Optimized human coagulation factor IX gene expression cassettes and their use

Abstract

To provide an improved method for treating hemorrhagic diseases.SOLUTION: The present invention discloses a polynucleotide encoding a human Factor IX that has been codon-optimized for expression in humans, a vector comprising the polynucleotide, and compositions comprising them.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] This application claims the benefits of U.S. Provisional Application No. 62 / 512,833, filed on 31 May 2017, which is incorporated herein by reference in its entirety.

[0002] This invention relates to synthetic liver-specific promoters and expression constructs for producing polypeptides and functional nucleic acids in a target liver. Furthermore, this invention relates to optimized polynucleotide sequences encoding factor IX protein, vectors containing the same, and methods for treating hemorrhagic disorders using compositions thereof. [Background technology]

[0003] Factor IX (FIX) plays a crucial role in the coagulation cascade by promoting the conversion of factor X to factor Xa. Deficiency in FIX activity causes hemophilia B, a bleeding disorder. Current treatment for hemophilia B is intravenous infusion of plasma-derived or recombinant FIX protein. Although this treatment is effective in controlling bleeding episodes, it is inherently expensive due to the need for frequent infusions caused by FIX's short half-life (8-12 hours). Gene therapy has emerged as an attractive strategy for finally curing this disease. However, progress in the technology of delivering the FIX gene using one of the most promising viral vectors, namely adeno-associated virus (AAV), has been hampered by insufficient levels of FIX expression.

[0004] The present invention overcomes the shortcomings in the art by providing short, synthetic, liver-specific promoters and expression constructs suitable for use in AAV vectors. Furthermore, the present invention provides optimized FIX coding sequences that can produce hyperphysiological levels of FIX over extended periods, and methods for their use in treating hemorrhagic disorders. [Overview of the project]

[0005] The present invention is, in part, based on the development of a synthetic liver-specific promoter having a length of less than 200 base pairs. This promoter can be used to produce polypeptides and functional nucleic acids in a liver-specific manner (particularly using AAV vectors that have a strict length limit and benefit from the availability of short but potent promoters).

[0006] Furthermore, the present invention is based in part on the development of an optimized FIX coding sequence that can produce hyperphysiological levels of FIX over a long period of time.

[0007] In one embodiment, the present invention relates to a polynucleotide comprising a synthetic liver-specific promoter containing the nucleotide sequence of SEQ ID NO: 1 or a sequence that is at least 90% identical thereto.

[0008] In a further embodiment, the present invention relates to a polynucleotide encoding human factor IX, whose codons are optimized for expression in humans.

[0009] In another aspect, the present invention relates to vectors, cells, and / or transgenic animals comprising the polynucleotides of the present invention.

[0010] In a further embodiment, the present invention relates to a method for producing polypeptides or functional nucleic acids in the liver of a target, comprising delivering the polynucleotides, vectors, and / or transformed cells of the present invention to the target, thereby causing the liver of the target to produce polypeptides or functional nucleic acids.

[0011] In a further embodiment, the present invention relates to a method for treating hemophilia B in a subject, comprising delivering a therapeutically effective amount of the polynucleotide, vector and / or transformed cells of the present invention to the subject, thereby treating hemophilia B in the subject.

[0012] In another aspect, the present invention relates to a method for enhancing the biological availability of factor IX polypeptide in a subject, comprising delivering to the subject an effective amount of the polynucleotide, vector and / or transformed cell of the present invention, thereby enhancing the biological availability of factor IX polypeptide in the subject.

[0013] In a further aspect, the present invention relates to the use of the polynucleotide, vector and / or transformed cell of the present invention in a method for producing a polypeptide or a functional nucleic acid in the liver of a subject.

[0014] In a further aspect, the present invention relates to the use of the polynucleotide, vector and / or transformed cell of the present invention in a method for treating hemophilia B in a subject.

[0015] In another aspect, the present invention relates to the use of the polynucleotide, vector and / or transformed cell of the present invention in a method for enhancing the biological availability of factor IX polypeptide in a subject.

[0016] In a further aspect, the present invention relates to the use of the polynucleotide, vector and / or transformed cell of the present invention in the preparation of a medicament for producing a polypeptide or a functional nucleic acid in the liver of a subject.

[0017] In a further aspect, the present invention relates to the use of the polynucleotide, vector and / or transformed cell of the present invention in the preparation of a medicament for treating hemophilia B in a subject.

[0018] In another aspect, the present invention relates to the use of the polynucleotide, vector and / or transformed cell of the present invention in the preparation of a medicament for enhancing the biological availability of factor IX polypeptide in a subject.

[0019] These and other aspects of the present invention are described in more detail in the description of the present invention below.

Brief Description of the Drawings

[0020] [Figure 1] Figure 1 shows the structure and sequence of the LXP2.1 promoter (SEQ ID NO: 1). Putative hepatic and house-keeping transcriptional factor binding sites are highlighted with an underline. [Figure 2] Figure 2 shows the construction of an optimized liver-specific human factor IX (FIX) gene expression cassette. [Figure 3] Figure 3 shows the FIX activity in Huh7 cell culture medium 48 hours after plasmid transfection. Huh7 hepatocellular carcinoma cells were seeded in 6-well plates and transfected with 2 μg of FIX gene expression construct. The cell culture medium was changed after 24 hours, and cells were collected 48 hours after transfection in serum-free Opti-MEM medium. FIX activity was measured by the APTT test using purified normal FIX protein as a reference standard. The wt FIX gene expression construct contained the original human FIX cDNA without codon optimization, but did not contain the R338L mutation. The Opti-FIX-1, 2, and 3 gene expression constructs all contained the R338L mutation, but different human codon optimization algorithms described in the text of Example 1 were used. [Figure 4] Figure 4 shows robust and long-term factor IX expression and coagulation activity assays after dose-increasing of the AAV8-LXP2.1-opti-FIX-1 vector in a FIX gene knockout mouse model. [Modes for carrying out the invention]

[0021] Next, the present invention will be described in more detail with reference to the accompanying drawings illustrating preferred embodiments of the invention. However, the present invention may be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided to make this disclosure thorough and complete and to fully convey the scope of the invention to those skilled in the art.

[0022] Unless the context indicates otherwise, it is particularly intended that the various features of the present invention described herein may be used in any combination. Furthermore, in some embodiments of the present invention, it is also intended that any feature or combination of features described herein may be excluded or omitted. For illustrative purposes, where it is stated herein that a complex comprises components A, B, and C, it is particularly intended that any one of A, B, or C, or any combination thereof, may be omitted and excluded individually or in any combination.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the invention pertains. Terms used herein in describing the invention are solely for the purpose of describing specific embodiments and are not intended to limit the invention.

[0024] Nucleotide sequences are shown herein only in a single strand, oriented from 5' to 3', i.e., left to right, unless otherwise indicated. Nucleotides and amino acids are represented herein by either the method recommended by the IUPAC-IUB Biochemical Nomenclature Committee or by either a single-letter or three-letter notation (for amino acids), both of which follow 37 C. FR §1.822 and established usage.

[0025] Unless otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, etc. Such techniques are publicly known to those skilled in the art. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual 2nd Ed.* (Cold Spring Harbor, NY, 1989); and Ausubel et al., *Current Protocols in Molecular Biology* (Green Publishing Associates and John Wiley & Sons, New York).

[0026] definition In the description of this invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to also include plural forms unless the context clearly indicates otherwise.

[0027] Furthermore, as used herein, "and / or" refers to and encompasses all possible combinations of one or more of the related enumerated items, as well as the absence of any combination when interpreted as a binary choice ("or").

[0028] As used herein, the term "about" is intended to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount when referring to measurable values ​​such as the amount, dose, time, temperature, enzyme activity, or other biological activity of a polypeptide.

[0029] The transitional phrase "essentially consisting of" should be interpreted to mean that the scope of the claim should include materials or processes specified in the claim that do not substantially affect the fundamental and novel characteristics of the claimed invention.

[0030] The term "essentially consisting of" (and its grammatical variations), when applied to the polynucleotide or polypeptide sequences of the present invention, means a polynucleotide or polypeptide consisting of both the described sequence (e.g., sequence number) and a total of 10 or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5' and / or 3' i.e., N-terminus and / or C-terminus of the described sequence, such that the function of the polynucleotide or polypeptide is substantially altered. The total of 10 or fewer additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids on both ends added together. The term "substantially altered," when applied to the polynucleotide of the present invention, means an increase or decrease of at least about 50% or more in the ability to express the encoded polypeptide compared to the expression level of the polynucleotide consisting of the described sequence. The term "substantially altered," when applied to the polypeptide of the present invention, means an increase or decrease of at least about 50% or more in coagulation stimulating activity compared to the activity of the polypeptide consisting of the enumerated sequences.

[0031] As used herein, the terms “enhance” or “increase,” or their grammatical variations thereof, refer to an increase of at least approximately 1.25, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, or even 15 times in the specified parameter.

[0032] As used herein, the terms “inhibit” or “reduce” or their grammatical variations refer to a reduction or reduction in the specified level or activity by at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, or higher. In certain embodiments, the inhibition or reduction results in a slightly detectable or essentially undetectable amount of activity (at most very small amounts, e.g., about 10% or even less than 5%).

[0033] As used herein, "effective" quantity refers to the quantity that produces the desired effect.

[0034] As used herein, a “therapeutably effective” amount is an amount that provides some improvement or benefit to a subject. In other words, a “therapeutably effective” amount is an amount that provides some relief, mitigation, or reduction of at least one clinical symptom in a subject. Those skilled in the art will understand that the therapeutic effect does not need to be complete or curative, as long as some benefit is provided to the subject.

[0035] As used herein, “preventively effective” means an amount sufficient to prevent (as defined herein) disease, impairment and / or clinical symptoms in a subject. Those skilled in the art will understand that the level of prevention does not need to be complete, as long as some benefit is provided to the subject.

[0036] The effectiveness of treating hemorrhagic disorders by the method of the present invention can be determined by detecting clinical improvement indicated by changes in the symptoms and / or clinical parameters of the subject, as is well known to those skilled in the art.

[0037] The terms “to treat,” “to treat,” or “treatment” are intended to reduce the severity of the disease in question, or at least partially improve or alleviate it, and to achieve some reduction, alleviation, or decrease in at least one clinical symptom.

[0038] The terms “prevent,” “prevention,” and “prevention” (and their grammatical variations) refer to a reduction or delay in the degree or severity of a disease, disorder, and / or clinical symptom after its onset, compared to what would occur if the methods of the present invention were not performed before the onset of the disease, disorder, and / or clinical symptom. With respect to hemophilia B, “prevention” refers to the occurrence of fewer bleeding episodes and / or less severe bleeding episodes than the number and / or severity of bleeding episodes that would occur in the absence of prophylactic treatment.

[0039] As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” are used synonymously and encompass both cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and RNA and DNA including RNA and DNA chimeras. The terms polynucleotide, nucleotide sequence, or nucleic acid refer to a chain of nucleotides regardless of chain length. Nucleic acids may be double-stranded or single-stranded. If single-stranded, the nucleic acid may be a sense strand or an antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Using such oligonucleotides, nucleic acids with altered base-pairing ability or increased resistance to nucleases can be prepared, for example. Furthermore, the present invention provides nucleic acids that are complements (may be full complements or partial complements) of the nucleic acids, nucleotide sequences, or polynucleotides of the present invention.

[0040] An "isolated polynucleotide" is a nucleotide sequence (e.g., DNA or RNA) that is not directly adjacent to any nucleotide sequence (one at the 5' end and one at the 3' end) that it is directly adjacent to in the naturally occurring genome of the organism from which it is obtained. Therefore, in one embodiment, the isolated nucleic acid contains some or all of the 5' non-coding (e.g., promoter) sequences directly adjacent to the coding sequence. Thus, this term includes recombinant DNA that exists, for example, embedded in a vector, a self-replicating plasmid or virus, or in the genomic DNA of a prokaryote or eukaryote, or as a separate molecule independent of other sequences (e.g., cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment). It also includes recombinant DNA that is part of a hybrid nucleic acid encoding a further polypeptide or peptide sequence. An isolated polynucleotide containing a gene is not a chromosome fragment containing such a gene, but rather contains the coding and regulatory regions associated with that gene, but does not include further genes that are not naturally present on that chromosome.

[0041] When applied to polynucleotides, the term “fragment” is understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence, comprising, essentially consisting of, and / or comprising, a sequence of consecutive nucleotides that are identical or nearly identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such nucleic acid fragments according to the present invention may, if necessary, be contained within a larger polynucleotide of which they are components. In some embodiments, such fragments may comprise, essentially consisting of, and / or comprising, an oligonucleotide having at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more consecutive nucleotide lengths of the nucleic acid or nucleotide sequence according to the present invention.

[0042] The term “isolated” can refer to a nucleic acid, nucleotide sequence, or polypeptide that is substantially free of cellular material, viral material, and / or culture medium (if produced by recombinant DNA technology) or chemical precursors or other chemicals (if chemically synthesized). Furthermore, “isolated fragment” is a fragment of a nucleic acid, nucleotide sequence, or polypeptide that does not occur naturally as a fragment and does not exist in its natural state. “Isolated” does not mean that its preparation is technically pure (homogeneous), but is pure enough to provide a polypeptide or nucleic acid in a form that can be used for its intended purpose.

[0043] When applied to a polypeptide, the term “fragment” is understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence, comprising, essentially consisting of, and / or comprising, an amino acid sequence of consecutive amino acids that are identical or nearly identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such polypeptide fragments according to the present invention may, if necessary, be contained within a larger polypeptide of which they are components. In some embodiments, such fragments may comprise, essentially consisting of, and / or comprising, a peptide having at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more consecutive amino acid lengths of the polypeptide or amino acid sequence according to the present invention.

[0044] A “vector” is any nucleic acid molecule used to clone and / or move nucleic acids into a cell. A vector may also be a replicon, to which another nucleotide sequence can be attached to enable replication of a bound nucleotide sequence. A “replicon” may also be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, i.e., can replicate under its own control. The term “vector” includes both viral and non-viral (e.g., plasmid) nucleic acid molecules for introducing nucleic acids into cells in vitro, ex vivo, and / or in vivo. Nucleic acids may be manipulated using numerous vectors known in the art, and response elements and promoters may be incorporated into genes, etc. For example, insertion of nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be achieved by ligating the appropriate nucleic acid fragments into a selected vector having complementary adherent ends. Alternatively, the ends of a nucleic acid molecule may be enzymatically modified, or any site may be produced by ligating a nucleotide sequence (linker) to the nucleic acid end. Such vectors may be engineered to include a sequence that contains the vector and / or codes for a selectable marker that gives a selection of cells to incorporate the nucleic acid of the vector into the cell genome. Such a marker allows for the identification and / or selection of host cells to incorporate and express the protein encoded by that marker. A “recombinant” vector refers to a viral or non-viral vector containing one or more heterogeneous nucleotide sequences (i.e., transgenes), e.g., two, three, four, five or more heterogeneous nucleotide sequences.

[0045] Viral vectors are used in a wide variety of gene delivery applications in cells and living animals. Available viral vectors include, but are not limited to, retroviruses, lentiviruses, adeno-associated viruses, poxviruses, alphaviruses, baculoviruses, vaccinia viruses, herpesviruses, Epstein-Barr viruses, and adenovirus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cytofectin), nucleic acid-protein complexes, and biopolymers. In addition to the nucleic acid of interest, the vector may also contain one or more regulatory regions and / or selectable markers useful for selecting, measuring, and monitoring the results of nucleic acid transport (such as delivery to specific tissues or duration of expression).

[0046] The vector may be introduced into the desired cells by methods known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or by a nucleic acid vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990). In various embodiments, other molecules such as cationic oligopeptides (e.g., International Publication No. 95 / 21931), peptides derived from nucleic acid-binding proteins (e.g., International Publication No. 96 / 25508), and / or cationic polymers (e.g., International Publication No. 95 / 21931) may be used to facilitate in vivo delivery of nucleic acids. It is also possible to introduce the vector in vivo as a naked nucleic acid (see U.S. Patents 5,693,622, 5,589,466, and 5,580,859). Receptor-mediated nucleic acid delivery methods can also be used (Curiel et al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem. 262:4429 (1987)).

[0047] As used herein, the terms “protein” and “polypeptide” are used synonymously and encompass both peptides and proteins unless otherwise indicated.

[0048] A "fusion protein" is a polypeptide produced when two heterogeneous nucleotide sequences or fragments thereof encoding two (or more) different polypeptides that do not inherently exist fused together are fused together in the correct translational frame. Exemplary fusion polypeptides include the fusion of all or part of the polypeptide (or fragment thereof) of the present invention to glutathione-S-transferase, maltose-binding protein, or reporter protein (e.g., green fluorescent protein, β-glucuronidase, β-galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc.

[0049] As used herein, “functional” polypeptides or “functional fragments” substantially retain at least one biological activity typically associated with that polypeptide (e.g., enzymatic activity, protein binding, ligand or receptor binding). In certain embodiments, a “functional” polypeptide or “functional fragment” substantially retains all the activities possessed by the unmodified peptide. To “substantially retain” biological activity means that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99% or more of the biological activity of the native polypeptide (and may even have higher levels of activity than the native polypeptide). A “non-functional” polypeptide typically exhibits little to no detectable biological activity typically associated with that polypeptide (e.g., at most very little, e.g., about 10% or even less than 5%). Biological activities such as protein binding and enzymatic activity are well known in the art and can be measured using assays described herein.

[0050] The terms "expressing" or "expressing" a polynucleotide coding sequence mean that the sequence is transcribed and optionally translated. Typically, according to the present invention, expression of the coding sequence of the present invention results in the production of the polypeptide of the present invention. The whole-expressed polypeptide or fragments can also function in unpurified intact cells.

[0051] In the context of this invention, the term “adeno-associated virus” (AAV) includes, but is not limited to, AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, cattle AAV, canine AAV, equine AAV, and sheep AAV, as well as any other AAV currently known or to be discovered later. See, for example, BERNARD N. FIELDS et al., VIROLOGY, VOL. 2, CE. 69 (4th edition, Lippincott-Raven Publishers). Many further AAV serotypes and clades have been identified (see, for example, Gao et al., (2004) J. Virol. 78:6381-6388 and Table 1), which are also encompassed by the term “AAV”.

[0052] The genome sequences of various AAVs and autonomous parvoviruses, as well as the sequences of ITRs, Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as the GenBank® database. For example, GenBank® access numbers NC002077, NC001401, NC001729, NC001863, NC001829, NC001862, NC000883, NC001701, NC001510, AF063497, U89790, AF043303, AF028705, AF028704, J02275 See J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC001358, NC001540, AF513851, AF513852, AY530579, AY631965, and AY631966, the entirety of which their disclosures are incorporated herein by reference. For example, Srivistava et al., (1983) J. Virol. 45:555; Chiorini et al., (1998) J. Virol. 71:6823; Chiorini et al., (1999) J. Virol. 73 :1309;Bantel-Schaal et al., (1999) J. Virol.73:939; Xiao et al., (1999) J. Virol.73:3994; Muramatsu et al., (1996) Virology 221:208;Shade et al.,(1986) J.Virol.58:921;Gao et al.,(2002)Proc.Nat.Acad.Sci.USA See also 99:11854; International Publications 00 / 28061, 99 / 61601, and 98 / 11244 of the international patent application; and U.S. Patent No. 6,156,303, the entirety of which disclosures are incorporated herein by reference. See also Table 1.An initial description of the AAV1, AAV2, and AAV3 terminal repeat sequences is provided by Xiao, X., (1996), "Characterization of Adeno-associated virus (AAV) DNA replication and integration," Doctoral dissertation, University of Pittsburgh, Pittsburgh, Pennsylvania (its entire contents are incorporated herein by reference).

[0053] A "recombinant AAV vector genome," or "rAAV genome," is an AAV genome (i.e., vDNA) containing at least one inverted terminal repeat (e.g., one, two, or three inverted terminal repeats) and one or more heterologous nucleotide sequences. While rAAV vectors generally contain 145 cis-terminal repeats (TRs) to produce the virus, modified AAV TRs and non-AAV TRs containing partially or completely synthetic sequences can also serve this purpose. Not all other viral sequences are necessarily required and may be supplied trans- (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). An rAAV vector may optionally contain two TRs (e.g., AAV TRs), which are generally located at the 5' and 3' ends of the heterologous nucleotide sequence, but do not need to be adjacent. These TRs may be the same or different from one another. The vector genome may also contain a single ITR at its 3' or 5' end.

[0054] The term “terminal repeat,” or “TR,” includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediating a desired function such as replication, viral packaging, integration, and / or proviral rescue). TRs may be AAV TRs or non-AAV TRs. For example, non-AAV TR sequences such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or non-AAV TR sequences of the SV40 hairpin that function as an SV40 replication origin can be used as TRs, and these can be further modified by cleavage, substitution, deletion, insertion, and / or addition. Furthermore, TRs may be partially or completely synthetic, such as the “double D sequence” described in U.S. Patent No. 5,478,745 to Samulski et al.

[0055] A "terminal AAV repeat," or "AAV TR," may be from any AAV, including, but not limited to, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any other AAV currently known or to be discovered later (see, for example, Table 1). A terminal AAV repeat does not need to have a native terminal repeat sequence, as long as the terminal repeat mediates the desired function, such as replication, viral packaging, integration, and / or proviral rescue (for example, the native AAV TR sequence may be altered by insertions, deletions, cleavage, and / or missense mutations).

[0056] Furthermore, the viral vector of the present invention may also be a “targeted” viral vector (e.g., directional) and / or a “hybrid” parvovirus (i.e., here the viral ITR and viral capsid are from different parvoviruses) as described in International Publication No. 00 / 28004 of the International Patent Application and Chao et al., (2000) Mol. Therapy 2:619.

[0057] Furthermore, the viral capsid or genomic element may include other modifications such as insertions, deletions, and / or substitutions.

[0058] As used herein, the term “amino acid” encompasses all naturally occurring amino acids, their modified forms, and synthetic amino acids.

[0059] Table 2 shows naturally occurring levorotatory (L-) amino acids. [Table 1] TIFF0007869828000002.tif64162 [Table 2]

[0060] Alternatively, the amino acid may be a modified amino acid residue (non-limiting examples are shown in Table 3), or an amino acid modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation). [Table 3]

[0061] Furthermore, amino acids that do not occur naturally may be "unnatural" amino acids as described by "Wang et al., (2006) Annu. Rev. Biophys. Biomol. Struct. 35:225-49". These unnatural amino acids can be advantageously used to chemically link the target molecule to the AAV capsid protein.

[0062] The terms “template” or “substrate” are used herein to refer to a polynucleotide sequence that can be replicated to produce parvovirus viral DNA. For vector production, the template is typically, but not limited to, incorporated into a larger nucleotide sequence or construct such as a plasmid, naked DNA vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or viral vector (e.g., adenovirus, herpesvirus, Epstein-Barr virus, AAV, baculovirus, retroviral vector, etc.). Alternatively, the template may be stably incorporated into the chromosome of a packaging cell.

[0063] As used herein, parvovirus or AAV "Rep-coding sequence" refers to a nucleic acid sequence encoding a parvovirus or AAV non-structural protein that mediates viral replication and the production of new viral particles. For information on parvovirus and AAV replication genes and proteins, see, for example, FIELDS et al., VIROLOGY, Vol. 2, Chapters 69 & 70 (4th edition, Lippincott-Raven Publishers).

[0064] A "Rep-coding sequence" does not need to encode all parvovirus or AAV Rep proteins. For example, with respect to AAV, the Rep-coding sequence does not need to encode all four AAV Rep proteins (Rep78, Rep68, Rep52, and Rep40); in fact, it is thought that only AAV5 expresses spliced ​​Rep68 and Rep40 proteins. In typical embodiments, the Rep-coding sequence encodes at least the replication proteins necessary for viral genome replication and packaging into new viral particles. Generally, the Rep-coding sequence encodes at least one large Rep protein (i.e., Rep78 / 68) and one small Rep protein (i.e., Rep52 / 40). In certain embodiments, the Rep-coding sequence encodes the AAV Rep78 protein and the AAV Rep52 and / or Rep40 protein. In other embodiments, the Rep-coding sequence encodes the Rep68 and Rep52 and / or Rep40 proteins. In further embodiments, the Rep coding sequence encodes Rep68 and Rep52 proteins, Rep68 and Rep40 proteins, Rep78 and Rep52 proteins, or Rep78 and Rep40 proteins.

[0065] As used herein, the term “large Rep protein” refers to Rep68 and / or Rep78. The large Rep protein of the claimed invention may be either wild-type or synthetic. Wild-type large Rep proteins may be from any parvovirus or AAV, including, but not limited to, serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, or any other AAV currently known or to be discovered later (see, for example, Table 1). Synthetic large Rep proteins may be altered by insertions, deletions, cleavage, and / or missense mutations.

[0066] Those skilled in the art will further understand that the replicating proteins do not need to be encoded by the same polynucleotide. For example, in MVM, NS-1 and NS-2 proteins (which are splice variants) may be expressed independently of each other. Similarly, in AAV, the p19 promoter may be deactivated, and the large Rep protein may be expressed from one type of polynucleotide, while the small Rep protein may be expressed from a different polynucleotide. However, it is typically more convenient to express the replicating proteins from a single construct. In some systems, the viral promoter (e.g., the AAVp 19 promoter) does not need to be recognized by the cell, and therefore it is necessary to express the large and small Rep proteins from separate expression cassettes. In other examples, it may be desirable to express the large and small Rep proteins separately, i.e., under the control of separate transcriptional and / or translational regulatory elements. For example, it may be desirable to control the expression of the large Rep protein in order to reduce the ratio of large to small Rep proteins. In insect cells, it may be advantageous to downregulate the expression of large Rep proteins (e.g., Rep78 / 68) to avoid cytotoxicity (see, for example, Urabe et al., (2002) Human Gene Therapy 13:1935).

[0067] As used herein, the parvovirus or AAV "cap coding sequence" encodes a structural protein that forms a functional parvovirus or AAV capsid (i.e., it can package DNA and infect target cells). Typically, the cap coding sequence encodes all of the parvovirus or AAV capsid subunits, but it may encode less than all of the capsid subunits, as long as a functional capsid is produced. Typically, but not always, the cap coding sequence resides on a single nucleic acid molecule.

[0068] The terms "rAAV particle" and "rAAV virus particle" are used synonymously here. An "rAAV particle" or "rAAV virus particle" contains an rAAV vector genome packaged within an AAV capsid.

[0069] The AAV capsid structure is described in more detail in Bernard N. Fields et al., Virology, Vol. 2, Chapters 69 & 70 (4th edition, Lippincott-Raven Publishers).

[0070] The term "pharmacokinetic properties" has its usual and common meaning and refers to the absorption, distribution, metabolism, and excretion of FIX protein.

[0071] The usual and customary meaning of “bioavailability” is the proportion or amount of a biologically active drug that reaches systemic circulation. In the context of the embodiments of the present invention, the term “bioavailability” includes the usual and customary meaning, but can also be interpreted to have a broader meaning as FIX protein is biologically active to some extent. In the case of FIX, for example, one measure of “bioavailability” is the procoagulant activity of the FIX protein obtained in circulation after injection.

[0072] "Post-translational modification" has its usual and common meaning, and is not limited to, but includes, for example, removal of the leader sequence, γ-carboxylation of glutamate residues, β-hydroxylation of aspartate residues, N-linked glycosylation of asparagine residues, O-linked glycosylation of serine and / or threonine residues, sulfation of tyrosine residues, phosphorylation of serine residues, and any combination thereof.

[0073] As used herein, "biological activity" is determined by reference to a reference material, for example, derived from human plasma or produced by recombinant processes. In the case of FIX, the reference material may be BeNEFIX® (Pfizer) or MONONINE® (CSL Behring). The biological activity of the reference material is assumed to be 100%.

[0074] As used herein, the terms “Factor IX protein” or “FIX protein” include wild-type FIX protein as well as naturally occurring or artificial proteins. The FIX protein of the present invention may further include mutant forms of FIX (e.g., Padua mutations) as known in the literature. The FIX protein of the present invention further includes any other naturally occurring or artificial human FIX protein that is currently known or will be identified later, and its derivatives and active fragments / active domains known in the art.

[0075] The amino acid sequences of FIX from multiple mammalian species are available from sequence databases such as Genbank. Examples of FIX sequences are listed in the table below. [Table 4]

[0076] The FIX protein of the present invention further comprises a pharmacologically active form of FIX, which is a molecule in which the signal peptide has been removed and the protein has been cleaved by the action of a protease (or by manipulating the protein to remove it at the nucleic acid level) to produce two discontinuous polypeptide chains linked by disulfide crosslinks.

[0077] The amino acid sequence of the human FIX protein is well known in the art and can be found, for example, in GenBank access number AAB59620.1. The human FIX protein is 461 amino acids long and consists of a signal peptide (residues 1-46), a Gla domain (residues 28-92), an EGF domain (residues 93-129), and a trypsin domain (residues 226-454).

[0078] The term “half-life” is a broad term encompassing both its common and conventional meanings, as well as its common and conventional meaning as found in the scientific literature on FIX. In particular, this definition includes measured values ​​of parameters associated with FIX that determine the time after infusion during which the FIX decreases from its initial value measured at infusion to half of its initial value. In some embodiments, the half-life of FIX can be measured in blood and / or blood components using antibodies against FIX in various immunological assays that are well known in the art and described herein. Alternatively, the half-life may be measured as a decrease in FIX activity using functional assays, including standard coagulation assays that are well known in the art and described herein.

[0079] As used herein, the term “recovery” includes, but is not limited to, the amount of FIX measured by any acceptable method, including FIX antigen levels or FIX protease or coagulation activity levels detected in a recipient animal or human subject (e.g., in circulation) at the earliest possible actual time of taking a biological sample (e.g., blood or blood product sample) to measure the level of FIX after its infusion, injection, delivery, or other administration. With current methodologies, the earliest biological sample collection time for measuring FIX recovery is typically within the first 15 minutes after the infusion, injection, or delivery / other administration of FIX, but it is reasonable to expect faster sample collection times as scientific and / or clinical techniques improve. Essentially, the FIX recovery value is intended herein to represent the maximum percentage of FIX delivered / administered by infusion, injection, or other means that can be measured in the recipient (e.g., in circulation) at the earliest possible time after infusion, injection, or other delivery to the recipient animal or patient.

[0080] As used herein, “transformed” cells are cells that have been transformed, transduced, and / or transfected with nucleic acid molecules encoding the FIX protein of the present invention, such as FIX protein vectors constructed using recombinant DNA technology, but are not limited to these.

[0081] As used herein, the term “hemorrhagic disorder” reflects any congenital, acquired, or induced defect of cellular, physiological, or molecular origin that manifests as bleeding. Examples include deficiencies in coagulation factors (e.g., hemophilia A and B or deficiencies in coagulation factors XI, VII, VIII, or IX), coagulation factor inhibitors, platelet dysfunction, thrombocytopenia, von Willebrand disease, or bleeding induced by surgery or trauma.

[0082] Excessive bleeding can occur even in individuals with a normally functioning blood coagulation cascade (no coagulation factor deficiencies or no inhibitors for any coagulation factors), and may be caused by platelet dysfunction, thrombocytopenia, or von Willebrand disease. In such cases, the bleeding may be likened to that caused by hemophilia because the hemostatic system lacks or is abnormal in essential coagulation "compounds" (such as platelets or von Willebrand factor proteins), leading to massive bleeding. In individuals experiencing extensive tissue damage associated with surgery or trauma, normal hemostatic mechanisms may be overwhelmed by the demand for immediate hemostasis, resulting in bleeding despite normal hemostatic mechanisms. Achieving adequate hemostasis is also a problem when bleeding occurs in organs such as the brain, inner ear, and eyes, where surgical hemostasis is limited. The same problem can arise in the process of biopsy from various organs (liver, lungs, tumor tissue, gastrointestinal tract) and in laparoscopic surgery. A common factor in all these situations is the difficulty in providing hemostasis through surgical techniques (suturing, clips, etc.), as well as when bleeding is diffuse (hemorrhagic gastritis and massive uterine bleeding). Acute and massive bleeding can also occur in patients receiving anticoagulation therapy, even if incomplete hemostasis is induced by the given treatment. Such patients may require surgical intervention if the anticoagulant effect must be quickly counteracted. Radical retropubic prostatectomy is a common procedure for patients with localized prostate cancer. This surgery is very often complicated by significant and sometimes massive blood loss. Significant blood loss during prostatectomy is mainly related to the complex anatomical situation, which has various densely vascularized areas that are not easily accessible for surgical hemostasis, and can result in diffuse bleeding over a wide area. Intracerebral hemorrhage is also the least treatable form of stroke and is associated with high mortality and hematoma enlargement in the first few hours after intracerebral hemorrhage. Another situation in which insufficient hemostasis can cause problems is when anticoagulant therapy is administered to a subject with a normal hemostatic mechanism to prevent thromboembolic disease.Such treatments include heparin, or other forms of proteoglycans; warfarin, or other forms of vitamin K antagonists; and aspirin and other platelet aggregation inhibitors.

[0083] In one embodiment, bleeding is associated with hemophilia. In another embodiment, bleeding is associated with hemophilia with acquired inhibitors. In another embodiment, bleeding is associated with thrombocytopenia. In another embodiment, bleeding is associated with von Willebrand disease. In another embodiment, bleeding is associated with serious tissue injury. In another embodiment, bleeding is associated with serious trauma. In another embodiment, bleeding is associated with surgery. In another embodiment, bleeding is associated with laparoscopic surgery. In another embodiment, bleeding is associated with hemorrhagic gastritis. In another embodiment, bleeding is massive uterine bleeding. In another embodiment, bleeding occurs in an organ where mechanical hemostasis is limited. In another embodiment, bleeding occurs in the brain, inner ear region, or eye. In another embodiment, bleeding is associated with the process of taking a biopsy. In another embodiment, bleeding is associated with anticoagulant therapy.

[0084] The “subjects” of the present invention include any animal having or susceptible to hemorrhagic disorders or bleeding diseases (such as hemophilia B and acquired FIX deficiency (e.g., caused by autoantibodies against FIX or hematological malignancies)) for which control of bleeding is necessary and / or desired, which can be treated, remitted or prevented by administration of FIX to the subject. Such subjects are generally mammalian subjects (e.g., laboratory animals such as rats, mice, guinea pigs, rabbits, and primates), farm or commercial animals (e.g., cattle, horses, goats, donkeys, sheep, etc.), or domestic animals (e.g., cats, dogs, ferrets, etc.). In certain embodiments, the subject is a primate subject, a non-human primate subject (e.g., chimpanzees, baboons, monkeys, gorillas, etc.) or a human. The subjects of the present invention may also be subjects known or considered to be at risk of hemorrhagic disorders or bleeding diseases for which control is necessary and / or desired. Alternatively, the subjects according to the present invention may include subjects who are not previously known or suspected to be at risk of hemorrhagic disorders or bleeding diseases that require or are desired to be controlled. As a further option, the subjects may be experimental animals and / or animal models of the disease.

[0085] The subjects include males and / or females of all ages, including newborns, young adults, adults, and elderly subjects. In typical embodiments with respect to human subjects, subjects may be infants (e.g., about 12 months, 10 months, 9 months, 8 months, 7 months, less than 6 months or less), toddlers (e.g., at least about 12, 18 or 24 months and / or less than about 36, 30 or 24 months), or children (e.g., at least about 1, 2, 3, 4 or 5 years and / or less than about 14, 12, 10, 8, 7, 6, 5 or 4 years). In embodiments of the present invention, the subjects are human subjects who are approximately 0-3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months old, approximately 3-6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months old, approximately 6-9, 12, 15, 18, 24, 30, 36, 48 or 60 months old, approximately 9-12, 15, 18, 24, 30, 36, 48 or 60 months old, approximately 12-18, 24, 36, 48 or 60 months old, approximately 18-24, 30, 36, 48 or 60 months old, or approximately 24-30, 36, 48 or 60 months old.

[0086] Promoter and expression cassette One aspect of the present invention relates to a polynucleotide comprising a synthetic liver-specific promoter, wherein the promoter comprises, essentially comprises, or comprises the nucleotide sequence of SEQ ID NO: 1 or a sequence identical thereto by at least about 90%. In some embodiments, the nucleotide sequence is identical to the nucleotide sequence of SEQ ID NO: 1 by at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The promoter is an ideal short (less than 200 base pairs) and potent liver-specific promoter for liver-specific expression of the polynucleotide of interest, and is particularly suitable for use in AAV vectors due to its short length and the limited volume of AAV vectors. The promoter is designed to include a conserved basic promoter element and a transcription start site. The basic promoter is ligated at its 5' end to many liver-specific transcription factor binding sites for liver-specific expression (Figure 1). This promoter was first identified in vitro using a luciferase reporter gene and transfection experiments in the human hepatocellular carcinoma cell line Huh7, and subsequently showed high activity when confirmed in vivo in mice.

[0087] The promoter may be operably ligated to the polynucleotide of interest. In some embodiments, the polynucleotide of interest encodes a polypeptide or functional nucleic acid. In certain embodiments, the polynucleotide of interest encodes a coagulation factor, such as FVIX. In some embodiments, the polynucleotide sequence encoding FIX has codons optimized for expression in humans. In certain embodiments, the codon-optimized sequence contains, essentially consists of, or comprises a sequence that is at least 90% identical to one of sequence numbers 14-16, for example, a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequences of sequence numbers 14-16. In some embodiments, the polynucleotide sequence encoding FIX encodes a FIX sequence that includes mutations or sequence changes known in the art. For example, the factor IX coding sequence includes a missense mutation that gives rise to the R338L mutation (Padua mutation) or other mutations at that site (numbering for human factor IX).

[0088] In some embodiments, the polynucleotide further comprises a synthetic 5' untranslated region (5'UTR) between the promoter and the polynucleotide of interest. The synthetic 5'UTR may contain, essentially consist of, or be composed of, the nucleotide sequence of SEQ ID NO: 3 or a sequence identical thereto by at least about 90%, for example, a sequence identical by at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the synthetic 5'UTR comprises a synthetic intron. The synthetic intron may contain, essentially consist of, or be composed of, the nucleotide sequence of SEQ ID NO: 13 or a sequence identical thereto by at least about 90%, for example, a sequence identical by at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the synthetic 5'UTR and synthetic intron contain, essentially consist of, or consist of, the nucleotide sequence of SEQ ID NO: 4 or a sequence that is at least about 90% identical thereto, for example, a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In certain embodiments, the promoter, synthetic 5'UTR, and synthetic intron contain, essentially consist of, or consist of, the nucleotide sequence of SEQ ID NO: 2 or a sequence that is at least about 90% identical thereto, for example, a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0089] In some embodiments, the target polynucleotide ligated to the promoter of the present invention or any promoter includes a synthetic intron. In some embodiments, the synthetic intron includes, essentially consists of, or consists of the nucleotide sequence of SEQ ID NO: 5 or a sequence that is at least about 90% identical thereto, for example, a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0090] In certain embodiments, the polynucleotide of interest is a codon-optimized factor IX coding sequence, and the codon-optimized factor IX coding sequence and the synthetic intron together contain, essentially consist of, or consist of one of the nucleotide sequences of sequence number 6, 7, or 8, or a sequence that is at least about 90% identical thereto, for example, a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0091] In certain embodiments, any of the polynucleotides of the present invention may be operably ligated to a polyadenylation site, for example, a bidirectional polyadenylation site. In some embodiments, the polyadenylation site includes, essentially consists of, or comprises the nucleotide sequence of SEQ ID NO: 9 or a sequence that is at least about 90% identical thereto, for example, a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0092] Another aspect of the present invention relates to a polynucleotide encoding human factor IX, which has codons optimized for expression in humans. In certain embodiments, the codon-optimized sequence includes, essentially consists of, or consists of, a sequence that is at least 90% identical to one of sequence numbers 14-16, for example, a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequences of sequence numbers 14-16. In some embodiments, the codon-optimized sequence includes a synthetic intron. In some embodiments, the synthetic intron includes, essentially consists of, or consists of, the nucleotide sequence of sequence number 5 or a sequence that is at least about 90% identical to it, for example, a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of sequence number 5. In certain embodiments, the codon-optimized factor IX coding sequence and the synthetic intron together contain, essentially consist of, or consist of one nucleotide sequence of sequence number 6, 7, or 8, or a sequence that is at least about 90% identical thereto, for example, a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto to the nucleotide sequence of sequence number 6, 7, or 8.

[0093] In certain embodiments, any of the polynucleotides of the present invention may be in the form of an expression cassette, for example, a cassette compressing a promoter, 5'UTR, the polynucleotide of interest, one or more synthetic introns and / or polyadenylation sites in any combination and any order. In some embodiments, the expression cassette contains, essentially consists of, or consists of, one nucleotide sequence of SEQ ID NOs: 10, 11, or 12 or a sequence that is at least about 90% identical thereto, for example, a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto to the nucleotide sequence of SEQ ID NOs: 10, 11, or 12.

[0094] Another aspect of the present invention is a vector comprising the polynucleotide of the present invention, for example, an expression vector. The vector may be any type of vector known in the art, such as plasmid vectors and viral vectors, but is not limited to these. In some embodiments, the viral vector is a retroviral or lentiviral vector. In some embodiments, the viral vector is an AAV vector from any known AAV serotype, including, but is not limited to, AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, and sheep AAV, as well as any other AAV currently known or to be discovered later. In some embodiments, the AAV vector is AAV8 or AAV9.

[0095] Further aspects of the present invention relate to cells (e.g., isolated cells, transformed cells, recombinant cells, in vitro or ex vivo cells, etc.) comprising the polynucleotides and / or vectors of the present invention. Accordingly, various embodiments of the present invention relate to recombinant host cells comprising vectors (e.g., expression cassettes). Such cells may be isolated and / or present in transgenic animals. Cell transformation is further described below.

[0096] Another aspect of the present invention relates to transgenic non-human animals comprising the polynucleotides, vectors, and / or transformed cells of the present invention. Transgenic animals are described further below.

[0097] The polynucleotides, vectors, and / or cells of the present invention may be included in pharmaceutical compositions. Some embodiments relate to kits including the polynucleotides, vectors, and / or cells, and / or reagents, and / or instructions for using a kit for carrying out, for example, the methods of the present invention.

[0098] Method of the present invention Further aspects of the present invention relate to the use of the promoter, optimized sequence, and expression cassette of the present invention for the specific production of polypeptides or functional nucleic acids, for example, in the liver. Thus, one aspect relates to a method for producing polypeptides or functional nucleic acids in the liver of a target, comprising delivering the polynucleotide, vector, and / or transformed cells of the present invention to a target to produce polypeptides or functional nucleic acids in the liver of the target. The polynucleotide, vector, and / or transformed cells are delivered under conditions that result in the expression of the polynucleotide of interest to produce polypeptides or functional nucleic acids. Such conditions are well known in the art and are further described below.

[0099] Another aspect of the present invention relates to a method for treating hemophilia B or acquired factor IX deficiency in a subject using the promoter, optimized sequence and expression cassette of the present invention, comprising delivering a therapeutically effective amount of the polynucleotide, vector and / or transformed cells of the present invention to the subject, thereby treating hemophilia B or acquired factor IX deficiency in the subject. In some embodiments, the polynucleotide of interest encodes the FIX polypeptide described above.

[0100] A further aspect of the present invention relates to a method for enhancing the bioavailability of FIX polypeptide in a subject using the promoter, optimized sequence, and expression cassette of the present invention, comprising delivering an effective amount of the polynucleotide, vector, and / or transformed cells of the present invention to the subject, thereby enhancing the bioavailability of FIX polypeptide in the subject. In this aspect, the polynucleotide of interest may encode the FIX polypeptide described above.

[0101] One aspect of the present invention relates to a method for producing FIX in the liver of a subject, comprising delivering a polynucleotide, vector, and / or transformed cells encoding the FIX polypeptide of the present invention to the subject, thereby causing the liver of the subject to produce FIX.

[0102] A further aspect of the present invention relates to a method for enhancing the bioavailability of FIX polypeptide in a subject, comprising delivering a polynucleotide, vector, and / or transformed cells encoding an effective amount of the FIX polypeptide of the present invention to the subject, thereby enhancing the bioavailability of FIX polypeptide in the subject.

[0103] Hemorrhagic disorders that can be treated according to the method of the present invention include all diseases that can be treated with FIX, such as hemophilia B and acquired FIX deficiency. Such therapeutic protocols and administration plans for administering or delivering the polynucleotide encoding the FIX protein of the present invention to a subject (e.g., a subject requiring it) are well known in the art.

[0104] In embodiments of the present invention, the dose of the vector encoding the FIX protein of the present invention (e.g., a viral vector or other nucleic acid vector) may be such that a therapeutic plasma concentration of the FIX protein is achieved. A therapeutic concentration of the FIX protein is considered to be more than 1% of the normal level in a healthy individual, measured at an average of 100%, and therefore 1 international unit (IU) of FIX in 1 mL of normal human plasma. Those skilled in the art will be able to determine the optimal dose for a given subject and given conditions.

[0105] For therapeutic interventions, the FIX protein of the present invention is typically administered within approximately 24 hours prior to the intervention and then for a period of at least 7 days thereafter. Administration as an anticoagulant may be via one of the various routes described herein.

[0106] This pharmaceutical composition is primarily intended for parenteral administration for preventive and / or therapeutic purposes. Preferably, this pharmaceutical composition may be administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly, or by continuous or pulsating infusion. Alternatively, this pharmaceutical composition may be formulated for administration by various methods, including, but not limited to, oral, subcutaneous, intravenous, intracerebral, intranasal, percutaneous, intraperitoneal, intramuscular, intrapulmonary, intravaginal, intrarectal, intraocular, or any other acceptable method.

[0107] The composition for parenteral administration comprises a polynucleotide, vector, or cell encoding the FIX protein of the present invention, combined with (e.g., dissolved in) a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers may be used, such as water, buffer water, 0.4% saline, or 0.3% glycine. The polynucleotide, vector, or cell encoding the FIX protein of the present invention may also be formulated with a composition that extends stability and storage, such as methionine and sucrose. The polynucleotide, vector, or cell encoding the FIX protein of the present invention may also be formulated in a liposome preparation for delivery to or targeting of a wound site. Liposome preparations are generally described, for example, in U.S. Patents 4,837,028, 4,501,728, and 4,975,282. The composition may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solution may be packaged for use, filtered under sterile conditions, and lyophilized, and the lyophilized preparation may be combined with the sterile aqueous solution before administration. This composition may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters, buffers, isotonic agents, and other substances such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. This composition may also contain preservatives, isotonic agents, nonionic surfactants or surfactants, antioxidants, and / or various other additives.

[0108] The concentrations of polynucleotides, vectors, or cells encoding the FIX protein in these formulations may vary considerably, ranging from approximately 0.5% by weight, typically about 1% by weight, or at least less than about 1% by weight, to approximately 15 or 20% by weight, and are selected according to the chosen specific mode of administration, primarily based on fluid volume and viscosity. The actual methods for preparing parenterally administered compositions are known or evident to those skilled in the art and are described in more detail, for example, in "Remington's Pharmaceutical Sciences, 21st Edition, Mack Publishing Company, Easton, Pennsylvania (2005)."

[0109] A composition comprising nucleic acid molecules, vectors, or cells encoding the FIX protein of the present invention can be administered for prophylactic and / or therapeutic purposes. For therapeutic use, the composition is administered to a subject already suffering from such a disease in an amount sufficient to cure, alleviate, or partially halt the disease and its complications. The amount appropriate to achieve this is defined as the "therapeutic effective dose." As will be understood by those skilled in the art, the effective dose for this purpose depends on the severity of the disease or injury, as well as the subject's weight and health condition.

[0110] For preventative purposes, compositions containing polynucleotides, vectors, or cells encoding the FIX protein of the present invention are administered to subjects prone to disease or injury, or at other risk thereof, to enhance their own clotting ability. Such a quantity is defined as a "preventively effective dose." The exact dose for preventative purposes will, in this case, also depend on the subject's health condition and body weight.

[0111] The composition can be administered as a single or multiple doses at dose levels and in patterns selected by the treating physician. For outpatients requiring routine management, the polynucleotide, vector, or cells encoding the FIX protein may be administered by continuous infusion, for example, using a portable pump system.

[0112] Furthermore, the polynucleotide, vector, or cell encoding the FIX protein of the present invention may be formulated as a sustained-release or sustained-release formulation. Methods for formulating sustained-release or sustained-release compositions are known in the art and are not limited thereto, but include a semipermeable matrix of solid hydrophobic particles containing the polynucleotide, vector, or cell.

[0113] Local delivery of the polynucleotide, vector, or cells encoding the FIX protein of the present invention, such as by topical application, may be carried out, for example, by spray, perfusion, using a double balloon catheter, a stent, or by incorporating it into an artificial blood vessel or stent, or by using a hydrogel or other well-established method used to coat a balloon catheter. In any case, the pharmaceutical composition must provide a sufficient amount of FIX protein to effectively treat the target.

[0114] In some embodiments, the target polynucleotide (e.g., FIX protein) is delivered to the target using an AAV vector. Accordingly, the present invention also provides AAV viral particles (i.e., virions) containing the target polynucleotide, where the viral particles package (i.e., capsidize) a vector genome, optionally an AAV vector genome.

[0115] In certain embodiments, the viral particle is a recombinant vector containing the desired heterologous polynucleotide for delivery to cells, for example. Therefore, the present invention is useful for delivering polynucleotides to cells in vitro, ex vivo, and in vivo. In typical embodiments, the recombinant vector of the present invention can be advantageously used to deliver or transport polynucleotides to animal (e.g., mammalian) cells, for example, to hepatocytes when using the liver-specific promoter of the present invention.

[0116] Any heterogeneous nucleotide sequence may be delivered by the viral vector of the present invention. Examples of polynucleotides of interest include polypeptides, optionally polynucleotides encoding therapeutic (e.g., for medical or veterinary use) and / or immunogenic (e.g., for vaccines) polypeptides.

[0117] Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulator (CFTR) proteins, dystrophin (including protein products of the dystrophin minigene or microgene, e.g., Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent Application Publication No. 2003017131; Wang et al., (2000) Proc. Natl. Acad. Sci. USA 97:13714-9 [mini-dystrophin]; Harper et al., (2002) Nature Med.8:253-61 [microdystrophin]), miniagrin, laminin-α2, sarcoglycans (α, β, γ or δ), fukutin-related proteins, myostatin propeptides, follistatin, dominant-negative myostatins, angiogenic factors (e.g., VEGF, angiopoietin-1 or 2), anti-apoptotic factors (e.g., heme oxygenase-1, TGF-β, pro-apoptotic signaling inhibitors such as caspases, proteases, kinases, death receptors [e.g., CD-095], cytochrome C release regulators, inhibitors of mitochondrial pore opening and expansion), activin type II soluble receptor, anti-inflammatory polypeptides such as Ikappa B dominant mutants, sarcospan, eutrophin, minieutrophin, antibodies or antibody fragments against myostatin or myostatin propeptides, cell cycle regulators, Rho kinase regulators such as Cethrin, a modified bacterial C3 extracellular enzyme [BioAxone Available from Therapeutics (Saint Laurent, Quebec, Canada), BCL-xL, BCL2, XIAP, FLICEc-s, dominant-negative caspase-8, dominant-negative caspase-9, SPI-6 (see, for example, U.S. Patent Application Publication No. 20070026076), transcription factors (PGC-α1, Pinch gene, ILK gene and thymosin β4 gene), coagulation factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, intracellular and / or extracellular superoxide dismutase, leptin, LDL receptor, neprilysin, lipoprotein lipase, ornithine transcarbamylase,β-globin, α-globin, spectrin, α1-antitrypsin, methylcytosine-binding protein 2, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, β-glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain keto acid dehydrogenase, RP65 protein, cytokines (e.g., α-interferon, β-interferon, interferon-γ, interleukin 1-14, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.) ), peptide growth factors, neurotrophic factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors including IGF-1 and IGF-2, GLP-1, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factors 3 and 4, brain-derived neurotrophic factor, glial growth factor, transforming growth factors α and β, etc.), bone morphogenetic proteins (including RANKL and VEGF), lysosomal proteins, glutamate receptors, lymphokines, soluble CD4, Fc receptor, T cell receptor, ApoE, A poC, protein phosphatase inhibitor 1 inhibitor 1 (I-1), phospholamban, serca2a, lysosomal acid α-glucosidase, α-galactosidase A, Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), calsarcin, receptor (e.g., tumor necrosis growth factor α-soluble receptor), anti-inflammatory factors such as IRAP, Pim-1, PGC-1α, SOD-1, SOD-2, ECF-SOD, kallikrein, cy Mosin-β4, hypoxia-inducible transcription factor [HIF], angiogenic factors, S100A1, parvalbumin, adenylyl cyclase type 6, molecules that affect G protein-coupled receptor kinase type 2 knockdown such as cleaved constitutively activated bARKct, phospholamban inhibitors such as phospholamban S16E or dominant-negative molecules, monoclonal antibodies (including single-chain monoclonal antibodies) or suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factors such as TNF-α),This also includes any other polypeptides that have a therapeutic effect in subjects requiring it.

[0118] Examples of heterogeneous nucleotide sequences encoding polypeptides include sequences encoding reporter polypeptides (e.g., enzymes). Reporter polypeptides are known in the art and are not limited to, but include fluorescent proteins (e.g., EGFP, GFP, RFP, BFP, YFP, or dsRED2), luciferases (e.g., derived from Gaussia, Renilla, or Photinus), β-galactosidase, β-glucuronidase, alkaline phosphatase, and enzymes that produce detectable products such as chloramphenicol acetyltransferase genes or proteins that can be directly detected. Substantially any protein can be directly detected, for example, using an antibody specific to that protein. Further markers (and associated antibiotics) suitable for either positive or negative selection of eukaryotic cells are disclosed in Sambrook and Russell (2001), Molecular Cloning, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al. (1992), Current Protocols in Molecular Biology, John Wiley & Sons (including periodic updates).

[0119] Alternatively, heterologous nucleic acids may encode functional RNA, such as antisense oligonucleotides, ribozymes (e.g., as described in U.S. Patent No. 5,877,022), RNA that affects spliceosome-mediated transsplicing (see Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Patent No. 6,013,487; U.S. Patent No. 6,083,702), interfering RNA (RNAi), including small interfering RNAs (siRNAs) that mediate gene silencing (see Sharp et al., (2000) Science 287:2431), microRNA, or other non-coding "functional" RNA such as "guide" RNA (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Patent No. 5,869,248 to Yuan et al.). Examples of non-coding RNAs include RNAi or antisense RNA against multidrug resistance (MDR) gene products (e.g., for administration to the heart to treat tumors and / or prevent chemotherapy-induced damage), RNAi or antisense RNA against myostatin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNA against tumor immunogens such as VEGF or, but not limited to, the tumor immunogens specifically described herein (for treating tumors), RNAi or antisense oligonucleotides targeting mutant dystrophin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNA against hepatitis B surface antigen genes (for preventing and / or treating hepatitis B infection), RNAi or antisense RNA against HIV tat and / or rev genes (for preventing and / or treating HIV), and / or RNAi or antisense RNA against any other immunogen from pathogens (for protecting a target from pathogens) or defective gene products (for preventing or treating disease). RNAi or antisense RNAs targeting the above targets or any other targets can also be used as research reagents.

[0120] As is known in the art, “exon skipping” can be induced in patients with muscular dystrophy resulting from a defect in the dystrophin gene using antisense nucleic acids (e.g., DNA or RNA) and repressive RNA (e.g., microRNA and RNAi such as siRNA or shRNA) sequences. Thus, heterologous nucleic acids can encode antisense nucleic acids or repressive RNA that induce appropriate exon skipping. Those skilled in the art will understand that the specific method for exon skipping depends on the nature of the underlying defect in the dystrophin gene, and that numerous such strategies are known in the art. Exemplary antisense nucleic acid and repressive RNA sequences target upstream branching points and / or downstream donor splice sites and / or internal splicing enhancer sequences of one or more dystrophin exons (e.g., exon 19 or 23). For example, in certain embodiments, heterologous nucleic acids encode antisense nucleic acids or repressive RNA targeting upstream branching points and / or downstream splice donor sites of exon 19 or 23 of the dystrophin gene. Such sequences can be incorporated into AAV vectors that deliver modified U7 snRNA and antisense nucleic acids or repressive RNA (see, e.g., Goyenvalle et al., (2004) Science 306:1796-1799). Alternatively, modified U1 snRNA can be incorporated into AAV vectors along with siRNA, microRNA, or antisense RNA complementary to the upstream and downstream splice sites of the dystrophin exon (e.g., exon 19 or 23) (see, e.g., Denti et al., (2006) Proc. Nat. Acad. Sci. USA 103:3758-3763). Furthermore, antisense nucleic acids and repressive RNAs can target splicing enhancer sequences within exons 19, 43, 45, or 53 (see, e.g., U.S. Patents 6,653,467, 6,727,355, and 6,653,466).

[0121] Ribozymes are RNA-protein complexes that site-specifically cleave nucleic acids. Ribozymes possess a specific catalytic domain with endonuclease activity (Kim et al., (1987) Proc. Natl. Acad. Sci. USA 84:8788; Gerlach et al., (1987) Nature 328:802; Forster and Symons, (1987) Cell 49:211). For example, many ribozymes promote phosphate transfer reactions with a high degree of specificity, often cleaving only one of several phosphate esters in an oligonucleotide substrate (Michel and Westof, (1990) J. Mol. Biol. 216:585; Reinhold-Hurek and Shub, (1992) Nature 357:173). This specificity stems from the requirement that the substrate binds to the ribozyme's internal guide sequence ("IGS") via specific base-pair interactions before the chemical reaction.

[0122] Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage / ligation reactions involving nucleic acids (Joyce, (1989) Nature 338:217). For example, U.S. Patent No. 5,354,855 reports that certain ribozymes can function as endonucleases with sequence specificity greater than that of known ribonucleases and approaching that of DNA restriction enzymes. Therefore, inhibition of nucleic acid expression mediated by sequence-specific ribozymes may be particularly suitable for therapeutic applications (Scanlon et al., (1991) Proc. Natl. Acad. Sci. USA 88:10591; Sarver et al., (1990) Science 247:1222; Sioud et al., (1992) J. Mol. Biol. 223:831).

[0123] MicroRNAs (mirs) are native cellular RNA molecules that can regulate the expression of multiple genes by controlling the stability of mRNA. Dysfunction can be treated using the overexpression or reduction of specific microRNAs, and this has been shown to be effective in many disease conditions and animal models of diseases (see, e.g., Couzin, (2008) Science 319:1782-4). Chimeric AAVs can be used to deliver microRNAs into cells, tissues, and subjects to treat hereditary and acquired diseases, or to enhance the functionality of specific tissues and promote their proliferation. For example, mir-1, mir-133, mir-206, and / or mir-208 can be used to treat heart and skeletal muscle diseases (e.g., Chen et al., (2006) Genet. 38:228-33; van Rooij et al., (2008) Trends Genet. 24:159-66). MicroRNAs can also be used to modulate the immune system after gene delivery (see Brown et al., (2007) Blood 110:4144-52).

[0124] As used herein, the term “antisense oligonucleotide” (including “antisense RNA”) refers to a nucleic acid that is complementary to and specifically hybridizes with a given DNA or RNA sequence. Antisense oligonucleotides and the nucleic acids encoding them can be prepared according to prior art. See, for example, U.S. Patent No. 5,023,243 to Tullis; U.S. Patent No. 5,149,797 to Pederson et al.

[0125] Those skilled in the art will understand that the antisense oligonucleotide does not need to be perfectly complementary to the target sequence, as long as the degree of sequence similarity is sufficient to specifically hybridize the antisense nucleotide sequence to its target (as defined above) and to reduce the production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more).

[0126] To determine the specificity of hybridization, hybridization of such oligonucleotides to target sequences can be carried out under low, moderate, or even severe conditions. Suitable conditions for achieving low, moderate, and severe hybridization are described herein.

[0127] In other words, in certain embodiments, the antisense oligonucleotides of the present invention have sequence identity with the complement of the target sequence of at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, or higher, and reduce the production of the protein product (as defined above). In some embodiments, the antisense sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches compared to the target sequence.

[0128] Methods for determining the percentage of nucleic acid sequence identity are described in more detail elsewhere in this specification.

[0129] The length of an antisense oligonucleotide is not critical as long as it specifically hybridizes to its intended target, reduces the production of the protein product (as defined above), and can be determined by routine procedures. Generally, antisense oligonucleotides have a nucleotide length of at least about 8, 10, 12, or 15, and / or less than about 20, 30, 40, 50, 60, 70, 80, 100, or 150.

[0130] RNA interference (RNAi) is another useful technique for reducing the production of protein products (e.g., shRNA or siRNA). RNAi is a post-transcriptional gene silencing mechanism in which a double-stranded RNA (dsRNA) corresponding to a target sequence of interest is introduced into a cell or organism, resulting in the degradation of the corresponding mRNA. The mechanism by which RNAi achieves gene silencing has been re-examined by Sharp et al., (2001) Genes Dev 15:485-490 and Hammond et al., (2001) Nature Rev. Gen. 2:110-119. The RNAi effect persists for multiple cell divisions before gene expression is restored. Therefore, RNAi is a powerful method for performing targeted knockout or "knockdown" at the RNA level. RNAi has been shown to be successful in human cells, including human fetal kidney and HeLa cells (see, e.g., Elbashir et al., Nature (2001) 411:494-8).

[0131] The first attempts to use RNAi in mammalian cells led to the development of antiviral defense mechanisms, including PKR, that respond to dsRNA molecules (see, e.g., Gil et al., (2000) Apoptosis 5:107). Since then, it has been demonstrated that short synthetic dsRNAs, consisting of approximately 21 nucleotides and known as "short interfering RNAs" (siRNAs), can mediate silencing in mammalian cells without inducing an antiviral response (see, e.g., Elbashir et al., Nature (2001) 411:494-8; Caplen et al., (2001) Proc. Nat. Acad. Sci. USA 98:9742).

[0132] RNAi molecules (including siRNA molecules) may also be small hairpin RNAs (shRNA; see Paddison et al., (2002), Proc. Nat. Acad. Sci. USA 99:1443-1448), which are thought to be processed in cells by the action of ribonuclease III, such as the enzyme Dicer, which degrades them into 20-25mer siRNA molecules. shRNAs generally have a stem-loop structure in which two reverse repeat sequences are separated by a short spacer sequence that loops out. ShRNAs with loops ranging in length from 3 to 23 nucleotides have been reported. The loop sequence is generally not important. Exemplary loop sequences include the following motifs: AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA.

[0133] RNAi may further include a cyclic molecule containing sense and antisense regions with two loop regions on one side, such that when dsRNA is formed between the sense and antisense regions, it forms a "dumbbell" shape. This molecule can be treated in vitro or in vivo to release a portion of the dsRNA, such as siRNA.

[0134] International Patent Application Publication No. 01 / 77350 describes a vector for bidirectional transcription to produce both heterologous sense and antisense transcripts in eukaryotic cells. This technique can be used to produce RNAi for use according to the present invention.

[0135] Shinagawa et al., (2003) Genes Dev. 17:1340, reported a method for expressing long dsRNA from the CMV promoter (pol II promoter), and this method is also applicable to tissue-specific pol II promoters. Similarly, the method by Xia et al., (2002) Nature Biotech. 20:1006 avoids poly(A) tailing and can be used in relation to tissue-specific promoters.

[0136] Methods for producing RNAi include chemosynthesis, in vitro transcription, digestion of long dsRNAs by a dicer (in vitro or in vivo), in vivo expression from a delivery vector, and in vivo expression from a PCR-derived RNAi expression cassette (see, for example, TechNotes 10(3) “Five Ways to Produce siRNAs” from Ambion, Inc. (Austin, Texas); available at www.ambion.com).

[0137] Guidelines for designing siRNA molecules are available (see, for example, literature from Ambion, Inc. in Austin, Texas; available at www.ambion.com). In certain embodiments, siRNA sequences have a G / C content of approximately 30–50%. Furthermore, when transcribing RNA using RNA polymerase III, long stretches greater than four T or A residues are generally avoided. Online siRNA target finders are available, for example, from Ambion, Inc. (www.ambion.com), through the Whitehead Institute of Biomedical Research (www.jura.wi.mit.edu), or from Dharmacon Research, Inc. (www.dharmacon.com).

[0138] The antisense region of the RNAi molecule may be perfectly complementary to the target sequence, but it does not have to be, as long as it specifically hybridizes to the target sequence (as defined above) and reduces the production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, hybridization of such oligonucleotides to the target sequence can be carried out under low, moderate, or even severe conditions as defined above.

[0139] In other embodiments, the RNAi antisense region has sequence identity with the complement of the target sequence of at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, or higher, and reduces the production of the protein product (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher). In some embodiments, the antisense region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches compared to the target sequence. Mismatches are generally more tolerable at the terminals of the dsRNA than in the central portion.

[0140] In certain embodiments, RNAi is formed by the formation of an intermolecular complex between two separate sense and antisense molecules. RNAi includes a ds region formed by intramolecular base pairing between two separate strands. In other embodiments, RNAi includes a ds region formed by intramolecular base pairing within a single nucleic acid molecule (e.g., shRNA or other stem-loop structure or cyclic RNAi molecule) that typically contains both sense and antisense regions as reverse repeats. RNAi may further include a spacer region between the sense and antisense regions.

[0141] Generally, RNAi molecules are highly selective. If desired, those skilled in the art can easily eliminate RNAi candidates that may interfere with the expression of non-target nucleic acids by searching relevant databases and identifying RNAi sequences that do not have significant sequence homology with other known sequences, for example, using BLAST (available at www.ncbi.nlm.nih.gov / BLAST).

[0142] Kits for RNAi production are commercially available from companies such as New England Biolabs and Ambion.

[0143] Furthermore, the recombinant viral vector may contain heterologous nucleotide sequences that share homology with and replace gene loci on the host chromosome. This method may be used to correct genetic defects in host cells.

[0144] The present invention also provides recombinant viral vectors that express immunogenic polypeptides, for example, for vaccination. Heterogeneous nucleic acids may encode any immunogen known in the art for any purpose, including, but not limited to, immunogens from human immunodeficiency virus, influenza virus, gag protein, tumor antigens, cancer antigens, bacterial antigens, viral antigens, etc. Alternatively, the immunogen may be provided encapsulated in a viral capsid (e.g., incorporated therein) or tethered to the viral capsid (e.g., by covalent modification).

[0145] The use of parvovirus as a vaccine is known in the art (see, for example, Miyamura et al., (1994) Proc. Nat. Acad. Sci. USA 91:8507; U.S. Patent No. 5,916,563 to Young et al., No. 5,905,040 and No. 5,882,652 to Mazzara et al., and No. 5,863,541 to Samulski et al.; the entirety of their disclosures is incorporated herein by reference). The antigen may be provided in a viral capsid. Alternatively, the antigen may be expressed from a heterologous nucleic acid introduced into a recombinant vector genome.

[0146] The immunogenic polypeptide, or immunogen, may be any polypeptide suitable for protecting the target from diseases such as microbial, bacterial, protozoan, parasitic, fungal, and viral diseases, but is not limited to these. For example, the immunogen may be an orthomyxovirus immunogen (e.g., influenza virus immunogen such as the influenza virus hemagglutinin (HA) surface protein or influenza virus nucleoprotein gene or equine influenza virus immunogen) or a lentivirus immunogen (e.g., equine infectious anemia virus immunogen, simian immunodeficiency virus (SIV) immunogen or human immunodeficiency virus (HIV) immunogen, e.g., HIV or SIV envelope GP160 protein, HIV or SIV matrix / capsid protein and HIV or SIV gag, pol and env gene products). The immunogen may also be an arenavirus immunogen (e.g., Lassa virus immunogen such as the Lassa virus nucleocapsid protein gene and the Lassa virus envelope glycoprotein gene), a poxvirus immunogen (e.g., vaccinia such as the vaccinia L1 or L8 gene), a flavivirus immunogen (e.g., yellow fever virus immunogen or Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., Ebola virus immunogen or Marburg virus immunogen such as the NP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF and SFS viruses), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen such as the human coronavirus envelope glycoprotein gene, or a porcine infectious gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen, or a severe acute respiratory syndrome (SARS) immunogen such as the S[S1 or S2], M, E or N protein or its immunogenic fragment). Furthermore, the immunogen may be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, B, or C) immunogen, or any other vaccine immunogen known in the art.

[0147] Alternatively, the immunogen may be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of cancer cells. Exemplary cancer and tumor cell antigens are described in SA Rosenberg, (1999) Immunity 10:281. Exemplary cancer and tumor antigens include, but are not limited to, BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, and melanoma tumor antigen (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res.54:3124), for example, MART-1 (Coulie et al., (1991) J.Exp.Med.180:35), gp100 (Wick et al., (1988) J.Cutan.Patel.4:201) and MAGE antigens (MAGE-1, MAGE-2 and MAGE-3) (Van der Bruggen et al., (1991) Science, 254:1643), CEA, TRP-1, TRP-2, P-15 and tyrosinase (Brichard et al., (1993) J.Exp.Med.178:489), HER-2 / neu gene product (US Patent No. 4,968,603), CA125, HE4, LK26, FB5 (endosialin), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, Span-1, CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2 protein, PSA, L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem.Examples include antigens associated with the following cancers: 62:623), mucin antigens (International Patent Application Publication No. 90 / 05142), telomerase, nuclear matrix proteins, prostatic acid phosphatase, papillomavirus antigens, and antigens related to the following cancers: melanoma, adenocarcinoma, thymoma, sarcoma, lung cancer, liver cancer, colorectal cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, renal cancer, gastric cancer, esophageal cancer, head and neck cancer, and others (see, for example, Rosenberg, (1996) Annu. Rev. Med. 47:481-91).

[0148] Alternatively, the heterologous nucleotide sequence may preferably encode any polypeptide produced in cells in vitro, ex vivo, or in vivo. For example, a viral vector may be introduced into cultured cells and expressed protein products isolated therefrom.

[0149] Those skilled in the art will understand that the desired heterologous polynucleotide may be operably associated with a suitable regulatory sequence. For example, heterologous nucleic acids may be operably associated with expression regulatory elements such as transcription / translation regulatory signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRESs), promoters, and enhancers.

[0150] Those skilled in the art will further understand that various promoter / enhancer elements can be used depending on the desired level and tissue-specific expression. The promoter / enhancer may be constitutive or inducible depending on the desired expression pattern. The promoter / enhancer may be constitutive or exogenous, and may be natural or synthetic sequences. "Exogenous" means that the transcription initiation region is not present in the wild-type host into which the transcription initiation region has been introduced.

[0151] The promoter / enhancer element may be innate to the target cell, treated, or / or innate to a heterologous nucleic acid sequence. The promoter / enhancer element is generally selected to function in the target cell of interest. In typical embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element may be constitutive or inducible.

[0152] Inducible expression regulatory elements are generally used in applications where it is desirable to regulate the expression of heterologous nucleic acid sequences. Inducible promoter / enhancer elements for gene delivery may be tissue-specific or tissue-preferred promoter / enhancer elements, and examples include muscle-specific or preferred (including cardiac muscle, skeletal muscle, and / or smooth muscle), nerve tissue-specific or preferred (including brain-specific), eye (including retina-specific and corneal-specific), liver-specific or preferred, bone marrow-specific or preferred, pancreas-specific or preferred, spleen-specific or preferred, and lung-specific or preferred promoter / enhancer elements. Other inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter / enhancer elements include, but are not limited to, Tet on / off elements, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, and metallothionein promoters.

[0153] In embodiments in which heterologous nucleic acid sequences are transcribed and subsequently translated into target cells, specific initiation signals are generally used for efficient translation of the inserted protein-coding sequences. These exogenous translational regulatory sequences, which may include an ATG start codon and adjacent sequences, may be of various origins and may be either natural or synthetic.

[0154] The present invention further provides a method for producing the viral vector of the present invention. In a typical embodiment, the present invention provides a method for producing a recombinant viral vector, comprising providing cells in vitro with (a) (i) a polynucleotide of interest, (ii) a template comprising a packaging signal sequence sufficient to capsidize the AAV template within a viral particle (e.g., one or more (e.g., two) terminal repeats, such as AAV terminal repeats), and (b) an AAV sequence sufficient to replicate the template and capsidize it within a viral particle (e.g., AAV Rep and AAV cap sequences). The template and AAV replication and capsid sequences are provided under conditions such that recombinant viral particles comprising the template packaged within a capsid are produced in the cell. The method may further include a step of recovering the viral particles from the cell. The viral particles may be recovered from the culture medium and / or by lysing the cell.

[0155] In one exemplary embodiment, the present invention provides a method for producing rAAV particles containing an AAV capsid, comprising the steps of: providing cells with a nucleic acid encoding an AAV capsid in vitro, an AAV Rep coding sequence, an AAV vector genome comprising a polynucleotide of choice, and a helper function for inducing a proliferative AAV infection; enabling the assembly of AAV particles containing the AAV capsid; and capsidizing the AAV vector genome.

[0156] The cells in question are typically cells that are tolerant to AAV virus replication. Any suitable cells known in the art, such as mammalian cells, may be used. Trans-complementary packaging cell lines that provide the function lost from replication-deficient helper viruses, such as 293 cells or other E1a trans-complementary cells, are also preferred.

[0157] The AAV replication and capsid sequences may be provided by any method known in the art. The current protocol typically expresses the AAV rep / cap gene on a single plasmid. The AAV replication and packaging sequences do not need to be provided together, but it may be convenient to do so. The AAV rep and / or cap sequences may be provided by any viral or nonviral vector. For example, the rep / cap sequence may be provided by a hybrid adenovirus or herpesvirus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus vector). Alternatively, the AAV cap and rep genes may be expressed using an EBV vector. One advantage of this method is that the EBV vector, while being an episome, still maintains a high copy number during successive cell divisions (i.e., it is stably incorporated into the cell as an extrachromosomal element referred to as an EBV-systemic nuclear episome).

[0158] As a further alternative, the rep / cap sequence may be stably maintained within the cell (either in the episome or integrated into it).

[0159] Typically, AAV rep / cap sequences are not adjacent to AAV packaging sequences (e.g., AAV ITRs) to prevent the rescue and / or packaging of these sequences.

[0160] The template (e.g., rAAV vector genome) can be delivered to cells using any method known in the art. For example, the template may be supplied by a non-viral (e.g., plasmid) or viral vector. In certain embodiments, the template is supplied by a herpesvirus or adenovirus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus). As another example, Palombo et al., (1998) J. Virol. 72:5025 describe a baculovirus vector carrying a reporter gene adjacent to the AAV ITR. Alternatively, the template may be delivered using an EBV vector with respect to the rep / cap gene as described above.

[0161] In another representative embodiment, a template is provided by a replicated rAAV virus. In yet another embodiment, an AAV provirus is stably incorporated into the chromosome of a cell.

[0162] To obtain the maximum viral titer, the cells are generally provided with the helper virus function (e.g., adenovirus or herpesvirus) essential for proliferative AAV infection. The helper virus sequences required for AAV replication are known in the art. Typically, these sequences are provided by helper adenovirus or herpesvirus vectors. Alternatively, the adenovirus or herpesvirus sequences can be provided by another non-viral or viral vector, for example, as a non-infectious adenovirus miniplasmid containing all the helper genes necessary for efficient AAV production, as described by Ferrari et al., (1997) Nature Med. 3:1295 and U.S. Patents 6,040,183 and 6,093,570.

[0163] Furthermore, helper virus function may be provided by packaging cells with the helper gene integrated into the chromosome or maintained as a stable extrachromosomal element. In a typical embodiment, the helper virus sequence cannot be packaged into the AAV virus particle and is not adjacent to, for example, the AAV ITR.

[0164] Those skilled in the art will understand that it may be advantageous to provide AAV replication and capsid sequences as well as helper virus sequences (e.g., adenovirus sequences) on a single helper construct. This helper construct may be a non-viral construct or a viral construct, or it may be a hybrid adenovirus or hybrid herpesvirus containing the AAV rep / cap gene.

[0165] In one particular embodiment, the AAV rep / cap sequence and the adenovirus helper sequence are supplied by a single adenovirus helper vector, which further includes an rAAV template. The AAV rep / cap sequence and / or the rAAV template may be inserted into an adenovirus deletion region (e.g., E1a or E3 region).

[0166] In further embodiments, the AAV rep / cap sequence and the adenovirus helper sequence are supplied by a single adenovirus helper vector. The rAAV template is provided as a plasmid template.

[0167] In another exemplary embodiment, the AAV rep / cap sequence and adenovirus helper sequence are provided by a single adenovirus helper vector, and the rAAV template is incorporated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector maintained intracellularly as an extrachromosomal element (see, for example, Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67) as an "EBV-system nuclear episome").

[0168] In further exemplary embodiments, the AAV rep / cap sequence and the adenovirus helper sequence are provided by a single adenovirus helper. The rAAV template is provided as a separate replicated viral vector. For example, the rAAV template may be provided by an rAAV particle or a second recombinant adenovirus particle.

[0169] According to the method described above, the hybrid adenovirus vector typically contains sufficient adenovirus 5' and 3' cis sequences (i.e., adenovirus terminal repeats and PAC sequences) for adenovirus replication and packaging. The AAV rep / cap sequence and, if present, the rAAV template are embedded within the adenovirus backbone and adjacent to the 5' and 3' cis sequences so that these sequences can be packaged within the adenovirus capsid. In the typical embodiments described above, the adenovirus helper sequence and the AAV rep / cap sequence are not adjacent to the AAV packaging sequence (e.g., AAV ITR) so that these sequences are not packaged within the AAV virus particle.

[0170] Herpesviruses can also be used as helper viruses in AAV packaging methods. Hybrid herpesviruses encoding AAV rep proteins can advantageously facilitate more scalable AAV vector production schemes. Hybrid herpes simplex virus type 1 (HSV-1) vectors expressing AAV-2 rep and cap genes are described (Conway et al., (1999) Gene Therapy 6:986 and International Publication No. 00 / 17377, the entirety of which disclosures are incorporated herein by reference).

[0171] As a further alternative, the viral vector of the present invention can be produced in insect cells using a baculovirus vector, as described by Urabe et al., (2002) Human Gene Therapy 13:1935-43, to deliver the rep / cap gene and rAAV template.

[0172] Another method for producing AAV is to use transformed packaging cells stably (see, for example, U.S. Patent No. 5,658,785).

[0173] AAV vector stocks free from helper virus contamination can be obtained by any method known in the art. For example, AAV and helper viruses can be easily identified based on size. AAV can also be isolated from helper viruses based on its affinity for heparin substrates (Zolotukhin et al., (1999) Gene Therapy 6:973). In typical embodiments, a deletion-deficient helper virus is used so that any contaminating helper virus is not replicable. As a further alternative, an adenovirus helper lacking late gene expression can be used, since only early adenovirus gene expression is required to mediate the packaging of the AAV virus. Adenovirus variants lacking late gene expression are known in the art (e.g., ts100K and ts149 adenovirus variants).

[0174] The packaging method of the present invention may be used to produce a high titer stock of virus particles. In certain embodiments, the virus stock is at least about 10 5 Transducing units (tu) / ml, at least about 10 6 tu / ml, at least about 10 7 tu / ml, at least about 10 8 tu / ml, at least about 10 9 tu / ml or at least about 10 10 It has a titer of tu / ml.

[0175] In certain embodiments, the present invention provides pharmaceutical compositions comprising the viral vector of the present invention and optionally other agents, pharmaceuticals, stabilizers, buffers, carriers, adjuvants, diluents, etc., in a pharmaceutically acceptable carrier. Injectable carriers may typically be liquid. Carriers for other administration methods may be solid or liquid. Carriers for inhalation administration are inhalable and preferably in solid or liquid particle form.

[0176] "Pharmacologically acceptable" means that a material is neither toxic nor otherwise undesirable; that is, it can be administered to a subject without causing any undesirable biological effects.

[0177] One aspect of the present invention is a method for transferring a target polynucleotide into cells in vitro. A viral vector can be introduced into cells with an appropriate degree of infection according to a standard transduction method suitable for specific target cells. The titer of the viral vector or capsid to be administered may vary depending on the target cell type and number, the specific viral vector or capsid, and can be determined by those skilled in the art without excessive experimental methods. In certain embodiments, at least about 10 3 Infectious units, more preferably at least about 10 5 The infectious units are introduced into the cells.

[0178] Cells into which a viral vector can be introduced include, but are not limited to, nerve cells (including cells of the peripheral and central nervous systems, particularly brain cells such as neurons, oligodendrocytes, glial cells, astrocytes, etc.), lung cells, eye cells (including retinal cells, retinal pigment epithelium, and corneal cells), epithelial cells (e.g., intestinal and respiratory epithelial cells), skeletal muscle cells (including myoblasts, myotubes, and muscle fibers), diaphragm muscle cells, dendritic cells, pancreatic cells (including islet cells), hepatocytes, gastrointestinal tract cells (including smooth muscle cells, epithelial cells), heart cells (including cardiomyocytes), bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, joint cells (including cartilage, meniscus, synovium, and bone marrow), germ cells, and the like. Alternatively, the cells may be any progenitor cells. As a further option, the cells may be stem cells (e.g., neural stem cells, liver stem cells). Still further, as an option, the cells may be cancer or tumor cells (cancers and tumors are described above). Further, the cells may be from any of the biological species shown above.

[0179] The viral vector may be introduced into the cells in vitro for administration to the modified cells. In certain embodiments, the cells are removed from the subject, the viral vector is introduced therein, and then the cells are returned into the subject. Methods for removing cells from the subject for ex vivo treatment and subsequent introduction into the subject are known in the art (see, e.g., U.S. Patent No. 5,399,346). Alternatively, a recombinant viral vector is introduced into cells from another subject, cultured cells, or cells from any other suitable source, and the cells are administered to the subject that requires them.

[0180] Cells suitable for ex vivo gene therapy are described above. The dosage of the cells for administration to the subject varies depending on the age, condition, and species of the subject, the type of cell, the nucleic acid expressed by the cell, the mode of administration, and the like. Typically, at least about 10 2 ~ about 108 or about 10 3 ~about 10 6 Individual cells are administered. In certain embodiments, cells transduced with a viral vector are administered to the subject in an effective amount combined with a pharmaceutical carrier.

[0181] In some embodiments, cells transduced with a viral vector may be administered to induce an immunogenic response to the delivered polypeptide (e.g., as a transgene or expressed within the capsid). Typically, a quantity of cells expressing an effective amount of polypeptide is administered in combination with a pharmaceutically acceptable carrier. Optionally, this dose is sufficient to produce a protective immune response (as defined above). The degree of protection provided does not need to be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any drawbacks.

[0182] A further aspect of the present invention is a method for administering the viral vector of the present invention to a target. In certain embodiments, the method includes a method for delivering the polynucleotide of interest to an animal target, and the method includes administering an effective amount of the viral vector according to the present invention to the animal target. Administration of the viral vector of the present invention to a human or animal subject requiring it may be by any means known in the art. Optionally, the viral vector may be delivered in an effective dose in a pharmaceutically acceptable carrier.

[0183] Furthermore, the viral vectors of the present invention can be administered to a target to induce an immunogenic response (e.g., as a vaccine). Typically, the vaccine of the present invention contains an effective amount of virus combined with a pharmaceutically acceptable carrier. Optionally, this dose is sufficient to produce a protective immune response (as defined above). The degree of protection provided does not need to be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any drawbacks. The target and immunogen are described above.

[0184] The dosage of the viral vector administered to the subject depends on the mode of administration, the disease or illness being treated, the individual subject's condition, the specific viral vector, and the nucleic acid being delivered, and can be determined in a routine manner. An exemplary dose to achieve a therapeutic effect is 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 , 10 17 , 10 18 Transduction units or more, preferably about 10 7 or 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 Trait introduction units, more preferably about 10 12 ~10 14 This is the viral titer of the transduction unit.

[0185] In certain embodiments, two or more doses (e.g., two, three, four or more doses) may be used to achieve the desired level of gene expression over various intervals, such as once a day, once a week, once a month, or once a year.

[0186] Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., by aerosol), oral (e.g., sublingual), vaginal, subarachnoid, intraocular, transdermal, intrauterine (or intraocular), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to the skeleton, diaphragm, and / or myocardium], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., administration to both skin and mucous membranes (including the airway surface) and transdermal), intralymphatic administration, and direct tissue or organ injection (e.g., injection into the liver, skeletal muscle, myocardium, diaphragmatic muscle, or brain). Administration may also be directed at tumors (e.g., in or near tumors or lymph nodes). The most preferred route in any given case depends on the nature and severity of the disease being treated and the properties of the specific vector being used.

[0187] Delivery to any of these tissues can also be achieved by delivering a depot agent containing a viral vector that can be embedded in these tissues, or by contacting these tissues with a film or other matrix containing the viral vector. Examples of such embeddable matrices or substrates are described in U.S. Patent No. 7,201,898.

[0188] The present invention can be used to treat diseases of tissues or organs. Alternatively, the present invention can be used to deliver nucleic acids to tissues or organs, for the production of protein products (e.g., enzymes) or non-coding RNA (e.g., RNAi, microRNA, antisense RNA) that circulate normally in the blood, or as a platform for systemic delivery to other tissues, to treat diseases (e.g., metabolic disorders such as diabetes (e.g., insulin), hemophilia (e.g., factor IX or factor VIII), or lysosomal storage disorders (e.g., Gaucher disease [glucocerebrosidase], Pompe disease [lysosomal α-glucosidase] or Fabry disease [α-galactosidase A]), or glycogen storage disorders (e.g., Pompe disease [lysosomal α-glucosidase]). Other suitable proteins for treating metabolic disorders are described above.

[0189] The injectable preparation can be prepared in any of the following forms: conventional form, liquid solution or suspension, solid form suitable for dissolution or suspension in liquid before injection, or emulsion. Alternatively, the viral vector may be administered locally rather than systemically, for example, in a depot or sustained-release formulation. Furthermore, the viral vector can be delivered in a dry state to surgically implantable matrices such as bone graft substitutes, sutures, and stents (see, for example, U.S. Patent No. 7,201,898).

[0190] Pharmaceutical compositions suitable for oral administration can be provided in individual units such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the composition of the present invention, as a powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion. Oral delivery can be achieved by complexing the viral vector of the present invention with a carrier that can withstand degradation by digestive enzymes in the intestines of animals. Examples of such carriers include plastic capsules or tablets as known in the art. Such formulations are prepared by any preferred compounding method, which includes the step of combining the composition with a suitable carrier (which may contain one or more minor components as described above). Generally, pharmaceutical compositions according to embodiments of the present invention are prepared by uniformly and densely mixing the composition with a liquid or finely powdered solid carrier or both, and then molding the resulting mixture if necessary. For example, tablets can be prepared by compressing or molding powder or granules containing the composition together with one or more minor components, optionally. Compressed tablets are prepared by compressing the composition, which is in a free-flowing form such as powder or granules mixed with a binder, lubricant, inert diluent and / or surfactant / dispersant, using a suitable machine. Molded tablets are prepared by molding a powder compound moistened with an inert liquid binder using a suitable machine.

[0191] Examples of pharmaceutical compositions suitable for oral (sublingual) administration include lozenges containing the composition of the present invention in a flavored base, usually sucrose and acacia or tragacanth, and scented tablets containing the composition in an inert base such as gelatin and glycerin or sucrose and acacia.

[0192] Pharmaceutical compositions suitable for parenteral administration may include sterile aqueous and non-aqueous injectable solutions of the compositions of the present invention, and these preparations may be isotonic with the blood of the intended recipient. These preparations may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the composition isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions, and emulsions may contain suspending agents and thickeners. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions (including saline and buffered media). Parenteral media include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's dextrose, or non-volatile oils. Intravenous media include fluids and nutritional replacement solutions, and electrolyte replacement solutions (such as those based on Ringer's dextrose). For example, preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, and inert gases may also be present.

[0193] This composition can be provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored under freeze-drying conditions requiring only the addition of a sterile liquid carrier, such as physiological saline or water for injection, immediately before use.

[0194] Immediate injection solutions and suspensions can be prepared from the sterile powders, granules, and tablets of the types described above. For example, the injectable, stable, sterile composition of the present invention can be provided as a unit dosage form in a sealed container. The composition can be provided in the form of a lyophilized product, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dosage form may be about 1 μg to about 10 grams of the composition of the present invention. If the composition is substantially water-insoluble, it can be emulsified in an aqueous carrier by including a sufficient amount of a physiologically acceptable emulsifier. One such useful emulsifier is phosphatidylcholine.

[0195] Pharmaceutical compositions suitable for rectal administration can be provided as unit-dose suppositories. These can be prepared by mixing the composition with one or more conventional solid carriers, such as cocoa butter, and then molding the resulting mixture.

[0196] The pharmaceutical compositions of the present invention, suitable for topical application to the skin, may be in the form of ointments, creams, lotions, pastes, gels, sprays, aerosols, or oils. Suitable carriers include, but are not limited to, petrolatum, lanolin, polyethylene glycol, alcohol, transdermal enhancers, and combinations of two or more thereof. In some embodiments, topical delivery can be achieved, for example, by mixing the pharmaceutical composition of the present invention with a lipophilic reagent (e.g., DMSO) that can penetrate the skin.

[0197] Pharmaceutical compositions suitable for transdermal administration may be in the form of individual patches configured to remain in close contact with the target epidermis for an extended period. Compositions suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., Pharm. Res. 3:318 (1986)) and may typically be in the form of an optionally buffered aqueous solution of the composition of the present invention. Preferred formulations may contain citrate or bis / tris buffer (pH 6) or ethanol / water and may contain 0.1 to 0.2 M of the active ingredient.

[0198] The viral vectors disclosed herein may be administered to the lungs of a subject by any suitable means, for example, by administering an aerosol suspension of inhalable particles consisting of the viral vector to be inhaled by the subject. The inhalable particles may be liquid or solid. Aerosols of liquid particles containing the viral vector may be generated by any suitable means, such as a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as known to those skilled in the art. See, for example, U.S. Patent No. 4,501,729. Aerosols of solid particles containing the viral vector may similarly be generated using any solid particulate medicinal aerosol generator by techniques known in the pharmaceutical industry.

[0199] Production of factor IX protein of the present invention Genetically modified cells can be produced using a variety of expression vectors. Several expression vectors can be designed to express large quantities of recombinant protein after amplifying transfected cells under various conditions favorable to selected high-expression cells. Several expression vectors can also be designed to express large quantities of recombinant protein without requiring amplification under selective pressure. This invention involves the production of genetically modified cells according to standard methods in the art and does not depend on the use of any specific expression vector or expression system.

[0200] To create genetically modified cells that produce large amounts of FIX protein, the cells are transfected with an expression vector containing the polynucleotide (e.g., cDNA) encoding the protein. In some embodiments, the FIX protein is expressed using a selected co-transfected enzyme that induces appropriate post-translational modifications of the FIX protein in a given cell line.

[0201] The cells in question may be selected from various sources, but they are also cells that can be transfected with an expression vector containing a nucleic acid molecule (e.g., cDNA) that encodes the FIX protein.

[0202] The implementation of this invention will utilize, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology within the scope of the art. Such techniques are fully described in the literature. For example, Sambrook et al., Molecular Cloning; A Laboratory Manual, 2nd edition (1989); DNA Cloning, Volumes I and II (edited by DN Glover, 1985); Oligonucleotide Synthesis (edited by MJ Gait, 1984); Nucleic Acid Hybridization (edited by B.D. Hames & S.J. Higgins, 1984); Transcription and Translation (edited by B.D. Hames & S.J. Higgins, 1984); Animal Cell Culture (edited by R.Freshney, 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); The Series, Methods in Enzymology (Academic Press, in particular volumes 154 and 155 (edited by Wu and Grossman and Wu, respectively); Gene Transfer Vectors for Mammalian Cells (JH Miller and MP)See also Calos (ed.), 1987, Cold Spring Harbor Laboratory; Immunochemical Methods in Cell and Molecular Biology, Mayer and Walker (eds.), Academic Press, London, 1987; Scopes, Protein Purification: Principles and Practice, 2nd edition, 1987 (Springer-Verlag, NY); and Handbook of Experimental Immunology, Volumes I-IV (eds., DMWeir and C.C.Blackwell, 1986). All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

[0203] Genetic engineering technology The production of cloned genes, recombinant DNA, vectors, transformed cells, proteins, and protein fragments by genetic engineering is well known. See, for example, U.S. Patent No. 4,761,371 to Bell et al., line 3 of column 6 to line 65 of column 9; U.S. Patent No. 4,877,729 to Clark et al., line 38 of column 4 to line 6 of column 7; U.S. Patent No. 4,912,038 to Schilling, line 26 of column 3 to line 12 of column 14; and U.S. Patent No. 4,879,224 to Wallner, line 8 of column 6 to line 59 of column 8.

[0204] A vector is a replicable DNA construct. Vectors are used here to amplify and / or express nucleic acids encoding the FIX protein. An expression vector is a replicable nucleic acid construct in which a nucleotide sequence encoding the FIX protein is operably linked to a suitable regulatory sequence that influences the expression of the nucleotide sequence to produce the FIX protein in a suitable host cell. The need for such a regulatory sequence depends on the selected host cell and the chosen conversion method. Common regulatory sequences include transcription promoters, arbitrary operator sequences for controlling transcription, sequences encoding a suitable mRNA-ribosome binding site, and sequences that control the termination of transcription and translation.

[0205] Vectors include plasmids, viruses (e.g., AAV, adenovirus, cytomegalovirus), phages, and embeddable DNA fragments (i.e., fragments that can be recombinatively incorporated into the host cell genome). Vectors can replicate and function independently of the host cell genome (e.g., by transient expression) or can be incorporated into the host cell genome itself (e.g., stable integration). Expression vectors may include promoters and RNA-binding sites that are operably ligated to the nucleic acid molecule to be expressed, and are operable in host cells and / or organisms.

[0206] DNA regions or nucleotide sequences are operably linked or operably associated if they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of that sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned to enable the translation of that sequence.

[0207] Suitable host cells include prokaryotes, yeast, or higher eukaryotic cells such as mammalian and insect cells. Cells derived from multicellular organisms are particularly suitable hosts for recombinant FIX protein synthesis, with mammalian cells being especially preferred. The proliferation of such cells in cell culture is a routine procedure (Tissue Culture, Academic Press, Kruse and Patterson (1973)). Examples of useful host cell lines include VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, HEK293, BHK, COS-7, CV, and MDCK cell lines. Expression vectors for such cells typically include a promoter located upstream of the nucleotide sequence encoding the FIX protein to be expressed, along with a replication origin, ribosome binding site (if necessary), a promoter operably linked thereto, an RNA splice site (if genomic DNA containing introns is used), a polyadenylation site, and a transcription termination sequence. In one embodiment, expression can be performed in Chinese hamster ovary (CHO) cells using the expression system described in U.S. Patent No. 5,888,809 (the entire disclosure of which is incorporated herein by reference).

[0208] Transcriptional and translational regulatory sequences in expression vectors used to transform vertebrate cells are often provided by viral sources. Non-exclusive examples include promoters derived from polyomas, adenovirus 2, and simian virus 40 (SV40). See, for example, U.S. Patent No. 4,599,308.

[0209] The origin of replication may be provided by constructing a vector that includes an exogenous origin, such as SV40 or another viral source (e.g., polyoma, adenovirus, VSV, or BPV), or by the host cell chromosome replication mechanism. When the vector is incorporated into the host cell chromosome, the latter is often sufficient.

[0210] Mammalian cells can be transformed by cotransfection using nucleic acid molecules encoding a selectable marker and a FIX protein, rather than using a vector containing a viral origin of replication. Non-limiting examples of suitable selectable markers include dihydrofolate reductase (DHFR) or thymidine kinase. This method is further described in U.S. Patent No. 4,399,216, which is incorporated herein by reference in its entirety.

[0211] Other methods suitable for adapting the synthesis of FIX protein in culture of recombinant vertebrate cells include those described in Gething et al. Nature 293:620 (1981); Mantei et al. Nature 281:40; and Levinson et al., EPO applications 117,060A and 117,058A, the entire contents of which are incorporated herein by reference.

[0212] Host cells such as insect cells (e.g., cultured armyworm cells) and expression vectors such as baculovirus expression vectors (e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be used when carrying out the present invention as described in U.S. Patents 4,745,051 and 4,879,236 to Smith et al. Generally, a baculovirus expression vector contains a baculovirus genome that includes an expressed nucleotide sequence inserted into the polyhedrin gene at a position from the polyhedrin transcription initiation signal to the ATG start site, under the transcriptional control of a baculovirus polyhedrin promoter.

[0213] Prokaryotic host cells include Gram-negative or Gram-positive organisms, such as Escherichia coli (E. coli) or bacilli, respectively. Higher eukaryotic cells include established mammalian cell lines as described herein. Exemplary bacterial host cells are Escherichia coli W3110 (ATCC 27,325), Escherichia coli B, Escherichia coli X1776 (ATCC 31,537), and Escherichia coli 294 (ATCC 31,446). A wide range of suitable prokaryotic and microbial vectors are available. Escherichia coli is typically transformed using pBR322. The most commonly used promoters in recombinant microbial expression vectors include the β-lactamase (penicillinase) and lactose promoter system (Chang et al. Nature 275:615 (1978); and Goeddel et al. Nature 281:544 (1979)), the tryptophan (trp) promoter system (Goeddel et al. Nucleic Acids Res. 8:4057 (1980) and EPO Publication No. 36,776), and the tac promoter (De Boer et al. Proc.Natl.Acad.Sci.USA 80:21 (1983)). These promoters and the Shine-Dalgano sequence (for prokaryotic host expression) are operably ligated to nucleic acids encoding FIX proteins; that is, they are positioned to promote the transcription of FIX messenger RNA from DNA.

[0214] Eukaryotic microorganisms, such as yeast cultures, may also be transformed with protein-coding vectors (see, for example, U.S. Patent No. 4,745,057). Budding yeast is the most commonly used lower eukaryotic host microorganism, but many other strains are generally available. Yeast vectors may contain a replication origin from a 2-micron yeast plasmid or self-replicating sequence (ARS), a promoter, nucleic acid encoding a FIX protein, sequences for polyadenylation and transcription termination, and a select gene. An exemplary plasmid is YRp7 (Stinchcomb et al. Nature 282:39 (1979); Kingsman et al. Gene 7:141 (1979); Tschemper et al. Gene 10:157 (1980)). Suitable promoter sequences in yeast vectors include promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem. 255:2073 (1980)), or other glycoseptic enzymes (Hess et al. J. Adv. Enzyme Reg. 7:149 (1968); and Holland et al. Biochemistry 17:4900 (1978)). Suitable vectors and promoters for yeast expression are further described in R. Hitzeman et al., EPO Publication No. 73,657.

[0215] The cloning coding sequences of the present invention may encode FIX from any species, including mice, rats, dogs, opossums, rabbits, cats, pigs, horses, sheep, cattle, guinea pigs, platypuses, and humans, but preferably human-derived FIX proteins. This also includes nucleic acids encoding FIX that can hybridize with the nucleic acids encoding the proteins disclosed herein. Hybridization of such sequences may be performed on nucleic acids encoding the FIX proteins disclosed herein under low or even harsh conditions in a standard in situ hybridization assay (e.g., harsh conditions represented by wash stringency of 0.3 M NaCl, 0.03 M sodium citrate, and 0.1% SDS at 60°C or even 70°C). See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd edition, 1989), Cold Spring Harbor Laboratory.

[0216] The FIX protein produced according to the present invention may be expressed in transgenic animals by known methods. See, for example, U.S. Patent No. 6,344,596, the entire disclosure of which is incorporated herein by reference. In short, transgenic animals include, but are not limited to, livestock (e.g., pigs, goats, sheep, cattle, horses, and rabbits), rodents (e.g., mice, rats, and guinea pigs), and domestic pets (e.g., cats and dogs). Livestock animals such as pigs, sheep, goats, and cattle are particularly preferred in some embodiments.

[0217] The transgenic animals of the present invention are prepared by introducing a suitable polynucleotide encoding the human FIX protein of the present invention into a single-cell embryo such that the polynucleotide is stably incorporated into the DNA of the germline cells of a mature animal and inherited according to normal Mendelian laws. The transgenic animals of the present invention have a phenotype of producing the FIX protein in body fluids and / or tissues. The FIX protein is extracted from these fluids and / or tissues and processed, for example, for therapeutic use. (For example, see Clark et al., “Expression of human anti-hemophilic factor IX in the milk of transgenic sheep,” Bio / Technology 7:487-492 (1989); and Van Cott et al., “Haemophilic factors produced by transgenic livestock: abundance can enable alternative therapies worldwide,” Haemophilia 10(4):70-77 (2004), the entire contents of which are incorporated herein by reference).

[0218] DNA molecules can be introduced into embryos by various means, including, but not limited to, microinjection, calcium phosphate-mediated precipitation, liposome fusion, or retroviral infection of totipotent or pluripotent stem cells. Transformed cells can then be introduced into embryos and incorporated therein to create transgenic animals. Methods for creating transgenic animals are described, for example, in "Transgenic Animal Generation and Use" by L.M. Houdebine, Harwood Academic Press, 1997. Transgenic animals can also be created using nuclear transfer or cloning methods with embryos or mature cell lines, as described, for example, in Campbell et al., Nature 380:64-66 (1996) and Wilmut et al., Nature 385:810-813 (1997). Furthermore, techniques utilizing intracytoplasmic injection of DNA can be used, as described in U.S. Patent No. 5,523,222.

[0219] FIX-producing transgenic animals can be obtained by introducing a chimeric construct containing the FIX coding sequence. Methods for obtaining transgenic animals are well known. For example, Hogan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Press 1986); Krimpenfort et al., Bio / Technology 9:88 (1991); Palmiter et al., Cell 41:343 (1985); Kraemer et al., Genetic Manipulation of the Early Mammalian Embryo (Cold Spring Harbor Laboratory Press 1985); Hammer et al., Nature See 315:680 (1985); Wagner et al., U.S. Patent No. 5,175,385; Krimpenfort et al., U.S. Patent No. 5,175,384; Janne et al., Ann. Med. 24:273 (1992); Brem et al., Chim. Oggi. 11:21 (1993); Clark et al., U.S. Patent No. 5,476,995, the entirety of which these disclosures are incorporated herein by reference.

[0220] In some embodiments, a cis-acting regulatory region that is “active” in mammary tissue in the sense that the promoter is more active in mammary tissue than in other tissues under the physiological conditions under which milk is synthesized may be used. Such promoters include, but are not limited to, short and long whey acid protein (WAP), short and long α, β, and κ casein, α-lactalbumin, and β-lactoglobulin ("BLG") promoters. Signal sequences according to the present invention that lead to the secretion of expressed proteins into other bodily fluids, particularly blood and urine, may also be used. Examples of such sequences include signal peptides of secreted coagulation factors such as FIX, protein C, and tissue plasminogen activator.

[0221] Useful sequences that regulate transcription include, in addition to the promoters discussed above, enhancers, splice signals, transcription termination signals, polyadenylation sites, buffering sequences, RNA processing sequences, and other sequences that regulate transgene expression.

[0222] Preferably, the expression system or construct includes a 3' untranslated region downstream of the nucleotide sequence encoding the desired recombinant protein. This region can increase the expression of the transgene. Among the 3' untranslated regions useful in this regard are sequences that provide a polyA signal.

[0223] Suitable heterologous 3' untranslated sequences may be derived, for example, from the SV40 small t antigen, the casein 3' untranslated region, or other 3' untranslated sequences well known in the art. Ribosome binding sites are also important for enhancing the efficiency of FIX expression. Similarly, sequences that modulate post-translational modifications of FIX are useful in the present invention.

[0224] Having described the present invention, it will be described in further detail in the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention. [Examples]

[0225] Example 1: Construction of a gene expression cassette To enhance the efficiency and lifespan of human factor IX gene expression, the inventors have made considerable efforts to achieve the following goals: 1) obtaining liver specificity and potent activity by synthesizing multiple designer promoters; 2) improving the efficiency of transgene mRNA processing by using small introns after the promoter and by novelly inserting a second small intron into the protein-coding region of the gene; 3) improving translation efficiency for protein synthesis of the transgene product by optimizing the 5' untranslated sequence for reduced secondary structure; 4) optimizing human codon usage frequency, reducing CpG motifs, and reducing long G and C tracks; and 5) using bidirectional polyadenylated sequences for efficient poly(A) synthesis and blocking residual antisense promoter activity from the 3' inverted terminal repeat (ITR) of AAV.

[0226] The inventors designed and fully synthesized many synthetic artificial promoters containing conserved basic promoter elements and transcription initiation sites. The basic promoter was ligated at its 5' end to numerous liver-specific transcription factor binding sites for liver-specific expression. Due to its small size (188 bp) and high activity, initially screened in vitro using transfection experiments with a luciferase reporter gene and the human hepatocellular carcinoma cell line Huh7 (Table 4), the promoter was selected and named LXP2.1 (Figure 1) (SEQ ID NO: 1). The inventors further investigated the LXP2.1 promoter in vivo with a ubiquitous and potent CMV promoter after packaging a Gaussian luciferase expression cassette within AAV8 virus particles and injecting it into the mouse tail vein. 5 × 10⁻⁶ 10 Luciferase expression was assayed using serum collected from mice two weeks after IV injection of a vector genome (vg) / mouse (Table 5). The LXP2.1 promoter was approximately four times stronger than the CMV promoter. [Table 5] [Table 6]

[0227] To enhance the expression of factor IX gene in vivo, the inventors employed two methods to achieve this goal. First, with the aim of maximizing the frequency of more effective codon use, the inventors completely synthesized the coding sequence of the human factor gene using the GeneArt (Invitrogen) human codon optimization program. The inventors also attempted to reduce or remove all CpG sequences within the synthesized factor IX gene. It has been demonstrated that CpG islands or motifs within the gene in question can induce an innate immune response (e.g., Toll-like receptor 9 (TLR9)-mediated immune response (Bauer et al., Proc. Natl. Acad. Sci. USA 98(16):9237 (2001))) and potentially cause gene silencing. In factor IX gene-1 (sequence number 6) and factor IX gene-2 ​​(sequence number 7), the CpG motif was completely removed. However, in factor IX gene-3 (sequence number 8), only the TCG and CGT sequences were removed. In human cells, the conserved CpG motif, namely GTCGTT, has been reported to be the most potent motif for inducing the TLR9 response. Removal of the TCG and CGT motifs effectively disabled the GTCGTT element.

[0228] Introns have been well demonstrated to exert their functions by facilitating mRNA precursor processing and enhancing gene expression. The native introns of the human factor IX gene are relatively large and unsuitable for our gene expression cassette. In attempting to further increase factor IX gene expression, we synthesized small human introns (SEQ ID NOs: 4 and 5). We inserted the first intron into an artificial 5' untranslated region (5'UTR) between AAG and G (SEQ ID NOs: 4). We inserted the second intron (SEQ ID NOs: 5) into the 5' coding region of the gene between nucleotides CAG and G, i.e., the consensus exon / intron junction site. The complete DNA sequences of the three genes with the insertion of the artificial intron (SEQ ID NOs: 5) are listed as SEQ ID NOs: 6, 7, and 8, respectively. Although we did not investigate the insertion of artificial intron 1 into the coding sequence of the factor IX gene and / or intron 2 into the 5'UTR, we predict that similar results can be obtained.

[0229] Example 2: In vitro expression of a gene expression cassette To investigate whether the designer human factor IX gene expression cassette functions in cells, the inventors transfected the expression cassette into Huh7 cell lines. Huh7 is often used to study promoters that exhibit activity in hepatocytes. Since Huh7 cells do not produce any endogenous coagulation factor IX, untransfected cells were used as a negative control. The purpose of this experiment was to confirm whether the inventors' novel factor IX constructs could produce extracellularly secreted functional factor proteins into the cell culture medium in human cells. Twenty-four hours after transfection, the cell culture medium was replaced with serum-free medium and cultured for another 24 hours. Subsequently, the cell culture medium was collected and subjected to the APTT test, a coagulation activity test commonly used in vitro. The inventors' results showed that all of the inventors' constructs expressed and secreted high levels of factor IX protein (Figure 3). Confirmation of the functionality of the inventors' gene expression cassette prompted them to conduct in vivo gene expression experiments in hemophilia B mice, a clinically relevant animal model.

[0230] Example 3: In vivo expression of a gene expression cassette In vitro cell culture transfection experiments have shown that artificial intron-2 (SEQ ID NO:) 5Since the gene expression cassette containing codon-optimized human factor IX gene-1, including ), was suggested to be more potent, we selected this construct packaged in the AAV8 serotype vector, a robust liver-directed AAV vector in mouse liver for high-level expression, adjacent to an AAV inverted terminal repeat (ITR). To investigate whether codon-optimized human factor IX gene-1 functions in other serotypes of the AAV vector, we packaged it in a novel AAV vector together with an engineered capsid (AAVXL14). This vector was purified by double CsCl density gradient ultracentrifugation, dialyzed with physiological saline, and titer was measured by DNA dot blotting and AAV capsid protein silver staining after PAGE gel separation. Simultaneously, a previously reported human factor IX gene expression cassette, named here F9-Zwu (Wu et al., Mol. Ther. 16(2):280 (2008)), was packaged in the AAV8 vector as a positive control. The cassette contained a liver-specific TTR promoter and a different codon-optimized human factor IX gene, but both had the same amino acid R338L mutation (the Padua mutation; Simioni et al., N.Engl.J.Med361(17):1671(2009)).

[0231] To investigate how efficiently the factor IX expression cassette functions in vivo, the inventors chose to use 6-week-old male factor IX gene knockout mice (a commonly used hemophilia B animal model). The aforementioned vector was administered at four different doses, namely 1 × 10⁶ 10 vg / kg (vector genome / kilogram body weight), 4 × 10 10 vg / kg, 1 × 10 11 vg / kg and 4×10 11Factor IX knockout mice were intravenously injected via the tail vein at vg / kg (Table 4). Untreated, age- and sex-matched factor IX knockout mice were used as negative controls. Plasma was collected every two weeks from the retroorbital vein using a standard protocol. Plasma samples were frozen at -80°C for further testing. To quantitatively evaluate human factor IX gene expression, plasma samples were subjected to a routine APTT test using purified recombinant human factor IX serially diluted in plasma from patients with severe hemophilia B, as a standard curve for factor IX activity.

[0232] Our results showed that all AAV vectors expressed human factor IX in KO mice (Table 6). However, our FIX gene-1 expression cassette was significantly more efficient than the previously reported positive control vector F9-ZWu (Wu et al., Mol. Ther. 16(2):280 (2008)). For example, 1 × 10 11 vg / kg and 4×10 11 At a vector dose of vg / kg, our vector achieved expression levels at approximately 25 and 34 hours that were comparable to those of the positive control vector. 1 × 10 10 At vector doses as low as vg / kg body weight, our vector was able to achieve approximately 140% of the normal physiological level of human factor activity. Furthermore, human factor gene expression remained stable at all tested doses for up to 20 weeks, the duration of the in vivo experiment (Figure 4 and Table 7). Thus, our results demonstrate that the gene expression is not only robust but also long-lasting. Sustained high levels of FIX-1 gene product expression in FIX KO mice did not cause any discernible adverse effects. [Table 7] Factor IX activity was measured two weeks after vector injection (n=7). Factor IX activity is shown as a percentage of normal human plasma concentrations. [Table 8] Factor IX activity is shown as a percentage of normal human plasma concentrations.

[0233] Those skilled in the art will understand that numerous modifications can be made without departing from the spirit of the present invention. Therefore, it should be clearly understood that the embodiments of the present invention are merely illustrative and not intended to limit the scope of the invention.

[0234] All publications, patent applications, patents, patent publications, sequences identified by their Genbank® database access numbers, and other references cited herein are incorporated by reference in their entirety for the purpose of teaching about the sentences and / or paragraphs to which they are referenced.

[0235] The above is illustrative of the present invention and should not be construed as limiting it. The present invention, along with equivalents of the claims contained herein, is defined by the following claims.

Claims

1. A polynucleotide encoding human factor IX, with codons optimized for expression in humans, The aforementioned polynucleotide contains synthetic introns, The polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6, Polynucleotide.

2. The polynucleotide according to claim 1, further comprising a promoter.

3. The polynucleotide according to claim 2, wherein the promoter is a synthetic liver-specific promoter comprising the nucleotide sequence of SEQ ID NO: 1 or a sequence identical to SEQ ID NO: 1 by at least 97%.

4. A vector comprising a polynucleotide according to any one of claims 1 to 3.

5. The vector according to claim 4, wherein the vector is a viral vector.

6. The vector according to claim 5, wherein the vector is an adeno-associated virus (AAV) vector.

7. The vector according to claim 6, wherein the AAV vector is an AAV8 vector or an AAV9 vector.

8. A transformed cell comprising a polynucleotide according to any one of claims 1 to 3 and / or a vector according to any one of claims 4 to 7.

9. A transgenic non-human animal comprising a polynucleotide according to any one of claims 1 to 3, a vector according to any one of claims 4 to 7, and / or a transformed cell according to claim 8.

10. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 3, a vector according to any one of claims 4 to 7 and / or a transformed cell according to claim 8, and a pharmaceutically acceptable carrier.

11. A composition for use in a method for producing factor IX in a target liver, comprising a polynucleotide according to any one of claims 1 to 3, a vector according to any one of claims 4 to 7, and / or transformed cells according to claim 8.

12. A composition for use in a method for treating hemophilia B or acquired factor IX deficiency in a subject, comprising a polynucleotide according to any one of claims 1 to 3, a vector according to any one of claims 4 to 7, and / or transformed cells according to claim 8.

13. A composition for use in a method for enhancing the bioavailability of factor IX polypeptide in a subject, comprising a polynucleotide according to any one of claims 1 to 3, a vector according to any one of claims 4 to 7, and / or transformed cells according to claim 8.

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