KR20210049129A - Nucleotides encoding factor IX - Google Patents

Abstract

The present invention relates to a polynucleotide comprising a factor IX nucleotide sequence, wherein the factor IX nucleotide sequence includes a coding sequence encoding a factor IX protein or fragment thereof, and some of the coding sequences are not wild-type. The present invention further relates to a viral particle comprising a recombinant genome comprising a polynucleotide of the present invention, a composition comprising the polynucleotide or viral particle, and a method and use of the polynucleotide, viral particle or composition.

Description

Nucleotides encoding factor IX

The present invention relates to a polynucleotide comprising a nucleotide sequence encoding factor IX, a viral particle comprising the polynucleotide, and a treatment using the polynucleotide.

Hemophilia type B, an X-linked life threatening bleeding disorder, affects men at 1:30,000. Current treatments include frequent intravenous injections of factor IX (FIX) protein (2-3 times per week). This treatment is very effective in controlling bleeding, but it is ineffective and very expensive (150,000 pounds per patient per year per year), which makes it unacceptable to most patients with hemophilia type B worldwide. Gene therapy for hemophilia type B offers the possibility of treatment by delivering a functional copy of the factor IX gene to the patient, allowing continued endogenous production of factor IX.

The present application relates to a gene therapy approach for treating hemophilia type B comprising administering a vector comprising a polynucleotide encoding factor IX. This gene therapy approach will avoid the need for frequent intravenous injections of factor IX. However, it is difficult to provide effective gene therapy vectors, i.e., vectors that allow high levels of factor IX expression and expression of highly active factor IX.

This application demonstrates that various modifications to a polynucleotide including 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 it can improve the efficacy of a polynucleotide comprising a factor IX nucleotide sequence for the treatment of hemophilia B in the following:

· Use of codon-optimized sequences;

· Site maintenance of the factor IX polypeptide that is not codon optimized;

Including introns or fragments of introns;

Providing sequences flanking introns or fragments of introns that are not codon optimized;

· Acquisition use of functional mutations;

· Use of specific promoters; And/or

· Maintaining the AAV genome containing nucleotides in single-stranded form.

These modifications provide a highly expressed, highly active Factor IX polypeptide or a Factor IX sequence encoding a fragment thereof. As demonstrated in the examples, the polynucleotides of the invention express and provide overall Factor IX activity at higher levels than other factor IX coding polynucleotides, for example those disclosed in WO16/075473.

Accordingly, according to the first aspect of the present invention, 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 the coding sequence The site of is not wild type.

According to a second aspect of the present invention, 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 fragment thereof, and the coding sequence is Includes: (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 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 15.

According to a third aspect of the present invention, the present invention provides a polynucleotide comprising a factor IX nucleotide sequence, wherein the factor IX nucleotide sequence encodes a factor IX protein or fragment thereof, and SEQ ID NO: 5 and at least 97%, 98 % Or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identity.

According to a fourth aspect of the present invention, the present invention provides a viral particle comprising a recombinant genome comprising the polynucleotide of the present invention.

According to a fifth aspect of the present invention, the present invention provides a composition comprising the polynucleotide or viral particles of the present invention and a pharmaceutically acceptable excipient.

According to a sixth aspect of the present invention, the present invention provides a method of treatment comprising administering to a patient an effective amount of a polynucleotide or viral particle of the present invention.

According to a seventh aspect of the invention, the invention provides the use of a polynucleotide, viral particle or composition of the invention in the manufacture of a medicament for use in a method of treatment.

According to the eighth aspect of the present invention, the polynucleotide, viral particle or composition of the present invention is twice the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7 It is intended for use in expressing the polypeptide encoded by the factor IX nucleotide sequence at an abnormal, or at least three-fold higher level, and thus treating a disease.

According to the ninth aspect of the present invention, the polynucleotide, viral particle or composition of the present invention is 2 than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7 It is intended for use in treating diseases by expressing the polypeptide encoded by the factor IX nucleotide sequence at a level of at least a fold, or at least three fold higher.

According to a tenth aspect of the present invention, the present invention comprises a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 comprising administering to a patient a polynucleotide, viral particle or composition of the present invention. It provides a method of expressing a polypeptide encoded by a factor IX nucleotide sequence at a level that is 2 times or more, or 3 times or more higher than the polypeptide encoded by the nucleotide sequence.

According to an eleventh aspect of the present invention, the present invention comprises a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 comprising administering to a patient a polynucleotide, viral particle or composition of the present invention. It provides a method of treating a disease by expressing a polypeptide encoded by a factor IX nucleotide sequence at a level that is 2 times or more, or 3 times or more than the polypeptide encoded by the nucleotide sequence.

According to the twelfth aspect of the present invention, the present invention is 2 times or more, or 3 times or more higher than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7 At a level, there is provided a use of a polynucleotide, viral particle or composition of the present invention, which expresses the polypeptide encoded by the factor IX nucleotide sequence and thus treats a disease.

According to the thirteenth aspect of the present invention, the present invention is 2 times or more, or 3 times or more higher than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7 At a level, there is provided a use of a polynucleotide, viral particle or composition of the present invention to treat a disease by expressing the polypeptide encoded by the factor IX nucleotide sequence.

According to the fourteenth aspect of the present invention, the polynucleotide, viral particle or composition of the present invention is twice the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6 It is intended for use in expressing the polypeptide encoded by the factor IX nucleotide sequence at an abnormal, or at least three-fold higher level, and thus treating a disease.

According to the fifteenth aspect of the present invention, the polynucleotide, viral particle or composition of the present invention is twice the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6 It is intended for use in the treatment of diseases by expressing the polypeptide encoded by the factor IX nucleotide sequence at an abnormal, or at least three-fold higher level.

According to a sixteenth aspect of the present invention, the present invention comprises a factor IX nucleotide sequence of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6, comprising administering a polynucleotide, viral particle or composition of the present invention to a patient. It provides a method of expressing a polypeptide encoded by a factor IX nucleotide sequence at a level that is 2 times or more, or 3 times or more higher than the polypeptide encoded by the nucleotide sequence.

According to the seventeenth aspect of the present invention, the present invention comprises the step of administering to a patient a polynucleotide, viral particle or composition of the present invention, comprising a factor IX nucleotide sequence of SEQ ID NO: 18 and a transcriptional control element of SEQ ID NO: 6 It provides a method of treating a disease by expressing a polypeptide encoded by a factor IX nucleotide sequence at a level that is 2 times or more, or 3 times or more than the polypeptide encoded by the nucleotide sequence.

According to the eighteenth aspect of the present invention, the present invention is 2 times or more, or 3 times or more higher than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6 At a level, there is provided a use of a polynucleotide, viral particle or composition of the present invention, which expresses the polypeptide encoded by the factor IX nucleotide sequence and thus treats a disease.

According to the 19th aspect of the present invention, the present invention is 2 times or more, or 3 times or more higher than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6 At a level, there is provided a use of a polynucleotide, viral particle or composition of the present invention to treat a disease by expressing the polypeptide encoded by the factor IX nucleotide sequence.

According to the twentieth aspect of the present invention, the polynucleotide, viral particle or composition of the present invention comprises a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional control element of SEQ ID NO: 7 and/or a factor IX nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: It is for use in treating a disease by expressing a factor IX protein or fragment thereof having a factor IX activity greater than that of factor IX expressed from a viral particle comprising a transcriptional regulatory element of 6.

According to a twenty-first aspect of the present invention, the present invention comprises the step of administering a polynucleotide, viral particle or composition of the present invention to a patient, comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 and/ Or by expressing a factor IX protein or fragment thereof having a factor IX activity greater than that of factor IX expressed from a viral particle comprising a factor IX nucleotide sequence of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6 Provides a way.

According to the 22nd aspect of the present invention, the present invention comprises a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcription control element of SEQ ID NO: 7 and/or a factor IX nucleotide sequence of SEQ ID NO: 18 and a transcription control element of SEQ ID NO: 6 Provided is the use of a polynucleotide, viral particle or composition of the present invention to treat a disease by expressing a factor IX protein or fragment thereof having a factor IX activity greater than that of factor IX expressed from the viral particle.

According to a twenty-third aspect of the present invention, the polynucleotide, viral particle or composition of the present invention is for use in achieving a stable factor IX activity level of at least 20% in a patient with hemophilia type B.

According to a twenty-fourth aspect of the present invention, the present invention achieves a stable factor IX activity level of at least 20% in a patient with hemophilia B, comprising administering to the patient an effective amount of a polynucleotide, viral particle or composition of the present invention. Provides a way.

According to a twenty-fifth aspect of the present invention, the present invention provides the use of the polynucleotide, viral particle or composition of the present invention to achieve a stable factor IX activity level of at least 20% in a patient with hemophilia type B.

According to a twenty-sixth aspect of the present invention, the present invention comprises administering to the patient a dose of said polynucleotide, viral particle or composition effective to achieve a stable factor IX activity level of 20% or more in the patient, hemophilia type B. It provides a polynucleotide, viral particle or composition of the present invention for use in the treatment of

According to a twenty-seventh aspect of the invention, the invention comprises administering to a patient a dose of said polynucleotide, viral particle or composition of the invention effective to achieve a level of stable factor IX activity of at least 20% in the patient, Provides a method of treating hemophilia type B.

According to a twenty-eighth aspect of the present invention, the present invention comprises administering to the patient a dose effective to achieve a stable factor IX activity level of 20% or more in the patient, hemophilia type B. It provides the use of the polynucleotide, viral particle or composition of the present invention to treat.

Figure 1-Schematic diagram of the 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 the transcriptional regulatory elements of SEQ ID NOs: 6 and 7, respectively; E = exon; T1 = Truncated Intron 1A; WT = wild type; CO = optimized codon; ACNP = codon optimized sequence of the present invention.
Figure 2-HUH7 transduction results using AAV2/Mut C vector-Experiment 1; (A) shows the level of FIX antigen in the supernatant; (B) shows the level of FIX antigen 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 10 ³ ; 1e4 = 1 x 10 ⁴ ; MOI = multiplicity of infection.
Figure 3-HUH7 transduction results using AAV2/Mut C vector-Experiment 2; The level of FIX antigen in the supernatant is shown. Error bars represent the mean ± SD of n=3. 1e3 = 1 x 10 ³ ; 5e3 = 5 x 10 ³ ; 1e4 = 1 x 10 ⁴ _, MOI = multiplicity of infection.
Figure 4-HUH7 transduction results using AAV2/Mut C vector-Experiment 2; A) shows the level of FIX antigen in the supernatant; (B) shows the level of FIX antigen after normalization using the number of vector genomes present in the cell lysate. Error bars represent the mean ± SD of n=3. 1e3 = 1 x 10 ³ ; 5e3 = 5 x 10 ³ ; 1e4 = 1 x 10 ⁴ _, MOI = multiplicity of infection.
Figure 5-Combined data for AAV2/Mut C transduction of HUH7 cells with MOI 5 x 10 ^{3 (Experiments 1 and 2).} Error bars represent mean±SD of n=12. P = 0.001 by Student's T-test. 5e3 = 5 x 10 ^3; MOI = multiplicity of infection.
Figure 6-In the case of MOI 1x 10 ³ , 5 x 10 ³ and 1 x 10 ⁴ , the activity of FIX appears after HUH7 transduction using AAV2/Mut C vector (Experiment 1). Error bars represent mean±SD of n=2 duplicate wells. 1e3 = 1 x 10 ³ ; 5e3 = 5 x 10 ³ ; 1e4 = 1 x 10 ^4; MOI = multiplicity of infection.
Figure 7-The activity of FIX appears after HUH7 transduction using AAV2/Mut C vector (Experiment 2). Error bars represent the mean ± SD of n=3. 1e3 = 1 x 10 ³ ; 5e3 = 5 x 10 ³ ; 1e4 = 1 x 10 ⁴ _; MOI = multiplicity of infection.
Figure 8-The activity of FIX appears after HUH7 transduction using AAV2/Mut C vector (Experiment 3). Error bars represent the mean ± SD of n=3. 1e3 = 1 x 10 ³ ; 5e3 = 5 x 10 ³ ; 1e4 = 1 x 10 ⁴ _; MOI = multiplicity of infection.
Figure 9-Combined data showing the activity of FIX shown after HUH7 transduction using AAV2/Mut C vector with MOI 5 x 10 ^{3 (Experiments 1 and 2; FIGS. 6 and 7).} Error bars represent mean±SD of n=12. 5e3 = 5 x 10 ^3; MOI = multiplicity of infection. Statistical significance is determined using Student's T-test (p=0.0195).
Figure 10-Normalized levels of human FIX in rat plasma after administration of AAV2/8.LP1.FIXco and AAV2/8.HLP2.HTFG (Experiment 3). FIX: Ag levels were normalized to vector copy/cell. Error bars represent the mean±SD of n=4 mice. P-value <0.05 (Student's T-test)
Figure 11-Comparison of alternative codon optimization of FIX in C57BL/6 mice (Experiment 4). Mice were injected with an AAV2/8 vector containing ssHLP2.HTFG, ssHLP2.HTAG or scLP1.FIXco (control). The level of FIX antigen (A) was assessed 3 weeks after injection (P=0.0007 between ssHLP2.HTAG and sc.LP1.FIXco and p=0.0198 between ssHLP2.HTAG and ssHLP2.HTFG). Antigen levels were normalized to 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 analysis of variance (multiple comparison).
Figure 12-Comparison of alternative codon optimization of FIX in C57BL/6 mice (Experiment 4). Mice were injected with an AAV2/8 vector containing ssHLP2.HTFG, ssHLP2.HTAG or scLP1.FIXco (control). The level of FIX activity was assessed 3 weeks after injection. n=4 mice. P=0.0008 between ssHLP2.HTAG and scLP1.FIXco and p=0.01 between ssHLP2.HTAG and ssHLP2.HTFG; The p value was determined using one-way analysis of variance (multiple comparison).
Figure 13-Schematic diagram of Factor IX structure. The numbers above the schematic represent the amino acid positions of the complete factor IX polypeptide, including the signal peptide and the pro-peptide region (encoded by SEQ ID NO: 9). The numbers under the scheme indicate the equivalent amino acid positions in the maturation factor IX (corresponding to the coding sequence region of SEQ ID NO: 19).
Figure 14-Sequence listing.
Figure 15-Mean factor IX activity of two hemophilia type B patients receiving the AAV2/Mut C vector containing ssHLP2.HTAG. Two hemophilia patients were administered 4.5 x 10 ¹¹ vg/kg of AAV2/Mut C vector containing ssHLP2.HTAG. Factor IX activity levels were assessed regularly for 0-55 weeks after dosing. Patients achieved a stable mean factor IX activity level of 40.5 +/- 4.5% at 52 weeks as assessed by a stage 1 coagulation assay using the ^{SynthASil™ kit.} In addition, patients received prophylactic prednisolone 6-12 weeks after vector administration (infusion). " FIX: C (%) " indicates the level of FIX activity; See left y-axis. “ ALT (IU/L) ” refers to the level of alanine aminotransferase; See right y-axis.

General definition

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In general, the term “comprising” is intended to mean including, but not limited to. For example, the phrase “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 additional nucleotides.

In certain embodiments of the invention, the word “comprising” is replaced with the phrase “consisting of”. The term “consisting of” is intended to be limiting. For example, the phrase “polynucleotide consisting of a factor IX nucleotide sequence” should be understood to mean that the polynucleotide has a factor IX nucleotide sequence and no additional nucleotides.

The terms “protein” and “polypeptide” are used interchangeably herein and are intended to refer to a polymeric chain of amino acids of any length.

For the purposes of the present invention, in order to determine the percent identity of two sequences (e.g., two polynucleotides or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g. , Gaps can be introduced in the first sequence for optimal alignment to the second sequence). Then, the nucleotide residue at the nucleotide position is compared. If the position of the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position of the second sequence, the nucleotide is identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequence (i.e.,% identity = number of identical positions/total number of positions (i.e., overlapping positions) × 100).

In general, sequence comparisons are performed over the entire length of the reference sequence. For example, if a user wishes to determine whether a given ("test") sequence is 95% identical to SEQ ID NO: 5, then SEQ ID NO: 5 becomes the reference sequence. For example, to assess whether a sequence is at least 80% identical to SEQ ID NO: 5 (example of a reference sequence), one skilled in the art will perform an alignment over the length of SEQ ID NO: 5, and how many positions in the test sequence are You can see if it is the same as in 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 should be considered non-identical positions.

One of skill in the art will recognize that several different computer programs are available to determine the homology or identity between two sequences. For example, comparison of sequences and determination of percent identity between two sequences can be achieved using a mathematical algorithm. In one embodiment, the percent identity between two amino acid or nucleic acid sequences is a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and It can be determined using the Needleman and Wunsch (1970) algorithm integrated into the GAP program of the Accelrys GCG software package, using length weights of 1, 2, 3, 4, 5, or 6 (http://wwwaccelryscom/products/ gcg/ available).

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 means 50 contiguous nucleotides of SEQ ID NO: 5.

Polynucleotide

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 fragment thereof, and the site of the factor IX nucleotide sequence is wild-type. no.

The polynucleotide may further comprise one or more of the following features. Polynucleotides may contain regions that are not codon optimized. Polynucleotides may include introns or fragments of introns. The polynucleotide may contain a mutation at the codon corresponding to codon 384 of wild-type factor IX.

The term “ polynucleotide ” refers to a polymeric form of nucleotides, deoxyribonucleotides, ribonucleotides or analogs thereof of any length. For example, polynucleotides may include DNA (deoxyribonucleotides) or RNA (ribonucleotides). Polynucleotides can be made up of DNA. The polynucleotide can be an mRNA. Since polynucleotides can contain RNA or DNA, all references to T (thymine) nucleotides can be replaced with U (uracil).

Factor IX nucleotide sequence

Polynucleotides contain a factor IX nucleotide sequence. The factor IX nucleotide sequence comprises a coding sequence encoding a factor IX protein or fragment thereof.

“ Coding sequence ” is a sequence that encodes a polynucleotide and excludes non-coding regions such as introns. The coding sequence may be interrupted by non-coding nucleotides (eg, introns), but only nucleotides encoding the polypeptide should be considered part of the coding sequence. For example, the coding sequence encoding a factor IX protein is an amino acid that forms part of the factor IX protein expressed from that coding sequence, regardless of whether these codons are contiguous in the sequence or separated by one or more non-coding nucleotides. Will include any codon that codes for. That is, a polynucleotide comprising a stretch of coding nucleotides interrupted by stretches of non-coding nucleotides is a "coding sequence" consisting of non-contiguous coding stretches arranged immediately side by side (i.e., minus the non-coding stretch). Is considered to contain. However, in this specification, the stop codon will be considered part of the full-length coding sequence.

The term "coding sequence" refers to a nucleotide sequence comprising a codon encoding an encoded polypeptide. For example, a nucleotide sequence encoding a factor IX protein or fragment thereof includes a codon encoding an amino acid sequence of a factor IX protein or fragment thereof. A suitable nucleotide sequence is provided in SEQ ID NO: 5.

The table below describes the codons encoding each amino acid:

The corresponding RNA codon includes U instead of T in the above table.

One aspect of the present invention provides a polynucleotide comprising a factor IX nucleotide sequence, wherein the factor IX nucleotide sequence encodes a factor IX protein or fragment thereof, and SEQ ID NO: 5 and 97% or more, 98% or more, 99% or more, 99.5% or higher, 99.8% or higher, or 100% identity. Optionally, the factor IX nucleotide sequence comprises a coding sequence and a region of the coding sequence is codon optimized.

In general, the Factor IX nucleotide sequence is 1200 or more, 1350 or more, or 1650 or more nucleotide fragments of SEQ ID NO: 5 and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% Or more, 99.5% or more, 99.8% or more, or 100% the same. The factor IX nucleotide sequence is 1200 or more, 1350 or more, or 1650 or more consecutive nucleotide fragments of SEQ ID NO: 5 and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more , 99.5% or more, 99.8% or more, or 100% the same. The factor IX nucleotide sequence may be 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 5. For example, the factor IX nucleotide sequence may be 98% or more identical to SEQ ID NO: 5.

Factor IX protein or fragment thereof

The polynucleotide comprises a factor IX nucleotide sequence comprising a coding sequence encoding a factor IX protein or fragment thereof.

Wild type factor IX is a serine protease that forms part of the coagulation cascade. Deficiency or mutation of factor IX can lead to decreased blood clotting and the disease, hemophilia type B. 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. Alternative wild-type factor IX polypeptides differ in that they are encoded by codon 194 of SEQ ID NO: 9, for example, may encode threonine ("Malmo A") instead of alanine.

As shown in FIG. 13, a precursor" comprising a hydrophobic signal peptide (amino acids 1-28 of SEQ ID NO: 16), a pro-peptide region (amino acids 29-46 of SEQ ID NO: 16) and a mature polypeptide region" Factor IX initially expressed in the "immature" form (eg, factor IX of SEQ ID NO: 16). The mature (zymogen) form of factor IX lacks a hydrophobic signal peptide and a pro-peptide region. The term " maturation factor IX " refers to a factor IX polypeptide that does not contain a hydrophobic signal peptide or a pro-peptide region, such as SEQ ID NO: 8.

During coagulation, the single-chain zymogen form is cleaved by factor XIa or factor VIIa to produce an active two-chain form (factor IXa), the two chains being linked by disulfide bridges. The activated form can catalyze the hydrolysis of the 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 responsible for catalytic cleavage.

The term “factor IX protein” refers to a single-chain zymogen form, an activated two-chain form and variants thereof of factor IX, and includes a maturation factor IX polypeptide or pro-peptide region and/or a signal peptide region. Factor IX polypeptide.

Preferably, the Factor IX fragment is 200 or more, 250 or more, 300 or more, 200 to 461, 250 to 461, or 300 to 461 amino acids in length. In one embodiment, the Factor IX protein or fragment thereof comprises the following sequence:

a) 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: 8; or

b) 200 or more, 250 or more, 300 or more, 200 to 415, 250 to 415, or 300 to 415 amino acids in length of a fragment of SEQ ID NO: 8 and 95% or more, 98% or more, 99 % Or more, 99.5% or more, 99.8% or more, or 100% identical sequences.

In one embodiment, the Factor IX protein or fragment thereof is of SEQ ID NO: 16 having a length of 200 or more, 250 or more, 300 or more, 200 to 461, 250 to 461, or 300 to 461 amino acids. 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to the fragment. In one embodiment, the Factor IX protein or fragment thereof is 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 16; 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 having a length of 300 or more, or 300 to 461 amino acids. The Factor IX protein or fragment thereof may have the sequence of SEQ ID NO: 16 or SEQ ID NO: 8.

Preferably, the Factor IX protein or fragment thereof is functional. The functional factor IX protein or fragment is one that undergoes hydrolysis of the arginine-isoleucine bond in factor X to form factor Xa.

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

For example, a suitable color development analysis is as follows. Factor IX is mixed with thrombin, phospholipid, calcium, thrombin activating factor VIII and factor XIa. Under these conditions, factor XIa activates factor IX to form factor IXa and the activity of factor IXa can catalyze the cleavage of the chromogenic substrate (SXa-11) that produces pNA. The level of pNA produced can be measured by determining the absorbance at 405 nm, which is proportional to the activity of factor IX in the sample.

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 factor IX concentration. For example, an antibody that binds to factor IX can be bound to a plate. A sample containing an unknown concentration of factor IX can be passed over the plate. A second detection antibody that binds to factor IX can be applied to the plate and the excess can be washed away. The remaining (ie, unwashed) detection antibody will bind to factor IX. The detection antibody can be linked to an enzyme such as horse radish peroxidase. By measuring the amount of detection antibody, the level of detection antibody that binds to factor IX on the plate can be determined. For example, if the detection antibody is linked to horse radish peroxidase, the horse radish peroxidase can catalyze the production of blue reaction products on substrates such as TMB (3,3', 5,5'-tetramethylbenzidine). In addition, the level of the blue product can be detected by absorbance at 450 nm. The level of the blue product is proportional to the amount of detection antibody remaining after the washing step, which is proportional to the amount of factor IX in the sample.

Optionally, the factor IX protein or fragment thereof has a greater activity than the factor IX polypeptide encoded by SEQ ID NO: 9, SEQ ID NO: 19 or SEQ ID NO: 12. Optionally, activity is measured using a chromogenic substrate specific for factor IX, ie a substrate that can be altered by factor IXa to provide a chromogenic signal. A suitable chromogenic substrate is SXa-11.

In one embodiment, the factor IX protein or fragment thereof comprises a mutation at a position corresponding to position 384 of wild-type factor IX. For example, position 384 of wild-type factor IX (numbering from the beginning of the signal peptide, that is, the position corresponding to amino acid 384 of SEQ ID NO: 16) is an arginine residue (R384), but it can be replaced with another residue. In one 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.

Mutation at the position corresponding to position 384 of the wild-type sequence can lead to a functional gain-of-function (GoF) mutation, resulting in the generation of hyperfunctional factor IX. The advantage of expressing the factor IX protein comprising the mutation at position 384 is that a relatively small increase in protein amount results in a larger increase in total protein activity.

It is within the ability of one of skill in the art to determine whether a given polypeptide has a mutation at the position corresponding to position 384. One skilled in the art aligns the sequence of the polypeptide sequence with that of the wild-type (precursor, immature) factor IX polypeptide, and determines whether the former residue aligned with the 384 th residue of the latter is arginine. Otherwise, the polypeptide has a mutation at the position corresponding to position 384 of wild-type factor IX. Alignment can be performed using any suitable algorithm such as Needleman and Wunsch described above.

The region of the coding sequence is not wild-type.

The region of the coding sequence may not be wild type. The wild-type factor IX-encoding nucleotide sequence is represented by SEQ ID NO: 9, and the coding sequence comprising a site different from SEQ ID NO: 9 contains a non-wild-type site (providing this part is also considered to be wild-type coding for other factor IX Sequence, e.g., is also different from the previously mentioned Malmo A variant).

In one embodiment, regions of the coding sequence that are not wild-type are codon optimized. In order to determine whether the coding sequence contains a codon optimized region, the coding sequence can be aligned with SEQ ID NO:9. If any part of the sequence is not identical to SEQ ID NO: 9, the user can determine whether the codon has been optimized, i.e., one or more codons replaced by the preferred codon, i.e., TTC, CTG, ATC, GTG, GTC, AGC, It should be determined whether it contains one of CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC, and GAG. Codon optimization is performed if the non-wild-type site contains more than one codon replaced with the preferred codon. Preferably, contiguous regions of the coding sequence are codon optimized. However, in certain embodiments, regions of the codon optimized coding sequence may be divided over 2, 3, 4 or 5 regions of the coding sequence. Optionally, regions of the coding sequence that are not codon optimized are divided over less than 3 or less than 2 regions of the coding sequence. The nucleotide sequence can be codon optimized by replacing codons with other codons that are preferred (i.e., reflecting codon bias) in a particular organ or organism (so-called preferred codon). This codon optimization improves the expression of nucleotide sequences in certain organs or in certain organisms. For example, if the nucleotide sequence is a codon optimized between humans, the nucleotide sequence is modified to increase the number of codons preferred in human liver. One of skill in the art will understand that the codons that optimize the sequence may not involve altering all codons because “ favoured codons” may already be present at some positions.

This codon optimization can be influenced by other factors. For example, the presence of CpG adversely affects expression, so a user can decide not to use a preferred codon if CpG is introduced into the sequence at a specific location; This will still be considered codon optimization. In one embodiment, if the next codon in the sequence starts with G, the preferred codon ending with C nucleotide will not be included in the portion of the codon optimized coding sequence. For example, the codon CTC codes for leucine. CTC should not be used to encode leucine if the next codon in the sequence begins with G, such as the codon GTT.

This application discloses that certain codons are preferred for expression in human liver and improve the expression of the coding sequence while maintaining a high proportion of these preferred codons while reducing the CpG content 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.

In one embodiment, the region of the codon optimized coding sequence is a codon optimized for expression in the liver, optionally human liver. The region of the coding sequence that is codon-optimized for expression in the liver is a higher proportion of codons preferred in the liver, e.g., preferred codons TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC, and GAG.

In one embodiment, the following codons are not wild-type or collectively overrepresent at the site of the codon-optimized coding sequence: TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG , AAC, AAA, AAG, GAC, TGC, AGG, GGC, and GAG. " Collectively overrepresented " means that the total number of preferred codons at the site of the codon-optimized or non-wild-type coding sequence corresponds to the wild-type factor IX nucleotide sequence (eg, SEQ ID NO: 9 or SEQ ID NO: 19). This means that it is higher than the total number of preferred codons at the site.

In a preferred embodiment, the frequency of the following codons is greater at the site of the codon optimized coding sequence compared to the corresponding site of the wild-type factor IX nucleotide sequence (e.g. 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 excessive at codon optimized coding sequence sites, except when their presence results in CpG: TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC, and GAG. Optionally, the region of the codon-optimized coding sequence is 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 73% or more of the codons selected from the group consisting of Includes: TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC, and GAG.

The codon usage in the codon optimization site (HLP2.T1-ACNP-FIX-GoF) of the polynucleotide of the present invention is compared with the codon usage in the corresponding stretch of the wild-type factor IX nucleotide sequence (SEQ ID NO: 9) in the table below.

amino acid Codon HTAG %Age of codon Wild type Phe TTT 5 26 12 TTC 14 74 9 Leu CTT 0 9 CTC One 5 5 CTA 0 2 CTG 19 95 3 TTA 0 6 TTG 0 3 Ile ATT 5 24 17 ATC 16 76 7 ATA 0 One Met TTG 0 0 ATG 2 100 6 Val GTT 0 22 GTC One 3 3 GTA 0 5 GTG 33 97 7 Ser TCT 6 25 6 TCC One 4 5 TCA 0 6 TCG 0 0 AGT 0 7 AGC 17 71 3 Pro CCT 3 23 4 CCC 8 62 3 CCA 2 15 8 CCG 0 0 Thr ACT 11 39 13 ACC 17 61 7 ACA 0 10 ACG 0 One Ala GCT 11 55 9 GCC 9 45 5 GCA 0 8 GCG 0 0 Tyr TAT 7 50 11 TAC 7 50 5 His CAT 3 33 6 CAC 6 67 4 Gln CAA One 8 7 CAG 11 92 7 Asn AAT 7 27 15 AAC 19 73 17 Lys AAA One 4 12 AAG 24 96 16 Asp GAT 7 39 12 GAC 11 61 7 Glu GAA One 3 33 GAG 35 97 10 Cys TGT 9 43 19 TGC 12 57 5 Trp TGG 7 88 7 TGA One 13 0 Arg CGT 0 One CGC 0 One CGA 0 6 CGG 0 3 AGA 3 20 8 AGG 12 80 One Gly GGT 0 8 GGC 21 66 9 GGA 0 15 GGG 11 34 4

The total number of preferred codons in SEQ ID NO: 9 in this region is 120 (30% of the sequence). On the other hand, the total number of preferred codons in the codon optimization site of HTAG is 293 (73% of codons). It is straightforward to determine whether a given region of a polynucleotide contains a preferred codon. To determine the frequency of each codon used in a site of a nucleotide sequence, one of ordinary skill in the art can enter the sequence of that part into one of the readily available algorithms to look at codon usage and review the results. Alternatively, the user can simply count.

The codons replaced in the codon optimization site of HTAG compared to the corresponding region of SEQ ID NO: 9 are shown in the table below.

amino acid Codon replacement 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 One TTA to TTG 0 Gly GGC to GGG One 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 One ATA to ATC One Val Gta to gtg 4 GTC to GTG 3 GTG to GTA GTT to GTG 20 GTA to GTC One Lys AAA to AAG 10 AAC to AAG AAG to AAA One Tyr TAT to TAC 3 TAC to TAT One Gln CAA to CAG 6 CAG to CAA One His CAT to CAC 2 Glu GAA to GAG 27 Cys TGT to TGC 9 TGC to TGT One Ser AGT to AGC 3 TCC to AGC 4 AGT to TCT 3 TCA to AGC 3 TCT to AGC 4 TCA to TCT One 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 One Cgg to agg One Cgg to aga One Thr ACA to ACC 6 ACT to ACC 4 ACA to ACT 3 ACG to ACC One Phe TTT to TTC 5 Asp GAT to GAC 5 GAC to GAT One Asn AAT to AAC 6 stop TAA to TGA One GoF mutation One

In one embodiment, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more, 4 or more or 5 or more codons encoding phenylalanine are replaced by TTC compared to the 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 rest are TTT; And/or

d) The codon encoding phenylalanine is TTC, except when the next codon starts with G.

For example, when one or more codon A is said to be replaced by one or more codon B, this means replacing codon A with codon B at one or more positions compared to a wild-type sequence such as SEQ ID NO:9. To determine whether this replacement has occurred, you can align the test sequence with the wild-type factor IX sequence and see which codon is different. If at least one codon in the test sequence corresponding to codon A of wild-type factor IX is codon B in the test sequence, at least one codon A has been replaced by codon B. For example, if the first codon of the test sequence is TTT and the wild-type factor IX sequence is TTC, the test sequence contains at least one codon TTC replaced by TTT.

In one embodiment, at the site of the codon optimized coding sequence,

a) 5 or more, 10 or more, 15 or more or 16 or more codons encoding leucine are replaced by CTGs compared to the reference wild-type factor IX sequence;

b) at least 90% or at least 94% codons encoding leucine are CTGs; And/or

c) More than 90% or more than 94% of the codons encoding leucine are CTGs and the rest are CTCs.

In one embodiment, at the site of the codon optimized coding sequence,

a) 5 or more, 10 or more, 11 or more or 12 or more codons encoding isoleucine are replaced by ATC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then at least one codon ATC is replaced with an ATT compared to the reference wild type factor IX sequence;

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 is ATT; And/or

e) The codon encoding isoleucine is ATC, except when the next codon starts with G.

In one embodiment, at the site of the codon optimized coding sequence,

a) 10 or more, 15 or more, 20 or more or 25 or more codons encoding valine are replaced by GTG compared to the reference wild-type factor IX sequence;

b) one or more codons encoding valine are replaced by GTC compared to the reference wild-type factor IX sequence;

c) at least 80%, at least 90% or at least 95% of the codons encoding valine are GTGs; 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.

In one embodiment, at the site of the codon optimized coding sequence,

a) 5 or more, 10 or more, 12 or more or 13 or more codons encoding serine are replaced by AGC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then one or more, two or more, or four or more codons encoding serine are replaced by TCT compared to the reference wild-type factor IX sequence;

c) at least 60%, at least 65% or at least 70% of the codons encoding serine are AGC; And/or

d) 60% or more, 65% or more, or 70% or more of the codons encoding serine are AGC and the rest are TCT or TCC.

In one embodiment, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more or 5 or more codons encoding proline are replaced by CCC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then one or more codons encoding proline are replaced by CCT compared to the reference wild-type factor IX sequence;

c) at least 50%, at least 55% or at least 60% of the codons encoding proline are CCCs;

d) 50%, 55% or more or 60% or more codons encoding proline are CCC and the remainder are CCA or CCT; And/or

e) The codon encoding proline is CCC, except when the next codon starts with G.

In one embodiment, at the site of the codon optimized coding sequence,

a) 6 or more, 7 or more, 8 or more, or 10 or more codons encoding threonine are replaced by ACC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then one or more or two or more codons encoding threonine are replaced by ACT compared to the reference wild-type factor IX sequence;

c) at least 45%, at least 50% or at least 55% of the codons encoding threonine are ACCs;

d) at least 45%, at least 50% or at least 55% of the codons encoding threonine are ACC and the remainder is ACT; And/or

e) The codon encoding threonine is ACC, except when the next codon starts with G.

In one embodiment, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more, 3 or more or 4 or more codons encoding alanine are replaced by GCC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then one or more, two or more, or three or more codons encoding alanine are replaced by GCT compared to the reference wild-type factor IX sequence;

c) 35% or more, 40% or more or 43% or more codons encoding alanine are GCC;

d) 35% or more, 40% or more or 45% or more encoding alanine are GCC and the remainder is GCT; And/or

e) The codon encoding alanine is GCC, except when the next codon starts with G.

In one embodiment, at the site of the codon optimized coding sequence,

a) one or more or two or more codons encoding tyrosine are replaced by TAC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then at least one codon TAC is replaced with a TAT compared to the reference wild-type factor IX sequence;

c) at least 40%, at least 45% or at least 48% codons encoding tyrosine are TACs;

d) 40% or more, 45% or more or 48% or more codons encoding tyrosine are TAC and the remainder is TAT; And/or

e) The codon encoding tyrosine is TAC, except when the next codon starts with G.

In one embodiment, at the site of the codon optimized coding sequence,

a) one or more codons encoding histidine are replaced by CAC compared to the reference wild-type factor IX sequence;

b) at least 50%, at least 60% or at least 65% of the codons encoding histidine are CACs;

c) 50% or more, 60% or more or 65% or more codons encoding histidine are CAC and the remainder is CAT; And/or

d) The codon encoding histidine is CAC, except when the next codon starts with G.

In one embodiment, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more, 4 or more or 5 or more codons encoding glutamine are replaced by CAG compared to the reference wild-type factor IX sequence;

b) at least one codon CAG is replaced by CAA compared to the reference wild-type factor IX sequence;

c) at least 80%, at least 85% or at least 90% of the codons encoding glutamine are CAGs; 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.

In one embodiment, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more, 4 or more or 5 or more codons encoding asparagine are replaced by AACs compared to the reference wild-type factor IX sequence;

b) at least 60%, at least 65% or at least 70% of the codons encoding asparagine are AAC;

c) at least 60%, at least 65% or at least 70% of the codons encoding asparagine are AAC and the rest are AAT; And/or

e) The codon coding for asparagine is AAC, except when the next codon starts with G.

In one embodiment, at the site of the codon optimized coding sequence,

a) 5 or more, 7 or more, 8 or more or 9 or more codons encoding lysine are replaced with AAG compared to the reference wild-type factor IX sequence;

b) at least one codon AAG is replaced by AAA compared to the 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 rest are AAA.

In one embodiment, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more, 3 or more or 4 or more codons encoding aspartate are replaced by GACs compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then at least one codon GAC is replaced by a GAT compared to the reference wild-type factor IX sequence;

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 rest are GAT; And/or

e) The codon coding for aspartate is GAC, except when the next codon starts with G.

In one embodiment, at the site of the codon optimized coding sequence,

a) 15 or more, 20 or more, 25 or more or 26 or more codons encoding glutamate are replaced by GAGs compared to the 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 codons encoding glutamate are GAG and the rest are GAA.

In one embodiment, at the site of the codon optimized coding sequence,

a) 5 or more, 6 or more, 7 or more or 8 or more codons encoding cysteine are replaced by TGCs compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then at least one codon TGC is replaced by a TGT compared to the reference wild-type factor IX sequence;

c) at least 40%, at least 50% or at least 55% codons encoding cysteine are TGCs;

d) at least 40%, at least 50% or at least 55% of the codons encoding cysteine are TGCs and the remainder are TGTs; And/or

e) The codon encoding cysteine is TGC, except when the next codon starts with G.

In one embodiment, at the site of the codon optimized coding sequence, the codon encoding tryptophan is TGG.

In one embodiment, at the site of the codon optimized coding sequence,

a) 5 or more, 8 or more, 10 or more or 11 or more codons encoding arginine are replaced by AGG compared to the reference wild-type factor IX sequence;

b) one or more codons encoding arginine are replaced by AGA compared to the 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 rest are AGA.

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

In one embodiment, at the site of the codon optimized coding sequence,

a) 5 or more, 10 or more, 12 or more or 13 or more codons encoding glycine are replaced by GGCs compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then 5 or more, 6 or more, 7 or more or 8 or more codons encoding glycine are replaced by GGG compared to the reference wild-type factor IX sequence;

c) at least 50%, at least 55% or at least 60% codons encoding glycine are GGCs;

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 codon encoding glycine is GGC, except when the next codon starts with G.

In one embodiment, the region of the codon-optimized coding sequence is phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartate, glutamate, cysteine, tryptophan, Includes codons encoding arginine and glycine.

In one embodiment, the region of the codon-optimized coding sequence is phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartate, glutamate, cysteine, tryptophan, It includes a codon encoding arginine and glycine, and the codon-optimized site,

a) 5 or more codons encoding phenylalanine are replaced by TTC compared to the reference wild-type factor IX sequence;

b) 16 or more codons encoding leucine are replaced by CTG compared to the reference wild-type factor IX sequence;

c) 12 or more codons encoding isoleucine are replaced by ATC compared to the reference wild-type factor IX sequence;

d) 25 or more codons encoding valine are replaced by GTG compared to the reference wild-type factor IX sequence;

e) 13 or more codons encoding serine are replaced by AGC compared to the reference wild-type factor IX sequence;

f) 5 or more codons encoding proline are replaced by CCC compared to the reference wild-type factor IX sequence;

g) 10 or more codons encoding threonine are replaced by ACC compared to the reference wild-type factor IX sequence;

h) 4 or more codons encoding alanine are replaced by GCC compared to the reference wild-type factor IX sequence;

i) two or more codons encoding tyrosine are replaced by TAC compared to the reference wild-type factor IX sequence;

j) one or more codons encoding histidine are replaced by CAC compared to the reference wild-type factor IX sequence;

k) 5 or more codons encoding glutamine are replaced by CAG compared to the reference wild-type factor IX sequence;

l) 5 or more codons encoding asparagine are replaced by AAC compared to the reference wild-type factor IX sequence;

m) 9 or more codons encoding lysine are replaced by AAG compared to the reference wild-type factor IX sequence;

n) 4 or more codons encoding aspartate are replaced by GAC compared to the reference wild-type factor IX sequence;

o) 26 or more codons encoding glutamate are replaced by GAG compared to the reference wild-type factor IX sequence;

p) 8 or more codons encoding cysteine are replaced by TGC compared to the reference wild-type factor IX sequence;

q) the codon encoding tryptophan is TGG;

r) 11 or more codons encoding arginine are replaced by AGG compared to the reference wild-type factor IX sequence; And

s) 13 or more codons encoding glycine are replaced by GGC compared to the reference wild-type factor IX sequence.

In one embodiment, the region of the codon-optimized coding sequence is phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartate, glutamate, cysteine, tryptophan, Including codons encoding arginine and glycine, and in the codon-optimized site,

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

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

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 CCCs;

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 codons encoding tyrosine are TAC;

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

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 encoding aspartate are GAC;

o) 95% or more of the codons encoding glutamate are GAG;

p) at least 55% of the codons encoding cysteine are TGC;

q) the codon encoding tryptophan is TGG;

r) at least 75% of the codons encoding arginine are AGG; And

s) At least 60% of the codons encoding glycine are GGCs.

In one embodiment, the region of the codon-optimized coding sequence is phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartate, glutamate, cysteine, tryptophan, It includes a codon encoding arginine and glycine, and the codon-optimized site,

a) at least 70% of the codons encoding phenylalanine are TTC and the remainder is TTT;

b) more than 94% of the codons encoding leucine are CTG and the rest are CTC;

c) 75% or more 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) 60% or more 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 is ACT;

h) 43% or more of the codons encoding alanine are GCC and the rest are GCT;

i) at least 48% of the codons encoding tyrosine are TAC and the rest are TAT;

j) 65% or more of the codons encoding histidine are CAC and the rest are CAT;

k) 90% or more 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) 95% or more 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) 95% or more of the codons encoding glutamate are GAG and the rest are GAA;

p) 55% or more of the codons encoding cysteine are TGC and the remainder are TGT;

q) the codon encoding tryptophan is TGG;

r) at least 75% of the codons encoding arginine are AGG and the rest are AGA; And

s) More than 60% of the codons encoding glycine are GGC and the rest are GGG.

The reference wild-type factor IX sequence may be SEQ ID NO: 9 or SEQ ID NO: 19.

The codon optimized region may correspond to a sequence encoding some or all of the Factor IX protein. For example, the Factor IX protein can be a full-length coding sequence (eg, a sequence encoding SEQ ID NO: 8 or SEQ ID NO: 16) or a variant thereof, and the entire coding sequence can be codon optimized. Thus, reference herein to “ a region of a coding sequence is codon optimized” should be understood to mean “ at least a portion of the codon sequence is codon optimized”. However, in certain embodiments, the region of the coding sequence is not codon optimized, e.g., the region of the coding sequence is not codon optimized for expression in the liver. In certain embodiments, the regions of the codon optimized coding sequence are 800 or more, 900 or more, 1100 or more, 1500 or less, 1300 or less, 1200 or less, 800 to 1500, 900 to 1300, 1100 to 1200 Dogs, or about 1191 nucleotides in length.

In one embodiment, the region of the codon optimized coding sequence is of exon 3 or exon 3 of 10 or more, 15 or more, 20 or more, less than 25, 10 to 25, 15 to 25, or 20 to 25 nucleotides. Includes the site. In another embodiment, the region of the codon optimized coding sequence is of exon 4 or exon 4 of 80 or more, 90 or more, 100 or more, less than 114, 80 to 114, 90 to 114, or 100 to 114 nucleotides. Includes the site. In another embodiment, the region of the codon-optimized coding sequence is exon 5 or exon 5 of 90 or more, 100 or more, 110 or more, less than 129, 90 to 129, 100 to 129, or 110 to 129 nucleotides. Includes the site. In another embodiment, the region of the codon optimized coding sequence is of exon 6 or exon 6 of 150 or more, 180 or more, 200 or more, less than 203, 150 to 203, 180 to 203, or 200 to 203 nucleotides. Includes the site. In another embodiment, the region of the codon-optimized coding sequence is exon 7 or exon 7 of 70 or more, 80 or more, 90 or more, 100 or more, less than 115, 70 to 115, 80 to 115, 90 to 115 , Or from 100 to 115 nucleotides. In another embodiment, the region of the codon optimized coding sequence is of exon 8 or exon 8 of 400 or more, 450 or more, 500 or more, less than 548, 400 to 548, 450 to 548, or 500 to 548 nucleotides. Includes the site.

Exon 3 comprises nucleotide 253-277 of wild-type factor IX (eg factor IX of SEQ ID NO: 9), or the corresponding sequence of non-wild-type factor IX nucleotide sequence. Exon 4 comprises nucleotides 278-391 of wild-type factor IX (eg factor IX of SEQ ID NO), or the corresponding sequence of non-wild-type factor IX nucleotide sequence. Exon 5 comprises nucleotides 392-520 of wild-type factor IX (eg factor IX of SEQ ID NO), or the corresponding sequence of the non-wild-type factor IX nucleotide sequence. Exon 6 comprises nucleotides 521-723 of wild-type factor IX (eg factor IX of SEQ ID NO: 9), or the corresponding sequence of the non-wild-type factor IX nucleotide sequence. Exon 7 comprises nucleotides 724-838 of wild-type factor IX (eg factor IX of SEQ ID NO:), or the corresponding sequence of the non-wild-type factor IX nucleotide sequence. Exon 8 comprises nucleotide 839-1386 of wild-type factor IX (eg factor IX of SEQ ID NO: 9), or the corresponding sequence of non-wild-type factor IX nucleotide sequence.

Preferably, a site of 20 or more nucleotides of exon 3, a site of 100 or more nucleotides of exon 4, a site of 110 or more nucleotides of exon 5, 180 or more nucleotides of exon 6, 100 or more nucleotides of exon 7 Sites and sites of more than 500 nucleotides of exon 8 are codon optimized. The region of the codon-optimized coding sequence may include exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8. In one embodiment, the region of the codon-optimized coding sequence includes exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8.

In one embodiment, the region of the codon-optimized coding sequence comprises a region of exon 2, and the region of exon 2 is less than 160, less than 150, less than 100, less than 75, less than 60, 20 or more, 30 or more , 40 or more, 50 or more, 20 to 160, 30 to 150, 30 to 100, 40 to 75, or about 56 nucleotides in length. Exon 2 comprises nucleotides 89-252 of wild-type factor IX (eg factor IX of SEQ ID NO: 9), or the corresponding sequence of the non-wild-type factor IX nucleotide sequence. In a preferred embodiment, the region of the codon-optimized coding sequence comprises a region of exon 2 that is 30 to 100 nucleotides long.

It is within the ability of one of skill in the art to determine whether a portion of the sequence encoding a factor IX protein or fragment thereof corresponds to, for example, exon 8 of wild-type factor IX. Those skilled in the art perform sequence alignment with exon 8 with a sequence encoding a factor IX protein or fragment thereof using a suitable alignment algorithm such as those of Needleman and Wunsch described above, and at least a portion of the nucleotide sequence is exon 8 of SEQ ID NO: 9 ( As described above, whether exon 8 of SEQ ID NO: 9 is composed of nucleotides 839-1386 of SEQ ID NO: 9) has 90% or more, 95% or more, or 98% or more identity.

As discussed above, providing a polynucleotide sequence comprising a partially or wholly codon-optimized coding sequence can ensure that the encoded polypeptide is expressed at a high level. In one embodiment, the polypeptide encoded by the factor IX nucleotide sequence is expressed in human liver cells at a higher level compared to a reference wild-type factor IX sequence. The reference wild type factor IX sequence may be SEQ ID NO:9. In one embodiment, the polypeptide encoded by the factor IX nucleotide sequence is a human liver cell compared to the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7 At a higher level. In one embodiment, the polypeptide encoded by the factor IX nucleotide sequence is at a higher level than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6 It is expressed in human liver cells.

In one embodiment, the polypeptide encoded by the factor IX nucleotide sequence is encoded by a nucleotide sequence comprising a factor IX nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 18 and a transcriptional control element of SEQ ID NO: 7 or SEQ ID NO: 6 It is expressed in human liver cells at a level of at least 1.5 times, at least 2 times, at least 2.5 times, or at least 3 times higher than that of the polypeptide. Optionally, the polypeptide encoded by the factor IX nucleotide sequence is 1.5 times or more, 2 times or more than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7 , At least 2.5 times, or at least 3 times higher levels in human liver cells. Optionally, the polypeptide encoded by the factor IX nucleotide sequence is 1.5 times or more, 2 times or more than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional control element of SEQ ID NO: 6 , At least 2.5 times, or at least 3 times higher levels in human liver cells.

In one embodiment, the polynucleotide, viral particle or composition of the present invention is at least two times more than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7, or 3 At a level that is more than twice as high, it is intended to express the polypeptide encoded by the factor IX nucleotide sequence, and thus for use in treating diseases. In one embodiment, the polynucleotide, viral particle or composition of the present invention is at least two times more than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7, or 3 It is intended for use in the treatment of diseases by expressing the polypeptide encoded by the factor IX nucleotide sequence at a more than fold higher level. In one embodiment, the present invention relates to a nucleotide sequence comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional control element of SEQ ID NO: 7 comprising administering a polynucleotide, viral particle or composition of the present invention to a patient. It provides a method of expressing a polypeptide encoded by a factor IX nucleotide sequence at a level of at least 2 times or more than 3 times higher than the polypeptide encoded by the factor IX nucleotide sequence. In one embodiment, the present invention relates to a nucleotide sequence comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional control element of SEQ ID NO: 7 comprising administering a polynucleotide, viral particle or composition of the present invention to a patient. A method of treating a disease by expressing a polypeptide encoded by a factor IX nucleotide sequence at a level of at least 2 times, or at least 3 times higher than the polypeptide encoded by is provided. In one embodiment, the present invention provides a factor IX nucleotide sequence of SEQ ID NO: 12 and a nucleotide sequence comprising a transcriptional regulatory element of SEQ ID NO: 7 at a level that is 2 times or more, or 3 times or more, higher than that of the polypeptide. It provides the use of a polynucleotide, viral particle or composition of the present invention, which expresses a polypeptide encoded by a factor IX nucleotide sequence and thus treats a disease. In one embodiment, the present invention provides a factor IX nucleotide sequence of SEQ ID NO: 12 and a nucleotide sequence comprising a transcriptional regulatory element of SEQ ID NO: 7 at a level that is 2 times or more, or 3 times or more, higher than that of the polypeptide. It provides the use of the polynucleotide, viral particle or composition of the present invention to treat a disease by expressing a polypeptide encoded by a factor IX nucleotide sequence. In one embodiment, expressing the polypeptide encoded by the factor IX nucleotide sequence comprises administering the polynucleotide, viral particle or composition of the present invention to a patient.

In one embodiment, the polynucleotide, viral particle or composition of the present invention is at least two times more than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6, or 3 At a level that is more than twice as high, it is intended to express the polypeptide encoded by the factor IX nucleotide sequence, and thus for use in treating diseases. In one embodiment, the polynucleotide, viral particle or composition of the present invention is at least two times more than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6, or 3 It is intended for use in the treatment of diseases by expressing the polypeptide encoded by the factor IX nucleotide sequence at a more than fold higher level. In one embodiment, the present invention relates to 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 a polynucleotide, viral particle or composition of the present invention to a patient. It provides a method of expressing a polypeptide encoded by a factor IX nucleotide sequence at a level of at least 2 times or more than 3 times higher than the polypeptide encoded by the factor IX nucleotide sequence. In one embodiment, the present invention relates to 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 a polynucleotide, viral particle or composition of the present invention to a patient. A method of treating a disease by expressing a polypeptide encoded by a factor IX nucleotide sequence at a level of at least 2 times, or at least 3 times higher than the polypeptide encoded by is provided. In one embodiment, the present invention provides a factor IX nucleotide sequence of SEQ ID NO: 18 and a nucleotide sequence comprising a transcriptional regulatory element of SEQ ID NO: 6 at a level 2 or more, or 3 or more times higher than that of the polypeptide encoded by the polypeptide. It provides the use of a polynucleotide, viral particle or composition of the present invention, which expresses a polypeptide encoded by a factor IX nucleotide sequence and thus treats a disease. In one embodiment, the present invention provides a factor IX nucleotide sequence of SEQ ID NO: 18 and a nucleotide sequence comprising a transcriptional regulatory element of SEQ ID NO: 6 at a level 2 or more, or 3 or more times higher than that of the polypeptide encoded by the polypeptide. It provides the use of the polynucleotide, viral particle or composition of the present invention to treat a disease by expressing a polypeptide encoded by a factor IX nucleotide sequence. In one embodiment, expressing the polypeptide encoded by the factor IX nucleotide sequence comprises administering the polynucleotide, viral particle or composition of the present invention to a patient. Optionally the disease is hemophilia, for example hemophilia type B.

One of skill in the art can determine whether the factor IX nucleotide sequence is expressed at a higher level compared to the reference sequence by transducing host cells with viral particles comprising a factor IX nucleotide sequence and some cells having a vector comprising a reference sequence. The cells can be cultured under conditions suitable for expressing the factor IX protein or fragment thereof encoded by the factor IX nucleotide sequence, and the levels of the expressed factor IX protein can be compared. The level of expressed Factor IX protein can be assessed using an ELISA as described in the section titled “Factor IX protein or fragment thereof”. Suitable host cells include cultured human liver cells such as Huh 7 cells.

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

In one embodiment, the region of the codon optimized coding sequence is at least 800, at least 900, at least 1100, at least 1191, less than 1100, less than 1000, 800 to 1191, 900 to 1191, or about 1191 of SEQ ID NO: 1. It is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to a canine nucleotide fragment. In one embodiment, the region of the codon-optimized coding sequence is SEQ ID NO: 1 and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical. In one embodiment, the region of the codon-optimized coding sequence is 95% or more identical to the 900 to 1191 nucleotide fragments of SEQ ID NO: 1. In one embodiment, the region of the codon-optimized coding sequence is 95% or more, or 98% or more identical to SEQ ID NO: 1.

The present invention provides a polynucleotide comprising a factor IX nucleotide sequence, the factor IX nucleotide sequence comprising a coding sequence encoding a factor IX protein or fragment thereof, the coding sequence 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical sequence to SEQ ID NO: 1 and SEQ ID NO: 15 and 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100 % Contains identical sequences. Optionally, the sequence 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 is codon optimized.

Codon-optimized coding sequence site

In one embodiment, the factor IX nucleotide sequence comprises a region that is not codon optimized. Sites that are not codon optimized may be contiguous. Including regions that are not codon-optimized can enhance the expression of the coding sequence, as regions that are not codon-optimized can beneficially interact with other regions of the coding sequence, such as introns or fragments of introns. For example, a factor IX nucleotide sequence may comprise an intron or a fragment of an intron, in which case flanking an intron or a fragment of an intron with a wild-type factor IX sequence will help to ensure correct splicing. I can.

Sites that are not codon optimized are not modified to contain a greater number of preferred codons compared to the wild-type sequence. For example, a region that is not codon optimized may contain a similar number of preferred codons as the wild-type sequence. Codon-optimized sites are 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 It may contain GAG. Optionally, the non-codon-optimized site consists of TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC, and GAG. It contains less than 50%, less than 45% or less than 40% of codons selected from the group.

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

Optionally, the non-codon optimized sites are 100 or more, 150 or more, 170 or more, 190 or more, 250 or less, 225 or less, 200 or less, or about 195 nucleotides in length.

As discussed in more detail below, the Factor IX nucleotide sequence may comprise an intron or a fragment of an intron. In this case, the intron or fragment of the intron may flank the site that is not codon optimized. That is, a portion of the codon-optimized portion may be adjacent to an intron or fragment of an intron at the 3'end, and a portion of the codon-optimized portion may be adjacent to an intron or a fragment of an intron at the 5'end. The intron or fragment of an intron may be between exon 1 and exon 2. In this case, it is advantageous to include non-codon-optimized sites including the site of exon 1 and the site of exon 2.

Optionally, the codon-optimized site comprises 60 or more, 70 or more, 80 or more, 60 to 88, 70 to 88, or 80 to 88 contiguous nucleotide sites of exon 1 or exon 1. . Exon 1 comprises nucleotides 1-88 of wild-type factor IX (eg factor IX of SEQ ID NO: 9), or the corresponding sequence of the non-wild-type factor IX nucleotide sequence. Part of exon 1 may encode a signal peptide region and a pro-peptide region. Optionally, the non-codon optimized site contains or does not contain a signal peptide and/or a pro-peptide region. In addition, exon 1 may contain an additional non-coding stretch of 29 nucleotides at the 5'end. When the factor IX nucleotide sequence includes an intron or a fragment of an intron, it is preferred that the non-codon-optimized site includes the site of exon 1 adjacent to the intron or fragment of the intron. For example, if an intron or a fragment of an intron is between exon 1 and exon 2, the non-codon optimized region is at nucleotides 80-88, 70-88, 60-88, 40-88, or 20- It is preferable to include the site of exon 1 corresponding to 88.

Optionally, the codon-optimized sites are 50 or more, 75 or more, 80 or more, 90 or more, 100 or more, less than 140, less than 120, 50 to 140, 75 to 120 of exon 2 , Or a site of about 107 nucleotides. For example, when an intron or a fragment of an intron is between exon 1 and exon 2, the codon-optimized region is at nucleotides 89-100, 89-120, 89-140, 89-160, 89-180 of SEQ ID NO: 9 Or, it is preferable to include the site of exon 2 corresponding to 89-196.

The codon-optimized site may include CpG, for example, the codon-optimized site may contain the same number of CpGs as the corresponding site of SEQ ID NO: 9. The codon-optimized site may contain 1 or more or 2 or more CpGs per 100 nucleotides. The codon-optimized site may include 1 or more, 2 or more, 3 or more, 1 to 5, 2 to 5, or about 5 CpGs.

The codon-optimized region is at least 100, at least 150, at least 175, less than 195, less than 190 or less than 180 fragments and 80% or more, 85% or more, 90% of SEQ ID NO: 15 or SEQ ID NO: 2 Or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% the same. The codon-optimized region is SEQ ID NO: 15 or SEQ ID NO: 2 and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% It can be the same. For example, the codon-optimized region may be at least 98% identical to SEQ ID NO: 15 or SEQ ID NO: 2.

Sites that are 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 non-codon optimized site may be 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to the corresponding site of SEQ ID NO: 9.

The factor IX nucleotide sequence may comprise an intron or a fragment of an intron.

The factor IX nucleotide sequence may comprise an intron or fragment of an intron that interferes with the coding sequence. Introns are nucleotide sequences that are removed during the expression process and do not form part of the coding sequence.

The genomic wild-type factor IX nucleotide sequence contains an intron that interferes with the factor IX coding sequence. The presence of introns can help maintain high levels of expression of wild-type factor IX. Accordingly, it may be advantageous to include an intron, or at least a fragment of an intron, in the factor IX nucleotide sequence of the present invention. For example, the factor IX nucleotide sequence may comprise an intron or a fragment of an intron corresponding to intron 1 in wild-type factor IX. Suitably, the intron is a fragment of intron 1A of wild-type factor IX as shown in SEQ ID NO: 3. It was found that truncating the sequence of intron 1 increased the expression of the factor IX nucleotide sequence. It is believed that cleavage of Intron 1 to form Intron 1A could delete the intron's repressor element. In addition, cleavage of the intron 1 sequence causes the factor IX nucleotide sequence to be shorter, allowing more efficient packaging of the factor IX nucleotide sequence into the viral delivery system in gene therapy embodiments.

The fragments of the introns are less than 500, less than 400, less than 350, less than 300, more than 100, more than 200, more than 250, more than 290, more than 100, 200 to 400, 250 to 350 Dogs, or about 299 nucleotides. The fragment of the intron is 100 or more, 200 or more, 250 or more, or 290 or more nucleotides of SEQ ID NO: 3 and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99 % Or more, 99.5% or more, 99.8% or more, or 100% the same. The intron or fragment of the intron may be 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 3. For example, the intron or fragment of the intron may be 95% or more, or 98% or more identical to SEQ ID NO: 3.

Preferably, the introns or fragments of introns interrupt regions that are not codon optimized. That is, the intron is 5'for the non-codon-optimized portion and 3'for the non-codon-optimized portion in the factor IX nucleotide sequence.

If the nucleotides immediately 3'and 5'of the intron or close to the 3'and 5'sections of the intron are not codon optimized, the intron is "flanked by " to the non-codon optimized sequence. "Close to the intron. to the intron )" means within 1, 2, 3, 4, 5, 6, 7, 8, 8 or 10 nucleotides of an intron As discussed above, flanking introns or fragments of introns with non-codon-optimized nucleotide sequences can help ensure correct splicing Optionally, introns or fragments of introns are not codon-optimized. 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more are flanked. For example, 40 nucleotides that are just 3'of the intron and 20 nucleotides that are just 5'of the intron are flanked. If the codon is not optimized, or if 30 nucleotides that are just 3'of the intron and 30 nucleotides that are just 5'of the intron are not codon optimized, the intron is flanked by 60 nucleotides that are not optimized. Or a fragment of an intron may be codon-optimized 110-120 nucleotides at the 5'end (e.g., just 5'of the intron) and 100-110 uncodon-optimized at the 3'end (e.g. just 3'of the intron). Flanked by dog nucleotides.

The intron or fragment of the intron may be located between the site of the coding sequence corresponding to exon 1 and exon 2 of the factor IX nucleotide sequence. When the intron or fragment of an intron corresponds to a fragment of intron 1 of wild-type factor IX, it is preferred that the intron or fragment of intron is between the site of the coding sequence corresponding to exon 1 and exon 2 of the factor IX nucleotide sequence.

The polynucleotide may further comprise a transcriptional regulatory element.

Polynucleotides may contain transcriptional regulatory elements.

In one embodiment, the transcriptional regulatory element is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 6. . In one 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.

Any suitable transcriptional regulatory elements such as HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT and LSP, which are total liver specific transcriptional regulatory elements can be used. These transcriptional regulatory elements are described in more detail in the following references: HLP1: McIntosh J. et al. , Blood 2013 Apr 25, 121(17):3335-44; LP1: Nathwani et al. , Blood. 2006 April 1, 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. 1999 March 30, 96(7): 3906-3910. The HLP2 transcriptional regulatory element has the sequence of SEQ ID NO: 6.

Transcriptional regulatory elements may include promoter elements and/or enhancer elements from promoters and/or enhancers such as HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT and LSP. Each of these transcriptional regulatory elements comprises a promoter, enhancer, and optionally a different nucleotide.

In one embodiment, the transcriptional regulatory element comprises an enhancer that is a human apolipoprotein E (ApoE) hepatic locus control region (HCR) (Miao et al (2000), Molecular Therapy 1(6): 522) or a fragment thereof. In one embodiment, the transcriptional regulatory element is 80 or more, 90 or more, 100 or more, less than 192, 80 to 192, 90 to 192, 100 to 250, or 117 to 192 nucleotides. It includes a fragment of the HCR enhancer, which is a fragment of length. Optionally, the fragment of the HCR enhancer is 100 to 250 nucleotides in length.

Suitable HCR enhancer element fragments are disclosed in SEQ ID NO: 13. Optionally, the transcriptional regulatory element is 80 or more, 90 or more, 100 or more, less than 192, 80 to 192, 90 to 192, 100 to 250, or 117 to 192 nucleotides in length. Including an enhancer, wherein the enhancer is a polynucleotide that is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 13 It contains the sequence. Optionally, the transcriptional regulatory element comprises an enhancer that is 117 to 192 nucleotides in length, and the enhancer is a polynucleotide that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 13 It contains the sequence.

Optionally, the transcriptional regulatory element is at least 90, at least 100, or at least 110 nucleotide fragments of SEQ ID NO: 13 and at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, It contains enhancers that are 99.5% or more, 99.8% or more, or 100% identical. Optionally, the polynucleotide comprises an enhancer that is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 13. do. Optionally, the polynucleotide comprises an enhancer that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 13. Optionally, the polynucleotide comprises an enhancer of SEQ ID NO: 13.

In one embodiment, the transcriptional regulatory element comprises a promoter, which is a human alpha-1 anti-trypsin (A1AT) (Miao et al (2000), Molecular Therapy 1(6):522) promoter or a fragment thereof. Optionally, fragments of the A1AT promoter are 100 or more, 120 or more, 150 or more, 180 or more, less than 255, 100 to 255, 150 to 225, 150 to 300, or 180 to It is 255 nucleotides long. Optionally, a fragment of the A1AT promoter is 150 to 300 nucleotides in length.

Suitable A1AT promoter fragments are disclosed in SEQ ID NO: 14. Optionally, the transcriptional regulatory elements are 100 or more, 120 or more, 150 or more, 180 or more, less than 255, 100 to 255, 150 to 225, 150 to 300, or 180 to It includes a promoter of 255 nucleotides in length, and the formatter is SEQ ID NO: 14 and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, Or 100% identical polynucleotide sequences. Optionally, the transcriptional regulatory element comprises a polynucleotide sequence that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 14. Optionally, the polynucleotide is 100 or more, 120 or more, or 150 or more nucleotide fragments of SEQ ID NO: 14 and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical promoters. Optionally, the polynucleotide comprises 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical promoters of SEQ ID NO: 14 do. Optionally, the polynucleotide comprises 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical promoters of SEQ ID NO: 14. Optionally, the polynucleotide comprises a promoter of SEQ ID NO: 14.

If the polynucleotide is intended to be expressed in the liver, the promoter may be a liver specific promoter. Optionally, the promoter is a human liver specific promoter. "Liver-specific promoter (liver-specific promoter)" is typically a cellular promoter to provide higher levels of expression in the liver relative to other cells. For example, one of skill in the art can determine whether the promoter is a liver-specific promoter by comparing the expression of the polynucleotide in liver cells (eg Huh 7 cells) to the expression of the polynucleotide in cells of other tissues. If the expression level is higher in liver cells compared to cells in other tissues, the promoter is a liver specific promoter.

Gain of Function Mutation

The factor IX protein or fragment thereof may contain a gain-of-function mutation. A gain-of-function mutation is a mutation that increases the activity of a factor IX protein or fragment thereof. For example, the acquisition of functional mutations is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, 4 times than wild type factor IX (e.g., factor IX encoded by SEQ ID NO: 9 or SEQ ID NO: 19). It may result in a Factor IX protein or fragment thereof having an activity greater than, 5 times or more, 6 times or more, 6.5 times or more, 7 times or more, 7.5 times or more, or 8 times or more.

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). The mutation at the position corresponding to position 384 of wild-type factor IX may be an gain-of-function mutation. For example, replacing arginine 384 with leucine can increase activity substantially.

Whether the factor IX protein contains a mutation at the position corresponding to position 384 of factor IX is determined by aligning the factor IX protein with SEQ ID NO: 16 using an appropriate algorithm such as Needleman and Wunsch's algorithm described above, and amino acid 384 (SEQ ID NO: 16) can be determined by determining whether the amino acid aligned to the arginine residue. When the amino acid aligned to amino acid 384 of SEQ ID NO: 16 is not an arginine residue, the factor IX protein has a mutation at the position corresponding to position 384 of wild-type factor IX.

Whether the mutation is a gain-of-function mutation can be determined by comparing the activity of the factor IX protein comprising the mutation with the activity of the same reference factor IX protein except for the presumption of the gain-of-function mutation. The relative activity of these two proteins can be determined using chromogenic assays such as those discussed under the heading “Factor IX protein or fragment thereof”. If the activity of the factor IX protein containing the mutation is higher than that of the reference protein, the mutation is a gain-of-function mutation.

Thus, the factor IX nucleotide sequence may comprise a codon encoding a mutation at a position corresponding to position 384 of factor IX. For example, the factor IX nucleotide sequence may include a codon encoding an amino acid at a position corresponding to position 384 of wild-type factor IX, a small hydrophobic amino acid. The small hydrophobic amino acids 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.

The codon encoding the mutation at the position corresponding to position 384 in wild-type factor IX may be a codon encoding a leucine such as CTX, wherein X is any nucleotide. Preferably, X is C or G. A codon encoding an amino acid at a position corresponding to position 384 of wild-type factor IX may be CTC as shown in SEQ ID NO: 4. In an alternative embodiment, the codon encoding the amino acid at the position corresponding to position 384 of wild-type factor IX is SEQ ID NO: 11, or TTG or CTG, as shown in SEQ ID NO: 26. For example, herein the reference for SEQ ID NO: 1 can be replaced with the reference for the corresponding site of SEQ ID NO: 26 or 11. That is, SEQ ID NO: 1 may be substituted with G at nucleotide 957 (C) or T and G at nucleotides 955 (C) and 957 (C), respectively.

Polynucleotide is at least 1200, at least 1350, or at least 1650 nucleotide fragments of SEQ ID NO: 5 and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical Factor IX sequences. For example, the factor IX nucleotide sequence is a factor that is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 5 IX sequence.

Suitably,

(i) the factor IX nucleotide sequence comprises a sequence that is 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 1; And

(ii) the factor IX nucleotide sequence includes a codon encoding an amino acid at a position corresponding to position 384 of wild-type factor IX, and the codon encoding an amino acid at position 384 of the wild-type factor IX encodes leucine. .

Suitably,

(i) the factor IX nucleotide sequence comprises a coding sequence that is 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 1;

(ii) the factor IX nucleotide sequence includes a codon encoding an amino acid at a position corresponding to position 384 of wild-type factor IX, and the codon encoding an amino acid at position 384 of the wild-type factor IX encodes leucine, and ; And

(iii) The polynucleotide comprises an enhancer element that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 13.

Suitably,

(i) the factor IX nucleotide sequence comprises a coding sequence that is 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 1;

(ii) the factor IX nucleotide sequence includes a codon encoding an amino acid at a position corresponding to position 384 of wild-type factor IX, and the codon encoding an amino acid at position 384 of the wild-type factor IX encodes leucine, and ; And

(iii) The polynucleotide comprises a promoter element that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 14.

Suitably,

(i) the factor IX nucleotide sequence comprises a sequence that is 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 1;

(ii) the factor IX nucleotide sequence includes a codon encoding an amino acid at a position corresponding to position 384 of wild-type factor IX, and the codon encoding an amino acid at position 384 of the wild-type factor IX encodes leucine, and ; And

(iii) The polynucleotide comprises a transcriptional regulatory element that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 6.

Suitably,

(i) the factor IX nucleotide sequence comprises a sequence that is 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 1;

(ii) the factor IX nucleotide sequence comprises a sequence that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 2; And

(ii) The factor IX nucleotide sequence includes a codon encoding an amino acid at a position corresponding to position 384 of wild-type factor IX, and the codon encoding an amino acid at position 384 of the wild-type factor IX encodes leucine. .

Suitably, the factor IX nucleotide sequence comprises an intron or a fragment of an intron, and the fragment of the intron is at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 3.

Viral particles containing polynucleotides

The present invention further provides a viral particle comprising a recombinant genome comprising the polynucleotide of the present invention. For the 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 this specification, the terms " viral particle " and " vector " are used interchangeably. For the purposes of this application, a " gene therapy " vector is a viral particle that can be used in gene therapy, ie, a viral particle comprising all functional elements necessary to express a transgene such as a factor IX nucleotide sequence in a host cell after administration. to be.

Suitable viral particles include parvovirus, retrovirus, lentivirus or herpes virus. The parvovirus can be an adeno-associated virus (AAV). The viral particles are preferably recombinant adeno-associated virus (AAV) vectors or lentiviral vectors. More preferably, the viral particles are AAV viral particles. The terms AAV and rAAV are used interchangeably herein.

The genomic structure of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding sequences of non-structural replication (Rep) and structural (VP) proteins. The VP proteins (VP1, VP2 and VP3) form a capsid. The terminal 145 nt is self-complementary and is configured to form an energetically stable intramolecular dimer forming a T-shaped hairpin structure. This hairpin structure functions as a starting point for viral DNA replication, and acts as a primer for the cellular DNA polymerase complex. Following wild-type (wt) AAV infection in mammalian cells, the Rep gene (i.e., encoding Rep78 and Rep52 proteins) is expressed from the P5 promoter and the P19 promoter, respectively, and both Rep proteins have viral genome replication functions. The splicing phenomenon of Rep ORF actually results in the expression of four Rep proteins (ie, Rep78, Rep68, Rep52 and Rep40). However, it was found that mRNA encoding the unspliced Rep78 and Rep52 proteins in mammalian cells is sufficient for AAV vector production. In addition, in insect cells, Rep78 and Rep52 proteins are sufficient for AAV vector production.

The recombinant viral genome of the present invention may include ITR. It is possible that the AAV vector of the present invention functions as only one ITR. Thus, the viral genome contains at least one ITR, but more typically two ITRs (generally on either side of the viral genome, i.e., the 5'end and the 3'end) are located on both sides. There may be intervening sequences between the polynucleotide and one or more ITRs. The polynucleotide of the present invention can be incorporated into viral particles located between two regular ITRs or located on both sides of an ITR engineered with two D regions.

The AAV sequences that can be used in the present invention for the production of AAV gene therapy vectors can be derived from any AAV serotype genome. In general, AAV serotypes have genomic sequences with significant homology at the amino acid and nucleic acid levels, provide the same set of genetic functions, produce essentially physically and functionally equivalent virions, and have virtually identical mechanisms. By replicating and assembling. For an overview of genomic sequence and genomic similarity of various AAV serotypes, see, eg, 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 serotypes 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11 or 12 can be used in the present invention. Sequences derived from AAV serotypes can be mutated or manipulated when used in the production of gene therapy vectors.

Optionally, the AAV vector comprises ITR sequences derived from AAV1, AAV2, AAV4 and/or AAV6. Preferably the ITR sequence is an AAV2 ITR sequence. In the present specification, the term AAVx /y refers to a viral particle comprising some components of AAVx (where x is the AAV serotype number) and some components of AAVy (where y is the number of the same or different serotypes). For example, the AAV2/8 vector may comprise a portion of the viral genome comprising the ITR from the AAV2 strain and a capsid derived from the AAV8 strain.

In one embodiment, the viral particle is an AAV viral particle comprising a capsid. AAV capsids are generally formed of three proteins: VP1, VP2 and VP3. The amino acid sequence of VP1 includes the sequence of VP2. A portion of VP1 that does not form part of VP2 is referred to as VP1unique or VP1U. The amino acid sequence of VP2 includes the sequence of VP3. A portion of VP2 that does not form part of VP3 is referred to as VP2unique or VP2U. Preferably the capsid is an AAV5 capsid or Mut C capsid. The Mut C capsid may have 96% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identity with SEQ ID NO: 10. The AAV capsid may have 96% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identity with SEQ ID NO: 17. In an alternative embodiment, the capsid is at least 96%, 98% or more with VP2U and/or VP3 of SEQ ID NO: 17 and 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, It has a VP1U sequence with at least 99%, at least 99.5%, at least 99.8% or at least 100% identity.

The viral particles of the present invention may be "hybrid " particles derived from different parvoviruses, such as AAV serotypes with different viral ITRs and viral capsids. Preferably, the viral ITR and capsid are derived from different serotypes of AAV, in which case such viral particles are known as transcapsidated or pseudotype. Likewise, parvoviruses can contain “ chimeric ” capsids (eg, containing sequences from different parvoviruses, preferably different AAV serotypes) or “ targeted ” capsids (eg, directional tropism). I can have it.

In certain embodiments, the recombinant AAV genome comprises intact ITRs comprising functional terminal resolution sites (TRS). These AAV genomes are capable of site-specific nicking to generate one or two degradable ITRs, i.e., free 3'hydroxyl groups that can serve as substrates for DNA polymerases to loosen and copy ITRs. It may include an ITR that includes a functional TRS. Preferably, the recombinant genome is single-stranded (ie, packaged in viral particles in single-stranded form). Optionally, the recombinant genome is not packaged in a self-complementary configuration. That is, the genome does not contain a single covalently bonded polynucleotide strand with a substantially self-complementary portion annealed in the viral particle. Alternatively, the recombinant genome can be packaged in a " monomeric duplex" form. The " monomer duplex " is described in WO 2011/122950. The genome can be packaged into two substantially complementary but non-shared polynucleotides annealed in the viral particle.

The viral particle may further comprise a poly A sequence. The poly A sequence may be located downstream of the nucleotide sequence encoding the functional factor IX protein. The poly A sequence may be a bovine growth hormone poly A sequence (bGHpA). The poly A sequence can be 250 to 270 nucleotides in length.

The viral particles of the invention are optionally highly expressed in host cells. For example, in the transduction in Huh7 cells, the viral particle is a viral particle comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory factor of SEQ ID NO: 7 and/or a factor IX nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: It expresses a factor IX protein or a fragment thereof having a greater factor IX activity than that of the factor IX protein expressed from the viral particle comprising the transcriptional regulatory factor of 6.

Optionally, after transduction into the Huh7 cell population, the viral particles are expressed from a comparable viral particle comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory factor of SEQ ID NO: 7 transduced with a comparable population of Huh7 cells. It expresses a factor IX protein or fragment thereof having a greater factor IX activity than that of the factor IX protein. Optionally, after transduction into the Huh7 cell population, the viral particles are expressed from a comparable viral particle comprising a factor IX nucleotide sequence of SEQ ID NO: 18 and a transcriptional regulatory factor of SEQ ID NO: 6 transduced with a comparable population of Huh7 cells. It expresses a factor IX protein or fragment thereof having a greater factor IX activity than that of the factor IX protein.

In one aspect, the polynucleotide, viral particle or composition of the present invention comprises a factor IX nucleotide sequence of SEQ ID NO: 12 and a viral particle comprising a transcriptional regulatory factor of SEQ ID NO: 7 and/or a factor IX nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: It is for use in treating a disease by expressing a factor IX protein or fragment thereof having a factor IX activity greater than that of factor IX expressed from a viral particle comprising a transcriptional regulatory factor of 6. In one aspect, the present invention is a viral particle comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory factor of SEQ ID NO: 7 comprising administering to a patient an effective amount of a polynucleotide, viral particle or composition of the present invention. And/or a factor IX protein or fragment thereof having a factor IX activity greater than that of factor IX expressed from a viral particle comprising a factor IX nucleotide sequence of SEQ ID NO: 18 and a transcriptional regulatory factor of SEQ ID NO: 6 Provides a method of treatment. In one aspect, the present invention comprises a viral particle comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory factor of SEQ ID NO: 7 and/or a factor IX nucleotide sequence of SEQ ID NO: 18 and a transcriptional regulatory factor of SEQ ID NO: 6 Provided is the use of a polynucleotide, viral particle or composition of the present invention for treating a disease by expressing a factor IX protein or fragment thereof having a factor IX activity greater than that of factor IX expressed from the viral particle. Optionally, a viral particle comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory factor 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 factor of SEQ ID NO: 6 is compared Possible viral particles. Optionally, expressing the Factor IX protein or fragment thereof comprises administering to the patient a polynucleotide, viral particle or composition of the present invention. Optionally the disease is hemophilia, for example hemophilia type B.

In this embodiment, the term " comparable viral particle " means that the comparable viral particle comprises a different factor IX nucleotide sequence and a different transcriptional regulatory element (SEQ ID NO: 12 and SEQ ID NO: 7 or those of SEQ ID NO: 18 and SEQ ID NO: 6). Except that, it refers to the same viral particles as the AAV viral particles of the present invention. Optionally, activity is assessed using a chromogenic assay, such as the chromogenic assay discussed above. However, in this case the activity is not normalized to the factor IX concentration, so the activity is a function of the expression level as well as the intrinsic activity of the factor IX protein.

Composition, method and use

In a further aspect of the invention there is provided a composition comprising the polynucleotide or vector/viral particles of the invention and a pharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients may include carriers, diluents and/or other drugs, drugs or adjuvants. Optionally, the pharmaceutically acceptable excipient includes saline. Optionally, pharmaceutically acceptable excipients include human serum albumin.

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

The invention further provides a method of treatment comprising administering to a patient an effective amount of a polynucleotide or vector/viral particle of the invention.

The invention further provides for the use of a polynucleotide, vector/viral particle or composition of the invention in the manufacture of a medicament for use in a method of treatment. Optionally, the method of treatment comprises administering to the patient an effective amount of a polynucleotide or vector/viral particle of the invention. Optionally, the treatment method is gene therapy. “ Genotherapy ” includes administering a vector/viral particle of the invention capable of expressing a transgene (eg, a factor IX nucleotide sequence) in a host to which it is administered.

Optionally, the method of treatment is a method of treating a coagulation condition such as hemophilia (eg hemophilia type A or B) or Van Willebrands' disease. Preferably, the coagulopathy is characterized by increased bleeding and/or decreased coagulation. Optionally, the method of treatment is a method of treating hemophilia, eg hemophilia type B. In certain embodiments, the patient is suffering from hemophilia type B. Optionally, the patient has an antibody or inhibitor against factor IX. Optionally, the polynucleotide and/or vector/viral particles are administered intravenously. Optionally, the polynucleotide and/or vector/viral particle is for administration to a patient only once (ie, a single dose).

"Treating " hemophilia type B in the above method means that one or more symptoms of hemophilia are improved. This does not mean that the symptoms of hemophilia are completely cured and are no longer present in the patient, but in some ways this may be true. Treatment methods can make one or more of the symptoms of hemophilia type B less severe than before treatment. Optionally, compared to the pre-administration situation, the treatment regimen is the amount/concentration of circulating factor IX in the patient's blood and/or the total level of factor IX activity detectable within a given volume in the patient's blood and/or Increases the specific activity of factor IX (activity per amount of factor IX protein).

A “therapeutically effective amount” refers to the dosage and duration necessary to achieve the desired treatment outcome, such as raising the level of functional factor IX in a subject (to induce production of functional factor IX to a level sufficient to improve symptoms of hemophilia B). Refers to the effective amount during.

Optionally, the vector/viral particle is ^{less than 1 x 10 11,} less than 1 x 10 ^12, less than 5 x 10 ^12, less than 2 x 10 ^12, less than 1.5 x 10 ^12, less than 3 x 10 ¹² , 1 x per kg of patient body weight. 10 is less than ^13, 2 x 10 ^13, is administered in less than, or less than the capacity of the vector genome of l3 x 10 ^13. Optionally, the polynucleotide, viral particle or composition is administered at a dose of 4.5 x 10 ¹¹ or greater or 4.5 x 10 ¹¹ to 1 x 10 ¹² vector genome (vg/kg) per kg of patient body weight. Optionally, the polynucleotide, viral particle or composition is 5 x 10 ¹¹ vg/kg It is administered in less than a dose. Optionally, the polynucleotide, viral particle or composition is administered at a dose ^{of 4.5 x 10 11} vg/kg to 5 x 10 ¹¹ vg/kg, or 4.5 x 10 ¹¹ vg/kg to 4.9 x 10 ^{11 vg/kg.} Optionally, the polynucleotide or viral particles to be administered are produced from mammalian cells and/or resulting from the use of mammalian viral vector producing cells and with vectors produced in insect viral vector producing cells (e.g., baculo virus systems). It has distinctive characteristics.

Optionally, administration of a given dose of vector/viral particles quantified by the number of vector genomes is achieved using qPCR to titrate the vector genome. In principle, qPCR primers can be designed to bind to any part of the recombinant vector genome that is not common to the wild-type genome, but it is not recommended to use a primer template sequence that is very close to the ITR because it can lead to an exaggerated vector genome titer measurement. Optionally, the vector genome is quantified by quantitative polymerase chain reaction (qPCR) using primers directed towards the promoter region, for example the promoter region of the transgene cassette (sometimes also known as the transgene expression cassette).

Methods of performing qPCR are known to those of skill in the art. Amplification of the initial double-stranded amplicon can be detected in real time using a real-time PCR cycler and a DNA binding dye such as SYBR Green (ThermoFisher Scientific). A standard curve can be made by serial dilution of a known amount of qPCR template genetic material (e.g., a promoter region), and sample vector genome titers interpolated from the standard curve can be made.

Optionally, the dose of vector/viral particle administered is 10%-90%, 20%-80%, 30%-70%, 25%-50%, 20%-150 of the factor IX activity of a healthy non-hemophilic subject. %, 30%-140%, 40%-130%, 50%-120%, 60%-110% or 70%-100% of activity are selected to express factor IX. Optionally, the polynucleotide, viral particle or composition is administered in a dose effective to achieve a stable factor IX activity level of at least 20% in hemophilia type B patients. Optionally, the polynucleotide, viral particle or composition is administered in a dose effective to achieve a stable level of factor IX activity of at least 25% in hemophilia type B patients. Optionally, the polynucleotide, viral particle or composition is administered in a dose effective to achieve a stable level of factor IX activity of at least 30% in hemophilia type B patients. Optionally, the polynucleotide, viral particle or composition is administered in a dose effective to achieve a stable factor IX activity level of 35% or greater in a hemophilia type B patient. Optionally, the polynucleotide, viral particle or composition is administered in a dose effective to achieve a stable factor IX activity level of 40% or more in a patient with hemophilia type B.

In one aspect, the polynucleotide, viral particle or composition of the invention is for use in achieving a stable factor IX activity level of 20% or greater in a patient with hemophilia type B. In one aspect, the present invention provides a method of achieving a stable factor IX activity level of at least 20% in a patient with hemophilia B, comprising administering to the patient an effective amount of a polynucleotide, viral particle or composition of the present invention. In another aspect, there is provided the use of a polynucleotide, viral particle or composition of the present invention, which achieves a level of stable factor IX activity of at least 20% in a patient with hemophilia type B. In another embodiment, the present invention provides for the treatment of hemophilia type B comprising administering to the patient a polynucleotide, viral particle or composition in a dose effective to achieve a stable factor IX activity level of 20% or more in a hemophilia type B patient. Polynucleotides, viral particles or compositions of the present invention for use. In another aspect, the invention provides a method of treating hemophilia type B comprising administering to a patient a polynucleotide, viral particle or composition of the invention in a dose effective to achieve a stable factor IX activity level of 20% or more. . In another embodiment, the present invention provides for the treatment of hemophilia type B comprising administering to the patient a polynucleotide, viral particle or composition in a dose effective to achieve a stable factor IX activity level of 20% or more in a hemophilia type B patient. The use of the polynucleotide, viral particle or composition of the present invention for use is provided. Optionally, treating hemophilia type B or achieving a stable level of factor IX activity comprises administering to the patient a polynucleotide, viral particle or composition of the present invention. Optionally, to achieve a stable factor IX activity level, e.g., at least 20%, at least 30% or at least 40%, a polynucleotide, viral particle or composition is ^{less than 5 x 10 11} vg/kg. Or 4.5 x 10 ¹¹ vg/kg to 4.9 x 10 ¹¹ vg/kg. Optionally, the polynucleotide or viral particles to be administered are produced from mammalian cells and/or produced from the use of mammalian viral vector producing cells and differentiated from vectors produced in insect viral vector producing cells (e.g. baculo virus systems). It possesses the characteristics of being.

A factor IX activity level of X% or greater (eg, 20% or greater) refers to a factor IX activity level that is X% or greater (eg, 20%) of the normal range of factor IX levels. Those of skill in the art will readily understand what the criteria for the% of normal factor IX activity levels determined in routine clinical practice with reference to international standards mean. Pooled normal (healthy) human plasma has a factor IX concentration of about 5 μg/ml.

The term “ stable factor IX activity level ” refers to a level of factor IX activity that maintains above a certain level for a period of at least 5 consecutive weeks. In certain embodiments, the factor IX activity level is maintained above a certain level for a continuous period of 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. For example, the patient has a stable ocular IX activity level of 20% or more if the activity level is maintained at 20% or more for at least 5 consecutive weeks. In this example, the factor IX activity level may continue at least 20% after at least 0 5 weeks, thus at least 20% for a cumulative continuous period of at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks. Can be maintained. Optionally, the patient has a stable factor IX activity level if the factor IX activity level remains above a certain level for at least 20 consecutive weeks. Optionally, the patient administered the polynucleotide, viral particle or composition may have a stable factor IX activity level of at least 20%, at least 25%, at least 30%, or at least 35%. Optionally, the patient administered the polynucleotide, viral particle or composition may have a stable level of factor IX activity of at least or about 40%.

Optionally, the factor IX activity level is stable after at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 weeks from administration of the polynucleotide, viral particle or composition. For example, if the patient has a stable factor IX activity level of 20% or more after 10 weeks or more of administration of the polynucleotide, viral particle or composition, then at least 5 weeks, at least 10 weeks after the initial at least 10 weeks after administration. , A factor IX activity level of at least 20% if maintained at 20% or more for a continuous period of at least 15 weeks, at least 20 weeks, at least 30 weeks or at least 40 weeks, or at least 50 weeks. Optionally, the patient has a stable factor IX activity level at least 10 weeks after administration of the polynucleotide, viral particle or composition, and the factor IX activity level (at least 10 weeks after administration) for a continuous period of at least 20 weeks or at least 40 weeks. While it is maintained at the specific level described above. Optionally, the level of stable Factor IX activity after at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration may be at least 20%, at least 25%, at least 30%, or at least 35%. Optionally, the level of stable Factor IX activity after at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration may be at least or about 40% or more. In certain embodiments, the patient has a stable factor IX activity level of at least 20% after at least 10 weeks from administration of the polynucleotide, viral particle or composition, the factor IX activity level (at least 10 weeks after administration) at least 20 weeks or It remains above 20% for at least 40 consecutive weeks. In certain embodiments, the patient has a stable factor IX activity level of 40% or more after at least 10 weeks from administration of the polynucleotide, viral particle or composition, the factor IX activity level (at least 10 weeks after administration) at least 20 weeks or It is maintained above 40% for at least 40 consecutive weeks.

Optionally, the factor IX activity level is at a specific level (e.g., at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks or at least 40 weeks or at least 50 weeks after administration of the polynucleotide, viral particle or composition) 20%, 25%, 30%, 35%, or 40%) or more. For example, the level of factor IX activity is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, after administration of the polynucleotide, viral particle or composition. 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, It is at or above a certain level (eg, 20%, 25%, 30%, 35%, or 40%) at 50, 51 or 52 weeks. Optionally, the factor IX activity level is at least 20% at about 10 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the factor IX activity level is at least 25% at about 10 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the factor IX activity level is at least 40% at about 10 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the factor IX activity level is at least 20% at about 20 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the factor IX activity level is at least 25% at about 20 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the factor IX activity level is at least 40% at about 20 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the factor IX activity level is at least 20% at about 52 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the factor IX activity level is at least 25% at about 52 weeks after administration of the polynucleotide, viral particle or composition. Optionally, the factor IX activity level is at least 40% at about 52 weeks after administration of the polynucleotide, viral particle or composition.

Factor IX activity level is at a certain level (e.g., 20%, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks or at least 40 weeks or at least 50 weeks after administration of the polynucleotide, viral particle or composition) 25%, 30%, 35%, or 40%) or higher, the level of factor IX activity may also be stable. In certain embodiments, when the factor IX activity level is stable, a continuous period of at least 5 weeks (or at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks or at least 40 weeks or at least 50 weeks) is a viral particle or composition. It may be placed immediately before, inclusion or immediately after administration of at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks or at least 50 weeks. Optionally, the factor IX activity level is at least 20% at about 10 weeks after administration of the polynucleotide, viral particle or composition, and the factor IX activity level is maintained at at least 20% for a continuous period of at least 40 weeks immediately after that time point. Optionally, the factor IX activity level is at least 25% at 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 after that time point. Optionally, the factor IX activity level is at least 40% at about 10 weeks after administration of the polynucleotide, viral particle or composition, and the factor IX activity level is maintained at at least 40% for a continuous period of at least 40 weeks immediately after that time point.

Optionally, the factor IX activity level is at least 20% at about 52 weeks after administration of the polynucleotide, viral particle or composition, and the factor IX activity level is maintained at at least 20% for a continuous period of at least 40 weeks immediately after that time point. Optionally, the factor IX activity level is at least 25% 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 after that time point. Optionally, the factor IX activity level is at least 40% at about 52 weeks after administration of the polynucleotide, viral particle or composition, and the factor IX activity level is maintained at at least 40% for a continuous period of at least 40 weeks immediately after that time point.

Factor IX activity in a patient such as a human patient can be measured by taking a blood sample from the patient or by performing an analysis on a blood sample taken from the patient.

Optionally, the level of factor IX activity can be measured using a coagulation assay. Optionally, the coagulation analysis is a one-step coagulation analysis. Optionally, coagulation assays, such as a one-step coagulation assay, use the ^{SynthASil™ kit.} In one embodiment, a suitable coagulation assay (one step coagulation assay) is as follows.

Because factor IX is part of the coagulation cascade, an increased activity factor IX polypeptide will catalyze blood clotting more rapidly than a less active factor IX polypeptide. Factor IX polypeptide is mixed with platelet defective plasma and incubated at 37°C. Then add phospholipids and ^{contact activation pathway activators such as SynthASil TM} APTT (Activated Partial Thromboplastin Time) reagent. Then calcium is added and the user measures the time it takes for clotting to occur. Coagulation formation can be assessed using either the spectrophotometric method or the magnetic steel ball method. For example, coagulation formation can be considered to have occurred if the optical density of the mixture exceeds a threshold value or if the mixture has solidified enough to change the motion of the magnetic balls sensed by the sensor.

Optionally, factor IX activity is measured using a ^SynthASil™ 1 step coagulation assay kit to determine factor IX activity in blood samples.

Optionally, the patient uses factor IX having an activity lower than that of factor IX expressed from viral particles comprising (i) a factor IX nucleotide sequence of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory factor of SEQ ID NO: 6 This is a patient who is refractory to treatment. Optionally, the patient has a factor IX polypeptide expressed at a lower level than that encoded by a nucleotide sequence comprising (i) a factor IX nucleotide sequence of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory factor of SEQ ID NO: 6 This is a patient who is refractory to treatment with As demonstrated in the examples, the viral particles comprising (i) a factor IX nucleotide sequence of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory factor of SEQ ID NO: 6 express a factor IX polypeptide at a high level, wherein the Polypeptides have higher activity compared to polypeptides expressed from other factor IX nucleotide sequences. Thus, the viral particles comprising (i) the factor IX nucleotide sequence of SEQ ID NO: 4 or 5 and (ii) the transcriptional regulatory factor of SEQ ID NO: 6 are patients in need of a lower dose of viral particles or a virus comprising a replacement factor It can be used to treat patients who have difficulty expressing the factor IX nucleotide sequence from the particles, for example, patients who have lost part of the liver and are unable to express high levels of the factor IX nucleotide sequence in the liver cells.

Optionally, the method or use includes prophylactic co-administration of a steroid, ie an appropriate dose of the steroid is administered to the patient. Co-administration may be involved. That is, the patient receives the dose of the polynucleotide, vector, or composition of the present invention at the same time the dose of the steroid. Alternatively, the steroid may be administered before or after the polynucleotide, vector or composition of the present invention is administered to the patient. Optionally, the steroid is administered 6 to 12 weeks after administration of the polynucleotide, vector or composition of the present invention to the patient.

Optionally, the steroid is selected from the group consisting of prednisone, betamethasone, prednisolone, triamcinolone, methyl prednisolone and dexamethasone. Optionally, the steroid is prednisolone.

Sequence list

Sequence identity number Sequence description One Codon optimization site of TI-ACNP-FIX-GoF coding sequence 2 Wild type of TI-ACNP-FIX-GoF coding sequence containing intron 3 Truncated FIX intron 1A 4 Coding sequence of TI-ACNP-FIX-GoF 5 Coding sequence of TI-ACNP-FIX-GoF factor IX sequence containing intron 6 HLP2 transcriptional regulatory element sequence 7 LP1 transcriptional regulatory element sequence 8 "Mature" factor IX amino acid sequence encoded by SEQ ID NO: 4 9 Wild type factor IX (Malmo B variant) coding sequence 10 Mut C capsid polypeptide sequence 11 FIXco coding sequence with TTG GoF codon 12 FIXco coding sequence 13 Enhancer element from HLP2 14 Promoter element from HLP2 15 Wild-type site of TI-ACNP-FIX-GoF coding sequence with intron removed 16 "Immature" Factor IX amino acid sequence encoded by SEQ ID NO: 4 17 AAV5 capsid polypeptide sequence 18 Coding sequence of TI-codop-FIX-GoF factor IX sequence comprising intron 19 Wild-type factor IX (Malmo B variant) coding sequence corresponding to mature FIX polypeptide 20 AAV2-5 hybrid VP1u variant 1 21 AAV2-5 hybrid VP1u variant2 22 AAV2-5 hybrid VP1u variant3 23 AAV2-5 hybrid VP1u variant4 24 AAV2-5 hybrid VP1u variant 5 25 AAV2-5 hybrid VP1u variant 6 26 FIXco coding sequence with CTG GoF codon

In the following examples, the experiments are the FIX transgene cassette ssLP1.FIXco (herein FIXco), ssHLP2.TI-codop-FIX-GoF (herein HTFG) and ssHLP2.TI-ACNP-FIX-GoF (herein). HTAG). SsHLP2.TI-codop-FIX-GoF is a version of the ssHLP2.TI-codop-FIX construct disclosed in WO2016/075473 which has been modified to encode leucine (L) instead of arginine (R) at position 384 of the encoded FIX polypeptide. to be. These cassettes are shown in FIG. 1. ssLP1.FIXco contains a fully codon-optimized FIX coding sequence (SEQ ID NO: 12) preceded by the SV40 intron 5'with expression driven by the LP1 promoter (SEQ ID NO: 7). ssHLP2.TI-codop-FIX-GoF and ssHLP2.TI-ACNP-FIX-GoF have a shorter HLP2 transcriptional regulatory element (SEQ ID NO: 6) 5'for the FIX coding sequence interrupted by the truncated version of native intron 1A. Share the structure having, which means that some of the exon 1 and exon 2 nucleotide sequences are wild-type (not codon optimized) and the remaining coding sequences are codon optimized (SEQ ID NO: 18 for HTFG and SEQ ID NO: 5 for HTAG). The nucleotide sequence of the codon-optimization site is different between each of the two constructs. Unlike the wild-type FIX protein encoded by ssLP1.FIXco, ssHLP2.TI-codop-FIX-GoF and ssHLP2.TI-ACNP-FIX-GoF are leucine (L) of arginine (R) at position 384 of the FIX polypeptide. It encodes a hyper-active FIX with a substitution of.

Example 1- Method

AAV vector production and quantification

1.The AAV vector stock was transfected with a standard triple plasmid into human embryonic kidney (HEK293) cells with a combination of vector genomic plasmids, adenovirus helper plasmids, and packaging plasmids containing AAV Rep and Cap (AAV8 or AAVMut) functions. Ready. Because the recombinant AAV particles contain a genome based on AAV2 and a synthetic capsid comprising a capsid of serotype 8 or a site from two serotypes ('Mut C'; SEQ ID NO: 10), it is'pseudotyped' It is called as'.

2. The vector was purified by density gradient centrifugation using iodixanol.

3. The vector genome was titrated by qPCR using primers that bind to the promoter region.

In vitro transduction and FIX expression detection

1. HUH7 cells were plated at ⁵ ×10 5 cells per well in 12-well plates.

2. Cells were stimulated with mitomycin C for 1 hour prior to transduction with AAV particles bearing the FIX-encoding transgene cassette.

3. Five days after transduction, the supernatant was collected and FIX levels were analyzed using a commercial ELISA kit (Stago Asserachrom IX: Ag kit Ref #00943). The activity of FIX was analyzed using a commercially available color development kit manufactured by Quadratech (Biophen FIX (6) kit Ref #221806).

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

FIX detection in vivo

^{1. Adult C57BL/6 mice were injected with a 5x10 10} vector genome (vg) of AAV particles carrying FIX-encoding transgene cassettes (n = 4 per group) through the tail vein.

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

3. For DNA extraction for vector genome analysis (Qiagen DNeasy Blood and Tissue kit, Ref #69506), livers were harvested and immediately frozen in liquid nitrogen before storage at -80°C.

4. The level of FIX present in rat plasma was determined using the FIX ELISA kit (Stago Asserachrom IX: Ag kit REF 00943). The activity of human FIX was determined using a commercially available color development kit from Quadratech (Biophen FIX (6) kit Ref #221806).

Example 2-In vitro FIX transgene expression analysis using ELISA

HUH7 (human hepatocyte) cells were cultured in standard cell culture conditions. The FIX transgene was expressed by treating HUH7 cells with mitomycin C for 1 hour and subsequently transducing pseudotyped ssAAV2/Mut C. In addition to the AAV helper and packaging plasmid, HEK293 cells were transduced with the recombinant vector genomic plasmid and cultured for an additional 48 hours to prepare AAV particles first. The ssAAV2/Mut C vector was purified from HEK293 cells by density gradient centrifugation and iodisanol. The vector genome was titrated by qPCR using primers for the promoter region of the transgene expression cassette. The relative ability to express the FIX transgene in vitro was determined by comparing the FIX expression cassette and measuring the FIX level 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. The FIX level of the culture supernatant was analyzed using a commercially available ELISA kit (Stago Asserachrom IX: Ag kit Ref #00943). In two separate experiments (see Figures 2B and 4B), FIX expression, derived from an ELISA assay utilizing the supernatant of HUH7 cultured cells, was performed using the Qiagen DNeasy Blood and Tissue Kit (Ref #69506) to detect HUH7 cell DNA. After harvesting, it was normalized to a copy of the vector genome per cell.

On day 5 after transduction with ssAAV2/Mut C.HLP2.TI-codop-FIX-GoF and ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF of ¹ ×10 3 vector genome (vg) MOI, ssAAV2/Mut C FIX levels were higher in HUH7 cells transduced with ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF than in HUH7 cells transduced with .HLP2.TI-codop-FIX-GoF (n=2; Fig. 2A) , 3 and 4A). Similarly, when the same FIX expression assay was performed in HUH7 cells with increased MOI (5x10 ³ and 1x10 ⁴ ), ssAAV2/Mut C compared to cells transduced with ssAAV2/Mut C.HLP2.TI-codop-FIX-GoF. FIX levels were higher in cells transduced with .HLP2.TI-ACNP-FIX-GoF (n=2; FIGS. 2A, 3 and 4A). When the FIX expression level was normalized to the viral vector genome copy per cell ^{, the FIX level at each of the three MOIs tested (1x10 3} , 5x10 3 and 1x10 ⁴ ) was ssAAV2/MutC.HLP2.TI-codop-FIX- It was consistently higher in HUH7 cells transduced with ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF compared to GoF (n=2, FIGS. 2B and 4B). ^{Combining the 5x10 3} transduction data obtained in the experiments of FIGS. 2, 3 and 4 in ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF compared to ssAAV2/Mut C.HLP2.TI-codop-FIX-GoF It shows significantly superior expression (Fig. 5).

Example 3-In vitro FIX transgene activity assay

FIX activity was evaluated by harvesting HUH7 cell supernatant 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 ssAAV2/Mut C transduction (MOI 1x10 ³ , 5x10 ³ and 1x10 ^The relative FIX activity according to 4) was determined. After 5 days of transduction, the supernatant was isolated from HUH7 cells and FIX activity was measured using the BIOPHEN Factor IX kit. Regardless of the MOI, a larger average FIX activity was observed in the supernatant derived from cells transduced with ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF (Figs. 6-8). ^{Combining the 5x10 3} transduction data obtained in the experiments of FIGS. 6 and 7 was significantly greater in ssAAV2/Mut C.HLP2.TI-ACNP-FIX-GoF compared to ssAAV2/Mut C.HLP2.TI-codop-FIX-GoF. It shows excellent expression (Fig. 9).

Example 4-Analysis of FIX transgene expression in vivo using ELISA

The FIX transgene was expressed in C57B1/6 mice after transfection with a pseudotyped ssAAV2/8 vector ^{of 5x10 10 vector genome (vg) by tail vein injection.} In addition to the AAV helper and packaging plasmid, HEK293 cells were transduced with the recombinant vector genomic plasmid and cultured for an additional 48 hours to prepare AAV particles first. The ssAAV2/8 vector was purified from HEK293 cells by density gradient centrifugation and iodisanol. The vector genome was titrated by qPCR using primers for the promoter region of the transgene expression cassette. The relative ability to express the FIX transgene was determined by comparing the FIX expression cassettes LP1.FIXco, HLP2.TI-codop-FIX-GoF and HLP2.TI-ACNP-FIX-GoF.

In the first experiment involving ssAAV2/8.HLP2.TI-codop-FIX-GoF and ssAAV2/8.LP1.FIXco, blood was collected from anesthetized mice via cardiac puncture 2 weeks after administration. Then, sodium citrate (1/10 dilution) was added and the plasma was separated by centrifugation at 3000xg for 15 minutes at 4°C. The circulating level of FIX was determined using the FIX ELISA kit (Stago Asserachrom IX: Ag kit Ref #00943).

The FIX expression level was normalized to the copy of the vector genome per cell after mouse liver harvest. It was determined that the normalized FIX expression level was significantly higher after transduction with ssAAV2/8.HLP2.TI-codop-FIX-GoF compared to ssAAV2/8.LP1.FIXco (p <0.05) (n=4 mice. ; Fig. 10).

In further experiments, C57B1/6 mice were transduced with ssAAV2/8 by comparing in vivo a partially codon-optimized FIX transgene (HLP2.TI-codop-FIX-GoF and HLP2.TI-ACNP-FIX-GoF) in vivo. Relative FIX expression after one was determined. At the same time, FIX expression was determined after transduction of C57Bl/6 mice with the scAAV2/8.LP1.FIXco vector. Plasma was isolated from mice 3 weeks after administration and FIX antigen levels were determined using ELISA analysis. Mean FIX expression levels were lowest in mice transduced with scAAV2/8.LP1.FIXco, while higher in mice transduced with ssAAV2/8.HLP2.TI-codop-FIX-GoF (n=4 mice; Fig. 11A). Mice transduced with ssAAV2/8.HLP2.TI-ACNP-FIX-GoF showed significantly greater FIX expression than mice transduced with ssAAV2/8.HLP2.TI-codop-FIX-GoF (n=4 Mouse; Figure 11A). The trend of FIX expression was maintained when the FIX expression level was normalized to the viral vector genomic copy per cell, whereby ssAAV2/8.HLP2.TI-ACNP-FIX-GoF became two ssAAV2/8.HLP2.TI-codop- Produces much more FIX than FIX-GoF and scAAV2/8.LP1.FIXco (n=4; Figure 11B). In addition, ssAAV2/8.HLP2.TI-codop-FIX-GoF showed greater FIX expression than scAAV2/8.LP1.FIXco (n=4 mice; Fig. 11B).

Example 5-In vivo FIX transgene activity assay

The BIOPHEN Factor IX kit (Quadratech #221806, #222101, #223201) is a color development assay for measuring Factor IX activity in human citrated plasma or Factor IX concentrate using standard color development methods.

In the presence of thrombin, phospholipid and calcium, the first factor XIa supplied to the assay in excess at a constant concentration activates FIX present in the tested sample with FIXa to form an enzyme complex with thrombin activating factor VIII:C, and further, Phospholipids (PLP) and calcium, which activate factor X present in the assay system as factor Xa, are supplied to the assay in a constant concentration in excess. This activity is directly related to the amount of limiting factor IX. The resulting factor Xa is accurately measured by the specific activity on the factor Xa chromogenic substrate (SXa-11). Factor Xa cleaves the substrate and releases pNA. The amount of pNA produced is directly proportional to factor IXa activity. Finally, there is a direct relationship between the amount of factor IX and the generated factor Xa activity in the analyzed sample, measured as the amount of pNA released, and determined by the color development at 405 nm.

In vivo comparison of partial codon-optimized FIX transgenes (HLP2.TI-codop-FIX-GoF and HLP2.TI-ACNP-FIX-GoF) to determine relative FIX activity after transduction of C57B1/6 mice with ssAAV2/8 I did. At the same time, FIX activity was determined after transduction of C57Bl/6 mice with scAAV2/8.LP1.FIXco. Plasma was isolated from mice 3 weeks after administration, and FIX activity was determined using the BIOPHEN Factor IX kit. Mean FIX activity was lowest in mice transduced with scAAV2/8.LP1.FIXco, whereas it was greater in mice transduced with ssAAV2/8.HLP2.TI-codop-FIX-GoF (n=4 mice; FIG. 12 ). Mice transduced with ssAAV2/8.HLP2.TI-ACNP-FIX-GoF had significantly greater FIX activity than mice transduced with ssAAV2/8.HLP2.TI-codop-FIX-GoF (n=4 Mouse; Fig. 12).

Example 6

Two hemophilia type B patients were treated with the AAV2/Mut C vector 4.5× ^{10 11} vg/kg containing ssHLP2.HTAG. Both patients were known to have severe hemophilia type B with an endogenous FIX activity of less than 1% at screening. Patient 1 was undergoing prophylaxis at baseline and continued to take prophylaxis during the first 4 days of the experiment. Within 4 weeks after receiving a single injection of the AAV2/Mut C vector containing ssHLP2.HTAG, both patients achieved a level of FIX activity in excess of 30% within 4 weeks. Patients achieved a stable factor IX activity level of 40.5 +/- 4.5% at 52 weeks as assessed by a stage 1 coagulation assay using the SynthASil ^{™ kit.} The prophylactic steroid (prednisolone) was administered for 6 to 12 weeks.

Neither patient experienced spontaneous or prolonged bleeding. Patient 1 had traumatic bleeding due to amputation of the right finger at week 10 after treatment. Importantly, the patient did not need an exogenous clotting factor and the bleeding resolved on the same day. At the time of the accident, the patient's FIX activity was measured as 38%.

In addition, the invention described herein relates to the following aspects:

1. A polynucleotide comprising a factor IX nucleotide sequence, wherein the factor IX nucleotide sequence includes a coding sequence encoding a factor IX protein or a fragment thereof, and the region of the coding sequence is not a wild-type polynucleotide.

2. The polynucleotide according to the first aspect, wherein the region of the non-wild-type coding sequence is codon-optimized.

3. A polynucleotide comprising a factor IX nucleotide sequence, wherein the factor IX nucleotide sequence includes a coding sequence encoding a factor IX protein or fragment thereof, and the coding sequence includes:

(i) a sequence that is 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 1; And

(ii) a sequence that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 15.

4. The polynucleotide according to the third aspect, wherein the sequence identical to SEQ ID NO: 1 and at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% is codon-optimized.

5. A polynucleotide comprising a factor IX nucleotide sequence, wherein the factor IX nucleotide sequence encodes a factor IX protein or fragment thereof, and SEQ ID NO: 5 and 97% or more, 98% or more, 99% or more, 99.5% or more, 99.8 % Or more, or 100% identity.

6. The polynucleotide according to the fifth aspect, wherein the factor IX nucleotide sequence comprises a coding sequence and a region of the coding sequence is codon-optimized.

7. The polynucleotide according to any one of the above-described aspects, wherein the polynucleotide comprises DNA or RNA.

8. The polynucleotide of any one of the 2nd, 4th, 6th or 7th aspect, wherein the region of the codon optimized coding sequence is a contiguous portion.

9. The polynucleotide of aspect 2, 4, 6, 7 or 8, wherein the region of the codon optimized coding sequence is a codon optimized for expression in human liver.

10. The polynucleotide according to any one of the preceding embodiments, wherein the polypeptide encoded by the factor IX nucleotide sequence is expressed in human liver cells at a higher level compared to the reference wild-type factor IX nucleotide sequence.

11.The 2nd, 4th, 6th or 7th aspect, wherein the region of the codon optimized coding sequence is at least 800, at least 900, at least 1100, less than 1500, less than 1300, less than 1200, 800 to 1500. , 900 to 1300, 1100 to 1200, or about 1191 nucleotides in length.

12. The polynucleotide of any of the 2, 4 or 6 to 11 aspects, wherein the region of the codon optimized coding sequence comprises 1, 2, 3, 4, 5 or all of the following:

a) a region of at least 10, at least 15, at least 20, less than 25, 10 to 25, 15 to 25, or 20 to 25 nucleotides of exon 3 or exon 3;

b) exon 4 or a portion of at least 80, at least 90, at least 100, less than 114, 80 to 114, 90 to 114, or 100 to 114 nucleotides of exon 4;

c) exon 5 or a region of 90 or more, 100 or more, 110 or more, less than 129, 90 to 129, 100 to 129, or 110 to 129 nucleotides of exon 5;

d) a region of at least 150, at least 180, at least 200, less than 203, 150 to 203, 180 to 203, or 200 to 203 nucleotides of exon 6 or exon 6;

e) a portion of exon 7 or exon 7 of 70 or more, 80 or more, 90 or more, 100 or more, less than 115, 70 to 115, 80 to 115, 90 to 115, or 100 to 115 nucleotides; And/or

f) Exon 8 or a region of 400 or more, 450 or more, 500 or more, less than 548, 400 to 548, 450 to 548, or 500 to 548 nucleotides of exon 8.

13. The polynucleotide of aspect 12, wherein the region of the codon optimized coding sequence comprises a), b), c), d), e) and f).

14. The method of embodiment 12 or 13, wherein the region of the codon-optimized coding sequence is a region of 20 or more nucleotides of exon 3, a region of 100 or more nucleotides of exon 4, a region of 110 or more nucleotides of exon 5, exon A polynucleotide comprising a region of at least 180 nucleotides of 6, a region of at least 100 nucleotides of exon 7 and a region of at least 500 nucleotides of exon 8.

15. The polynucleotide of any one of embodiments 12-14, wherein the region of the codon optimized coding sequence comprises exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8.

16. The 2nd, 4th or 6th to 15th aspect, wherein the region of the codon optimized coding sequence comprises a region of exon 2, wherein the region of exon 2 is less than 160, less than 150, less than 100, 75 A polynucleotide of less than, less than 60, 20 or more, 30 or more, 40 or more, 50 or more, 20 to 160, 30 to 150, 30 to 100, 40 to 75, or about 56 nucleotides in length.

17. The polynucleotide of any one of embodiments 2, 4 or 6 to 16, wherein the region of the codon optimized coding sequence comprises an exon 2 region that is 30 to 100 nucleotides in length.

18. The polynucleotide of any of embodiments 2, 4 or 6-17, wherein the region of the codon optimized coding sequence comprises a reduced number of CpGs compared to the corresponding portion of the reference wild-type factor IX sequence. .

19. The polynucleotide of aspect 18, wherein the region of the codon optimized coding sequence comprises less than 40, less than 20, less than 18, less than 10, less than 5 or less than 1 CpG.

20. The polynucleotide of aspect 18 or 19, wherein the region of the codon optimized coding sequence is CpG-free.

21.The 2nd, 4th or any one of 6-20 aspect, wherein the region of the codon optimized coding sequence is 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, A polynucleotide, wherein at least 70% or at least 73% 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.

22. The 2nd, 4th or 6th to 21st aspect, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more, 4 or more or 5 or more codons encoding phenylalanine are replaced by TTC compared to the 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 rest are TTT; And/or

d) The codon encoding phenylalanine is TTC, except when the next codon starts with G. Polynucleotide.

23. The 2nd, 4th or 6th to 22nd aspect, at the site of the codon optimized coding sequence,

a) 5 or more, 10 or more, 15 or more or 16 or more codons encoding leucine are replaced by CTGs compared to the reference wild-type factor IX sequence;

b) at least 90% or at least 94% codons encoding leucine are CTGs; And/or

c) At least 90% or at least 94% of the codons encoding leucine are CTGs and the rest are CTCs.

24. The 2nd, 4th or 6th to 23rd aspect, at the site of the codon optimized coding sequence,

a) 5 or more, 10 or more, 11 or more or 12 or more codons encoding isoleucine are replaced by ATC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then at least one codon ATC is replaced with an ATT compared to the reference wild type factor IX sequence;

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 is ATT; And/or

e) The codon encoding isoleucine is ATC, except when the next codon starts with G. Polynucleotide.

25. The method according to any one of the second, 4 or 6 to 24 aspects, at the site of the codon optimized coding sequence,

a) 10 or more, 15 or more, 20 or more or 25 or more codons encoding valine are replaced by GTG compared to the reference wild-type factor IX sequence;

b) one or more codons encoding valine are replaced by GTC compared to the reference wild-type factor IX sequence;

c) at least 80%, at least 90% or at least 95% of the codons encoding valine are GTGs; And/or

d) At least 80%, at least 90% or at least 95% of codons encoding valine are GTG and the remainder is GTC.

26. The method of any one of the 2nd, 4th or 6-25th embodiments, at the site of the codon optimized coding sequence,

a) 5 or more, 10 or more, 12 or more or 13 or more codons encoding serine are replaced by AGC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then one or more, two or more, or four or more codons encoding serine are replaced by TCT compared to the reference wild-type factor IX sequence;

c) at least 60%, at least 65% or at least 70% of the codons encoding serine are AGC; And/or

d) 60% or more, 65% or more, or 70% or more of the codons encoding serine are AGC and the remainder is TCT or TCC.

27. The 2nd, 4th or 6th to 26th aspect, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more or 5 or more codons encoding proline are replaced by CCC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then one or more codons encoding proline are replaced by CCT compared to the reference wild-type factor IX sequence;

c) at least 50%, at least 55% or at least 60% of the codons encoding proline are CCCs;

d) 50%, 55% or more or 60% or more codons encoding proline are CCC and the remainder are CCA or CCT; And/or

e) The codon encoding proline is CCC, except when the next codon starts with G. Polynucleotide.

28. The 2nd, 4th or 6th to 27th aspect, at the site of the codon optimized coding sequence,

a) 6 or more, 7 or more, 8 or more, or 10 or more codons encoding threonine are replaced by ACC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then one or more or two or more codons encoding threonine are replaced by ACT compared to the reference wild-type factor IX sequence;

c) at least 45%, at least 50% or at least 55% of the codons encoding threonine are ACCs;

d) at least 45%, at least 50% or at least 55% of the codons encoding threonine are ACC and the remainder is ACT; And/or

e) A polynucleotide, wherein the codon encoding threonine is ACC, except when the next codon starts with G.

29. The 2nd, 4th or 6th to 28th aspect, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more, 3 or more or 4 or more codons encoding alanine are replaced by GCC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then one or more, two or more, or three or more codons encoding alanine are replaced by GCT compared to the reference wild-type factor IX sequence;

c) 35% or more, 40% or more or 43% or more codons encoding alanine are GCC;

d) 35% or more, 40% or more or 45% or more encoding alanine are GCC and the remainder is GCT; And/or

e) A polynucleotide, wherein the codon encoding alanine is GCC, except when the next codon starts with G.

30. The 2nd, 4th or 6th to 29th aspect, at the site of the codon optimized coding sequence,

a) one or more or two or more codons encoding tyrosine are replaced by TAC compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then at least one codon TAC is replaced with a TAT compared to the reference wild-type factor IX sequence;

c) at least 40%, at least 45% or at least 48% codons encoding tyrosine are TACs;

d) 40% or more, 45% or more or 48% or more codons encoding tyrosine are TAC and the remainder is TAT; And/or

e) Polynucleotide, wherein the codon encoding tyrosine is TAC, except when the next codon starts with G.

31.The 2nd, 4th or 6th to 30th aspect, at the site of the codon optimized coding sequence,

a) one or more codons encoding histidine are replaced by CAC compared to the reference wild-type factor IX sequence;

b) at least 50%, at least 60% or at least 65% of the codons encoding histidine are CACs;

c) 50% or more, 60% or more or 65% or more codons encoding histidine are CAC and the remainder is CAT; And/or

d) A polynucleotide, wherein the codon encoding histidine is CAC, except when the next codon starts with G.

32. The method of any one of the 2nd, 4th or 6-31 embodiments, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more, 4 or more or 5 or more codons encoding glutamine are replaced by CAG compared to the reference wild-type factor IX sequence;

b) at least one codon CAG is replaced by CAA compared to the reference wild-type factor IX sequence;

c) at least 80%, at least 85% or at least 90% of the codons encoding glutamine are CAGs; And/or

d) At least 80%, at least 85% or at least 90% of codons encoding glutamine are CAG and the remainder is CAA.

33. The 2nd, 4th or 6th to 32nd embodiment, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more, 4 or more or 5 or more codons encoding asparagine are replaced by AACs compared to the reference wild-type factor IX sequence;

b) at least 60%, at least 65% or at least 70% of the codons encoding asparagine are AAC;

c) at least 60%, at least 65% or at least 70% of the codons encoding asparagine are AAC and the rest are AAT; And/or

e) A polynucleotide, wherein the codon encoding asparagine is AAC, except when the next codon starts with G.

34. The method according to any one of the second, 4 or 6 to 33 aspects, at the site of the codon optimized coding sequence,

a) 5 or more, 7 or more, 8 or more or 9 or more codons encoding lysine are replaced with AAG compared to the reference wild-type factor IX sequence;

b) at least one codon AAG is replaced by AAA compared to the 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 is AAA.

35. The method according to any one of the 2, 4 or 6 to 34 aspects, at the site of the codon optimized coding sequence,

a) 1 or more, 2 or more, 3 or more or 4 or more codons encoding aspartate are replaced by GACs compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then at least one codon GAC is replaced by a GAT compared to the reference wild-type factor IX sequence;

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 rest are GAT; And/or

e) A polynucleotide, wherein the codon encoding aspartate is GAC, except when the next codon starts with G.

36. The second, fourth or any one of embodiments 6 to 35, at the site of the codon optimized coding sequence,

a) 15 or more, 20 or more, 25 or more or 26 or more codons encoding glutamate are replaced by GAGs compared to the 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 codons encoding glutamate are GAG and the remainder is GAA.

37.The according to any one of embodiments 2, 4 or 6 to 36, at the site of the codon optimized coding sequence,

a) 5 or more, 6 or more, 7 or more or 8 or more codons encoding cysteine are replaced by TGCs compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then at least one codon TGC is replaced by a TGT compared to the reference wild-type factor IX sequence;

c) at least 40%, at least 50% or at least 55% codons encoding cysteine are TGCs;

d) at least 40%, at least 50% or at least 55% of the codons encoding cysteine are TGCs and the remainder are TGTs; And/or

e) Polynucleotide, wherein the codon encoding cysteine is TGC, except when the next codon starts with G.

38. The polynucleotide of any of embodiments 2, 4 or 6 to 37, wherein at the site of the codon optimized coding sequence, the codon encoding tryptophan is TGG.

39. The second, fourth or any one of embodiments 6 to 38, at the site of the codon optimized coding sequence,

a) 5 or more, 8 or more, 10 or more or 11 or more codons encoding arginine are replaced by AGG compared to the reference wild-type factor IX sequence;

b) one or more codons encoding arginine are replaced by AGA compared to the 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) A polynucleotide, wherein at least 60%, at least 70% or at least 75% of the codons encoding arginine are AGG and the remainder is AGA.

40. The 2nd, 4th or 6th to 39th aspect, at the site of the codon optimized coding sequence,

a) 5 or more, 10 or more, 12 or more or 13 or more codons encoding glycine are replaced by GGCs compared to the reference wild-type factor IX sequence;

b) if the next codon starts with G, then 5 or more, 6 or more, 7 or more or 8 or more codons encoding glycine are replaced by GGG compared to the reference wild-type factor IX sequence;

c) at least 50%, at least 55% or at least 60% codons encoding glycine are GGCs;

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) A polynucleotide, wherein the codon encoding glycine is GGC, except when the next codon starts with G.

41.The 2nd, 4th or any one of 6-40 embodiments, wherein the region of the codon optimized coding sequence is phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, A polynucleotide comprising codons encoding lysine, aspartate, glutamate, cysteine, tryptophan, arginine and glycine.

42. The 2nd, 4th or 6th to 41th aspect, wherein the region of the codon optimized coding sequence is phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, Including codons encoding lysine, aspartate, glutamate, cysteine, tryptophan, arginine and glycine, and the codon-optimized site,

a) 5 or more codons encoding phenylalanine are replaced by TTC compared to the reference wild-type factor IX sequence;

b) 16 or more codons encoding leucine are replaced by CTG compared to the reference wild-type factor IX sequence;

c) 12 or more codons encoding isoleucine are replaced by ATC compared to the reference wild-type factor IX sequence;

d) 25 or more codons encoding valine are replaced by GTG compared to the reference wild-type factor IX sequence;

e) 13 or more codons encoding serine are replaced by AGC compared to the reference wild-type factor IX sequence;

f) 5 or more codons encoding proline are replaced by CCC compared to the reference wild-type factor IX sequence;

g) 10 or more codons encoding threonine are replaced by ACC compared to the reference wild-type factor IX sequence;

h) 4 or more codons encoding alanine are replaced by GCC compared to the reference wild-type factor IX sequence;

i) two or more codons encoding tyrosine are replaced by TAC compared to the reference wild-type factor IX sequence;

j) one or more codons encoding histidine are replaced by CAC compared to the reference wild-type factor IX sequence;

k) 5 or more codons encoding glutamine are replaced by CAG compared to the reference wild-type factor IX sequence;

l) 5 or more codons encoding asparagine are replaced by AAC compared to the reference wild-type factor IX sequence;

m) 9 or more codons encoding lysine are replaced by AAG compared to the reference wild-type factor IX sequence;

n) 4 or more codons encoding aspartate are replaced by GAC compared to the reference wild-type factor IX sequence;

o) 26 or more codons encoding glutamate are replaced by GAG compared to the reference wild-type factor IX sequence;

p) 8 or more codons encoding cysteine are replaced by TGC compared to the reference wild-type factor IX sequence;

q) the codon encoding tryptophan is TGG;

r) 11 or more codons encoding arginine are replaced by AGG compared to the reference wild-type factor IX sequence; And

s) 13 or more codons encoding glycine are replaced by GGC compared to the reference wild-type factor IX sequence.

43. The 2nd, 4th or 6th to 42th aspect of the invention, wherein the region of the codon optimized coding sequence is phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, Including codons encoding lysine, aspartate, glutamate, cysteine, tryptophan, arginine and glycine, and the codon-optimized site,

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

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

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 CCCs;

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 codons encoding tyrosine are TAC;

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

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 encoding aspartate are GAC;

o) 95% or more of the codons encoding glutamate are GAG;

p) at least 55% of the codons encoding cysteine are TGC;

q) the codon encoding tryptophan is TGG;

r) at least 75% of the codons encoding arginine are AGG; And

s) A polynucleotide, wherein at least 60% of the codons encoding glycine are GGC.

44. The 2nd, 4th or 6th to 43th aspect, wherein the region of the codon optimized coding sequence is phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, Including codons encoding lysine, aspartate, glutamate, cysteine, tryptophan, arginine and glycine, and the codon-optimized site,

a) at least 70% of the codons encoding phenylalanine are TTC and the remainder is TTT;

b) more than 94% of the codons encoding leucine are CTG and the rest are CTC;

c) 75% or more 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) 60% or more 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 is ACT;

h) 43% or more of the codons encoding alanine are GCC and the rest are GCT;

i) at least 48% of the codons encoding tyrosine are TAC and the rest are TAT;

j) 65% or more of the codons encoding histidine are CAC and the rest are CAT;

k) 90% or more 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) 95% or more 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) 95% or more of the codons encoding glutamate are GAG and the rest are GAA;

p) 55% or more of the codons encoding cysteine are TGC and the remainder are TGT;

q) the codon encoding tryptophan is TGG;

r) at least 75% of the codons encoding arginine are AGG and the rest are AGA; And

s) At least 60% of the codons encoding glycine are GGC and the remainder is GGG.

45. The polynucleotide of any of embodiments 10-44, wherein the reference wild-type factor IX sequence is SEQ ID NO: 9 or SEQ ID NO: 19.

46. The 2nd, 4th or 6th to 45th aspect, wherein the region of the codon optimized coding sequence is at least 800, at least 900, at least 1100, at least 1191, less than 1100, less than 1000 of SEQ ID NO: 1. , 800 to 1191, 900 to 1191, or about 1191 nucleotide fragments and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% The same thing, polynucleotide.

47. The method of aspect 46, wherein the region of the codon-optimized coding sequence is SEQ ID NO: 1 and at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, and 99.8. % Or more or 100% identical.

48. The polynucleotide of aspect 46 or 47, wherein the region of the codon optimized coding sequence is 95% or more identical to the 900 to 1191 nucleotide fragment of SEQ ID NO: 1.

49. The polynucleotide of any one of embodiments 46 to 48, wherein the region of the codon optimized coding sequence is at least 95%, or at least 98% identical to SEQ ID NO: 1.

50. The polynucleotide of any of the preceding embodiments, wherein the coding sequence comprises a region that is not codon optimized.

51.The embodiment 50, wherein the non-codon optimized site is 100 or more, 150 or more, 170 or more, 190 or more, less than 250, less than 225, less than 200, or about 195 nucleotides. Which is a polynucleotide.

52. The method of any one of embodiments 50 or 51, wherein the non-codon optimized site is exon 1 or exon 1 at least 60, at least 70, at least 80, at least 60 to 88, at 70 to 88, or at 80 A polynucleotide comprising a region of to 88 nucleotides.

53. The method of any one of embodiments 50 to 52, wherein the non-codon optimized sites are at least 50, at least 75, at least 80, at least 90, at least 100, less than 140, less than 120 of exon 2. , A polynucleotide comprising a region of 50 to 140, 75 to 120, or about 107 nucleotides.

54. The polynucleotide of any of embodiments 50-53, wherein the codon-optimized site comprises CpG.

55. The polynucleotide of aspect 54, wherein the non-codon optimized site comprises at least 1 or at least 2 CpGs per 100 nucleotides.

56. The polynucleotide of any one of embodiments 50-55, wherein the non-codon optimized site 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.

57. The method of any one of embodiments 50 to 56, wherein the codon-optimized region is at least 100, at least 150, at least 175, less than 195, less than 190, or less than 180 nucleotide fragments of SEQ ID NO: 15. And 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.

58. The method of embodiment 57, wherein the codon-optimized region 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% with SEQ ID NO: 15. , Or 100% identical.

59. The polynucleotide of any of embodiments 50-58, wherein the codon-optimized site is wild-type.

60. The method of any one of embodiments 50 to 59, wherein the codon-optimized region is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100 with SEQ ID NO: 15. % Identical.

61. The polynucleotide of any one of the preceding embodiments, wherein the polynucleotide further comprises an intron and a fragment of an intron that interrupts the coding sequence.

62. The polynucleotide of aspect 61, wherein the intron or fragment of an intron is a region of a wild-type factor IX intron.

63. The embodiment 61 or 62, wherein the fragments of the introns are less than 500, less than 400, less than 350, less than 300, 100 or more, 200 or more, 250 or more, 290 or more, 100 to 500. Polynucleotides of which are dogs, 200 to 400, 250 to 350, or about 299 nucleotides.

64. The method of any one of embodiments 61 to 63, wherein the fragment of the intron is at least 100, at least 200, at least 250, or at least 290 nucleotides of SEQ ID NO: 3 and 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.

65. The method of any one of embodiments 61 to 64, wherein the intron or fragment of the intron is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, and at least 99.8% of SEQ ID NO: 3 Polynucleotides that are more than or equal to 100%.

66. The polynucleotide of aspect 65, wherein the intron or fragment of the intron is 95% or more, or 98% or more identical to SEQ ID NO: 3.

67. The polynucleotide of any one of embodiments 61 to 66, wherein the intron or fragment of an intron cleaves a region that is not codon optimized.

68. The polynucleotide of aspect 67, wherein the intron or fragment of the 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.

69. The poly according to embodiment 68, wherein the intron or fragment of the intron is flanked by 110 to 120 nucleotides not codon-optimized at the 5'end and 100 to 110 nucleotides not codon-optimized at the 3'end. Nucleotides.

70. The polynucleotide of any one of embodiments 61 to 69, wherein the intron or fragment of an intron is located between exon 1 and exon 2.

71. The polynucleotide of any one of embodiments 61 to 70, wherein the intron or fragment of an intron is a fragment of native intron 1 (Intron 1a).

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

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

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

75. The embodiment of 74, wherein the fragments of the A1AT promoter are 100 or more, 120 or more, 150 or more, 180 or more, less than 255, 100 to 255, 150 to 225, 150 to 300 Polynucleotides, or 180 to 255 nucleotides in length.

76. The polynucleotide of aspect 75, wherein the fragment of the A1AT promoter is 150 to 300 nucleotides in length.

77. The polynucleotide of any of embodiments 72-76, wherein the transcriptional regulatory element comprises an enhancer.

78. The polynucleotide according to aspect 77, wherein the enhancer is an HCR enhancer or a fragment of an HCR enhancer.

79.The embodiment 78, wherein the fragments of the HCR enhancer are 80 or more, 90 or more, 100 or more, less than 192, 80 to 192, 90 to 192, 100 to 250, or 117. A polynucleotide that is between dogs and 192 nucleotides in length.

80. The polynucleotide of aspect 79, wherein the fragment of the HCR enhancer is 100 to 250 nucleotides in length.

81.The method of any of embodiments 72 to 80, wherein the transcriptional 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.

82. The polynucleotide of aspect 81, wherein the transcriptional regulatory element is SEQ ID NO: 6.

83. According to any one of the foregoing embodiments, the polynucleotide is SEQ ID NO: 13 and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more , Or a polynucleotide comprising an enhancer that is 100% identical.

84. The polynucleotide of any one of the preceding embodiments, wherein the polynucleotide comprises an enhancer that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 13.

85. The polynucleotide according to any one of the preceding embodiments, wherein the polynucleotide comprises an enhancer of SEQ ID NO: 13.

86. According to any one of the foregoing embodiments, the polynucleotide is SEQ ID NO: 14 and 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 a polynucleotide comprising a promoter that is 100% identical.

87. The polynucleotide according to any one of the preceding embodiments, wherein the polynucleotide comprises a promoter that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 14.

88. The polynucleotide according to any one of the foregoing aspects, wherein the polynucleotide comprises a promoter of SEQ ID NO: 14.

89. According to any one of the foregoing embodiments, 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 the amino acid at a position corresponding to position 384 of wild-type factor IX. The codon encoding for is that encoding alanine or leucine, polynucleotide.

90. The polynucleotide according to embodiment 89, wherein the codon encoding the amino acid at the position corresponding to position 384 of the wild-type factor IX is CTX, and X is an arbitrary nucleotide.

91. The method of any one of the foregoing embodiments, wherein the polynucleotide is at least 1200, at least 1350, or at least 1650 nucleotide fragments of SEQ ID NO: 5 and 80% or more, 85% or more, 90% or more, 95% or more, A polynucleotide comprising at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical Factor IX nucleotide sequences.

92. The method of any one of the foregoing embodiments, wherein the polynucleotide 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% with SEQ ID NO: 5 , Or 100% identical Factor IX nucleotide sequence comprising a polynucleotide.

93. In any one of the foregoing aspects,

(i) the factor IX nucleotide sequence comprises a sequence that is 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 1; And

(ii) The factor IX nucleotide sequence includes a codon encoding an amino acid at a position corresponding to the 384 position of wild-type factor IX, and the codon encoding an amino acid at a position corresponding to the 384 position of the wild-type factor IX encodes leucine. That is, polynucleotides.

94. In any one of the foregoing aspects,

(i) the factor IX nucleotide sequence comprises a coding sequence that is 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 1;

(ii) The factor IX nucleotide sequence includes a codon encoding an amino acid at a position corresponding to the 384 position of wild-type factor IX, and the codon encoding an amino acid at a position corresponding to the 384 position of the wild-type factor IX encodes leucine. and; And

(iii) the polynucleotide sequence comprises a promoter element that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 14, and/or 98% or more, 99% or more, A polynucleotide comprising at least 99.5%, at least 99.8% or at least 100% identical enhancer elements.

95. In any one of the foregoing aspects,

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

(ii) The factor IX nucleotide sequence includes a codon encoding an amino acid at a position corresponding to the 384 position of wild-type factor IX, and the codon encoding an amino acid at a position corresponding to the 384 position of the wild-type factor IX encodes leucine. and; And

(iii) The polynucleotide sequence is a polynucleotide comprising a transcriptional regulatory element that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 6.

96. In any one of the foregoing aspects,

(i) the factor IX nucleotide sequence comprises a sequence that is 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 1;

(ii) the Factor IX nucleotide sequence contains a sequence that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to the corresponding site of SEQ ID NO: 2;

(iii) The factor IX nucleotide sequence includes a codon encoding an amino acid at a position corresponding to the 384 position of wild-type factor IX, and the codon encoding an amino acid at a position corresponding to the 384 position of the wild-type factor IX encodes leucine. That is, polynucleotide.

97. The method of embodiment 95 or 96, wherein the factor IX nucleotide sequence comprises an intron or a fragment of an intron, and the fragment of the intron is SEQ ID NO: 3 and 98% or more, 99% or more, 99.5% or more, 99.8% or more, or A polynucleotide comprising a sequence that is 100% identical.

98. In any one of the foregoing aspects

(i) the Factor IX nucleotide sequence comprises a coding sequence, and the region of the coding sequence is not codon optimized; And

(ii) The factor IX nucleotide sequence includes a codon encoding an amino acid at a position corresponding to the 384 position of wild-type factor IX, and the codon encoding an amino acid at a position corresponding to the 384 position of the wild-type factor IX encodes leucine. That is, polynucleotides.

99. According to any one of the above embodiments, the polypeptide encoded by the factor IX nucleotide sequence is a poly encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional control element of SEQ ID NO: 7 The polynucleotide, which is expressed at a higher level in human liver cells compared to the peptide.

100. The polypeptide according to any one of the above embodiments, wherein the polypeptide encoded by the factor IX nucleotide sequence is a polypeptide encoded by a nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and a transcriptional control element of SEQ ID NO: 6 The polynucleotide, which is expressed in human liver cells at a higher level than.

101. The method according to any one of the preceding embodiments, wherein the polypeptide encoded by the factor IX nucleotide sequence comprises a factor IX nucleotide sequence of SEQ ID NO: 12 or 18 and a transcriptional control element of SEQ ID NO: 7 or SEQ ID NO: 6. A polynucleotide that is expressed in human liver cells at a level that is 2 times or more, or 3 times or more than the polypeptide encoded by the nucleotide sequence.

102. A viral particle comprising a recombinant genome comprising the polynucleotide of any one of the foregoing embodiments.

103. The viral particle of aspect 102, wherein the viral particle is an AAV, adenovirus or lentiviral virus particle.

104. The viral particle of aspect 103, wherein the viral particle is an AAV viral particle.

105. The viral particle of any of embodiments 102-104, wherein the recombinant genome further comprises:

a) AAV2 ITRs;

b) peptide A sequence;

c) origin of replication; And/or

d) two resolvable ITRs.

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

107. The viral particle of any of embodiments 102-106, wherein the viral particle comprises a capsid selected from the group consisting of:

(i) a capsid having 96% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identity with SEQ ID NO: 10;

(ii) a capsid having 96% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identity with SEQ ID NO: 17;

(iii) AAVMutC; And

(iv) AAV5.

108. The method according to any of embodiments 102 to 107, wherein upon transduction into Huh7 cells, the viral particle is a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory factor of SEQ ID NO: 7 and/or factor IX nucleotide of SEQ ID NO: 18. A viral particle that expresses a factor IX protein or fragment thereof having a greater factor IX activity than that of factor IX expressed from the viral particle comprising the sequence and the transcriptional regulatory factor of SEQ ID NO: 6.

109. The viral particle of embodiment 108, wherein the activity is measured using a chromogenic substrate specific for factor Xa.

110. The method of any one of the preceding embodiments, wherein the Factor IX protein fragment is 200 or more, 250 or more, 300 or more, 200 to 415, 250 to 415, or 300 to 415 amino acids in length. Which, polynucleotides or virus particles.

111. The polynucleotide or viral particle according to any one of the preceding embodiments, wherein the Factor IX protein or fragment thereof comprises the following sequence:

a) 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: 8; or

b) 200 or more, 250 or more, 300 or more, 200 to 415, 250 to 415, or 300 to 415 amino acids in length of a fragment of SEQ ID NO: 8 and 95% or more, 98% or more, 99 % Or more, 99.5% or more, 99.8% or more, or 100% identical sequences.

112. A composition comprising the polynucleotide or viral particles of any of the foregoing embodiments and a pharmaceutically acceptable excipient.

113. The polynucleotide, viral particle or composition of any one of the foregoing embodiments for use in a method of treatment.

114. The polynucleotide, viral particle or composition of aspect 113, wherein the method of treatment comprises administering to the patient an effective amount of the polynucleotide, composition, or viral particle of any one of embodiments 1-112.

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

116. Use of the polynucleotide, viral particle or composition of any one of embodiments 1 to 112 in the manufacture of a medicament for use in a method of treatment.

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

118. The polynucleotide, viral particle, composition, use or method of any one of embodiments 113 to 117, wherein the method of treatment is a method of treating hemophilia.

119. The polynucleotide, viral particle, composition, use or method of aspect 118, wherein the hemophilia is hemophilia type B.

120. The polynucleotide, viral particle, composition, use or method of embodiment 119, wherein the patient has an antibody or inhibitor against factor IX.

121. The polypeptide according to any one of embodiments 1 to 112, which is at least 2 times, or at least 3 times more than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7 At a high level, a polynucleotide, viral particle or composition for expressing the polypeptide encoded by the factor IX nucleotide sequence and thus for use in treating a disease.

122. The method according to any one of embodiments 1 to 112, which is at least 2 times, or at least 3 times more than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7 A polynucleotide, viral particle or composition for use in treating a disease by expressing a polypeptide encoded by the factor IX nucleotide sequence at a high level.

123. In the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional control element of SEQ ID NO: 7 comprising administering the polynucleotide, viral particle or composition of any one of embodiments 1 to 112 to a patient. A method of expressing a polypeptide encoded by a factor IX nucleotide sequence at a level that is 2 times or more, or 3 times or more than the polypeptide encoded by.

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

125. By the factor IX nucleotide sequence at a level that is at least 2 times or 3 times higher than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional regulatory element of SEQ ID NO: 7 The use of the polynucleotide, viral particle or composition of any one of embodiments 1 to 112, wherein the encoded polypeptide is expressed and thus a disease is treated.

126. By the factor IX nucleotide sequence of SEQ ID NO: 12 at a level that is at least 2 times or 3 times higher than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence and the transcriptional regulatory element of SEQ ID NO: 7 The use of the polynucleotide, viral particle or composition of any one of embodiments 1 to 112, wherein the disease is treated by expressing the encoded polypeptide.

127. The polypeptide according to any one of embodiments 1 to 112, which is at least 2 times, or at least 3 times more than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6 At a high level, a polynucleotide, viral particle or composition for expressing the polypeptide encoded by the factor IX nucleotide sequence and thus for use in treating a disease.

128. The method according to any one of embodiments 1 to 112, which is at least 2 times, or at least 3 times more than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6 A polynucleotide, viral particle or composition for use in treating a disease by expressing a polypeptide encoded by the factor IX nucleotide sequence at a high level.

129. In the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6, comprising administering the polynucleotide, viral particle or composition of any one of embodiments 1 to 112 to a patient A method of expressing a polypeptide encoded by a factor IX nucleotide sequence at a level of at least 2 times, or at least 3 times higher than the polypeptide encoded by.

130. In the nucleotide sequence comprising the step of administering to a patient the polynucleotide, viral particle or composition of any one of embodiments 1 to 112, and comprising a factor IX nucleotide sequence of SEQ ID NO: 18 and a transcriptional regulatory element of SEQ ID NO: 6 A method of treating a disease by expressing a polypeptide encoded by a factor IX nucleotide sequence at a level of at least 2 times, or at least 3 times higher than the polypeptide encoded by.

131. By the factor IX nucleotide sequence of SEQ ID NO: 18 at a level that is at least 2 times or 3 times higher than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence and the transcriptional regulatory element of SEQ ID NO: 6 The use of the polynucleotide, viral particle or composition of any one of embodiments 1 to 112, wherein the encoded polypeptide is expressed and thus a disease is treated.

132. At a level 2 or more, or 3 or more times higher than the polypeptide encoded by the nucleotide sequence comprising the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional regulatory element of SEQ ID NO: 6, by the factor IX nucleotide sequence The use of the polynucleotide, viral particle or composition of any one of embodiments 1 to 112, wherein the disease is treated by expressing the encoded polypeptide.

133. The method of any one of embodiments 121-122, 125-128, or 131-132, wherein expressing the polypeptide encoded by the factor IX nucleotide sequence is a polynucleotide or virus of any one of the first to 112 aspects. A polynucleotide, viral particle or composition, or use thereof, comprising administering the particle or composition to a patient.

134. The method according to any one of embodiments 1 to 112, comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional control element of SEQ ID NO: 7 and/or a factor IX nucleotide sequence of SEQ ID NO: 18 and a transcription control element of SEQ ID NO: 6 A polynucleotide, viral particle or composition for use in treating a disease by expressing a factor IX protein or fragment thereof having a factor IX activity greater than that of factor IX expressed from the viral particle.

135. A step of administering to a patient the polynucleotide, viral particle or composition of any one of embodiments 1 to 112, comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory element of SEQ ID NO: 7 and/or SEQ ID NO: 18. A method of treating a disease by expressing a factor IX protein or fragment thereof having a factor IX activity greater than that of factor IX expressed from a viral particle comprising a factor IX nucleotide sequence of and a transcriptional regulatory element of SEQ ID NO: 6.

136. Than the activity of factor IX expressed from viral particles comprising the factor IX nucleotide sequence of SEQ ID NO: 12 and the transcriptional control element of SEQ ID NO: 7 and/or the factor IX nucleotide sequence of SEQ ID NO: 18 and the transcriptional control element of SEQ ID NO: 6 The use of the polynucleotide, viral particle or composition of any one of embodiments 1 to 112, wherein the disease is treated by expressing a factor IX protein or fragment thereof having a greater factor IX activity.

137.The method of 134 or 136, wherein expressing the Factor IX protein or fragment thereof comprises administering to the patient the polynucleotide, viral particle or composition of any one of embodiments 1 to 112, Polynucleotides, viral particles or compositions, or uses thereof.

138. The polynucleotide, viral particle or composition according to any one of embodiments 121 to 122, 124 to 128, 130 to 137, wherein the disease is hemophilia, a method of treating or use thereof,

139. The method of embodiment 138, wherein the disease is hemophilia type B, a polynucleotide, a viral particle or composition, a method of treating the same or a use thereof,

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

141. A method of achieving a stable factor IX activity level of at least 20% in a hemophilia B patient comprising administering to the patient an effective amount of the polynucleotide, viral particle or composition of any one of embodiments 1-112.

142. Use of the polynucleotide, viral particle or composition of any one of embodiments 1 to 112 to achieve a level of stable factor IX activity of at least 20% in a patient with hemophilia type B.

143. Hemophilia B according to any one of embodiments 1-112, comprising administering to the patient a dose of the polynucleotide, viral particle or composition effective to achieve a level of stable Factor IX activity of at least 20% in the patient. Polynucleotides, viral particles or compositions for use in the treatment of a type.

144. A hemophilia type B comprising administering to the patient a dose of the polynucleotide, viral particle or composition of any one of embodiments 1-112 in an effective dose to achieve a level of stable factor IX activity of at least 20% in the patient. Method of treatment.

145. Any of the first to 112 aspects of treating hemophilia type B comprising administering to the patient a dose effective to achieve a level of stable factor IX activity of at least 20% in the patient. Use of a single polynucleotide, viral particle or composition.

146. The method of 140, 142, 143 or 145, wherein the treatment of hemophilia type B or achieving a stable factor IX activity level comprises administering to a patient the polynucleotide, viral particle or composition of any one of the first to 112 aspects. Containing, polynucleotides, viral particles or compositions or uses thereof.

147. The embodiment of 141, 144, 146 to 148, wherein the factor IX activity level is at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks from administration of the polynucleotide, viral particle or composition. Or a polynucleotide, viral particle or composition, or a method, or use thereof, which is stabilized after at least 50 weeks.

148. The method of embodiment 123, 124, 129, 130, 133, 135, 137 to 147, wherein the patient has (i) a factor IX nucleotide sequence of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory factor of SEQ ID NO: 6 A polynucleotide, viral particle or composition, or a method, or use thereof, that is a patient refractory to treatment with factor IX having an activity lower than that of factor IX expressed from the viral particle comprising.

149.The method of embodiment 123, 124, 129, 130, 133, 135, 137 to 148, wherein the patient has (i) a factor IX nucleotide sequence of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory factor of SEQ ID NO: 6 A polynucleotide, a viral particle or composition, or a method, or use thereof, that is a patient refractory to treatment with a factor IX polypeptide expressed at a lower level than that encoded by the containing nucleotide sequence.

150. The polynucleotide, virus of any one of embodiments 121-149, wherein the polynucleotide, viral particle or composition is administered in a dose effective to achieve a stable factor IX activity level of 20% or more in a hemophilia type B patient. Particles or compositions, or methods, or uses thereof.

151.The method of any one of embodiments 121 to 150, wherein the polynucleotide, viral particle or composition is at a ^{dose of at least 4.5 x 10 11} vg/kg, or at ^{a dose of less than 5 x 10 11} vg/kg, or from 4.5 x 10 ¹¹ to 1 x 10 ¹² vg/kg, or 4.5 x 10 ¹¹ vg/kg to 4.9 x 10 ¹¹ vg/kg, which is administered at a dose of a polynucleotide, viral particle or composition, or a method, or use thereof.

152. The method of embodiment 151, wherein the dose is determined by quantifying the number of vector genomes using qPCR, and optionally, the qPCR uses a primer that binds to a promoter region, and optionally, the promoter region is a transgene. ) The promoter region of the cassette, polynucleotide, viral particle or composition, or a method, or use thereof.

153. The method of any one of embodiments 121 to 152, wherein the administered polynucleotide or viral particle is produced from a mammalian cell and/or is characterized by the use of a mammalian viral vector producing cell, and has an insect viral vector producing cell (e.g. For example, a polynucleotide, a virus particle or composition, or a method, or use thereof, which is distinct from a vector produced in a baculovirus system).

154. The polynucleotide, viral particle or composition, or method, or use thereof, according to any one of embodiments 140 to 153, wherein the level of stable factor IX activity is at least 25% or at least 30% of stable factor IX activity. .

155. The polynucleotide, viral particle or composition, or method, or use thereof, according to any of embodiments 140 to 154, wherein the level of stable factor IX activity is a level of stable factor IX activity of at least 35%.

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

157.The polynucleotide, viral particle or any one of embodiments 140-156, wherein the level of Factor IX activity is at least 20% at 50 weeks or more or about 52 weeks after administration of the polynucleotide, viral particle or composition. A composition, or a method, or use thereof.

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

159.The polynucleotide, viral particle, or any of embodiments 140-156, wherein the level of Factor IX activity is at least 25% at 50 or about 52 weeks after administration of the polynucleotide, viral particle or composition. A composition, or method, or use thereof.

160. The polynucleotide, viral particle or composition, or method, or use thereof, according to aspect 159, wherein the level of factor IX activity is maintained at least 25% for a continuous period of at least 40 weeks immediately prior to the time point.

161.The poly of any of embodiments 140-156, wherein the level of Factor IX activity is 40% or more or about 40% at 50 or more or about 52 weeks after administration of the polynucleotide, viral particle or composition. Nucleotides, viral particles or compositions, or methods, or uses thereof.

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

163. The method of any one of embodiments 140 to 162, wherein the factor IX activity is determined using a coagulation assay to determine factor IX activity in a patient's blood sample, and optionally, the coagulation assay is using a ^{SynthASil™ kit.} Phosphorus, polynucleotide, viral particle or composition, or a method, or use thereof.

164. The factor IX activity of any one of embodiments 140-162, wherein the factor IX activity is determined in the blood sample by taking a blood sample from the patient and measuring it using a coagulation assay, and optionally the coagulation assay is a SynthASil ^TM kit. Using a polynucleotide, a virus particle or composition, or a method, or use thereof.

165.The method of 163 or 164, wherein the coagulation assay is a one-stage clotting assay, and optionally, the one-step coagulation assay is ^{using a SynthASil TM} kit, polynucleotide, viral particle, or A composition, or a method, or use thereof.

166. The polynucleotide, viral particle or composition, or method, or use thereof, according to any of embodiments 113 to 165, wherein the method or use comprises co-administration of a steroid.

167. The polynucleotide, viral particle or composition, or method, or use thereof, according to aspect 166, wherein the steroid is prednisolone.

SEQUENCE LISTING <110> UCL BUSINESS PLC <120> FACTOR IX ENCODING NUCLEOTIDES <130> N411562WO <150> GB1813528.5 <151> 2018-08-20 <150> US16/105,583 <151> 2018-08-20 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 1191 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 1 gaggagaagt gcagctttga ggaggccagg gaggtgtttg agaacactga gaggaccact 60 gagttctgga agcagtatgt ggatggggac cagtgtgaga gcaacccctg cctgaatggg 120 ggcagctgca aggatgacat caacagctat gagtgctggt gcccctttgg ctttgagggc 180 aagaactgtg agctggatgt gacctgcaac atcaagaatg gcagatgtga gcagttctgc 240 aagaactctg ctgacaacaa ggtggtgtgc agctgcactg agggctacag gctggctgag 300 aaccagaaga gctgtgagcc tgctgtgcca ttcccatgtg gcagagtgtc tgtgagccag 360 accagcaagc tgaccagggc tgaggctgtg ttccctgatg tggactatgt gaacagcact 420 gaggctgaaa ccatcctgga caacatcacc cagagcaccc agagcttcaa tgacttcacc 480 agggtggtgg ggggggagga tgccaagcct ggccagttcc cctggcaagt ggtgctgaat 540 ggcaaggtgg atgccttctg tgggggcagc attgtgaatg agaagtggat tgtgactgct 600 gcccactgtg tggagactgg ggtgaagatc actgtggtgg ctggggagca caacattgag 660 gagactgagc acactgagca gaagaggaat gtgatcagga tcatccccca ccacaactac 720 aatgctgcca tcaacaagta caaccatgac attgccctgc tggagctgga tgagcccctg 780 gtgctgaaca gctatgtgac ccccatctgc attgctgaca aggagtacac caacatcttc 840 ctgaagtttg gctctggcta tgtgtctggc tggggcaggg tgttccacaa gggcaggtct 900 gccctggtgc tgcagtacct gagggtgccc ctggtggaca gggccacctg cctgctcagc 960 accaagttca ccatctacaa caacatgttc tgtgctggct tccatgaggg gggcagggac 1020 agctgccagg gggactctgg gggcccccat gtgactgagg tggagggcac cagcttcctg 1080 actggcatca tcagctgggg ggaggagtgt gccatgaagg gcaagtatgg catctacacc 1140 aaagtctcca gatatgtgaa ctggatcaag gagaagacca agctgacctg a 1191 <210> 2 <211> 494 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 2 atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60 ggatatctac tcagtgctga atgtacaggt ttgtttcctt ttttaaaata cattgagtat 120 gcttgccttt tagatataga aatatctgat gctgtcttct tcactaaatt ttgattacat 180 gatttgacag caatattgaa gagtctaaca gccagcacgc aggttggtaa gtactgtggg 240 aacatcacag attttggctc catgccctaa agagaaattg gctttcagat tatttggatt 300 aaaaacaaag actttcttaa gagatgtaaa attttcatga tgttttcttt tttgctaaaa 360 ctaaagaatt attcttttac atttcagttt ttcttgatca tgaaaacgcc aacaaaattc 420 tgaatcggcc aaagaggtat aattcaggta aattggaaga gtttgttcaa gggaaccttg 480 agagagaatg tatg 494 <210> 3 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 3 gtttgtttcc ttttttaaaa tacattgagt atgcttgcct tttagatata gaaatatctg 60 atgctgtctt cttcactaaa ttttgattac atgatttgac agcaatattg aagagtctaa 120 cagccagcac gcaggttggt aagtactgtg ggaacatcac agattttggc tccatgccct 180 aaagagaaat tggctttcag attatttgga ttaaaaacaa agactttctt aagagatgta 240 aaattttcat gatgttttct tttttgctaa aactaaagaa ttattctttt acatttcag 299 <210> 4 <211> 1386 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 4 atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60 ggatatctac tcagtgctga atgtacagtt tttcttgatc atgaaaacgc caacaaaatt 120 ctgaatcggc caaagaggta taattcaggt aaattggaag agtttgttca agggaacctt 180 gagagagaat gtatggagga gaagtgcagc tttgaggagg ccagggaggt gtttgagaac 240 actgagagga ccactgagtt ctggaagcag tatgtggatg gggaccagtg tgagagcaac 300 ccctgcctga atgggggcag ctgcaaggat gacatcaaca gctatgagtg ctggtgcccc 360 tttggctttg agggcaagaa ctgtgagctg gatgtgacct gcaacatcaa gaatggcaga 420 tgtgagcagt tctgcaagaa ctctgctgac aacaaggtgg tgtgcagctg cactgagggc 480 tacaggctgg ctgagaacca gaagagctgt gagcctgctg tgccattccc atgtggcaga 540 gtgtctgtga gccagaccag caagctgacc agggctgagg ctgtgttccc tgatgtggac 600 tatgtgaaca gcactgaggc tgaaaccatc ctggacaaca tcacccagag cacccagagc 660 ttcaatgact tcaccagggt ggtggggggg gaggatgcca agcctggcca gttcccctgg 720 caagtggtgc tgaatggcaa ggtggatgcc ttctgtgggg gcagcattgt gaatgagaag 780 tggattgtga ctgctgccca ctgtgtggag actggggtga agatcactgt ggtggctggg 840 gagcacaaca ttgaggagac tgagcacact gagcagaaga ggaatgtgat caggatcatc 900 ccccaccaca actacaatgc tgccatcaac aagtacaacc atgacattgc cctgctggag 960 ctggatgagc ccctggtgct gaacagctat gtgaccccca tctgcattgc tgacaaggag 1020 tacaccaaca tcttcctgaa gtttggctct ggctatgtgt ctggctgggg cagggtgttc 1080 cacaagggca ggtctgccct ggtgctgcag tacctgaggg tgcccctggt ggacagggcc 1140 acctgcctgc tcagcaccaa gttcaccatc tacaacaaca tgttctgtgc tggcttccat 1200 gaggggggca gggacagctg ccagggggac tctgggggcc cccatgtgac tgaggtggag 1260 ggcaccagct tcctgactgg catcatcagc tggggggagg agtgtgccat gaagggcaag 1320 tatggcatct acaccaaagt ctccagatat gtgaactgga tcaaggagaa gaccaagctg 1380 acctga 1386 <210> 5 <211> 1685 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 5 atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60 ggatatctac tcagtgctga atgtacaggt ttgtttcctt ttttaaaata cattgagtat 120 gcttgccttt tagatataga aatatctgat gctgtcttct tcactaaatt ttgattacat 180 gatttgacag caatattgaa gagtctaaca gccagcacgc aggttggtaa gtactgtggg 240 aacatcacag attttggctc catgccctaa agagaaattg gctttcagat tatttggatt 300 aaaaacaaag actttcttaa gagatgtaaa attttcatga tgttttcttt tttgctaaaa 360 ctaaagaatt attcttttac atttcagttt ttcttgatca tgaaaacgcc aacaaaattc 420 tgaatcggcc aaagaggtat aattcaggta aattggaaga gtttgttcaa gggaaccttg 480 agagagaatg tatggaggag aagtgcagct ttgaggaggc cagggaggtg tttgagaaca 540 ctgagaggac cactgagttc tggaagcagt atgtggatgg ggaccagtgt gagagcaacc 600 cctgcctgaa tgggggcagc tgcaaggatg acatcaacag ctatgagtgc tggtgcccct 660 ttggctttga gggcaagaac tgtgagctgg atgtgacctg caacatcaag aatggcagat 720 gtgagcagtt ctgcaagaac tctgctgaca acaaggtggt gtgcagctgc actgagggct 780 acaggctggc tgagaaccag aagagctgtg agcctgctgt gccattccca tgtggcagag 840 tgtctgtgag ccagaccagc aagctgacca gggctgaggc tgtgttccct gatgtggact 900 atgtgaacag cactgaggct gaaaccatcc tggacaacat cacccagagc acccagagct 960 tcaatgactt caccagggtg gtgggggggg aggatgccaa gcctggccag ttcccctggc 1020 aagtggtgct gaatggcaag gtggatgcct tctgtggggg cagcattgtg aatgagaagt 1080 ggattgtgac tgctgcccac tgtgtggaga ctggggtgaa gatcactgtg gtggctgggg 1140 agcacaacat tgaggagact gagcacactg agcagaagag gaatgtgatc aggatcatcc 1200 cccaccacaa ctacaatgct gccatcaaca agtacaacca tgacattgcc ctgctggagc 1260 tggatgagcc cctggtgctg aacagctatg tgacccccat ctgcattgct gacaaggagt 1320 acaccaacat cttcctgaag tttggctctg gctatgtgtc tggctggggc agggtgttcc 1380 acaagggcag gtctgccctg gtgctgcagt acctgagggt gcccctggtg gacagggcca 1440 cctgcctgct cagcaccaag ttcaccatct acaacaacat gttctgtgct ggcttccatg 1500 aggggggcag ggacagctgc cagggggact ctgggggccc ccatgtgact gaggtggagg 1560 gcaccagctt cctgactggc atcatcagct ggggggagga gtgtgccatg aagggcaagt 1620 atggcatcta caccaaagtc tccagatatg tgaactggat caaggagaag accaagctga 1680 cctga 1685 <210> 6 <211> 334 <212> DNA <213> Artificial Sequence <220> <223> Synthetic regulatory element <400> 6 ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60 tgaccttgga gctggggcag aggtcagaca cctctctggg cccatgccac ctccaactgg 120 acacaggacg ctgtggtttc tgagccaggg ggcgactcag atcccagcca gtggacttag 180 cccctgtttg ctcctccgat aactggggtg accttggtta atattcacca gcagcctccc 240 ccgttgcccc tctggatcca ctgcttaaat acggacgagg acagggccct gtctcctcag 300 cttcaggcac caccactgac ctgggacagt gaat 334 <210> 7 <211> 447 <212> DNA <213> Artificial Sequence <220> <223> Synthetic regulatory element <400> 7 ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60 tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc 120 cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg tggtttaggt 180 agtgtgagag gggaatgact cctttcggta agtgcagtgg aagctgtaca ctgcccaggc 240 aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact tagcccctgt 300 ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct cccccgttgc 360 ccctctggat ccactgctta aatacggacg aggacagggc cctgtctcct cagcttcagg 420 caccaccact gacctgggac agtgaat 447 <210> 8 <211> 415 <212> PRT <213> Homo sapiens <400> 8 Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg 1 5 10 15 Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe 20 25 30 Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly 35 40 45 Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp 50 55 60 Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys 65 70 75 80 Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu 85 90 95 Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr 100 105 110 Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val 115 120 125 Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr 130 135 140 Arg Ala Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu 145 150 155 160 Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn 165 170 175 Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe 180 185 190 Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly 195 200 205 Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu 210 215 220 Thr Gly Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu 225 230 235 240 Thr Glu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His 245 250 255 His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu 260 265 270 Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile 275 280 285 Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser 290 295 300 Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala 305 310 315 320 Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys 325 330 335 Leu Leu Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly 340 345 350 Phe His Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro 355 360 365 His Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser 370 375 380 Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys 385 390 395 400 Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr 405 410 415 <210> 9 <211> 1386 <212> DNA <213> Homo sapiens <400> 9 atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60 ggatatctac tcagtgctga atgtacagtt tttcttgatc atgaaaacgc caacaaaatt 120 ctgaatcggc caaagaggta taattcaggt aaattggaag agtttgttca agggaacctt 180 gagagagaat gtatggaaga aaagtgtagt tttgaagaag cacgagaagt ttttgaaaac 240 actgaaagaa caactgaatt ttggaagcag tatgttgatg gagatcagtg tgagtccaat 300 ccatgtttaa atggcggcag ttgcaaggat gacattaatt cctatgaatg ttggtgtccc 360 tttggatttg aaggaaagaa ctgtgaatta gatgtaacat gtaacattaa gaatggcaga 420 tgcgagcagt tttgtaaaaa tagtgctgat aacaaggtgg tttgctcctg tactgaggga 480 tatcgacttg cagaaaacca gaagtcctgt gaaccagcag tgccatttcc atgtggaaga 540 gtttctgttt cacaaacttc taagctcacc cgtgctgagg ctgtttttcc tgatgtggac 600 tatgtaaatt ctactgaagc tgaaaccatt ttggataaca tcactcaaag cacccaatca 660 tttaatgact tcactcgggt tgttggtgga gaagatgcca aaccaggtca attcccttgg 720 caggttgttt tgaatggtaa agttgatgca ttctgtggag gctctatcgt taatgaaaaa 780 tggattgtaa ctgctgccca ctgtgttgaa actggtgtta aaattacagt tgtcgcaggt 840 gaacataata ttgaggagac agaacataca gagcaaaagc gaaatgtgat tcgaattatt 900 cctcaccaca actacaatgc agctattaat aagtacaacc atgacattgc ccttctggaa 960 ctggacgaac ccttagtgct aaacagctac gttacaccta tttgcattgc tgacaaggaa 1020 tacacgaaca tcttcctcaa atttggatct ggctatgtaa gtggctgggg aagagtcttc 1080 cacaaaggga gatcagcttt agttcttcag taccttagag ttccacttgt tgaccgagcc 1140 acatgtcttc gatctacaaa gttcaccatc tataacaaca tgttctgtgc tggcttccat 1200 gaaggaggta gagattcatg tcaaggagat agtgggggac cccatgttac tgaagtggaa 1260 gggaccagtt tcttaactgg aattattagc tggggtgaag agtgtgcaat gaaaggcaaa 1320 tatggaatat ataccaaggt atcccggtat gtcaactgga ttaaggaaaa aacaaagctc 1380 acttaa 1386 <210> 10 <211> 736 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 10 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly 145 150 155 160 Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr 435 440 445 Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser 450 455 460 Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro 465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn 485 490 495 Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn 500 505 510 Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly 530 535 540 Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile 545 550 555 560 Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln 565 570 575 Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr 580 585 590 Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640 Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 11 <211> 1386 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 11 atgcagaggg tgaacatgat catggctgag agccctggcc tgatcaccat ctgcctgctg 60 ggctacctgc tgtctgctga gtgcactgtg ttcctggacc atgagaatgc caacaagatc 120 ctgaacaggc ccaagagata caactctggc aagctggagg agtttgtgca gggcaacctg 180 gagagggagt gcatggagga gaagtgcagc tttgaggagg ccagggaggt gtttgagaac 240 actgagagga ccactgagtt ctggaagcag tatgtggatg gggaccagtg tgagagcaac 300 ccctgcctga atgggggcag ctgcaaggat gacatcaaca gctatgagtg ctggtgcccc 360 tttggctttg agggcaagaa ctgtgagctg gatgtgacct gcaacatcaa gaatggcaga 420 tgtgagcagt tctgcaagaa ctctgctgac aacaaggtgg tgtgcagctg cactgagggc 480 tacaggctgg ctgagaacca gaagagctgt gagcctgctg tgccattccc atgtggcaga 540 gtgtctgtga gccagaccag caagctgacc agggctgagg ctgtgttccc tgatgtggac 600 tatgtgaaca gcactgaggc tgaaaccatc ctggacaaca tcacccagag cacccagagc 660 ttcaatgact tcaccagggt ggtggggggg gaggatgcca agcctggcca gttcccctgg 720 caagtggtgc tgaatggcaa ggtggatgcc ttctgtgggg gcagcattgt gaatgagaag 780 tggattgtga ctgctgccca ctgtgtggag actggggtga agatcactgt ggtggctggg 840 gagcacaaca ttgaggagac tgagcacact gagcagaaga ggaatgtgat caggatcatc 900 ccccaccaca actacaatgc tgccatcaac aagtacaacc atgacattgc cctgctggag 960 ctggatgagc ccctggtgct gaacagctat gtgaccccca tctgcattgc tgacaaggag 1020 tacaccaaca tcttcctgaa gtttggctct ggctatgtgt ctggctgggg cagggtgttc 1080 cacaagggca ggtctgccct ggtgctgcag tacctgaggg tgcccctggt ggacagggcc 1140 acctgcctgt tgagcaccaa gttcaccatc tacaacaaca tgttctgtgc tggcttccat 1200 gaggggggca gggacagctg ccagggggac tctgggggcc cccatgtgac tgaggtggag 1260 ggcaccagct tcctgactgg catcatcagc tggggggagg agtgtgccat gaagggcaag 1320 tatggcatct acaccaaagt ctccagatat gtgaactgga tcaaggagaa gaccaagctg 1380 acctga 1386 <210> 12 <211> 1386 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 12 atgcagaggg tgaacatgat catggctgag agccctggcc tgatcaccat ctgcctgctg 60 ggctacctgc tgtctgctga gtgcactgtg ttcctggacc atgagaatgc caacaagatc 120 ctgaacaggc ccaagagata caactctggc aagctggagg agtttgtgca gggcaacctg 180 gagagggagt gcatggagga gaagtgcagc tttgaggagg ccagggaggt gtttgagaac 240 actgagagga ccactgagtt ctggaagcag tatgtggatg gggaccagtg tgagagcaac 300 ccctgcctga atgggggcag ctgcaaggat gacatcaaca gctatgagtg ctggtgcccc 360 tttggctttg agggcaagaa ctgtgagctg gatgtgacct gcaacatcaa gaatggcaga 420 tgtgagcagt tctgcaagaa ctctgctgac aacaaggtgg tgtgcagctg cactgagggc 480 tacaggctgg ctgagaacca gaagagctgt gagcctgctg tgccattccc atgtggcaga 540 gtgtctgtga gccagaccag caagctgacc agggctgagg ctgtgttccc tgatgtggac 600 tatgtgaaca gcactgaggc tgaaaccatc ctggacaaca tcacccagag cacccagagc 660 ttcaatgact tcaccagggt ggtggggggg gaggatgcca agcctggcca gttcccctgg 720 caagtggtgc tgaatggcaa ggtggatgcc ttctgtgggg gcagcattgt gaatgagaag 780 tggattgtga ctgctgccca ctgtgtggag actggggtga agatcactgt ggtggctggg 840 gagcacaaca ttgaggagac tgagcacact gagcagaaga ggaatgtgat caggatcatc 900 ccccaccaca actacaatgc tgccatcaac aagtacaacc atgacattgc cctgctggag 960 ctggatgagc ccctggtgct gaacagctat gtgaccccca tctgcattgc tgacaaggag 1020 tacaccaaca tcttcctgaa gtttggctct ggctatgtgt ctggctgggg cagggtgttc 1080 cacaagggca ggtctgccct ggtgctgcag tacctgaggg tgcccctggt ggacagggcc 1140 acctgcctga ggagcaccaa gttcaccatc tacaacaaca tgttctgtgc tggcttccat 1200 gaggggggca gggacagctg ccagggggac tctgggggcc cccatgtgac tgaggtggag 1260 ggcaccagct tcctgactgg catcatcagc tggggggagg agtgtgccat gaagggcaag 1320 tatggcatct acaccaaagt ctccagatat gtgaactgga tcaaggagaa gaccaagctg 1380 acctga 1386 <210> 13 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> Synthetic enhancer element <400> 13 ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60 tgaccttgga gctggggcag aggtcagaca cctctctggg cccatgccac ctccaac 117 <210> 14 <211> 185 <212> DNA <213> Artificial Sequence <220> <223> Synthetic promoter element <400> 14 gggcgactca gatcccagcc agtggactta gcccctgttt gctcctccga taactggggt 60 gaccttggtt aatattcacc agcagcctcc cccgttgccc ctctggatcc actgcttaaa 120 tacggacgag gacagggccc tgtctcctca gcttcaggca ccaccactga cctgggacag 180 tgaat 185 <210> 15 <211> 195 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 15 atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60 ggatatctac tcagtgctga atgtacagtt tttcttgatc atgaaaacgc caacaaaatt 120 ctgaatcggc caaagaggta taattcaggt aaattggaag agtttgttca agggaacctt 180 gagagagaat gtatg 195 <210> 16 <211> 461 <212> PRT <213> Homo sapiens <400> 16 Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr 1 5 10 15 Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu 20 25 30 Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn 35 40 45 Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys 50 55 60 Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn 65 70 75 80 Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln 85 90 95 Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile 100 105 110 Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys 115 120 125 Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe 130 135 140 Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly 145 150 155 160 Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe 165 170 175 Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala 180 185 190 Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu 195 200 205 Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe 210 215 220 Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp 225 230 235 240 Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile 245 250 255 Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly 260 265 270 Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu 275 280 285 His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn 290 295 300 Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu 305 310 315 320 Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile 325 330 335 Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr 340 345 350 Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val 355 360 365 Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Leu 370 375 380 Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His 385 390 395 400 Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val 405 410 415 Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly 420 425 430 Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser 435 440 445 Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr 450 455 460 <210> 17 <211> 724 <212> PRT <213> adeno-associated virus 5 <400> 17 Met Ser Phe Val Asp His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu 1 5 10 15 Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys 20 25 30 Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly 35 40 45 Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val 50 55 60 Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu 65 70 75 80 Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp 85 90 95 Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn 100 105 110 Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe 115 120 125 Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile 130 135 140 Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser 145 150 155 160 Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln 165 170 175 Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr 180 185 190 Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala 195 200 205 Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp 210 215 220 Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro 225 230 235 240 Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp 245 250 255 Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr 260 265 270 Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln 275 280 285 Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val 290 295 300 Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr 305 310 315 320 Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp 325 330 335 Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys 340 345 350 Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr 355 360 365 Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser 370 375 380 Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn 385 390 395 400 Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser 405 410 415 Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp 420 425 430 Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln 435 440 445 Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp 450 455 460 Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly 465 470 475 480 Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu 485 490 495 Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr 500 505 510 Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile 515 520 525 Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu 530 535 540 Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg 545 550 555 560 Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser 565 570 575 Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro 580 585 590 Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp 595 600 605 Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met 610 615 620 Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn 625 630 635 640 Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser 645 650 655 Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu 660 665 670 Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln 675 680 685 Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp 690 695 700 Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu 705 710 715 720 Thr Arg Pro Leu <210> 18 <211> 1685 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 18 atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60 ggatatctac tcagtgctga atgtacaggt ttgtttcctt ttttaaaata cattgagtat 120 gcttgccttt tagatataga aatatctgat gctgtcttct tcactaaatt ttgattacat 180 gatttgacag caatattgaa gagtctaaca gccagcacgc aggttggtaa gtactgtggg 240 aacatcacag attttggctc catgccctaa agagaaattg gctttcagat tatttggatt 300 aaaaacaaag actttcttaa gagatgtaaa attttcatga tgttttcttt tttgctaaaa 360 ctaaagaatt attcttttac atttcagttt ttcttgatca tgaaaacgcc aacaaaattc 420 tgaatcggcc aaagaggtat aattcaggta aattggaaga gtttgttcaa gggaaccttg 480 agagagaatg tatggaggag aagtgttctt tcgaggaggc gagagaggtt ttcgagaata 540 ctgagcgaac aaccgaattc tggaaacaat atgtggatgg cgaccaatgt gaatctaatc 600 cctgcctcaa cggtggctca tgcaaagacg atatcaacag ctacgagtgt tggtgcccct 660 ttggtttcga gggaaagaat tgcgagcttg atgtaacctg taacattaag aatgggcgct 720 gcgaacagtt ttgcaagaac agcgccgaca ataaggtcgt ctgcagttgt accgaaggct 780 ataggcttgc agagaatcag aagagttgcg agcctgctgt gccgttccca tgtggcagag 840 tcagtgtgtc ccaaactagc aagctgacaa gagcagaagc cgttttcccc gatgtggact 900 acgtgaattc cactgaagcc gaaacgatcc tggacaatat cacacagagc actcagtctt 960 tcaacgactt cacacgggtt gtgggaggag aggacgccaa acccggccag tttccttggc 1020 aagtcgttct taacggcaag gtcgacgcct tttgtggagg gagtattgtg aacgagaaat 1080 ggattgtcac cgctgctcat tgtgttgaaa ctggggtgaa aatcactgtt gtcgcaggag 1140 agcacaatat cgaagagaca gaacacaccg agcagaaacg caacgttatt cggatcattc 1200 cacatcacaa ctacaatgct gccatcaaca agtacaacca cgacattgcg ctgctggagt 1260 tggatgaacc tctcgtgctc aactcctatg tgaccccaat ctgcatagca gataaggagt 1320 ataccaacat cttcctgaag tttgggtcag gttatgtgtc aggctgggga cgagtgtttc 1380 ataaagggag atcagcactg gtgttgcagt atctgcgcgt accactggtg gatcgggcta 1440 cttgcctgct aagcacaaaa ttcaccatct acaacaacat gttttgtgcc ggttttcacg 1500 aaggcggcag ggacagctgt cagggagatt ccggagggcc tcatgtcaca gaggtcgagg 1560 gcacctcctt tctcactggg attataagct ggggagaaga atgcgccatg aaagggaagt 1620 acggcatata cacgaaagtg tctagatacg tgaattggat taaggaaaag accaaactga 1680 cgtga 1685 <210> 19 <211> 1248 <212> DNA <213> Homo sapiens <400> 19 tataattcag gtaaattgga agagtttgtt caagggaacc ttgagagaga atgtatggaa 60 gaaaagtgta gttttgaaga agcacgagaa gtttttgaaa acactgaaag aacaactgaa 120 ttttggaagc agtatgttga tggagatcag tgtgagtcca atccatgttt aaatggcggc 180 agttgcaagg atgacattaa ttcctatgaa tgttggtgtc cctttggatt tgaaggaaag 240 aactgtgaat tagatgtaac atgtaacatt aagaatggca gatgcgagca gttttgtaaa 300 aatagtgctg ataacaaggt ggtttgctcc tgtactgagg gatatcgact tgcagaaaac 360 cagaagtcct gtgaaccagc agtgccattt ccatgtggaa gagtttctgt ttcacaaact 420 tctaagctca cccgtgctga ggctgttttt cctgatgtgg actatgtaaa ttctactgaa 480 gctgaaacca ttttggataa catcactcaa agcacccaat catttaatga cttcactcgg 540 gttgttggtg gagaagatgc caaaccaggt caattccctt ggcaggttgt tttgaatggt 600 aaagttgatg cattctgtgg aggctctatc gttaatgaaa aatggattgt aactgctgcc 660 cactgtgttg aaactggtgt taaaattaca gttgtcgcag gtgaacataa tattgaggag 720 acagaacata cagagcaaaa gcgaaatgtg attcgaatta ttcctcacca caactacaat 780 gcagctatta ataagtacaa ccatgacatt gcccttctgg aactggacga acccttagtg 840 ctaaacagct acgttacacc tatttgcatt gctgacaagg aatacacgaa catcttcctc 900 aaatttggat ctggctatgt aagtggctgg ggaagagtct tccacaaagg gagatcagct 960 ttagttcttc agtaccttag agttccactt gttgaccgag ccacatgtct tcgatctaca 1020 aagttcacca tctataacaa catgttctgt gctggcttcc atgaaggagg tagagattca 1080 tgtcaaggag atagtggggg accccatgtt actgaagtgg aagggaccag tttcttaact 1140 ggaattatta gctggggtga agagtgtgca atgaaaggca aatatggaat atataccaag 1200 gtatcccggt atgtcaactg gattaaggaa aaaacaaagc tcacttaa 1248 <210> 20 <211> 724 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 20 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Glu Val Gly Glu 1 5 10 15 Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys 20 25 30 Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly 35 40 45 Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val 50 55 60 Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu 65 70 75 80 Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp 85 90 95 Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn 100 105 110 Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe 115 120 125 Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile 130 135 140 Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser 145 150 155 160 Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln 165 170 175 Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr 180 185 190 Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala 195 200 205 Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp 210 215 220 Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro 225 230 235 240 Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp 245 250 255 Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr 260 265 270 Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln 275 280 285 Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val 290 295 300 Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr 305 310 315 320 Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp 325 330 335 Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys 340 345 350 Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr 355 360 365 Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser 370 375 380 Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn 385 390 395 400 Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser 405 410 415 Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp 420 425 430 Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln 435 440 445 Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp 450 455 460 Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly 465 470 475 480 Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu 485 490 495 Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr 500 505 510 Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile 515 520 525 Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu 530 535 540 Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg 545 550 555 560 Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser 565 570 575 Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro 580 585 590 Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp 595 600 605 Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met 610 615 620 Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn 625 630 635 640 Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser 645 650 655 Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu 660 665 670 Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln 675 680 685 Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp 690 695 700 Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu 705 710 715 720 Thr Arg Pro Leu <210> 21 <211> 725 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 21 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro 50 55 60 Val Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn 65 70 75 80 Glu Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg 130 135 140 Ile Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp 145 150 155 160 Ser Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser 165 170 175 Gln Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp 180 185 190 Thr Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly 195 200 205 Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr 210 215 220 Trp Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu 225 230 235 240 Pro Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val 245 250 255 Asp Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 260 265 270 Tyr Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp 275 280 285 Gln Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg 290 295 300 Val Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser 305 310 315 320 Thr Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr 325 330 335 Asp Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly 340 345 350 Cys Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly 355 360 365 Tyr Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser 370 375 380 Ser Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly 385 390 395 400 Asn Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser 405 410 415 Ser Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val 420 425 430 Asp Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val 435 440 445 Gln Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn 450 455 460 Trp Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser 465 470 475 480 Gly Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met 485 490 495 Glu Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met 500 505 510 Thr Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met 515 520 525 Ile Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu 530 535 540 Glu Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn 545 550 555 560 Arg Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser 565 570 575 Ser Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val 580 585 590 Pro Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile 595 600 605 Trp Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala 610 615 620 Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys 625 630 635 640 Asn Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val 645 650 655 Ser Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met 660 665 670 Glu Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile 675 680 685 Gln Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro 690 695 700 Asp Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr 705 710 715 720 Leu Thr Arg Pro Leu 725 <210> 22 <211> 725 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 22 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn 65 70 75 80 Glu Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg 130 135 140 Ile Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp 145 150 155 160 Ser Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser 165 170 175 Gln Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp 180 185 190 Thr Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly 195 200 205 Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr 210 215 220 Trp Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu 225 230 235 240 Pro Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val 245 250 255 Asp Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 260 265 270 Tyr Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp 275 280 285 Gln Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg 290 295 300 Val Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser 305 310 315 320 Thr Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr 325 330 335 Asp Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly 340 345 350 Cys Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly 355 360 365 Tyr Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser 370 375 380 Ser Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly 385 390 395 400 Asn Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser 405 410 415 Ser Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val 420 425 430 Asp Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val 435 440 445 Gln Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn 450 455 460 Trp Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser 465 470 475 480 Gly Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met 485 490 495 Glu Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met 500 505 510 Thr Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met 515 520 525 Ile Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu 530 535 540 Glu Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn 545 550 555 560 Arg Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser 565 570 575 Ser Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val 580 585 590 Pro Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile 595 600 605 Trp Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala 610 615 620 Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys 625 630 635 640 Asn Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val 645 650 655 Ser Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met 660 665 670 Glu Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile 675 680 685 Gln Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro 690 695 700 Asp Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr 705 710 715 720 Leu Thr Arg Pro Leu 725 <210> 23 <211> 725 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 23 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg 130 135 140 Ile Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp 145 150 155 160 Ser Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser 165 170 175 Gln Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp 180 185 190 Thr Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly 195 200 205 Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr 210 215 220 Trp Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu 225 230 235 240 Pro Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val 245 250 255 Asp Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 260 265 270 Tyr Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp 275 280 285 Gln Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg 290 295 300 Val Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser 305 310 315 320 Thr Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr 325 330 335 Asp Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly 340 345 350 Cys Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly 355 360 365 Tyr Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser 370 375 380 Ser Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly 385 390 395 400 Asn Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser 405 410 415 Ser Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val 420 425 430 Asp Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val 435 440 445 Gln Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn 450 455 460 Trp Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser 465 470 475 480 Gly Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met 485 490 495 Glu Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met 500 505 510 Thr Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met 515 520 525 Ile Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu 530 535 540 Glu Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn 545 550 555 560 Arg Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser 565 570 575 Ser Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val 580 585 590 Pro Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile 595 600 605 Trp Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala 610 615 620 Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys 625 630 635 640 Asn Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val 645 650 655 Ser Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met 660 665 670 Glu Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile 675 680 685 Gln Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro 690 695 700 Asp Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr 705 710 715 720 Leu Thr Arg Pro Leu 725 <210> 24 <211> 725 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 24 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg 130 135 140 Ile Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp 145 150 155 160 Ser Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser 165 170 175 Gln Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp 180 185 190 Thr Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly 195 200 205 Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr 210 215 220 Trp Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu 225 230 235 240 Pro Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val 245 250 255 Asp Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 260 265 270 Tyr Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp 275 280 285 Gln Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg 290 295 300 Val Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser 305 310 315 320 Thr Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr 325 330 335 Asp Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly 340 345 350 Cys Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly 355 360 365 Tyr Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser 370 375 380 Ser Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly 385 390 395 400 Asn Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser 405 410 415 Ser Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val 420 425 430 Asp Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val 435 440 445 Gln Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn 450 455 460 Trp Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser 465 470 475 480 Gly Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met 485 490 495 Glu Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met 500 505 510 Thr Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met 515 520 525 Ile Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu 530 535 540 Glu Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn 545 550 555 560 Arg Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser 565 570 575 Ser Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val 580 585 590 Pro Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile 595 600 605 Trp Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala 610 615 620 Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys 625 630 635 640 Asn Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val 645 650 655 Ser Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met 660 665 670 Glu Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile 675 680 685 Gln Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro 690 695 700 Asp Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr 705 710 715 720 Leu Thr Arg Pro Leu 725 <210> 25 <211> 725 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 25 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Thr Gly Lys Arg 130 135 140 Ile Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp 145 150 155 160 Ser Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser 165 170 175 Gln Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp 180 185 190 Thr Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly 195 200 205 Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr 210 215 220 Trp Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu 225 230 235 240 Pro Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val 245 250 255 Asp Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 260 265 270 Tyr Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp 275 280 285 Gln Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg 290 295 300 Val Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser 305 310 315 320 Thr Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr 325 330 335 Asp Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly 340 345 350 Cys Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly 355 360 365 Tyr Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser 370 375 380 Ser Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly 385 390 395 400 Asn Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser 405 410 415 Ser Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val 420 425 430 Asp Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val 435 440 445 Gln Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn 450 455 460 Trp Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser 465 470 475 480 Gly Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met 485 490 495 Glu Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met 500 505 510 Thr Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met 515 520 525 Ile Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu 530 535 540 Glu Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn 545 550 555 560 Arg Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser 565 570 575 Ser Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val 580 585 590 Pro Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile 595 600 605 Trp Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala 610 615 620 Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys 625 630 635 640 Asn Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val 645 650 655 Ser Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met 660 665 670 Glu Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile 675 680 685 Gln Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro 690 695 700 Asp Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr 705 710 715 720 Leu Thr Arg Pro Leu 725 <210> 26 <211> 1386 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 26 atgcagaggg tgaacatgat catggctgag agccctggcc tgatcaccat ctgcctgctg 60 ggctacctgc tgtctgctga gtgcactgtg ttcctggacc atgagaatgc caacaagatc 120 ctgaacaggc ccaagagata caactctggc aagctggagg agtttgtgca gggcaacctg 180 gagagggagt gcatggagga gaagtgcagc tttgaggagg ccagggaggt gtttgagaac 240 actgagagga ccactgagtt ctggaagcag tatgtggatg gggaccagtg tgagagcaac 300 ccctgcctga atgggggcag ctgcaaggat gacatcaaca gctatgagtg ctggtgcccc 360 tttggctttg agggcaagaa ctgtgagctg gatgtgacct gcaacatcaa gaatggcaga 420 tgtgagcagt tctgcaagaa ctctgctgac aacaaggtgg tgtgcagctg cactgagggc 480 tacaggctgg ctgagaacca gaagagctgt gagcctgctg tgccattccc atgtggcaga 540 gtgtctgtga gccagaccag caagctgacc agggctgagg ctgtgttccc tgatgtggac 600 tatgtgaaca gcactgaggc tgaaaccatc ctggacaaca tcacccagag cacccagagc 660 ttcaatgact tcaccagggt ggtggggggg gaggatgcca agcctggcca gttcccctgg 720 caagtggtgc tgaatggcaa ggtggatgcc ttctgtgggg gcagcattgt gaatgagaag 780 tggattgtga ctgctgccca ctgtgtggag actggggtga agatcactgt ggtggctggg 840 gagcacaaca ttgaggagac tgagcacact gagcagaaga ggaatgtgat caggatcatc 900 ccccaccaca actacaatgc tgccatcaac aagtacaacc atgacattgc cctgctggag 960 ctggatgagc ccctggtgct gaacagctat gtgaccccca tctgcattgc tgacaaggag 1020 tacaccaaca tcttcctgaa gtttggctct ggctatgtgt ctggctgggg cagggtgttc 1080 cacaagggca ggtctgccct ggtgctgcag tacctgaggg tgcccctggt ggacagggcc 1140 acctgcctgc tgagcaccaa gttcaccatc tacaacaaca tgttctgtgc tggcttccat 1200 gaggggggca gggacagctg ccagggggac tctgggggcc cccatgtgac tgaggtggag 1260 ggcaccagct tcctgactgg catcatcagc tggggggagg agtgtgccat gaagggcaag 1320 tatggcatct acaccaaagt ctccagatat gtgaactgga tcaaggagaa gaccaagctg 1380 acctga 1386

Claims (27)

As a polynucleotide comprising a factor IX nucleotide sequence,
(i) the factor IX nucleotide sequence comprises a coding sequence encoding a factor IX protein or fragment thereof;
(ii) the portion of the coding sequence is codon optimized;
(iii) the region of the codon optimized coding sequence is 1100 or more nucleotides in length;
(iv) At least 60% of codons in the codon-optimized site are TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, Selected from the group consisting of GGC and GAG;
(v) the codon optimized site contains a reduced number of CpGs compared to the corresponding site of SEQ ID NO:9;
(vi) the polynucleotide further comprises the following transcriptional regulatory elements:
(a) the A1AT promoter or a fragment of the A1AT promoter; And/or
(b) an HCR enhancer or fragment of an HCR enhancer; And;
(vii) The factor IX nucleotide sequence comprises a codon encoding leucine at a position corresponding to the 384 position of wild-type factor IX,
A polynucleotide comprising a factor IX nucleotide sequence.
The method of claim 1,
The region of the codon-optimized coding sequence is codon-optimized for expression in human liver,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 1 to 2,
(i) the 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 nucleotide sequence; And/or
(ii) The region of the codon-optimized coding sequence is 800 or more, 900 or more, 1100 or more, 1500 or less, 1300 or less, 1200 or less, 800 to 1500, 900 to 1300, 1100 to 1200 , Or about 1191 nucleotides in length; And/or
(iii) the region of the codon optimized coding sequence comprises 1, 2, 3, 4, 5 or all of the following:
a) a region of 10 or more, 15 or more, 20 or more, less than 25, 10 to 25, 15 to 25, or 20 to 25 nucleotides of exon 3 or exon 3;
b) exon 4 or a portion of at least 80, at least 90, at least 100, less than 114, 80 to 114, 90 to 114, or 100 to 114 nucleotides of exon 4;
c) exon 5 or a region of 90 or more, 100 or more, 110 or more, less than 129, 90 to 129, 100 to 129, or 110 to 129 nucleotides of exon 5;
d) a site of 150 or more, 180 or more, 200 or more, less than 203, 150 to 203, 180 to 203, or 200 to 203 nucleotides of exon 6 or exon 6 nucleotides;
e) a portion of exon 7 or exon 7 of 70 or more, 80 or more, 90 or more, 100 or more, less than 115, 70 to 115, 80 to 115, 90 to 115, or 100 to 115 nucleotides; And/or
f) a region of at least 400, at least 450, at least 500, less than 548, 400 to 548, 450 to 548, or 500 to 548 nucleotides of exon 8 or exon 8; And/or
(iv) the region of the codon-optimized coding sequence is a region of 20 or more nucleotides of exon 3, a region of 100 or more nucleotides of exon 4, a region of 110 or more nucleotides of exon 5, 180 or more nucleotides of exon 6, A region of at least 100 nucleotides of exon 7 and a region of at least 500 nucleotides of exon 8; And/or
(v) the region of the codon optimized coding sequence includes exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8; And/or
(vi) the region of the codon-optimized coding sequence includes a region of exon 2, and the region of exon 2 is less than 160, less than 150, less than 100, less than 75, less than 60, 20 or more, 30 or more, 40 At least 50, 20 to 160, 30 to 150, 30 to 100, 40 to 75, or about 56 nucleotides in length; And/or
(vii) the region of the codon-optimized coding sequence comprises an exon 2 region having a length of 30 to 100 nucleotides in length,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 1 to 3,
(i) the region of the codon optimized coding sequence comprises less than 40, less than 20, less than 18, less than 10, less than 5 or less than 1 CpG; And/or
(ii) the region of the codon-optimized coding sequence is CpG-free,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 1 to 4,
The region of the codon-optimized coding sequence,
(i) a) 1 or more, 2 or more, 4 or more or 5 or more codons encoding phenylalanine are replaced by TTC compared to the 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 rest are TTT; And/or
d) the codon encoding phenylalanine is TTC, except when the next codon starts with G; And/or

(ii) a) 5 or more, 10 or more, 15 or more or 16 or more codons encoding leucine are replaced by CTGs compared to the reference wild-type factor IX sequence;
b) at least 90% or at least 94% codons encoding leucine are CTGs; And/or
c) at least 90% or at least 94% of the codons encoding leucine are CTGs and the remainder are CTCs; And/or

(iii) a) 5 or more, 10 or more, 11 or more or 12 or more codons encoding isoleucine are replaced by ATC compared to the reference wild-type factor IX sequence;
b) if the next codon starts with G, then at least one codon ATC is replaced by an ATT compared to the reference wild-type factor IX sequence;
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 is ATT; And/or
e) the codon encoding isoleucine is ATC, except when the next codon starts with G; And/or

(iv) a) 10 or more, 15 or more, 20 or more or 25 or more codons encoding valine are replaced by GTG compared to the reference wild-type factor IX sequence;
b) one or more codons encoding valine are replaced by GTC compared to the reference wild-type factor IX sequence;
c) at least 80%, at least 90% or at least 95% of the codons encoding valine are GTGs; 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) 5 or more, 10 or more, 12 or more or 13 or more codons encoding serine are replaced by AGC compared to the reference wild-type factor IX sequence;
b) if the next codon starts with G, then one or more, two or more, or four or more codons encoding serine are replaced by TCT compared to the reference wild-type factor IX sequence;
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; And/or

(vi) a) one or more, two or more or 5 or more codons encoding proline are replaced by CCC compared to the reference wild-type factor IX sequence;
b) if the next codon starts with G, then one or more codons encoding proline are replaced by CCT compared to the reference wild-type factor IX sequence;
c) at least 50%, at least 55% or at least 60% of the codons encoding proline are CCCs;
d) 50%, 55% or more or 60% or more codons encoding proline are CCC and the remainder are CCA or CCT; And/or
e) the codon encoding proline is CCC, except when the next codon starts with G; And/or

(vii) a) 6 or more, 7 or more, 8 or more, or 10 or more codons encoding threonine are replaced by ACCs compared to the reference wild-type factor IX sequence;
b) if the next codon starts with G, then one or more or two or more codons encoding threonine are replaced by ACT compared to the reference wild-type factor IX sequence;
c) at least 45%, at least 50% or at least 55% of the codons encoding threonine are ACCs;
d) at least 45%, at least 50% or at least 55% of the codons encoding threonine are ACC and the remainder is ACT; And/or
e) the codon encoding threonine is ACC, except when the next codon starts with G; And/or

(viii) a) one or more, two or more, three or more or four or more codons encoding alanine are replaced by GCC compared to the reference wild-type factor IX sequence;
b) if the next codon starts with G, then one or more, two or more, or three or more codons encoding alanine are replaced by GCT compared to the reference wild-type factor IX sequence;
c) 35% or more, 40% or more or 43% or more codons encoding alanine are GCC;
d) 35% or more, 40% or more or 45% or more encoding alanine are GCC and the remainder is GCT; And/or
e) the codon encoding alanine is GCC, except when the next codon starts with G; And/or

(ix) a) one or more or two or more codons encoding tyrosine are replaced by TAC compared to the reference wild-type factor IX sequence;
b) if the next codon starts with G, then at least one codon TAC is replaced with a TAT compared to the reference wild-type factor IX sequence;
c) at least 40%, at least 45% or at least 48% codons encoding tyrosine are TACs;
d) 40% or more, 45% or more or 48% or more codons encoding tyrosine are TAC and the remainder is TAT; And/or
e) the codon encoding tyrosine is TAC, except when the next codon starts with G; And/or

(x) a) one or more codons encoding histidine are replaced by CAC compared to the reference wild-type factor IX sequence;
b) at least 50%, at least 60% or at least 65% of the codons encoding histidine are CACs;
c) 50% or more, 60% or more or 65% or more codons encoding histidine are CAC and the remainder is CAT; And/or
d) the codon encoding histidine is CAC, except when the next codon starts with G; And/or

(xi) a) 1 or more, 2 or more, 4 or more or 5 or more codons encoding glutamine are replaced by CAG compared to the reference wild-type factor IX sequence;
b) at least one codon CAG is replaced by CAA compared to the reference wild-type factor IX sequence;
c) at least 80%, at least 85% or at least 90% of the codons encoding glutamine are CAGs; 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; And/or

(xii) a) 1 or more, 2 or more, 4 or more or 5 or more codons encoding asparagine are replaced by AAC compared to the reference wild-type factor IX sequence;
b) at least 60%, at least 65% or at least 70% of the codons encoding asparagine are AAC;
c) at least 60%, at least 65% or at least 70% of the codons encoding asparagine are AAC and the rest are AAT; And/or
e) the codon encoding asparagine is AAC, except when the next codon starts with G; And/or

(xiii) a) 5 or more, 7 or more, 8 or more or 9 or more codons encoding lysine are replaced with AAG compared to the reference wild-type factor IX sequence;
b) at least one codon AAG is replaced by AAA compared to the 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 rest are AAA; And/or

(xiv) a) 1 or more, 2 or more, 3 or more or 4 or more codons encoding aspartate are replaced by GAC compared to the reference wild-type factor IX sequence;
b) if the next codon starts with G, then at least one codon GAC is replaced by a GAT compared to the reference wild-type factor IX sequence;
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 rest are GAT; And/or
e) the codon encoding aspartate is GAC, except when the next codon starts with G; And/or

(xv) a) 15 or more, 20 or more, 25 or more or 26 or more codons encoding glutamate are replaced by GAGs compared to the 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) 80% or more, 90% or more or 95% or more codons encoding glutamate are GAG and the rest are GAA; And/or

(xvi) a) 5 or more, 6 or more, 7 or more or 8 or more codons encoding cysteine are replaced by TGCs compared to the reference wild-type factor IX sequence;
b) if the next codon starts with G, then at least one codon TGC is replaced by a TGT compared to the reference wild-type factor IX sequence;
c) at least 40%, at least 50% or at least 55% codons encoding cysteine are TGCs;
d) at least 40%, at least 50% or at least 55% of the codons encoding cysteine are TGCs and the remainder are TGTs; And/or
e) the codon encoding cysteine is TGC, except when the next codon starts with G; And/or

(xvii) the codon encoding tryptophan is TGG; And/or

(xviii)a) 5 or more, 8 or more, 10 or more or 11 or more codons encoding arginine are replaced by AGG compared to the reference wild-type factor IX sequence;
b) one or more codons encoding arginine are replaced by AGA compared to the 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 is AGA; And/or

(xix) a) 5 or more, 10 or more, 12 or more or 13 or more codons encoding glycine are replaced by GGCs compared to the reference wild-type factor IX sequence;
b) if the next codon starts with G, then 5 or more, 6 or more, 7 or more or 8 or more codons encoding glycine are replaced by GGG compared to the reference wild-type factor IX sequence;
c) at least 50%, at least 55% or at least 60% codons encoding glycine are GGCs;
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 codon encoding glycine is GGC, except when the next codon starts with G,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 1 to 5,
The region of the codon-optimized coding sequence encodes phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartate, glutamate, cysteine, tryptophan, arginine, and glycine. Contains a codon that,
The codon-optimized site,
a) 5 or more codons encoding phenylalanine are replaced by TTC compared to the reference wild-type factor IX sequence;
b) 16 or more codons encoding leucine are replaced by CTG compared to the reference wild-type factor IX sequence;
c) 12 or more codons encoding isoleucine are replaced by ATC compared to the reference wild-type factor IX sequence;
d) 25 or more codons encoding valine are replaced by GTG compared to the reference wild-type factor IX sequence;
e) 13 or more codons encoding serine are replaced by AGC compared to the reference wild-type factor IX sequence;
f) 5 or more codons encoding proline are replaced by CCC compared to the reference wild-type factor IX sequence;
g) 10 or more codons encoding threonine are replaced by ACC compared to the reference wild-type factor IX sequence;
h) 4 or more codons encoding alanine are replaced by GCC compared to the reference wild-type factor IX sequence;
i) two or more codons encoding tyrosine are replaced by TAC compared to the reference wild-type factor IX sequence;
j) one or more codons encoding histidine are replaced by CAC compared to the reference wild-type factor IX sequence;
k) 5 or more codons encoding glutamine are replaced by CAG compared to the reference wild-type factor IX sequence;
l) 5 or more codons encoding asparagine are replaced by AAC compared to the reference wild-type factor IX sequence;
m) 9 or more codons encoding lysine are replaced by AAG compared to the reference wild-type factor IX sequence;
n) 4 or more codons encoding aspartate are replaced by GAC compared to the reference wild-type factor IX sequence;
o) 26 or more codons encoding glutamate are replaced by GAG compared to the reference wild-type factor IX sequence;
p) 8 or more codons encoding cysteine are replaced by TGC compared to the reference wild-type factor IX sequence;
q) the codon encoding tryptophan is TGG;
r) 11 or more codons encoding arginine are replaced by AGG compared to the reference wild-type factor IX sequence; And
s) 13 or more codons encoding glycine are replaced by GGC compared to the reference wild-type factor IX sequence,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 1 to 6,
The region of the codon-optimized coding sequence encodes phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartate, glutamate, cysteine, tryptophan, arginine, and glycine. Contains a codon that,
The codon-optimized site,
a) at least 70% of the codons encoding phenylalanine are TTC;
b) at least 94% of the codons encoding leucine are CTGs;
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 CCCs;
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 codons encoding tyrosine are TAC;
j) at least 65% of the codons encoding histidine are CACs;
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 encoding aspartate are GAC;
o) 95% or more of the codons encoding glutamate are GAG;
p) at least 55% of the codons encoding cysteine are TGC;
q) the codon encoding tryptophan is TGG;
r) at least 75% of the codons encoding arginine are AGG; And
s) at least 60% of the codons encoding glycine are GGC,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 1 to 7,
The region of the codon-optimized coding sequence encodes phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartate, glutamate, cysteine, tryptophan, arginine, and glycine. Contains a codon that,
The codon-optimized site,
a) at least 70% of the codons encoding phenylalanine are TTC and the remainder is TTT;
b) more than 94% of the codons encoding leucine are CTG and the rest are CTC;
c) 75% or more 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) 60% or more 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 is ACT;
h) 43% or more of the codons encoding alanine are GCC and the rest are GCT;
i) at least 48% of the codons encoding tyrosine are TAC and the rest are TAT;
j) 65% or more of the codons encoding histidine are CAC and the rest are CAT;
k) 90% or more 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) 95% or more 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) 95% or more of the codons encoding glutamate are GAG and the rest are GAA;
p) 55% or more of the codons encoding cysteine are TGC and the remainder are TGT;
q) the codon encoding tryptophan is TGG;
r) at least 75% of the codons encoding arginine are AGG and the rest are AGA; And
s) 60% or more of the codons encoding glycine are GGC and the rest are GGG,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 4 to 8,
The reference wild-type factor IX sequence is SEQ ID NO: 9 or SEQ ID NO: 19,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 1 to 9,
The electronic regulatory element is one comprising a liver-specific promoter,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 1 to 10,
A polynucleotide comprising a factor IX nucleotide sequence, wherein the fragment of the A1AT promoter is:
(i) 100 or more, 120 or more, 150 or more, 180 or more, less than 255, 100 to 255, 150 to 225, 150 to 300, or 180 to 255 nucleotides in length ; And/or
(ii) 150 to 300 nucleotides in length.
The method according to any one of claims 1 to 11,
A polynucleotide comprising a factor IX nucleotide sequence, wherein the fragment of the HCR enhancer is:
(i) 80 or more, 90 or more, 100 or more, less than 192, 80 to 192, 90 to 192, 100 to 250, or 117 to 192 nucleotides in length; And/or
(ii) 100 to 250 nucleotides in length.
The method according to any one of claims 1 to 12,
(i) the transcriptional 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; And/or
(ii) the transcriptional regulatory element is SEQ ID NO: 6,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 1 to 13,
(i) The polynucleotide contains an enhancer that is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 13. and; And/or
(ii) the polynucleotide comprises an enhancer that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 13; And/or
(iii) the polynucleotide comprises an enhancer of SEQ ID NO: 13; And/or
(iv) The polynucleotide contains a promoter that is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 14 and; And/or
(v) the polynucleotide comprises a promoter that is 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 100% identical to SEQ ID NO: 14; And/or
(vi) the polynucleotide comprises a promoter of SEQ ID NO: 14,
A polynucleotide comprising a factor IX nucleotide sequence.
The method according to any one of claims 1 to 14,
The codon encoding the amino acid at the position corresponding to the 384 position of the wild-type factor IX is CTX, wherein X is an arbitrary nucleotide, and optionally, X is C or G,
A polynucleotide comprising a factor IX nucleotide sequence.
As a polynucleotide comprising a factor IX nucleotide sequence,
The factor IX nucleotide sequence comprises a coding sequence encoding a factor IX protein or fragment thereof,
The region of the coding sequence is not wild-type,
A polynucleotide comprising a factor IX nucleotide sequence.
A viral particle comprising a recombinant genome comprising the polynucleotide of claim 1.
The method of claim 17,
(i) the viral particles are AAV, adenovirus or lentiviral virus particles; And/or
(ii) the viral particles are AAV viral particles; And/or
(iii) the recombinant genome further comprises;
a) AAV2 ITRs;
b) peptide A sequence;
c) origin of replication; And/or
d) two resolvable ITRs; And/or
(iv) the recombinant genome is single-stranded,
Virus particles.
The method of claim 17 or 18,
Upon transduction into Huh7 cells, the viral particle is a factor IX protein having a greater factor IX activity than that of factor IX expressed from a viral particle comprising a factor IX nucleotide sequence of SEQ ID NO: 12 and a transcriptional regulatory factor of SEQ ID NO: 7 Or expressing a fragment thereof,
Virus particles.
The method according to any one of claims 1 to 19,
The Factor IX fragment is 200 or more, 250 or more, 300 or more, 200 to 415, 250 to 415, or 300 to 415 amino acids in length,
Polynucleotide or viral particles.
A composition comprising the polynucleotide or virus particle of any one of claims 1 to 20 and a pharmaceutically acceptable excipient.
The polynucleotide, viral particle or composition of any one of claims 1 to 21 for use in a method of treatment.
The method of claim 22
The method of treatment comprises administering to a patient an effective amount of the polynucleotide, viral particle or composition of any one of claims 1 to 21,
A polynucleotide, viral particle or composition for use in a method of treatment.
The method of claim 22 or 23,
(i) the treatment method is a hemophilia treatment method; And/or
(ii) the treatment method is a method of treating hemophilia type B; And/or
(iii) the patient has an antibody or inhibitor against factor IX,
A polynucleotide, viral particle or composition for use in a method of treatment.
The method of any one of claims 1-21, wherein optionally achieving the level of stable factor IX activity comprises administering to the patient the polynucleotide, viral particle or composition of any one of claims 1-21. A polynucleotide, viral particle or composition for use in achieving a stable factor IX activity level of at least 20% in a hemophilia type B patient comprising a.
The method of any one of the preceding claims, comprising administering to the patient a dose of said polynucleotide, viral particle or composition effective to achieve a level of stable Factor IX activity of at least 20% in the patient. , Polynucleotide, virus particle or composition for use in the treatment of hemophilia type B.
The method of claim 25 or 26,
(a) said factor IX activity level stabilizes 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;
(b) the patient has a factor IX having an activity lower than that of factor IX expressed from viral particles comprising (i) a factor IX nucleotide sequence of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory factor of SEQ ID NO: 6 The patient is refractory to the treatment used;
(c) The patient is a factor IX polypeptide that is expressed at a lower level than that encoded by a nucleotide sequence comprising (i) a factor IX nucleotide sequence of SEQ ID NO: 4 or 5 and (ii) a transcriptional regulatory factor of SEQ ID NO: 6 Is a patient refractory to treatment with
(d) the polynucleotide, viral particle or composition is administered in a dose effective to achieve a stable factor IX activity level of at least 20% in hemophilia type B patients;
(e) the polynucleotide, viral particle or composition is at a ^{dose of 4.5 x 10 11} vg/kg or more, or ^{a dose of less than 5 x 10 11} vg/kg, or 4.5 x 10 ¹¹ to 1 x 10 ¹² vg/kg, or 4.5 It is administered at a dose of x 10 ¹¹ vg/kg to 4.9 x 10 ¹¹ vg/kg, and optionally, the dose is determined by quantifying the number of vector genomes using qPCR, and optionally, the qPCR uses a primer that binds to the promoter region. And, optionally, the promoter region is a promoter region of a transgene cassette;
(f) The administered polynucleotide or viral particles are produced from mammalian cells and/or have characteristics due to the use of mammalian viral vector producing cells and are produced in insect viral vector producing cells (e.g., baculovirus systems). Differentiated from the vector;
(g) the stable factor IX activity level is at least 25% or at least 30% stable factor IX activity level, the selectively stable factor IX activity level is at least 35% stable factor IX activity level, and optionally the stable factor IX activity level is At least or about 40%;
(h) the factor IX activity level is at least 20% at 50 weeks or more or about 52 weeks after administration of the polynucleotide, viral particle or composition, and optionally the factor IX activity level is 20 for a continuous period of at least 40 weeks immediately before the time point. Keep% or more;
(i) the factor IX activity level is at least 25% at 50 weeks or more or about 52 weeks after administration of the polynucleotide, viral particle or composition, and optionally, the factor IX activity level is 25 for a continuous period of at least 40 weeks immediately before the time point. Keep% or more;
(j) the factor IX activity level is 40% or more or about 40% at 50 weeks or more or about 52 weeks after administration of the polynucleotide, viral particle or composition, and optionally, the factor IX activity level is 40 weeks or more immediately before the time point. Maintaining 40% or more for continuous periods;
(k) the factor IX activity is determined using a coagulation assay to determine the factor IX activity in a patient's blood sample, optionally the coagulation assay using the ^{SynthASil™ kit;}
(l) the factor IX activity is determined by taking a blood sample from the patient and measuring using a coagulation assay to determine the factor IX activity in the blood sample, optionally using a ^{SynthASil™ kit for coagulation assay;}
(m) the coagulation assay is a one-step coagulation assay, optionally using a ^{SynthASil™ kit for the one-step coagulation assay;} And/or
(n) the use comprises co-administration of a steroid, optionally wherein the steroid is prednisolone,
Polynucleotide, virus particle or composition.

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