BR112021002998A2 - polynucleotides, viral particle and composition - Google Patents

Abstract

“polynucleotides, viral particle and composition”. The present invention relates to polynucleotides comprising a factor ix nucleotide sequence, wherein the factor ix nucleotide sequence comprises a coding sequence encoding a factor ix protein or a fragment thereof, and wherein a portion of the coding sequence not the wild type. the present invention further relates to viral particles comprising a recombinant genome comprising the polynucleotide of the invention, compositions comprising the polynucleotides or the viral particles, and methods and uses of the polynucleotides, viral particles or compositions.

Description

"POLYNUCLEOTIDES, VIRAL PARTICLE AND COMPOSITION" FIELD OF THE INVENTION

[001] The present invention relates to polynucleotides comprising a nucleotide sequence encoding Factor IX, viral particles comprising the polynucleotides and treatments using the polynucleotides.

BACKGROUND OF THE INVENTION

[002] Haemophilia B, a life-threatening X-related bleeding disorder, affects 1:30,000 men. Current treatment involves frequent intravenous injections (2-3 times a week) of the Factor IX protein (FIX).

This treatment is highly effective in stopping bleeding, but it is not curative and is extremely expensive (£150,000/patient/year), making it unaffordable for the majority of hemophilia B patients worldwide. Gene therapy for hemophilia B offers the potential for healing through persistent endogenous production of Factor IX after transfer of a working copy of the Factor IX gene to an affected patient.

[003] The present application concerns a gene therapy approach for the treatment of hemophilia B, involving the administration of a vector comprising a polynucleotide encoding Factor IX. This gene therapy approach would avoid the need for frequent intravenous Factor IX injections. However, it is difficult to provide a vector for effective gene therapy, that is, a vector that allows a high level of expression of Factor IX and expression of Factor IX that is highly active.

BRIEF DESCRIPTION OF THE INVENTION

[004] The present application demonstrates that various modifications in a polynucleotide comprising a Factor IX nucleotide sequence can help to improve the expression level and activity of the expressed Factor IX polypeptide. For example, the present application demonstrates that the following approaches can improve the effectiveness of a polynucleotide comprising a Factor IX nucleotide sequence for the treatment of hemophilia B: • the use of a codon-optimized sequence; • retaining a portion of the Factor IX polypeptide that is not codon-optimized; • the inclusion of an intron or fragment of an intron; • providing sequences that flank the intron or fragment of an intron that are not codon-optimized; • the use of a gain-of-function mutation; • the use of a specific promoter; and/or • maintaining an AAV genome, comprising the nucleotide, in single-stranded form.

[005] These modifications provide a Factor IX sequence that is highly expressed, and that encodes a highly active Factor IX polypeptide or fragment. As demonstrated in the Examples, the polynucleotide of the invention expresses and provides the general activity of Factor IX, at higher levels than other polynucleotides encoding Factor IX, for example those disclosed in WO16/075473.

[006] Accordingly, in a first aspect of the invention, there is provided a polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence comprises a coding sequence encoding a Factor IX protein or a fragment thereof , and in which a portion of the coding sequence is not wild-type.

[007] In a second aspect of the invention, there is provided a polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence comprises a coding sequence encoding a Factor IX protein or a fragment thereof and the sequence coding comprises: (i) a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 1; and (ii) a sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 15.

[008] In a third aspect of the invention, there is provided a polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence encodes a Factor IX protein or a fragment thereof and is at least 97%, at least at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identity with SEQ ID NO: 5.

[009] In a fourth aspect of the invention, there is provided a viral particle comprising a recombinant genome comprising the polynucleotide of the invention.

[010] In a fifth aspect of the invention, a composition comprising the polynucleotide or viral particle of the invention and a pharmaceutically acceptable excipient is provided.

[011] In a sixth aspect of the invention, a method of treatment is provided which comprises administering an effective amount of the polynucleotide or viral particle of the invention to a patient.

[012] In a seventh aspect of the invention, a use of the polynucleotide, viral particle or composition of the invention in the manufacture of a medicament for use in a method of treatment is provided.

[013] In an eighth aspect, the polynucleotide, viral particle or composition of the invention is for use in the expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, thus treating a disease.

[014] In a ninth aspect, the polynucleotide, viral particle or composition of the invention is for use in treating a disease by expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7.

[015] In a tenth aspect, a method of expressing a polypeptide encoded by a Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, comprising administering the polynucleotide, viral particle or composition of the invention to a patient.

[016] In an eleventh aspect, a method of treating a disease by expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of, is provided. a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, comprising administering the polynucleotide, viral particle or composition of the invention to a patient.

[017] In a twelfth aspect, a use of the polynucleotide, viral particle or composition of the invention in the expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater is provided. than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, thus treating a disease.

[018] In a thirteenth aspect, a use of the polynucleotide, viral particle or composition of the invention in the treatment of a disease by expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7.

[019] In a fourteenth aspect, the polynucleotide, viral particle or composition of the invention is for use in the expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, thus treating a disease.

[020] In a fifteenth aspect, the polynucleotide, viral particle or composition of the invention is for use in the treatment of a disease by expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6.

[021] In a sixteenth aspect, a method of expressing a polypeptide encoded by a Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by, is provided. a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, comprising administering the polynucleotide, viral particle or composition of the invention to a patient.

[022] In a seventeenth aspect, a method of treating a disease by expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of, is provided. a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, comprising administering the polynucleotide, viral particle or composition of the invention to a patient.

[023] In an eighteenth aspect, a use of the polynucleotide, viral particle or composition of the invention in the expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater is provided. than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, thus treating a disease.

[024] In a nineteenth aspect, a use of the polynucleotide, viral particle or composition of the invention in the treatment of a disease by expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6.

[025] In a twentieth aspect, the polynucleotide, viral particle or composition of the invention is for use in the treatment of a disease by the expression of the Factor IX protein or a fragment thereof having an activity of Factor IX greater than the activity of Factor IX expressed from a viral particle comprising a nucleotide sequence of factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: :18 and a transcriptional regulatory element of SEQ ID NO:6.

[026] In a twenty-first aspect, a method of treating a disease by expressing the Factor IX protein or a fragment thereof having a greater Factor IX activity than the Factor IX activity expressed from a viral particle is provided. comprising a nucleotide sequence of Factor IX of SEQ ID NO:12 and a transcriptional regulatory element of SEQ ID NO:7 and/or a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO:18 and a regulatory element of the transcript of SEQ ID NO: 6, comprising administering an effective amount of the polynucleotide, viral particle or composition of the invention to a patient.

[027] In a twenty-second aspect, a use of the polynucleotide, viral particle or composition of the invention in the treatment of a disease by expression of the Factor IX protein or a fragment thereof having a Factor IX activity greater than the Factor IX activity is provided. IX expressed from a viral particle comprising a nucleotide sequence of factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6.

[028] In a twenty-third aspect, the polynucleotide, viral particle or composition of the invention is used to achieve a stable Factor IX activity level of at least 20% in a patient with hemophilia B.

[029] In a twenty-fourth aspect, a method is provided for achieving a stable Factor IX activity level of at least 20% in a patient with hemophilia B, comprising administering an effective amount of the polynucleotide, viral particle or composition of the invention for a patient.

[030] In a twenty-fifth aspect, a use of the polynucleotide, viral particle or composition of the invention to achieve a stable Factor IX activity level of at least 20% in a patient with hemophilia B is provided.

[031] In a twenty-sixth aspect, the polynucleotide, viral particle or composition of the invention is provided for use in the treatment of hemophilia B, comprising administering the polynucleotide, viral particle or composition to a patient in an effective dose to achieve a level of stable Factor IX activity of at least 20% in one patient.

[032] In a twenty-seventh aspect, a method of treating hemophilia B comprising administering the polynucleotide, viral particle or composition of the invention to a patient in a dose effective to achieve a stable Factor IX activity level of at least 20% in one patient.

[033] In a twenty-eighth aspect, a use of the polynucleotide, viral particle or composition of the invention in the treatment of hemophilia B is provided, comprising administering the polynucleotide, viral particle or composition to a patient in an effective dose to achieve a level of stable Factor IX activity of at least 20% in one patient.

BRIEF DESCRIPTION OF THE FIGURES

[034] Figure 1 - Scheme of FIX transgene cassettes; ssLP1.FIXco, ssHLP2.TI-codop-FIX-GOF (HTFG) and ssHLP2.TI -ACNP-FIX-GOF (HTAG). ITR = Inverted Terminal Repeat; HLP2 and LP1 are transcriptional regulatory elements of SEQ ID NOs: 6 and 7, respectively; E = Exon; T1 = Intron truncated 1A; WT = wild type; CO = Codon-Optimized; ACNP = a codon-optimized sequence of the invention.

[035] Figure 2 - Results of HUH7 transduction with AAV2/Mut C vectors - Experiment 1; (A) shows FIX antigen level in supernatant; (B) shows the FIX antigen level after normalization using the number of vector genomes present in the cell lysate; Error bars represent mean ± SD of n = 2. 1e3 = 1 x 10 3; 5e3 = 5 x 103; 1e4 = 1 x 104; MOI = multiplicity of infection.

[036] Figure 3 - Results of HUH7 transduction with AAV2/Mut C vectors - Experiment 2, which shows the level of FIX antigen in the supernatant. Error bars represent mean ± SD of n = 3. 1e3 = 1 x 103; 5e3 = 5 x 103; 1e4 = 1 x 104; MOI = multiplicity of infection.

[037] Figure 4 - Results of HUH7 transduction with AAV2/Mut C vectors - Experiment 2; (A) shows FIX antigen level in supernatant; (B) shows the FIX antigen level after normalization using the number of vector genomes present in the cell lysate. Error bars represent mean ± SD of n = 3. 1e3 = 1 x 10 3; 5e3 = 5 x 103; 1e4 = 1 x 104; MOI = multiplicity of infection.

[038] Figure 5 - Combined data (from Experiments 1 and 2) for AAV2/Mut C transduction in HUH7 cells at an MOI of 5 x 10 3.

Error bars represent mean ± SD of n = 12. P = 0.001 by Student's t test. 5e3 = 5 x 103; MOI = multiplicity of infection.

[039] Figure 6 - FIX activity for MOI 1x 103, 5x103 and 1x

104 is shown after transduction into HUH7 with AAV2/Mut C vectors (Experiment 1). Error bars represent mean ± SD of n = 2 wells in duplicates. 1e3 = 1 x 103; 5e3 = 5 x 103; 1e4 = 1 x 104; MOI = multiplicity of infection.

[040] Figure 7 - FIX activity is shown after HUH7 transduction with AAV2/Mut C vectors (Experiment 2). Error bars represent mean ± SD of n = 3. 1e3 = 1 x 103; 5e3 = 5 x 103; 1e4 = 1 x 104; MOI = multiplicity of infection.

[041] Figure 8 - FIX activity is shown after HUH7 transduction with AAV2/Mut C vectors (Experiment 3). Error bars represent mean ± SD of n = 3. 1e3 = 1 x 10 3; 5e3 = 5 x 103; 1e4 = 1 x 104; MOI = multiplicity of infection.

[042] Figure 9 - Combined data (from Experiments 1 and 2; Figures 6 and 7) that show FIX activity for MOI of 5 x 10 3 shown after transduction in HUH7 with AAV2/Mut C vectors. error represent the mean ± SD of n = 12. 5e3 = 5 x 10 3; MOI = multiplicity of infection. Statistical significance determined by Student's t test (p = 0.0195).

[043] Figure 10 - Normalized level of human FIX in murine plasma after administration of AAV2/8.LP1.FIXco and AAV2/8.HLP2.HTFG (Experiment 3). FIX:Ag levels were normalized to vector/cell copies. Error bars represent mean ± SD of n = 4 mice. P value < 0.05 (Student's t test).

[044] Figure 11 - Comparison of alternative codon-optimization of FIX in C57BL/6 mice (Experiment 4). Mice were injected with AAV2/8 vectors containing ssHLP2.HTFG, ssHLP2.HTAG or scLP1.FIXco (control). FIX (A) antigen level was assessed 3 weeks after injection (P = 0.0007 between ssHLP2.HTAG and sc.LP1.FIXco and P-value =

0.0198 between ssHLP2.HTAG and ssHLP2.HTFG). Antigen levels were normalized for the vector genome (B) (p = 0.0009 between ssHLP2.HTAG and sc.LP1.FIXco and p = 0.0039 between ssHLP2.HTAG and ssHLP2.HTFG). n = 4 mice. P values were determined using one-way ANOVA (multiple comparison).

[045] Figure 12 - Comparison of alternative codon-optimization of FIX in C57BL/6 mice (Experiment 4). Mice were injected with AAV2/8 vectors containing ssHLP2.HTFG, ssHLP2.HTAG or scLP1.FIXco (control). FIX activity level was assessed 3 weeks after injection. n = 4 mice. P-value = 0.0008 between ssHLP2.HTAG and scLP1.FIXco and P-value = 0.01 between ssHLP2.HTAG and ssHLP2.HTFG; P-values were determined using one-way ANOVA (multiple comparison).

[046] Figure 13 - Scheme of the structure of Factor IX. The numbers above the scheme represent the amino acid positions in the complete Factor IX polypeptide, including the signal peptide and propeptide region (encoded by SEQ ID NO: 9). The numbers below the scheme represent equivalent amino acid positions in mature Factor IX (which corresponds to the portion of the coding sequence in SEQ ID NO:19).

[047] Figure 14 - Listing of sequences.

[048] Figure 15 - Mean Factor IX activity in two patients with hemophilia B who received AAV2/Mut C vectors containing ssHLP2.HTAG.

Two hemophilia patients received 4.5 x 1011 vg/kg of AAV2/Mut C vectors containing ssHLP2.HTAG. Factor IX activity levels were assessed regularly for 0-55 weeks after administration. Patients achieved mean stable Factor IX activity levels of 40.5 ± 4.5% at 52 weeks, as assessed by a clotting assay using the commercial SynthASil® kit. Patients also received prophylactic prednisolone at 6-12 weeks after vector administration (infusion). "FIX:C (%)"

describes the FIX activity level; look at the left y axis. "ALT (IU/L)" describes the level of alanine aminotransferase; look at the right y axis.

DETAILED DESCRIPTION OF THE INVENTION GENERAL DEFINITIONS

[049] Unless otherwise defined, technical and scientific terms used in the present disclosure have the same meaning as is commonly understood by one of ordinary skill in the subject to which the present invention belongs.

[050] In general, the term "comprising" is intended to mean ‘including, but not limited to’. For example, the phrase “a polynucleotide comprising a Factor IX nucleotide sequence” should be interpreted to mean that the polynucleotide has a Factor IX nucleotide sequence, but the polynucleotide may contain other additional nucleotides.

[051] In some embodiments of the invention, the word "comprising" is replaced by the phrase "consisting of". The term “consisting of” is intended to be limiting. For example, the phrase “a polynucleotide consisting of a Factor IX nucleotide sequence” should be understood as 'the polynucleotide has a Factor IX nucleotide sequence and no additional nucleotides'.

[052] The terms "protein" and "polypeptide" are used interchangeably in the present invention and are intended to refer to a polymeric chain of amino acids of any length.

[053] For the purpose of the present invention, in order to determine the percentage identity of two sequences (such as two polynucleotide sequences or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps) can be introduced into a first sequence for optimal alignment with a second sequence). Nucleotide residues at nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide residue as the corresponding position in the second sequence, then the nucleotides are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (ie % identity = number of identical positions/total number of positions in the reference sequence x 100).

[054] Typically, sequence comparison is performed along the length of the reference sequence. For example, if the user wants to determine if a particular sequence (“test”) is 95% identical to SEQ ID NO: 5, then SEQ ID NO: 5 would be the reference sequence. For example, to assess whether a sequence is at least 80% identical to SEQ ID NO:5 (an example of a reference sequence), one skilled in the art would perform an alignment along the length of SEQ ID NO:5, and identify how many positions in the test sequence were identical to SEQ ID NO: 5. If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO: 5. If the sequence is shorter than SEQ ID NO: 5, gaps or missing positions are to be considered non-identical positions.

[055] The person skilled in the art is aware of the different computer programs that are available to determine the homology or identity between two sequences. For example, a string comparison and determination of percent identity between two strings can be performed using a mathematical algorithm. In an example embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the algorithm of Needleman and Wunsch (1970) which has been incorporated into the program

GAP in the Accelrys GCG software package (available from http://www.accelrys.com/products/gcg/), using a Blosum 62 matrix or PAM250 matrix and a gap weight of 16, 14, 12, 10, 8 , 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[056] For the purposes of the present invention, the term "fragment" refers to a contiguous portion of a sequence. For example, a fragment of SEQ ID NO: 5 of 50 nucleotides refers to 50 contiguous nucleotides of SEQ ID NO: 5.

A POLYNUCLEOTIDE

[057] In one aspect, the present invention provides a polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence comprises a coding sequence encoding a Factor IX protein or a fragment thereof; and wherein a portion of the Factor IX nucleotide sequence is not wild-type.

[058] The polynucleotide may further comprise one or more of the following features. The polynucleotide can comprise a portion that is not codon-optimized. The polynucleotide can comprise an intron or a fragment of an intron. The polynucleotide may comprise a mutation at a codon corresponding to codon 384 of wild-type Factor IX.

[059] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides or analogues thereof. For example, the polynucleotide can comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides).

The polynucleotide can consist of DNA. The polynucleotide can be an mRNA. Since the polynucleotide can comprise RNA or DNA, all references to nucleotides T (thymine) can be replaced by U (uracil).

A SEQUENCE OF FACTOR IX NUCLEOTIDES

[060] The polynucleotide comprises a nucleotide sequence of Factor IX. The Factor IX nucleotide sequence comprises a coding sequence that encodes the Factor IX protein or a fragment thereof.

[061] A "coding sequence" is a sequence that encodes a polynucleotide and excludes non-coding regions such as introns. A coding sequence may be interrupted by non-coding nucleotides (eg an intron), but only the nucleotides encoding the polypeptide should be considered as part of the coding sequence. For example, a coding sequence encoding a Factor IX protein will comprise any codons that encode an amino acid forming part of the Factor IX protein that is expressed from that coding sequence, regardless of whether those codons are contiguous in sequence or separated by one or more non-nucleotide nucleotides. encodings. In other words, a polynucleotide that contains stretches of coding nucleotides interrupted by a stretch of non-coding nucleotides will be considered to comprise a "coding sequence" consisting of immediately juxtaposed non-contiguous coding stretches (ie, minus the non-coding stretch). However, here the stop codon will be considered as part of the full-length (full) coding sequence.

[062] The term "coding sequence" refers to a sequence of nucleotides comprising codons that encode the encoded polypeptide. For example, a nucleotide sequence encoding a Factor IX protein or fragment thereof comprises codons encoding the amino acid sequence of the Factor IX protein or fragment thereof. A suitable nucleotide sequence is provided in SEQ ID NO:

5.

[063] The following table describes codons that encode each amino acid: Amino Acid Codon Amino Acid Codon Amino Acid Codon Phenylalanine TTC Proline CCT Aspargin AAT

TTT CCC AAC CCA

CCG Leucine TTA Threonine ACT Lysine AAA

TTG ACC AAG CTT ACA CTC ACG CTA

CTG Isoleucine ATT Alanine GCT Aspartic Acid GAT

ATC GCC GAC ATA GCA

GCG Methionine ATG Tyrosine TAT Glutamic acid GAA

TAC GAG Valine GTT Histidine CAT Cysteine TGT

GTC CAC TGC GTA

GTG Serine TCT Glutamine CAA Tryptophan TGG

TCC CAG TCA TCG AGT

AGC Arginine CGT Glycine GGT

CGC GGC CGA GGA CGG GGG AGA AGG

[064] The corresponding RNA codons will contain U in place of T in the Table above.

[065] In one aspect of the present invention there is provided a polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence encodes a Factor IX protein or a fragment thereof and has at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identity to SEQ ID NO: 5. Optionally, the Factor IX nucleotide sequence comprises a coding sequence and a portion of the coding sequence is codon-optimized.

[066] In general, the Factor IX nucleotide sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99, 5%, at least 99.8%, or 100% identical to a fragment of at least 1200, at least 1350, or at least 1650 nucleotides of SEQ ID NO: 5. The Factor IX nucleotide sequence can be at least 80% , at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a contiguous fragment of at least minus 1200, at least 1350, or at least 1650 nucleotides of SEQ ID NO: 5. The nucleotide sequence of Factor IX can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 5. For example, the nucleotide sequence of Factor IX can be at least 98% identical to SEQ ID NO: 5.

FACTOR IX PROTEIN OR FRAGMENT THEREOF

[067] The polynucleotide comprises a Factor IX nucleotide sequence comprising a coding sequence encoding a Factor IX protein or fragment thereof.

[068] Wild-type Factor IX is a serine protease that is part of the coagulation cascade. The lack or mutation of Factor IX can lead to reduced blood clotting and hemophilia B disease. A typical wild-type Factor IX polypeptide is encoded by SEQ ID NO: 9 (sometimes referred to as Factor IX Malmo B) or SEQ ID NO: 19. An alternative wild-type Factor IX polypeptide differs from that encoded by SEQ ID NO: 9 at codon 194, for example, codon 194 may encode threonine ("Malmo A") instead of alanine.

[069] Factor IX (for example, a Factor IX of SEQ ID NO: 16),

as initially expressed as an "immature" precursor form, comprising a hydrophobic signal peptide (amino acids 1-28 of SEQ ID NO: 16), a propeptide region (amino acids 29-46 of SEQ ID NO: 16) and a region mature polypeptide, as set out in Figure 13. The mature (zymogen) form of Factor IX lacks the hydrophobic signal peptide and propeptide region. The term "mature Factor IX" refers to a Factor IX polypeptide that does not comprise the hydrophobic signal peptide or propeptide region, such as SEQ ID NO: 8.

[070] During coagulation the single-chain form of zymogen is cleaved by Factor XIa or Factor VIIa to produce an active two-chain form (Factor IXa), with the two chains linked by a disulfide bridge. The activated form can catalyze the hydrolysis of an arginine-isoleucine bond in Factor X to form Factor Xa. Wild-type Factor IX is inhibited by thrombin. The wild-type Factor IX protein has four protein domains, a Gla domain, two tandem copies of the EGF domain, and a C-terminal trypsin-like peptidase domain that is responsible for catalytic cleavage.

[071] The term "Factor IX protein" refers to the single-chain zymogenic form of Factor IX, the activated two-chain form and its variants, and may refer to the mature Factor IX polypeptide or a Factor IX polypeptide comprising the region of the pro-peptide and/or the signal peptide region.

[072] Preferably, the Factor IX fragment is at least 200, at least 250, at least 300, between 200 and 461, between 250 and 461, or between 300 and 461 amino acids in length. In an exemplary embodiment, the Factor IX protein or fragment thereof comprises a sequence: a) at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100 % identical to SEQ ID NO: 8; or b) at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to a fragment of SEQ ID NO: 8 at least 200, at least 250, at least 300, between 200 and 415, between 250 and 415, or between 300 and 415 amino acids in length.

[073] In an example embodiment, the Factor IX protein or fragment thereof is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 16; or at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to a fragment of SEQ ID NO: 16 with at least 200, at least 250 , at least 300, between 200 and 461, between 250 and 461, or between 300 and 461 amino acids in length. In an example embodiment, the Factor IX protein or fragment thereof is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to the SEQ ID NO: 16; or at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of SEQ ID NO: 16 with at least 300, or between 300 and 461 amino acids in length. The Factor IX protein or fragment thereof may have a sequence of SEQ ID NO: 16 or SEQ ID NO: 8.

[074] Preferably, the Factor IX protein or its fragment is functional. A Factor IX protein or functional fragment is one that hydrolyzes an arginine-isoleucine bond in Factor X to form Factor Xa.

[075] It is within the ability of the skilled person to determine whether a Factor IX protein or fragment encoded by a Factor IX nucleotide sequence is functional. A person skilled in the art need only express the nucleotide sequence of Factor IX and test whether the expressed protein is active. For example, one skilled in the art can prepare a viral particle of the invention comprising the Factor IX nucleotide sequence linked to an operable promoter and transduce cells with the viral particle under conditions suitable for the expression of the Factor IX protein or fragment thereof. The activity of the expressed Factor IX protein or fragment thereof can be analyzed using a chromogenic assay, such as the activity assay described in Example 3.

[076] For example, a suitable chromogenic assay is as follows. Factor IX is mixed with thrombin, phospholipids, calcium, thrombin activated Factor VIII and Factor XIa. Under these conditions, Factor XIa activates Factor IX to form Factor IXa, and Factor IXa activity can catalyze the cleavage of a chromogenic substrate (SXa-11) to produce pNA. The level of pNA generated can be measured by determining the absorbance at 405 nm, and this is proportional to the activity of Factor IX in the sample.

[077] Activity can be normalized to compensate for different concentrations of Factor IX in the sample by measuring the concentration of Factor IX in the sample using a standard ELISA assay, such as the assay described in Example 4, and dividing the activity by the concentration of Factor IX . For example, an antibody that binds to Factor IX can be bound to a plate. The sample, which comprises Factor IX in unknown concentration, can be passed over the plate. A second detection antibody that binds to Factor IX can be applied to the plate and any excess washed away. Any detection antibody that remains (ie is not washed away) will be bound to Factor IX. The detection antibody can be linked to an enzyme such as horseradish peroxidase. The level of detection antibody that binds to Factor IX on the plate can be measured by measuring the amount of detection antibody.

For example, if the detection antibody is linked to horseradish peroxidase, the horseradish peroxidase can catalyze the production of a blue reaction product from a substrate such as TMB (3,3',5,5'-tetramethylbenzidine), and the blue product level can be detected by absorbance at 450 nm. The level of blue product is proportional to the amount of detection antibody that remained after the wash step, which is proportional to the amount of Factor IX in the sample.

[078] Optionally, the Factor IX protein or fragment thereof has an activity greater than that of the Factor IX polypeptide encoded by SEQ ID NO: 9, SEQ ID NO: 19 or SEQ ID NO: 12. Optionally, the activity is measured using a chromogenic substrate that is specific for Factor IX, that is, a substrate that can be altered by Factor IXa to provide a chromogenic signal. A suitable chromogenic substrate is SXa-

11.

[079] In an embodiment example, the Factor IX protein or fragment thereof comprises a mutation at a position corresponding to position 384 of the wild-type Factor IX. For example, position 384 (numbering from the beginning of the signal peptide, i.e., a position corresponding to amino acid 384 of SEQ ID NO: 16) of the wild-type Factor IX is an arginine residue (R384), but it can be replaced by a different residue. In an exemplary embodiment, R384 is replaced with a small hydrophobic amino acid. For example, the small hydrophobic amino acid can be alanine, isoleucine, leucine, valine or glycine. Preferably, the Factor IX protein or fragment thereof comprises a leucine at a position corresponding to position 384 in wild-type Factor IX, as shown in SEQ ID NO:16.

[080] A mutation at a position corresponding to position 384 of the wild-type sequence can cause a gain-of-function (GoF) mutation, resulting in Factor IX that is hyperfunctional. The advantage of expressing a Factor IX protein containing a mutation at position 384 is that a relatively small increase in the amount of protein produces a greater increase in overall protein activity.

[081] It is within the skill of the person skilled in the art to determine whether a particular polypeptide has a mutation at a position corresponding to position 384. The man in the art simply needs to align the polypeptide sequence sequence with the wild-type Factor IX polypeptide sequence (precursor, immature), and determine if the residue of the former that aligns with the 384th residue of the latter is an arginine.

Otherwise, the polypeptide has a mutation at a position corresponding to position 384 in wild-type Factor IX. Alignment can be performed using any suitable algorithm, such as the Needleman and Wunsch described above.

A PORTION OF THE ENCODING SEQUENCE IS NOT OF THE WILD TYPE

[082] A portion of the coding sequence may not be wild-type. The nucleotide sequence encoding wild-type Factor IX is represented by SEQ ID NO: 9, and a coding sequence comprising a portion other than that of SEQ ID NO: 9 comprises a portion which is not wild-type (provided such portion is also different from other Factor IX coding sequences which are also considered to be wild type, e.g. the Malmo A variant mentioned above).

[083] In an example embodiment, the non-wild-type portion of the coding sequence is codon-optimized. To identify whether a coding sequence comprises a portion that is codon-optimized, one can align the coding sequence with SEQ ID NO: 9. If any part of the sequence is not identical to SEQ ID NO: 9, the user must then determine if they are codon optimized, that is, if they comprise at least one codon that has been replaced by a favored codon, that is, a TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG,

GAC, TGC, AGG, GGC and GAG. If the non-wild-type portion comprises at least one codon that has been replaced by a favored codon, then it is codon-optimized. Preferably, a contiguous portion of the coding sequence is codon-optimized. However, in some embodiment examples, the portion of the coding sequence that is codon-optimized can be divided into 2, 3, 4 or 5 regions of the coding sequence. Optionally, the portion of the coding sequence that is not codon-optimized is divided into less than 3 or less than 2 regions of the coding sequence. A nucleotide sequence can be codon-optimized by replacing codons with other codons that are favored (ie, reflecting the polarization of codons) in a particular organ or organism (so-called favored codons). This codon optimization improves the expression of the nucleotide sequence in the specific organ or organism. For example, if a nucleotide sequence is codon-optimized for the human liver, the nucleotide sequence is modified to increase the number of codons that are favored in the human liver. The person skilled in the art will recognize that codon optimization in a sequence may not imply changing all codons, as in some positions a “favored codon” may already be present.

[084] Such codon optimizations may be subject to other factors. For example, it can be seen that the presence of CpGs has an adverse effect on expression and therefore the user may decide not to use favored codons if their use in certain positions introduces CpGs into the sequence; this will still be considered codon optimization. In an example embodiment, a favored codon ending with a C nucleotide will not be included in the portion of the coding sequence that is codon-optimized, where the next codon in the sequence starts with a G. For example, the CTC codon encodes leucine.

CTC should not be used to encode leucine where the next codon in the sequence starts with a G, such as the GTT codon.

[085] The present application discloses that certain codons are favored for expression in the human liver and that reducing the CpG content of a coding sequence, while maintaining a high proportion of these favored codons, improves the expression of the coding sequence. Preferred codons are TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC and GAG.

[086] In an example embodiment, the portion of the coding sequence that is codon-optimized is codon-optimized for expression in the liver, optionally in the human liver. A portion of the coding sequence that is codon-optimized for expression in the liver may comprise a greater proportion of codons that are favored in the liver, such as the favored codons TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC , CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC and GAG.

[087] In an example embodiment, the following codons are collectively over-represented in the portion of the coding sequence that is not wild-type or is codon-optimized: TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC , TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC and GAG. By "collectively over-represented", it is meant that the total number of favored codons in the portion of the coding sequence that is codon-optimized or non-wild-type is greater than the total number of favored codons in the corresponding portion of a nucleotide sequence of the wild-type Factor IX (such as SEQ ID NO: 9 or SEQ ID NO: 19).

[088] In a preferred embodiment, in the portion of the coding sequence that is codon-optimized, there is a higher frequency of the following codons compared to the corresponding portion of a wild-type Factor IX nucleotide sequence (such as that of SEQ ID NO: 9): TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG,

AAC, AAA, AAG, GAC, TGC, AGG, GGC and GAG. Optionally, the following codons are collectively over-represented in the portion of the coding sequence that is codon-optimized, except where their presence results in a CpG: TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC and GAG. Optionally, the portion of the coding sequence that is codon-optimized comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65%, at least 70% or at least at least 73% of the codons selected from the group consisting of: TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC, and GAG.

[089] The preferential use of codons (codon usage) in a codon-optimized portion of a polynucleotide of the invention (HLP2.T1-ACNP-FIX-GoF) is compared with the preferential use of codons (codon usage) in a corresponding stretch of the wild-type Factor IX nucleotide sequence (SEQ ID NO: 9) in the table below.

TABLE 1 Amino Acid Codon HTAG % Codons Wild Type TTT 5 26 12 Phe TTC 14 74 9 CTT 0 9 CTC 1 5 5 CTA 0 2 Leu CTG 19 95 3 TTA 0 6 TTG 0 3 ATT 5 24 17 Ile ATC 16 76 7 ATA 0 1

Amino Acid Codon HTAG % Wild Type Codons

Met TTG 0 0

ATG 2 100 6

GTT 0 22

GTC 1 3 3 Val GTA 0 5

GTG 33 97 7

TCT 6 25 6

TCC 1 4 5

TCA 0 6 Be TCG 0 0

AGT 0 7

AGC 17 71 3

CCT 3 23 4

CCC 8 62 3 Pro CCA 2 15 8

CCG 0 0

ACT 11 39 13

ACC 17 61 7 Thr ACA 0 10

ACG 0 1

GCT 11 55 9

GCC 9 45 5 Wing GCA 0 8

GCG 0 0

TAT 7 50 11 Tyr TAC 7 50 5

CAT 3 33 6 His CAC 6 67 4

CAA 1 8 7 Gln CAG 11 92 7

Asn AAT 7 27 15

Amino Acid Codon HTAG % of codons Wild type AAC 19 73 17 AAA 1 4 12 Lys AAG 24 96 16 GAT 7 39 12 Asp GAC 11 61 7 GAA 1 3 33 Glu GAG 35 97 10 TGT 9 43 19 Cys TGC 12 57 5 TGG 7 88 7 Trp TGA 1 13 0 CGT 0 1 CGC 0 1 CGA 0 6 Arg CGG 0 3 AGA 3 20 8 AGG 12 80 1 GGT 0 8 GGC 21 66 9 Gly GGA 0 15 GGG 11 34 4

[090] The total number of codons favored in SEQ ID NO: 9 in this region is 120 (30% of the sequence). On the other hand, the total number of codons favored in the codon-optimized portion of HTAG is 293 (73% of the codons).

[091] It is simple to determine whether a particular portion of a polynucleotide comprises favored codons. In order to determine the frequency of each codon used in a part of a nucleotide sequence, the person skilled in the art simply needs to input the sequence of that part into one of the readily available algorithms that analyzes codon usage and analyzes the results. Alternatively, the user can simply count them.

[092] The codons that are substituted in the codon-optimized portion of HTAG compared to the corresponding region of SEQ ID NO:9 are shown in the table below.

TABLE 2 Amino Acid Codon Substitutions Frequency Pro CCA to CCC 2 CCA to CCT 2 CCT to CCC 3 Leu TTA to CTG 5 CTC to CTG 3 CTT to CTG 6 TTG to CTG 2 CTA to CTG 1 TTA to TTG 0 Gly GGC to GGG 1 GGA to GGC 9 GGT to GGG 4 GGT to GGC 3 GGG to GGC 2 GGA to GGG 5 Ile ATT to ATC 12 ATC to ATT 1 ATA to ATC 1 Val GTA to GTG 4 GTC to GTG 3 GTG to GTA GTT to GTG 20 GTA for GTC 1 Lys AAA to AAG 10 AAC to AAG AAG to AAA 1

Amino Acid Codon Substitutions Frequency Tyr TAT to TAC 3 TAC to TAT 1 Gln CAA to CAG 6 CAG to CAA 1 His CAT to CAC 2 Glu GAA to GAG 27 Cys TGT to TGC 9 TGC to TGT 1 Ser AGT to AGC 3 TCC to AGC 4 AGT to TCT 3 TCA to AGC 3 TCT to AGC 4 TCA to TCT 1 Ala GCA to GCC 3 GCA to GCT 4 GCT to GCC 2 Arg CGA to AGG 5 AGA to AGG 5 CGT to AGG 1 CGG to AGG 1 CGG to AGA 1 Thr ACA to ACC 6 ACT to ACC 4 ACA to ACT 3 ACG to ACC 1 Phe TTT to TTC 5 Asp GAT to GAC 5 GAC to GAT 1 Asn AAT to AAC 6 stop (stop) TAA to TGA 1 GoF 1 mutation

[093] In an example embodiment, in the portion of the coding sequence that is codon-optimized:

a) at least 1, at least 2, at least 4 or at least 5 codons encoding phenylalanine is/are replaced by TTC compared to a reference wild type Factor IX sequence; b) at least 60%, at least 65% or at least 70% of the codons encoding phenylalanine are TTC; c) at least 60%, at least 65%, or at least 70% of the codons encoding phenylalanine are TTC and the remainder are TTT; and/or d) the codons encoding phenylalanine are TTC, except when the next codon starts with G.

[094] For example, when we say that at least 1 of codon A is replaced by at least 1 of codon B, it refers to the replacement of codon A by codon B in at least 1 position compared to a wild-type sequence such as to SEQ ID NO: 9. To determine if such a substitution has occurred, it is only necessary to align the test sequence with a wild-type Factor IX sequence and see which codons are different.

If at least 1 codon in the test sequence corresponding to codon A of wild-type Factor IX is codon B in the test sequence, then at least 1 of codon A has been replaced by codon B. For example, if the first codon is TTT in the sequence test and TTC in the wild-type Factor IX sequence, the test sequence comprises at least 1 of the TTC codon replaced by TTT.

[095] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 10, at least 15, or at least 16 codons encoding leucine is/are replaced by CTG compared to a wild-type Factor IX reference sequence; b) at least 90%, or at least 94% of the codons encoding leucine are CTG; and/or c) at least 90%, or at least 95% of the codons encoding leucine are CTG and the remainder are CTC.

[096] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 10, at least 11, or at least 12 codons encoding isoleucine is/are replaced by ATC compared to a wild-type Factor IX reference sequence; b) at least 1 ATC codon is (are) replaced by ATT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 60%, at least 70%, or at least 75% of the codons encoding isoleucine are ATC; d) at least 60%, at least 70%, or at least 75% of the codons encoding isoleucine are ATC and the remainder are ATT; and/or e) the codons encoding isoleucine are ATC, except when the next codon begins with a G.

[097] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 10, at least 15, at least 20, or at least 25 codons encoding valine are replaced by GTG compared to a reference wild-type Factor IX sequence; b) at least 1 codon encoding valine is (are) replaced by GTC compared to a reference wild-type Factor IX sequence; c) at least 80%, at least 90%, or at least 95% of the codons encoding valine are GTG; and/or d) at least 80%, at least 90%, or at least 95% of the codons encoding valine are GTG and the remainder are GTC.

[098] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 10, at least 12, or at least 13 codons encoding serine are replaced by AGC compared to a reference wild-type Factor IX sequence; b) at least 1, at least 2, or at least 4 serine-encoding codons is/are replaced by TCT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 60%, at least 65%, or at least 70% of the codons encoding serine are AGC; and/or d) at least 60%, at least 65%, or at least 70% of the codons encoding serine are AGC and the remainder are TCT or TCC.

[099] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 1, at least 2, or at least 5 codons encoding proline is (are) replaced by CCC in comparison with a reference wild-type Factor IX sequence; b) at least 1 codon encoding proline is replaced by CCT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 50%, at least 55%, or at least 60% of the codons encoding proline are CCC; d) in 50%, at least 55%, or at least 60% of the codons encoding proline are CCC and the remainder are CCA or CCT; and/or e) the codons encoding proline are CCC, except when the next codon starts with a G.

[100] In an example embodiment, in the portion of the coding sequence that is codon-optimized:

a) at least 6, at least 7, at least 8, or at least 10 codons encoding threonine are replaced by ACC compared to a reference wild-type Factor IX sequence; b) at least 1, or at least 2, codons encoding threonine is/are replaced by ACT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 45%, at least 50%, or at least 55% of the codons encoding threonine are ACC; d) at least 45%, at least 50%, or at least 55% of the codons encoding threonine are ACC and the remainder are ACT; and/or e) the codons encoding threonine are ACC, except when the next codon starts with a G.

[101] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 1, at least 2, at least 3, or at least 4 codons encoding alanine is (are) replaced by GCC compared to a wild-type Factor IX reference sequence; b) at least 1, at least 2, or at least 3 codons encoding alanine is/are replaced by GCT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 35%, at least 40%, or at least 43% of the codons encoding alanine are GCC; d) at least 35%, at least 40%, or at least 45% of the codons encoding alanine are GCC and the remainder are GCT; and/or e) the codons encoding alanine are GCC, except when the next codon starts with a G.

[102] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 1, or at least 2 tyrosine-encoding codons is (are) replaced by TAC compared to a sequence of the Factor IX wild-type reference; b) at least 1 TAC codon is replaced by TAT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 40%, at least 45%, or at least 48% of the tyrosine-encoding codons are TAC; d) at least 40%, at least 45%, or at least 48% of the codons encoding tyrosine are TAC and the remainder are TAT; and/or e) the codons encoding tyrosine are TAC, except when the next codon starts with a G.

[103] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 1 codon encoding histidine is replaced by CAC compared to a reference wild-type Factor IX sequence; b) at least 50%, at least 60%, or at least 65% of the codons encoding histidine are CAC; c) at least 50%, at least 60%, or at least 65% of the codons encoding histidine are CAC and the remainder are CAT; and/or d) the codons encoding histidine are CAC, except when the next codon begins with a G.

[104] In an example embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 1, at least 2, at least 4, or at least

5 codons encoding glutamine is(are) replaced by CAG compared to a wild-type Factor IX reference sequence; b) at least 1 CAG codon is replaced by CAA compared to a reference wild-type Factor IX sequence; c) at least 80%, at least 85%, or at least 90% of the codons encoding glutamine are CAG; and/or d) at least 80%, at least 85%, or at least 90% of the codons encoding glutamine are CAG and the remainder are CAA.

[105] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 1, at least 2, at least 4, or at least 5 codons encoding aspargine is (are) replaced by AAC compared to a wild-type Factor IX reference sequence; b) at least 60%, at least 65%, or at least 70% of the codons encoding aspargine are AAC; c) at least 60%, at least 65%, or at least 70% of the codons encoding asparagine are AAC and the remainder are AAT; and/or d) the codons encoding asparagine are AAC, except when the next codon begins with a G.

[106] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 7, at least 8, or at least 9 codons encoding lysine are replaced by AAG compared to a reference wild-type Factor IX sequence; b) at least 1 AAG codon is replaced by AAA compared to a reference wild-type Factor IX sequence; c) at least 80%, at least 90%, or at least 95% of the codons encoding lysine are AAG; and/or d) at least 80%, at least 90%, or at least 95% of the codons encoding lysine are AAG and the remainder are AAA.

[107] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 1, at least 2, at least 3, or at least 4 codons encoding aspartate is (are) substituted by GAC compared to a wild-type Factor IX reference sequence; b) at least 1 GAC codon is replaced by GAT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 45%, at least 50%, or at least 60% of the codons encoding aspartate are GAC; d) at least 45%, at least 50%, or at least 60% of the codons encoding aspartate are GAC and the remainder are GAT; and/or e) the codons that encode aspartate are GAC, except when the next codon starts with a G.

[108] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 15, at least 20, at least 25, or at least 26 codons encoding glutamate are replaced by GAG compared to a reference wild-type Factor IX sequence; b) at least 80%, at least 90%, or at least 95% of the codons encoding glutamate are GAG; and/or c) at least 80%, at least 90%, or at least 95% of the codons encoding glutamate are GAG and the remainder are GAA.

[109] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 6, at least 7, or at least

8 codons encoding cysteine are replaced by TGC compared to a reference wild-type Factor IX sequence; b) at least 1 TGC codon is replaced by TGT compared to a wild-type Factor IX reference sequence, where the next codon starts with a G; c) at least 40%, at least 50%, or at least 55% of the cysteine-encoding codons are TGC; d) at least 40%, at least 50%, or at least 55% of the cysteine-encoding codons are TGC and the remainder are TGT; and/or e) the codons encoding cysteine are TGC, except when the next codon starts with a G.

[110] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized in the portion of the coding sequence that is codon-optimized the codons encoding tryptophan are TGG.

[111] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 8, at least 10, or at least 11 codons encoding arginine are replaced by AGG compared to one reference wild-type Factor IX sequence; b) at least 1 codon encoding arginine is replaced by AGA compared to a reference wild-type Factor IX sequence; c) at least 60%, at least 70%, or at least 75% of the codons encoding arginine are AGG; and/or d) at least 60%, at least 70%, or at least 75% of the codons encoding arginine are AGG and the remainder are AGA.

[112] Preferably at least 60%, at least 70%, or at least 75% of the codons encoding arginine are AGG.

[113] In an exemplary embodiment, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 10, at least 12, or at least 13 codons encoding glycine are replaced by GGC compared to a reference wild-type Factor IX sequence; b) at least 5, at least 6, at least 7, or at least 8 glycine-encoding codons are replaced by GGG compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 50%, at least 55%, or at least 60% of the codons encoding glycine are GGC; d) at least 50%, at least 55%, or at least 60% of the codons encoding glycine are GGC and the remainder are GGG; and/or e) the codons encoding glycine are GGC, except when the next codon starts with a G.

[114] In an exemplary embodiment, the portion of the coding sequence that is codon-optimized comprises codons that encode phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, aspargine, lysine, aspartate , glutamate, cysteine, tryptophan, arginine and glycine.

[115] In an exemplary embodiment, the portion of the coding sequence that is codon-optimized comprises codons encoding phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, aspargine, lysine, aspartate , glutamate, cysteine, tryptophan, arginine and glycine and in the codon-optimized portion: a) at least 5 codons encoding phenylalanine are replaced by TTC compared to a reference wild-type Factor IX sequence;

b) at least 16 codons encoding leucine are replaced by CTG compared to a reference wild-type Factor IX sequence;

c) at least 12 codons encoding isoleucine are replaced by ATC compared to a reference wild-type Factor IX sequence;

d) at least 25 codons encoding valine are replaced by GTG compared to a reference wild-type Factor IX sequence;

e) at least 13 serine-encoding codons are replaced by AGC compared to a reference wild-type Factor IX sequence;

f) at least 5 codons encoding proline are replaced by CCC compared to a reference wild-type Factor IX sequence;

g) at least 10 codons encoding threonine are replaced by ACC compared to a reference wild-type Factor IX sequence;

h) at least 4 codons encoding alanine are replaced by GCC compared to a reference wild-type Factor IX sequence;

i) at least 2 tyrosine-encoding codons are replaced by TAC compared to a reference wild-type Factor IX sequence;

j) at least 1 codon encoding histidine is replaced by

CAC compared to a reference wild-type Factor IX sequence;

k) at least 5 glutamine-encoding codons are replaced by CAG compared to a reference wild-type Factor IX sequence; l) at least 5 codons encoding asparagine are replaced by AAC compared to a reference wild-type Factor IX sequence; m) at least 9 lysine-encoding codons are replaced by AAG compared to a reference wild-type Factor IX sequence; n) at least 4 codons encoding aspartate are replaced by GAC compared to a reference wild-type Factor IX sequence; o) at least 26 glutamate-encoding codons are replaced by GAG compared to a reference wild-type Factor IX sequence; p) at least 8 cysteine-encoding codons are replaced by TGC compared to a reference wild-type Factor IX sequence; q) the codons encoding tryptophan are TGG; r) at least 11 arginine-encoding codons are replaced by AGG compared to a reference wild-type Factor IX sequence; and s) at least 13 glycine-encoding codons are replaced by GGC compared to a reference wild-type Factor IX sequence.

[116] In an exemplary embodiment, the portion of the coding sequence that is codon-optimized comprises codons encoding phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, aspargine, lysine, aspartate , glutamate, cysteine,

tryptophan, arginine and glycine and in the codon-optimized portion:

a) at least 70% of the codons encoding phenylalanine are

TTC;

b) at least 94% of the codons encoding leucine are

CTG;

c) at least 75% of the codons encoding isoleucine are

ATC;

d) at least 95% of the codons encoding valine are

GTG;

e) at least 70% of the serine encoding codons are

AGC;

f) at least 60% of the codons encoding proline are

CCC;

g) at least 55% of the codons encoding threonine are

ACC;

h) at least 43% of the codons encoding alanine are

GCC;

i) at least 48% of the tyrosine-encoding codons are

TAC;

j) at least 65% of the codons encoding histidine are

CAC;

k) at least 90% of the codons encoding glutamine are

CAG;

l) at least 70% of the codons encoding asparagine are

AAC;

m) at least 95% of the codons encoding lysine are AAG; n) at least 60% of the codons that encode aspartate are

GAC;

o) at least 95% of the codons encoding glutamate are GAG; p) at least 55% of the codons encoding cysteine are TGC; q) the codons encoding tryptophan are TGG; r) at least 75% of the codons encoding arginine are AGG; and s) at least 60% of the glycine encoding codons are GGC.

[117] In an exemplary embodiment, the portion of the coding sequence that is codon-optimized comprises codons encoding phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, aspargine, lysine, aspartate , glutamate, cysteine, tryptophan, arginine and glycine and in the codon-optimized portion: a) at least 70% of the codons encoding phenylalanine are TTC and the rest are TTT; b) at least 94% of the codons encoding leucine are CTG and the remainder are CTC; c) at least 75% of the codons encoding isoleucine are ATC and the remainder are ATT; d) at least 95% of the codons encoding valine are GTG; e) at least 70% of the codons encoding serine are AGC; f) at least 60% of the codons encoding proline are CCC and the rest are CCA or CCT; g) at least 55% of the codons encoding threonine are ACC and the remainder are ACT;

h) at least 43% of the codons encoding alanine are GCC and the remainder are GCT; i) at least 48% of the tyrosine-encoding codons are TAC and the remainder are TAT; j) at least 65% of the codons encoding histidine are CAC and the remainder are CAT; k) at least 90% of the codons encoding glutamine are CAG and the rest are CAA; l) at least 70% of the codons encoding asparagine are AAC and the rest are AAT; m) at least 95% of the codons encoding lysine are AAG and the rest are AAA; n) at least 60% of the codons encoding aspartate are GAC and the rest are GAT; o) at least 95% of the codons encoding glutamate are GAG and the rest are GAA; p) at least 55% of the cysteine-encoding codons are TGC and the rest are TGT; q) the codons encoding tryptophan are TGG; r) at least 75% of the codons encoding arginine are AGG and the rest are AGA; and s) at least 60% of the glycine encoding codons are GGC and the remainder are GGG.

[118] The reference wild-type Factor IX sequence may be SEQ ID NO: 9 or SEQ ID NO: 19.

[119] The portion that is codon-optimized can correspond to a sequence that encodes part of a Factor IX protein or an entire (complete) Factor IX protein. For example, the Factor IX protein can be a full-length coding sequence (such as a sequence encoding SEQ ID NO: 8 or SEQ ID NO: 16) or a variant thereof, and the entire (full) coding sequence can be codon-optimized. Therefore, reference herein to "a portion of the coding sequence is codon-optimised" is to be understood to mean "at least a part of the coding sequence is codon-optimised". In some embodiment examples, however, a portion of the coding sequence is not codon-optimized, for example, a portion of the coding sequence is not codon-optimized for expression in the liver. In some embodiment examples, the portion of the coding sequence that is codon-optimized is at least 800, at least 900, at least 1100, less than 1500, less than 1300, less than 1200, between 800 and 1500, between 900 and 1300, between 1100 and 1200, or about 1191 nucleotides in length.

[120] In an exemplary embodiment, the portion of the coding sequence that is codon-optimized comprises exon 3 or a portion of at least 10, at least 15, at least 20, less than 25, between 10 and 25, between 15 and 25, or between 20 and 25 nucleotides of exon 3. In a further example embodiment, the portion of the coding sequence that is codon-optimized comprises exon 4 or a portion of at least 80, at least 90, at least 100 , less than 114, between 80 and 114, between 90 and 114, or between 100 and 114 nucleotides of exon 4. In a further embodiment example, the portion of the coding sequence that is codon-optimized comprises exon 5 or a portion of at least 90, at least 100, at least 110, less than 129, between 90 and 129, between 100 and 129, or between 110 and 129 exon nucleotides

5. In a further exemplary embodiment, the portion of the coding sequence that is codon-optimized comprises exon 6 or a portion of at least 150, at least 180, at least 200, less than 203, between 150 and 203, between 180 and 203, or between 200 and 203 nucleotides of exon 6. In an additional embodiment example, the portion of the coding sequence that is codon-

optimized comprises exon 7 or a portion of at least 70, at least 80, at least 90, at least 100, less than 115, between 70 and 115, between 80 and 115, between 90 and 115, or between 100 and 115 nucleotides of exon 7. In a further embodiment example, the portion of the coding sequence that is codon-optimized comprises exon 8 or a portion of at least 400, at least 450, at least 500, less than 548, between 400 and 548, between 450 and 548, or between 500 and 548 nucleotides of exon 8.

[121] Exon 3 comprises nucleotides 253-277 of wild-type Factor IX (such as a Factor IX of SEQ ID NO: 9), or a corresponding sequence in a non-wild-type Factor IX nucleotide sequence. Exon 4 comprises nucleotides 278-391 of wild-type Factor IX (such as a Factor IX of SEQ ID NO: 9), or a corresponding sequence in a non-wild-type Factor IX nucleotide sequence. Exon 5 comprises nucleotides 392-520 of wild-type Factor IX (such as a Factor IX of SEQ ID NO: 9), or a corresponding sequence in a non-wild-type Factor IX nucleotide sequence. Exon 6 comprises nucleotides 521-723 of wild-type Factor IX (such as a Factor IX of SEQ ID NO: 9), or a corresponding sequence in a non-wild-type Factor IX nucleotide sequence. Exon 7 comprises nucleotides 724-838 of wild-type Factor IX (such as a Factor IX of SEQ ID NO: 9), or a corresponding sequence in a non-wild-type Factor IX nucleotide sequence. Exon 8 comprises nucleotides 839-1386 of wild-type Factor IX (such as a Factor IX of SEQ ID NO: 9), or a corresponding sequence in a non-wild-type Factor IX nucleotide sequence.

[122] Preferably a portion of at least 20 nucleotides from exon 3, a portion of at least 100 nucleotides from exon

4, a portion of at least 110 nucleotides of exon 5, a portion of at least 180 nucleotides of exon 6, a portion of at least 100 nucleotides of exon 7 and a portion of at least 500 nucleotides of exon 8 is codon-optimized. The portion of the coding sequence that is codon-optimized may comprise exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8. In an exemplary embodiment, the portion of the coding sequence that is codon-optimized comprises the exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8.

[123] In an example embodiment, the portion of the coding sequence that is codon-optimized comprises a portion of exon 2 and the portion of exon 2 is less than 160, less than 150, less than 100, less than 75, less than 60, at least 20, at least 30, at least 40, at least 50, between 20 and 160, between 30 and 150, between 30 and 100, between 40 and 75, or about 56 nucleotides in length . Exon 2 comprises nucleotides 89-252 of wild-type Factor IX (such as a Factor IX of SEQ ID NO: 9), or a corresponding sequence in a non-wild-type Factor IX nucleotide sequence. In a preferred example embodiment, the portion of the coding sequence that is codon-optimized comprises a portion of exon 2 that is between 30 and 100 nucleotides in length.

[124] It is within the skill of a person skilled in the art to determine whether a portion of a sequence encoding a Factor IX protein or a fragment thereof corresponds, for example, to exon 8 of wild-type Factor IX. One skilled in the art need only perform a sequence alignment of the sequence encoding the Factor IX protein or its fragment with exon 8 using a suitable alignment algorithm, such as that of Needleman and Wunsch described above, and determine whether at least part of the sequence of nucleotides has greater than 90%, greater than 95% or greater than 98% identity with exon 8 of SEQ ID NO:9 (as described above, exon 8 of SEQ ID NO:9 consists of nucleotides 839-1386 of SEQ ID NO: 9).

[125] As discussed above, providing a polynucleotide sequence that comprises a coding sequence that is partially or fully codon-optimized can ensure that the encoded polypeptide is expressed at a high level. In an exemplary embodiment, a polypeptide encoded by the Factor IX nucleotide sequence is expressed in human liver cells at higher levels compared to the reference wild-type Factor IX sequence. The reference wild-type Factor IX sequence may be the sequence of SEQ ID NO: 9. In an example embodiment, a polypeptide encoded by the Factor IX nucleotide sequence is expressed in human liver cells at higher levels compared to a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7. In an example embodiment, a polypeptide encoded by the nucleotide sequence of Factor IX is expressed in human liver cells at higher levels compared to a polypeptide encoded by a nucleotide sequence comprising a Factor IX nucleotide sequence of SEQ ID NO: 18 and a transcription regulatory element of SEQ ID NO: 6.

[126] In an example embodiment, a polypeptide encoded by the Factor IX nucleotide sequence is expressed in human liver cells at a level of at least 1.5, at least 2, at least 2.5, or at least 3 times greater. than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 or SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 7 or SEQ ID NO: 6. Optionally, a polypeptide encoded by the Factor IX nucleotide sequence is expressed in human liver cells at a level that is at least 1.5, at least 2, at least 2.5, or at least 3 times greater than an encoded polypeptide by a nucleotide sequence comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcription regulatory element of SEQ ID NO: 7. Optionally, a polypeptide encoded by the Factor IX nucleotide sequence is expressed in cells of human liver in u a level that is at least 1.5, at least 2, at least 2.5, or at least 3 times greater than a polypeptide encoded by a nucleotide sequence comprising a factor IX nucleotide sequence of SEQ ID NO:18 and a transcriptional regulatory element of SEQ ID NO:6.

[127] In one aspect, the polynucleotide, viral particle or composition of the invention is for use in expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than the of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, thus treating a disease. In one aspect, the polynucleotide, viral particle or composition of the invention is for use in treating a disease by expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7. In one aspect, a method of expressing a polypeptide is provided. encoded by a Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a Factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, comprising administering the polynucleotide, viral particle or composition of the invention to a patient. In one aspect, a method of treating a disease by expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, comprising administering the polynucleotide, viral particle or composition of the invention to a patient. In one aspect, a use of the polynucleotide, viral particle or composition of the invention in expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, thus treating a disease. In one aspect, a use of the polynucleotide, viral particle or composition of the invention in treating a disease by expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater is provided. than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7. In an example embodiment, expression of an encoded polypeptide by the nucleotide sequence of Factor IX comprises administering the polynucleotide, viral particle or composition of the invention to a patient.

[128] In one aspect, the polynucleotide, viral particle or composition of the invention is for use in expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than the of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, thereby treating a disease.

In one aspect, the polynucleotide, viral particle or composition of the invention is for use in treating a disease by expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of the

Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID

NO: 6. In one aspect, provided is a method of expressing a polypeptide encoded by a Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, comprising administering the polynucleotide, viral particle or composition of the invention to a patient.

In one aspect, a method of treating a disease by expressing a polypeptide encoded by the nucleotide sequence of the disease is provided.

Factor IX at a level that is at least 2, or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a Factor IX nucleotide sequence of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, comprising administering the polynucleotide, viral particle or composition of the invention to a patient.

In one aspect, a use of the polynucleotide, viral particle or composition of the invention in expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2 is provided.

or at least 3 times greater than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, thus treating a disease. In one aspect, a use of the polynucleotide, viral particle or composition of the invention in treating a disease by expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level that is at least 2, or at least 3 times greater is provided. than that of a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6. In an example embodiment, expression of an encoded polypeptide by the nucleotide sequence of Factor IX comprises administering the polynucleotide, viral particle or composition of the invention to a patient.

Optionally, the disease is hemophilia, eg hemophilia B.

[129] One of skill in the art can determine whether the Factor IX nucleotide sequence is expressed at higher levels compared to a reference sequence by transducing host cells with a viral particle comprising the Factor IX nucleotide sequence and some cells with a vector comprising the reference sequence. Cells can be cultured under conditions suitable for expression of the Factor IX protein or fragment thereof encoded by the Factor IX nucleotide sequence, and the level of expressed Factor IX protein can be compared. The level of expressed Factor IX protein can be assessed using an ELISA assay such as that described in the section entitled “Factor IX protein or fragment thereof”. Suitable host cells include cultured human liver cells, such as Huh7 cells.

[130] As discussed above, the presence of CpG (ie, CG dinucleotides) can reduce the efficiency of expression. This is because CpGs can be methylated and their methylation can lead to gene silencing, thus reducing expression. For this reason, it is preferred that the portion of the coding sequence that is codon-optimized comprises a reduced number of CpGs compared to a corresponding portion of a reference wild-type Factor IX sequence. In a preferred embodiment example, the portion of the coding sequence that is codon-optimized comprises less than 40, less than 20, less than 10, less than 5 or less than 1 CpG. Preferably, the portion of the coding sequence which is codon-optimized is free of CpG, i.e. it does not contain (0) CG dinucleotides.

[131] In an example embodiment, the portion of the coding sequence that is codon-optimized is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% , at least 99.5%, at least 99.8% or 100% identical to a fragment of at least 800, at least 900, at least 1100, less than 1191, less than 1100, less than 1000, between 800 and 1191, between 900 and 1191, or about 1191 nucleotides of SEQ ID NO: 1. In an example embodiment, the portion of the coding sequence that is codon-optimized is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 1. In an example embodiment, the portion of the coding sequence that is codon-optimized is at least 95% identical to a fragment that is between 900 and 1191 nucleotides of SEQ ID NO: 1. In an example embodiment, the portion of the coding sequence that is codon-optimized is at least 95%, or at least 98% identical to SEQ ID NO: 1.

[132] The present invention provides a polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence comprises a coding sequence that encodes a Factor IX protein or a fragment thereof and the coding sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1; and a sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 15 Optionally, the sequence that is at least 95%, at least at least 98%, at least 99%, at least 99.5% or at least 99.8% identical to SEQ ID NO: 1 is codon-optimized.

PORTION OF THE CODING SEQUENCE THAT IS NOT CODON-OPTIMIZED

[133] In an example embodiment, the Factor IX nucleotide sequence comprises a portion that is not codon-optimized.

The portion that is not codon-optimized can be a contiguous portion. Including a non-codon-optimized portion can improve expression of the coding sequence, as the non-codon-optimized portion can beneficially interact with other portions of the coding sequence, such as an intron or a fragment of an intron. For example, the Factor IX nucleotide sequence may comprise an intron or a fragment of an intron and, in such cases, flanking the intron or fragment of an intron with the wild-type Factor IX sequence may help ensure correct splicing .

[134] The non-codon-optimized portion is not modified to include a greater number of favored codons compared to the wild-type sequence. For example, the portion that is not codon-optimized can comprise a similar number of favored codons from a wild-type sequence. The non-codon-optimized portion may comprise less than 50% of the codons TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC and GAG. Optionally, the non-codon-optimized portion comprises less than 50%, less than 45% or less than 40% of codons selected from the group consisting of: TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC and GAG.

[135] Optionally, the portion that is not codon-optimized is not codon-optimized for expression in human liver cells. In an exemplary embodiment, the non-codon-optimized portion comprises substantially the same number of favored codons as a corresponding portion of SEQ ID NO: 9. For example, the non-codon-optimized portion may comprise at least 90% of the number of codons favored as a portion of the corresponding SEQ ID NO:9.

[136] Optionally, the non-codon-optimized portion is at least 100, at least 150, at least 170, at least 190, less than 250, less than 225, less than 200, or about 195 nucleotides of lenght.

[137] As discussed in detail below, the nucleotide sequence of Factor IX can comprise an intron or a fragment of an intron. In such cases, the intron or fragment of an intron may be flanked by the non-codon-optimized portion, that is, part of the non-codon-optimized portion may be adjacent to the 3' end of the intron or intron fragment; and part of the portion that is not codon-optimized may be adjacent to the 5' end of the intron or intron fragment. The intron or intron fragment may be between exon 1 and exon 2. In such cases, it is advantageous to include a portion that is not codon-optimized, which portion comprises a portion of exon 1 and a portion of exon 2.

[138] Optionally, the non-codon-optimized portion comprises exon 1 or a portion of at least 60, at least 70, at least 80, between 60 and 88, between 70 and 88, or between 80 and 88 contiguous nucleotides of exon 1. Exon 1 comprises nucleotides 1-88 of wild-type Factor IX (such as a Factor IX of SEQ ID NO: 9), or a corresponding sequence in a non-wild-type Factor IX nucleotide sequence. Part of exon 1 can encode the signal peptide region and the pro-peptide region. Optionally, the non-codon-optimized portion comprises or does not comprise the signal peptide and/or pro-peptide regions. Exon 1 may also comprise an additional non-coding extension of 29 nucleotides at the 5' end. If the Factor IX nucleotide sequence comprises an intron or fragment of an intron, it is preferable that the portion that is not codon-optimized comprise a portion of exon 1 that is adjacent to the intron or fragment of an intron. For example, if the intron or fragment of an intron is between exon 1 and exon 2, it is preferable that the non-codon-optimized portion comprises a portion of exon 1 that corresponds to nucleotides 80-88, 70-88, 60-88, 40-88 or 20-88 of SEQ ID NO: 9.

[139] Optionally, the portion that is not codon-optimized includes a portion of at least 50, at least 75, at least 80, at least 90, at least 100, less than 140, less than 120, between 50 and 140, between 75 and 120, or about 107 nucleotides of exon 2. For example, if the intron or fragment of an intron is between exon 1 and exon 2, it is preferable that the non-codon-optimized portion comprises a portion of exon 2 that corresponds to nucleotides 89-100, 89-120, 89-140, 89-160, 89-180 or 89-196 of SEQ ID NO: 9.

[140] The portion that is not codon-optimized may comprise CpGs. For example, the non-codon-optimized portion may comprise the same number of CpGs as a corresponding portion of SEQ ID NO: 9. The non-codon-optimized portion may comprise at least 1, at least 1.5, or at least minus 2 CpGs per 100 nucleotides. The portion that is not codon-optimized can comprise at least 1, at least 2, at least 3, between 1 and 5, between 2 and 5, or about 5 CpGs.

[141] The portion that is not codon-optimized can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% , at least 99.8%, or 100% identical to a fragment of at least 100, at least 150, at least 175, less than 195, less than 190 or less than 180 nucleotides of SEQ ID NO: 15 or SEQ ID NO: 2. The non-codon-optimized portion may be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99 .5%, at least 99.8%, or 100% identical to SEQ ID NO: 15 or SEQ ID NO: 2. For example, the portion that is not codon-optimized can be at least 98% identical to SEQ ID NO: 15 or SEQ ID NO: 2

[142] The portion that is not codon-optimized may be wild type. SEQ ID NO: 9 is an example of a wild type Factor IX nucleotide coding sequence. Thus, the portion that is not codon-optimized can be at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a corresponding portion of SEQ ID NO: 9.

THE FACTOR IX NUCLEOTIDE SEQUENCE MAY COMPRISE AN INTRON OR A FRAGMENT OF AN INTRON

[143] The Factor IX nucleotide sequence may comprise an intron or a fragment of an intron that interrupts the coding sequence. An intron is a sequence of nucleotides that is excised during the expression process and is not part of the coding sequence.

[144] A wild-type Factor IX genomic nucleotide sequence comprises introns that interrupt the Factor IX coding sequence. The presence of an intron can help maintain a high level of wild-type Factor IX expression. Thus, it may be advantageous to include an intron, or at least a fragment of an intron, in a Factor IX nucleotide sequence of the invention. For example, the nucleotide sequence of Factor IX can comprise an intron or a fragment of an intron that corresponds to intron 1 in wild-type Factor IX.

Suitably, the intron is a fragment of wild-type Factor IX intron 1A, such as SEQ ID NO: 3. It has been found that truncating the intron 1 sequence causes the expression of the Factor IX nucleotide sequence to increase. It is believed that the truncation of intron 1 to form intron 1A can eliminate a repressor element in the intron. Truncation of the intron 1 sequence also results in a shorter Factor IX nucleotide sequence, which allows for more efficient packaging of the Factor IX nucleotide sequence into a viral delivery system in examples of gene therapy realizations.

[145] The fragment of an intron can be less than 500, less than 400, less than 350, less than 300, at least 100, at least 200, at least 250, at least 290, between 100 and 500, between 200 and 400, between 250 and 350, or about 299 nucleotides. The fragment of an intron can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to a fragment of at least 100, at least 200, at least 250, or at least 290 nucleotides of SEQ ID NO: 3. The intron or fragment of an intron can be at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 3. For example, the intron or fragment of an intron can be at least 95%, or at least 98% identical to SEQ ID NO: 3.

[146] Preferably, the intron or the fragment of an intron interrupts the portion that is not codon-optimized, that is, the intron is at 5’

for a non-codon-optimized portion and 3' for a non-codon-optimized portion in the Factor IX nucleotide sequence. An intron is "flanked by" a sequence that is not codon-optimized if the nucleotides immediately 3' and 5' of the intron or near the 3' and 5' sections of the intron are not codon-optimized. "Near the intron" refers to 1, 2, 3, 4, 5, 6, 7, 8, 8 or 10 nucleotides of the intron. As discussed above, flanking the intron or fragment of an intron with a nucleotide sequence that is not codon-optimized can help ensure correct splicing. Optionally, the intron or fragment of an intron is flanked by at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides that are not codon-optimized. For example, an intron is flanked by 60 nucleotides that are not codon-optimized if 40 nucleotides that are immediately 3' to the intron and 20 nucleotides that are immediately 5' to the intron are not codon-optimized, or if 30 nucleotides that are immediately 3' to the intron and 30 nucleotides that are immediately 5' to the intron are not codon-optimized. Optionally, the intron or fragment of an intron is flanked by between 110 and 120 nucleotides that are not codon-optimized at the 5' end (eg, immediately 5' of the intron) and between 100 and 110 nucleotides that are not codon-optimized at the 3' end (eg immediately 3' from the intron).

[147] The intron or fragment of an intron can be positioned between portions of the coding sequence corresponding to exon 1 and exon 2 of a Factor IX nucleotide sequence. If the intron or fragment of an intron corresponds to a fragment of intron 1 in wild-type Factor IX, it is preferable that the intron or fragment of an intron be between portions of the coding sequence corresponding to exon 1 and exon 2 of a nucleotide sequence of factor IX.

POLYNUCLEOTIDE MAY ALSO INCLUDE A REGULATORY ELEMENT OF TRANSCRIPTION

[148] The polynucleotide may comprise a transcriptional regulatory element.

[149] In an example embodiment, the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5 %, at least 99.8%, or 100% identical to SEQ ID NO: 6. In an example embodiment, the polynucleotide comprises a transcriptional regulatory element that is at least 98%, at least 99%, at least 99, 5%, at least 99.8% or 100% identical to SEQ ID NO: 6. Optionally, the polynucleotide comprises a transcriptional regulatory element of SEQ ID NO: 6.

[150] A suitable transcriptional regulatory element can be used, such as HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT and LSP, which are all liver-specific transcriptional regulatory elements. These transcriptional regulatory elements are described in more detail in the following references: HLP1: McIntosh J. et al., Blood Apr 25, 2013, 121(17):3335-44; LP1: Nathwani et al., Blood. April 1, 2006, 107(7): 2653–2661; HCR-hAAT: Miao et al., Mol Ther. 2000;1: 522-532; ApoE-hAAT: Okuyama et al., Human Gene Therapy, 7, 637-645 (1996); and LSP: Wang et al., Proc Natl Acad Sci US A. March 30, 1999, 96(7): 3906–3910. The HLP2 transcriptional regulatory element has a sequence of SEQ ID NO:6.

[151] The transcriptional regulatory element may comprise a promoter and/or enhancer/enhancer element, such as the promoter and/or enhancer element HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT and LSP.

Each of these transcriptional regulatory elements comprises a promoter, an enhancer/enhancer and, optionally, other nucleotides.

[152] In an exemplary embodiment, the transcriptional regulatory element comprises an enhancer that is the control region of the human apolipoprotein E (ApoE) hepatic locus (HCR; Miao et al (2000), Molecular Therapy 1 (6): 522), or a fragment thereof. In an exemplary embodiment, the transcriptional regulatory element comprises an enhancer fragment HCR which is a fragment with at least 80, at least 90, at least 100, less than 192, between 80 and 192, between 90 and 192 , between 100 and 250, or between 117 and 192 nucleotides in length.

Optionally, the HCR enhancer fragment is between 100 and 250 nucleotides in length.

[153] A suitable HCR enhancer element fragment is described in SEQ ID NO: 13. Optionally, the transcriptional regulatory element comprises an enhancer that has at least 80, at least 90, at least 100, less than 192, between 80 and 192, between 90 and 192, between 100 and 250, or between 117 and 192 nucleotides in length and the enhancer comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO:

13. Optionally, the transcription regulatory element comprises an enhancer that is between 117 and 192 nucleotides in length and the enhancer comprises a polynucleotide sequence that is at least 98%, at least 99%, at least 99.5% , at least 99.8%, or 100% identical to SEQ ID NO: 13.

[154] Optionally, the transcriptional regulatory element comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5 %, at least 99.8%, or 100% identical to a fragment of at least 90, at least 100, or at least 110 nucleotides of SEQ ID NO: 13.

Optionally, the polynucleotide comprises an enhancer that is at least

80%, at least 85%, at least 90%, at least 95% at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 13. Optionally, the polynucleotide comprises an enhancer that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 13. Optionally, the polynucleotide comprises an enhancer of SEQ ID NO: 13.

[155] In an exemplary embodiment, the transcriptional regulatory element comprises a promoter that is a human alpha-1 anti-trypsin promoter (A1AT; Miao et al (2000), Molecular Therapy 1(6):522), or a fragment of it. Optionally, a fragment of an A1AT promoter is at least 100, at least 120, at least 150, at least 180, less than 255, between 100 and 255, between 150 and 225, between 150 and 300, or between 180 and 255 nucleotides of lenght. Optionally, the fragment of an A1AT promoter is between 150 and 300 nucleotides in length.

[156] A suitable A1AT promoter fragment is described in SEQ ID NO: 14. Optionally, the transcriptional regulatory element comprises a promoter having at least 100, at least 120, at least 150, at least 180, less than 255, between 100 and 255, between 150 and 300, or between 180 and 255 nucleotides in length and the promoter comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO:

14. Optionally, the transcriptional regulatory element comprises a promoter that is between 180 and 255 nucleotides in length and the promoter comprises a polynucleotide sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 14. Optionally, the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 90, at least 100, or at least 150 nucleotides of SEQ ID NO: 14. Optionally, the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100 % identical to SEQ ID NO: 14. Optionally, the polynucleotide comprises a promoter that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 14. Option additionally, the polynucleotide comprises a promoter of SEQ ID NO:14.

[157] If the polynucleotide is intended for expression in the liver, the promoter may be a liver-specific promoter (liver-specific promoter). Optionally, the promoter is a human liver specific promoter.

[158] A "liver-specific promoter" (or "liver-specific promoter") is a promoter that provides a higher level of expression in liver cells compared to other cells in general. For example, one skilled in the art can determine whether a promoter is a liver-specific promoter by comparing polynucleotide expression in liver cells (such as Huh 7 cells) with polynucleotide expression in cells from other tissues. If the expression level is higher in liver cells compared to cells from other tissues, the promoter is a liver-specific promoter.

FUNCTION GAIN MUTATION

[159] The Factor IX protein or its fragment may comprise a gain-of-function mutation. A gain-of-function mutation is a mutation that increases the activity of the Factor IX protein or a fragment thereof. For example, the gain-of-function mutation can result in a Factor IX protein or fragment thereof that has an activity that is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least at least 4 times, at least 5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, or at least 8 times or more greater than wild-type Factor IX activity (such as Factor IX encoded by SEQ ID NO: 9 or SEQ ID NO: 19).

[160] The Factor IX protein or fragment thereof may comprise a mutation at a position corresponding to position 384 of wild-type Factor IX (corresponding to codon 384 of SEQ ID NO:9 or amino acid 384 of the immature polypeptide encoded by SEQ ID NO:9 ). A mutation at a position corresponding to position 384 of the wild-type Factor IX may be a gain-of-function mutation. For example, replacing arginine 384 with leucine can lead to a substantial increase in activity.

[161] Whether or not a Factor IX protein comprises a mutation at a position corresponding to position 384 in Factor IX can be determined by aligning the Factor IX protein with SEQ ID NO: 16 using a suitable algorithm such as that of Needleman and Wunsch described above, and determining whether the amino acid that aligns with amino acid 384 (which is leucine in SEQ ID NO: 16) is an arginine residue. If the amino acid that aligns with amino acid 384 of SEQ ID NO: 16 is not an arginine residue, then the Factor IX protein has a mutation at a position corresponding to position 384 of the wild-type Factor IX.

[162] One can determine whether or not a mutation is a gain-of-function mutation by comparing the activity of a Factor IX protein that comprises the mutation with the activity of a reference Factor IX protein that is identical except for the gain mutation of putative function. The relative activities of these two proteins can be determined using a chromogenic assay such as the one discussed in the section “Protein Factor IX or

Fragment Of The Same”. If the activity of the Factor IX protein comprising the mutation is greater than the activity of the reference protein, the mutation is a gain-of-function mutation.

[163] Consequently, the Factor IX nucleotide sequence may comprise a codon that encodes a mutation at a position corresponding to position 384 in Factor IX. For example, the Factor IX nucleotide sequence may comprise a codon that encodes an amino acid at a position corresponding to position 384 of wild-type Factor IX which is a small hydrophobic amino acid. The small hydrophobic amino acid can be alanine, leucine, isoleucine, glycine or valine. For example, the small hydrophobic amino acid can be alanine or leucine. Preferably, the small hydrophobic amino acid is leucine.

[164] The codon that encodes a mutation at a position corresponding to position 384 in wild-type Factor IX may be a codon that encodes leucine, such as CTX, where X is any nucleotide. Preferably, X is C or G. The codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX may be CTC, as in SEQ ID NO: 4. In alternative embodiment examples, the codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX is TTG or CTG, such as in SEQ ID NO: 11 or SEQ ID NO: 26. For example, the reference to SEQ ID NO: 1 herein may be substituted by reference to the corresponding portions of SEQ ID NOs: 26 or 11. In other words, SEQ ID NO: 1 can be substituted at nucleotide 957 (C) by G, or at nucleotides 955 (C) and 957 (C) by T and G, respectively.

[165] In general, the Factor IX nucleotide sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99, 5%, at least 99.8%, or

100% identical to a fragment of at least 1200, at least 1350, or at least 1650 nucleotides of SEQ ID NO: 5. For example, the Factor IX nucleotide sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 5.

[166] Suitably, (i) the Factor IX nucleotide sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1; and (ii) the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX, where the codon encoding an amino acid at a position corresponding to position 384 of Factor Wild type IX encodes leucine.

[167] Suitably, (i) the Factor IX nucleotide sequence comprises a coding sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1; (ii) the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX, where the codon encoding an amino acid at a position corresponding to position 384 of Factor IX wild type encodes leucine; and (iii) the polynucleotide comprises an enhancer element that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 13.

[168] Appropriately,

(i) the Factor IX nucleotide sequence comprises a coding sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1; (ii) the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX, where the codon encoding an amino acid at a position corresponding to position 384 of Factor IX wild type encodes leucine; and (iii) the polynucleotide comprises a promoter element that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 14.

[169] Suitably, (i) the Factor IX nucleotide sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1; (ii) the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX, where the codon encoding an amino acid at a position corresponding to position 384 of Factor IX wild type encodes leucine; and (iii) the polynucleotide comprises a transcription regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 6.

[170] Suitably, (i) the Factor IX nucleotide sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1;

(ii) the Factor IX nucleotide sequence comprises a sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 2; and (iii) the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX, where the codon encoding an amino acid at a position corresponding to position 384 of Factor Wild type IX encodes leucine.

[171] Suitably, the Factor IX nucleotide sequence comprises an intron or a fragment of an intron, and the fragment of an intron is at least 98%, at least 99%, at least 99.5%, at least 99, 8% or 100% identical to SEQ ID NO: 3.

A VIRAL PARTICLE THAT COMPRISES POLYNUCLEOTIDE

[172] The invention further provides a viral particle comprising a recombinant genome comprising polynucleotides of the invention. For purposes of the present invention, the term "viral particle" refers to all or part of a virion. For example, the viral particle comprises a recombinant genome and may further comprise a capsid. The viral particle can be a gene therapy vector. In the present invention, the terms "viral particle" and "vector" are used interchangeably. For the purpose of the present application, a "gene therapy" vector is a viral particle that can be used in gene therapy, that is, a viral particle that comprises all the functional elements necessary to express a transgene, such as a nucleotide sequence of the Factor IX, in a host cell after administration.

[173] Suitable viral particles include a parvovirus, a retrovirus, a lentivirus, or a herpes simplex virus. Parvovirus may be an adeno-associated virus (AAV). The viral particle is preferably a recombinant adeno-associated viral vector (AAV) or a lentiviral vector. Most preferably, the viral particle is an AAV viral particle. The terms AAV and rAAV are used interchangeably in the present invention.

[174] The genomic organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule less than 5,000 nucleotides in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for non-structural replication proteins (Rep) and structural proteins (VP). The VP proteins (VP1, -2 and -3) form the capsid.

The nt 145 terminals are self-complementary and arranged so that an energetically stable intramolecular duplex forming a T-shaped staple can be formed. These hairpin structures function as an origin for viral DNA replication, serving as initiators for the cellular DNA polymerase complex. After wild-type (wt) AAV infection in mammalian cells, the Rep genes (i.e., encoding the Rep78 and Rep52 proteins) are expressed from the P5 promoter and the P19 promoter, respectively, and both Rep proteins have a role in viral genome replication. A splicing event in the Rep ORF results in the expression of four Rep proteins (ie, Rep78, Rep68, Rep52, and Rep40). However, unprocessed mRNA encoding the Rep78 and Rep52 proteins in mammalian cells has been shown to be sufficient for the production of the AAV vector. Furthermore, in insect cells, the Rep78 and Rep52 proteins are sufficient for the production of the AAV vector.

[175] The recombinant viral genome of the invention may comprise ITRs. It is possible that an AAV vector of the invention works with only one ITR. Thus, the viral genome comprises at least one ITR, or more typically two ITRs (usually one at each end of the viral genome, that is, one at the 5' end and one at the 3' end). There may be sequences intermediate between the polynucleotide and one or more of the ITRs. The polynucleotide of the invention can be incorporated into a viral particle located between two regular ITRs or located on either side of an ITR designed with two D regions.

[176] The AAV sequences that can be used in the present invention for the production of AAV vectors can be derived from the genome of any AAV serotype. Generally, AAV serotypes have genomic sequences of significant homology at the amino acid and nucleic acid levels, provide an identical set of genetic functions, produce virions that are essentially physical and functionally equivalent, and replicate and assemble by practically mechanisms. identical.

For the genomic sequence of the various AAV serotypes and an overview of genomic similarities, see, for example, GenBank accession number U89790; GenBank accession number J01901; GenBank accession number AF043303; GenBank accession number AF085716; Chiorini et al, 1997; Srivastava et al, 1983; Chiorini et al, 1999; Rutledge et al, 1998; and Wu et al,

2000. AAV serotype 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11 or 12 can be used in the present invention. Sequences of AAV serotypes can be mutated or manipulated when used in the production of vectors for gene therapy.

[177] Optionally, an AAV vector comprises ITR sequences that are derived from AAV1, AAV2, AAV4 and/or AAV6. Preferably, the ITR sequences are AAV2 ITR sequences. Here, the term AAVx/y refers to a viral particle comprising some AAVx components (where x is an AAV serotype number) and some AAVy components (where y is the number of the same or a serotype different). For example, an AAV2/8 vector can comprise a portion of a viral genome, including the ITRs from an AAV2 strain and a capsid derived from an AAV8 strain.

[178] In an exemplary embodiment, the viral particle is an AAV viral particle comprising a capsid (or capsid). AAV capsids are generally formed from three proteins, VP1, VP2 and VP3. The amino acid sequence of VP1 comprises the sequence of VP2. The portion of VP1 that is not part of VP2 is referred to as VP1unique (VP1-unique) or VP1U. The amino acid sequence of VP2 comprises the sequence of VP3. The portion of VP2 that is not part of VP3 is referred to as VP2unique (VP2-unique) or VP2U. Preferably, the capsid is an AAV5 capsid or a Mut C capsid. The Mut C capsid may have at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8% of identity or 100% identity with SEQ ID NO: 10. The AAV capsid may have at least 96%, at least 98%, at least 99%, at 99.5%, at least 99.8% or 100% of identity with SEQ ID NO: 17. In an alternative embodiment example, the capsid has a VP2U and/or VP3 sequence of SEQ ID NO: 17 and a VP1U sequence of at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identity with SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.

[179] A viral particle of the invention may be a "hybrid" particle in which the viral ITRs and the viral capsid are from different parvoviruses, such as from different AAV serotypes. Preferably, the viral ITRs and capsid are from different AAV serotypes, in which case such viral particles are known as transcapsidated or pseudotyped. Likewise, the parvovirus may have a "chimeric" capsid (eg, containing different parvovirus sequences, preferably different AAV serotypes) or a "targeted" capsid (eg, a targeted tropism).

[180] In some embodiment examples, the recombinant AAV genome comprises intact ITRs, comprising functional terminal resolution sites (TRS). Such an AAV genome may contain one or two resolvable ITRs, that is, ITRs containing a functional TRS in which site-specific nicking can occur to create a free 3' hydroxyl group that can serve as a substrate for the DNA polymerase to unfold and copy the ITR. Preferably, the recombinant genome is single-stranded (that is, it is packaged into the viral particle in a single-stranded form). Optionally, the recombinant genome is not packaged in a self-complementary configuration, i.e., the genome does not comprise a single strand polynucleotide covalently linked with substantial self-complementary portions that loop in the viral particle.

Alternatively, the recombinant genome can be packaged as a "monomeric duplex". "Monomeric duplexes" are described in WO 2011/122950. The genome can be packaged as two substantially complementary, but not covalently linked, polynucleotides that anneal in the viral particle.

[181] The viral particle may further comprise a poly A sequence. The poly A sequence may be positioned downstream of the nucleotide sequence encoding a functional Factor IX protein. The poly A sequence can be a bovine growth hormone (bGHpA) poly A sequence. The poly A sequence can be between 250 and 270 nucleotides in length.

[182] The viral particle of the invention, optionally, is strongly expressed in host cells. For example, in transduction in Huh7 cells, the viral particle expresses the Factor IX protein or a fragment thereof with a greater Factor IX activity than the Factor IX protein activity expressed from a viral particle comprising a Factor nucleotide sequence IX of SEQ ID NO: 12 and a transcription regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcription regulatory element of SEQ ID NO: 6.

[183] Optionally, after transduction into a population of Huh7 cells, the viral particle expresses the Factor IX protein or a fragment thereof, having greater Factor IX activity than the Factor IX activity expressed from a comparable viral particle which comprises a Factor IX nucleotide sequence of SEQ ID NO:12 and a transcriptional regulatory element of SEQ ID NO:7 transduced into a comparable population of Huh7 cells. Optionally, after transduction into a population of Huh7 cells, the viral particle expresses the Factor IX protein or a fragment thereof, having a greater Factor IX activity than the Factor IX activity expressed from a comparable viral particle comprising a Factor IX nucleotide sequence of SEQ ID NO:18 and a transcriptional regulatory element of SEQ ID NO:6 transduced in a comparable population of Huh7 cells.

[184] In one aspect, the polynucleotide, viral particle or composition of the invention is for use in treating a disease by expressing the Factor IX protein or a fragment thereof having a greater Factor IX activity than the Factor IX activity expressing the from a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6. In one aspect, a method of treating a disease by expressing the Factor IX protein or a fragment thereof having a greater Factor IX activity than the activity of the disease is provided. Factor IX expressed from a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a sequence of nucleotides of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, comprising administering an effective amount of the polynucleotide, viral particle or composition of the invention to a patient. In one aspect, a use of the polynucleotide, viral particle or composition of the invention in treating a disease by expressing the Factor IX protein or a fragment thereof having a greater Factor IX activity than the Factor IX activity expressed from is provided. a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcription regulatory element of SEQ ID NO: 6. Optionally, the viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcription regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a Factor IX nucleotide sequence of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, is a comparable viral particle.

Optionally, expressing the Factor IX protein or a fragment thereof comprises administering the polynucleotide, viral particle or composition of the invention to a patient. Optionally, the disease is hemophilia, eg hemophilia B.

[185] In such embodiment examples, the term "comparable viral particle" refers to a viral particle that is the same as an AAV viral particle of the invention, except that the comparable viral particle comprises a different Factor IX nucleotide sequence and a different transcription regulatory element (those of SEQ ID NO: 12 and SEQ ID NO: 7 or SEQ ID NO: 18 and SEQ ID NO: 6). Optionally, activity is assessed using a chromogenic assay, such as the chromogenic assay discussed above. In this case, however, the activity is not normalized to the Factor IX concentration,

so activity is a function of the expression level as well as the inherent activity of the Factor IX protein.

COMPOSITIONS, METHODS AND USES

[186] In a further aspect of the invention, a composition comprising the polynucleotide or viral particle/vector of the invention and a pharmaceutically acceptable excipient is provided.

[187] Pharmaceutically acceptable excipients may comprise carriers, diluents and/or other medicinal agents, pharmaceutical agents or adjuvants, etc. Optionally, pharmaceutically acceptable excipients comprise saline solution. Optionally, pharmaceutically acceptable excipients comprise human serum albumin.

[188] The invention further provides a polynucleotide, viral vector/particle or composition of the invention for use in a method of treatment. Optionally, the method of treatment comprises administering an effective amount of the polynucleotide or vector/viral particle of the invention to a patient.

[189] The invention further provides a method of treatment which comprises administering an effective amount of the polynucleotide or vector/viral particle of the invention to a patient.

[190] The invention further provides the use of a polynucleotide, viral vector/particle or composition of the invention in the manufacture of a drug for use in a method of treatment. Optionally, the method of treatment comprises administering an effective amount of the polynucleotide or vector/viral particle of the invention to a patient.

[191] Optionally the method of treatment is gene therapy. A “gene therapy” involves the administration of a vector/viral particle of the invention that is capable of expressing a transgene (such as a Factor IX nucleotide sequence) in the host to which it is administered.

[192] Optionally, the method of treatment is a method of treating a coagulopathy such as hemophilia (eg, hemophilia A or B) or Van Willebrand's disease. Preferably, the coagulopathy is characterized by increased bleeding and/or reduced coagulation.

Optionally, the method of treatment is a method of treating hemophilia, e.g., hemophilia B. In some embodiment examples, the patient is a patient suffering from hemophilia B. Optionally, the patient has antibodies or inhibitors to Factor IX. Optionally, the polynucleotide and/or viral vector/particle is administered intravenously. Optionally, the polynucleotide and/or vector/viral particle is for one-time administration (i.e., a single dose) to a patient.

[193] Although hemophilia B is “treated” in the above method, this means that one or more symptoms of hemophilia are ameliorated. This does not mean that the symptoms of hemophilia are completely remedied so that they are no longer present in the patient, although in some methods this can occur. The treatment method may result in making one or more of the symptoms of hemophilia B less severe than before treatment. Optionally, relative to the pre-administration situation, the treatment method results in an increase in the amount/concentration of Factor IX circulating in the patient's blood and/or the overall level of detectable Factor IX activity within a given blood volume of the patient. patient and/or the specific activity (activity per amount of Factor IX protein) of Factor IX in the patient's blood.

[194] A "therapeutically effective amount" refers to an effective amount, in dosages and for periods of time necessary, to achieve the desired therapeutic result, such as increasing the level of functional factor IX in a subject (so as to lead to production of functional factor IX at a level sufficient to improve the symptoms of hemophilia B).

[195] Optionally, the viral vector/particle is administered at a dose less than 1 x 1011, less than 1 x 1012, less than 5 x 1012, less than 2 x 1012, less than 1.5 x 1012, less than 3 x 1012, less than 1 x 1013, less than 2 x 1013, or less than 3 x 1013 vector genomes per kg of patient weight. Optionally, the polynucleotide, viral particle or composition is administered at a dose of at least less than 4.5 x 1011, or between 4.5 x 1011 and 1 x 1012 vector genomes per kg of patient weight (vg/kg) . Optionally, the polynucleotide, or viral particle composition is administered at a dose less than 5 x 10 11 vg/kg.

Optionally, the polynucleotide, viral particle or composition is administered at a dose between 4.5 x 1011 vg/kg and 5 x 1011 vg/kg, or between 4.5 x 1011 vg/kg and 4.9 x 1011 vg/kg . Optionally, the polynucleotide or viral particle that is administered is produced from a mammalian cell and/or has characteristics that result from the use of mammalian viral vector production cells and are distinguished from vectors produced in viral vector production cells of insect (eg baculovirus system).

[196] Optionally, administration of a given dose of vectors/viral particles - quantified in terms of the number of vector genomes - is achieved using qPCR for titration of vector genomes. In principle, qPCR primers can be designed to bind to any part of the recombinant vector genome that is not common to wild-type genomes, but the use of primer template sequences to regions in close proximity to the ITRs is not recommended as this may lead to an over-measurement of the vector's genome titer. Optionally, vector genomes are quantified by quantitative polymerase chain reaction (qPCR) using primers targeted to a promoter region, e.g., the promoter region of the transgene cassette (also known as the transgene expression cassette).

[197] Methods for performing qPCR are known to those skilled in the art. Using a real-time PCR cycler and a DNA-binding dye such as SYBR Green (ThermoFisher Scientific), amplification of nascent double-stranded amplicons can be detected in real-time. Known amounts of the qPCR template genetic material (eg, the promoter region) can be serially diluted to create a standard curve and the genome titre of the sample vector interpolated from the standard curve.

[198] Optionally, the dose of vector/viral particle that is administered is selected such that the subject expresses Factor IX at an activity that is 10% -90%, 20% -80%, 30% -70% , 25% -50%, 20% - 150%, 30% -140%, 40% -130%, 50% -120%, 60% -110% or 70% -100% a subject's Factor IX activity healthy non-haemophiliac. Optionally, the polynucleotide, viral particle or composition is administered at a dose effective to achieve a stable Factor IX activity level of at least 20% in a patient with hemophilia B. Optionally, the polynucleotide, viral particle or composition is administered in one effective dose to achieve a stable Factor IX activity level of at least 25% in a patient with hemophilia B. Optionally, the polynucleotide, viral particle or composition is administered at an effective dose to achieve a stable Factor IX activity level of at least 30% in a patient with hemophilia B. Optionally, the polynucleotide, viral particle or composition is administered in an effective dose to achieve a stable Factor IX activity level of at least 35% in a patient with hemophilia B. the polynucleotide, viral particle or composition is administered in an effective dose to achieve a stable Factor IX activity level of at least or approximately 40 % in a patient with hemophilia B.

[199] In one aspect, the polynucleotide, viral particle or composition of the invention is used to achieve a stable Factor IX activity level of at least 20% in a patient with hemophilia B. In one aspect, a method is provided for achieving a stable Factor IX activity level of at least 20% in a patient with hemophilia B, comprising administering an effective amount of the polynucleotide, viral particle or composition of the invention to a patient. In a further aspect, a use of the polynucleotide, viral particle or composition of the invention to achieve a stable Factor IX activity level of at least 20% in a patient with hemophilia B is provided. In a further aspect, the polynucleotide, is provided, viral particle or composition of the invention for use in the treatment of hemophilia B, comprising administering the polynucleotide, viral particle or composition to a patient at a dose effective to achieve a stable Factor IX activity level of at least 20% in a patient with hemophilia B. In a further aspect, there is provided a method of treating hemophilia B comprising administering the polynucleotide, viral particle or composition of the invention to a patient at a dose effective to achieve a stable Factor IX activity level of at least 20 % in a patient who has hemophilia B. In a further aspect, the use of the polynucleotide, viral particle or composition of the invention in the treatment of he is provided. mophilia B, comprising administering the polynucleotide, viral particle or composition to a patient at a dose effective to achieve a stable Factor IX activity level of at least 20% in a patient with haemophilia B. Optionally, treating haemophilia B or achieving a stable Factor IX activity level comprises administering the polynucleotide, viral particle or composition of the invention to a patient.

Optionally, to achieve a stable Factor IX activity level, such as a stable Factor IX activity level of at least 20%, at least 30%, or at least 40%, the polynucleotide, viral particle or composition is administered in a dose less than 5 x 10 11 vg/kg or between 4.5 x 1011 vg/kg and 4.9 x 1011 vg/kg. Optionally, the polynucleotide or viral particle that is administered is produced from a mammalian cell and/or has characteristics that result from the use of mammalian viral vector production cells and are distinguished from vectors produced in viral vector production cells of insect (eg baculovirus system).

[200] A Factor IX activity level of at least X% (eg at least 20%) refers to a Factor IX activity level that is at least X% (eg 20 %) of normal Factor IX level variation. The person skilled in the art will readily understand what is meant by reference to % normal Factor IX activity level, which is determined in routine clinical practice by reference to international standards. Normal (healthy) pooled human plasma has a Factor IX concentration of approximately 5 µg/mL.

[201] The term “stable Factor IX activity level” refers to a level of Factor IX activity that remains at or above a given level for a continuous period of at least 5 weeks. In some embodiment examples, the Factor IX activity level remains at or above a given level for a continuous period of at least 10, at least 15, at least 20, at least 30, at least 40, or at least at least 50 weeks. For example, a patient has a stable Factor IX activity level of at least 20% if the activity level remains at least 20% for a continuous period of at least 5 weeks. In such an example, the Factor IX activity level may continue to be at least 20% after at least 5 weeks and therefore remains at least 20% for a continuous cumulative period of at least 10, at least 15 , at least 20,

at least 30 or at least 40, or at least 50 weeks. Optionally, a patient has a stable Factor IX activity level if the Factor IX activity level remains at or above a certain level for a continuous period of at least 20 weeks. Optionally, a patient administered the polynucleotide, viral particle or composition can have a stable Factor IX activity level of at least 20%, at least 25%, at least 30% or at least 35%. Optionally, a patient administered the polynucleotide, viral particle or composition can have a stable Factor IX activity level of at least or about 40%.

[202] Optionally, Factor IX activity level is stable after at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks from administration of the polynucleotide, viral particle or composition. For example, when a patient has a stable Factor IX activity level of at least 20% after at least 10 weeks from when the patient is administered with the polynucleotide, viral particle or composition, there is a Factor IX activity level of at least 20% which remains at least 20% for a continuous period of at least 5, at least 10, at least 15, at least 20, at least 30 or at least 40, or at least 50 weeks after the period at least 10 weeks from administration. Optionally, a patient has a stable Factor IX activity level after at least 10 weeks of administration of the polynucleotide, viral particle or composition, and the Factor IX activity level remains at or above a certain level for a period of time. continuous for at least 20 weeks or at least 40 weeks (after the initial period of at least 10 weeks after administration). Optionally, the stable Factor IX activity level after at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks from administration can be at least 20%, at least 25 %, at least 30% or at least 35%. Optionally, the stable Factor IX activity level after at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks from administration can be at least or about 40%.

In some embodiment examples, a patient has a stable Factor IX activity level of at least 20% after at least 10 weeks of administration of the polynucleotide, viral particle or composition, and the Factor IX activity level remains at least at least 20% for a continuous period of at least 20 weeks or at least 40 weeks (after the initial period of at least 10 weeks after administration). In some embodiment examples, a patient has a stable Factor IX activity level of at least 40% after at least 10 weeks of administration of the polynucleotide, viral particle or composition, and the Factor IX activity level remains at least at least 40% for a continuous period of at least 20 weeks or at least 40 weeks (after the initial period of at least 10 weeks after administration).

[203] Optionally, the Factor IX activity level is, or is above, a certain level (eg, 20%, 25%, 30%, 35%, or 40%) at a point in time of at least 10, at least 20, at least 30, at least 40 or at least 50 weeks after administration of the polynucleotide, viral particle or composition. For example, the Factor IX activity level is at or above a certain level (eg 20%, 25%, 30%, 35% or 40%) at a time point of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the activity level of Factor IX is at least 20% at a time point in about 10 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the activity level of Factor IX is at least 25% at a time point in about 10 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the Factor IX activity level is at least 40% at one time point in about 10 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the Factor IX activity level is at least 20% at a time point in about 20 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the activity level of Factor IX is at least 25% at one time point in about 20 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the activity level of Factor IX is at least or about 40% at one time point in about 20 weeks after administration of the polynucleotide, viral particle or composition.

Optionally, the Factor IX activity level is at least 20% at a time point in about 52 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the activity level of Factor IX is at least 25% at a time point in about 52 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the activity level of Factor IX is at least or about 40% at one time point in about 52 weeks after administration of the polynucleotide, viral particle or composition.

[204] When the Factor IX activity level is, or is above, a certain level (eg, 20%, 25%, 30%, 35%, or 40%) at a point in time of at least 10 , at least 20, at least 30, at least 40, or at least 50 weeks after administration of the polynucleotide, viral particle or composition, the level of Factor IX activity may also be stable. In such examples of achievement where the activity level of the Factor

IX is also stable, the continuous period of at least 5 weeks (or at least 10, at least 15, at least 20, at least 30 or at least 40 or at least 50 weeks) can immediately precede, understand or immediately follow the point of time at least 10, at least 20, at least 30, at least 40 or at least 50 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the Factor IX activity level is at least 20% at a time point in about 10 weeks after administration of the polynucleotide, viral particle or composition, and the Factor IX activity level remains at least 20% for a continuous period of at least 40 weeks immediately following the time point. Optionally, the Factor IX activity level is at least 25% at one time point in about 10 weeks after administration of the polynucleotide, viral particle or composition, and the Factor IX activity level is maintained at at least 25% for a continuous period of at least 40 weeks immediately following the time point.

Optionally, the Factor IX activity level is at least 40% at one time point in about 10 weeks after administration of the polynucleotide, viral particle or composition, and the Factor IX activity level remains at least 40% for a continuous period of at least 40 weeks immediately following the time point. Optionally, the Factor IX activity level is at least 20% at one time point at about 52 weeks after administration of the polynucleotide, viral particle or composition, and the Factor IX activity level remains at least 20% for a continuous period of at least 40 weeks immediately preceding the point in time. Optionally, the Factor IX activity level is at least 25% at one time point at about 52 weeks after administration of the polynucleotide, viral particle or composition, and the Factor IX activity level remains at least 25% for a continuous period of at least 40 weeks immediately preceding the point in time.

Optionally, the Factor IX activity level is at least 40% at one time point at about 52 weeks after administration of the polynucleotide, viral particle or composition, and the Factor IX activity level remains at least 40% for a continuous period of at least 40 weeks immediately preceding the point in time.

[205] In patients, such as human patients, Factor IX activity can be measured either by taking a blood sample from the patient or by performing an assay on a blood sample taken from the patient.

[206] Optionally, the level of Factor IX activity can be measured using a coagulation assay. Optionally, the clotting assay is a one-stage clotting assay. Optionally, the clotting assay, like a one-stage clotting assay, uses a SynthASil® kit. In one example, a suitable clotting assay (a one-stage clotting assay) is as follows.

[207] Since Factor IX is part of the coagulation cascade, a Factor IX polypeptide that has greater blood clotting activity will catalyze faster than a Factor IX polypeptide that has a lower activity. Factor IX polypeptide is mixed with platelet-poor plasma and incubated at 37°C. Then phospholipids and a contact activation pathway activator such as SynthASil® APTT (Activated Partial Thromboplastin Time) reagent are added. Calcium is then added and the user measures the time it takes for clotting to occur. Clot formation can be assessed by spectrophotometry or by a magnetic steel ball method. For example, clot formation can be considered to have occurred when the optical density of the mixture exceeds a threshold or when the mixture has gelled enough to alter the movement of the magnetic ball, which is detected by a sensor.

[208] Optionally, Factor IX activity is measured using a SynthASil® one-stage coagulation test kit to determine Factor IX activity in the blood sample.

[209] Optionally, the patient is a patient who is refractory to Factor IX treatment, with an activity lower than the activity of Factor IX, expressed from a viral particle that comprises (i) a nucleotide sequence of Factor IX of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory element of SEQ ID NO: 6. Optionally, the patient is a patient refractory to treatment with a Factor IX polypeptide that is expressed at a level lower than that encoded. by a nucleotide sequence comprising (i) a nucleotide sequence of Factor IX of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory element of SEQ ID NO:

6. As demonstrated in the examples, a viral particle comprising (i) a nucleotide sequence of Factor IX of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory sequence of SEQ ID NO: 6 expresses a Factor IX polypeptide in higher levels and said polypeptide has higher activity compared to a polypeptide expressed from other Factor IX nucleotide sequences. Thus, a viral particle comprising (i) a nucleotide sequence of Factor IX of SEQ ID NO: 4 or 5 and (ii) a transcription regulatory sequence of SEQ ID NO: 6 can be used to treat patients who need higher levels. of Factor IX, lower doses of viral particle, or who have problems expressing Factor IX from a viral particle comprising the alternative Factor IX nucleotide sequences, eg, patients who have lost part of their liver and are therefore less able to express high levels of the Factor IX nucleotide sequence from liver cells.

[210] Optionally, the method or use comprises prophylactic co-administration of a steroid, that is, an adequate dose of a steroid is administered to the patient. Co-administration can be concurrent, that is, the patient receives the dose of steroid at the same time as receiving a dose of the polynucleotide, vector or composition of the invention.

Alternatively, the steroid can be administered before or after the polynucleotide, vector or composition of the invention is administered to the patient. Optionally, the steroid is administered within 6 to 12 weeks after administering the polynucleotide, vector or composition of the invention to a patient.

[211] Optionally, the steroid is selected from the group consisting of prednisone, betamethasone, prednisolone, triamcinolone, methylprednisolone, and dexamethasone. Optionally, the steroid is prednisolone.

SEQUENCE LISTING TABLE 3 Sequence Description Identification Number Sequence (SEQ ID NO) Codon-optimized portion of the TI-1 coding sequence ACNP-FIX-GoF Portion of the coding sequence of wild type TI-ACNP-FIX-GoF, including intron 3 Intron 1A of truncated FIX 4 Coding sequence of TI-ACNP-FIX-GoF Coding sequence of Factor IX sequence TI-ACNP-5 FIX-GoF, including intron 6 Sequence of transcription regulatory element HLP2 7 Sequence of transcription regulatory element LP1 "Mature" Factor IX amino acid sequence encoded by SEQ ID NO: 4 Wild-type factor IX (Malmo B variant) 9 coding sequence 10 Mut C capsid polypeptide sequence 11 FIXco coding sequence with TTG GoF codon 12 FIXco coding sequence 13 Enhancer element of HLP2 14 Promoter element of HLP2 Portion of the coding sequence of wild type TI-ACNP-FIX-GoF, excluding the intron. "Immature" Factor IX amino acid sequence encoded by SEQ ID NO: 4 17 AAV5 capsid polypeptide sequence

Sequence Description identification number Sequence (SEQ ID NO) Coding sequence of the sequence of Factor IX TI-codop-18 FIX-GoF, including wild-type Factor IX intron (Malmo B variant) coding sequence corresponding to the mature FIX polypeptide 20 AAV2-5 hybrid VP1u variant 1 21 AAV2-5 hybrid VP1u variant 2 22 AAV2-5 hybrid VP1u variant 3 23 AAV2-5 hybrid VP1u variant 4 24 AAV2-5 hybrid VP1u variant 5 25 AAV2-5 hybrid VP1u variant 6 26 Coding sequence of FIXco with CTG GoF codon

EXAMPLES

[212] In the following examples, experiments were performed with recombinant AAV carrying the FIX transgene cassettes ssLP1.FIXco (herein called FIXco), ssHLP2.TI-codop-FIX-GoF (herein called HTFG), and ssHLP2.TI-ACNPFG -FIX-GoF (herein called HTAG). SsHLP2.TI-codop-FIX-GoF is a version of the ssHLP2.TI-codop-FIX construct disclosed in WO2016/075473 modified to encode leucine (L) instead of arginine (R) at position 384 of the encoded FIX polypeptide. These cassettes are shown in Figure 1. ssLP1.FIXco contains a fully codon-optimized FIX coding sequence (SEQ ID NO: 12) preceded 5' by an SV40 intron, with expression driven by the LP1 promoter (SEQ ID NO: 7). ssHLP2.TI-codop-FIX-GoF and ssHLP2.TI-ACNP-FIX-GoF share the structure of having the shorter HLP2 transcriptional regulatory element (SEQ ID NO:6) 5' to a FIX coding sequence that is interrupted by a truncated version of native intron 1A and in which exon 1 and part of the nucleotide sequence of exon 2 is wild type (non-codon-optimized), with the remainder of the coding sequence codon-optimized (SEQ ID NO: 18 for HTFG and SEQ ID NO: 5 for HTAG). The nucleotide sequence of the codon-optimized portions is what differs between the two respective constructs. Unlike the wild-type FIX protein encoded by ssLP1.FIXco, ssHLP2.TI-codop-FIX-GoF and ssHLP2.TI-ACNP-FIX-GoF encode a hyperactive FIX with a substitution of arginine (R) for leucine (L) in position 384 of the FIX polypeptide.

EXAMPLE 1

METHODS

[213] Production and quantification of the AAV vector:

1. AAV vector stocks were prepared by standard triple plasmid transfection of human embryonic kidney cells (HEK293) with a plasmid combination consisting of a vector genome plasmid, an adenoviral helper plasmid and a packaging plasmid containing Rep functions and Cap of AAV (AAV8 or AAVMut C).

As the recombinant AAV particles contained an AAV2-based genome and either a serotype 8 capsid or a synthetic capsid comprising portions of two serotypes ('Mut C'; SEQ ID NO: 10), they are referred to as 'pseudotyped';

2. Vectors were purified by density gradient centrifugation with iodixanol; and

3. The vector genomes were titrated by qPCR with primers directed to the promoter region.

[214] In vitro transduction and detection of FIX expression:

1. HUH7 cells were seeded at 5x105 cells per well in 12-well plates;

2. Cells were then stimulated with mitomycin C for 1 hour before transduction with AAV particles carrying a transgene cassette encoding FIX;

3. Five days after transduction, the supernatant was collected and analyzed for FIX level using a commercially available ELISA kit (Stago Asserachrom IX:Ag kit Ref #00943). FIX activity was analyzed using the commercially available chromogenic kit from

Quadratech (Biophen FIX (6) kit Ref # 221806); and

4. Vector genome DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (Ref #69506) according to the manufacturer's instructions and quantified by qPCR.

[215] In vivo detection of FIX:

1. Adult C57BL/6 mice received injections with 5x1010 vector genomes (vg) of AAV particles carrying a transgene cassette encoding FIX via the tail vein (n = 4 per group);

2. Two weeks after injection, mice were anesthetized and blood collected by cardiac puncture, added to sodium citrate (1/10 dilution) and centrifuged at 3000 xg for 15 minutes at 4ºC to collect plasma, which was frozen at -80°C until analysis;

3. The liver was collected and snap-frozen in liquid nitrogen before storage at -80°C for DNA extraction (Qiagen DNeasy Blood and Tissue Kit, Ref # 69506) for vector genome analysis; and

4. The level of FIX present in murine plasma was determined using a FIX ELISA kit (Stago Asserachrom IX:Ag kit; reference; 00943). Human FIX activity was determined using the commercially available chromogenic kit from Quadratech (Biophen FIX (6) kit Ref # 221806).

EXAMPLE 2

IN VITRO TRANSGENE FIX EXPRESSION ANALYSIS USING ELISA

[216] HUH7 cells (human hepatocytes) were cultured under standard cell culture conditions. FIX transgenes were expressed by treating HUH7 cells with mitomycin C for 1 hour, then subsequently transducing with pseudotyped ssAAV2/Mut C.

AAV particles were first generated by transfection of cells

HEK293 with recombinant vector genome plasmid, plus AAV helper and packaging plasmids, and culture for an additional 48 hours. ssAAV2/Mut C vectors were purified from HEK293 cells by density gradient centrifugation and iodixanol. Vector genomes were titrated by qPCR using primers directed to the promoter region of the transgene expression cassette. FIX expression cassettes were compared to determine their relative ability to express a FIX transgene in vitro by measuring FIX levels 5 days after transduction.

The vectors evaluated were ssAAV2/Mut C.HLP2.TI-codop-FIX-GoF and ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF. FIX levels in the culture supernatant were analyzed using a commercially available ELISA kit (Stago Asserachrom IX:Ag kit Ref # 00943). In two separate experiments (see Figure 2B and 4B), FIX expression levels - as derived from ELISA assays using supernatant from cultured HUH7 cells - were normalized against vector genome copies per cell after DNA collection from cells HUH7 using the Qiagen DNeasy Blood and Tissue Kit (Ref #69506).

[217] 5 days after transduction with ssAAV2/Mut C.HLP2.TI-codop-FIX-GoF and ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF at an MOI of 1x103 vector genomes (vg), FIX levels were higher in HUH7 cells transduced with ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF than in HUH7 cells transduced with ssAAV2/Mut C.HLP2.TI-codop-FIX-GoF (n = 2 ; Figure 2A, 3 and 4A). Similarly, when identical FIX expression assays were performed in HUH7 cells with increased MOI (5x103 and 1x104), FIX levels were higher in cells transduced with ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF do than in cells transduced with ssAAV2/Mut C.HLP2.TI-codop-FIX-GoF (n = 2; Figure 2A, 3 and 4A). When FIX expression levels were normalized against viral vector genome copies per cell at each of the three MOIs tested (1x103, 5x103 and 1x104), FIX levels were consistently higher in HUH7 cells transduced with ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF versus ssAAV2/MutC.HLP2.TI-codop-FIX-GoF (n = 2; Figure 2B and 4B). When the 5x103 transduction data from the experiments in Figures 2, 3, and 4 are combined, they show significantly higher expression of ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF relative to ssAAV2/Mut C.HLP2.TI - codop-FIX-GoF (Figure 5).

EXAMPLE 3

ANALYSIS OF IN VITRO TRANSGENE FIX ACTIVITY

[218] FIX activity was assessed by harvesting supernatant from HUH7 cells and using the BIOPHEN Factor IX kit (Quadratech # 221806, # 222101, # 223201). Partially codon-optimized FIX transgenes (HLP2.TI-codop-FIX-GoF and HLP2.TI-ACNP-FIX-GoF) were compared in vitro to determine the relative activity of FIX after ssAAV2/Mut C transduction (at an MOI of 1x103, 5x103 and 1x104) in HUH7 cells. The supernatant was isolated from HUH7 cells 5 days after transduction and FIX activity was determined using the BIOPHEN Factor IX kit. Regardless of the MOI, a higher mean FIX activity was observed in the supernatant derived from cells transduced with ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF (Figure 6-8). When the 5x103 transduction data from the experiments in Figures 6 and 7 are combined, they show significantly higher expression of ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF relative to ssAAV2/Mut C.HLP2.TI -codop-FIX-GoF (Figure 9).

EXAMPLE 4

IN VIVO TRANSGENE FIX EXPRESSION ANALYSIS USING ELISA

[219] FIX transgenes were expressed in C57BL/6 mice after transduction by tail vein injection with 5x1010 vector genomes (VG) of ssAAV2/8 pseudotype vectors. AAV particles were first generated by transfection of HEK293 cells with recombinant vector genome plasmid, in addition to AAV helper and packaging plasmids, and cultured for an additional 48 hours. ssAAV2/8 vectors were purified from HEK293 cells by density gradient centrifugation and iodixanol. Vector genomes were titrated by qPCR using primers directed to the promoter region of the transgene expression cassette. The FIX expression cassettes LP1.FIXco, HLP2.TI-codop-FIX-GoF and HLP2.TI-ACNP-FIX-GoF were compared to determine their relative ability to express a FIX transgene.

[220] In a first experiment involving ssAAV2/8.HLP2.TI-codop-FIX-GoF and ssAAV2/8.LP1.FIXco, 2 weeks after dosing, blood was collected from anesthetized mice via cardiac puncture. Subsequently, the plasma was isolated by adding sodium citrate (1/10 dilution) and centrifuging at 3000 x g for 15 minutes at 4ºC.

Circulating FIX levels were determined using a FIX ELISA kit (Stago Asserachrom IX:Ag kit Ref # 00943).

[221] FIX expression levels were normalized against vector genome copies per cell after harvest from mouse liver.

Normalized FIX expression levels were determined to be significantly higher (p < 0.05) after transduction with ssAAV2/8.HLP2.TI-codop-FIX-GoF relative to ssAAV2/8.LP1.FIXco (n = 4 mice; Figure 10).

[222] In another experiment, partially codon-optimized FIX transgenes (HLP2.TI-codop-FIX-GoF and HLP2.TI-ACNP-FIX-GoF) were compared for relative in vivo expression of FIX after transduction in mice C57Bl/6 with ssAAV2/8. Concomitantly, FIX expression was determined after transduction of C57Bl/6 mice with scAAV2/8.LP1.FIXco vector. Plasma was isolated from mice 3 weeks after dosing, and FIX antigen levels were determined using the ELISA assay. Mean FIX expression levels were lower in mice transduced with scAAV2/8.LP1.FIXco, whereas levels were higher in mice transduced with ssAAV2/8.HLP2.TI-codop-FIX-GoF (n = 4 mice ; Figure 11A). Mice transduced with ssAAV2/8.HLP2.TI-ACNP-FIX-GoF had significantly higher FIX expression than mice transduced with ssAAV2/8.HLP2.TI-codop-FIX-GoF (n = 4 mice; Figure 11A) . When FIX expression levels were normalized against viral vector genome copies per cell, the trend in FIX expression was maintained, such that ssAAV2/8.HLP2.TI-ACNP-FIX-GoF produces significantly more FIX than ssAAV2/8.HLP2.TI-codop-FIX-GoF and scAAV2/8.LP1.FIXco (n = 4; Figure 11B). In addition, ssAAV2/8.HLP2.TI-codop-FIX-GoF exhibited greater FIX expression than scAAV2/8.LP1.FIXco (n = 4 mice; Figure 11B).

EXAMPLE 5

ANALYSIS OF TRANSGENE FIX IN VIVO ACTIVITY

[223] The BIOPHEN Factor IX kit (Quadratech # 221806, # 222101, # 223201) is a chromogenic assay for measuring Factor IX activity in human citrated plasma or in Factor IX concentrates using a manual chromogenic method.

[224] In the presence of thrombin, phospholipids and calcium, first Factor XIa, provided in the assay at a constant concentration and in excess, activates FIX present in the tested sample, in FIXa, which forms an enzyme complex with thrombin or activated Factor VIII:C , also provided in the assay in constant concentration and in excess, phospholipids (PLPs) and Calcium, which activates Factor X, present in the assay system, in Factor Xa. This activity is directly related to the amount of Factor IX, which is the limiting factor.

The Factor Xa generated is then measured exactly by its specific activity on the Factor Xa chromogenic substrate (SXa-11). Factor Xa cleaves the substrate and releases pNA. The amount of pNA generated is directly proportional to the activity of Factor IXa. Finally, there is a direct relationship between the amount of Factor IX in the sample tested and the activity of Factor Xa generated, measured by the amount of pNA released, determined by color development at 405 nm.

[225] Partially codon-optimized FIX transgenes (HLP2.TI-codop-FIX-GoF and HLP2.TI-ACNP-FIX-GoF) were compared for relative FIX activity in vivo after transduction in C57Bl/6 mice with ssAAV2/8. Concomitantly, FIX activity was determined after transduction of C57Bl/6 mice with scAAV2/8.LP1.FIXco. Plasma was isolated from mice 3 days after treatment, and FIX activity was determined using the BIOPHEN Factor IX kit. The mean FIX activity in mice was lower in mice transduced with scAAV2/8.LP1.FIXco, whereas the activity was higher in mice transduced with ssAAV2/8.HLP2.TI-codop-FIX-GoF (n = 4 mice ; Figure 12). Mice transduced with ssAAV2/8.HLP2.TI-ACNP-FIX-GoF had significantly higher FIX activity than mice transduced with ssAAV2/8.HLP2.TI-codop-FIX-GoF (n = 4 mice; Figure 12) .

EXAMPLE 6

[226] Two patients with hemophilia B were treated with 4 x 1011 vg/kg of AAV2/Mut C vectors containing ssHLP2.HTAG. Both patients were known to have severe hemophilia B with endogenous FIX activity < 1% at screening. Patient 1 was receiving prophylaxis at baseline and continued to do so for the first four days of the study. Within 4 weeks of receiving a single infusion of AAV2/Mut C vectors containing ssHLP2.HTAG, both patients achieved FIX activity levels > 30%. Patients achieved stable Factor IX activity levels of 40.5 ± 4.5% at 52 weeks, as assessed by a one-stage clotting assay using the SynthASil® kit. Prophylactic steroids (prednisolone) were administered during weeks 6 to 12.

[227] None of the patients had spontaneous or prolonged bleeding events. Patient 1 had a traumatic bleeding due to a cut in the right finger at week 10 post-treatment.

Significantly, the patient did not need any exogenous clotting factor and the event resolved on the same day. The patient's FIX activity at the time of the event was measured to be 38%.

[228] The invention described herein also relates to the following aspects.

[229] 1. A polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence comprises a coding sequence encoding a Factor IX protein or a fragment thereof and wherein a portion of the coding sequence is not of the wild type.

[230] 2. The polynucleotide of aspect 1, wherein the non-wild-type portion of the coding sequence is codon-optimized.

[231] 3. A polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence comprises a coding sequence encoding a Factor IX protein or a fragment thereof, and the coding sequence comprises: (i) a sequence which is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1; and (ii) a sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 15.

[232] 4. The polynucleotide of aspect 3, wherein the sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to the SEQ ID NO:1 is codon-optimized.

[233] 5. A polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence encodes a Factor IX protein or a fragment thereof and is at least 97%, at least 98%, at least 99% , at least 99.5%, at least 99.8%, or 100% identity with SEQ ID NO: 5.

[234] 6. The polynucleotide of aspect 5, wherein the Factor IX nucleotide sequence comprises a coding sequence and a portion of the coding sequence is codon-optimized.

[235] 7. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide comprises DNA or RNA.

[236] 8. The polynucleotide of any one of aspects 2, 4, 6, or 7, wherein the portion of the coding sequence that is codon-optimized is a contiguous portion.

[237] 9. The aspect 2, 4, 6, 7, or 8 polynucleotide, wherein the portion of the coding sequence that is codon-optimized is codon-optimized for expression in the human liver.

[238] 10. The polynucleotide of any of the foregoing aspects, wherein a polypeptide encoded by the Factor IX nucleotide sequence is expressed in human liver cells at higher levels compared to a wild-type Factor IX nucleotide sequence of reference.

[239] 11. The polynucleotide of any one of aspects 2, 4, 6, or 7, wherein the portion of the coding sequence that is codon-optimized is at least 800, at least 900, at least 1100, less than 1500, less than 1300, less than 1200, between 800 and 1500, between 900 and 1300,

between 1100 and 1200, or about 1191 nucleotides in length.

[240] 12. The polynucleotide of any of aspects 2, 4, or 6-11, wherein the portion of the coding sequence that is codon-optimized comprises 1, 2, 3, 4, 5 or all of: a) exon 3 or a portion of at least 10, at least 15, at least 20, less than 25, between 10 and 25, between 15 and 25, or between 20 and 25 nucleotides of exon 3; b) exon 4 or a portion of at least 80, at least 90, at least 100, less than 114, between 80 and 114, between 90 and 114, or between 100 and 114 nucleotides of exon 4; c) exon 5 or a portion of at least 90, at least 100, at least 110, less than 129, between 90 and 129, between 100 and 129, or between 110 and 129 nucleotides of exon 5; d) exon 6 or a portion of at least 150, at least 180, at least 200, less than 203, between 150 and 203, between 180 and 203, or between 200 and 203 nucleotides of exon 6; e) exon 7 or a portion of at least 70, at least 80, at least 90, at least 100, less than 115, between 70 and 115, between 80 and 115, between 90 and 115, or between 100 and 115 nucleotides from exon 7; and/or f) exon 8 or a portion of at least 400, at least 450, at least 500, less than 548, between 400 and 548, between 450 and 548, or between 500 and 548 nucleotides of exon 8.

[241] 13. The polynucleotide of aspect 12, wherein the portion of the coding sequence that is codon-optimized comprises a), b), c), d), e) and f).

[242] 14. The polynucleotide of aspect 12 or aspect 13, wherein the portion of the coding sequence that is codon-optimized comprises a portion of at least 20 nucleotides from exon 3, a portion of at least 100 nucleotides from exon 4, a at least 110 nucleotide portion of exon 5, at least 180 nucleotide portion of exon 6, at least 100 nucleotide portion of exon 7 and at least 500 nucleotide portion of exon 8.

[243] 15. The polynucleotide of any of aspects 12-14, wherein the portion of the coding sequence that is codon-optimized comprises exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8.

[244] 16. The polynucleotide of any of aspects 2, 4, or 6-15, wherein the portion of the coding sequence that is codon-optimized comprises a portion of exon 2 and the portion of exon 2 is less than 160, less than 150, less than 100, less than 75, less than 60, at least 20, at least 30, at least 40, at least 50, between 20 and 160, between 30 and 150, between 30 and 100 , between 40 and 75, or about 56 nucleotides in length.

[245] 17. The polynucleotide of any one of aspects 2, 4, or 6-16, wherein the portion of the coding sequence that is codon-optimized comprises a portion of exon 2 that is between 30 and 100 nucleotides in length.

[246] 18. The polynucleotide of any one of aspects 2, 4, or 6-17, wherein the portion of the coding sequence that is codon-optimized comprises a reduced number of CpGs as compared to a portion of a Factor IX-like sequence corresponding reference wildflower.

[247] 19. The polynucleotide of aspect 18, wherein the portion of the coding sequence that is codon-optimized comprises less than less than 40, less than 20, less than 18, less than 10, less than 5, or less than 1 CpG.

[248] 20. The polynucleotide of aspect 18 or 19, wherein the portion of the coding sequence that is codon-optimized is free of CpG.

[249] 21. The polynucleotide of any one of aspects 2, 4, or 6-20, wherein, in the portion of the coding sequence that is codon-optimized, at least 40%, at least 45%, at least 50%, at least at least 55%, at least 60%, at least 65%, at least 70%, or at least 73% of the codons are selected from the group consisting of: a) TTC; b) CTG; c) ATC; d) GTG; e) GTC; f) AGC; g) CCC; h) ACC; i) GCC; j) TAC; k) CAC; l) CAG; m) AAC; n) AAA; o) AAG; p) GAC; q) TGC; r) AGG; s) GGC; and t) GAG.

[250] 22. The polynucleotide of any one of aspects 2, 4, or 6-21, wherein, in the portion of the coding sequence that is codon-optimized:

a) at least 1, at least 2, at least 4, or at least 5 codons encoding phenylalanine is(are) replaced by TTC compared to a reference wild-type Factor IX sequence; b) at least 60%, at least 65%, or at least 70% of the codons encoding phenylalanine are TTC; c) at least 60%, at least 65%, or at least 70% of the codons encoding phenylalanine are TTC and the remainder are TTT; and/or d) the codons encoding phenylalanine are TTC, except when the next codon begins with a G.

[251] 23. The polynucleotide of any one of aspects 2, 4, or 6-22, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 10, at least 15, or at least 16 fewer codons encoding leucine are replaced by CTG compared to a reference wild-type Factor IX sequence; b) at least 90%, or at least 94% of the codons encoding leucine are CTG; and/or c) at least 90%, or at least 94% of the codons encoding leucine are CTG and the remainder are CTC.

[252] 24. The polynucleotide of any one of aspects 2, 4, 6-23, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 10, at least 11, or at least 12 fewer codons encoding isoleucine are replaced by ATC compared to a wild-type Factor IX reference sequence; b) at least 1 ATC codon is replaced by ATT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 60%, at least 70%, or at least 75% of the codons encoding isoleucine are ATC;

d) at least 60%, at least 70%, or at least 75% of the codons encoding isoleucine are ATC and the remainder are ATT; and/or e) the codons encoding isoleucine are ATC, except when the next codon begins with a G.

[253] 25. The polynucleotide of any one of aspects 2, 4, or 6-24, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 10, at least 15, at least 20, or at least at least 25 codons encoding valine are replaced by GTG compared to a wild-type Factor IX reference sequence; b) at least 1 codon encoding valine is replaced by GTC compared to a reference wild-type Factor IX sequence; c) at least 80%, at least 90%, or at least 95% of the codons encoding valine are GTG; and/or d) at least 80%, at least 90%, or at least 95% of the codons encoding valine are GTG and the remainder are GTC.

[254] 26. The polynucleotide of any one of aspects 2, 4, or 6-25, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 10, at least 12, or at least 13 fewer serine-encoding codons are replaced by AGC compared to a reference wild-type Factor IX sequence; b) at least 1, at least 2, or at least 4 serine-encoding codons is/are replaced by TCT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 60%, at least 65%, or at least 70% of the codons encoding serine are AGC; and/or d) at least 60%, at least 65%, or at least 70%

of the codons encoding serine are AGC and the rest are TCT or TCC.

[255] 27. The polynucleotide of any one of aspects 2, 4, or 6-26, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 1, at least 2, or at least 5 codons that encode proline is (are) replaced by CCC compared to a reference wild-type Factor IX sequence; b) at least 1 codon encoding proline is replaced by CCT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 50%, at least 55%, or at least 60% of the codons encoding proline are CCC; d) 50%, at least 55%, or at least 60% of the codons encoding proline are CCC and the remainder are CCA or CCT; and/or e) the codons encoding proline are CCC, except when the next codon starts with a G.

[256] 28. The polynucleotide of any one of aspects 2, 4, or 6-27, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 6, at least 7, at least 8, or at least 10 fewer codons encoding threonine are replaced by ACC compared to a reference wild-type Factor IX sequence; b) at least 1, or at least 2, codons encoding threonine is/are replaced by ACT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 45%, at least 50%, or at least 55% of the codons encoding threonine are ACC; d) at least 45%, at least 50%, or at least 55% of the codons encoding threonine are ACC and the remainder are ACT; and/or e) the codons encoding threonine are ACC, except when the next codon starts with a G.

[257] 29. The polynucleotide of any one of aspects 2, 4, or 6-28, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 1, at least 2, at least 3, or at least at least 4 codons encoding alanine is (are) replaced by GCC compared to a reference wild-type Factor IX sequence; b) at least 1, at least 2, or at least 3 codons encoding alanine is/are replaced by GCT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 35%, at least 40%, or at least 43% of the codons encoding alanine are GCC; d) at least 35%, at least 40%, or at least 45% of the codons encoding alanine are GCC and the remainder are GCT; and/or e) the codons encoding alanine are GCC, except when the next codon starts with a G.

[258] 30. The polynucleotide of any one of aspects 2, 4, or 6-29, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 1, or at least 2 tyrosine-encoding codons is( are) substituted by TAC compared to a reference wild-type Factor IX sequence; b) at least 1 TAC codon is replaced by TAT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 40%, at least 45%, or at least 48% of the tyrosine-encoding codons are TAC; d) at least 40%, at least 45%, or at least 48%

of the codons that encode tyrosine are TAC and the rest are TAT; and/or e) the codons encoding tyrosine are TAC, except when the next codon starts with a G.

[259] 31. The polynucleotide of any one of aspects 2, 4, or 6-30, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 1 codon encoding histidine is replaced by CAC compared to a wild-type Factor IX reference sequence; b) at least 50%, at least 60%, or at least 65% of the codons encoding histidine are CAC; c) at least 50%, at least 60%, or at least 65% of the codons encoding histidine are CAC and the remainder are CAT; and/or d) the codons encoding histidine are CAC, except when the next codon begins with a G.

[260] 32. The polynucleotide of any one of aspects 2, 4, or 6-31, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 1, at least 2, at least 4, or at least at least 5 codons encoding glutamine is/are replaced by CAG compared to a reference wild-type Factor IX sequence; b) at least 1 CAG codon is replaced by CAA compared to a reference wild-type Factor IX sequence; c) at least 80%, at least 85%, or at least 90% of the codons encoding glutamine are CAG; and/or d) at least 80%, at least 85%, or at least 90% of the codons encoding glutamine are CAG and the remainder are CAA.

[261] 33. The polynucleotide of any one of aspects 2, 4, or 6-32, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 1, at least 2, at least 4, or at least any less

5 codons encoding aspargine is (are) replaced by AAC compared to a reference wild-type Factor IX sequence; b) at least 60%, at least 65%, or at least 70% of the codons encoding aspargine are AAC; c) at least 60%, at least 65%, or at least 70% of the codons encoding asparagine are AAC and the remainder are AAT; and/or d) the codons encoding asparagine are AAC, except when the next codon begins with a G.

[262] 34. The polynucleotide of any one of aspects 2, 4, or 6-33, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 7, at least 8, or at least 9 fewer codons encoding lysine are replaced by AAG compared to a wild-type Factor IX reference sequence; b) at least 1 AAG codon is replaced by AAA compared to a reference wild-type Factor IX sequence; c) at least 80%, at least 90%, or at least 95% of the codons encoding lysine are AAG; and/or d) at least 80%, at least 90%, or at least 95% of the codons encoding lysine are AAG and the remainder are AAA.

[263] 35. The polynucleotide of any one of aspects 2, 4, or 6-34, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 1, at least 2, at least 3, or at least at least 4 codons encoding aspartate is (are) replaced by GAC compared to a reference wild-type Factor IX sequence; b) at least 1 GAC codon is replaced by GAT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 45%, at least 50%, or at least 60%

of the codons that encode aspartate are GAC; d) at least 45%, at least 50%, or at least 60% of the codons encoding aspartate are GAC and the remainder are GAT; and/or e) the codons that encode aspartate are GAC, except when the next codon starts with a G.

[264] 36. The polynucleotide of any one of aspects 2, 4, or 6-35, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 15, at least 20, at least 25, or at least at least 26 glutamate-encoding codons are replaced by GAG compared to a wild-type Factor IX reference sequence; b) at least 80%, at least 90%, or at least 95% of the codons encoding glutamate are GAG; and/or c) at least 80%, at least 90%, or at least 95% of the codons encoding glutamate are GAG and the remainder are GAA.

[265] 37. The polynucleotide of any one of aspects 2, 4, or 6-36, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 6, at least 7, or at least 8 cysteine-encoding codons are replaced by TGC compared to a reference wild-type Factor IX sequence; b) at least 1 TGC codon is replaced by TGT compared to a wild-type Factor IX reference sequence, where the next codon starts with a G; c) at least 40%, at least 50%, or at least 55% of the cysteine-encoding codons are TGC; d) at least 40%, at least 50%, or at least 55% of the cysteine-encoding codons are TGC and the remainder are TGT; and/or e) the codons encoding cysteine are TGC, except when the next codon starts with a G.

[266] 38. The polynucleotide of any one of aspects 2, 4, or 6-37, wherein, in the portion of the coding sequence that is codon-optimized, the codons encoding tryptophan are TGG.

[267] 39. The polynucleotide of any one of aspects 2, 4, or 6-38, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 8, at least 10, or at least 11 arginine-encoding codons are replaced by AGG compared to a wild-type Factor IX reference sequence; b) at least 1 codon encoding arginine is replaced by AGA compared to a reference wild-type Factor IX sequence; c) at least 60%, at least 70%, or at least 75% of the codons encoding arginine are AGG; and/or d) at least 60%, at least 70%, or at least 75% of the codons encoding arginine are AGG and the remainder are AGA.

[268] 40. The polynucleotide of any one of aspects 2, 4, or 6-39, wherein, in the portion of the coding sequence that is codon-optimized: a) at least 5, at least 10, at least 12, or at least 13 glycine-encoding codons are replaced by GGC compared to a reference wild-type Factor IX sequence; b) at least 5, at least 6, at least 7, or at least 8 glycine-encoding codons are replaced by GGG compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 50%, at least 55%, or at least 60% of the codons encoding glycine are GGC; d) at least 50%, at least 55%, or at least 60% of the codons encoding glycine are GGC and the remainder are GGG; and/or e) the codons encoding glycine are GGC, except when the next codon starts with a G.

[269] 41. The polynucleotide of any one of aspects 2, 4, or 6-40, wherein the portion of the coding sequence that is codon-optimized comprises codons encoding phenylalanine, leucine, isoleucine, valine, serine, proline, threonine , alanine, tyrosine, histidine, glutamine, aspargine, lysine, aspartate, glutamate, cysteine, tryptophan, arginine and glycine.

[270] 42. The polynucleotide of any one of aspects 2, 4, or 6-41, wherein the portion of the coding sequence that is codon-optimized comprises codons encoding phenylalanine, leucine, isoleucine, valine, serine, proline, threonine , alanine, tyrosine, histidine, glutamine, aspargine, lysine, aspartate, glutamate, cysteine, tryptophan, arginine and glycine and in the codon-optimized portion: a) at least 5 codons encoding phenylalanine are replaced by TTC compared to a sequence of the Factor IX wild-type reference; b) at least 16 codons encoding leucine are replaced by CTG compared to a reference wild-type Factor IX sequence; c) at least 12 codons encoding isoleucine are replaced by ATC compared to a reference wild-type Factor IX sequence; d) at least 25 codons encoding valine are replaced by GTG compared to a reference wild-type Factor IX sequence; e) at least 13 serine-encoding codons are replaced by AGC compared to a reference wild-type Factor IX sequence;

f) at least 5 codons encoding proline are replaced by CCC compared to a reference wild-type Factor IX sequence;

g) at least 10 codons encoding threonine are replaced by ACC compared to a reference wild-type Factor IX sequence;

h) at least 4 codons encoding alanine are replaced by GCC compared to a reference wild-type Factor IX sequence;

i) at least 2 tyrosine-encoding codons are replaced by TAC compared to a reference wild-type Factor IX sequence;

j) at least 1 codon encoding histidine is replaced by

CAC compared to a reference wild-type Factor IX sequence;

k) at least 5 glutamine-encoding codons are replaced by CAG compared to a reference wild-type Factor IX sequence;

l) at least 5 codons encoding asparagine are replaced by AAC compared to a reference wild-type Factor IX sequence;

m) at least 9 lysine-encoding codons are replaced by AAG compared to a reference wild-type Factor IX sequence;

n) at least 4 codons encoding aspartate are replaced by GAC compared to a reference wild-type Factor IX sequence;

o) at least 26 glutamate-encoding codons are replaced by GAG compared to a reference wild-type Factor IX sequence; p) at least 8 cysteine-encoding codons are replaced by TGC compared to a reference wild-type Factor IX sequence; q) the codons encoding tryptophan are TGG; r) at least 11 arginine-encoding codons are replaced by AGG compared to a reference wild-type Factor IX sequence; and s) at least 13 glycine-encoding codons are replaced by GGC compared to a reference wild-type Factor IX sequence.

[271] 43. The polynucleotide of any one of aspects 2, 4, or 6-42, wherein the portion of the coding sequence that is codon-optimized comprises codons encoding phenylalanine, leucine, isoleucine, valine, serine, proline, threonine , alanine, tyrosine, histidine, glutamine, aspargine, lysine, aspartate, glutamate, cysteine, tryptophan, arginine and glycine and in the codon-optimized portion: a) at least 70% of the codons encoding phenylalanine are TTC; b) at least 94% of the codons encoding leucine are CTG; c) at least 75% of the codons encoding isoleucine are ATC; d) at least 95% of the codons encoding valine are GTG; e) at least 70% of the codons encoding serine are AGC;

f) at least 60% of the codons encoding proline are CCC; g) at least 55% of the codons encoding threonine are ACC; h) at least 43% of the codons encoding alanine are GCC; i) at least 48% of the tyrosine-encoding codons are TAC; j) at least 65% of the codons encoding histidine are CAC; k) at least 90% of the codons encoding glutamine are CAG; l) at least 70% of the codons that encode asparagine are AAC; m) at least 95% of the codons encoding lysine are AAG; n) at least 60% of the codons that encode aspartate are GAC; o) at least 95% of the codons encoding glutamate are GAG; p) at least 55% of the codons encoding cysteine are TGC; q) the codons encoding tryptophan are TGG; r) at least 75% of the codons encoding arginine are AGG; and s) at least 60% of the glycine encoding codons are GGC.

[272] 44. The polynucleotide of any one of aspects 2, 4, or 6-43, wherein the portion of the coding sequence that is codon-optimized comprises codons encoding phenylalanine, leucine, isoleucine, valine,

serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, lysine,

aspartate, glutamate, cysteine, tryptophan, arginine and glycine and in the codon-

optimized:

a) at least 70% of the codons encoding phenylalanine are

TTC and the rest are TTT;

b) at least 94% of the codons encoding leucine are

CTG and the rest are CTC;

c) at least 75% of the codons encoding isoleucine are

ATC and the rest are ATT;

d) at least 95% of the codons encoding valine are

GTG;

e) at least 70% of the serine encoding codons are

AGC;

f) at least 60% of the codons encoding proline are

CCC and the rest are CCA or CCT;

g) at least 55% of the codons encoding threonine are

ACC and the rest are ACT;

h) at least 43% of the codons encoding alanine are

GCC and the rest are GCT;

i) at least 48% of the tyrosine-encoding codons are

TAC and the rest are TAT;

j) at least 65% of the codons encoding histidine are

CAC and the rest are CAT;

k) at least 90% of the codons encoding glutamine are

CAG and the rest are CAA; l) at least 70% of the codons encoding asparagine are

AAC and the rest are AAT;

m) at least 95% of the codons encoding lysine are AAG and the rest are AAA; n) at least 60% of the codons encoding aspartate are GAC and the rest are GAT; o) at least 95% of the codons encoding glutamate are GAG and the rest are GAA; p) at least 55% of the cysteine-encoding codons are TGC and the rest are TGT; q) the codons encoding tryptophan are TGG; r) at least 75% of the codons encoding arginine are AGG and the rest are AGA; and s) at least 60% of the glycine encoding codons are GGC and the remainder are GGG.

[273] 45. The polynucleotide of any one of aspects 10-44, wherein the reference wild-type Factor IX sequence is from SEQ ID NO: 9 or SEQ ID NO: 19.

[274] 46. The polynucleotide of any one of aspects 2, 4, or 6-45, wherein the portion of the coding sequence that is codon-optimized is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment with at least 800, at least 900, at least 1100, less than 1191, less than 1100, less than 1000, between 800 and 1191, between 900 and 1191, or about 1191 nucleotides of SEQ ID NO:1.

[275] 47. The polynucleotide of aspect 46, wherein the portion of the coding sequence that is codon-optimized is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least minus 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO:

1.

[276] 48. The polynucleotide of aspect 46 or 47, wherein the portion of the coding sequence that is codon-optimized is at least 95% identical to a fragment having between 900 and 1191 nucleotides of SEQ ID NO: 1.

[277] 49. The polynucleotide of any one of aspects 46-48, wherein the portion of the coding sequence that is codon-optimized is at least 95%, or at least 98% identical to SEQ ID NO: 1.

[278] 50. The polynucleotide of any of the foregoing aspects, wherein the coding sequence comprises a portion that is not codon-optimized.

[279] 51. The polynucleotide of aspect 50, wherein the non-codon-optimized portion is at least 100, at least 150, at least 170, at least 190, less than 250, less than 225, less than that 200, or about 195 nucleotides.

[280] 52. The polynucleotide of any of aspects 50 or 51, wherein the portion that is not codon-optimized comprises exon 1 or a portion of at least 60, at least 70, at least 80, between 60 and 88 , between 70 and 88, or between 80 and 88 nucleotides of exon 1.

[281] 53. The polynucleotide of any one of aspects 50-52, wherein the portion that is not codon-optimized comprises a portion of at least 50, at least 75, at least 80, at least 90, at least 100, less than 140, less than 120, between 50 and 140, between 75 and 120, or about 107 nucleotides of exon 2.

[282] 54. The polynucleotide of any of aspects 50-53, wherein the portion that is not codon-optimized comprises CpGs.

[283] 55. The polynucleotide of aspect 54, wherein the portion that is not codon-optimized comprises at least 1 or at least 2 CpGs per 100 nucleotides.

[284] 56. The polynucleotide of any of aspects 50-55, wherein the portion that is not codon-optimized comprises less than 50%, less than 45%, less than 40%, or less than 35 % codons selected from the group consisting of: a) TTC; b) CTG; c) ATC; d) GTG; e) GTC; f) AGC; g) CCC; h) ACC; i) GCC; j) TAC; k) CAC; l) CAG; m) AAC; n) AAA; o) AAG; p) GAC; q) TGC; r) AGG; s) GGC; and t) GAG.

[285] 57. The polynucleotide of any of aspects 50-56, wherein the portion that is not codon-optimized is at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least

99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 100, at least 150, at least 175, less than 195, less than 190, or less than that 180 nucleotides of SEQ ID NO:15.

[286] 58. The polynucleotide of aspect 57, wherein the portion that is not codon-optimized is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99 %, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 15.

[287] 59. The polynucleotide of any of aspects 50-58, wherein the portion that is not codon-optimized is wild type.

[288] 60. The polynucleotide of any of aspects 50-59, wherein the portion that is not codon-optimized is at least 90%, at least 95%, at least 98%, at least 99%, at least 99 .5%, at least 99.8%, or 100% identical to SEQ ID NO: 15.

[289] 61. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide further comprises an intron or a fragment of an intron that interrupts the coding sequence.

[290] 62. The aspect 61 polynucleotide, wherein the intron or fragment of an intron is the wild-type Factor IX portion of the intron.

[291] 63. The polynucleotide of aspect 61 or 62, wherein the fragment of an intron is less than 500, less than 400, less than 350, less than 300, at least 100, at least 200, at least minus 250, at least 290, between 100 and 500, between 200 and 400, between 250 and 350, or about 299 nucleotides.

[292] 64. The polynucleotide of any one of aspects 61-63, wherein the fragment of an intron is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 100, at least 200, at least 250, or at least 290 nucleotides of SEQ ID NO: 3.

[293] 65. The polynucleotide of any of aspects 61-64, wherein the intron or fragment of an intron is at least 85%, at least 90%, at least 95%, at least 98%, at least 99 %, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 3.

[294] 66. The polynucleotide of aspect 65, wherein the intron or fragment of an intron is at least 95%, or at least 98% identical to SEQ ID NO: 3.

[295] 67. The polynucleotide of any of aspects 61-66, wherein the intron or fragment of an intron interrupts the portion that is not codon-optimized.

[296] 68. The aspect 67 polynucleotide, wherein the intron or fragment of an intron is flanked by at least 60, at least 70, at least 80, at least 90, or at least 100 non-codon nucleotides. optimized.

[297] 69. The aspect 68 polynucleotide, wherein the intron or fragment of an intron is flanked by between 110 and 120 nucleotides that are not codon-optimized at the 5' end and between 100 and 110 nucleotides that are not codon- optimized at the 3' end.

[298] 70. The polynucleotide of any of aspects 61-69, wherein the intron or fragment of an intron is positioned between exon 1 and exon 2.

[299] 71. The polynucleotide of any of aspects 61-70, wherein the intron or intron fragment is a fragment of native intron 1 (intron 1a).

[300] 72. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide further comprises a transcriptional regulatory element.

[301] 73. The polynucleotide of aspect 72, wherein the transcriptional regulatory element comprises a liver-specific promoter.

[302] 74. The polynucleotide of aspect 72 or aspect 73, wherein the transcriptional regulatory element comprises an A1AT promoter or a fragment of an A1AT promoter.

[303] 75. The polynucleotide of aspect 74, wherein the fragment of an A1AT promoter is at least 100, at least 120, at least 150, at least 180, less than 255, between 100 and 255, between 150 and 225 , between 150 and 300, or between 180 and 255 nucleotides in length.

[304] 76. The aspect 75 polynucleotide, wherein the fragment of an A1AT promoter is between 150 and 300 nucleotides in length.

[305] 77. The polynucleotide of any of aspects 72-76, wherein the regulatory element of transcription comprises an enhancer.

[306] 78. The polynucleotide of aspect 77, wherein the enhancer is an HCR enhancer or a fragment of an HCR enhancer.

[307] 79. The polynucleotide of aspect 78, wherein the fragment of an enhancer (enhancer) HCR is a fragment of at least 80, at least 90, at least 100, less than 192, between 80 and 192, between 90 and 192, between 100 and 250, or between 117 and 192 nucleotides in length.

[308] 80. The aspect 79 polynucleotide, wherein the fragment of an enhancer (enhancer) HCR is between 100 and 250 nucleotides in length.

[309] 81. The polynucleotide of any one of aspects 72-80, wherein the transcriptional regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least any less

99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO:

6.

[310] 82. The polynucleotide of aspect 81, wherein the transcriptional regulatory element has a sequence of SEQ ID NO:6.

[311] 83. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95% at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 13.

[312] 84. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide comprises an enhancer that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 13.

[313] 85. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide comprises an enhancer of SEQ ID NO: 13.

[314] 86. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99 %, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 14.

[315] 87. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide comprises a promoter that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 14.

[316] 88. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide comprises a promoter of SEQ ID NO:

14.

[317] 89. The polynucleotide of any of the foregoing aspects, wherein the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to codon 384 of wild-type Factor IX and wherein the codon encoding a amino acid at a position corresponding to position 384 of wild-type Factor IX encodes alanine or leucine.

[318] 90. The polynucleotide of aspect 89, where the codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX is CTX, where X is any nucleotide.

[319] 91. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide comprises a Factor IX nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1200, at least 1350, or at least 1650 nucleotides of SEQ ID NO: 5.

[320] 92. The polynucleotide of any of the foregoing aspects, wherein the polynucleotide comprises a Factor IX nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 5.

[321] 93. The polynucleotide of any of the foregoing aspects, wherein: (i) the Factor IX nucleotide sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99 .5%, at least 99.8%, or 100% identical to SEQ ID NO: 1; and (ii) the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX wherein the codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX encodes leucine.

[322] 94. The polynucleotide of any of the foregoing aspects, wherein: (i) the Factor IX nucleotide sequence comprises a coding sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1; (ii) the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX wherein the codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX encodes leucine; and (iii) the polynucleotide comprises a promoter element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 14 and/or a enhancer element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 13.

[323] 95. The polynucleotide of any of the foregoing aspects, wherein: (i) the Factor IX nucleotide sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99 .5%, at least 99.8%, or 100% identical to SEQ ID NO: 1; (ii) the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX wherein the codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX encodes leucine; and

(iii) the polynucleotide comprises a transcription regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 6.

[324] 96. The polynucleotide of any of the foregoing aspects, wherein: (i) the Factor IX nucleotide sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99 .5%, at least 99.8%, or 100% identical to SEQ ID NO: 1; (ii) the Factor IX nucleotide sequence comprises a sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a corresponding portion of SEQ ID NO: 2; and (iii) the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX wherein the codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX encodes leucine.

[325] 97. The polynucleotide of either aspect 95 or 96, wherein the Factor IX nucleotide sequence comprises an intron or a fragment of an intron and the fragment of an intron is at least 98%, at least 99% , at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 3.

[326] 98. The polynucleotide of any of the foregoing aspects, wherein: (i) the Factor IX nucleotide sequence comprises a coding sequence and a portion of the coding sequence is not codon-optimized; and (ii) the Factor IX nucleotide sequence comprises a codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX wherein the codon encoding an amino acid at a position corresponding to position 384 of wild-type Factor IX encodes leucine.

[327] 99. The polynucleotide of any of the foregoing aspects, wherein a polypeptide encoded by a Factor IX nucleotide sequence is expressed in human liver cells at higher levels compared to a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7.

[328] 100. The polynucleotide of any of the foregoing aspects, wherein a polypeptide encoded by a Factor IX nucleotide sequence is expressed in human liver cells at higher levels compared to a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6.

[329] 101. The polynucleotide of any of the foregoing aspects, wherein a polypeptide encoded by the Factor IX nucleotide sequence is expressed in human liver cells at a level at least 2, or at least 3 times greater than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 or SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 7 or SEQ ID NO: 6.

[330] 102. A viral particle comprising a recombinant genome that comprises the polynucleotide of any of the foregoing aspects.

[331] 103. The aspect 102 viral particle, which is an AAV, adenoviral, or lentiviral viral particle.

[332] 104. The aspect 103 viral particle, which is an AAV viral particle.

[333] 105. The viral particle of any one of aspects 102-104, wherein the recombinant genome further comprises: a) AAV2 ITRs; b) a poly A sequence; c) an origin of replication; and/or d) two resolvable ITRs.

[334] 106. The viral particle of aspect 105, wherein the recombinant genome is single-stranded and/or comprises two resolvable ITRs.

[335] 107. The viral particle of any one of aspects 102- 106, wherein the viral particle comprises a capsid selected from the group consisting of: (i) a capsid having at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8% identity or 100% identity to SEQ ID NO: 10; (ii) a capsid having at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity with SEQ ID NO: 17; (iii) AAVMutC; and (iv) AAV5.

[336] 108. The viral particle of any of aspects 102-107, in which on transduction in Huh7 cells, the viral particle expresses the Factor IX protein or a fragment thereof having a greater Factor IX activity than the Factor IX activity. IX expressed from a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID

NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6.

[337] 109. The aspect 108 viral particle, in which activity is measured using a chromogenic substrate that is specific for Factor Xa.

[338] 110. The polynucleotide or viral particle of any of the foregoing aspects, wherein the Factor IX protein fragment is at least 200, at least 250, at least 300, between 200 and 415, between 250 and 415, or between 300 and 415 amino acids in length.

[339] 111. The polynucleotide or viral particle of any of the foregoing aspects, wherein the Factor IX protein or a fragment thereof comprises a sequence: a) at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 8; or b) at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of SEQ ID NO: 8 at least 200, at least minus 250, at least 300, between 200 and 415, between 250 and 415, or between 300 and 415 amino acids in length.

[340] 112. A composition comprising the polynucleotide or viral particle of any of the foregoing aspects and a pharmaceutically acceptable excipient.

[341] 113. The polynucleotide, viral particle, or composition of any of the foregoing aspects for use in a method of treatment.

[342] 114. The polynucleotide, viral particle or composition for use of aspect 113, wherein the method of treatment comprises administering an effective amount of the polynucleotide, viral particle or composition of any one of aspects 1-112 to a patient.

[343] 115. A method of treatment comprising administering an effective amount of the polynucleotide, viral composition or particle of any one of aspects 1-112 to a patient.

[344] 116. The use of the polynucleotide, viral particle, or composition of any of aspects 1-112 in the manufacture of a medicament for use in a method of treatment.

[345] 117. The use of aspect 116, wherein the method of treatment comprises administering an effective amount of the polynucleotide, viral composition or particle of any one of aspects 1-112 to a patient.

[346] 118. The polynucleotide, viral particle, composition, use, or method of any one of aspects 113-117, wherein the method of treatment is a method of treating hemophilia.

[347] 119. The polynucleotide, viral particle, composition, use, or method of aspect 118, where hemophilia is hemophilia B.

[348] 120. The polynucleotide, viral particle, composition, use, or method of aspect 119, where the patient has antibodies or inhibitors to Factor IX.

[349] 121. The polynucleotide, viral particle, or composition of any of aspects 1-112 for use in the expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level of at least 2, or at least 3 times greater than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, thus treating a disease.

[350] 122. The polynucleotide, viral particle or composition of any of aspects 1-112 for use in treating a disease by expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level of at least 2, or at least 3 times larger than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7.

[351] 123. A method of expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level at least 2, or at least 3 times greater than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of the Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 comprising administering the polynucleotide, viral particle or composition of any one of aspects 1-112 to a patient.

[352] 124. A method of treating a disease by expressing a polypeptide encoded by a Factor IX nucleotide sequence at a level at least 2, or at least 3 times greater than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, comprising administering the polynucleotide, viral particle or composition of any one of aspects 1-112 to a patient.

[353] 125. The use of the polynucleotide, viral particle, or composition of any of aspects 1-112 in the expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level of at least 2, or at least 3 times greater than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7, thus treating a disease.

[354] 126. The use of the polynucleotide, viral particle or composition of any of aspects 1-112 in the treatment of a disease by expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level of at least 2, or at least 3 times larger than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7.

[355] 127. The polynucleotide, viral particle, or composition of any of aspects 1-112 for use in expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level of at least 2, or at least 3 times greater than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, thereby treating a disease.

[356] 128. The polynucleotide, viral particle or composition of any of aspects 1-112 for use in treating a disease by expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level of at least 2, or at least 3 times larger than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6.

[357] 129. A method of expressing a polypeptide encoded by the Factor IX nucleotide sequence at a level at least 2, or at least 3 times greater than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of the Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6 comprising the polynucleotide, viral particle or composition of any one of aspects 1-112 for a patient.

[358] 130. A method of treating a disease by expressing a polypeptide encoded by a Factor IX nucleotide sequence at a level at least 2, or at least 3 times greater than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6 comprising administering the polynucleotide, viral particle or composition of any one of aspects 1-112 to a patient.

[359] 131. The use of the polynucleotide, viral particle, or composition of any of aspects 1-112 in the expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level of at least 2, or at least 3 times greater than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, thereby treating a disease.

[360] 132. The use of the polynucleotide, viral particle or composition of any of aspects 1-112 in the treatment of a disease by expression of a polypeptide encoded by the Factor IX nucleotide sequence at a level of at least 2, or at least 3 times larger than a polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6.

[361] 133. The polynucleotide, viral particle or composition for use, or use of any of aspects 121-122, 125-128, or 131-132, wherein the expression of a polypeptide encoded by the nucleotide sequence of Factor IX comprises administering the polynucleotide, viral particle or composition of any one of aspects 1-112 to a patient.

[362] 134. The polynucleotide, viral particle or composition of any of aspects 1-112 for use in the treatment of a disease by expression of the Factor IX protein or a fragment thereof having a greater Factor IX activity than the Factor IX activity. IX expressed from a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6.

[363] 135. A method of treating a disease by expressing a Factor IX protein or a fragment thereof having a greater Factor IX activity than the Factor IX activity expressed from a viral particle comprising a Factor IX nucleotide sequence of SEQ ID NO: 12 and a transcription regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcription regulatory element of SEQ ID NO: 6 , comprising administering an effective amount of the polynucleotide, viral particle or composition of any one of aspects 1-112 to a patient.

[364] 136. The use of the polynucleotide, viral particle, or composition of any of aspects 1-112 in the treatment of a disease by expression of the Factor IX protein or a fragment thereof having a greater Factor IX activity than the Factor IX activity. IX expressed from a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 and/or a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6.

[365] 137. The polynucleotide, viral particle or composition for use, or use in any of aspects 134 or 136, wherein expression of the Factor IX protein or a fragment thereof comprises the administration of the polynucleotide, viral particle or composition of any one of aspects 1-112 for a patient.

[366] 138. The polynucleotide, viral particle or composition for use, method of treatment, or use of any one of aspects 121-122, 124-1228, 130-137, wherein the disease is hemophilia.

[367] 139. The polynucleotide, viral particle or composition for use, method of treatment, or use of aspect 138, wherein the disease is hemophilia B.

[368] 140. The polynucleotide, viral particle, or composition of any of aspects 1-112 for use in achieving a stable Factor IX activity level of at least 20% in a patient with hemophilia B.

[369] 141. A method of achieving a stable Factor IX activity level of at least 20% in a patient with hemophilia B, comprising administering an effective amount of the polynucleotide, viral particle or composition of any of the aspects 1- 112 for a patient.

[370] 142. The use of the polynucleotide, viral particle, or composition of any of aspects 1-112 to achieve a stable Factor IX activity level of at least 20% in a patient with hemophilia B.

[371] 143. The polynucleotide, viral particle or composition of any of aspects 1-112 for use in treating hemophilia B comprising administering the polynucleotide, viral particle or composition to a patient in a dose effective to achieve a level of activity of the Stable factor IX of at least 20% in one patient.

[372] 144. A method of treating hemophilia B comprising administering the polynucleotide, viral particle or composition of any of aspects 1-112 to a patient at a dose effective to achieve a stable Factor IX activity level of at least 20% in one patient.

[373] 145. The use of the polynucleotide, viral particle or composition of any of aspects 1-112 in the treatment of hemophilia B comprising administering the polynucleotide, viral particle or composition to a patient in an effective dose to achieve a level of activity of at least 20% stable Factor IX in one patient.

[374] 146. The polynucleotide, viral particle, or composition for use or use of aspect 140, 142, 143 or 145, wherein treating hemophilia B or achieving a stable Factor IX activity level comprises administering the polynucleotide, viral particle, or composition of any of aspects 1-112 for a patient.

[375] 147. The polynucleotide, viral particle or composition for use, method, or use of any of aspects 141, 144, 146-148, wherein the Factor IX activity level is stable after at least 10 weeks, at least at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks since administration of the polynucleotide, viral particle or composition.

[376] 148. The polynucleotide, viral particle or composition for use, method, or use of any of aspects 123, 124, 129, 130, 133, 135, 137-147, wherein the patient is a patient who is refractory to treatment with Factor IX having an activity lower than the activity of Factor IX expressed from a viral particle comprising (i) a Factor IX nucleotide sequence of SEQ ID NO: 4 or 5 and (ii) a transcription regulatory element of SEQ ID NO:6.

[377] 149. The polynucleotide, viral particle or composition for use, method, or use of any of aspects 123, 124, 129, 130, 133, 135, 137-148, wherein the patient is a patient who is refractory to treatment with a Factor IX polypeptide that is expressed at a level lower than that encoded by a nucleotide sequence comprising (i) a Factor IX nucleotide sequence of SEQ ID NO: 4 or 5 and (ii) a transcription regulatory sequence of SEQ ID NO:6.

[378] 150. The polynucleotide, viral particle or composition for use, method, or use of any of aspects 121-149, wherein the polynucleotide, viral particle or composition is administered in a dose effective to achieve a level of activity of the Stable factor IX of at least 20% in a patient with hemophilia B.

[379] 151. The polynucleotide, viral particle or composition for use, method, or use of any of aspects 121-150, wherein the polynucleotide, viral particle or composition is administered in a dose of at least 4.5 x 1011 vg/kg, or at a dose less than 5 x 10 11 vg/kg, or at a dose between 4.5 x 1011 and 1 x 1012 vg/kg, or at a dose between 4.5 x 1011 vg/kg and 4.9 x 1011 vg/kg.

[380] 152. The polynucleotide, viral particle or composition for use, method, or use of aspect 151, wherein the dose is determined by quantifying the number of vector genomes using qPCR, wherein qPCR optionally uses primers that bind to a promoter region, wherein optionally the promoter region is the promoter region of a transgene cassette.

[381] 153. The polynucleotide, viral particle or composition for use, method, or use of any of aspects 121-152, wherein the polynucleotide or viral particle that is administered is produced from a mammalian cell and/or it has characteristics that result from the use of mammalian viral vector producing cells and are distinguished from vectors produced in insect viral vector producing cells (eg baculovirus system).

[382] 154. The polynucleotide, viral particle or composition for use, method, or use of any of the aspects 140-153 where the stable Factor IX activity level is a stable Factor IX activity level of at least 25 % or at least 30%.

[383] 155. The polynucleotide, viral particle or composition for use, method, or use of any of the aspects 140-154 wherein the stable Factor IX activity level is a stable Factor IX activity level of at least 35 %.

[384] 156. The polynucleotide, viral particle, or composition for use, method, or use of any one of aspects 140-155, wherein the stable Factor IX activity level is at least or about 40%.

[385] 157. The polynucleotide, viral particle, or composition for use, method, or use of any one of aspects 140-156, wherein the Factor IX activity level is at least 20% at a time point of at least at least 50 weeks or about 52 weeks after administration of the polynucleotide, viral particle or composition.

[386] 158. The polynucleotide, viral particle, or composition for use, method, or use of aspect 157, wherein the level of Factor IX activity is maintained at at least 20% for a continuous period of at least 40 weeks immediately previous to the time point.

[387] 159. The polynucleotide, viral particle, or composition for use, method, or use of any one of aspects 140-156, wherein the Factor IX activity level is at least 25% at a time point of at least at least 50 weeks or about 52 weeks after administration of the polynucleotide, viral particle or composition.

[388] 160. The polynucleotide, viral particle, or composition for use, method, or use of aspect 159, wherein the level of Factor IX activity is maintained at least 25% for a continuous period of at least 40 weeks immediately previous to the time point.

[389] 161. The polynucleotide, viral particle, or composition for use, method, or use of any one of aspects 140-156, wherein the Factor IX activity is at least or about 40% at one time point at least 50 weeks or about 52 weeks after administration of the polynucleotide, viral particle or composition.

[390] 162. The polynucleotide, viral particle, or composition for use, method, or use of aspect 161, wherein the level of Factor IX activity is maintained at at least 40% for a continuous period of at least 40 weeks immediately previous to the time point.

[391] 163. The polynucleotide, viral particle or composition for use, method, or use of any of the aspects 140-162, wherein Factor IX activity is measured using a clotting assay to determine Factor IX activity in a blood sample from the patient, optionally where the clotting assay uses a SynthASil® kit.

[392] 164. The polynucleotide, viral particle or composition for use, method, or use of any of the aspects 140-162, in which Factor IX activity is measured by taking a blood sample from the patient and using a clotting assay to determine Factor IX activity in the blood sample, where, optionally, the clotting assay utilizes a SynthASil® kit.

[393] 165. The polynucleotide, viral particle or composition for the use, method, or use of aspect 163 or 164, wherein the clotting assay is a one-stage clotting assay, wherein, optionally, the clotting assay of one stage uses a SynthASil® kit.

[394] 166. The polynucleotide, viral particle or composition for use, method, or use of any of aspects 113-165, wherein the method or use comprises co-administration of a steroid ee.

[395] 167. The polynucleotide, viral particle, or composition for use, method, or use of aspect 166, wherein the steroid is prednisolone.

Claims (27)

1. POLYNUCLEOTIDE comprising a Factor IX nucleotide sequence, characterized: (i) in that the Factor IX nucleotide sequence comprises a coding sequence encoding a Factor IX protein or a fragment thereof; (ii) that the portion of the coding sequence is codon-optimized; (iii) that the portion of the coding sequence that is codon-optimized is at least 1100 nucleotides in length; (iv) by the portion that is codon-optimized, at least 60% of the codons are selected from the group consisting of TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC and GAG; (v) that the portion that is codon-optimized comprises a reduced number of CpGs as compared to a corresponding portion of SEQ ID NO:9; (vi) by the polynucleotide further comprising a transcriptional regulatory element comprising: (a) an A1AT promoter or a fragment of an A1AT promoter; and/or (b) an HCR enhancer or a fragment of an HCR enhancer; and (vii) by the Factor IX nucleotide sequence comprising a codon encoding leucine at a position corresponding to position 384 of wild-type Factor IX. 2. POLYNUCLEOTIDE, according to the preceding claim, characterized in that the portion of the coding sequence that is codon-optimized is codon-optimized for expression in the human liver. 3. POLYNUCLEOTIDE, according to any one of the preceding claims, characterized in: (i) a polypeptide encoded by the Factor IX nucleotide sequence is expressed in human liver cells at higher levels when compared to a reference wild-type Factor IX nucleotide sequence; and/or (ii) by the portion of the coding sequence that is codon- optimized to have at least 800, at least 900, at least 1100, less than 1500, less than 1300, less than 1200, between 800 and 1500, between 900 and 1300, between 1100 and 1200, or about 1191 nucleotides in length; and/or (iii) by the portion of the coding sequence that is codon- optimized understand 1, 2, 3, 4, 5 or all of: a) exon 3 or a portion of at least 10, at least 15, at least 20, less than 25, between 10 and 25, between 15 and 25, or between 20 and 25 nucleotides from exon 3; b) exon 4 or a portion of at least 80, at least 90, at least 100, less than 114, between 80 and 114, between 90 and 114, or between 100 and 114 nucleotides of exon 4; c) exon 5 or a portion of at least 90, at least 100, at least 110, less than 129, between 90 and 129, between 100 and 129, or between 110 and 129 nucleotides of exon 5; d) exon 6 or a portion of at least 150, at least 180, at least 200, less than 203, between 150 and 203, between 180 and 203, or between 200 and 203 nucleotides of exon 6; e) exon 7 or a portion of at least 70, at least 80, at least 90, at least 100, less than 115, between 70 and 115, between 80 and 115, between 90 and 115, or between 100 and 115 nucleotides of exon 7; and/or f) exon 8 or a portion of at least 400, at least 450, at least 500, less than 548, between 400 and 548, between 450 and 548, or between 500 and 548 nucleotides of exon 8; and/or (iv) that the portion of the coding sequence that is codon-optimized comprises a portion of at least 20 nucleotides of exon 3, a portion of at least 100 nucleotides of exon 4, a portion of at least 110 nucleotides of exon 5, a portion of at least 180 nucleotides of exon 6, a portion of at least 100 nucleotides of exon 7 and a portion of at least 500 nucleotides of exon 8; and/or (v) by the portion of the coding sequence that is codon-optimized comprising exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8; and/or (vi) that the portion of the coding sequence that is codon-optimized comprises a portion of exon 2 and the portion of exon 2 is less than 160, less than 150, less than 100, less than 75, less than 60, at least 20, at least 30, at least 40, at least 50, between 20 and 160, between 30 and 150, between 30 and 100, between 40 and 75, or about 56 nucleotides in length; and/or (vii) that the portion of the coding sequence that is codon-optimized comprises a portion of exon 2 that is between 30 and 100 nucleotides in length. 4. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in: (i) that the portion of the coding sequence which is codon-optimized comprises less than less than 40, less than 20, less than 18, less than 10, less than 5, or less than 1 CpG; and/or (ii) by the portion of the coding sequence which is codon- optimized to be CpG-free. 5. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in that the portion of the coding sequence that is codon-optimized: (i) a) at least 1, at least 2, at least 4, or at least 5 codons encoding phenylalanine is(are) replaced by TTC compared to a reference wild-type Factor IX sequence; b) at least 60%, at least 65%, or at least 70% of the codons encoding phenylalanine are TTC; c) at least 60%, at least 65%, or at least 70% of the codons encoding phenylalanine are TTC and the remainder are TTT; and/or d) the codons encoding phenylalanine are TTC, except when the next codon starts with a G; and/or (ii) a) at least 5, at least 10, at least 15, or at least 16 codons encoding leucine are replaced by CTG compared to a reference wild-type Factor IX sequence; b) at least 90%, or at least 94% of the codons encoding leucine are CTG; and/or c) at least 90%, or at least 94% of the codons encoding leucine are CTG and the remainder are CTC; and/or (iii) a) at least 5, at least 10, at least 11, or at least 12 codons encoding isoleucine are replaced by ATC compared to a reference wild-type Factor IX sequence; b) at least 1 ATC codon is replaced by ATT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 60%, at least 70%, or at least 75% of the codons encoding isoleucine are ATC; d) at least 60%, at least 70%, or at least 75% of the codons encoding isoleucine are ATC and the rest are ATT; and/or e) the codons encoding isoleucine are ATC, except when the next codon begins with a G; and/or (iv) a) at least 10, at least 15, at least 20, or at least 25 codons encoding valine are replaced by GTG compared to a reference wild-type Factor IX sequence; b) at least 1 codon encoding valine is replaced by GTC compared to a wild-type Factor IX reference sequence; c) at least 80%, at least 90%, or at least 95% of the codons that encode valine are GTG; and/or d) at least 80%, at least 90%, or at least 95% of the codons encoding valine are GTG and the rest are GTC; and/or (v) a) at least 5, at least 10, at least 12, or at least 13 serine encoding codons are replaced by AGC compared to a reference wild-type Factor IX sequence; b) at least 1, at least 2, or at least 4 serine-encoding codons is/are replaced by TCT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 60%, at least 65%, or at least 70% of the codons that encode serine are AGC; and/or d) at least 60%, at least 65%, or at least 70% of the codons encoding serine are AGC and the rest are TCT or TCC; and/ or (saw) a) at least 1, at least 2, or at least 5 codons encoding proline is/are replaced by CCC compared to a reference wild-type Factor IX sequence; b) at least 1 codon encoding proline is replaced by CCT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 50%, at least 55%, or at least 60% of the codons that encode proline are CCC; d) in 50%, at least 55%, or at least 60% of the codons encoding proline are CCC and the remainder are CCA or CCT; e/or e) the codons encoding proline are CCC, except when the next codon starts with a G; and/or (vii) a) at least 6, at least 7, at least 8, or at least 10 codons encoding threonine are replaced by ACC compared to a reference wild-type Factor IX sequence; b) at least 1, or at least 2, codons encoding threonine is(are) replaced by ACT compared to a sequence of the Wild-type reference factor IX, where the next codon starts with a G; c) at least 45%, at least 50%, or at least 55% of the codons that encode threonine are ACC; d) at least 45%, at least 50%, or at least 55% of the codons that encode threonine are ACC and the rest are ACT; e/or e) the codons encoding threonine are ACC, except when the next codon starts with a G; and/or (viii) a) at least 1, at least 2, at least 3, or at least 4 codons encoding alanine is (are) replaced by GCC compared to a reference wild-type Factor IX sequence; b) at least 1, at least 2, or at least 3 codons encoding alanine is/are replaced by GCT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 35%, at least 40%, or at least 43% of the codons encoding alanine are GCC; d) at least 35%, at least 40%, or at least 45% of the codons encoding alanine are GCC and the rest are GCT; e/or e) the codons encoding alanine are GCC, except when the next codon starts with a G; and/or (ix) a) at least 1, or at least 2 tyrosine-encoding codons is (are) replaced by TAC compared to a sequence of the Factor IX wild-type reference; b) at least 1 TAC codon is replaced by TAT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 40%, at least 45%, or at least 48% of the codons that encode tyrosine are TAC; d) at least 40%, at least 45%, or at least 48% of the codons encoding tyrosine are TAC and the remainder are TAT; and/or e) the codons encoding tyrosine are TAC, except when the next codon starts with a G; and/or (x) a) at least 1 codon encoding histidine is replaced by CAC compared to a reference wild-type Factor IX sequence; b) at least 50%, at least 60%, or at least 65% of the codons encoding histidine are CAC; c) at least 50%, at least 60%, or at least 65% of the codons encoding histidine are CAC and the rest are CAT; and/or d) the codons encoding histidine are CAC, except when the next codon begins with a G; and/or (xi) a) at least 1, at least 2, at least 4, or at least 5 codons encoding glutamine is(are) replaced by CAG compared to a wild-type Factor IX reference sequence; b) at least 1 CAG codon is replaced by CAA compared to a reference wild-type Factor IX sequence; c) at least 80%, at least 85%, or at least 90% of the codons encoding glutamine are CAG; and/or d) at least 80%, at least 85%, or at least 90% of the codons encoding glutamine are CAG and the rest are CAA; and/or (xii) a) at least 1, at least 2, at least 4, or at least 5 codons encoding aspargine is (are) replaced by AAC compared to a reference wild-type Factor IX sequence; b) at least 60%, at least 65%, or at least 70% of the codons encoding aspargine are AAC; c) at least 60%, at least 65%, or at least 70% of the codons that encode asparagine are AAC and the rest are AAT; e/or e) the codons encoding asparagine are AAC, except when the next codon begins with a G; and/or (xiii) a) at least 5, at least 7, at least 8, or at least 9 codons encoding lysine are replaced by AAG compared to a reference wild-type Factor IX sequence; b) at least 1 AAG codon is replaced by AAA compared to a reference wild-type Factor IX sequence; c) at least 80%, at least 90%, or at least 95% of the codons that encode lysine are AAG; and/or d) at least 80%, at least 90%, or at least 95% of the codons that encode lysine are AAG and the rest are AAA; and/or (xiv) a) at least 1, at least 2, at least 3, or at least 4 codons encoding aspartate is (are) replaced by GAC compared to a reference wild-type Factor IX sequence; b) at least 1 GAC codon is replaced by GAT compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 45%, at least 50%, or at least 60% of the codons that encode aspartate are GAC; d) at least 45%, at least 50%, or at least 60% of the codons that encode aspartate are GAC and the rest are GAT; e/or e) the codons encoding aspartate are GAC, except when the next codon begins with a G; and/or (xv) a) at least 15, at least 20, at least 25, or at least 26 glutamate-encoding codons are replaced by GAG compared to a reference wild-type Factor IX sequence; b) at least 80%, at least 90%, or at least 95% of the codons that encode glutamate are GAG; and/or c) at least 80%, at least 90%, or at least 95% of the codons encoding glutamate are GAG and the rest are GAA; and/or (xvi) a) at least 5, at least 6, at least 7, or at least 8 codons encoding cysteine are replaced by TGC compared to a reference wild-type Factor IX sequence; b) at least 1 TGC codon is replaced by TGT compared to a wild-type Factor IX reference sequence, where the next codon starts with a G; c) at least 40%, at least 50%, or at least 55% of the codons that encode cysteine are TGC; d) at least 40%, at least 50%, or at least 55% of the codons that encode cysteine are TGC and the rest are TGT; e/or e) the codons encoding cysteine are TGC, except when the next codon starts with a G; and/or (xvii) the codons encoding tryptophan are TGG; and/or (xviii) a) at least 5, at least 8, at least 10, or at least 11 arginine encoding codons are replaced by AGG compared to a reference wild-type Factor IX sequence; b) at least 1 codon encoding arginine is replaced by AGA compared to a wild-type Factor IX reference sequence; c) at least 60%, at least 70%, or at least 75% of the codons that encode arginine are AGG; and/or d) at least 60%, at least 70%, or at least 75% of the codons encoding arginine are AGG and the remainder are AGA; and/or (xix) a) at least 5, at least 10, at least 12, or at least 13 codons encoding glycine are replaced by GGC compared to a reference wild-type Factor IX sequence; b) at least 5, at least 6, at least 7, or at least 8 glycine-encoding codons are replaced by GGG compared to a reference wild-type Factor IX sequence, where the next codon starts with a G; c) at least 50%, at least 55%, or at least 60% of the codons encoding glycine are GGC; d) at least 50%, at least 55%, or at least 60% of the codons encoding glycine are GGC and the remainder are GGG; and/or e) the codons encoding glycine are GGC, except when the next codon starts with a G. 6. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in that the portion of the coding sequence that is codon-optimized comprises codons that encode phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, aspargine, lysine, aspartate, glutamate, cysteine, tryptophan, arginine and glycine and in the codon-optimized portion: a) at least 5 codons encoding phenylalanine are replaced by TTC compared to a wild-type Factor IX reference sequence; b) at least 16 codons encoding leucine are replaced by CTG compared to a reference wild-type Factor IX sequence; c) at least 12 codons encoding isoleucine are replaced by ATC compared to a reference wild-type Factor IX sequence; d) at least 25 codons encoding valine are replaced by GTG compared to a reference wild-type Factor IX sequence; e) at least 13 serine-encoding codons are replaced by AGC compared to a reference wild-type Factor IX sequence; f) at least 5 codons encoding proline are replaced by CCC compared to a reference wild-type Factor IX sequence; g) at least 10 codons encoding threonine are replaced by ACC compared to a reference wild-type Factor IX sequence; h) at least 4 codons encoding alanine are replaced by GCC compared to a reference wild-type Factor IX sequence; i) at least 2 tyrosine-encoding codons are replaced by TAC compared to a reference wild-type Factor IX sequence; j) at least 1 codon encoding histidine is replaced by CAC compared to a reference wild-type Factor IX sequence; k) at least 5 glutamine-encoding codons are replaced by CAG compared to a reference wild-type Factor IX sequence; l) at least 5 codons encoding asparagine are replaced by AAC compared to a reference wild-type Factor IX sequence; m) at least 9 lysine-encoding codons are replaced by AAG compared to a reference wild-type Factor IX sequence; n) at least 4 codons encoding aspartate are replaced by GAC compared to a reference wild-type Factor IX sequence; o) at least 26 glutamate-encoding codons are replaced by GAG compared to a reference wild-type Factor IX sequence; p) at least 8 cysteine-encoding codons are replaced by TGC compared to a reference wild-type Factor IX sequence; q) the codons encoding tryptophan are TGG; r) at least 11 arginine-encoding codons are replaced by AGG compared to a reference wild-type Factor IX sequence; and s) at least 13 glycine-encoding codons are replaced by GGC compared to a reference wild-type Factor IX sequence. 7. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in that the portion of the coding sequence that is codon-optimized comprises codons that encode phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, aspargine, lysine, aspartate, glutamate, cysteine, tryptophan, arginine and glycine and in the codon-optimized portion: a) at least 70% of the codons encoding phenylalanine are TTC; b) at least 94% of the codons encoding leucine are CTG; c) at least 75% of the codons encoding isoleucine are ATC; d) at least 95% of the codons encoding valine are GTG; e) at least 70% of the serine encoding codons are AGC; f) at least 60% of the codons encoding proline are CCC; g) at least 55% of the codons encoding threonine are ACC; h) at least 43% of the codons encoding alanine are GCC; i) at least 48% of the tyrosine-encoding codons are TAC; j) at least 65% of the codons encoding histidine are CAC; k) at least 90% of the codons encoding glutamine are CAG; l) at least 70% of the codons encoding asparagine are AAC; m) at least 95% of the codons encoding lysine are AAG; n) at least 60% of the codons that encode aspartate are GAC; o) at least 95% of the codons encoding glutamate are GAG; p) at least 55% of the codons encoding cysteine are TGC; q) the codons encoding tryptophan are TGG; r) at least 75% of the codons encoding arginine are AGG; and s) at least 60% of the glycine encoding codons are GGC. 8. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in that the portion of the coding sequence that is codon-optimized comprises codons that encode phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, aspargine, lysine, aspartate, glutamate, cysteine, tryptophan, arginine and glycine and in the codon-optimized portion: a) at least 70% of the codons encoding phenylalanine are TTC and the rest are TTT; b) at least 94% of the codons encoding leucine are CTG and the remainder are CTC; c) at least 75% of the codons encoding isoleucine are ATC and the remainder are ATT; d) at least 95% of the codons encoding valine are GTG; e) at least 70% of the codons encoding serine are AGC; f) at least 60% of the codons encoding proline are CCC and the rest are CCA or CCT; g) at least 55% of the codons encoding threonine are ACC and the remainder are ACT; h) at least 43% of the codons encoding alanine are GCC and the remainder are GCT; i) at least 48% of the tyrosine-encoding codons are TAC and the remainder are TAT; j) at least 65% of the codons encoding histidine are CAC and the remainder are CAT; k) at least 90% of the codons encoding glutamine are CAG and the rest are CAA; l) at least 70% of the codons encoding asparagine are AAC and the rest are AAT; m) at least 95% of the codons encoding lysine are AAG and the rest are AAA; n) at least 60% of the codons encoding aspartate are GAC and the rest are GAT; o) at least 95% of the codons encoding glutamate are GAG and the rest are GAA; p) at least 55% of the cysteine-encoding codons are TGC and the rest are TGT; q) the codons encoding tryptophan are TGG; r) at least 75% of the codons encoding arginine are AGG and the rest are AGA; and s) at least 60% of the glycine encoding codons are GGC and the remainder are GGG. A POLYNUCLEOTIDE according to any one of claims 4 to 8, characterized in that the reference wild type Factor IX sequence is SEQ ID NO: 9 or SEQ ID NO: 19. 10. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in that the transcriptional regulatory element comprises a liver-specific promoter. 11. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in that the fragment of an A1AT promoter: (i) has at least 100, at least 120, at least 150, at least 180, less than 255, between 100 and 255 , between 150 and 225, between 150 and 300, or between 180 and 255 nucleotides in length; and/or (ii) be between 150 and 300 nucleotides in length. 12. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in that the fragment of an HCR enhancer: (i) has a fragment of at least 80, at least 90, at least 100, less than 192, between 80 and 192, between 90 and 192, between 100 and 250, or between 117 and 192 nucleotides in length; and/or (ii) be between 100 and 250 nucleotides in length. 13. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in: (i) that the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 6; and/or (ii) by the transcriptional regulatory element having a sequence of SEQ ID NO: 6. 14. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in: (i) that the polynucleotide comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95% at least 98 %, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 13; and/or (ii) the polynucleotide comprises an enhancer that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 13; and/or (iii) the polynucleotide comprises an enhancer of SEQ ID NO: 13; and/or (iv) by the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least minus 99.8%, or 100% identical to SEQ ID NO: 14; and/or (v) the polynucleotide comprises a promoter that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 14; and/or (vi) that the polynucleotide comprises a promoter of SEQ ID NO:14. 15. POLYNUCLEOTIDE according to any one of the preceding claims, characterized in that the codon that encodes an amino acid in a position corresponding to position 384 of the wild-type Factor IX is CTX, where X is any nucleotide, where, optionally, X is C or G. 16. POLYNUCLEOTIDE comprising a Factor IX nucleotide sequence, characterized in that the Factor IX nucleotide sequence comprises a coding sequence encoding a Factor IX protein or a fragment thereof and wherein a portion of the coding sequence is non-wild type. 17. VIRAL PARTICLE, characterized in that it comprises a recombinant genome comprising the polynucleotide according to any one of the preceding claims. 18. VIRAL PARTICLE, according to claim 17, characterized in: (i) being an AAV, adenoviral or lentiviral viral particle; and/or (ii) being an AAV viral particle; and/or (iii) the recombinant genome further comprises: a) AAV2 ITRs; b) a poly A sequence; c) an origin of replication; and/or d) two resolvable ITRs; and/or (iv) by the recombinant genome being single-stranded. 19. VIRAL PARTICLE, according to claim 17 or 18, characterized in that in transduction in Huh7 cells the viral particle expresses the Factor IX protein or a fragment thereof having a Factor IX activity greater than the Factor IX activity expressed from of a viral particle comprising a nucleotide sequence of Factor IX of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7. 20. POLYNUCLEOTIDE or viral particle, according to any one of the preceding claims, characterized in that the Factor IX fragment has at least 200, at least 250, at least 300, between 200 and 415, between 250 and 415, or between 300 and 415 long amino acids. 21. COMPOSITION, characterized in that it comprises the polynucleotide or viral particle according to any one of the preceding claims and a pharmaceutically acceptable excipient. 22. POLYNUCLEOTIDE, viral particle or composition, according to any one of the preceding claims, characterized in that it is for use in a method of treatment. 23. POLYNUCLEOTIDE, viral particle or composition for use according to claim 22, characterized in that the method of treatment comprises administering an effective amount of the polynucleotide, viral particle or composition according to any one of claims 1 to 21 to a patient. 24. POLYNUCLEOTIDE, viral particle or composition for use according to claim 22 or 23, characterized in: (i) the method of treatment is a method of treating hemophilia; and/or (ii) the method of treatment is a method of treating hemophilia B; and/or (iii) the patient has antibodies or inhibitors to Factor IX. 25. POLYNUCLEOTIDE, viral particle or composition, according to any one of claims 1 to 21 for use in obtaining a stable Factor IX activity level of at least 20% in a patient with hemophilia B, in which optionally obtaining a stable Factor IX activity level comprises administering the polynucleotide, viral particle or composition according to any one of claims 1 to 21 to the patient. 26. POLYNUCLEOTIDE, viral particle or composition, according to any one of claims 1 to 21, characterized in that it is for use in the treatment of hemophilia B, characterized in that it comprises the administration of the polynucleotide, viral particle or composition to a patient in one dose effective in achieving a stable Factor IX activity level of at least 20% in a patient. 27. POLYNUCLEOTIDE, viral particle or composition for use, according to claim 25 or 26, characterized in: (a) the activity level of Factor IX is stable after at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks since administration of the polynucleotide, viral particle or composition; (b) by the patient being a patient who is refractory to treatment with Factor IX having an activity lower than the activity of the Factor IX expressed from a viral particle comprising (i) a nucleotide sequence of Factor IX of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory element of SEQ ID NO: 6; (c) by the patient being a patient who is refractory to treatment with a Factor IX polypeptide that is expressed at a level lower than that encoded by a nucleotide sequence comprising (i) a nucleotide sequence of Factor IX of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory sequence of SEQ ID NO: 6; (d) the polynucleotide, viral particle or composition is administered in an effective dose to achieve a level of activity of the Stable factor IX of at least 20% in a patient with hemophilia B; (e) because the polynucleotide, viral particle or composition is administered at a dose of at least 4.5 x 1011 vg/kg, or at a dose less than 5 x 1011 vg/kg, or at a dose between 4, 5 x 1011 and 1 x 1012 vg/kg, or at a dose between 4.5 x 1011 vg/kg and 4.9 x 1011 vg/kg, in which, optionally, the dose is determined by quantifying the number of vector genomes using qPCR, wherein, optionally, qPCR uses primers that bind to a promoter region, wherein, optionally, the promoter region is the promoter region of a cassette. transgene; (f) the polynucleotide or viral particle that is administered is produced from a mammalian cell and/or has characteristics that result from the use of mammalian viral vector producing cells and are distinguished from vectors produced in cells production of insect viral vectors (eg baculovirus system); (g) where the stable Factor IX activity level is a stable Factor IX activity level of at least 25% or at least 30%, where, optionally, the stable Factor IX activity level is an activity level Factor IX stable at least 35%, where, optionally, the activity level of Factor IX stable is at least or about 40%; (h) by the activity level of Factor IX being at least 20% at a time point of at least 50 weeks or about 52 weeks after administration of the polynucleotide, viral particle or composition, where, optionally, the Factor IX activity level is maintained at at least 20% for a continuous period of at least 40 weeks immediately prior to the time point; (i) by the activity level of Factor IX being at least 25% at a time point of at least 50 weeks or about 52 weeks after administration of the polynucleotide, viral particle or composition, where, optionally, the Factor IX activity level is maintained at at least 25% for a continuous period of at least 40 weeks immediately prior to the time point; (j) because the activity of Factor IX is at least or about 40% at a time point of at least 50 weeks or about 52 weeks after administration of the polynucleotide, viral particle or composition, where, optionally, the Factor IX activity level is maintained at at least 40% for a period continuous for at least 40 weeks immediately preceding the time point; (k) Factor IX activity is measured using a clotting assay to determine Factor IX activity in a blood sample from the patient, where, optionally, the clotting assay utilizes a SynthASil® kit; (l) Factor IX activity is measured by collecting a blood sample from the patient and using a clotting assay to determine Factor IX activity in the blood sample, wherein, optionally, the clotting assay uses a SynthASil ® kit; (m) in that the clotting assay is a one-stage clotting assay, wherein, optionally, the one-stage clotting assay utilizes a SynthASil® kit; and/or (n) the use comprises co-administration of a steroid and, optionally, the steroid is prednisolone.