RU2831751C2 - Codon-optimized nucleic acid which codes blood coagulation factor ix protein, and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present application relates to biotechnology and can be used in medicine. More specifically, the present invention relates to an isolated codon-optimized nucleic acid which codes a FIX (blood coagulation factor IX) protein, an expression cassette and a vector based thereon, as well as to a recombinant virus based on AAV5 (serotype 5 adeno-associated virus) for increasing FIX gene expression in target cells and their use.

EFFECT: disclosed is a codon-optimized nucleic acid which codes a coagulation factor protein ix, and its use.

10 cl, 5 ex, 1 tbl, 4 dwg

Description

AREA OF TECHNOLOGY

The present application relates to the field of genetics, gene therapy and molecular biology. More specifically, the present invention relates to an isolated codon-optimized nucleic acid that encodes the FIX protein (coagulation factor IX), an expression cassette and a vector based on it, as well as a recombinant virus based on AAV5 (adeno-associated virus 5 serotype) for increasing the expression of the FIX gene in target cells, and their use.

LEVEL OF TECHNOLOGY

Gene therapy is one of the promising branches of modern medicine. Its main focus is the development of effective solutions for the treatment of hereditary diseases, since only gene therapy methods allow us to influence the cause of these diseases. Of the large number of hereditary diseases, one of the most common is a group of pathologies associated with hemostasis disorders.

Hemophilia is an X-linked disorder associated with the absence or severe deficiency of plasma coagulation factors, characterized by a blood clotting disorder clinically manifested in the form of spontaneous or provoked, often uncontrolled, bleeding into joints, muscles and internal organs, etc.

Hemophilia B is caused by the absence or deficiency of plasma coagulation factor IX. In most cases, the disease has a family history, but in some cases sporadic mutations are detected. The overwhelming majority of patients with hemophilia are males; cases of hemophilia in women have been established, but are extremely rare. Blood coagulation factor IX (FIX, Christmas factor) is a proenzyme of serine protease, which in the presence of Ca2+ and membrane phospholipids hydrolyzes the arginine-isoleucine bond in the factor X molecule to form activated factor X (FXa). The catalytic efficiency of factor 1Xa increases upon binding of the cofactor - activated coagulation factor VIII (FVIIIa).

Factor IX is synthesized in the liver as an inactive precursor protein that is processed in the endoplasmic reticulum and Golgi apparatus, where it undergoes multiple post-translational modifications of various types and is secreted into the circulation after proteolytic cleavage of the propeptide. In the blood coagulation cascade, factor IX is activated after proteolytic cleavage by activated factor XI (intrinsic pathway) or activated factor VII (extrinsic pathway) to form two disulfide-linked polypeptide chains. Activated factor IX is gradually inactivated, mainly by slow binding to antithrombin III, nexin-2, protein Z-dependent protease inhibitor, and hepatocyte endocytosis receptors, and by cleavage by neutrophil elastase.

Currently, replacement therapy is used. After the development of the cryoprecipitation method in 1966, the first coagulation factor preparation was registered, which was obtained from donor blood plasma. In the 1980s, it was found that coagulation factor preparations obtained from plasma could be infected with viruses (HIV, hepatitis C), which led to the infection of about 20,000 patients. This circumstance served as an impetus for the development of methods for the elimination and inactivation of viruses in the production of plasma preparations and the creation of new preparations obtained without the use of plasma. The production process for obtaining preparations from plasma (plasma derived - pdFIX) included a heat treatment stage, which made it possible to eliminate contamination of the preparations. In parallel with the improvement of the technological process for obtaining preparations from plasma, research was conducted on the development of coagulation factors using recombinant DNA technology. Based on this technology, recombinant blood coagulation factor IX (rFIX) preparations were obtained and registered in 1997. The technology for obtaining preparations based on recombinant DNA allows to significantly reduce the risk of viral contamination of preparations. Currently, the preparations for replacement therapy of hemophilia are plasma and recombinant preparations, however, they have a number of disadvantages.

The main problem with the production of plasma preparations is the need for large volumes of plasma. At the same time, despite the fact that since the late 1980s there have been no cases of infection of patients using pdFIX, the manufacturers of these preparations cannot theoretically exclude the possibility of their viral contamination.

Among the disadvantages of FIX drugs currently used to treat hemophilia, the following should be highlighted:

• theoretical probability of viral infection of patients with plasma preparations;

• high immunogenicity of plasma and recombinant preparations;

• lower (in comparison with plasma preparations) effectiveness of recombinant preparations;

• low period of circulation of blood clotting factors in the blood;

• need for frequent intravenous infusions (2-3 times a week);

• lack of wide availability of lifelong replacement therapy.

The use of gene therapy drugs to transduce the factor IX gene is a fundamentally new and promising approach compared to existing treatment options: a gene therapy drug administered by intravenous infusion restores the production of the clotting factor in the patient's body.

One of the main methods of gene therapy is the delivery of a target gene into the body's cells using viral vectors, such as an AAV-based vector.

Adeno-associated virus (AAV) is a small (25 nm), non-enveloped, non-spontaneous virus. Many different AAV serotypes have been described in humans and primates. The adeno-associated virus genome contains (+ or -) single-stranded DNA (ssDNA) approximately 4.7 thousand nucleotides long. At the ends of the genomic DNA molecule are inverted terminal repeats (ITRs). The genome contains two open reading frames (ORFs): Rep and Cap, which contain several alternative reading frames encoding different protein products. The Rep products are essential for AAV replication, and the Cap gene, in addition to other alternative products, encodes three capsid proteins (VP1, VP2, and VP3). The VP1, VP2, and VP3 proteins are present in a 1:1:10 ratio to form an icosahedral capsid (Xie Q. et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA, 2002; 99:10405-10410). To form a recombinant AAV vector (rAAV), an expression cassette flanked by an ITR is packaged into the AAV capsid. The genes required for AAV replication are not included in the cassette. Recombinant AAV is considered the safest and one of the most widely used viral vectors for in vivo gene transfer. The vectors can infect cells from a variety of tissue types, providing potent and robust transgene expression. They are also nonpathogenic and have a low immunogenicity profile (High KA et al., "rAAV human trial experience"). Methods Mol Biol. 2011; 807:429-57).

One of the pressing goals of research in the field of developing effective gene therapy is codon optimization of genes of interest in vectors to obtain the maximum level of expression of genes of interest, which, in turn, will allow the use of lower doses of the vector to achieve a significant effect.

One of the properties of the genetic code is degeneracy - the ability of different codons (trinucleotides) to code the same amino acid. Such codons, which give the same amino acid in the process of translation, are called synonymous. In natural sequences, the choice of one of the synonymous codons is carried out randomly in the process of evolution, but the frequencies of using synonymous codons differ: for each amino acid there are more and less preferable ones. Codon optimization is a technique widely used in the world aimed at increasing the productivity of protein molecule production, which consists in rationally matching each amino acid in the protein sequence with one of the suitable synonymous codons. One of the common principles of codon optimization involves the use of the most frequent codons; other approaches were subsequently proposed, such as harmonization (reproduction of the distribution of frequencies of used codons), but they do not always increase productivity. In addition to codon frequencies, the efficiency of production can be affected by the GC content of the sequence (the ratio of the number of guanines and cytosines to the total length of the sequence); in particular, it has been shown that an elevated GC content is associated with an increase in the amount of mRNA in mammalian cells (Grzegorz Kudla ET AL., High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells, June 2006, Volume 4, Issue 6, el80, pp.933-942). It is also worth noting that stable elements of the secondary structure of mRNA, i.e. those with low free energy of folding, can reduce efficiency.

Different codon optimization options for the sequence of the gene of interest can lead to the following (in comparison with the wild-type gene):

a) the expression level of the genes of interest will be slightly increased;

b) the level of expression of the genes of interest will be significantly increased;

c) the expression level of the genes of interest will remain approximately at the same level;

d) the level of expression of the genes of interest will be reduced.

Thus, there is a need to obtain a codon-optimized FIX gene sequence to increase the expression of the FIX gene in target cells and create a gene therapy drug based on it.

DESCRIPTION OF THE INVENTION

The authors of this group of inventions have established that the codon-optimized nucleic acid of the invention, which encodes the FIX protein (coagulation factor IX), with the nucleotide sequence of SEQ ID NO: 2 (hFIXco-v1) or SEQ ID NO: 4 (hFIXco-v2) unexpectedly shows an increase in the expression level of the FIX gene and an increase in the level of production of the coagulation factor IX protein by several times compared to the wild-type gene encoding the coagulation factor IX (hFIX-wt). These variants of the codon-optimized nucleic acid of the invention with the nucleotide sequence of SEQ ID NO: 2 (hFIXco-v1) and SEQ ID NO: 4 (hFIXco-v2) are part of an expression cassette and a vector based on it, as well as part of a recombinant virus based on AAV5 (adeno-associated virus serotype 5).

Brief description of the invention

In one aspect, the present invention relates to an isolated codon-optimized nucleic acid that encodes a FIX (coagulation factor IX) protein with the amino acid sequence of SEQ ID NO: 1, and which comprises a nucleotide sequence selected from the group: SEQ ID NO: 2 or SEQ ID NO: 4.

In one aspect, the present invention relates to an expression cassette that comprises the above-mentioned codon-optimized nucleic acid.

In some embodiments, the expression cassette includes the following elements in the 5' to 3' direction:

left (first) ITR (inverted terminal repeats);

TTR promoter (transthyretin promoter);

intron of the hBG1 gene (a fragment of the human β globin gene carrying an intron);

the above-mentioned codon-optimized nucleic acid;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

right (second) ITR.

In some embodiments, the expression cassette comprises a nucleotide sequence selected from the group: SEQ ID NO: 3 or SEQ ID NO: 5.

In one aspect, the present invention relates to an expression vector that comprises the above-mentioned codon-optimized nucleic acid or any of the above-mentioned expression cassettes.

In one aspect, the present invention relates to an isolated recombinant virus based on AAV5 (adeno-associated virus 5 serotype) for increasing the expression of the FIX gene in target cells, which comprises the above-mentioned codon-optimized nucleic acid or any of the above-mentioned expression cassettes.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes the AAV5 VP1 protein.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes the AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 11 with one or more point mutations.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes the AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 11 or the amino acid sequence of SEQ ID NO: 11 with one or more point mutations, and the expression cassette includes the following elements in the 5'-to-3'-direction:

left (first) ITR (inverted terminal repeats); TTR promoter (transthyretin promoter);

intron of the hBG1 gene (a fragment of the human β globin gene carrying an intron); the above-mentioned codon-optimized nucleic acid;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

right (second) ITR.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 11 or the amino acid sequence of SEQ ID NO: 11 with one or more point mutations, and the expression cassette includes a nucleotide sequence selected from the group: SEQ ID NO: 3 or SEQ ID NO: 5.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 11 with one or more point mutations, is the amino acid sequence of SEQ ID NO: 14.

In one aspect, the present invention relates to a pharmaceutical composition for delivering a FIX gene to target cells, which comprises any of the above-mentioned recombinant AAV5-based viruses in combination with one or more pharmaceutically acceptable excipients.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition for delivering the FIX gene to target cells.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition for providing FIX protein to a subject who has hemophilia B and/or lacks fully functional copies of the FIX gene.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition for the treatment of hemophilia B in a subject who has hemophilia B.

In one aspect, the present invention relates to a method of providing FIX protein to a subject with hemophilia B, comprising administering a therapeutically effective amount of any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition to cells of the subject in need thereof.

In one aspect, the present invention relates to a method of delivering a FIX gene to target cells of a subject with hemophilia B, comprising administering any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition to the cells of the subject.

In one aspect, the present invention relates to a method of treating hemophilia B in a subject, which comprises administering a therapeutically effective amount of any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition to a subject who has hemophilia B.

Brief description of the drawings

Figure 1 is a schematic of the plasmids pAAV-hFIXco-v1 and pAAV-hFIXco-v2, designed to produce AAV vectors with an expression cassette that contains codon-optimized sequences of the human blood coagulation factor IX (FIX) gene hFIXco-v1 and hFIXco-v2, respectively, where

hFIXco-v1 - codon-optimized sequence of the human blood coagulation factor IX gene (variant No. 1);

hFIXco-v2 - codon-optimized sequence of the human blood coagulation factor IX gene (variant No. 2);

AmpR is a beta-lactamase gene that provides resistance to ampicillin;

pUC origin - pUC origin of replication in bacteria;

ITR - inverted terminal repeats;

TTR Promoter - transthyretin gene promoter;

Poly A - polyadenylation signal sequence to increase mRNA stability;

HBG Intron - (human beta globine intron), a fragment of the human β globin gene carrying an intron.

Figure 2 is a graph showing the expression level of the FIX gene in Huh7 cells 7 days after transduction of the cells with an AAV5-FIX virus preparation carrying the wild-type FIX gene (AAV5-hFIX-wt), an AAV5-FIX preparation carrying the codon-optimized hFIXco-v1 gene (AAV5-hFIXco-v1), and an AAV5-FIX preparation carrying the codon-optimized hFIXco-v2 gene (AAV5-hFIXco-v2) (all FIX gene variants in the AAV-FIX preparations contain the natural Padua mutation, R338L). * - p-value < 0.05. Statistical analysis was performed using two-way ANOVA with Dunnett's test.

Figure 3A is a graph showing the FIX protein concentration in culture fluid 7 days after transduction of Huh7 cells with an AAV5-FIX virus preparation carrying the wild-type FIX gene (AAV5-hFIX-wt), an AAV5-FIX preparation carrying the codon-optimized hFIXco-v1 gene (AAV5-hFIXco-v1), and an AAV5-FIX preparation carrying the codon-optimized hFIXco-v2 gene (AAV5-hFIXco-v2) (all FIX gene variants in the AAV-FIX preparations contain the natural Padua mutation, R338L). Control - untransduced Huh7 cells. *** - p-value < 0.001. Statistical analysis was performed using two-way ANOVA with Dunnett's test.

Figure 3B is a graph showing the FIX protein activity in culture fluid 7 days after transduction of Huh7 cells with the AAV5-FIX virus preparation carrying the wild-type FIX gene (AAV5-hFIX-wt), the AAV5-FIX preparation carrying the codon-optimized hFIXco-v1 gene (AAV5-hFIXco-v1), and the AAV5-FIX preparation carrying the codon-optimized hFIXco-v2 gene (AAV5-hFIXco-v2) (all FIX gene variants in the AAV-FIX preparations contain the natural Padua mutation, R338L). Control - untransduced Huh7 cells. *** - p-value < 0.001. Statistical analysis was performed using two-way ANOVA with Dunnett's test.

Figure 4A is a graph showing the plasma protein content of factor IX in experimental animals after intravenous administration of an AAV5-FIX preparation carrying the wild-type FIX gene (AAV5- hFIX-wt) and an AAV5-FIX preparation carrying the codon-optimized hFIXco-v1 gene (AAV5-hFIXco-v1) (all FIX gene variants in AAV-FIX preparations contain the natural Padua mutation, R338L). Mean values±standard deviation (n=10). *** - p-value < 0.001; ** - p-value < 0.01; * - p-value < 0.05. Statistical analysis was performed using one-way ANOVA with Dunnett's test.

Figure 4B is a graph showing the plasma protein content of coagulation factor IX in experimental animals after intravenous administration of the AAV5-FIX preparation carrying the wild-type FIX gene (AAV5- hFIX-wt) and the AAV5-FIX preparation carrying the codon-optimized hFIXco-v2 gene (AAV5-hFIXco-v2) (all FIX gene variants in the AAV-FIX preparations contain the natural Padua mutation, R338L). Mean values ± standard deviation (n=10). *** - p-value < 0.001; ** - p-value < 0.01; * - p-value < 0.05. Statistical analysis was performed using one-way ANOVA with Dunnett's test.

Definitions and General Methods

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art.

In addition, unless the context otherwise requires, singular terms include plural terms, and plural terms include singular terms. Generally, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art and are widely used in the art. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is customary in the art, or as described herein.

"Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in an animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the materials accompanying it in its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment, such as, for example, a genetically modified cell.

The terms "naturally occurring," "native," or "wild-type" are used to describe something that can be found in nature as distinct from something produced artificially. For example, a protein or nucleotide sequence that is present in an organism (including a virus), that can be isolated from its source in nature, and that has not been intentionally modified by a technician in a laboratory, is naturally occurring.

The term "genome" refers to the complete genetic material of an organism.

In the present description and in the following claims, unless the context otherwise requires, the words “include” and “comprise” or variations thereof such as “having,” “includes,” “including,” “comprises,” or “containing,” are to be understood as including the stated whole or group of wholes, but not excluding any other whole or group of wholes.

Protein (Peptide)

In the present description, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that a protein or peptide sequence may contain. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and longer chains, typically referred to in the art as proteins, of which there are many types. "Polypeptides" include, but are not limited to, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include naturally occurring peptides, recombinant peptides, synthetic peptides, or a combination thereof.

Nucleic acid molecules

The terms "nucleic acid", "nucleic sequence" or "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", which are used interchangeably in this specification, denote a distinct sequence of nucleotides, modified or unmodified, defining a fragment or section of nucleic acid, containing or not containing non-natural nucleotides and being either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of said DNAs.

As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any methods available in the art, including, but not limited to, recombinant methods, i.e., cloning nucleic acid sequences from a recombinant library or cell genome, using conventional cloning and PCR technology, etc., and synthetic methods.

It should also be mentioned here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in their natural state. The sequences of the present invention have been isolated and/or purified, i.e. taken directly or indirectly, for example by copying, whereby their environment has been at least partially modified. Thus, isolated nucleic acids obtained by genetic recombination, for example with the help of host cells, or obtained by chemical synthesis should also be understood here.

The term nucleotide sequence includes its complement unless otherwise specified. Thus, a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

Adeno-associated virus (AAV)

Viruses of the family Parvoviridae are small DNA viruses of animals. The family Parvoviridae can be divided into two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect insects. By 2006, 11 serotypes of adeno-associated virus had been described (Mori, S. ET AL., 2004, “Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein,” Virology, T. 330 (2): 375–83). All known serotypes can infect cells of many tissue types. Tissue specificity is determined by the serotype of the capsid proteins, so adeno-associated virus-based vectors are constructed by specifying the required serotype. Additional information on parvoviruses and other members of Parvoviridae is described in the literature (Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication)), Chapter 69 in Fields Virology (3d Ed. 1996)).

The genomic organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule that is less than approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (1TR) flank unique nucleotide coding sequences for the replication nonstructural proteins (Rep) and structural proteins (Cap). The Cap gene encodes the VP proteins (VP1, VP2, and VP3) that form the capsid. The terminal 145 nt are self-complementary and are organized in such a way that an energetically stable intramolecular duplex can be formed, forming a T-shaped hairpin structure. These hairpin structures function as origins of viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following infection of mammalian cells with wild-type AAV (wtAAV), Rep genes (e.g., Rep78 and Rep52) are expressed through the P5 promoter and P19 promoter, respectively, and both Rep proteins have a defined function in viral genome replication. Splicing within the Rep open reading frame (Rep ORF) results in the expression of virtually four Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep40). However, unspliced mRNA encoding the Rep78 and Rep52 proteins has been shown to be sufficient for AAV vector production in mammalian cells.

Vector

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Additionally, the term "vector" as used herein means a viral particle capable of transporting a nucleic acid.

As used herein, the term "expression" is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

Application

"Gene therapy" is the insertion of genes into a subject's cells and/or tissues to treat a disease, usually a hereditary disease, whereby a defective mutant allele is replaced or supplemented with a functional allele.

"Treat," "treatment," and "therapy" refer to a method of alleviating or eliminating a biological disorder and/or at least one of its associated symptoms. As used herein, the term "alleviate" a disease, disorder, or condition means to reduce the severity and/or frequency of the symptoms of the disease, disorder, or condition. Additionally, references herein to "treatment" include references to curative, palliative, and prophylactic therapy.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The said subject may be male or female of any age.

The term “disorder” means any condition that can be improved by treatment according to the present invention.

"Disease" is a state of health of a subject where the subject cannot maintain homeostasis, and where, unless the disease is alleviated, the subject's health continues to deteriorate.

The term "subject", "patient", "individual" and the like are used interchangeably herein and refer to any animal that is amenable to the methods described herein. In certain non-limiting embodiments, the subject, patient or individual is a human. The aforementioned subject may be male or female of any age.

A "therapeutically effective amount" or "effective amount" is the amount of a therapeutic agent administered that will relieve to some degree one or more symptoms of the disease being treated.

Detailed description of the invention

Nucleic acid

In one aspect, the present invention relates to an isolated codon-optimized nucleic acid that encodes a FIX (coagulation factor IX) protein with an amino acid sequence

An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in a natural source of the nuclease nucleic acid. An isolated nucleic acid molecule is different from the form or set in which it is naturally found. Thus, an isolated nucleic acid molecule is different from a nucleic acid molecule that naturally exists in cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule that is found in cells that normally express the nuclease, such as when the nucleic acid molecule has a chromosomal location that is different from its location in cells in natural conditions.

To obtain the above codon-optimized nucleic acid, codon optimization was performed on a wild-type nucleic acid with the nucleotide sequence

Codon optimization of the wild-type nucleic acid encoding the FIX protein with the nucleotide sequence SEQ ID NO: 17 yielded a series of codon-optimized nucleic acids that were further tested for protein production levels compared to the control (wild-type nucleic acid with SEQ ID NO: 17).

All codon-optimized nucleic acids showed an increase in the level of FIX protein production compared to the wild type, with the codon-optimized nucleic acid of the invention with the nucleotide sequence of SEQ ID NO: 2 (hFIXco-v1) and the codon-optimized nucleic acid of the invention with the nucleotide sequence of SEQ ID NO: 4 (hFIXco-v2) unexpectedly showing the best indicator, namely an increase in the level of FIX gene expression, an increase in the level of FIX protein production by several times compared to the wild type (see examples 2, 3 and 4).

Expression cassette. Expression vector.

In one aspect, the present invention relates to an expression cassette that comprises the above-mentioned codon-optimized nucleic acid that encodes a FIX (coagulation factor IX) protein.

The term "expression cassette" or "expression cassette" as used herein, in particular, refers to a fragment of DNA that is capable, under appropriate circumstances, of driving the expression of a polynucleotide encoding a polypeptide of interest that is included in said expression cassette. When introduced into a host cell, the expression cassette is, among other things, capable of engaging the cellular machinery to transcribe the polynucleotide encoding the polypeptide of interest into RNA, which is then typically further processed and finally translated into the polypeptide of interest. The expression cassette may be contained in an expression vector.

The expression cassette of the present invention comprises a promoter element. The term "promoter" as used herein particularly refers to a DNA element that promotes transcription of a polynucleotide to which the promoter is operably linked. A promoter may also form part of a "promoter/enhancer" element. Although the physical boundaries between the "promoter" and "enhancer" elements are not always clear, the term "promoter" generally refers to a site on a nucleic acid molecule to which RNA polymerase and/or associated factors bind and from which transcription is initiated. Enhancers enhance promoter activity temporally as well as spatially. A variety of promoters are known in the art that are transcriptionally active in a wide range of cell types. Promoters can be divided into two classes: those that function constitutively and those that are regulated by induction or release of repression. Both classes are useful for protein expression. Promoters that are used for the production of high levels of polypeptides in eukaryotic cells and in particular in mammalian cells should be strong and preferably should be active in a wide range of cell types. Strong constitutive promoters that are capable of driving expression in many cell types are well known in the art and therefore need not be described in detail herein. In accordance with the teachings of the present invention, it is preferred to use the TTR promoter. The TTR promoter or promoter/enhancer is particularly suitable as a promoter in the expression cassette of the present invention. According to one embodiment of the invention, the TTR promoter is used in the expression cassette of the present invention.

In some embodiments of the invention, the expression cassette comprises the following elements in the 5' to 3' direction:

left (first) 1TR (inverted terminal repeats);

TTR promoter (transthyretin promoter);

intron of the hBG1 gene (a fragment of the human |3 globin gene carrying an intron);

the above-mentioned codon-optimized nucleic acid, which encodes the FIX protein (coagulation factor IX);

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

right (second) ITR.

In some embodiments of the invention, the left (first) ITR has the following nucleic acid sequence:

In some embodiments of the invention, the TTR promoter has the following nucleic acid sequence:

In some embodiments of the invention, the intron of the hBG1 gene has the following nucleic acid sequence:

In some embodiments, the hGH1 polyadenylation signal has the following nucleic acid sequence:

In some embodiments of the invention, the right (second) ITR has the following nucleic acid sequence:

In some embodiments of the invention, the expression cassette comprises a nucleotide sequence selected from the group:

or

In one aspect, the present invention relates to an expression vector that comprises the above-mentioned codon-optimized nucleic acid or any of the above-mentioned expression cassettes.

In some embodiments of the invention, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated.

In some embodiments of the invention, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.

In some embodiments, vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). In other embodiments, vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell, and thus replicate along with the host gene. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors" ("expression vector" or "expression vector")).

Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic viruses, cosmids, YAC, EBV derived episomes, and the like. DNA molecules can be ligated into a vector such that transcription and translation control sequences in the vector perform the intended function of regulating transcription and translation of DNA. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules can be introduced into the expression vector by standard methods (e.g., ligation of complementary restriction sites or blunt end ligation if restriction sites are not present).

The recombinant expression vector may also encode a leader peptide (or signal peptide) that facilitates production of the protein of interest by the host cell. The gene for the protein of interest may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the protein of interest. The leader peptide (or signal peptide) may be an immunoglobulin leader peptide or a different leader peptide (i.e., a leader peptide of a protein that is not immunoglobulin in nature).

In addition to the FIX gene of the present invention, the recombinant expression vectors of the present invention may carry regulatory sequences that control expression of the FIX gene in a host cell. Those skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on factors such as the selection of the host cell for transformation, the level of expression of the desired protein, etc. Preferred regulatory sequences for the mammalian expression host cell include viral elements that provide high level expression of proteins in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR, cytomegalovirus (CMV) (e.g., the CMV promoter/enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter/enhancer), adenovirus (e.g., the adenovirus large late promoter (AdMLP)), polyoma virus, as well as strong mammalian promoters such as the TTR promoter, the native immunoglobulin promoter, or the actin promoter.

The expression "control sequences" refers to DNA sequences required for the expression of a functionally linked coding sequence in a given host organism. Suitable control sequences for prokaryotes are, for example, a promoter, optionally an operator, and a ribosome binding site. Promoters, polyadenylation signals, and enhancers are known to be present in eukaryotic cells.

As used herein, the term "promoter" or "transcriptional regulatory sequence" or "regulatory sequence" refers to a nucleic acid fragment that controls the transcription of one or more coding sequences and that is located upstream of the transcription initiation site of the coding sequence and that is structurally identifiable by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that directly or indirectly regulate the level of transcription from the promoter. A "constitutive" promoter is one that is active in most tissues under normal physiological and developmental conditions. An "inducible" promoter is one that is subject to physiological or developmental regulation, such as by exposure to a chemical inducer. A "tissue-specific" promoter is active only in specific tissue or cell types.

The terms "enhancers" or "enhancer" as used herein may refer to a DNA sequence that is located adjacent to a DNA sequence encoding a recombinant product. Enhancer elements are typically located 5' of a promoter element or may be located downstream of or within a coding DNA sequence (e.g., a DNA sequence transcribed or translated into a recombinant product or products). Thus, an enhancer element may be located 100 base pairs, 200 base pairs, or 300 base pairs or more upstream of or after a DNA sequence encoding a recombinant product. Enhancer elements may increase the amount of recombinant product expressed from a DNA sequence beyond that resulting from a single promoter element. A variety of enhancer elements are available to those skilled in the art.

In addition to the above genes and control sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, typically a selectable marker gene confers resistance to drugs, such as G418, ampicillin, hygromycin, or methotrexate, to a host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells for methotrexate selection/amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

The term "expression control sequence" as used herein refers to polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., the Kozak consensus sequence); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequences" includes, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion sequences.

As used herein, the term "operably linked" refers to the association of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" if it is in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of said coding sequence. The term "operably linked" means that the linked DNA sequences are generally contiguous and, when necessary to connect two protein coding regions, are also contiguous and in reading frame.

In one embodiment of the present invention, an "expression vector" refers to a vector comprising one or more polynucleotide sequences of interest, genes of interest, or "transgenes" that are flanked by parvovirus or inverted terminal repeat (ITR) sequences.

Neither the cassette nor the vector according to the invention contains nucleotide sequences of genes encoding non-structural proteins (Rep) and structural proteins (Cap) of the adeno-associated virus.

Recombinant virus based on AAV5 (adeno-associated virus 5 serotype)

In one aspect, the present invention relates to an isolated recombinant virus based on AAV5 (adeno-associated virus 5 serotype) for increasing the expression of the FIX gene in target cells, which comprises the above-mentioned codon-optimized nucleic acid or any of the above-mentioned expression cassettes.

The term "recombinant AAV-based virus" (or "AAV-based virus-like particle", or "recombinant AAV viral strain", or "recombinant AAV vector", or "rAAV vector") as used herein refers to the above expression cassette (or the above expression vector), which is contained within the AAV capsid.

The Cap gene, in addition to other alternative products, encodes three capsid proteins (VP1, VP2, and VP3). The VP1, VP2, and VP3 proteins are in a 1:1:10 ratio, forming an icosahedral capsid (Xie Q. et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA, 2002; 99:10405-10410). Transcription of these genes begins from a single promoter, p40. The molecular weight of the corresponding proteins (VP1, VP2, and VP3) is 87, 72, and 62 kDa, respectively. All three proteins are translated from a single mRNA. After transcription, the pre-mRNA can be spliced in two different ways, with a longer or shorter intron being excised and mRNAs of different nucleotide lengths being formed.

In the formation of a recombinant AAV-based virus (rAAV), an expression cassette flanked by an ITR is packaged into the AAV capsid. The genes required for AAV replication, as noted above, are not included in the cassette.

The expression cassette DNA is packaged into the viral capsid as a single-stranded DNA (ssDNA) molecule approximately 3,000 nucleotides long. Once a cell is infected with the virus, the single-stranded DNA is converted to a double-stranded DNA (dsDNA) form. Only dsDNA can be used by the cell's proteins, which transcribe the gene or genes it contains into RNA.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes the AAV5 VP1 protein.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes the AAV5 VP1 protein having the amino acid sequence

In some embodiments, the recombinant AAV5-based virus has a capsid that includes the AAV5 VP2 protein.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP2 protein having the following amino acid sequence:

In some embodiments, the recombinant AAV5-based virus has a capsid that includes the AAV5 VP3 protein.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes the AAV5 VP3 protein having the following amino acid sequence:

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes the VP1, VP2, and VP3 proteins of AAV5.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes proteins VP1 with the amino acid sequence of SEQ ID NO: 11, VP2 with the amino acid sequence of SEQ ID NO: 12, and VP3 with the amino acid sequence of SEQ ID NO: 13.

In some embodiments of the invention, a recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 11 with one or more point mutations.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having an amino acid sequence that includes amino acid substitutions at positions S2A and T711S of wild-type AAV5 VP1 (SEQ ID NO: 11), and has an amino acid sequence

In some embodiments of the invention, a recombinant AAV5-based virus has a capsid that includes an AAV5 VP2 protein having the amino acid sequence of SEQ ID NO: 12 with one or more point mutations.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP2 protein that has an amino acid sequence that includes an amino acid substitution at position T575S of wild-type AAV5 VP2 (SEQ ID NO: 12) and has an amino acid sequence

In some embodiments of the invention, a recombinant AAV5-based virus has a capsid that includes an AAV5 VP3 protein having the amino acid sequence of SEQ ID NO: 13 with one or more point mutations.

In some embodiments, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP3 protein that has an amino acid sequence that includes an amino acid substitution at position T519S of wild-type AAV5 VP3 (SEQ ID NO: 13) and has an amino acid sequence

In some embodiments of the invention, a recombinant AAV5-based virus has a capsid that includes proteins VP1 with the amino acid sequence of SEQ ID NO: 11 with one or more point mutations, VP2 with the amino acid sequence of SEQ ID NO: 12 with one or more point mutations, and VP3 with the amino acid sequence of SEQ ID NO: 13 with one or more point mutations.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes proteins VP1 with the amino acid sequence of SEQ ID NO: 14, VP2 with the amino acid sequence of SEQ ID NO: 15, and VP3 with the amino acid sequence of SEQ ID NO: 16.

By "multiple point mutations" we mean two, three, four, five, six, seven, eight, nine, or ten point substitutions.

Particularly preferred variants include substitutions (mutations) that are conservative in nature, i.e., those substitutions that occur within a family of amino acids that are grouped together by their side chains. In particular, amino acids are usually divided into four families: (1) acidic - aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) nonpolar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar - glycine, asparagine, glutamine, cysteine, threonine series, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that a selected substitution of leucine for isoleucine or valine, aspartate for glutamate, threonine for serine, or a similar conservative substitution of an amino acid for a structurally related amino acid will not have an important effect on biological activity. For example, a polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, provided that the desired function of the molecule remains intact.

A variant of point mutations in the sequences of the AAV5 VP1, VP2 or VP3 proteins by amino acid substitutions is a replacement of at least one amino acid residue in the AAV5 VP1, VP2 or VP3 protein with another amino acid residue.

Conservative substitutions are shown in Table A under the heading "preferred substitutions."

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 11 or the amino acid sequence of SEQ ID NO: 11 with one or more point mutations, and the expression cassette includes the following elements in the 5'-to-3'-direction:

left (first) ITR (inverted terminal repeats);

TTR promoter (transthyretin promoter);

intron of the hBG1 gene (a fragment of the human β globin gene carrying an intron);

the above-mentioned codon-optimized nucleic acid;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

right (second) ITR.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 11, an AAV5 VP2 protein having the amino acid sequence of SEQ ID NO: 12, and an AAV5 VP3 protein having the amino acid sequence of SEQ ID NO: 13, and the expression cassette includes the following elements in the 5'-to-3'-direction:

left (first) ITR (inverted terminal repeats);

TTR promoter (transthyretin promoter);

intron of the hBG1 gene (a fragment of the human β globin gene carrying an intron);

the above-mentioned codon-optimized nucleic acid;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

right (second) ITR.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 11 with one or more point mutations, an AAV5 VP2 protein having the amino acid sequence of SEQ ID NO: 12 with one or more point mutations, and an AAV5 VP3 protein having the amino acid sequence of SEQ ID NO: 13 with one or more point mutations, and the expression cassette includes the following elements in the 5'-to-3'-direction:

left (first) ITR (inverted terminal repeats);

TTR promoter (transthyretin promoter);

intron of the hBG1 gene (a fragment of the human β globin gene carrying an intron);

the above-mentioned codon-optimized nucleic acid;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

right (second) ITR.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 14, an AAV5 VP2 protein having the amino acid sequence of SEQ ID NO: 15, and an AAV5 VP3 protein having the amino acid sequence of SEQ ID NO: 16, and the expression cassette includes the following elements in the 5'-to-3'-direction:

left (first) ITR (inverted terminal repeats);

TTR promoter (transthyretin promoter);

intron of the hBG1 gene (a fragment of the human β globin gene carrying an intron);

the above-mentioned codon-optimized nucleic acid;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

right (second) ITR.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes an AAV5 VP1 protein having the amino acid sequence of SEQ ID NO: 11 or the amino acid sequence of SEQ ID NO: 11 with one or more point mutations, and the expression cassette includes a nucleotide sequence selected from the group: SEQ ID NO: 3 or SEQ ID NO: 5.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes proteins VP1 with the amino acid sequence of SEQ ID NO: 11, VP2 with the amino acid sequence of SEQ ID NO: 12 and VP3 with the amino acid sequence of SEQ ID NO: 13, and the expression cassette includes a nucleotide sequence that is selected from the group: SEQ ID NO: 3 or SEQ ID NO: 5.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes VP1 proteins with the amino acid sequence of SEQ ID NO: 11 with one or more point mutations, VP2 with the amino acid sequence of SEQ ID NO: 12 with one or more point mutations, and VP3 with the amino acid sequence of SEQ ID NO: 13 with one or more point mutations, and the expression cassette includes a nucleotide sequence selected from the group: SEQ ID NO: 3 or SEQ ID NO: 5.

In some embodiments of the invention, the recombinant AAV5-based virus has a capsid that includes proteins VP1 with the amino acid sequence of SEQ ID NO: 14, VP2 with the amino acid sequence of SEQ ID NO: 15 and VP3 with the amino acid sequence of SEQ ID NO: 16, and the expression cassette includes a nucleotide sequence that is selected from the group: SEQ ID NO: 3 or SEQ ID NO: 5.

Pharmaceutical composition

In one aspect, the present invention relates to a pharmaceutical composition for delivering a FIX gene to target cells, which comprises any of the above-mentioned recombinant AAV5-based viruses in combination with one or more pharmaceutically acceptable excipients.

The active substance in the above composition is in an effective amount, for example, in a biologically effective amount.

In particular embodiments, the present invention relates to a pharmaceutical composition comprising a recombinant AAV5-based virus of the invention in a pharmaceutically acceptable carrier or in other pharmaceutical agents, adjuvants, diluents, etc. The carrier for injection is usually liquid. The carrier for other modes of administration can be either solid or liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline. For administration by inhalation, the carrier is respirable and is preferably in solid or liquid dispersed form. As an injection medium, it is preferable to use water containing additives conventional for injection solutions, such as stabilizing agents, salts or saline solutions and/or buffers.

"Pharmaceutical composition" means a composition comprising the above-mentioned recombinant AAV5-based virus of the invention and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients such as fillers, solvents, diluents, carriers, auxiliary, distributing, delivery vehicles, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, regulators of prolonged delivery, the choice and ratio of which depends on the nature and route of administration and dosage. The pharmaceutical compositions of the present invention and the methods for their manufacture will be undoubtedly obvious to those skilled in the art. The manufacture of pharmaceutical compositions should preferably comply with the requirements of GMP (good manufacturing practice). The composition may include a buffer composition, tonic agents, stabilizers and solubilizers.

"Pharmaceutically acceptable" means a material that does not have biological or other contraindications, for example, the material can be administered to a subject without any undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, to transfect a cell ex vivo or to administer in vivo a recombinant AAV5-based virus of the invention directly to a subject.

The term "excipient" or "auxiliary substance" is used in this document to describe any component different from those previously described in this invention. These are substances of inorganic or organic origin used in the process of production, manufacturing of medicinal products to impart the necessary physicochemical properties to them.

A "stabilizer" is an excipient or a mixture of two or more excipients that ensures the physical and/or chemical stability of the active agent.

The term "buffer", "buffer composition", "buffering agent" means a solution capable of maintaining a pH value due to the interaction of acidic and alkaline components included in its composition, which enables the rAAV5-based vector preparation to exhibit resistance to pH changes. In general, the pH values of the pharmaceutical composition from 4.0 to 8.0 are preferable. For example, acetate, phosphate, citrate, histidine, succinate, etc. buffer solutions can be used as buffering agents, but are not limited to them.

A pharmaceutical composition is "stable" if the active agent retains its physical stability and/or chemical stability and/or biological activity during the stated shelf life at a storage temperature of, for example, 2-8°C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The shelf life is selected based on the results of stability studies under accelerated and natural storage.

The pharmaceutical composition of the present invention may be manufactured, packaged, or commercially sold in a single unit dose or multiple single unit doses in a finished dosage form. As used herein, the term "unit dose" means a discrete quantity of the pharmaceutical composition containing a predetermined amount of the active ingredient. The quantity of the active ingredient is typically equal to the dosage of the active ingredient to be administered to the subject, or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

Application

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition for delivering the FIX gene to target cells.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition for providing FIX protein to a subject who has hemophilia B and/or lacks fully functional copies of the FIX gene.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition for the treatment of hemophilia B in a subject who has hemophilia B.

In one aspect, the present invention relates to a method of providing FIX protein to a subject with hemophilia B, comprising administering a therapeutically effective amount of any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition to cells of the subject in need thereof.

In one aspect, the present invention relates to a method of delivering a FIX gene to target cells of a subject with hemophilia B, comprising administering any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition to the cells of the subject.

In one aspect, the present invention relates to a method of treating hemophilia B in a subject, which comprises administering a therapeutically effective amount of any of the above-mentioned recombinant AAV5-based viruses or the above-mentioned composition to a subject who has hemophilia B.

Hemophilia B is an inherited bleeding disorder caused by a deficiency or absence of clotting factor IX (FIX). The incidence of hemophilia B is about 1 in 40,000 male births. The deficiency of the clotting factor causes spontaneous or induced bleeding into joints, muscles, and internal organs.

Absence of fully functional copies of the FIX gene refers to inactivating mutations or deletions in all copies of the FIX gene in the genome that result in loss or defective function of the FIX gene.

By subject is meant any animal that is susceptible to the methods described herein. In certain non-limiting embodiments, the subject is a human. The aforementioned subject may be male or female of any age.

A subject in need of delivery of a FIX gene to target cells or a subject in need of provision of a FIX protein means a subject who has hemophilia B, or a subject who has a deficiency of coagulation factor IX, or a subject who has inactivating mutations or deletions in the FIX gene that result in a loss or defect in the function of the FIX gene.

Examples of routes of administration include topical, intranasal, inhalational, transmucosal, transdermal, enteral (e.g., oral, rectal), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular) administration, and injection directly into tissue or into an organ.

Injectable preparations can be prepared in conventional dosage forms: as liquid solutions or suspensions, solid forms suitable for preparing solutions or suspensions in liquid prior to injection, or as emulsions. Alternatively, the above-mentioned recombinant AAV5-based virus of the present invention can be administered locally rather than systemically, for example, as a depot or in a sustained-release composition.

The recombinant AAV5-based virus is administered to the body in an effective amount. The recombinant AAV5-based virus is preferably administered to the body in a biologically effective amount. A "biologically effective" amount of a recombinant virus is an amount that is sufficient to cause transduction and expression of a nucleic acid sequence in a cell. If the virus is administered to a cell in vivo (e.g., the virus is administered to a subject as described below), a "biologically effective" amount of the viral vector is an amount that is sufficient to cause transduction and expression of the nucleic acid sequence in the target cell.

The dosages of the above-mentioned recombinant AAV5-based virus of the present invention will depend on the route of administration of the particular viral vector, and can be determined by routine methods. Exemplary dosages for achieving a therapeutic effect are viral titers of at least about 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , 10 ¹⁶ transducing units or more, preferably about 10 ⁹ to 10 ¹⁵ transducing units, even more preferably 10 ¹⁴ transducing units per kilogram.

The cell for administering the above-mentioned recombinant AAV5-based virus of the invention may be any cell type, including, but not limited to, epithelial cells (e.g., epithelial cells of the skin, respiratory tract, and intestine), liver cells, muscle cells, spleen cells, fibroblasts, endothelial cells, and the like.

The above-mentioned recombinant AAV5-based virus according to the invention is not used to modify the genetic integrity of human germline cells.

Examples

For a better understanding of the invention, the following examples are given. These examples are given for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference. Although the above invention has been described in some detail by way of illustration and example for the purpose of avoiding ambiguity, it will be readily apparent to those skilled in the art, based on the teachings disclosed in this invention, that certain changes and modifications may be made without departing from the spirit and scope of the accompanying embodiments of the invention.

Materials and General Methods Recombinant DNA Methods

DNA manipulations followed standard procedures described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturers' instructions. Briefly, plasmid DNA for further manipulations was produced in E. coli cells grown under selective pressure with antibiotics to ensure that plasmids were not lost in the cell population. Plasmid DNA was isolated from the cells using commercial kits, measured for concentration, and used for cloning by restriction endonuclease digestion or PCR amplification. DNA fragments were ligated together using ligases and transformed into bacterial cells for clone selection and further development. All resulting genetic constructs were confirmed by restriction patterns and complete Sanger sequencing.

Gene synthesis

The desired gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments of 300 to 1000 bp in length, flanked by unique restriction sites, were assembled by annealing the oligonucleotides on each other, followed by PCR amplification from the outer primers. The result was a mixture of fragments, including the desired one. The fragments were cloned into intermediate vectors by restriction sites, after which the DNA sequences of the subcloned fragments were confirmed by DNA sequencing.

Determination of DNA sequences

DNA sequences were determined by Sanger sequencing. DNA and protein sequence analysis and sequence data processing were performed using SnapGene 4.2 and higher for sequence generation, mapping, analysis, annotation, and illustration.

Cultivation of cell cultures

The HEK293 (Human Embryonic Kidney clone 293) and HUH7 (human hepatocellular carcinoma cell lines) cell lines were used in the experiments. HEK293 suspension cells used for AAV production were cultured under standard conditions at 37°C and 5% _CO2 , in a complete nutrient medium without FBS and antibiotic. Adherent HUH7 cells used to test the efficiency of AAV preparations were cultured under standard conditions at 37°C and 5% _CO2 , in a complete DMEM nutrient medium with the addition of 10% FBS, antibiotic/antimycotic. HUH7 cells were subcultured when 80-90% confluency was reached. TrypLE Select enzyme (10x) was used to dissociate the cell monolayer. Cell viability was assessed using Trypan Blue staining and disposable cell counting chambers using a Countess II automated cell counter.

Assembly and purification of viral particles of recombinant AAV vectors

To assemble AAV viral particles containing codon-optimized variants of the FIX gene (hFIXco-v1 and hFIXco-v2), HEK293 producer cells were used, which were transfected with three plasmids:

Plasmids pAAV-hFIXco-v1 and pAAV-hFIXco-v2 containing the AAV expression cassette for expression of the hFIXco-v1 and hFIXco-v2 transgenes, respectively (Fig. 1.);

Plasmid for expression of the AAV5 serotype Cap gene and the AAV2 serotype Rep gene. Each gene encodes several protein products using alternative reading frames;

Plasmid for expression of adenovirus Ad5 genes required for assembly and packaging of AAV capsids.

After 72 hours, the cells were lysed and the viral particles were purified and concentrated using filtration, chromatography, and ultracentrifugation. The titer of viral particles was determined using quantitative PCR with primers and probe specific to a region of the recombinant viral genome and was expressed as the number of copies of viral genomes per 1 ml.

Transduction of cell cultures

The HUH7 cell line was pre-seeded into wells of 12-well plates at a density of 10,000 cells/cm ² . After cell attachment to the adhesive substrate, AAV preparations were added at an MOI of 500,000 vg/cell. On day 7 post-transduction, the FIX protein content and activity in the culture fluid were determined by ELISA, as well as the expression level of the coagulation factor IX gene in the cells by quantitative reverse transcription PCR, as described above. The FIX protein level in the culture fluid was assessed in 6 independent experiments. The activity of the secreted FIX protein and the expression level of the coagulation factor IX gene in the cells were assessed in 3 independent experiments. Intact cells were used as a negative control.

Determination of the expression level of the blood coagulation factor IX gene

The expression level of the FIX gene in cell cultures after transduction (or transfection) was determined by quantitative PCR with reverse transcription. Briefly, RNA was isolated from cell pellets using the RNeasy Plus mini kit (Qiagen) according to the manufacturer's protocol. Reverse transcription was performed using the GoScript reagent kit (Promega) according to the manufacturer's protocol, namely, 500 ng of RNA were initially taken for the reverse transcription reaction, cDNA was obtained using random and oligo-dT primers, after reverse transcription, the cDNA volume was brought to 50 μl with sterile water. Quantitative PCR was performed using TaqMan technology using the qPCRmix-HS HighROX reaction mixture (Eurogen), according to the manufacturer's protocol, on a StepOne device (Applied Biosystems). Specific primers and probes were selected for the target gene (FIX) and for the housekeeping gene (GAPDH). Plasmid DNA (plDNA) samples carrying the corresponding gene were used to construct standard curves. Seven standard samples were prepared for each standard curve. Dilution series: from 20 million to 200 plDNA copies per reaction (dilution step - 1:10). For each studied sample, the analysis was carried out in three technical replicates and was accompanied by the "RT-minus" control (to control for the absence of DNA impurity in the studied RNA sample) and a negative control. The results of determining the mRNA expression level were presented as: the number of mRNA copies of the target gene in the sample, normalized to the number of mRNA copies of the GAPDH gene.

Determination of the level of protein of blood coagulation factor IX by the ELISA method

The content of factor IX protein in the culture fluid after transduction of HUH7 with the target candidates AAV5-FIX, as well as in the blood plasma of animals after administration of the target viral preparations, was assessed using a sandwich method of non-competitive solid-phase enzyme-linked immunosorbent assay (ELISA) with a commercial kit. Briefly, samples of culture fluid and blood plasma diluted in dilution buffer were added to the wells of a 96-well plate sensitized with primary antibodies specific for factor IX. Standards for constructing a calibration curve, positive and negative controls were also added to the same plate. The plate was incubated for 2 hours at room temperature. The wells of the plate were washed with washing buffer before adding biotinylated antibodies, streptavidin peroxidase conjugate solution and TMB. A solution with specific biotinylated detection antibodies to factor IX was added and the plate was incubated for 1 hour at room temperature. Then a solution of streptavidin-peroxidase conjugate was added to the formed complex and the plate was incubated for 30 minutes at room temperature. TMB solution was added to visualize the enzymatic reaction. Upon reaching the desired degree of color intensity, stop solution was added to all wells to stop the reaction. Then the optical density of the solutions in the wells of the plate was measured. The concentration of blood coagulation factor IX in the studied samples was determined using a calibration curve taking into account the preliminary dilution of the samples.

Determination of the activity of the protein coagulation factor IX

Blood coagulation factor IX is vitamin K-dependent and for the synthesis of the active form of factor IX by the cell culture, the presence of vitamin K in the growth medium is necessary. Therefore, during HUH7 transduction, vitamin K1 was added to the complete growth medium at a concentration of 500 ng/ml.

Evaluation of the activity of the blood coagulation factor IX protein in the culture fluid after transduction of HUH7 with target drugs was carried out by the chromogenic method using a commercial kit.

Briefly, culture fluid samples, standards, and controls diluted in Tris-BSA reaction buffer were added to the wells of a 96-well plate. Reagent 1 (FX-FVIII) was then added. After 2 min of incubation at 37°C, reagent 2 (activating agent) was added. The plate was incubated for 3 min at 37°C. Reagent 3 (chromogenic substrate) was then added to the wells of the plate and the plate was incubated for 2 min. Then, 20% acetic acid was added to stop the reaction. The resulting FXa hydrolyzes the chromogenic substrate, which leads to the release of p-nitroaniline, the amount of which (detected by optical density) is directly proportional to the concentration of factor IX (FIX) in the sample.

Conducting in vivo studies on laboratory animals

C57BL/6 mice (6-8 week old males) were used for the experiments. The drugs were administered to the animals once by intravenous injection into the tail vein. A group of negative control animals was administered a buffer solution that did not contain AAV. Blood plasma was collected on the day of injection before administration of the drugs, then on days 7, 14, 21, 28, 35 and 42 after administration of the drugs.

Example 1. Modification of the FIX gene sequence using the codon optimization algorithm

The drug we developed is a suspension of modified recombinant capsids of adeno-associated virus 5 (AAV5) carrying an expression cassette encoding the human coagulation factor IX (hFIX) gene under the control of the liver-specific tissue-specific transthyretin (TTR) promoter.

The wild-type nucleic acid used was a nucleic acid encoding the wild-type human coagulation factor IX protein with the natural mutation R338L (the so-called Padua mutation) and comprising the nucleotide sequence SEQ ID NO: 17. This wild-type nucleic acid was used as a control.

Additionally, to improve expression efficiency, the natural sequence of the factor IX gene was modified using a codon optimization algorithm.

Codon optimization of the wild-type factor IX nucleic acid with the nucleotide sequence of SEQ ID NO: 17 yielded a series of codon-optimized nucleic acids that were subsequently tested for factor IX protein expression levels and activity compared to a control (wild-type nucleic acid with SEQ ID NO: 17) in an AAV5-based formulation.

All codon-optimized nucleic acids showed an increase in the level of coagulation factor IX protein production compared to the wild type, with most codon-optimized nucleic acids showing a slight increase in the level of coagulation factor IX protein production compared to the wild type and only two codon-optimized variants of the nucleic acid of the invention with the nucleotide sequence of SEQ ID NO: 2 (hFIXco-v1) and SEQ ID NO: 4 (hFIXco-v2) unexpectedly showed the best indicator, namely an increase in the level of expression of the coagulation factor IX gene and an increase in the level of coagulation factor IX protein production by several times, which, in turn, led to an increase in the activity of the AAV5-based drug, which contained SEQ ID NO: 2 (hFIXco-v1) or SEQ ID NO: 4 (hFIXco-v2) (see Examples 2, 3 and 4).

Codon-optimized nucleic acids with the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 4 are characterized by an increased codon adaptation index (a standard metric for assessing a sequence for the frequencies of codons used) for mammalian cells compared to the wild-type nucleic acid with the nucleotide sequence of SEQ ID NO: 17.

Codon-optimized nucleic acids with the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 4 are referred to hereinafter in the examples as hFIXco-v1 and hFIXco-v2, respectively.

Example 2. Assembly of a genetic construct containing an AAV expression cassette with variants of the recombinant codon-optimized gene of blood coagulation factor IX (hFIXco-v1 and hFIXco-v2).

The target plasmids pAAV-hFIXco-v1 or pAAV-hFIXco-v2 (Fig. 1), designed to produce AAV5 viral vectors with an expression cassette containing a codon-optimized version of the factor IX gene (SEQ ID NO: 2 or SEQ ID NO: 4), were obtained by sequentially replacing the modified green fluorescent protein sequence and the CMV promoter in the original pAAV-GFP construct using the restriction enzyme ligation cloning method with codon-optimized sequences of the factor IX gene and the TTR promoter, respectively, synthesized de novo from oligonucleotides created by chemical synthesis.

The final vector contains all the necessary elements for gene expression and assembly into the recombinant AAV genome:

1) terminal ITR repeats at the ends of the sequence that is encapsidated into the viral capsid;

2) elements for expression of the target gene (promoter, enhancer, intron, Kozak sequence, transgene, polyadenylation site);

3) the origin of bacterial replication and the antibiotic resistance gene for the production of plasmid DNA in bacterial cells.

Example 3. Creation of viral preparations expressing blood coagulation factor IX

The target plasmids pAAV-hFIXco-v1 and pAAV-hFIXco-v2 (Fig. 1) together with the other plasmids required for the production of recombinant AAV viral particles (see above) were used to produce AAV5-hFIXco-v1 and AAV5-hFIXco-v2 preparations carrying, respectively, codon-optimized versions of the factor IX gene (hFIXco-v1 and hFIXco-v2). The bioprocess resulted in the production of recombinant viral particles AAV5-hFIXco-v1 and AAV5-hFIXco-v2 containing expression cassettes with codon-optimized versions of the factor IX gene (hFIXco-v1 and hFIXco-v2). Purified AAV5-hFIXco-v1 and AAV5-hFIXco-v2 preparations used for in vitro and in vivo studies were prepared using standard buffers and excipients that are safe and do not alter the properties of AAV. As a comparison preparation, AAV5-hFIX-wt preparation containing an expression cassette with the natural gene of blood coagulation factor IX (wild-type gene including the natural mutation R338L, see Example 1) was produced using the technology described above.

Example 4. In vitro testing of the performance of AAV5-hFIXco-v1 and AAV5-hFIXco-v2 preparations

Prior to animal studies, purified AAV5-hFIXco-v1 and AAV5-hFIXco-v2 preparations were tested in vitro. These experiments were performed using the adherent cell line HUH7 (Figs. 2 and 3). The cells of the line were seeded at a density of 10,000 cells/cm ² in 12-well plates. After cell attachment to the adherent substrate, AAV preparations were added at an MOI of 500,000 vg/cell. On day 7 post-transduction, FIX protein content and activity in the culture fluid were determined by ELISA, as well as the level of factor IX gene expression in the cells by quantitative reverse transcription-PCR as described above. All samples were supplied in triplicate. Intact cells were used as a negative control.

Our developed AAV5-hFIXco-v1 and AAV5-hFIXco-v2 preparations, carrying codon-optimized versions of the factor IX gene [hFIXco-v1 and hFIXco-v1], were shown to efficiently deliver the factor IX transgene into cells and ensure the production of the target protein, as confirmed by quantitative real-time PCR, ELISA, and factor IX protein activity analysis (Figs. 2 and 3). Thus, when using the drugs AAV5-hFIXco-v1 and AAV5-hFIXco-v2, containing a codon-optimized sequence of the gene of blood coagulation factor IX, the level of expression of the gene of blood coagulation factor IX is 1.8 times higher in the case of the drug AAV5-hFIXco-v1 and 2.8 times higher in the case of the drug AAV5-hFIXco-v2 compared to the use of the drug with the natural version of the gene of blood coagulation factor IX (AAV5-hFIX-wt) (Fig. 2). In addition, when using the AAV5-hFIXco-v1 and AAV5-hFIXco-v2 preparations containing a codon-optimized sequence of the factor IX gene, the level of factor IX protein production was 1.6 times higher in the case of the AAV5-hFLXco-v1 preparation and 2.1 times higher in the case of the AAV5-hFIXco-v2 preparation compared to using the preparation with the natural version of the factor IX gene (AAV5-hFIX-wt) (Fig. 3A). It is also important to note that when using the AAV5-hFIXco-v1 and AAV5-hFIXco-v2 preparations containing a codon-optimized sequence of the factor IX gene, the level of factor IX protein activity was 2.1 times higher in the case of the AAV5-hFIXco-v1 preparation and 2.9 times higher in the case of the AAV5-hFIXco-v2 preparation compared to using the preparation with the natural version of the factor IX gene (AAV5-hFIX-wt) (Fig. 3B).

Example 5. Testing the performance of AAV5-hFIXco-v1 and AAV5-hFIXco-v2 in vivo

Laboratory mice of the C57BL/6 line were used to conduct in vivo studies of the AAV5-hFIXco-v1 and AAV5-hFIXco-v2 preparations. The study used a dose of AAV preparations equal to 4×10 ¹¹ VG/mouse. A control solution without AAV was used as a negative control. The preparations were administered to animals once by intravenous injection into the tail vein. Blood plasma was collected on the day of injection before administration of the preparations, then on days 7, 14, 21, 28, 35 and 42 after administration of the preparations. The level of coagulation factor IX protein was determined in the blood plasma samples by ELISA, as described above.

In vivo studies have shown that when using the AAV5-hFIXco-v1 preparation containing the codon-optimized sequence of the coagulation factor IX gene hFIXco-v1, a significantly higher level of coagulation factor IX protein in the blood of animals (2.2-2.3 times) is observed on days 21 and 28 after administration of the preparation than when using the preparation with the natural version of the coagulation factor IX gene (AAV5-hFIX-wt) (Fig. 4A). In the case of using the AAV5-hFIXco-v2 preparation containing the codon-optimized sequence of the coagulation factor IX gene hFIXco-v2, a significantly higher level of coagulation factor IX protein in the blood of animals (by 1.8-2.5 times) was observed on days 14, 21, 28, 35 and 42 after administration of the preparation than in the case of using the preparation with the natural version of the coagulation factor FIX gene (AAV5-hFIX-vvt) (Fig. 4A).

Thus, the developed recombinant AAV5-based viruses carrying codon-optimized versions of the factor IX gene (AAV5-hFIXco-v1 or AAV5-hFIXco-v2) have an advantage over the AAV5 vector with the natural version of the factor IX gene and have a high potential for gene therapy of Hemophilia B.

--->

SEQUENCE LIST

<110> OOO "ANABION"

<120> A codon-optimized nucleic acid that encodes a protein

coagulation factor IX and its uses

<160> 17

<170> BiSSAP 1.3.6

<210> 1

<211> 461

<212> PRT

<213> Natural Sequence

<220>

<223> Amino acid sequence of the recombinant factor protein

blood coagulation disorder IX with mutation R338L

<400> 1

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 Thr 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> 2

<211> 1383

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleic acid encoding

recombinant blood coagulation factor IX (hFIXco-v1 variant)

<400> 2

atgcagcggg tcaacatgat catggcggag tcgccggggcc tgatcacgat ctgcctcctc 60

gggtacctgc tctccgccga gtgcaccgtg ttcctggacc acgagaacgc caacaagatc 120

ctcaaccggc ccaagcgcta caactccggc aagctggagg agttcgtgca ggggaacctc 180

gagcgcgagt gcatggagga gaagtgctcg ttcgaggagg cgcgggaggt gttcgagaac 240

accgagcgca ccacggagtt ctggaagcag tacgtggacg gggaccagtg cgagtcgaac 300

ccgtgcctca acggggggtc gtgcaaggac gacatcaact cgtacgagtg ctggtgcccc 360

ttcggcttcg agggcaagaa ctgcgagctg gacgtgacct gcaacatcaa gaacgggcgc 420

tgcgagcagt tctgcaagaa cagcgccgac aacaaggtgg tctgctcctg caccgagggg 480

taccgcctcg cggagaacca gaagtcctgc gagccggccg tgcccttccc ctgcggccgc 540

gtgtccgtca gccagacgtc gaagctgacg cgcgccgaga ccgtcttccc ggacgtggac 600

tacgtgaact cgacggaggc cgagaccatc ctggacaaca tcacccagag cacccagtcc 660

ttcaacgact tcacgcgggt ggtcggcggc gagacgcca agcccgggca gttcccgtgg 720

caggtcgtcc tcaacgggaa ggtcgacgcg ttctgcggcg ggagcatcgt gaacgagaag 780

tggatcgtga ccgccgcgca ctgcgtcgag acgggcgtga agatcaccgt ggtggccggg 840

gagcacaaca tcgaggagac ggagcacacc gagcagaagc ggaacgtgat ccgcatcatc 900

ccgcaccaca actacaacgc cgccatcaac aagtacaacc acgacatcgc gctcctcgag 960

ctggacgagc cgctggtcct caactcctac gtcacgccga tctgcatcgc cgacaaggag 1020

tacacgaaca tcttcctgaa gttcgggagc ggctacgtct cgggctgggg ccgcgtgttc 1080

cacaaggggc gcagcgcgct cgtgctccag tacctgcggg tccccctggt cgaccgcgcg 1140

acctgcctcc tctccacgaa gttcacgatc tacaacaaca tgttctgcgc ggggttccac 1200

gagggcggcc gggacagctg ccagggcgac agcggggggcc cgcacgtgac ggaggtggag 1260

ggcacgagct tcctgaccgg gatcatctcg tggggcgagg agtgcgcgat gaaggggaag 1320

tacggcatct acaccaaggt cagccggtac gtgaactgga tcaaggagaa gacgaagctg 1380

acg 1383

<210> 3

<211> 3122

<212> DNA

<213> Artificial sequence

<220>

<223> codon-optimized gene expression cassette

recombinant blood coagulation factor IX (version

AAV5-hFIXco-v1)

<400> 3

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct gcggccgcac gcgtgccgcc accatggtcg agcttgggct gcaggtcgag 180

ggcactggga ggatgttgag taagatggaa aactactgat gacccttgca gagacagagt 240

attaggacat gtttgaacag gggccggcga tcagcaggta gctctagagg atccccgtct 300

gtctgcacat ttcgtagagc gagtgttccg atactctaat ctcctaggc aaggttcata 360

tttgtgtagg ttacttattc tccttttgtt gactaagtca ataatcagaa tcagcaggtt 420

tggagtcagc ttggcaggga tcagcagcct gggttggaag gagggggtat aaaagcccct 480

tcaccaggag aagccgtcac acagatccac aagctcctga caggaagctc taggtgactc 540

tcttaaggta gcctccgcgg attcgaatcc cggccgggaa cggtgcattg gaacgcggat 600

tccccgtgcc aagagtgacg taagtaccgc ctatagagtc tataggccca caaaaaatgc 660

tttcttcttt taatatactt ttttgtttat cttatttcta atactttccc taatctcttt 720

ctttcagggc aataatgata caatgtatca tgcctctttg caccattcta aagaataaca 780

gtgataattt ctgggttaag gcaatagcaa tatttctgca tataaatatt tctgcatata 840

aattgtaact gatgtaagag gtttcatatt gctaatagca gctacaatcc agctaccatt 900

ctgcttttat tttatggttg ggataaggct ggattattct gagtccaagc taggcccttt 960

tgctaatcat gttcatacct cttatcttcc tcccacagct cctgggcaac gtgctggtct 1020

gtgtgctggc ccatcacttt ggcaaagaat tgggattcga acatcgatat gcagcgggtc 1080

aacatgatca tggcggagtc gccggggcctg atcacgatct gcctcctcgg gtacctgctc 1140

tccgccgagt gcaccgtgtt cctggaccac gagaacgcca acaagatcct caaccggccc 1200

aagcgctaca actccggcaa gctggaggag ttcgtgcagg ggaacctcga gcgcgagtgc 1260

atggaggaga agtgctcgtt cgaggaggcg cgggaggtgt tcgagaacac cgagcgcacc 1320

acggagttct ggaagcagta cgtggacggg gaccagtgcg agtcgaaccc gtgcctcaac 1380

ggggggtcgt gcaaggacga catcaactcg tacgagtgct ggtgcccctt cggcttcgag 1440

ggcaagaact gcgagctgga cgtgacctgc aacatcaaga acgggcgctg cgagcagttc 1500

tgcaagaaca gcgccgacaa caaggtggtc tgctcctgca ccgaggggta ccgcctcgcg 1560

gagaaccaga agtcctgcga gccggccgtg cccttcccct gcggccgcgt gtccgtcagc 1620

cagacgtcga agctgacgcg cgccgagacc gtcttcccgg acgtggacta cgtgaactcg 1680

acggaggccg agaccatcct ggacaacatc acccagagca cccagtcctt caacgacttc 1740

acgcgggtgg tcggcggcga ggacgccaag cccggggcagt tcccgtggca ggtcgtcctc 1800

aacgggaagg tcgacgcgtt ctgcggcggg agcatcgtga acgagaagtg gatcgtgacc 1860

gccgcgcact gcgtcgagac gggcgtgaag atcaccgtgg tggccgggga gcacaacatc 1920

gaggagacgg agcacaccga gcagaagcgg aacgtgatcc gcatcatccc gcaccacaac 1980

tacaacgccg ccatcaacaa gtacaaccac gacatcgcgc tcctcgagct ggacgagccg 2040

ctggtcctca actcctacgt cacgccgatc tgcatcgccg acaaggagta cacgaacatc 2100

ttcctgaagt tcgggagcgg ctacgtctcg ggctggggcc gcgtgttcca caaggggcgc 2160

agcgcgctcg tgctccagta cctgcgggtc cccctggtcg accgcgcgac ctgcctcctc 2220

tccacgaagt tcacgatcta caacaacatg ttctgcgcgg ggttccacga gggcggccgg 2280

gacagctgcc agggcgacag cggggggcccg cacgtgacgg aggtggaggg cacgagcttc 2340

ctgaccggga tcatctcgtg gggcgaggag tgcgcgatga aggggaagta cggcatctac 2400

accaaggtca gccggtacgt gaactggatc aaggagaaga cgaagctgac gtgatgaaga 2460

tctacgggtg gcatccctgt gacccctccc cagtgcccct cctggccctg gaagttgcca 2520

ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg 2580

tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga 2640

agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat 2700

cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg 2760

agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga 2820

gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc 2880

caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc 2940

ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga 3000

gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 3060

ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca 3120

gg 3122

<210> 4

<211> 1383

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleic acid encoding

recombinant coagulation factor IX (hFIXco-v2 variant)

<400> 4

atgcagcggg tgaacatgat catggccgag tccccaggcc tgattaccat ctgtctgctg 60

ggctacctgc tgagcgccga atgcaccgtg tttctggacc acgagaacgc caacaagatc 120

ctgaaccgcc ctaagcggta caactccggc aagctggagg agtttgtgca gggcaatctg 180

gagcgggagt gtatggagga gaagtgcagc ttcgaggagg ccagggaggt gttcgagaac 240

accgagagga ccaccgagtt ctggaagcag tatgtggacg gcgaccagtg cgagtctaat 300

ccttgtctga atggcgggag ctgcaaggac gacatcaaca gctacgagtg ctggtgccct 360

ttcggcttcg agggcaagaa ttgcgagctg gacgtgacct gcaacatcaa gaacggccgg 420

tgtgagcagt tctgcaagaa cagcgccgac aacaaggtgg tgtgctcctg caccgaggga 480

tacaggctgg ccgagaatca gaagagctgt gagcccgccg tgccattccc ctgtggcaga 540

gtgtctgtga gccagaccag caagctgacc agagccgaga ccgtgtttcc agacgtggac 600

tacgtgaaca gcaccgaggc cgagaccatc ctggataata tcacccagtc cacccagagc 660

ttcaacgact tcaccagagt ggtgggaggc gaggatgcca agccaggaca gtttccctgg 720

caggtggtgc tgaatggcaa ggtggacgcc ttctgcggag gcagcatcgt gaacgagaag 780

tggattgtga ccgcagccca ctgcgtggag actggcgtga agattaccgt ggtcgccggc 840

gagcacaata tcgaagagac cgagcacacc gagcagaagc gcaacgtgat ccggatcatc 900

cctcaccaca actacaacgc agccatcaac aagtacaacc acgacatcgc cctgctggag 960

ctggacgagc cactggtgct gaactcttac gtgaccccta tctgcatcgc cgacaaggag 1020

tacaccaaca tcttcctgaa gttcggcagc ggctacgtga gcggatgggg cagagtgttt 1080

cacaagggca ggagcgccct ggtgctgcag tatctgagag tgccactggt ggacagagct 1140

acctgcctgc tgagcaccaa gttcaccatc tacaacaaca tgttctgcgc cggcttccac 1200

gaggggggaa gagactcttg ccagggcgat tccggcggac cacacgtgac cgaagtggag 1260

ggcaccagct tcctgaccgg catcatctcc tggggcgagg aatgcgccat gaagggcaag 1320

tacggcatct acaccaaggt gagcaggtac gtgaactgga tcaaggagaa gaccaagctg 1380

acc 1383

<210> 5

<211> 3123

<212> DNA

<213> Artificial sequence

<220>

<223> codon-optimized gene expression cassette

recombinant blood coagulation factor IX (version

AAV5-hFIXco-v2)

<400> 5

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct gcggccgcac gcgtgccgcc accatggtcg agcttgggct gcaggtcgag 180

ggcactggga ggatgttgag taagatggaa aactactgat gacccttgca gagacagagt 240

attaggacat gtttgaacag gggccggcga tcagcaggta gctctagagg atccccgtct 300

gtctgcacat ttcgtagagc gagtgttccg atactctaat ctcctaggc aaggttcata 360

tttgtgtagg ttacttattc tccttttgtt gactaagtca ataatcagaa tcagcaggtt 420

tggagtcagc ttggcaggga tcagcagcct gggttggaag gagggggtat aaaagcccct 480

tcaccaggag aagccgtcac acagatccac aagctcctga caggaagctc taggtgactc 540

tcttaaggta gcctccgcgg attcgaatcc cggccgggaa cggtgcattg gaacgcggat 600

tccccgtgcc aagagtgacg taagtaccgc ctatagagtc tataggccca caaaaaatgc 660

tttcttcttt taatatactt ttttgtttat cttatttcta atactttccc taatctcttt 720

ctttcagggc aataatgata caatgtatca tgcctctttg caccattcta aagaataaca 780

gtgataattt ctgggttaag gcaatagcaa tatttctgca tataaatatt tctgcatata 840

aattgtaact gatgtaagag gtttcatatt gctaatagca gctacaatcc agctaccatt 900

ctgcttttat tttatggttg ggataaggct ggattattct gagtccaagc taggcccttt 960

tgctaatcat gttcatacct cttatcttcc tcccacagct cctgggcaac gtgctggtct 1020

gtgtgctggc ccatcacttt ggcaaagaat tgggattcga acatcgatat aaatgcagcg 1080

ggtgaacatg atcatggccg agtccccagg cctgattacc atctgtctgc tgggctacct 1140

gctgagcgcc gaatgcaccg tgtttctgga ccacgagaac gccaacaaga tcctgaaccg 1200

ccctaagcgg tacaactccg gcaagctgga ggagtttgtg cagggcaatc tggagcggga 1260

gtgtatggag gagaagtgca gcttcgagga ggccagggag gtgttcgaga acaccgagag 1320

gaccaccgag ttctggaagc agtatgtgga cggcgaccag tgcgagtcta atccttgtct 1380

gaatggcggg agctgcaagg acgacatcaa cagctacgag tgctggtgcc ctttcggctt 1440

cgagggcaag aattgcgagc tggacgtgac ctgcaacatc aagaacggcc ggtgtgagca 1500

gttctgcaag aacagcgccg acaacaaggt ggtgtgctcc tgcaccgagg gatacaggct 1560

ggccgagaat cagaagagct gtgagcccgc cgtgccattc ccctgtggca gagtgtctgt 1620

gagccagacc agcaagctga cgagccga gaccgtgttt cgacgtgg actacgtgaa 1680

cagcaccgag gccgagacca tcctggataa tatcacccag tccacccaga gcttcaacga 1740

cttcaccaga gtggtgggag gcgaggatgc caagccagga cagtttccct ggcaggtggt 1800

gctgaatggc aaggtggacg ccttctgcgg aggcagcatc gtgaacgaga agtggattgt 1860

gaccgcagcc cactgcgtgg agactggcgt gaagattacc gtggtcgccg gcgagcacaa 1920

tatcgaagag accgagcaca ccgagcagaa gcgcaacgtg atccggatca tccctcacca 1980

caactacaac gcagccatca acaagtacaa ccacgacatc gccctgctgg agctggacga 2040

gccactggtg ctgaactctt acgtgacccc tatctgcatc gccgacaagg agtacaccaa 2100

catcttcctg aagttcggca gcggctacgt gagcggatgg ggcagagtgt ttcacaaggg 2160

caggagcgcc ctggtgctgc agtatctgag agtgccactg gtggacagag ctacctgcct 2220

gctgagcacc aagttcacca tctacaacaa catgttctgc gccggcttcc acgagggggg 2280

aagagactct tgccagggcg attccggcgg accacacgtg accgaagtgg agggcaccag 2340

cttcctgacc ggcatcatct cctggggcga ggaatgcgcc atgaagggca agtacggcat 2400

ctacaccaag gtgagcaggt acgtgaactg gatcaaggag aagaccaagc tgacctgaag 2460

atctacgggt ggcatccctg tgacccctcc ccagtgcccc tcctggccct ggaagttgcc 2520

actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag 2580

gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg 2640

aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa 2700

tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc 2760

gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag 2820

agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac 2880

ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc 2940

cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg 3000

agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg 3060

cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc 3120

agg 3123

<210> 6

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> Left (first) ITR (inverted terminal repeats)

<400> 6

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct 130

<210> 7

<211> 372

<212> DNA

<213> Natural Sequence

<220>

<223> TTR promoter sequence (transthyretin promoter)

<400> 7

tcgagcttgg gctgcaggtc gagggcactg ggaggatgtt gagtaagatg gaaaactact 60

gatgaccctt gcagagacag agtattagga catgtttgaa caggggccgg cgatcagcag 120

gtagctctag aggatccccg tctgtctgca catttcgtag agcgagtgtt ccgatactct 180

aatctcccta ggcaaggttc atatttgtgt aggttactta ttctcctttt gttgactaag 240

tcaataatca gaatcagcag gtttggagtc agcttggcag ggatcagcag cctgggttgg 300

aaggagggg tataaaagcc ccttcaccag gagaagccgt cacacagatc cacaagctcc 360

tgacaggaag ct 372

<210> 8

<211> 493

<212> DNA

<213> Natural Sequence

<220>

<223> intron of the hBG1 gene (a fragment of the human β globin gene carrying

intron)

<400> 8

cgaatcccgg ccgggaacgg tgcattggaa cgcggattcc ccgtgccaag agtgacgtaa 60

gtaccgccta tagagtctat aggcccacaa aaaatgcttt cttcttttaa tatacttttt 120

tgtttatctt atttctaata ctttccctaa tctctttctt tcagggcaat aatgatacaa 180

tgtatcatgc ctctttgcac cattctaaag aataacagtg ataatttctg ggttaaggca 240

atagcaatat ttctgcatat aaatatttct gcatataaat tgtaactgat gtaagaggtt 300

tcatattgct aatagcagct acaatccagc taccattctg cttttatttt atggttggga 360

taaggctgga ttattctgag tccaagctag gcccttttgc taatcatgtt catacctctt 420

atcttcctcc cacagctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc 480

aaagaattgg gat 493

<210> 9

<211> 479

<212> DNA

<213> Natural Sequence

<220>

<223> hGH1 polyadenylation signal (polyadenylation signal of the gene

human growth hormone)

<400> 9

acgggtggca tccctgtgac ccctccccag tgcccctcct ggccctggaa gttgccactc 60

cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt 120

ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga 180

caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt 240

ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt 300

tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac 360

ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac 420

cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtcctt 479

<210> 10

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> right (second) ITR

<400> 10

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag ctgcctgcag g 141

<210> 11

<211> 724

<212> PRT

<213> Natural Sequence

<220>

<223>Natural sequence of wild-type AAV5 capsid protein VP1

<400> 11

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> 12

<211> 588

<212> PRT

<213> Natural Sequence

<220>

<223>Natural sequence of wild-type AAV5 capsid VP2 protein

<400> 12

Thr Ala Pro Thr Gly Lys Arg Ile Asp Asp His Phe Pro Lys Arg Lys

1 5 10 15

Lys Ala Arg Thr Glu Glu Asp Ser Lys Pro Ser Thr Ser Ser Asp Ala

20 25 30

Glu Ala Gly Pro Ser Gly Ser Gln Gln Leu Gln Ile Pro Ala Gln Pro

35 40 45

Ala Ser Ser Leu Gly Ala Asp Thr Met Ser Ala Gly Gly Gly Gly Pro

50 55 60

Leu Gly Asp Asn Asn Gln Gly Ala Asp Gly Val Gly Asn Ala Ser Gly

65 70 75 80

Asp Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Val Thr Lys

85 90 95

Ser Thr Arg Thr Trp Val Leu Pro Ser Tyr Asn Asn His Gln Tyr Arg

100 105 110

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

115 120 125

Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His Ser

130 135 140

His Trp Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Tyr Trp Gly

145 150 155 160

Phe Arg Pro Arg Ser Leu Arg Val Lys Ile Phe Asn Ile Gln Val Lys

165 170 175

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

180 185 190

Ser Thr Val Gln Val Phe Thr Asp Asp Asp Tyr Gln Leu Pro Tyr Val

195 200 205

Val Gly Asn Gly Thr Glu Gly Cys Leu Pro Ala Phe Pro Pro Gln Val

210 215 220

Phe Thr Leu Pro Gln Tyr Gly Tyr Ala Thr Leu Asn Arg Asp Asn Thr

225 230 235 240

Glu Asn Pro Thr Glu Arg Ser Ser Phe Phe Cys Leu Glu Tyr Phe Pro

245 250 255

Ser Lys Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Thr Tyr Asn Phe

260 265 270

Glu Glu Val Pro Phe His Ser Ser Phe Ala Pro Ser Gln Asn Leu Phe

275 280 285

Lys Leu Ala Asn Pro Leu Val Asp Gln Tyr Leu Tyr Arg Phe Val Ser

290 295 300

Thr Asn Asn Thr Gly Gly Val Gln Phe Asn Lys Asn Leu Ala Gly Arg

305 310 315 320

Tyr Ala Asn Thr Tyr Lys Asn Trp Phe Pro Gly Pro Met Gly Arg Thr

325 330 335

Gln Gly Trp Asn Leu Gly Ser Gly Val Asn Arg Ala Ser Val Ser Ala

340 345 350

Phe Ala Thr Thr Asn Arg Met Glu Leu Glu Gly Ala Ser Tyr Gln Val

355 360 365

Pro Pro Gln Pro Asn Gly Met Thr Asn Asn Leu Gln Gly Ser Asn Thr

370 375 380

Tyr Ala Leu Glu Asn Thr Met Ile Phe Asn Ser Gln Pro Ala Asn Pro

385 390 395 400

Gly Thr Thr Ala Thr Tyr Leu Glu Gly Asn Met Leu Ile Thr Ser Glu

405 410 415

Ser Glu Thr Gln Pro Val Asn Arg Val Ala Tyr Asn Val Gly Gly Gln

420 425 430

Met Ala Thr Asn Asn Gln Ser Ser Thr Thr Ala Pro Ala Thr Gly Thr

435 440 445

Tyr Asn Leu Gln Glu Ile Val Pro Gly Ser Val Trp Met Glu Arg Asp

450 455 460

Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro Glu Thr Gly Ala

465 470 475 480

His Phe His Pro Ser Pro Ala Met Gly Gly Phe Gly Leu Lys His Pro

485 490 495

Pro Pro Met Met Leu Ile Lys Asn Thr Pro Val Pro Gly Asn Ile Thr

500 505 510

Ser Phe Ser Asp Val Pro Val Ser Ser Phe Ile Thr Gln Tyr Ser Thr

515 520 525

Gly Gln Val Thr Val Glu Met Glu Trp Glu Leu Lys Lys Glu Asn Ser

530 535 540

Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Asn Asn Tyr Asn Asp Pro

545 550 555 560

Gln Phe Val Asp Phe Ala Pro Asp Ser Thr Gly Glu Tyr Arg Thr Thr

565 570 575

Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

580 585

<210> 13

<211> 532

<212> PRT

<213> Natural Sequence

<220>

<223>Natural sequence of wild-type AAV5 capsid VP3 protein

<400> 13

Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala

1 5 10 15

Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp

20 25 30

Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro

35 40 45

Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp

50 55 60

Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr

65 70 75 80

Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln

85 90 95

Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val

100 105 110

Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr

115 120 125

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

130 135 140

Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys

145 150 155 160

Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr

165 170 175

Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser

180 185 190

Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn

195 200 205

Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser

210 215 220

Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp

225 230 235 240

Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln

245 250 255

Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp

260 265 270

Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly

275 280 285

Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu

290 295 300

Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr

305 310 315 320

Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile

325 330 335

Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu

340 345 350

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

355 360 365

Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser

370 375 380

Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro

385 390 395 400

Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp

405 410 415

Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met

420 425 430

Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn

435 440 445

Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser

450 455 460

Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu

465 470 475 480

Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln

485 490 495

Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp

500 505 510

Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu

515 520 525

Thr Arg Pro Leu

530

<210> 14

<211> 724

<212> PRT

<213> Artificial sequence

<220>

<223> A modified AAV5 capsid protein VP1 that includes substitutions

S2A and T711S

<400> 14

Met Ala 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 Ser Thr Arg Pro Ile Gly Thr Arg Tyr Leu

705 710 715 720

Thr Arg Pro Leu

<210> 15

<211> 588

<212> PRT

<213> Artificial sequence

<220>

<223> A modified AAV5 capsid protein VP2 that includes a substitution

T575S

<400> 15

Thr Ala Pro Thr Gly Lys Arg Ile Asp Asp His Phe Pro Lys Arg Lys

1 5 10 15

Lys Ala Arg Thr Glu Glu Asp Ser Lys Pro Ser Thr Ser Ser Asp Ala

20 25 30

Glu Ala Gly Pro Ser Gly Ser Gln Gln Leu Gln Ile Pro Ala Gln Pro

35 40 45

Ala Ser Ser Leu Gly Ala Asp Thr Met Ser Ala Gly Gly Gly Gly Pro

50 55 60

Leu Gly Asp Asn Asn Gln Gly Ala Asp Gly Val Gly Asn Ala Ser Gly

65 70 75 80

Asp Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Val Thr Lys

85 90 95

Ser Thr Arg Thr Trp Val Leu Pro Ser Tyr Asn Asn His Gln Tyr Arg

100 105 110

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

115 120 125

Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His Ser

130 135 140

His Trp Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Tyr Trp Gly

145 150 155 160

Phe Arg Pro Arg Ser Leu Arg Val Lys Ile Phe Asn Ile Gln Val Lys

165 170 175

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

180 185 190

Ser Thr Val Gln Val Phe Thr Asp Asp Asp Tyr Gln Leu Pro Tyr Val

195 200 205

Val Gly Asn Gly Thr Glu Gly Cys Leu Pro Ala Phe Pro Pro Gln Val

210 215 220

Phe Thr Leu Pro Gln Tyr Gly Tyr Ala Thr Leu Asn Arg Asp Asn Thr

225 230 235 240

Glu Asn Pro Thr Glu Arg Ser Ser Phe Phe Cys Leu Glu Tyr Phe Pro

245 250 255

Ser Lys Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Thr Tyr Asn Phe

260 265 270

Glu Glu Val Pro Phe His Ser Ser Phe Ala Pro Ser Gln Asn Leu Phe

275 280 285

Lys Leu Ala Asn Pro Leu Val Asp Gln Tyr Leu Tyr Arg Phe Val Ser

290 295 300

Thr Asn Asn Thr Gly Gly Val Gln Phe Asn Lys Asn Leu Ala Gly Arg

305 310 315 320

Tyr Ala Asn Thr Tyr Lys Asn Trp Phe Pro Gly Pro Met Gly Arg Thr

325 330 335

Gln Gly Trp Asn Leu Gly Ser Gly Val Asn Arg Ala Ser Val Ser Ala

340 345 350

Phe Ala Thr Thr Asn Arg Met Glu Leu Glu Gly Ala Ser Tyr Gln Val

355 360 365

Pro Pro Gln Pro Asn Gly Met Thr Asn Asn Leu Gln Gly Ser Asn Thr

370 375 380

Tyr Ala Leu Glu Asn Thr Met Ile Phe Asn Ser Gln Pro Ala Asn Pro

385 390 395 400

Gly Thr Thr Ala Thr Tyr Leu Glu Gly Asn Met Leu Ile Thr Ser Glu

405 410 415

Ser Glu Thr Gln Pro Val Asn Arg Val Ala Tyr Asn Val Gly Gly Gln

420 425 430

Met Ala Thr Asn Asn Gln Ser Ser Thr Thr Ala Pro Ala Thr Gly Thr

435 440 445

Tyr Asn Leu Gln Glu Ile Val Pro Gly Ser Val Trp Met Glu Arg Asp

450 455 460

Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro Glu Thr Gly Ala

465 470 475 480

His Phe His Pro Ser Pro Ala Met Gly Gly Phe Gly Leu Lys His Pro

485 490 495

Pro Pro Met Met Leu Ile Lys Asn Thr Pro Val Pro Gly Asn Ile Thr

500 505 510

Ser Phe Ser Asp Val Pro Val Ser Ser Phe Ile Thr Gln Tyr Ser Thr

515 520 525

Gly Gln Val Thr Val Glu Met Glu Trp Glu Leu Lys Lys Glu Asn Ser

530 535 540

Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Asn Asn Tyr Asn Asp Pro

545 550 555 560

Gln Phe Val Asp Phe Ala Pro Asp Ser Thr Gly Glu Tyr Arg Ser Thr

565 570 575

Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

580 585

<210> 16

<211> 532

<212> PRT

<213> Artificial sequence

<220>

<223> A modified AAV5 capsid protein VP3 that includes a substitution

T519S

<400> 16

Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala

1 5 10 15

Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp

20 25 30

Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro

35 40 45

Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp

50 55 60

Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr

65 70 75 80

Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln

85 90 95

Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val

100 105 110

Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr

115 120 125

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

130 135 140

Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys

145 150 155 160

Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr

165 170 175

Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser

180 185 190

Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn

195 200 205

Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser

210 215 220

Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp

225 230 235 240

Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln

245 250 255

Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp

260 265 270

Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly

275 280 285

Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu

290 295 300

Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr

305 310 315 320

Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile

325 330 335

Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu

340 345 350

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

355 360 365

Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser

370 375 380

Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro

385 390 395 400

Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp

405 410 415

Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met

420 425 430

Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn

435 440 445

Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser

450 455 460

Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu

465 470 475 480

Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln

485 490 495

Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp

500 505 510

Ser Thr Gly Glu Tyr Arg Ser Thr Arg Pro Ile Gly Thr Arg Tyr Leu

515 520 525

Thr Arg Pro Leu

530

<210> 17

<211> 1383

<212> DNA

<213> Natural Sequence

<220>

<223> nucleic acid encoding recombinant coagulation factor

blood IX wild type (hFIX-wt) with R338L mutation

<400> 17

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 cgtgctgaga ctgtttttcc tgatgtggac 600

tatgtaaatt ctactgaagc tgaaaccatt ttggataaca tcactcaaag cacccaatca 660

tttaatgact tcactcgggt tgttggtgga gaagatgcca aaccaggtca attcccttgg 720

caggttgttt tgaatggtaa agttgatgca ttctgtggag gctctatcgt taatgaaaaa 780

tggattgtaa ctgctgccca ctgtgttgaa actggtgtta aaattacagt tgtcgcaggt 840

gaacataata ttgaggagac agaacataca gagcaaaagc gaaatgtgat tcgaattatt 900

cctcaccaca actacaatgc agctattaat aagtacaacc atgacattgc ccttctggaa 960

ctggacgaac ccttagtgct aaacagctac gttacaccta tttgcattgc tgacaaggaa 1020

tacacgaaca tcttcctcaa atttggatct ggctatgtaa gtggctgggg aagagtcttc 1080

cacaaaggga gatcagcttt agttcttcag taccttagag ttccacttgt tgaccgagcc 1140

acatgtcttc tatctacaaa 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

act 1383

<---

Claims (16)

1. An isolated codon-optimized nucleic acid encoding the FIX protein (coagulation factor IX) with the amino acid sequence of SEQ ID NO: 1, which includes a nucleotide sequence selected from the group: SEQ ID NO: 2 or SEQ ID NO: 4. 2. An expression cassette that includes a codon-optimized nucleic acid according to claim 1. 3. An expression cassette according to claim 2, comprising the following elements in the direction from the 5'-end to the 3'-end: first ITR (inverted terminal repeats); TTR promoter (transthyretin promoter); intron of the hBG1 gene (a fragment of the human β globin gene carrying an intron); a codon-optimized nucleic acid according to claim 1; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal); second ITR. 4. The expression cassette according to claim 3, which comprises a nucleotide sequence selected from the group: SEQ ID NO: 3 or SEQ ID NO: 5. 5. An expression vector that comprises a codon-optimized nucleic acid according to claim 1 or an expression cassette according to any one of claims 2-4. 6. An isolated recombinant virus based on AAV5 (adeno-associated virus 5 serotype) for increasing the expression of the FIX gene in target cells, which comprises the nucleic acid of claim 1 or the expression cassette of any one of claims 2-4. 7. A recombinant virus based on AAV5 according to claim 6, wherein the capsid comprises the AAV5 VP1 protein. 8. A pharmaceutical composition for delivering the FIX gene to target cells, comprising a recombinant virus based on AAV5 according to any one of claims 6, 7 in combination with one or more pharmaceutically acceptable excipients. 9. Use of a recombinant AAV5-based virus according to claims 6, 7 or a composition according to claim 8 for delivering the FIX gene to target cells. 10. A method for delivering a FIX gene to target cells of a subject with hemophilia B, comprising introducing a recombinant AAV5-based virus according to any one of claims 6, 7 or a composition according to claim 8 into the cells of the subject.

Images