQ ID NO:25), CS03-SP-NA (SEQ ID NO:26), CS04-SP- NA (SEQ ID NO:27), CS05-SP-NA (SEQ ID NO:28), CS06-SP-NA (SEQ ID NO:29), CS02-PP-NA (SEQ ID NO:31), CS03- PP-NA (SEQ ID NO:32), CS04-PP-NA (SEQ ID NO:33), CS05-PP-NA (SEQ ID NO:34), CS06-PP-NA (SEQ ID NO:35), CS02-PPP-NA (SEQ ID NO:19), CS03-PPP-NA (SEQ ID NO:20), CS04-PPP-NA (SEQ ID NO:21), CS05-PPP-NA (SEQ ID NO:22 ) and CS06-PPP-NA (SEQ ID NO:23).

CS02 signal and propeptides

[00301] In one embodiment, the codon-altered polynucleotide encoding the factor IX signal peptide has at least 95% sequence identity with CS02-SP-NA (SEQ ID NO: 25). In other embodiments, the codon-altered polynucleotide encoding the factor IX signal peptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS02-SP-NA (SEQ ID NO: 25).

[00302] In one embodiment, the codon-altered polynucleotide encoding the factor IX propeptide has at least 95% sequence identity with CS02-PP-NA (SEQ ID NO: 31). In other embodiments, the codon-altered polynucleotide encoding the factor IX propeptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS02-PP-NA (SEQ ID NO: 31).

[00303] In one embodiment, the codon-altered polynucleotide encoding the factor IX prepropeptide has at least 95% sequence identity with CS02-PPP-NA (SEQ ID NO: 19). In other embodiments, the codon-altered polynucleotide encoding the factor IX prepropeptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS02-PPP-NA (SEQ ID NO: 19).

CS03 signal and propeptides

[00304] In one embodiment, the codon-altered polynucleotide encoding the factor IX signal peptide has at least 95% sequence identity with CS03-SP-NA (SEQ ID NO: 26). In other embodiments, the codon-altered polynucleotide encoding the factor IX signal peptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS03-SP-NA (SEQ ID NO: 26).

[00305] In one embodiment, the codon-altered polynucleotide encoding the factor IX propeptide has at least 95% sequence identity with CS03-PP-NA (SEQ ID NO: 32). In other embodiments, a codon-altered polynucleotide encoding a factor IX propeptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS03-PP-NA (SEQ ID NO: 32).

[00306] In one embodiment, the codon-altered polynucleotide encoding the factor IX prepropeptide has at least 95% sequence identity with CS03-PPP-NA (SEQ ID NO: 20). In other embodiments, the codon-altered polynucleotide encoding the factor IX prepropeptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS03-PPP-NA (SEQ ID NO: 20).

CS04 signal and propeptides

[00307] In one embodiment, the codon-altered polynucleotide encoding the factor IX signal peptide has at least 95% sequence identity with CS04-SP-NA (SEQ ID NO: 27). In other embodiments, the codon-altered polynucleotide encoding the factor IX signal peptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS04-SP-NA (SEQ ID NO: 27).

[00308] In one embodiment, the codon-altered polynucleotide encoding the factor IX propeptide has at least 95% sequence identity with CS04-PP-NA (SEQ ID NO: 33). In other embodiments, a codon-altered polynucleotide encoding a factor IX propeptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS04-PP-NA (SEQ ID NO: 33).

[00309] In one embodiment, the codon-altered polynucleotide encoding the factor IX prepropeptide has at least 95% sequence identity with CS04-PPP-NA (SEQ ID NO: 21). In other embodiments, the codon-altered polynucleotide encoding the factor IX prepropeptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS04-PPP-NA (SEQ ID NO: 21).

CS05 signal and propeptides

[00310] In one embodiment, the codon-altered polynucleotide encoding the factor IX signal peptide has at least 95% sequence identity with CS05-SP-NA (SEQ ID NO: 28). In other embodiments, the codon-altered polynucleotide encoding the factor IX signal peptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS05-SP-NA (SEQ ID NO: 28).

[00311] In one embodiment, the codon-altered polynucleotide encoding the factor IX propeptide has at least 95% sequence identity with CS05-PP-NA (SEQ ID NO: 34). In other embodiments, the codon-altered polynucleotide encoding the factor IX propeptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS05-PP-NA (SEQ ID NO: 34).

[00312] In one embodiment, the codon-altered polynucleotide encoding the factor IX prepropeptide has at least 95% sequence identity with CS05-PPP-NA (SEQ ID NO: 22). In other embodiments, the codon-altered polynucleotide encoding the factor IX prepropeptide has at least 96%, 97%, 98%, 99% or 100% identity with CS05-PPP-NA (SEQ ID NO: 22).

CS06 signal and propeptides

[00313] In one embodiment, the codon-altered polynucleotide encoding the factor IX signal peptide has at least 95% sequence identity with CS06-SP-NA (SEQ ID NO: 29). In other embodiments, the codon-altered polynucleotide encoding the factor IX signal peptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS06-SP-NA (SEQ ID NO: 29).

[00314] In one embodiment, the codon-altered polynucleotide encoding the factor IX propeptide has at least 95% sequence identity with CS06-PP-NA (SEQ ID NO: 35). In other embodiments, the codon-altered polynucleotide encoding the factor IX propeptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS06-PP-NA (SEQ ID NO: 35).

[00315] In one embodiment, the codon-altered polynucleotide encoding the factor IX prepropeptide has at least 95% sequence identity with CS06-PPP-NA (SEQ ID NO: 23). In other embodiments, the codon-altered polynucleotide encoding the factor IX prepropeptide has at least 96%, 97%, 98%, 99%, or 100% identity with CS06-PPP-NA (SEQ ID NO: 23).

Factor IX expression vectors

[00316] In some embodiments, the codon-altered polynucleotides described herein are integrated into expression vectors. As one of skill in the art will appreciate, many forms of vectors can be used to effect factor IX gene therapy using the codon-altered factor IX polynucleotide sequences disclosed herein. Non-limiting examples of expression vectors include viral vectors (eg, vectors suitable for gene therapy), plasmid vectors, bacteriophage vectors, cosmids, phagemids, artificial chromosomes, and the like.

[00317] In some embodiments, the codon-altered polynucleotides described herein are integrated into a viral gene therapy vector. Non-limiting examples of viral vectors include: a retrovirus such as Moloney mouse leukemia virus (MMLV), Harvey mouse sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; viruses of type SV40; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; and polio viruses.

[00318] Under natural conditions, factor IX is synthesized primarily in the liver. Thus, hepatocytes were selected as suitable host cells for factor IX gene therapy constructs. Several classes of viral vectors have been shown to be suitable for targeted delivery of a gene therapy construct to the liver, including retroviral vectors (see, e.g., Axelrod et al., 1990; Kay et al., 1992; Van den Driessche et al., 1999 and Xu et al., 2003, 2005, the disclosures of which are expressly incorporated herein by reference in their entirety for all purposes), lentivirus (see e.g. Ward et al., 2011, Brown et al., 2007 and Matrai et al., 2011, the disclosures of which are expressly incorporated herein by reference in their entirety for all purposes), adeno-associated virus (AAV) (see, for example, Herzog et al., 1999, the disclosure of which is expressly incorporated herein by reference references in its entirety for all purposes), and adenoviral vectors (see, for example, Brown et al., 2004 and Ehrhardt & Kay, 2002, the disclosures of which are expressly incorporated herein by reference in their entirety for all purposes).

[00319] In some embodiments, the gene therapy vector is a retrovirus, and in particular a replication-deficient retrovirus. Protocols for obtaining replication-deficient retroviruses are known in the art. For a 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).

[00320] In one embodiment, the gene therapy vector is an adeno-associated virus (AAV) 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., PNAS). U.S.A., 89(13):6094-98 (1992); Curiel, Nat Immun, 13(2-3):141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A, et al., Mol. Ther., 20(4): 699-708 (2012), each of which is hereby incorporated by reference in their entirety for all purposes). Details regarding the generation and use of rAAV vectors are described, for example, in US Pat. 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, the AAV vector is an AAV-8 vector.

[00321] An exemplary AAV delivery vector for liver-specific factor IX expression is described in WO 2009/130208, the contents of which are expressly incorporated herein by reference in its entirety for all purposes. The vector is a single stranded AAV vector encoding human factor IX and includes the TTR Serp regulatory sequences driving the factor cDNA. The vector also includes intron I of the human factor IX gene and a polyadenylation signal.

[00322] 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 particular 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; U.S. Patent Nos. 6,013,516 and 5,994,136).

[00323] In some embodiments, the codon-altered polynucleotides described herein can be administered to a subject in a non-viral manner. For example, deproteinized DNA can be introduced into a cell by electroporation, sonoporation, particle bombardment, or hydrodynamic delivery. The DNA may also be encapsulated or associated with polymers, eg liposomes, polysomes, polypeloses, dendrimers, and administered to the subject as a complex. Similarly, DNA can be linked to inorganic nanoparticles, such as gold, silicon, iron oxide, or calcium phosphate, or attached to cell-penetrating peptides for delivery to cells in vivo .

[00324] Codon-altered polynucleotides encoding factor IX can also be incorporated into artificial chromosomes, such as artificial chromosome expression (ACE) (see, e.g., Lindenbaum et al., Nucleic Acids Res., 32 (21): e172 (2004)) and mammalian artificial chromosomes (MAC). For a review, see, for example, Pérez-Luz and Diaz-Nido, J Biomed Biotechnol. 2010: Article ID 642804 (2010).

[00325] Various vectors can be used to express a factor IX polypeptide from a codon-altered polypeptide in cell culture, including eukaryotic and prokaryotic expression vectors. In certain embodiments, a plasmid vector is contemplated for expression of a factor IX polypeptide in cell culture. Typically, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vector may carry a replication site as well as marking sequences that are capable of conferring phenotypic selection in transformed cells. The plasmid will include a codon-altered polynucleotide encoding a Factor IX polypeptide operably linked to one or more control sequences, such as a promoter.

[00326] Non-limiting examples of prokaryotic expression vectors include plasmids such as pRSET, pET, pBAD, etc., where promoters used in prokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc. Examples vectors for eukaryotic expression include: (i) vectors such as pAO, pPIC, pYES, pMET using promoters such as AOX1, GAP, GAL1, AUG1, etc. for expression in yeast; (ii) vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc. using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc., for expression in insect cells and (iii) 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 for expression in mammalian cells.

[00327] In some embodiments, the invention provides an AAV gene therapy vector that includes a codon-altered Factor IX polynucleotide as described herein, internal terminal repeat (ITR) sequences at the 5' and 3' ends of the vector, one or more promoters and/or enhancer sequences that are operably linked to the factor IX polynucleotide; and a polyadenylation signal following the 3' end of the factor IX polynucleotide sequence. In some embodiments, one or more promoter and/or enhancer sequences comprise one or more copies of a liver-specific regulatory control element.

[00328] Figure 1 shows several exemplary factor IX gene therapy vector architectures, in accordance with some embodiments. Figure 1A shows a self-complementary AAV vector having a mutated 5'-ITR, truncated TTR enhancer/promoter sequences, an MVM viral intron sequence, a codon-altered factor IX sequence, a polyadenylation sequence, and a 3'-ITR. Figure 1B shows a self-complementary AAV vector encoding a factor IX polypeptide similar to that shown in Figure 1A, but additionally including one or more (eg, one, two, three or more) liver-specific regulatory controls. Figure 1C shows a single stranded vector having the same elements as shown in Figure 1A, except that the 5'-ITR is not mutated, which prevents self complementarity. Figure 1D shows a single chain AAV vector encoding a factor IX polypeptide similar to that shown in Figure 1A, but additionally including one or more (eg, one, two, three or more) liver-specific regulatory controls. Although these illustrations are for a factor IX protein that includes the R384L "Padua" amino acid substitution in Figure 1, in some embodiments, the Factor IX nucleotide construct has the general structure as depicted in Figure 1 (e.g., structure A, B, C, or D) encoding a factor IX protein that does not include the amino acid substitution R384L "Padua".

[00329] Figure 25 shows the nucleotide sequence of AAV factor IX gene therapy vector CS06-CRM8.3-ssV (SEQ ID NO: 40), which represents the gene therapy vector architecture illustrated in Figure 1D. Nucleotides 1-145 of CS06-CRM8.3-ssV (SEQ ID NO: 40) represent the 5'-ITR sequence of AAV2 (SEQ ID NO: 51). The 5'-ITR sequence is followed by three copies of the liver-specific regulatory control element CRM8 CRM8 (SEQ ID NO: 39) at nucleotides 165-236, 238-309 and 311-382. The following CRM8 sequence is a truncated TTR enhancer/promoter sequence (SEQ ID NO: 52) at nucleotides 383-712. The vector then includes a mouse small virus (MVM) intron (SEQ ID NO: 53) at nucleotides 724-800. Nucleotides 814-2199 of the vector are the codon-altered factor IX (R384L) CS06 coding sequence (CS06-FL-NA (SEQ ID NO: 9)). The Factor IX polynucleotide sequence is followed by the BGH polyadenylation signal at nucleotides 2208-2441 and finally the AAV2 3'-ITR sequence (SEQ ID NO: 55) at nucleotides 2458-2602.

[00330] In some embodiments, the disclosure relates to a factor IX polynucleotide comprising a sequence having at least 95% identity with nucleotides 1-2602 of SEQ ID NO: 40. In some embodiments, the disclosure relates to a factor IX polynucleotide comprising a sequence having at least 99% identity with nucleotides 1-2602 of SEQ ID NO: 40. In some embodiments, the disclosure relates to a factor IX polynucleotide containing a sequence having at least 99.5% identity with nucleotides 1-2602 of SEQ ID NO: 40 In some embodiments, the disclosure relates to a Factor IX polynucleotide comprising the nucleotide sequence 1-2602 of SEQ ID NO: 40.

[00331] Several AAV serotypes have been characterized, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In general, any AAV serotype can be used for the factor IX gene therapy constructs described herein. However, serotypes have different tropisms, for example, they preferentially affect different tissues. In one embodiment, since factor IX is produced primarily in the liver, the AAV serotype for the disclosed gene therapy constructs is selected based on the liver tropism found in at least the AAV7, AAV8 and AAV9 serotypes. Accordingly, in one embodiment, the factor IX gene therapy construct is an AAV7 serotype vector. In another embodiment, the factor IX gene therapy construct is an AAV8 serotype vector. In yet another embodiment, the factor IX gene therapy construct is an AAV9 serotype vector.

[00332] Factor IX gene therapy constructs described herein can be single stranded (e.g. ssAAV vector as shown in Figures 1C and 1D) or self-complementary (e.g. scAAV vector as shown in Figures 1A and 1B) . Although studies and theory suggest that self-complementary AAV vectors should promote better transgene expression by bypassing the requirement of second strand synthesis prior to translation, single-stranded AAV vectors have been identified to promote better expression of factor IX compared to comparable self-complementary vectors, as reported in Example 5.

Promoters and enhancers

[00333] Factor IX gene therapy constructs described herein typically include one or more promoter and/or enhancer elements that direct gene expression in vivo , such as regulatory elements. In some embodiments, the promoter or enhancer element stimulates expression in a tissue-dependent manner, eg, preferentially in a particular tissue. Because factor IX is primarily synthesized in the liver, in some embodiments, the gene therapy vectors described herein include a liver-specific regulatory element that substantially limits expression of the gene therapy vector to liver cells.

[00334] In general, liver-specific regulatory elements can be derived from any gene known to be exclusively expressed in the liver. WO 2009/130208 identified several genes expressed in a liver-specific manner, including serpinopeptidase inhibitor, clade A member 1, also known as α-antitrypsin (SERPINA1; GeneID 5265), apolipoprotein C-I (APOC1; GeneID 341), apolipoprotein C-IV (APOC4; GeneID 346), apolipoprotein H (APOH; GeneID 350); transthyretin (TTR; GeneID 7276), albumin (ALB; GeneID 213), aldolase B (ALDOB; GeneID 229), cytochrome P450 family 2 E subfamily polypeptide 1 (CYP2E1; GeneID 1571), fibrinogen alpha chain (FGA; GeneID 2243) , transferrin (TF; GeneID 7018), haptoglobin-related protein (HPR; GeneID 3250). In some embodiments, the factor IX gene therapy constructs described herein comprise a liver-specific regulatory element derived from genomic loci of one or more of these proteins. Several examples of such elements are described in WO 2009/130208, the contents of which are expressly incorporated herein by reference in their entirety for all purposes.

[00335] One example of a liver-specific regulatory element is the transthyretin (TTR) gene, commonly referred to as "TTRe" or "TTREnh". See Hsieh JL, et al., Cancer Sci., 100(3):537-45 (2009), the contents of which are expressly incorporated herein by reference in their entirety for all purposes. In some embodiments, the factor IX gene therapy constructs described herein include truncated TTR enhancer and promoter elements. An example of these elements is provided at nucleotides 383-712 of CS06-CRM8.3-ssV (SEQ ID NO: 40) shown in FIG. 25. In some embodiments, the truncated TTR enhancer and promoter element has at least 85% sequence identity with nucleotides 383-712 of CS06-CRM8.3-ssV (SEQ ID NO: 40). In other embodiments, the truncated TTR enhancer and promoter element have at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with nucleotides 383-712 of CS06-CRM8. 3-ssV (SEQ ID NO: 40).

[00336] Another example of a liver-specific regulatory element is the SERPINA1 gene as described in PCT Publication No. WO 2016/146757, the contents of which are specifically incorporated herein by reference in its entirety for all purposes. An example of such an element is the CRM8 regulatory control element (SEQ ID NO: 39) provided at nucleotides 165-236 of CS06-CRM8.3-ssV (SEQ ID NO: 40). In some embodiments, a regulatory control element derived from SERPINA1 has at least 85% sequence identity with CRM8 (SEQ ID NO: 39). In other embodiments, the truncated regulatory element derived from SERPINA1 has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with CRM8 (SEQ ID NO: 39).

[00337] In some embodiments, the factor IX gene therapy construct includes one or more regulatory control elements derived from SERPINA1, exemplified by the constructs shown in FIG. 1B and 1D. In one embodiment, the construct includes a single regulatory control element derived from SERPINA1 (eg, CRM8). In another embodiment, the construct includes two regulatory control elements derived from SERPINA1 (eg, CRM8). In another embodiment, the construct includes three regulatory control elements derived from SERPINA1 (eg, CRM8). In yet other embodiments, the construct includes 4, 5, 6 or more SERPINA1-derived regulatory control elements (eg, CRM8).

[00338] In one embodiment, the factor IX gene therapy construct includes one or more regulatory control elements derived from SERPINA1 (eg, CRM8) and a truncated TTR enhancer and promoter element as illustrated in FIG. 1B, 1D and 25.

introns

[00339] In some embodiments, the factor IX gene therapy constructs described herein include an intron, eg, a virus-derived intron, to increase factor IX gene expression. Suitable introns for expression of gene therapy constructs are known in the art. Typically, the intron is located 5' from the transgene coding sequence, as shown in the factor IX constructs shown in FIG. 1 and FIG. 25. However, in some embodiments, the intron may be located within the transgene coding sequence, eg, in a naturally occurring factor IX intron junction or otherwise, or 3' to the transgene coding sequence. Non-limiting examples of introns that can be used in the factor IX gene therapy constructs described herein include introns derived from mouse small virus (MVM) introns, beta-globin (betalVS-ll) introns, factor IX A (FIX) introns , small T-introns of simian virus 40 (SV40) and beta-actin introns.

[00340] In one embodiment, the Factor IX gene therapy constructs described herein include an MVM-derived intron, eg, as shown in FIG. 1, exemplified by the MVM intron (SEQ ID NO: 53) at nucleotides 724-800 of CS06-CRM8.3-ssV (SEQ ID NO: 40) in FIG. 25. In some embodiments, the intron used in the gene therapy constructs described herein has at least 85% sequence identity with MVM (SEQ ID NO: 53). In other embodiments, the intron used in the gene therapy constructs described herein has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with MVM (SEQ ID NO : 53).

Polyadenylation signals

[00341] In some embodiments, the factor IX gene therapy constructs described herein include a polyadenylation signal, for example, as shown in FIG. 1. The polyadenylation signal directs the synthesis of the poly-A tail at the 3' end of the mRNA transcript derived from the factor IX transgene. Accordingly, the polyadenylation signal is located 3' to the Factor IX coding sequence. Non-limiting examples of polyadenylation signals that can be used in the factor IX gene therapy constructs described herein include polyadenylation signals derived from the simian virus 40 (SV40) late gene, bovine growth hormone (BGH) polyadenylation signal. ) and the rabbit β-globin minimal gene (mRBG).

[00342] In one embodiment, the factor IX gene therapy constructs described herein include a polyadenylation signal derived from a bovine growth hormone (BGH) polyadenylation signal, for example, as illustrated in FIG. 1 and exemplified by the BGHpA signal (SEQ ID NO: 54) at nucleotides 2208-2441 CS06-CRM8.3-ssV (SEQ ID NO: 40) in Figure 25. In some embodiments, a polyadenylation signal used in gene therapy constructs described herein has at least 85% sequence identity with the BGHpA signal (SEQ ID NO: 54). In other embodiments, the polyadenylation signal used in the gene therapy constructs described herein has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the BGHpA signal ( SEQ ID NO: 54).

Ways

Receipt

[00343] The codon-altered polynucleotides and factor IX viral vectors described herein (eg, nucleic acid compositions) are prepared according to conventional nucleic acid amplification and vector production methods. Two main platforms have been developed for the large-scale production of recombinant AAV vectors. The first platform is based on replication in mammalian cells and the second is based on replication in invertebrate cells. For a review, see Kotin R.M., Hum. Mol. Genet., 20 (R1): R2-6 (2011), the contents of which are expressly incorporated herein by reference in their entirety for all purposes.

[00344] Accordingly, the present invention relates to methods for producing adeno-associated virus (AAV) particles. In some embodiments, the methods include introducing a codon-altered Factor IX polynucleotide construct having high nucleotide sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9 % or 100%) with one of the sequences CS02, CS03, CS04, CS05 or CS06, as described herein, into a host cell, where the polynucleotide construct is competent to replicate in the host cell.

[00345] In some embodiments, the host cell is a mammalian host cell, such as a HEK, CHO, or BHK cell. In specific embodiments, the host cell is a HEK 293 cell. In some embodiments, the host cell is an invertebrate cell, such as an insect cell. In a specific embodiment, the host cell is an SF9 cell.

Lineups

[00346] This document provides compositions for use in the treatment of bleeding disorders. Such compositions contain a therapeutically effective amount of a codon-altered factor IX polynucleotide, for example, an AAV gene therapy vector, comprising a codon-altered polynucleotide encoding factor IX, as described herein. A therapeutically effective amount of a codon-altered FIX polynucleotide (eg, an AAV gene therapy vector comprising a codon-altered Factor IX sequence) is mixed with a suitable pharmaceutical carrier or carrier for systemic, topical, or topical administration. The final composition of the codon-altered Factor IX polynucleotides disclosed herein will be within the ability of those skilled in the art.

Dosages

[00347] The nucleic acid compositions of the invention are administered to patients in need thereof. The amount or dose of the gene therapy therapeutic agent administered will depend on such factors as the particular codon-altered FIX polynucleotide construct, the delivery vector used, the severity of the disease, and the general characteristics of the subject. The exact dosage will depend on the goal of treatment and will be determined by those skilled in the art using known techniques ( see e.g. Lieberman, Pharmaceutical Dosage Forms (vols. 13, 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). An experienced physician can determine the specific dosage and dosage regimen for the treatment of a particular subject.

[00348] In some embodiments, a gene therapy vector (e.g., an AAV gene therapy vector particle) containing a codon-altered Factor IX polynucleotide is administered intravenously at a therapeutically effective dose to a subject in need thereof (e.g., a subject with mild, moderate, or severe hemophilia B). In some embodiments, a therapeutically effective dose is from about 2x10E11 to 2x10E14 vector genomes per kilogram of subject body weight. In a specific embodiment, a therapeutically effective dose is from about 2x10E12 to 2x10E13 vector genomes per kilogram of the subject's body weight. In some embodiments, the subject is administered about 2x10E11, 3x10E11, 4x10E11, 5x10E11, 6x10E11, 7x10E11, 8x10E11, 9x10E11, 1x10E12, 2x10E12, 3x10E12, 4×10E12, 5×10E12, 6×10E12, 7×10E12, 8×10E12, 9×10E12, 1×10E13, 2×10E13, 3×10E13, 4×10E13, 5×10E13, 6×10E13, 7× 10E13, 8x10E13, 9x10E13, 1x10E14, or 2x10E14 vector genomes per kilogram of subject's body weight.

[00349] Accordingly, the disclosure provides methods for treating factor IX deficiency (eg, hemophilia B). In some embodiments, the methods include administering to a patient in need thereof a codon-altered Factor IX polynucleotide construct having high nucleotide sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99%, 99, 5%, 99.9% or 100%) with one of the sequences CS02, CS03, CS04, CS05 or CS06 as described herein. In some embodiments, the codon-altered factor polynucleotide has high sequence identity with the codon-altered Factor IX prepropolypeptide coding sequence, e.g., high sequence identity with one of CS02-FL-NA (SEQ ID NO: 5), CS03-FL-NA (SEQ ID NO: 6), CS04-FL-NA (SEQ ID NO: 7), CS05-FL-NA (SEQ ID NO: 8) or CS06-FL-NA (SEQ ID NO: 9). In some embodiments, the codon-altered factor polynucleotide has high sequence identity with the codon-altered mature factor IX single-stranded coding sequence, e.g., high sequence identity with one of CS02-MP-NA (SEQ ID NO: 13), CS03-MP-NA ( SEQ ID NO: 14), CS04-MP-NA (SEQ ID NO: 15), CS05-MP-NA (SEQ ID NO: 16) or CS06-MP-NA (SEQ ID NO: 17).

[00350] In some embodiments, the treatment comprises administering to a patient in need thereof a gene therapy vector comprising a codon-altered Factor IX polynucleotide construct having high nucleotide sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99% 99.5%, 99.9% or 100%) with one of the sequences CS02, CS03, CS04, CS05 or CS06 as described herein. In one embodiment, the gene therapy vector is a mammalian gene therapy vector. In a specific embodiment, the mammalian gene therapy vector is a viral vector, such as a lentivirus, retrovirus, adenovirus, or adeno-associated viral vector.

[00351] In one embodiment, the gene therapy vector is an adeno-associated virus (AAV) particle containing a viral vector encoding a codon-altered Factor IX coding sequence. Typically, the viral vector includes inverted terminal repeats (ITRs) at each end, one or more expression control elements, a codon-altered factor IX coding sequence, and a poly-A signal sequence. In a particular embodiment, the gene therapy vector includes a liver-specific regulatory control element (eg, one or more copies of the CRM8 element).

Receipt

[00352] The codon-altered polynucleotides and factor IX viral vectors described herein (eg, nucleic acid compositions) are prepared according to conventional nucleic acid amplification and vector production methods. Two main platforms have been developed for the large-scale production of recombinant AAV vectors. The first platform is based on replication in mammalian cells and the second is based on replication in invertebrate cells. For a review, see Kotin R.M., Hum. Mol. Genet., 20 (R1): R2-6 (2011), the contents of which are expressly incorporated herein by reference in their entirety for all purposes.

[00353] Accordingly, the present invention relates to methods for producing adeno-associated virus (AAV) particles. In some embodiments, the methods include introducing a codon-altered Factor IX polynucleotide construct having high nucleotide sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9 % or 100%) with one of the sequences CS02, CS03, CS04, CS05 or CS06, as described herein, into a host cell, where the polynucleotide construct is competent to replicate in the host cell.

[00354] In some embodiments, the host cell is a mammalian host cell, such as a HEK, CHO, or BHK cell. In specific embodiments, the host cell is a HEK 293 cell. In some embodiments, the host cell is an invertebrate cell, such as an insect cell. In a specific embodiment, the host cell is an SF9 cell.

Treatment

[00355] In some embodiments, the nucleic acid compositions (eg, codon-altered polynucleotides) described herein are administered to a subject in need thereof, according to known methods of administration. Methods for introducing gene therapy vectors are well known in the art. These include, without limitation, intravenous administration, intramuscular injection, interstitial injection, and intrahepatic administration (eg, via an intrahepatic artery or vein). For example, see Chuah MK et al., Hum Gene Ther., 23(6):557-65 (2012); Chuah MK et al., J Thromb Haemost., 10(8):1566-69 (2012); Chuah MK et al., J Thromb Haemost. 11 Suppl 1:99-110 (2013); VandenDriessche et al., Hum Gene Ther. 23(1):4-6 (2012); High KA, Blood, 120(23):4482-87 (2012); Matrai et al., Mol Ther., 18(3):477-90 (2010); and Matrai et al., Curr Opin Hematol., 17(5):387-92 (2010), each of which is incorporated herein by reference for review.

Assessment of therapeutic efficacy

[00356] The therapeutic efficacy of treating hemophilia B can be assessed, for example, by measuring factor IX dependent coagulation potential in a treated subject. Metrics for evaluating coagulation potential include, but are not limited to, in vitro partial thromboplastin time (APPT) assay, factor IX chromogenic activity assays, clotting time, and factor IX antigen levels (eg, using factor IX-specific ELISA). It should be noted that the therapeutic dose should not result in wild-type FIX levels in the patient; but rather, sufficient expression to reduce symptoms in a significant or measurable way, which is considered therapeutic for the purposes of the invention.

[00357] According to the National Hemophilia Foundation, a subject is classified as having mild hemophilia B when their blood plasma contains 6 to 49% factor IX activity relative to normal human plasma. Subjects with mild hemophilia B typically experience bleeding only after a major injury, trauma, or surgery. In many cases, mild hemophilia is not diagnosed until trauma, surgery, or tooth extraction results in prolonged bleeding. The first episode may not occur until adulthood. Women with mild hemophilia often experience menorrhagia, heavy menstrual periods, and may experience bleeding after childbirth.

[00358] According to the National Hemophilia Foundation, a subject is classified as having moderate hemophilia B when their blood plasma contains 1 to 5% factor IX activity relative to normal human plasma. Subjects with moderate hemophilia B tend to bleed after trauma. Bleeding that occurs without an obvious cause is called a spontaneous bleeding episode.

[00359] According to the National Hemophilia Foundation, a subject is classified as having severe hemophilia B when their blood plasma contains less than 1% factor IX activity relative to normal human plasma. Subjects with severe hemophilia B experience bleeding after trauma and may have frequent episodes of spontaneous bleeding, often in the joints and muscles.

[00360] In some embodiments, normal human plasma is defined as containing 1 IU of factor IX activity per ml. Thus, in some embodiments, the blood plasma of a subject classified as having mild hemophilia B contains 0.06 to 0.49 IU of factor IX activity per ml. In some embodiments, the blood plasma of a subject classified as having moderate hemophilia B contains 0.01 to 0.05 IU of factor IX activity per ml. In some embodiments, the blood plasma of a subject classified as having severe hemophilia B contains 0.01 to 0.05 IU of factor IX activity per ml.

[00361] Accordingly, in some embodiments, the implementation of therapy for hemophilia B is therapeutically effective when it increases the average level of factor IX activity in the blood/plasma of the subject. In some embodiments, the therapeutically effective treatment increases the subject's average blood/plasma factor IX activity level by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10 %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more. In a specific embodiment, a therapeutically effective therapy for hemophilia increases the average factor IX activity in the subject's blood/plasma by at least 5%. In another specific embodiment, a therapeutically effective therapy for hemophilia increases the mean factor IX activity in the subject's blood/plasma by at least 10%. In another specific embodiment, a therapeutically effective therapy for hemophilia increases the average activity of factor IX in the blood/plasma of the subject by at least 15%. In another specific embodiment, a therapeutically effective therapy for hemophilia increases the mean factor IX activity in the subject's blood/plasma by at least 20%. In another specific embodiment, a therapeutically effective therapy for hemophilia increases the average factor IX activity in the subject's blood/plasma by at least 25%. In another specific embodiment, a therapeutically effective therapy for hemophilia increases the average factor IX activity in the subject's blood/plasma by at least 30%.

[00362] In some embodiments, the therapeutically effective treatment increases the subject's mean blood level of factor IX activity such that the subject is classified as having less severe hemophilia B. For example, in one embodiment, the subject is initially classified as having severe hemophilia B, is reclassified as having moderate hemophilia B or mild hemophilia B after receiving therapeutically effective treatment. In another embodiment, a subject initially classified as having mild hemophilia B is reclassified as having mild hemophilia B after undergoing therapeutically effective treatment.

Examples

Example 1 Codon-Changed Factor IX Expression Sequences Increase FIX Expression Levels

[00363] To create constructs for gene therapy that provide improved expression of heterologous factor IX in vivo , a panel of self-complementary AAV8-based vectors encoding a full-length factor IX pre-proprotein with the amino acid substitution R384L (FIXp-FL-AA SEQ ID NO: 4)) was constructed . The Factor IX coding sequence of each construct was modified to improve expression in humans in several steps. Each factor IX coding sequence was modified according to an algorithm designed to account for preferred/unwanted sequence motifs and to bias codon usage towards preferred human codons. Several algorithms were used in this first step, as shown in Table 2. The intermediate codon-changed sequences resulting from the algorithms presented in Table 2 were then further manually modified to reduce or eliminate CpG dinucleotides, after which adjustments were made to the final GC content, correction for preferred codon pairs, correction to avoid unfavorable codon pairs, and adjustment of final codon usage. For more information on these considerations, see, for example, Fath S. et al., PLoS. One., 6, e17596 (2011); Haas J. et al., Curr. Biol., 6, 315-324 (1996); Tats A., BMC Genomics. 9:463 (2008), Grote A. et al., Nucleic Acids Research, 33(Web Server issue), W526-W531 (2005), Mirsafian H. et al., Scientific World Journal., 639682 (2014) and Pechmann S. et al., Nat Struct Mol Biol. 20(2):237-43 (2013), the contents of which are expressly incorporated herein by reference, in their entirety, for all purposes, especially with respect to the teaching of codon change considerations.

[00364] Each generated codon-changed coding sequence (e.g., CS02, CS03, CS04, CS05, and CS06 shown in Figures 5-9, respectively) is encoded for an identical FIX(R384L) protein (FIXp-FL-AA (SEQ ID NO : four)). Constructs CS02, CS03 and CS04 do not contain CpG motifs, while CS05 and CS06 contain 11 and 3 CpGs, respectively.

[00365] Vector constructs were also generated for use as controls, including sequences encoding wild-type FIX with and without the R384L Padova amino acid substitutions. Construct WH01 encodes a wild type FIX preproprotein without the R384L Padua mutation and contains 20 CpG dinucleotides. The WH02 construct encodes a wild-type FIX preproprotein with the R384L Padua mutation and includes 19 CpG dinucleotides.

[00366] The coding sequences for constructs WH01 and WH02 include 20 and 19 CpGs, respectively. In contrast, constructs CS02, CS03, and CS04 contain no CpG motifs, while constructs CS05 and CS06 contain 11 and 3 CpGs, respectively.

[00367] As shown in FIG. 1A, codon-altered factor IX coding sequences were inserted into an adeno-associated virus (“AAV”) transgenic cassette containing a mouse transthyrethrin enhancer/promoter (SEQ ID NO: 52), mouse small virus intron (“MVM”), (SEQ ID NO : 53), a codon-altered FIX construct comprising the amino acid substitution R384L "Padua" (U.S. Patent No. 6,531,298; Simione et al., NEJM 361:1671-75 (2009); the R384L mutation is usually described as an R338L mutation referring to the arginine position wild type in the single chain human FIX protein lacking signal and propeptide), followed by a poly-A bovine growth hormone element (SEQ ID NO: 54). The gene cassette is flanked by AAV2 inverted terminal repeats ("ITR") (SEQ ID NOS: 51 and 55). The left repeat ITR includes a mutation at the terminal site of concatemer resolution resulting in a self-complementary (sc) phenotype of the vectors. The basic construction of the vector is described in detail in Wu et al., Mol. Ther. 16:280-89 (2008) and PCT Publication No. WO 2014/064277 A1, the contents of which are incorporated herein by reference in their entirety for all purposes.

[00368] Factor IX AAV CS and WH constructs were administered to FIX B6/129P2-F9tm1Dws knockout mice (described in Lin et al., Blood, 90:3962-66 (1997), the contents of which are incorporated herein by reference in its entirely, for any purpose). AAV vector dilutions were administered to animals (4-8 animals per group) via the lateral tail vein based on the body weight of individual animals (4×10E11 vector genomes/kilogram (vg/kg) body weight). Blood samples were collected at regular intervals post-dose by retroorbital puncture according to known procedures using glass capillaries. Then the blood was transferred into a test tube pre-filled with sodium citrate anticoagulant, plasma was obtained by standard procedures and frozen at -20°C.

Expression of various factor IX constructs was determined and FIX levels in plasma at day 14 in FIX knockout mice were used to evaluate the efficacy of the constructs after injection of vectors into mice in the tail vein, as shown in Table 2. By day 14, expression levels in the knockout mouse model were almost reached maximum FIX expression. As shown in Table 2, the WH02 FIX control construct (R384L) was expressed at 1.03 FIX units on day 14 after administration of 4×10E11 vector genomes/kilogram (wg/kg) body weight. This expression level was used as a baseline to determine the fold enrichment of codon-altered factor IX constructs. As shown in Table 2, the codon-altered CS constructs provided approximately 2-fold and 4-fold increased expression compared to the WH02 control construct encoded by the wild-type polynucleotide sequence. In particular, the codon-altered construct CS06 provided 4.2 times more factor IX activity than the WH02 control construct and 21.6 times more factor IX activity than the WH01 control construct (wt factor IX).

Table 2 Factor IX expression from contracts with wild-type (WH01-wtFIX; WH02-FIX (R384L) and codon-changed (CS02-CS06) codon sequences.

AAV
Design Vector genome modification Number of CpGs Day 14 expression levels
[% of people FIX] Expression fold compared to WH01 Expression fold compared to WH02 WH01 Wild type human FIX sequence (GeneBank NM000133.3) without R338L mutation (Padua) twenty 0.20 1.0 0.19 WH02 Human wild-type FIX sequence with mutation R338L (Padua) 19 1.03 5.2 1.0 CS02 Human FIX sequence with mutation R338L; Geneart's core algorithm is further optimized for human serum albumin codons. 0 2.12 10.6 2.1 CS03 Human FIX sequence with mutation R338L; the basic Geneart algorithm is further optimized for the most commonly used human codons (Haas et al., 1996. Curr Biol. 6, 315-324). 0 1.98 9.9 1.9 CS04 Human FIX sequence with mutation R338L; the underlying Geneart algorithm is further optimized to use liver codons as described in Uhlen et al., 2015 Science 347, 6220. 0 2.77 13.9 2.7 CS05 Human FIX sequence with mutation R338L; JCAT algorithm modified to reduce CpG; (Grote et al., 2005. Nucleic Acids Res 1.33). eleven 3.93 19.7 3.8 CS06 Human FIX sequence with mutation R338L; the basic Geneart algorithm is further optimized for the most commonly used human codons (Haas et al., 1996. Curr Biol. 6, 315-324). 3 4.32 21.6 4.2

Example 2 Liver-Specific CRM8 Elements Enhance FIX Expression in Mice

[00369] To further increase factor IX expression and activity of codon-altered constructs, one to three copies of a liver-specific cis-regulatory control element (CRM8 (SEQ ID NO: 39)), as reported in Nair et al., Blood 123 :3195-99 (2014) were inserted into the gene cassette to create the construct shown in Figure 1B. AAV vectors containing a codon-altered FIX CS02 coding sequence with zero (CS02-CRM8.0-V), one (CS02-CRM8.1-V), two (CS02-CRM8.2-V) or three (CS02-CRM8 .3-V) CRM8 controls were administered to wild-type mice via the tail vein. The level of human anti-FIX antigen in mouse plasma was then measured over time by human FIX-specific ELISA.

[00370] As shown in Table 3, the use of the CRM8 regulatory elements increased expression of factor IX in vivo by about 2 and 4 times compared to the expression of the control construct lacking the CRM8 element at 21 days post infection. For example, the CS02-CRM8.1-V vector containing a single CRM8 element provides dual FIX expression, as does the control vector CS02-CRM8.0-V. The inclusion of multiple copies of the CRM8 element led to a further improvement in this expression. For example, vectors containing 2 copies of the CRM8 element provided 3-fold expression, and vectors containing 3 copies of the CRM8 element provided 3.4-fold expression relative to the control vector.

Table 3 Plasma levels of factor IX expression in wild-type mice injected with codon-altered AAV vectors with 0-3 copies of the CRM8 regulatory control element.

No. AAV construction Number of CRM8 elements FIX (ng/ml)
Day 4 FIX (ng/ml)
Day 11 FIX (ng/ml)
Day 21 Multiple magnification
Day 21 one CS02-CRM8.0-scV 0 65.8 133.4 239.2 1.0 2 CS02-CRM8.1-scV one 120.7 250.7 442.8 1.9 3 CS02-CRM8.2-scV 2 152.9 417.3 713.8 3.0 four CS02-CRM8.3-scV 3 130.9 432.6 800.9 3.4

Example 3 Liver-Specific CRM8 Elements Enhance FIX Expression in Human Liver Cells

[00371] Factor IX CS02 gene therapy constructs containing 0-3 copies of the liver-specific regulatory control element CRM8 as described in Example 2 were further tested by in vitro biopotency assays using the human liver cell line HepG2. Briefly, HepG2 cells were infected with one of the AAV CS02-CRM8-V vectors as described in Example 2 and FIX activity was measured by chromogenic substrate assay three days post infection. Consistent with the results presented in Example 2, all vectors containing the CRM8 regulatory control element provided higher FIX expression, as shown in Table 4. Strikingly, the effect of using multiple CRM8 elements in human liver HepG2 cells was even more pronounced. than in the mouse model. For example, vectors containing 2 copies of the CRM8 element provided 6.7-fold expression, and vectors containing 3 copies of the CRM8 element provided 12.8-fold expression relative to the control vector. This confirms the beneficial effects that the CRM8 regulatory control element has on FIX expression in these vectors.

Table 4 Factor IX expression levels in human liver HepG2 cells injected with codon-altered AAV vectors with 0-3 copies of the CRM8 regulatory control element.

AAV construction Number of CRM8 elements FIX Activity
[Units of biological activity] Multiple magnification CS02-CRM8.0-scV 0 0.35 one CS02-CRM8.1-scV one 0.82 2.3 CS02-CRM8.2-scV 2 2.36 6.7 CS02-CRM8.3-scV 3 4.48 12.8

Example 4 Single stranded AAV8 FIX vectors provide similar in vivo expression to comparable self complementary vectors.

[00372] In some instances, self-complementary (sc) AAV vectors express the transgene cassette more efficiently than a similar single-stranded (ss) AAV vector. This is probably due to the faster formation of double-stranded strands after decoupling of the self-complementary vector genome in the cell nucleus. For a review, see McCarty D., Mol. Ther., (16): 1648-56 (2008), the contents of which are incorporated herein by reference in their entirety for all purposes.

[00373] A recent study confirmed this effect using the EGFP vector. Bell et al., Hum. Gene Ther. Methods, (27):228-37 (2016). However, the study also showed that this effect was transgenic and dose dependent. For example, the human ornithine transcarbamylase (hOTC) gene cassette in the self-complementary AAV8 vector showed better expression at low dose in mouse liver compared to the corresponding single-stranded vector. However, this effect could not be demonstrated at high doses, suggesting that the effect, at least in the non-secreted gene studied, was transgenic and dose dependent. Id.

[00374] In order to investigate the properties of the disclosed codon-altered FIX gene constructs in the context of single-stranded and self-complementary design, single-stranded constructs carrying the codon-altered FIX(R338L) CS06 gene and two intact ITRs were constructed with and without CRM8 regulatory control elements. them, as shown in Fig. 1D and 1C, respectively. Single-stranded (ss) vectors were generated in the HEK293 cell system and expression of factor IX was compared to that of the self-complementary constructs described in Examples 1-3.

[00375] First, self-complementary (sc) and single-stranded (ss) CS06-CRM8.0-V constructs were tested in vivo after injection into FIX B6/129P2-F9tm1Dws knockout mice as described above. Surprisingly, as shown in Table 5, self-complementary (sc) and single-stranded (ss) CS06 vector constructs showed very similar levels of plasma FIX activity, indicating that the reported advantage of sc vectors over ss vectors does not observed in the codon-altered factor IX constructs described herein. Expression depends on many parameters, including the transgene construct, transcript stability, promoters used in the construct, timing, and dose. As shown in Table 5, under the conditions chosen to correct bleeding and obtain long-term expression in FIX ko mice, the respective sc and ss vectors provided substantially similar levels of expression.

[00376] The effects of the liver-specific regulatory control element CRM8 on FIX expression were also investigated based on a single-stranded vector. As shown in Table 5, incorporation of one CRM8 element into the single stranded vector improved FIX expression in FIX B6/129P2-F9tm1Dws knockout mice. Inclusion of three CRM8 elements further improved FIX expression of the CS06 single chain construct to levels slightly more than 2-fold higher than the self-complementary control CS06 without the CRM8 element. Compared to the wild-type WH02 construct, CS06 single-stranded vectors provided 7-fold greater expression when combined with three CRM8 regulatory control elements.

Table 5. Factor IX expression levels in FIX knockout mice injected with various single-stranded (ss) and self-complementary (sc) factor IX AAV vectors.

AAV construction Number of CRM8 elements Expression level ¹
(D 7) expression level
(d. 14) Expression level (d. 28) Multiple increase rel. CS06
(d. 7/d. 14/d. 28) Multiple increase rel. WH02
(d. 7/d. 14/d. 28) CS06-CRM8.0-ssV 0 1.38 2.73 2.99 0.7/0.9/0.9 1.5/2.7 /2.7 CS06-CRM8.1-ssV one 1.92 3.57 3.47 1.0/1.1/1.0 2.1/3.5/3.2 CS06-CRM8.3-ssV (SEQ ID NO:40) 3 4.43 6.65 7.78 2.3/2.1/2.2 4.9/6.5/7.1 CS06-CRM8.0-scV 0 1.89 3.17 3.50 1.0/1.0/1.0 2.1/3.1/3.2 WH02-CRM8.0-scV 0 0.90 1.03 1.10 0.5/0.3/0.3 1.0 ¹ FIX activity in international units (average value based on the results of 7-8 mice); d., day;

Example 5 AAV8 single strand FIX vectors provide better FIX expression in human liver cells than comparable self complementary vectors.

[00377] Factor IX expression of the single-stranded CS06 vectors described in Example 4 was then examined in human HepG2 cell culture and compared with similar self-complementary vector constructs. Consistent with the in vivo results presented in Example 4, in HepG2 cells, single-stranded CS06 vectors lacking the CRM8 element provided FIX expression at somewhat lower levels than the comparative self-complementary vector. However, inclusion of a single CRM8 element increased FIX expression of the single-stranded CS06 vector to 2.6-fold the expression of the self-complementary CS06 vector, as shown in Table 6.

[00378] Most surprisingly, however, incorporation of three CRM8 elements into the single-stranded CS06 vector increased FIX expression to 16.8 times that of the self-complementary CS06 vector. The increased expression of FIX was more than 100 times greater than the FIX expression of the control vector WH02. Thus, the single-stranded CS06 vector containing three CRM8 elements provides the highest levels of expression in both in vivo and in vitro biopotency assays.

[00379] Table 6. Expression levels of factor IX single-stranded (ss) and self-complementary vectors in human liver cells.

AAV construction Number of CRM8 elements FIX Activity
[Biopotency Units] Multiple increase rel.
CS06-CRM8.0-scV Multiple increase rel. WH02-CRM8.0-scV CS06-CRM8.0-ssV 0 0.24 0.6 3.9 CS06-CRM8.1-ssV one 0.95 2.6 15.9 CS06-CRM8.3-ssV (SEQ ID NO: 40) 3 6.20 16.8 103.3 CS06-CRM8.0-scV 0 0.37 1.0 6.2 WH02-CRM8.0-scV 0 0.06 0.2 1.0

Materials and methods for examples 1-5.

[00380] Animal experiments. For experiments on the FIX knockout model, the FIX mouse strain ko B6/129P2-F9tm1Dws (developed by Lin et al., 1997. Blood 90:3962-6) was used. In the wild-type mouse model, male C57BL6-J Bl6 mice were used at 4-5 weeks of age. Both strains were obtained from commercial breeders. Dilutions of the AAV vector were administered to animals (4-8 animals per group) via the lateral tail vein based on the body weight of the individual animals. Blood sampling was performed at certain intervals after dosing by retroorbital puncture in accordance with known procedures using glass capillaries. Then the blood was transferred into a test tube pre-filled with sodium citrate anticoagulant, plasma was obtained by standard procedures and frozen at -20°C.

[00381] In vitro biopotency assay in HepG2 cells, including analysis of the chromogenic substrate FIX. In vitro biopotency analysis of the gene therapy vector preparations was performed on the human liver cell line HepG2 (ATCC HB-8065). Cells were treated with hydroxyurea, then infected with AAV8FIX vectors and incubated for approximately 96 hours. During incubation, FIX was expressed and released into the cell supernatant, and FIX activity was determined by measuring the chromogenic endpoint (Rossix AB, Sweden). Each assay includes a standard curve of purified AAV-FIX vector material using an MOI ranging from 700 to 7000. The activity of the FIX standard at MOI 3270 is taken as 1 biopotential unit (BPU).

Quantification of human FIX in mouse plasma. To quantify human FIX in the plasma of knockout mice, FIX coagulation assays were performed using standard FIX coagulation assays. A commercially available ELISA kit (ASSERACHROM IX: AG (Cat. No. 00943 Stago BNL)), which specifically detects human FIX, was used to quantify human anti-FIX antigen in wild-type mouse plasma.

Example 6 Improved Transcriptional Efficiency by Incorporating CRM8 Elements.

[00382] To investigate whether the increase in biopotency of CRM8-containing vectors is the result of increased transcriptional efficiency, a human liver cell line (HepG2) and mouse liver cells (FIX knockout mice) were transduced with single-stranded CS06 vectors containing 0, 1, or 3 CRM8 elements. FIX mRNA and DNA levels were determined and presented as the ratio between normalized FIX mRNA and DNA levels.

[00383] In an in vitro model, inclusion of one CRM8 element (CS06-CRM8.1-ssV) or three CRM8 elements (CS06-CRM8.3-ssV (SEQ ID NO: 40)) resulted in a 5-fold and a 23-fold increase human FIX mRNA levels in transduced human liver cells compared to cells transduced with a vector lacking the CRM8 element (CS06-CRM8.0-ssV) (Table 6), respectively. Similarly, in an in vivo model, FIX expression in mouse liver from vectors containing one or three CRM8 elements was 2.0-fold and 2.8-fold higher than FIX expression from a vector lacking the CRM8 element (Table 6) , respectively. Both models confirm that CRM8 elements have a positive effect by improving the transcriptional activity of the FIX construct.

[00384] Table 7. FIX mRNA levels following transduction of AAV8-FIX human or mouse liver cell lines.

Liver cell line (HepG2) in vitro Mouse liver tissue in vitro AAV construction Normalized FIX:mRNA/DNA Ratio Multiple increase rel. CS06-CRM8.0-ssV Normalized FIX:mRNA/DNA Ratio Multiple increase rel. CS06-CRM8.0-ssV CS06-CRM8.0-ssV 0.025 one 0.38 one CS06-CRM8.1-ssV 0.14 5.4 0.78 2.0 CS06-CRM8.3-ssV 0.58 23.4 1.09 2.8

Methods for Example 6

[00385] Quantitative real-time polymerase chain reaction, including RNA and DNA extraction. Genomic DNA and total RNA were extracted from frozen liver (see animal experiments) or HepG2 cells (see in vitro biopotency assay in HepG2 cells) using standard procedures. For the analysis of in vivo experiments, a subgroup of three animals per treatment group was selected that was close to the mean FIX activity of the corresponding group on day 14 (calculated from the mean ± standard deviation). cDNA synthesis was performed using oligo(dT20) primer, SuperScript III reverse transcriptase (RT) and DNase-treated total RNA according to the manual (DNeasy Blood & Tissue Kit, Qiagen, Germany; RNeasy mini kit, Qiagen).

[00386] The copy number of transgenic FIX in both gDNA and cDNA samples was determined using quantitative real-time fluorescence polymerase chain reaction (qPCR) amplifying the 96 bp FIX exon 6 sequence. Mouse β-actin was used as an endogenous control, which was quantified using a commercially available TaqMan assay. Analysis of qPCR data was performed using device-specific software, calculating the number of copies of FIX or β-actin per reaction based on the linear regression parameters of the standard curve. In addition, the results were normalized to 1 µg of RNA or DNA and the mRNA:DNA ratio was calculated.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be made to those skilled in the art and should be included within the spirit and 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 and for all purposes.

--->

SEQUENCE LIST

<110> Baxalta Incorporated

Baxalta GmbH

Horling, Franziska

Lengler, Johannes

Falkner, Falko-Guenther

Rottensteiner, Hanspeter

Scheiflinger, Friedrich

<120> VIRAL VECTORS ENCODING RECOMBINANT FIX WITH INCREASED EXPRESSION

FOR HEMOPHILIA B GENE THERAPY

<130> 008073-5117-WO

<150> US 62/509 616

<151> 05/22/2017

<160> 64

<170> PatentIn version 3.5

<210> 1

<211> 1386

<212> DNA

<213> Artificial sequence

<220>

<223> FIX-FL-NA

<400> 1

atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60

ggatatctac tcagtgctga atgtacagtt ttttcttgatc atgaaaacgc caacaaaatt 120

ctgaatcggc caaagaggta taattcaggt aaattggaag agtttgttca agggaacctt 180

gagagagaat gtatggaaga aaagtgtagt tttgaagaag cacgagaagt ttttgaaaac 240

actgaaagaa caactgaatt ttggaagcag tatgttgatg gagatcagtg tgagtccaat 300

ccatgtttaa atggcggcag ttgcaaggat gacattaatt cctatgaatg ttggtgtccc 360

tttggatttg aaggaaagaa ctgtgaatta gatgtaacat gtaacattaa gaatggcaga 420

tgcgagcagt tttgtaaaaa tagtgctgat aacaaggtgg tttgctcctg tactgaggga 480

tatcgacttg cagaaaacca gaagtcctgt gaaccagcag tgccatttcc atgtggaaga 540

gtttctgttt cacaaacttc taagctcacc cgtgctgaga ctgtttttcc tgatgtggac 600

tatgtaaatt ctactgaagc tgaaaccatt ttggataaca tcactcaaag cacccaatca 660

tttaatgact tcactcgggt tgttggtgga gaagatgcca aaccaggtca attcccttgg 720

caggttgttt tgaatggtaa agttgatgca ttctgtggag gctctatcgt taatgaaaaa 780

tggattgtaa ctgctgccca ctgtgttgaa actggtgtta aaattacagt tgtcgcaggt 840

gaacataata ttgaggagac agaacataca gagcaaaagc gaaatgtgat tcgaattatt 900

cctcaccaca actacaatgc agctattaat aagtacaacc atgacattgc ccttctggaa 960

ctggacgaac ccttagtgct aaacagctac gttacaccta tttgcattgc tgacaaggaa 1020

tacacgaaca tcttcctcaa atttggatct ggctatgtaa gtggctgggg aagagtcttc 1080

cacaaaggga gatcagcttt agttcttcag taccttagag ttccacttgt tgaccgagcc 1140

acatgtcttc gatctacaaa gttcaccatc tataacaaca tgttctgtgc tggcttccat 1200

1260

gggaccagtt tcttaactgg aattattagc tggggtgaag agtgtgcaat gaaaggcaaa 1320

tatggaatat ataccaaggt atcccggtat gtcaactgga ttaaggaaaa aacaaagctc 1380

acttaa 1386

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Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile

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Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe

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Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala

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<210> 5

<211> 1385

<212> DNA

<213> Artificial sequence

<220>

<223> CS02-FL-NA

<400> 5

atgcagaggg tgaacatgat catggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtcagcaga gtgcacagtg ttcctggacc atgagaatgc caacaagatc 120

ctgaacaggc ccaagagata caactcaggc aagctggagg agtttgtgca gggcaacctg 180

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acagagagga ccacagagtt ctggaagcag tatgtggatg gagaccagtg tgagagcaac 300

ccttgcctga atggaggcag ctgcaaggat gacatcaaca gctatgagtg ctggtgccct 360

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tgtgagcagt tctgcaagaa ctcagctgac aacaaagtgg tgtgtagctg cacagaggggc 480

tacagactgg ctgagaacca gaagagctgt gagcctgctg tgcccttccc ctgtggcaga 540

gtgtcagtgt cccagaccag caagctgacc agagctgaga cagtgttccc tgatgtggac 600

tatgtgaata gcacagaggc tgagaccatc ctggacaaca tcaccgaga cacccagtcc 660

ttcaatgact tcaccagagt tgtgggagga gaggatgcca agcctggcca gttcccctgg 720

caggtggtgc tgaatggcaa agtggatgcc ttctgtggag gcagcattgt gaatgagaag 780

tggattgtga cagctgccca ctgtgtggag acaggagtga agatcacagt ggtggctgga 840

gaacacaata ttgaggagac agagcacaca gagcagaaga ggaatgtcat caggattatc 900

ccccaccaca actacaatgc tgccatcaac aagtacaacc atgacattgc cctgctggag 960

ctggatgagc ctctggtgct gaatagctat gtgaccccca tctgcattgc tgacaaggag 1020

tacaccaaca tcttcctgaa gtttggctca ggctatgtgt caggctgggg cagagtgttc 1080

cacaagggca gatcagccct ggtgctgcag tacctgagag tgcccctggt ggacagagcc 1140

acctgcctgt tgagcaccaa gttcaccatc tacaacaaca tgttctgtgc tggcttccat 1200

gagggaggca gagacagctg ccagggagac tcaggaggac cccatgtgac agaagtggag 1260

ggcaccagct tcctgacagg catcatcagc tggggagagg agtgtgccat gaagggcaag 1320

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acctg 1385

<210> 6

<211> 1386

<212> DNA

<213> Artificial sequence

<220>

<223> CS03-FL-NA

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atgcagaggg tgaacatgat catggctgag agccctggcc tgatcaccat ctgcctgctg 60

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ctgaacaggc ccaagagata caactctggc aagctggagg agtttgtgca gggcaacctg 180

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ccttgcctga atgggggcag ctgcaaggat gacatcaaca gctatgagtg ctggtgccct 360

tttggctttg agggcaagaa ctgtgagctg gatgtgacct gcaacatcaa gaatggcagg 420

tgtgagcagt tctgcaagaa ctctgctgac aacaaagtgg tgtgtagctg cactgagggc 480

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gtgtctgtgt cccagaccag caagctgacc agagctgaga ctgtgttccc tgatgtggac 600

tatgtgaata gcactgaggc tgagaccatc ctggacaaca tcaccaggag cacccagtcc 660

ttcaatgact tcaccagagt ggtggggggg gaggatgcca agcctggcca gttcccctgg 720

caggtggtgc tgaatggcaa agtggatgcc ttctgtgggg gcagcattgt gaatgagaag 780

tggattgtga ctgctgccca ctgtgtggag actggggtga agatcactgt ggtggctggg 840

gaacacaata ttgaggagac tgagcacact gagcagaaga ggaatgtcat caggattatc 900

ccccaccaca actacaatgc tgccatcaac aagtacaacc atgacattgc cctgctggag 960

ctggatgagc ctctggtgct gaatagctat gtgaccccca tctgcattgc tgacaaggag 1020

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ggcaccagct tcctgactgg catcatcagc tgggggggagg agtgtgccat gaagggcaag 1320

tatggcatct acaccaaagt gagcaggtat gtgaactgga tcaaggagaa aaccaagctg 1380

acctga 1386

<210> 7

<211> 1386

<212> DNA

<213> Artificial sequence

<220>

<223> CS04-FL-NA

<400> 7

atgcagaggg tgaacatgat tatggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgcacagtg ttcctggacc atgagaatgc caacaagatc 120

ctgaacaggc ccaagagata caactctggc aagctggagg agtttgtgca gggcaacctg 180

gagagggagt gcatggagga gaagtgcagc tttgaggagg ccagggaggt gtttgagaac 240

acagagagga ccacagagtt ctggaagcag tatgtggatg gtgaccagtg tgagagcaac 300

ccttgcctga atggaggcag ctgcaaggat gacatcaaca gctatgagtg ctggtgccct 360

tttggctttg agggcaagaa ctgtgagctg gatgtgacct gcaacatcaa gaatggcagg 420

tgtgagcagt tctgcaagaa ctctgctgac aacaaggtgg tgtgtagctg cacagaggggc 480

tacagactgg ctgagaacca gaagagctgt gagcctgctg tgcccttccc ctgtggcaga 540

gtgtctgtgt cccagaccag caagctgacc agagctgaga cagtgttccc tgatgtggac 600

tatgtgaaca gcacagaggc tgagaccatc ctggacaaca tcaccgaac cacccagtcc 660

ttcaatgact tcaccagagt ggtgggagga gaggatgcca agcctggcca gttcccctgg 720

caggtggtgc tgaatggcaa ggtggatgcc ttctgtggag gcagcattgt gaatgagaag 780

tggattgtga cagctgccca ctgtgtggag acaggagtga agatcacagt ggtggctgga 840

gagcacaaca ttgaggagac agagcacaca gagcagaaga ggaatgtgat caggatcatc 900

cctcaccaca actacaatgc tgccatcaac aagtacaacc atgacattgc cctgctggag 960

ctggatgagc ctctggtgct gaacagctat gtgaccccta tctgcattgc tgacaaggag 1020

tacaccaaca tcttcctgaa gtttggctct ggctatgtgt ctggctgggg cagagtgttc 1080

cacaagggca ggtctgccct ggtgctgcag tacctgagag tgcccctggt ggacagagcc 1140

acctgcctgt tgagcaccaa gttcaccatc tacaacaaca tgttctgtgc tggcttccat 1200

gagggaggca gagacagctg ccagggtgac tctggaggac cccatgtgac agaggtggag 1260

ggcaccagct tcctgacagg catcatcagc tggggagagg agtgtgccat gaagggcaag 1320

tatggcatct acaccaaagt gagcagatat gtgaactgga tcaaggagaa gaccaagctg 1380

acctga 1386

<210> 8

<211> 1386

<212> DNA

<213> Artificial sequence

<220>

<223> CS05-FL-NA

<400> 8

atgcagaggg tgaacatgat tatggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgcactgtg ttcctggacc atgagaatgc caacaagatc 120

ctgaaccgcc ccaagcgcta caactctggc aagctggagg agtttgtgca gggcaacctg 180

gagagggagt gcatggagga gaagtgcagc tttgaggagg ccagggaggt gtttgagaac 240

actgagcgca ccactgagtt ctggaagcag tatgtggatg gggaccagtg tgagagcaac 300

ccctgcctga atggggggag ctgcaaggat gacatcaaca gctatgagtg ctggtgcccc 360

tttggctttg agggcaagaa ctgtgagctg gatgtgacct gcaacatcaa gaatggccgc 420

tgtgagcagt tctgcaagaa ctctgctgac aacaaggtgg tgtgctcttg cactgagggc 480

taccgcctgg ctgagaacca gaagagctgt gagcctgctg tgcccttccc ctgtggcagg 540

gtgtctgtga gccagaccag caagctgacc agggctgaga ctgtgttccc tgacgtggac 600

tatgtgaaca gcactgaggc tgagaccatc ctggacaaca tcaccgaag cacccagagc 660

ttcaatgact tcaccagggt ggtgggagga gaggatgcca agcctggcca gttcccctgg 720

caggtggtgc tgaatggcaa ggtggatgcc ttctgtggag gcagcattgt gaatgagaag 780

tggattgtga ccgctgccca ctgtgtggag actggagtga agatcactgt ggtggctggg 840

gagcacaaca ttgaggagac agagcacaca gagcagaagc gcaatgtgat caggatcatc 900

ccccaccaca actacaatgc tgccatcaac aagtacaacc atgacattgc cctgctggag 960

ctggatgagc ccctggtgct gaacagctac gtgaccccca tctgcattgc agacaaggag 1020

tacaccaaca tcttcctgaa gtttggctct ggctatgtgt ctggctgggg cagggtgttc 1080

cacaagggca ggtctgccct ggtgctgcag tacctgagggg tgcccctggt ggacagggcc 1140

acctgcctgc tgagcaccaa gttcaccatc tacaacaaca tgttctgcgc tggcttccat 1200

gagggaggaa gggacagctg ccagggagac tctggaggcc cccatgtgac agaggtggag 1260

ggcaccagct tcctgacagg catcatcagc tgggggggagg agtgtgccat gaagggcaag 1320

tatggcatct acaccaaagt gtcccgctat gtgaactgga tcaaggagaa gaccaagctg 1380

acctga 1386

<210> 9

<211> 1386

<212> DNA

<213> Artificial sequence

<220>

<223> CS06-FL-NA

<400> 9

atgcagaggg tcaacatgat catggctgag tcccctggcc tcatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgcactgtc ttcctggacc atgagaatgc caacaagatc 120

ctcaacaggc ccaagagata caactctggc aaactggagg agtttgtcca gggcaacctg 180

gagagggagt gcatggagga gaagtgctcc tttgaggagg ccagggaggt ctttgagaac 240

actgagcgca ccactgagtt ctggaaacag tatgtggatg gggaccagtg tgagtccaac 300

ccctgcctga atgggggcag ctgcaaggat gacatcaaca gctatgagtg ctggtgcccc 360

tttggctttg agggcaagaa ctgtgagctg gatgtgacct gcaacatcaa gaatggcaga 420

tgtgagcagt tctgcaagaa ctctgctgac aacaaggtgg tgtgctcctg cactgagggc 480

taccgcctgg ctgagaacca gaagagctgt gagcctgctg tgccattccc atgtggcaga 540

gtctctgtga gccagaccag caagctcacc agggctgaga ctgtgttccc tgatgtggac 600

tatgtgaaca gcactgaggc tgaaaccatc ctggacaaca tcaccgaag cacccagagc 660

ttcaatgact tcaccagagt ggtgggagga gaggatgcca agcctggcca gttcccctgg 720

caagtggtgc tcaatggcaa ggtggatgcc ttctgtgggg gctccattgt gaatgagaag 780

tggattgtca ctgctgccca ctgtgtggag actggggtca agatcactgt ggtggctggg 840

gagcacaaca ttgaggagac tgagcacact gagcagaagc gcaatgtgat caggatcatc 900

ccccaccaca actacaatgc tgccatcaac aagtacaacc atgacattgc cctgctggag 960

ctggatgagc ccctggtcct caacagctat gtgaccccca tctgcattgc tgacaaggag 1020

tacaccaaca tcttcctcaa gtttggctct ggctatgtct ctggctgggg cagagtgttc 1080

cacaaaggca ggtctgccct ggtgctccag tacctgagag tgcccctggt ggacagggcc 1140

acctgcctct tgagcaccaa gttcaccatc tacaacaaca tgttctgtgc tggcttccat 1200

gagggaggaa gagacagctg ccagggggac tctggaggac cccatgtcac tgaggtggag 1260

ggcacctcct tcctcactgg catcatctcc tggggagagg agtgtgccat gaaaggcaaa 1320

tatggcatct acaccaaagt ctccagatat gtcaactgga tcaaggagaa gaccaagctg 1380

acctga 1386

<210> 10

<211> 415

<212> Protein

<213> Artificial sequence

<220>

<223> FIX-MP-AA

<400> 10

Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg

1.5 10 15

Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe

20 25 30

Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly

35 40 45

Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp

50 55 60

Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys

65 70 75 80

Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu

85 90 95

Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr

100 105 110

Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val

115 120 125

Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr

130 135 140

Arg Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu

145 150 155 160

Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn

165 170 175

Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe

180 185 190

Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly

195 200 205

Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu

210 215 220

Thr Gly Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu

225 230 235 240

Thr Glu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His

245 250 255

His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu

260 265 270

Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile

275 280 285

Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser

290 295 300

Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala

305 310 315 320

Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys

325 330 335

Leu Arg Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly

340 345 350

Phe His Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

355 360 365

His Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser

370 375 380

Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys

385 390 395 400

Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr

405 410 415

<210> 11

<211> 377

<212> Protein

<213> Artificial sequence

<220>

<223> FIX2-MP-AA

<400> 11

Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg

1.5 10 15

Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe

20 25 30

Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Val

35 40 45

Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe Cys Lys Asn Ser

50 55 60

Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly Tyr Arg Leu Ala

65 70 75 80

Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe Pro Cys Gly Arg

85 90 95

Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe

100 105 110

Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu Thr Ile Leu Asp

115 120 125

Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe Thr Arg Val Val

130 135 140

Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp Gln Val Val Leu

145 150 155 160

Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile Val Asn Glu Lys

165 170 175

Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly Val Lys Ile Thr

180 185 190

Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu His Thr Glu Gln

195 200 205

Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn Tyr Asn Ala Ala

210 215 220

Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu Leu Asp Glu Pro

225 230 235 240

Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile Ala Asp Lys Glu

245 250 255

Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr Val Ser Gly Trp

260 265 270

Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val Leu Gln Tyr Leu

275 280 285

Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg Ser Thr Lys Phe

290 295 300

Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His Glu Gly Gly Arg

305 310 315 320

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val Thr Glu Val Glu

325 330 335

Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly Glu Glu Cys Ala

340 345 350

Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser Arg Tyr Val Asn

355 360 365

Trp Ile Lys Glu Lys Thr Lys Leu Thr

370 375

<210> 12

<211> 415

<212> Protein

<213> Artificial sequence

<220>

<223> FIXp-MP-AA

<400> 12

Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg

1.5 10 15

Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe

20 25 30

Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly

35 40 45

Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp

50 55 60

Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys

65 70 75 80

Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu

85 90 95

Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr

100 105 110

Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val

115 120 125

Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr

130 135 140

Arg Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu

145 150 155 160

Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn

165 170 175

Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe

180 185 190

Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly

195 200 205

Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu

210 215 220

Thr Gly Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu

225 230 235 240

Thr Glu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His

245 250 255

His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu

260 265 270

Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile

275 280 285

Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser

290 295 300

Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala

305 310 315 320

Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys

325 330 335

Leu Leu Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly

340 345 350

Phe His Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

355 360 365

His Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser

370 375 380

Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys

385 390 395 400

Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr

405 410 415

<210> 13

<211> 1248

<212> DNA

<213> Artificial sequence

<220>

<223> CS02-MP-NA

<400> 13

tacaactcag gcaagctgga ggagtttgtg cagggcaacc tggagaggga gtgcatggag 60

120

ttctggaagc agtatgtgga tggagaccag tgtgagagca acccttgcct gaatggaggc 180

agctgcaagg atgacatcaa cagctatgag tgctggtgcc cttttggctt tgagggcaag 240

aactgtgagc tggatgtgac ctgcaacatc aagaatggca ggtgtgagca gttctgcaag 300

aactcagctg acaacaaagt ggtgtgtagc tgcacagagg gctacagact ggctgagaac 360

cagaagagct gtgagcctgc tgtgcccttc ccctgtggca gagtgtcagt gtcccagacc 420

agcaagctga ccagagctga gacagtgttc cctgatgtgg actatgtgaa tagcacagag 480

gctgagacca tcctggacaa catcacccag agcacccagt ccttcaatga cttcaccaga 540

gttgtgggag gagaggatgc caagcctggc cagttcccct ggcaggtggt gctgaatggc 600

aaagtggatg ccttctgtgg aggcagcatt gtgaatgaga agtggattgt gacagctgcc 660

cactgtgtgg agacaggagt gaagatcaca gtggtggctg gagaacacaa tattgaggag 720

acagagcaca cagagcagaa gaggaatgtc atcaggatta tcccccacca caactacaat 780

gctgccatca acaagtacaa ccatgacatt gccctgctgg agctggatga gcctctggtg 840

ctgaatagct atgtgacccc catctgcatt gctgacaagg agtacaccaa catcttcctg 900

aagtttggct caggctatgt gtcaggctgg ggcagagtgt tccacaaggg cagatcagcc 960

ctggtgctgc agtacctgag agtgcccctg gtggacagag ccacctgcct gttgagcacc 1020

aagttcacca tctacaacaa catgttctgt gctggcttcc atgagggagg cagagacagc 1080

tgccagggag actcaggagg accccatgtg acagaagtgg agggcaccag cttcctgaca 1140

ggcatcatca gctggggaga ggagtgtgcc atgaagggca agtatggcat ctacaccaaa 1200

gtgagcagat atgtgaactg gatcaaggag aaaaccaagc tgacctga 1248

<210> 14

<211> 1248

<212> DNA

<213> Artificial sequence

<220>

<223> CS03-MP-NA

<400> 14

tacaactctg gcaagctgga ggagtttgtg cagggcaacc tggagaggga gtgcatggag 60

gagaagtgca gctttgagga ggccagggaa gtgtttgaga acactgagag gaccactgag 120

ttctggaagc agtatgtgga tggggaccag tgtgagagca acccttgcct gaatgggggc 180

agctgcaagg atgacatcaa cagctatgag tgctggtgcc cttttggctt tgagggcaag 240

aactgtgagc tggatgtgac ctgcaacatc aagaatggca ggtgtgagca gttctgcaag 300

aactctgctg acaacaaagt ggtgtgtagc tgcactgagg gctacagact ggctgagaac 360

cagaagagct gtgagcctgc tgtgcccttc ccctgtggca gagtgtctgt gtcccagacc 420

agcaagctga ccagagctga gactgtgttc cctgatgtgg actatgtgaa tagcactgag 480

gctgagacca tcctggacaa catcacccag agcacccagt ccttcaatga cttcaccaga 540

gtggtggggg gggaggatgc caagcctggc cagttcccct ggcaggtggt gctgaatggc 600

aaagtggatg ccttctgtgg gggcagcatt gtgaatgaga agtggattgt gactgctgcc 660

cactgtgtgg agactggggt gaagatcact gtggtggctg gggaacacaa tattgaggag 720

actgagcaca ctgagcagaa gaggaatgtc atcaggatta tcccccacca caactacaat 780

gctgccatca acaagtacaa ccatgacatt gccctgctgg agctggatga gcctctggtg 840

ctgaatagct atgtgacccc catctgcatt gctgacaagg agtacaccaa catcttcctg 900

aagtttggct ctggctatgt gtctggctgg ggcagagtgt tccacaaggg caggtctgcc 960

ctggtgctgc agtacctgag agtgcccctg gtggacagag ccacctgcct gctgagcacc 1020

aagttcacca tctacaacaa catgttctgt gctggcttcc atgagggggg cagagacagc 1080

tgccagggggg actctggggg cccccatgtg actgaagtgg agggcaccag cttcctgact 1140

ggcatcatca gctgggggga ggagtgtgcc atgaagggca agtatggcat ctacaccaaa 1200

gtgagcaggt atgtgaactg gatcaaggag aaaaccaagc tgacctga 1248

<210> 15

<211> 1250

<212> DNA

<213> Artificial sequence

<220>

<223> CS04-MP-NA

<400> 15

cstacaactc tggcaagctg gaggagtttg tgcagggcaa cctggagagg gagtgcatgg 60

120 aggagaagtg cagctttgag gaggccaggg

agttctggaa gcagtatgtg gatggtgacc agtgtgagag caacccttgc ctgaatggag 180

gcagctgcaa ggatgacatc aacagctatg agtgctggtg cccttttggc tttgagggca 240

agaactgtga gctggatgtg acctgcaaca tcaagaatgg caggtgtgag cagttctgca 300

agaactctgc tgacaacaag gtggtgtgta gctgcacaga gggctacaga ctggctgaga 360

accagaagag ctgtgagcct gctgtgccct tcccctgtgg cagagtgtct gtgtcccaga 420

ccagcaagct gaccagagct gagacagtgt tccctgatgt ggactatgtg aacagcacag 480

aggctgagac catcctggac aacatcaccc aggcaccca gtccttcaat gacttcacca 540

gagtggtggg aggagaggat gccaagcctg gccagttccc ctggcaggtg gtgctgaatg 600

gcaaggtgga tgccttctgt ggaggcagca ttgtgaatga gaagtggatt gtgacagctg 660

cccactgtgt ggagacagga gtgaagatca cagtggtggc tggagagcac aacattgagg 720

agacagagca cacagagcag aagaggaatg tgatcaggat catccctcac cacaactaca 780

atgctgccat caacaagtac aaccatgaca ttgccctgct ggagctggat gagcctctgg 840

tgctgaacag ctatgtgacc cctatctgca ttgctgacaa ggagtacacc aacatcttcc 900

tgaagtttgg ctctggctat gtgtctggct ggggcagagt gttccacaag ggcaggtctg 960

ccctggtgct gcagtacctg agagtgcccc tggtggacag agccacctgc ctgttgagca 1020

ccaagttcac catctacaac aacatgttct gtgctggctt ccatgaggga ggcagagaca 1080

gctgccaggg tgactctgga ggaccccatg tgacagaggt ggagggcacc agcttcctga 1140

caggcatcat cagctgggga gaggagtgtg ccatgaaggg caagtatggc atctacacca 1200

aagtgagcag atatgtgaac tggatcaagg agagaccaa gctgacctga 1250

<210> 16

<211> 1248

<212> DNA

<213> Artificial sequence

<220>

<223> CS05-MP-NA

<400> 16

tacaactctg gcaagctgga ggagtttgtg cagggcaacc tggagaggga gtgcatggag 60

gagaagtgca gctttgagga ggccagggag gtgtttgaga acactgagcg caccactgag 120

ttctggaagc agtatgtgga tggggaccag tgtgagagca acccctgcct gaatgggggg 180

agctgcaagg atgacatcaa cagctatgag tgctggtgcc cctttggctt tgagggcaag 240

aactgtgagc tggatgtgac ctgcaacatc aagaatggcc gctgtgagca gttctgcaag 300

aactctgctg acaacaaggt ggtgtgctct tgcactgagg gctaccgcct ggctgagaac 360

cagaagagct gtgagcctgc tgtgcccttc ccctgtggca gggtgtctgt gagccagacc 420

agcaagctga ccagggctga gactgtgttc cctgacgtgg actatgtgaa cagcactgag 480

gctgagacca tcctggacaa catcacccag agcacccaga gcttcaatga cttcaccagg 540

gtggtgggag gagaggatgc caagcctggc cagttcccct ggcaggtggt gctgaatggc 600

aaggtggatg ccttctgtgg aggcagcatt gtgaatgaga agtggattgt gaccgctgcc 660

cactgtgtgg agactggagt gaagatcact gtggtggctg gggagcacaa cattgaggag 720

acagagcaca cagagcagaa gcgcaatgtg atcaggatca tcccccacca caactacaat 780

gctgccatca acaagtacaa ccatgacatt gccctgctgg agctggatga gcccctggtg 840

ctgaacagct acgtgacccc catctgcatt gcagacaagg agtacaccaa catcttcctg 900

aagtttggct ctggctatgt gtctggctgg ggcagggtgt tccacaaggg caggtctgcc 960

ctggtgctgc agtacctgag ggtgcccctg gtggacaggg ccacctgcct gctgagcacc 1020

aagttcacca tctacaacaa catgttctgc gctggcttcc atgagggagg aagggacagc 1080

tgccagggag actctggagg cccccatgtg acagaggtgg agggcaccag cttcctgaca 1140

ggcatcatca gctgggggga ggagtgtgcc atgaagggca agtatggcat ctacaccaaa 1200

gtgtcccgct atgtgaactg gatcaaggag aagaccaagc tgacctga 1248

<210> 17

<211> 1248

<212> DNA

<213> Artificial sequence

<220>

<223> CS06-MP-NA

<400> 17

tacaactctg gcaaactgga ggagtttgtc cagggcaacc tggagaggga gtgcatggag 60

gagaagtgct cctttgagga ggccagggag gtctttgaga acactgagcg caccactgag 120

ttctggaaac agtatgtgga tggggaccag tgtgagtcca acccctgcct gaatgggggc 180

agctgcaagg atgacatcaa cagctatgag tgctggtgcc cctttggctt tgagggcaag 240

aactgtgagc tggatgtgac ctgcaacatc aagaatggca gatgtgagca gttctgcaag 300

aactctgctg acaacaaggt ggtgtgctcc tgcactgagg gctaccgcct ggctgagaac 360

cagaagagct gtgagcctgc tgtgccattc ccatgtggca gagtctctgt gagccagacc 420

agcaagctca ccagggctga gactgtgttc cctgatgtgg actatgtgaa cagcactgag 480

gctgaaacca tcctggacaa catcacccag agcacccaga gcttcaatga cttcaccaga 540

gtggtgggag gagaggatgc caagcctggc cagttcccct ggcaagtggt gctcaatggc 600

aaggtggatg ccttctgtgg gggctccatt gtgaatgaga agtggattgt cactgctgcc 660

cactgtgtgg agactggggt caagatcact gtggtggctg gggagcacaa cattgaggag 720

actgagcaca ctgagcagaa gcgcaatgtg atcaggatca tcccccacca caactacaat 780

gctgccatca acaagtacaa ccatgacatt gccctgctgg agctggatga gcccctggtc 840

ctcaacagct atgtgacccc catctgcatt gctgacaagg agtacaccaa catcttcctc 900

aagtttggct ctggctatgt ctctggctgg ggcagagtgt tccacaaagg caggtctgcc 960

ctggtgctcc agtacctgag agtgcccctg gtggacaggg ccacctgcct cttgagcacc 1020

aagttcacca tctacaacaa catgttctgt gctggcttcc atgagggagg aagagacagc 1080

tgccagggggg actctggagg accccatgtc actgaggtgg agggcacctc cttcctcact 1140

ggcatcatct cctggggaga ggagtgtgcc atgaaaggca aatatggcat ctacaccaaa 1200

gtctccagat atgtcaactg gatcaaggag aagaccaagc tgacctga 1248

<210> 18

<211> 138

<212> DNA

<213> Artificial sequence

<220>

<223> FIX-PPP-NA

<400> 18

atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60

ggatatctac tcagtgctga atgtacagtt ttttcttgatc atgaaaacgc caacaaaatt 120

ctgaatcggc caaagagg 138

<210> 19

<211> 138

<212> DNA

<213> Artificial sequence

<220>

<223> CS02-PPP-NA

<400> 19

atgcagaggg tgaacatgat catggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtcagcaga gtgcacagtg ttcctggacc atgagaatgc caacaagatc 120

ctgaacaggc ccaagaga 138

<210> 20

<211> 138

<212> DNA

<213> Artificial sequence

<220>

<223> CS03-PPP-NA

<400> 20

atgcagaggg tgaacatgat catggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgcactgtg ttcctggacc atgagaatgc caacaagatc 120

ctgaacaggc ccaagaga 138

<210> 21

<211> 138

<212> DNA

<213> Artificial sequence

<220>

<223> CS04-PPP-NA

<400> 21

atgcagaggg tgaacatgat tatggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgcacagtg ttcctggacc atgagaatgc caacaagatc 120

ctgaacaggc ccaagaga 138

<210> 22

<211> 138

<212> DNA

<213> Artificial sequence

<220>

<223> CS05-PPP-NA

<400> 22

atgcagaggg tgaacatgat tatggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgcactgtg ttcctggacc atgagaatgc caacaagatc 120

ctgaaccgcc ccaagcgc 138

<210> 23

<211> 138

<212> DNA

<213> Artificial sequence

<220>

<223> CS06-PPP-NA

<400> 23

atgcagaggg tcaacatgat catggctgag tcccctggcc tcatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgcactgtc ttcctggacc atgagaatgc caacaagatc 120

ctcaacaggc ccaagaga 138

<210> 24

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> FIX-SP-NA

<400> 24

atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60

ggatatctac tcagtgctga atgt 84

<210> 25

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> CS02-SP-NA

<400> 25

atgcagaggg tgaacatgat catggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtcagcaga gtgc 84

<210> 26

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> CS03-SP-NA

<400> 26

atgcagaggg tgaacatgat catggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgc 84

<210> 27

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> CS04-SP-NA

<400> 27

atgcagaggg tgaacatgat tatggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgc 84

<210> 28

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> CS05-SP-NA

<400> 28

atgcagaggg tgaacatgat tatggctgag agccctggcc tgatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgc 84

<210> 29

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> CS06--SP-NA

<400> 29

atgcagaggg tcaacatgat catggctgag tcccctggcc tcatcaccat ctgcctgctg 60

ggctacctgc tgtctgctga gtgc 84

<210> 30

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> FIX-PP-NA

<400> 30

acagtttttc ttgatcatga aaacgccaac aaaattctga atcggccaaa gagg 54

<210> 31

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> CS02-PP-NA

<400> 31

acagtgttcc tggaccatga gaatgccaac aagatcctga acaggcccaa gaga 54

<210> 32

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> CS03-PP-NA

<400> 32

actgtgttcc tggaccatga gaatgccaac aagatcctga acaggcccaa gaga 54

<210> 33

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> CS04-PP-NA

<400> 33

acagtgttcc tggaccatga gaatgccaac aagatcctga acaggcccaa gaga 54

<210> 34

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> CS05-PP-NA

<400> 34

actgtgttcc tggaccatga gaatgccaac aagatcctga accgccccaa gcgc 54

<210> 35

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> CS06-PP-NA

<400> 35

actgtcttcc tggaccatga gaatgccaac aagatcctca acaggcccaa gaga 54

<210> 36

<211> 46

<212> Protein

<213> Artificial sequence

<220>

<223> FIX-PPP-AA

<400> 36

Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr

1.5 10 15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu

20 25 30

Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg

35 40 45

<210> 37

<211> 28

<212> Protein

<213> Artificial sequence

<220>

<223> FIX-SP-AA

<400> 37

Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr

1.5 10 15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys

20 25

<210> 38

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> FIX-PP-AA

<400> 38

Thr Val Phe Leu Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro

1.5 10 15

Lys Arg

<210> 39

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> CRM8

<400> 39

gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcgggagg agcaaacagg 60

ggctaagtcc ac 72

<210> 40

<211> 5276

<212> DNA

<213> Artificial sequence

<220>

<223> CS06-CRM8.3-ssV

<400> 40

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcctgagtt taaacttcgt cgacggggga ggctgctggt 180

gaatattaac caaggtcacc ccagttatcg gaggagcaaa caggggctaa gtccaccgggg 240

ggaggctgct ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc 300

taagtccacc gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcgggagg 360

agcaaacagg ggctaagtcc accgagggca ctgggaggat gttgagtaag atggaaaact 420

actgatgacc cttgcagaga cagagtatta ggacatgttt gaacaggggc cgggcgatca 480

gcaggtagct ctagaggatc cccgtctgtc tgcacatttc gtagagcgag tgttccgata 540

ctctaatctc cctaggcaag gttcatattt gtgtaggtta cttattctcc ttttgttgac 600

taagtcaata atcagaatca gcaggtttgg agtcagcttg gcagggatca gcagcctggg 660

ttggaaggag ggggtataaa agccccttca ccaggagaag ccgtcacaca gactaggcgc 720

gccctaaggt aagttggcgc cgtttaaggg atggttggtt ggtggggtat taatgtttaa 780

ttaccttttt tacaggcctg aagatctgcc accatgcaga gggtcaacat gatcatggct 840

gagtcccctg gcctcatcac catctgcctg ctgggctacc tgctgtctgc tgagtgcact 900

gtcttcctgg accatgagaa tgccaacaag atcctcaaca ggcccaagag atcaactct 960

ggcaaactgg aggagtttgt ccagggcaac ctggagaggg agtgcatgga ggagaagtgc 1020

tcctttgagg aggccaggga ggtctttgag aacactgagc gcaccactga gttctggaaa 1080

cagtatgtgg atggggacca gtgtgagtcc aacccctgcc tgaatggggg cagctgcaag 1140

gatgacatca acagctatga gtgctggtgc ccctttggct ttgagggcaa gaactgtgag 1200

ctggatgtga cctgcaacat caagaatggc agatgtgagc agttctgcaa gaactctgct 1260

gacaacaagg tggtgtgctc ctgcactgag ggctaccgcc tggctgagaa ccagaagagc 1320

tgtgagcctg ctgtgccatt cccatgtggc agagtctctg tgagccagac cagcaagctc 1380

accagggctg agactgtgtt ccctgatgtg gactatgtga acagcactga ggctgaaacc 1440

atcctggaca acatcaccca gagcacccag agcttcaatg acttcaccag agtggtggga 1500

ggagaggatg ccaagcctgg ccagttcccc tggcaagtgg tgctcaatgg caaggtggat 1560

gccttctgtg ggggctccat tgtgaatgag aagtggattg tcactgctgc ccactgtgtg 1620

gagactgggg tcaagatcac tgtggtggct ggggagcaca acattgagga gactgagcac 1680

actgagcaga agcgcaatgt gatcaggatc atcccccacc acaactacaa tgctgccatc 1740

aacaagtaca accatgacat tgccctgctg gagctggatg agcccctggt cctcaacagc 1800

tatgtgaccc ccatctgcat tgctgacaag gagtacacca acatcttcct caagtttggc 1860

tctggctatg tctctggctg gggcagagtg ttccacaaag gcaggtctgc cctggtgctc 1920

cagtacctga gagtgcccct ggtggacagg gccacctgcc tcttgagcac caagttcacc 1980

atctacaaca acatgttctg tgctggcttc catgagggag gaagagacag ctgccagggg 2040

gactctggag gaccccatgt cactgaggtg gagggcacct ccttcctcac tggcatcatc 2100

tcctggggag aggagtgtgc catgaaaggc aaatatggca tctacaccaa agtctccaga 2160

tatgtcaact ggatcaagga gaagaccaag ctgacctgat gagcatgcct agagctcgct 2220

gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 2280

cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 2340

catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 2400

agggggagga ttgggaagac aatagcaggc atgctgggga attaattaag ctcgcgaagg 2460

aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 2520

ggcgaccaaa ggtcgcccga cgccggggct ttgcccggggc ggcctcagtg agcgagcgag 2580

cgcgcagaga gggagtggcc aagacgattt aaatgacaag cttggcgtaa tcatggtcat 2640

agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 2700

gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 2760

gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 2820

aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 2880

cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 2940

ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 3000

3060

acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 3120

gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 3180

catagctcac 3240

gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 3300

3360

3420

atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 3480

3540

cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 3600

ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 3660

ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 3720

3780

aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 3840

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 3900

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 3960

atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 4020

tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 4080

ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 4140

ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 4200

tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 4260

ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 4320

ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 4380

tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 4440

gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 4500

taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 4560

cttttacttt caccagcgtt tctggggtgag caaaaacagg aaggcaaaat gccgcaaaaa 4620

agggaataag ggcgacacgg aaatgttgaa tactcatact cttcctttttt caatattatt 4680

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 4740

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 4800

ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg 4860

cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag 4920

cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg 4980

gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc 5040

atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt 5100

cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac 5160

gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt 5220

cccagtcacg acgttgtaaa acgacggcca gtgaattcct cgagatttaa atgacg 5276

<210> 41

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> CS02-HC-NA

<400> 41

gttgtgggag gagaggatgc caagcctggc cagttcccct ggcaggtggt gctgaatggc 60

aaagtggatg ccttctgtgg aggcagcatt gtgaatgaga agtggattgt gacagctgcc 120

cactgtgtgg agacaggagt gaagatcaca gtggtggctg gagaacacaa tattgaggag 180

acagagcaca cagagcagaa gaggaatgtc atcaggatta tcccccacca caactacaat 240

gctgccatca acaagtacaa ccatgacatt gccctgctgg agctggatga gcctctggtg 300

ctgaatagct atgtgacccc catctgcatt gctgacaagg agtacaccaa catcttcctg 360

aagtttggct caggctatgt gtcaggctgg ggcagagtgt tccacaaggg cagatcagcc 420

ctggtgctgc agtacctgag agtgcccctg gtggacagag ccacctgcct gttgagcacc 480

aagttcacca tctacaacaa catgttctgt gctggcttcc atgagggagg cagagacagc 540

tgccagggag actcaggagg accccatgtg acagaagtgg agggcaccag cttcctgaca 600

ggcatcatca gctggggaga ggagtgtgcc atgaagggca agtatggcat ctacaccaaa 660

gtgagcagat atgtgaactg gatcaaggag aaaaccaagc tg 702

<210> 42

<211> 435

<212> DNA

<213> Artificial sequence

<220>

<223> CS02-LC-NA

<400> 42

tacaactcag gcaagctgga ggagtttgtg cagggcaacc tggagaggga gtgcatggag 60

120

ttctggaagc agtatgtgga tggagaccag tgtgagagca acccttgcct gaatggaggc 180

agctgcaagg atgacatcaa cagctatgag tgctggtgcc cttttggctt tgagggcaag 240

aactgtgagc tggatgtgac ctgcaacatc aagaatggca ggtgtgagca gttctgcaag 300

aactcagctg acaacaaagt ggtgtgtagc tgcacagagg gctacagact ggctgagaac 360

cagaagagct gtgagcctgc tgtgcccttc ccctgtggca gagtgtcagt gtcccagacc 420

agcaagctga ccaga 435

<210> 43

<211> 705

<212> DNA

<213> Artificial sequence

<220>

<223> CS03-HC-NA

<400> 43

gtggtggggg gggaggatgc caagcctggc cagttcccct ggcaggtggt gctgaatggc 60

aaagtggatg ccttctgtgg gggcagcatt gtgaatgaga agtggattgt gactgctgcc 120

cactgtgtgg agactggggt gaagatcact gtggtggctg gggaacacaa tattgaggag 180

actgagcaca ctgagcagaa gaggaatgtc atcaggatta tcccccacca caactacaat 240

gctgccatca acaagtacaa ccatgacatt gccctgctgg agctggatga gcctctggtg 300

ctgaatagct atgtgacccc catctgcatt gctgacaagg agtacaccaa catcttcctg 360

aagtttggct ctggctatgt gtctggctgg ggcagagtgt tccacaaggg caggtctgcc 420

ctggtgctgc agtacctgag agtgcccctg gtggacagag ccacctgcct gctgagcacc 480

aagttcacca tctacaacaa catgttctgt gctggcttcc atgagggggg cagagacagc 540

tgccagggggg actctggggg cccccatgtg actgaagtgg agggcaccag cttcctgact 600

ggcatcatca gctgggggga ggagtgtgcc atgaagggca agtatggcat ctacaccaaa 660

gtgagcaggt atgtgaactg gatcaaggag aaaaccaagc tgacc 705

<210> 44

<211> 435

<212> DNA

<213> Artificial sequence

<220>

<223> CS03-LC-NA

<400> 44

tacaactctg gcaagctgga ggagtttgtg cagggcaacc tggagaggga gtgcatggag 60

gagaagtgca gctttgagga ggccagggaa gtgtttgaga acactgagag gaccactgag 120

ttctggaagc agtatgtgga tggggaccag tgtgagagca acccttgcct gaatgggggc 180

agctgcaagg atgacatcaa cagctatgag tgctggtgcc cttttggctt tgagggcaag 240

aactgtgagc tggatgtgac ctgcaacatc aagaatggca ggtgtgagca gttctgcaag 300

aactctgctg acaacaaagt ggtgtgtagc tgcactgagg gctacagact ggctgagaac 360

cagaagagct gtgagcctgc tgtgcccttc ccctgtggca gagtgtctgt gtcccagacc 420

agcaagctga ccaga 435

<210> 45

<211> 705

<212> DNA

<213> Artificial sequence

<220>

<223> CS04-HC-NA

<400> 45

gtggtgggag gagaggatgc caagcctggc cagttcccct ggcaggtggt gctgaatggc 60

aaggtggatg ccttctgtgg aggcagcatt gtgaatgaga agtggattgt gacagctgcc 120

cactgtgtgg agacaggagt gaagatcaca gtggtggctg gagagcacaa cattgaggag 180

acagagcaca cagagcagaa gaggaatgtg atcaggatca tccctcacca caactacaat 240

gctgccatca acaagtacaa ccatgacatt gccctgctgg agctggatga gcctctggtg 300

ctgaacagct atgtgacccc tatctgcatt gctgacaagg agtacaccaa catcttcctg 360

aagtttggct ctggctatgt gtctggctgg ggcagagtgt tccacaaggg caggtctgcc 420

ctggtgctgc agtacctgag agtgcccctg gtggacagag ccacctgcct gttgagcacc 480

aagttcacca tctacaacaa catgttctgt gctggcttcc atgagggagg cagagacagc 540

tgccagggtg actctggagg accccatgtg acagaggtgg agggcaccag cttcctgaca 600

ggcatcatca gctggggaga ggagtgtgcc atgaagggca agtatggcat ctacaccaaa 660

gtgagcagat atgtgaactg gatcaaggag aagaccaagc tgacc 705

<210> 46

<211> 435

<212> DNA

<213> Artificial sequence

<220>

<223> CS04-LC-NA

<400> 46

tacaactctg gcaagctgga ggagtttgtg cagggcaacc tggagaggga gtgcatggag 60

120

ttctggaagc agtatgtgga tggtgaccag tgtgagagca acccttgcct gaatggaggc 180

agctgcaagg atgacatcaa cagctatgag tgctggtgcc cttttggctt tgagggcaag 240

aactgtgagc tggatgtgac ctgcaacatc aagaatggca ggtgtgagca gttctgcaag 300

aactctgctg acaacaaggt ggtgtgtagc tgcacagagg gctacagact ggctgagaac 360

cagaagagct gtgagcctgc tgtgcccttc ccctgtggca gagtgtctgt gtcccagacc 420

agcaagctga ccaga 435

<210> 47

<211> 705

<212> DNA

<213> Artificial sequence

<220>

<223> CS05-HC-NA

<400> 47

gtggtgggag gagaggatgc caagcctggc cagttcccct ggcaggtggt gctgaatggc 60

aaggtggatg ccttctgtgg aggcagcatt gtgaatgaga agtggattgt gaccgctgcc 120

cactgtgtgg agactggagt gaagatcact gtggtggctg gggagcacaa cattgaggag 180

acagagcaca cagagcagaa gcgcaatgtg atcaggatca tcccccacca caactacaat 240

gctgccatca acaagtacaa ccatgacatt gccctgctgg agctggatga gcccctggtg 300

ctgaacagct acgtgacccc catctgcatt gcagacaagg agtacaccaa catcttcctg 360

aagtttggct ctggctatgt gtctggctgg ggcagggtgt tccacaaggg caggtctgcc 420

ctggtgctgc agtacctgag ggtgcccctg gtggacaggg ccacctgcct gctgagcacc 480

aagttcacca tctacaacaa catgttctgc gctggcttcc atgagggagg aagggacagc 540

tgccagggag actctggagg cccccatgtg acagaggtgg agggcaccag cttcctgaca 600

ggcatcatca gctgggggga ggagtgtgcc atgaagggca agtatggcat ctacaccaaa 660

gtgtcccgct atgtgaactg gatcaaggag aagaccaagc tgacc 705

<210> 48

<211> 435

<212> DNA

<213> Artificial sequence

<220>

<223> CS05-LC-NA

<400> 48

tacaactctg gcaagctgga ggagtttgtg cagggcaacc tggagaggga gtgcatggag 60

gagaagtgca gctttgagga ggccagggag gtgtttgaga acactgagcg caccactgag 120

ttctggaagc agtatgtgga tggggaccag tgtgagagca acccctgcct gaatgggggg 180

agctgcaagg atgacatcaa cagctatgag tgctggtgcc cctttggctt tgagggcaag 240

aactgtgagc tggatgtgac ctgcaacatc aagaatggcc gctgtgagca gttctgcaag 300

aactctgctg acaacaaggt ggtgtgctct tgcactgagg gctaccgcct ggctgagaac 360

cagaagagct gtgagcctgc tgtgcccttc ccctgtggca gggtgtctgt gagccagacc 420

agcaagctga ccagg 435

<210> 49

<211> 705

<212> DNA

<213> Artificial sequence

<220>

<223> CS06-HC-NA

<400> 49

gtggtgggag gagaggatgc caagcctggc cagttcccct ggcaagtggt gctcaatggc 60

aaggtggatg ccttctgtgg gggctccatt gtgaatgaga agtggattgt cactgctgcc 120

cactgtgtgg agactggggt caagatcact gtggtggctg gggagcacaa cattgaggag 180

actgagcaca ctgagcagaa gcgcaatgtg atcaggatca tcccccacca caactacaat 240

gctgccatca acaagtacaa ccatgacatt gccctgctgg agctggatga gcccctggtc 300

ctcaacagct atgtgacccc catctgcatt gctgacaagg agtacaccaa catcttcctc 360

aagtttggct ctggctatgt ctctggctgg ggcagagtgt tccacaaagg caggtctgcc 420

ctggtgctcc agtacctgag agtgcccctg gtggacaggg ccacctgcct cttgagcacc 480

aagttcacca tctacaacaa catgttctgt gctggcttcc atgagggagg aagagacagc 540

tgccagggggg actctggagg accccatgtc actgaggtgg agggcacctc cttcctcact 600

ggcatcatct cctggggaga ggagtgtgcc atgaaaggca aatatggcat ctacaccaaa 660

gtctccagat atgtcaactg gatcaaggag aagaccaagc tgacc 705

<210> 50

<211> 435

<212> DNA

<213> Artificial sequence

<220>

<223> CS06-LC-NA

<400> 50

tacaactctg gcaaactgga ggagtttgtc cagggcaacc tggagaggga gtgcatggag 60

gagaagtgct cctttgagga ggccagggag gtctttgaga acactgagcg caccactgag 120

ttctggaaac agtatgtgga tggggaccag tgtgagtcca acccctgcct gaatgggggc 180

agctgcaagg atgacatcaa cagctatgag tgctggtgcc cctttggctt tgagggcaag 240

aactgtgagc tggatgtgac ctgcaacatc aagaatggca gatgtgagca gttctgcaag 300

aactctgctg acaacaaggt ggtgtgctcc tgcactgagg gctaccgcct ggctgagaac 360

cagaagagct gtgagcctgc tgtgccattc ccatgtggca gagtctctgt gagccagacc 420

agcaagctca ccagg 435

<210> 51

<211> 145

<212> DNA

<213> Artificial sequence

<220>

<223>AAV2_5'-ITR

<400> 51

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcct 145

<210> 52

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> truncated_enhancer/promoter_TTR

<400> 52

cgagggcact gggaggatgt tgagtaagat ggaaaactac tgatgaccct tgcagagaca 60

gagtattagg acatgtttga acaggggccg ggcgatcagc aggtagctct agaggatccc 120

cgtctgtctg cacatttcgt agagcgagtg ttccgatact ctaatctccc taggcaaggt 180

tcatatttgt gtaggttact tattctcctt ttgttgacta agtcaataat cagaatcagc 240

300

ccccttcacc aggagaagcc gtcacacaga 330

<210> 53

<211> 77

<212> DNA

<213> Artificial sequence

<220>

<223> intron_MVM

<400> 53

ctaaggtaag ttggcgccgt ttaagggatg gttggttggt ggggtattaa tgtttaatta 60

ccttttttac aggcctg 77

<210> 54

<211> 234

<212> DNA

<213> Artificial sequence

<220>

<223> BGH_polyadenylation_signal

<400> 54

cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc 60

ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa 120

aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg 180

gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggaa 234

<210> 55

<211> 145

<212> DNA

<213> Artificial sequence

<220>

<223> AAV2 3'-ITR

<400> 55

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag agagggagtg gccaa 145

<210> 56

<211> 35

<212> Protein

<213> Artificial sequence

<220>

<223> FIX-AP-AA

<400> 56

Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala

1.5 10 15

Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp

20 25 30

Phe Thr Arg

35

<210> 57

<211> 105

<212> DNA

<213> Artificial sequence

<220>

<223> CS02-AP-NA

<400> 57

gctgagacag tgttccctga tgtggactat gtgaatagca cagaggctga gaccatcctg 60

gacaacatca ccagagcac ccagtccttc aatgacttca ccaga 105

<210> 58

<211> 105

<212> DNA

<213> Artificial sequence

<220>

<223> CS03-AP-NA

<400> 58

gctgagactg tgttccctga tgtggactat gtgaatagca ctgaggctga gaccatcctg 60

gacaacatca ccagagcac ccagtccttc aatgacttca ccaga 105

<210> 59

<211> 105

<212> DNA

<213> Artificial sequence

<220>

<223> CS04-AP-NA

<400> 59

gctgagacag tgttccctga tgtggactat gtgaacagca cagaggctga gaccatcctg 60

gacaacatca ccagagcac ccagtccttc aatgacttca ccaga 105

<210> 60

<211> 105

<212> DNA

<213> Artificial sequence

<220>

<223> CS05-AP-NA

<400> 60

gctgagactg tgttccctga cgtggactat gtgaacagca ctgaggctga gaccatcctg 60

gacaacatca ccagagcac ccagagcttc aatgacttca ccagg 105

<210> 61

<211> 105

<212> DNA

<213> Artificial sequence

<220>

<223> CS06-AP-NA

<400> 61

gctgagactg tgttccctga tgtggactat gtgaacagca ctgaggctga aaccatcctg 60

gacaacatca ccagagcac ccagagcttc aatgacttca ccaga 105

<210> 62

<211> 145

<212> Protein

<213> Artificial sequence

<220>

<223> FIX-LC-AA

<400> 62

Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg

1.5 10 15

Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe

20 25 30

Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly

35 40 45

Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp

50 55 60

Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys

65 70 75 80

Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu

85 90 95

Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr

100 105 110

Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val

115 120 125

Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr

130 135 140

Arg

145

<210> 63

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> FIX-HC-AA

<400> 63

Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp Gln Val

1.5 10 15

Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile Val Asn

20 25 30

Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly Val Lys

35 40 45

Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu His Thr

50 55 60

Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn Tyr Asn

65 70 75 80

Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu Leu Asp

85 90 95

Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile Ala Asp

100 105 110

Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr Val Ser

115 120 125

Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val Leu Gln

130 135 140

Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg Ser Thr

145 150 155 160

Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His Glu Gly

165 170 175

Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val Thr Glu

180 185 190

Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly Glu Glu

195 200 205

Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser Arg Tyr

210 215 220

Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr

225 230 235

<210> 64

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> FIXp-HC-AA

<400> 64

Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp Gln Val

1.5 10 15

Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile Val Asn

20 25 30

Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly Val Lys

35 40 45

Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu His Thr

50 55 60

Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn Tyr Asn

65 70 75 80

Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu Leu Asp

85 90 95

Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile Ala Asp

100 105 110

Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr Val Ser

115 120 125

Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val Leu Gln

130 135 140

Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Leu Ser Thr

145 150 155 160

Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His Glu Gly

165 170 175

Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val Thr Glu

180 185 190

Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly Glu Glu

195 200 205

Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser Arg Tyr

210 215 220

Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr

225 230 235

<---

Claims (26)

1. A polynucleotide encoding a factor IX protein, wherein said polynucleotide contains a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 17. 2. A polynucleotide according to claim 1, wherein the polynucleotide has no more than 10 CpG dinucleotides. 3. A polynucleotide according to claim 1, wherein the polynucleotide has no more than 3 CpG dinucleotides. 4. The polynucleotide according to claim 1, wherein the polynucleotide contains the nucleic acid sequence of SEQ ID NO: 17. 5. The polynucleotide of claim 1, wherein the polynucleotide encodes leucine at the amino acid position corresponding to residue 384 of the full-length wild-type factor IX polypeptide having the nucleic acid sequence of SEQ ID NO: 2. 6. The polynucleotide of claim 2, wherein the polynucleotide encodes leucine at the amino acid position corresponding to residue 384 of the full-length wild-type factor IX polypeptide having the nucleic acid sequence of SEQ ID NO: 2. 7. The polynucleotide of claim 3, wherein the polynucleotide encodes leucine at the amino acid position corresponding to residue 384 of the full-length wild-type factor IX polypeptide having the nucleic acid sequence of SEQ ID NO: 2. 8. Polynucleotide according to any one of paragraphs. 1-7, wherein the factor IX protein encoded by the polynucleotide has 1 to 10 amino acid substitutions compared to SEQ ID NO: 12. 9. Polynucleotide according to any one of paragraphs. 1-7, wherein the factor IX protein encoded by the polynucleotide has the amino acid sequence of SEQ ID NO: 12. 10. Polynucleotide according to any one of paragraphs. 1-7, wherein the polynucleotide further comprises a preproleader polynucleotide encoding a preproleader peptide, said preproleader peptide comprising the amino acid sequence of SEQ ID NO: 36. 11. The polynucleotide of claim 10, wherein the preprolider polynucleotide has the nucleic acid sequence of SEQ ID NO: 23. 12. Polynucleotide according to any one of paragraphs. 1-7, wherein the polynucleotide has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 9. 13. Polynucleotide according to any one of paragraphs. 1-7, in which the polynucleotide has the nucleic acid sequence of SEQ ID NO: 9. 14. Polynucleotide according to any one of paragraphs. 1-7, additionally containing a liver-specific promoter element operably linked to a nucleic acid sequence encoding a factor IX protein. 15. A polynucleotide according to claim 14, wherein the liver-specific promoter element contains one copy of a promoter polynucleotide, wherein said promoter polynucleotide contains a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 39. 16. The polynucleotide of claim 14, wherein the liver-specific promoter element contains three copies of the promoter polynucleotide, wherein said promoter polynucleotide contains a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 39. 17. A polynucleotide according to claim 15, wherein said promoter polynucleotide contains the nucleic acid sequence of SEQ ID NO: 39. 18. Polynucleotide according to any one of paragraphs. 1-7, additionally containing an intron operably linked to a nucleic acid sequence encoding a factor IX protein. 19. The polynucleotide of claim 18, wherein the intron comprises an MVM intron polynucleotide comprising a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 53. 20. The polynucleotide of claim 19, wherein said MVM intron polynucleotide contains the nucleic acid sequence of SEQ ID NO: 53. 21. The polynucleotide of claim 18, wherein said intron is located between the promoter element and the translation initiation site of the nucleotide sequence encoding the factor IX polypeptide. 22. A polynucleotide according to claim 1, containing the nucleic acid sequence of SEQ ID NO: 40. 23. A vector for gene therapy in mammals containing a polynucleotide according to any one of paragraphs. 1-22. 24. The mammalian gene therapy vector of claim 23, wherein the mammalian gene therapy vector is an adeno-associated virus (AAV) vector. 25. The mammalian gene therapy vector of claim 24, wherein said adeno-associated virus vector is an adeno-associated virus serotype 8 (AAV-8) vector. 26. The mammalian gene therapy vector of claim 23, wherein the polynucleotide is a single stranded polynucleotide encoding a factor IX protein.

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