Modified Factor IX and Compositions, Methods and Uses for Gene Transfer into Cells, Organs and Tissues

Abstract

To provide nucleic acid sequences, expression vectors (e.g., vector genomes) and plasmids, compositions and viral vectors in which the nucleic acid encodes Factor IX (e.g. human Factor IX).SOLUTION: A nucleic acid sequence encodes human Factor IX protein, where the nucleic acid has a reduced number of CpG di-nucleotides compared to a wild-type sequence encoding human Factor IX.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This patent application is a continuation of U.S. patent application Ser. No. 62 / 183,555, filed June 23, 2015. No. 99, U.S. Patent Application No. 62 / 315,453, filed March 30, 2016, 2 U.S. Patent Application No. 62 / 338,315, filed May 18, 2016, No. 62 / 348,781 filed on June 10, 2016, and U.S. Patent Application No. 62 / 348,781 filed on June 1, 2016. This application claims the benefit of U.S. Patent Application No. 62 / 349,572, filed on the 3rd of this application. The entire application is expressly incorporated herein by reference. Introduction

[0002] Desirable gene deletion or deficiency (loss of function) or undesirable gene or Genetic disorders caused by the expression of defective genes (gain of function) can lead to a variety of diseases. An example of a genetic disorder in which clotting factor VIII (FVIII, hemophilia A) or factor IX (FVIII) is lost is Hemophilia is an inherited bleeding disorder caused by a deficiency of one of the following genes: IX, hemophilia B An example of a gain-of-function genetic disorder is the accumulation of ATP, particularly in the basal ganglia and cerebral cortex, leading to the slow degeneration of neurons. The pathological "HTT" gene (huntingtin protein) encodes a mutant protein that leads to the destruction of various The most common cause of Huntington's disease is the mitochondrial leukemia (MLE) which encodes the mitochondrial protein ATP.

[0003] Current treatment for hemophilia involves recombinant coagulants, either as needed when bleeding occurs or prophylactically. However, this therapeutic approach is difficult to achieve with repeated The need for repeated infusions, the cost of treatment, the risk of developing an anti-treatment factor immune response, and the potential These limitations make it difficult to administer steroids to patients with hemophilia. This has prompted the development of gene-based therapies. To this end, hemophilia is characterized by: 1) therapeutic concentrations; The range is very wide, and levels above 1% of normal already change the phenotype from severe to moderate. 100% levels are not associated with any side effects, and 2) tissue specificity of the therapeutic transgene is maintained. 3) there is a lot of experience in measuring endpoints of therapeutic efficacy. It is ideal for gene transfer-based therapy.

[0004] Currently, adeno-associated virus (AAV) vectors are the most popular vectors for in vivo gene delivery. It has a good safety and efficacy profile and is therefore considered an ideal gene transfer vector. Of the AAV serotypes isolated to date, AAV2 and AAV8 are the most common causes of severe HIV infection. It has been used to target the liver in humans with hemophilia B.

[0005] The present invention relates to nucleic acid sequences, expression vectors (e.g., vector genomes) and plasmids, compositions and viral vectors, the nucleic acid of which encodes factor IX (e.g., human factor IX). Nucleic acids encoding factor IX are provided. modified to reduce the number of CpG (cytosine-guanine) dinucleotides compared to the In a particular embodiment, the modified nucleic acid encoding Factor IX is human Factor IX. A reduced number of CpG dinucleotides compared to the wild-type or native sequence encoding Factor X. It has a

[0006] FIX modified to reduce the number of CpG (cytosine-guanine) dinucleotides Such modified nucleic acids encoding Factor IX may be contained in a vector, such as a viral vector. Representative viral vectors may be used to transfect, for example, the liver, among other cell types. Cell-targeted lentiviral and parvoviral vectors (e.g., adenoviral vectors or adeno-associated virus (AAV) vectors). As a delivery vector, AAV vectors express polynucleotides in cells. Polynucleotides encoding proteins, such as modified nucleic acids encoding factor IX, After administration, the leutide can optionally be expressed at therapeutic levels.

[0007] Thus, the vector genome includes a modified nucleic acid encoding Factor IX (capsules). Recombinant AAV vectors (encapsidated with a nucleotide) are provided. In certain embodiments, Therefore, recombinant AAV particles encapsidate or encase the vector genome. Such recombinant AAV particles of the invention may contain heterologous polynucleotide sequences (e.g., , FIX modified to reduce the number of CpG (cytosine-guanine) dinucleotides, etc. a viral vector genome containing a modified nucleic acid encoding factor IX, such as In one embodiment, the number of CpG (cytosine-guanine) dinucleotides is reduced. A vector containing a modified nucleic acid encoding factor IX, such as a FIX modified as follows: The genome is encapsidated by an AAV capsid or an AAV capsid mutant. It is wrapped or enclosed.

[0008] In the recombinant AAV vectors of the present invention, the heterologous polynucleotide sequence is transcribed In various embodiments, the heterologous polynucleotide can be synthesized and subsequently translated into a protein. The nucleotide sequence encodes a therapeutic protein. In particular embodiments, the protein is a therapeutic protein. Fluid coagulation factors (e.g., factor IX, factor XIII, factor X, factor VIII, factor VII) In a further specific embodiment, the vector is Factor IX. Modified nucleic acids (e.g., CpG (cytosine-guanine) dinucleotides) encoding the factor a modified nucleic acid encoding factor IX, such as factor IX modified to reduce the number of Includes:

[0009] AAV and variants of AAV, such as capsid variants, provide desirable or therapeutic benefits. It is possible to deliver polynucleotides and / or proteins that inhibit the growth of various diseases. Treating diseases. For example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 , AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 and variants thereof, as well as AAV capsid variants (e.g., 4-1) is a method for transducing therapeutic genes (e.g., factor IX) for treating hemophilia A, B, etc., into cells. It is a useful vector for delivery to cells, tissues and organs.

[0010] The present invention includes (encapsidates, packages) vector genomes (viruses or AAVs). Recombinant viruses and AAV vectors contain additional factors that function in cis or trans. In certain embodiments, the vector genome is contained (encapsidated, packaged). Recombinant viral (e.g., AAV) vectors may further comprise: (e.g., CpG (cytosine Factor IX, such as FIX, modified to reduce the number of (guanine-guanine) dinucleotides adjacent to the 5' or 3' end of the heterologous polynucleotide sequence one or more inverted terminal repeat (ITR) sequences that Such heterologous polynucleotide sequences (e.g., CpG (cytosine-guanine) nucleotides) Modified factor IX, such as FIX, modified to reduce the number of peptides an expression control element (e.g., a promoter or enhancer) that causes transcription of the nucleic acid Tissue-specific expression regulators; intron sequences, stuffer or filler polynucleotides and / or a polyadenylation sequence located 3' of the heterologous polynucleotide sequence. It has.

[0011] Thus, vectors may further contain introns, expression control elements (e.g., structural elements or is a controllable regulatory element, or a tissue-specific expression regulatory element or promoter (e.g., Human α1-antitrypsin (hAAT) promoter and / or apolipoprotein E ( ApoE) HCR-1 and / or HCR-2 enhancers for hepatic expression )), one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) (e.g., A AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, A AAV serum of AV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 and / or filler polynucleotide sequences. The location of such additional factors may vary. The gene is present in the sequence encoding the human FIX protein, and / or the expression control element is operably linked to a sequence encoding a human FIX protein, and / or The TR is adjacent to the 5' or 3' of the sequence encoding the human FIX protein and / or The end of the filler polynucleotide sequence is 5' of the sequence encoding the human FIX protein. Adjacent to the end or 3' end.

[0012] In some embodiments, the number of CpG (cytosine-guanine) dinucleotides is reduced. The FIX nucleic acid modified to resemble the native or wild-type sequence encoding human factor IX. 1 to 5 fewer, 5 to 10 fewer, 10 to 15 fewer, 15 to 20 fewer than the array Fewer, 20-25 fewer, 25-30 fewer, 30-40 fewer, 40-55 Fewer, 55-75 fewer, 75-100 fewer, 100-150 fewer, 150 In particular embodiments, the present invention may have 200 fewer CpG dinucleotides. The modified FIX nucleic acids described herein have a reduced number of CpG dinucleotides. The wild-type or naturally occurring sequence encoding human factor IX is not present in the present invention. Encodes the human FIX protein expressed in the bell

[0013] In further embodiments, introns, expression control elements (e.g., structural elements or Regulatory elements, or tissue-specific expression regulators or promoters (e.g., human human alpha 1-antitrypsin (hAAT) promoter and / or apolipoprotein E (A poE) HCR-1 and / or HCR-2 enhancers for hepatic expression ), one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) (e.g., , AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 , AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 The ITR sequences of any of the serotypes) and / or filler polynucleotide sequences are equivalent The native or wild-type expression regulator of adeno-associated virus (AAV) inverted terminal repeats (IT R), and / or CpG (cytosine-guanine) compared to the filler polynucleotide sequence In particular embodiments, the amino acid sequence may be modified to reduce the number of amino acid dinucleotides. expression control elements, adeno-associated virus (AAV) inverted terminal repeats (ITRs), and / or The filler polynucleotide sequence may be 1 to 5 less than the native or wild-type equivalent sequence. ~10 fewer, 10-15 fewer, 15-20 fewer, 20-25 fewer, 25 ~30 fewer, 30-40 fewer, 40-55 fewer, 55-75 fewer, 75 -100 fewer, 100-150 fewer, 150-200 fewer CpG dinucleotides It has chid.

[0014] Typical AAV vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AAV26, AV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8, or AAV1, AAV2 , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV1 0, AAV11, Rh10, Rh74, or AAV-2i8 capsid mutants The recombinant AAV particles of the present invention are also AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV22, AAV33, AAV44, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10 , Rh74, or AAV-2i8, as well as variants thereof. Capsid variants include capsid sequences with amino acid substitutions, deletions, or insertions / additions. , AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8 In particular embodiments, the substitution is in the Rh74 VP1 capsid sequence ( In SEQ ID NO: 1, for example, any of amino acid positions 195, 199, 201, and 202 In a further particular embodiment, the substituted residue is Rh74 V at any one of amino acid positions 195, 199, 201, or 202 of the P1 capsid sequence In a more particular embodiment, the capsid corresponds to an amino acid A, V, P, or N in The sequence is: A residue at amino acid position 195 of the Rh74 VP1 capsid sequence; V residue at amino acid position 199, P residue at amino acid position 201, or N residue at amino acid position 202 In a further specific embodiment, the capsid sequence is the Rh74 VP1 capsid sequence. The A residue at amino acid position 195, the V residue at amino acid position 199, and the V residue at amino acid position 201 P residue or any two, three, or all four of the N residues at amino acid position 202 do.

[0015] In further specific embodiments, the capsid mutant comprises any of SEQ ID NOs: 4-9 In a further embodiment, the AAV vector comprises any VP1 of any AAV serotype, AAV VPs with 90% or greater sequence identity to VP2 and / or VP3 VP1, VP2 and / or VP3 sequences. In the present invention, the AAV vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV22, AAV33, AAV44, AAV5, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, A AV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or 90% or against the VP1 sequence, VP2 sequence and / or VP3 sequence of AAV-2i8 VP1 capsid sequence, VP2 capsid sequence or VP3 capsid sequence with higher identity In a further specific embodiment, the AAV vector has a capsid sequence. The target is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, A AV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 VP1 capsid sequence, VP2 capsid sequence selected from any of the AAV serotypes or has a capsid sequence having a VP3 capsid sequence.

[0016] In a further embodiment, the recombinant vector genome comprises CpG (cytosine-guanine) ) Factor IX coding fragments, such as FIX modified to reduce the number of dinucleotides. and a filler polynucleotide sequence or a stuffer polynucleotide sequence. In particular embodiments, the modified nucleic acid encoding Factor IX comprises a sequence of about 4 In a more particular embodiment, the modified Factor IX encoding The nucleic acid has a length of less than 4.7 kb and is flanked by one or more AAV ITRs. or located within two adeno-associated virus (AAV) ITR sequences. In particular embodiments, the filler or stuffer polynucleotide sequence is a sequence encoding Factor IX. a nucleic acid sequence that encodes Factor IX when combined with a modified nucleic acid encoding the factor IX polypeptide; The total combined length of the filler or stuffer polynucleotide sequence is approximately 3.0 Between kb and 5.5kb, approximately 4.0kb to 5.0kb, or approximately 4.3kb to 4.8kb b.

[0017] The filler or stuffer polynucleotide sequence may be used to enhance the function or activity of the vector. It can be placed at any desired location in the non-interfering vector sequence. In this case, the filler or stuffer polynucleotide sequence is CpG (cytosine guanine). Each of the factor IX-encoding nucleic acid sequences, such as FIX, has a reduced number of dinucleotides. The 5' and / or 3' ends of each ITR are flanked by 5' and / or 3' ITRs (e.g., AAV 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV 9, ITR of AAV10, AAV11, Rh10, Rh74 or AAV-2i8 and In another embodiment, a filler sequence or stuffer sequence is located between the The oligonucleotide sequence is a variant of factor IX, such as FIX, which has a reduced number of CpG dinucleotides. 5' and / or 3' adjacent to the 5' and / or 3' ends of each of the coding nucleic acid sequences In a further embodiment, the filler sequence or stuffer is located within the ITR. The polynucleotide sequence may be a factor IX-like FIX with a reduced number of CpG dinucleotides. 5' and / or 3' adjacent to the 5' and / or 3' ends of each of the coding nucleic acid sequences In a further embodiment, the filler sequence or stuff is located adjacent to the ITR. The polynucleotide sequence may be a nucleotide sequence similar to FIX, such as FIX, that has a reduced number of CpG dinucleotides. Within a modified nucleic acid (e.g., similar to an intron within a genomic nucleic acid) encoding a factor are located in the department.

[0018] Thus, in various embodiments, the filler or stuffer polynucleotide sequence are located adjacent to the AAV ITR sequences; two adeno-associated virus (AAV) ITRs Located within the R sequence; located outside the two adeno-associated virus (AAV) ITR sequences or two filler or stuffer polynucleotide sequences are present, the first filler The lar sequence or stuffer polynucleotide sequence is a sequence of two adeno-associated viruses (AAVs) A second filler or stuffer polynucleotide sequence located within the ITR sequence. is located outside the two adeno-associated virus (AAV) ITR sequences.

[0019] In a further specific embodiment, the filler or stuffer polynucleotide sequence is When placed within two adeno-associated virus (AAV) ITR sequences, CpG dimers and modified nucleic acids encoding factor IX, such as factor IX with a reduced number of nucleotides. When combined, the heterologous polynucleotide sequence and the filler or stuffer sequence The total length of the combined nucleotide sequence is approximately 3.0 kb to 5.5 kb, approximately 4. The length is 0 kb to 5.0 kb or approximately 4.3 kb to 4.8 kb. In another specific embodiment, the filler or stuffer polynucleotide sequence comprises two If placed outside the adeno-associated virus (AAV) ITR sequences, it may exceed 4.7 kb. The length of the fragment is approximately 5.0 kb to 10.0 kb or approximately 6.0 kb to 8.0 kb.

[0020] Typically, the filler or stuffer polynucleotide sequences are inert or It is non-toxic and has no function or activity. the sequence or stuffer polynucleotide sequence is not a bacterial polynucleotide sequence, Filler or stuffer polynucleotide sequences are not protein-coding sequences. or a filler or stuffer polynucleotide sequence, a modified nucleic acid encoding factor IX, an AAV inverted terminal repeat (I TR) sequence, expression control element, replication origin, selectable marker or polyadenylation (poly A) A sequence that is distinct from any signal sequence.

[0021] In various additional specific embodiments, the filler or stuffer polynucleotide sequence The columns represent heterologous polynucleotide sequences (e.g., F with a reduced number of CpG dinucleotides). a modified nucleic acid encoding factor IX, such as factor IX, In particular embodiments, the intron sequence is a heterologous polynucleotide. a sequence (e.g., encoding factor IX, such as FIX, with a reduced number of CpG dinucleotides) In other specific embodiments, the intron is located within a heterologous modified nucleic acid. by a species-origin polynucleotide sequence (e.g., a modified nucleic acid encoding Factor IX) Present in genomic DNA, such as genomic DNA that encodes proteins encoded by Thus, intron sequences may be inserted into heterologous polynucleotide sequences (e.g., CpG dinucleotides). It is associated with a modified nucleic acid encoding factor IX, such as factor IX with a reduced number of nucleotides. do.

[0022] AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 , such as AAV9, AAV10, AAV11, Rh10, Rh74, and AAV-2i8 Recombinant lentiviral and parvoviral (e.g., AAV) vectors and recombinant Capsid mutants (e.g., encapsidated) containing recombinant AAV vector genomes For example, a variant such as 4-1) particle may be contained within a cell. In this state, the cells are capable of producing viral (AAV) particles or are lysed. It also consists of packaging cells that can produce AAV particles. or in a target cell in which it is desired to express the heterologous polynucleotide sequence. Thus, factor IX, such as FIX with a reduced number of CpG dinucleotides, may be used. and vectors encoding modified nucleic acids, such as AAV1, AAV2, AAV3, AA V4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Lentiviruses and parvoviruses such as Rh10, Rh74, and AAV-2i8 (e.g. Cells containing particles of the mutant (e.g., AAV) vectors and variants (e.g., 4-1, etc.) are provided. .

[0023] In a further embodiment, a nucleic acid sequence encoding a human FIX protein is Has a reduced number of CpG dinucleotides compared to the native sequence encoding factor IX or an expression vector or plasmid containing a nucleic acid sequence encoding the human FIX protein. and a reduced number of CpG residues compared to the native sequence encoding human Factor IX. Nucleic acids having nucleotides are included in the composition. In particular embodiments, such nucleic acids The nucleic acid sequences may be included in pharmaceutical compositions. , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, Rh10, Rh74, and AAV-2i8, and recombinant lentiviruses and gene transfer vectors. Aerovirus (e.g., AAV) vectors, as well as vector genomes (capsids) Mutant (e.g., 4-1, etc.) particles may be included in pharmaceutical compositions. Such compositions include recombinant vectors (e.g., AAV) and AAV1, AAV2, and , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV1 viral particles such as AAV-0, AAV11, Rh10, Rh74, or AAV-2i8; and , a mutant (e.g., encapsidated) containing a vector (e.g., AAV) genome For example, it is useful for administration to a subject of 4-1).

[0024] AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 , such as AAV9, AAV10, AAV11, Rh10, Rh74, and AAV-2i8 Recombinant lentiviral and parvoviral (e.g., AAV) vectors and vectors Mutant (e.g., 4-1) particles containing the targenome (encapsidated, enveloped) It can be used in a variety of methods and applications. In an organism or cell such as a cell, a heterologous polynucleotide sequence (e.g., a CpG dinucleotide) is introduced. a modified nucleic acid encoding factor IX, such as factor IX with a reduced number of nucleotides Methods and uses for transferring or transporting the same are provided.

[0025] In one embodiment, the method or use comprises the steps of: AV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10 , lentiviruses such as Rh74 and AAV-2i8 or parvoviruses (e.g. , AAV) vectors or heterologous polynucleotide sequences (e.g., CpG dinucleotides) a modified nucleic acid particle encoding factor IX, such as factor IX with a reduced number of nucleotides; The mutants contained in the vector genome are transfected into mammals under appropriate conditions. and administering the heterologous polynucleotide to a mammal or mammalian cells to induce the production of the heterologous polynucleotide in the mammal or mammalian cells. In one embodiment, the method or use comprises delivering or transferring a nucleotide sequence to a mammal. and / or a heterologous polynucleotide (e.g., a polynucleotide having a high number of CpG dinucleotides) in the cell Transfer / deliver a modified nucleic acid encoding factor IX, such as reduced FIX. In embodiments, the method comprises the step of: and the introduction into cells of a modified nucleic acid encoding factor IX, such as factor IX with a reduced number of amino acids. Transfer / delivery, transcription to form subsequent transcripts, and subsequent gene products (e.g. This allows translation to form a factor (e.g., factor IX).

[0026] In a further embodiment, the method or use is directed to the treatment of a deficiency or defect in the expression or function of a protein. for treating a subject (e.g., a mammal) in need of expression or function of a protein Yes, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AA V8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 Such lentiviral or parvoviral (e.g., AAV) vectors or variants , a plurality of such viral (e.g., AAV) particles, or AAV1, AAV2, AA V3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, A Lentiviruses such as AV11, Rh10, Rh74, or AAV-2i8, or Parvovirus (e.g., AAV) vectors or variants, particles, or multiple thereof and providing pharmaceutical compositions of such viral (e.g., AAV) particles. a virus particle, a plurality of virus particles, or a pharmaceutical composition of a virus particle or a plurality of virus particles The heterologous polynucleotide thus administered includes administering to a subject (e.g., a mammal) Nucleotide sequences (e.g., FIX, such as FIX, with a reduced number of CpG dinucleotides) The modified nucleic acid encoding the factor is then expressed in a subject (e.g., a mammal). It is possible.

[0027] The methods and uses for administration or delivery include any mode compatible with the subject. In embodiments, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 and AA lentiviral or parvoviral (e.g., AAV) vectors such as V-2i8, and The mutant or multiple such viral particles may be administered intravenously, intraarterially, intramuscularly, subcutaneously, or intravenously. The drug may be administered or delivered parenterally, such as into the body or via a catheter.

[0028] Subjects include mammals, such as humans and non-humans (e.g., primates). In some embodiments, the subject benefits from expression of a heterologous polynucleotide sequence, or In further specific embodiments, the expression of heterologous polynucleotide sequences may be required. In this case, the subject may be, for example, a subject suffering from hemophilia B who expresses reduced amounts of factor IX. A subject, such as a subject, would benefit from Factor IX expression or function.

[0029] According to the present invention, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, A AV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 and AAV Recombinant lentiviral and parvoviral (e.g., AAV) vectors such as -2i8 To produce mutants containing the vector genome (encapsidated, enveloped) In one embodiment, a method is provided for producing a productive viral infection. To achieve this, a recombinant vector (e.g., AAV) plasmid is introduced into the packaging cells. and incubating the packaging cells under conditions that produce recombinant viral particles. In another embodiment, the recombinant viral vector is Recombinant virus or AA containing the introduced nucleic acid and having a reduced amount of recombinant virus particles. 1. A method for producing V particles, the method comprising: injecting a recombinant vector ( introducing a plasmid (e.g., AAV); and conditions for producing recombinant viral particles. and incubating the packaging cells under a temperature of 100° C. for 1 hour to produce recombinant human ovarian milk. The virus particle is a recombinant virus vector in which the filler sequence or stuffer polynucleotide sequence is The number of viral particles containing contaminating nucleic acids compared to the number of viral particles produced in the absence of the vector The number of viral particles carrying the vector genome containing the nucleic acid is reduced. In embodiments, the contaminating nucleic acid is a bacterial nucleic acid; or ITR, promoter, enhancer, replication origin, polyA sequence, or is a sequence other than the selectable marker.

[0030] The packaging cell comprises a mammalian cell. In certain embodiments, the packaging cell The cells contain a (heterologous polynucleotide) sequence (e.g., a reduced number of CpG dinucleotides). a modified nucleic acid encoding factor IX, such as a modified factor IX; an expression vector (e.g., a vector genome) into a viral particle (e.g., an AAV particle) In particular embodiments, the packaging cells contain AAV V Rep proteins and / or Cap proteins (e.g., Rep78 and / or Re p68 protein); the packaging cell provides the Rep protein sequence and / or Ca a polynucleotide encoding the p protein sequence is stably or transiently introduced; and / or Alternatively, the packaging cells may be modified with Rep78 and / or Rep68 protein polynucleotides. The coding sequence can be introduced stably or transiently.

[0031] In the present invention, recombinant lentiviral or parvoviral (e.g., AAV) vectors - and associated cis elements (e.g., expression control elements, ITR, polyA), or trans (e.g., packaging functions such as capsid proteins, Rep / Cap proteins, etc.) The agent may be based on any organism, species, strain or serotype. (e.g., AAV) particles are typically AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh7 Hybridization of multiple different serotypes based on AAV-2i8 and AAV-2i8 variants Representative AAV serotypes include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, This includes the serotypes AAV10, AAV11, Rh10, Rh74, and AAV-2i8. Therefore, recombinant viral (e.g., AAV) particles containing vector genomes are classified as AAV1, AAV 2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV VP1, VP2 or VP3 of serotypes 10, AAV11, Rh10, Rh74 or AAV-2i8 can be a mixture of different serotypes, such as the VP3 capsid protein, or a mixture of different serotypes. Contains capsid proteins derived from hybrids or chimeras of multiple serotypes Furthermore, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV 7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 and AAV-2 recombinant lentiviral or parvoviral (e.g., AAV) vectors such as i8, The sequences, plasmids, and vector genomes may be of one serotype, a mixture of serotypes, or different serotypes. It may also contain factors derived from hybrids or chimeras of multiple serotypes. In some embodiments, the recombinant AAV vector comprises ITRs, Cap, Rep, and / or Or AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AA V8, AAV9, AAV10, AAV11, Rh10, Rh74 and / or AAV- 2i8 serotype, or mixtures, hybrids, or chimeras of the aforementioned AAV serotypes. Contains an array of [Brief explanation of the drawings]

[0032] Figure 1 shows the amino acid sequence of Rh74 VP1.

[0033] Figure 2 shows the amino acid sequence of Rh74 VP2.

[0034] FIG. 3 shows the amino acid sequence of Rh74 VP3.

[0035] FIG. 4 shows the amino acid sequence of the capsid mutant 4-1 VP1 protein.

[0036] FIG. 5 shows the amino acid sequence of capsid mutant 15-1.

[0037] FIG. 6 shows the amino acid sequence of capsid mutant 15-2.

[0038] FIG. 7 shows the amino acid sequences of capsid mutants 15-3 / 15-5.

[0039] FIG. 8 shows the amino acid sequence of capsid mutant 15-4.

[0040] FIG. 9 shows the amino acid sequence of capsid mutant 15-6.

[0041] FIG. 10 shows the nucleic acid sequence of FIX39.

[0042] FIG. 11 shows the nucleic acid sequence of FIX19.

[0043] FIG. 12A shows the sequence of the FIX39 plasmid.

[0044] FIG. 12B shows the sequence of the phFIX39v2 plasmid.

[0045] FIG. 13 shows a map of the FIX39 plasmid.

[0046] FIG. 14 shows the Intron A nucleic acid sequence.

[0047] FIG. 15 shows the nucleic acid sequence of FIX39+intron A.

[0048] FIG. 16 shows the AAV-4-1 capsid variant analyzed in an in vitro setting. The transduction efficiency of the variant (SEQ ID NO: 4) is shown.

[0049] Figure 17 shows AAV-FIX39-Padua (square / circle) and AAV-FIX19-P Adua (diamond / hexagon) 1×10 11 or 1 x 10 12 vg / kg The levels of hFIX in the plasma of wild-type mice after intravenous injection at 8 weeks are shown. Plasma concentrations were analyzed by ELISA and were measured in the same groups of animals throughout the study. Multiple measurements obtained by bleeding are shown, and error bars indicate standard error.

[0050] Figure 18 shows the results of 5 μg of pFIX19-Padua plasmid or pFIX39-Padua a) The blood concentration of human FIX in mouse plasma 24 hours after hydraulic tail vein injection of the plasmid. P=0.3337

[0051] FIG. 19 shows a vector carrying the AAV-FIX Padua mutant (FIX39) according to the present invention. Summary data from four human hemophilia B patients who received a single infusion of acetaminophen and a subsequent evaluation period FIX activity (%) over a period of 183 days, 102 days, 69 days, and 50 days, respectively, is shown. .

[0052] Figure 20A shows the efficacy of AAV-FIX Padua mutant (FI) over a 183-day evaluation period. FIX activity (% ) of the first human hemophilia B patient who received a single injection of a vector carrying X39 ) data shown.

[0053] Figure 20B shows the efficacy of the AAV-FIX Padua mutant (FIX) over a 183-day evaluation period. Liver function tests (A) of the first human hemophilia B patient who received a single injection of vector containing 39 The LDH values ​​(L DH1) is divided by 10 to show together the ALT and AST values.

[0054] Figure 21A shows the efficacy of AAV-FIX Padua mutant (FIX) over a 102-day evaluation period. FIX activity (%) in a second human hemophilia B patient who received a single injection of a vector containing 39 Show the data.

[0055] Figure 21B shows the efficacy of AAV-FIX Padua mutant (FIX) over a 102-day evaluation period. Liver function tests (A) of a second human hemophilia B patient who received a single infusion of vector bearing 39 The LDH values ​​(L DH1) is divided by 10 to show together the ALT and AST values.

[0056] Figure 22A shows the efficacy of the AAV-FIX Padua mutant (FIX3) over a 69-day evaluation period. FIX activity (%) data from a third human hemophilia B patient who received a single injection of a vector containing 9 Indicates the data.

[0057] Figure 22B shows the efficacy of AAV-FIX Padua mutant (FIX3) over a 69-day evaluation period. Liver function tests (AL) of a third human hemophilia B patient who received a single infusion of vector bearing 9 The data shows the LDH values ​​(LD H1) is divided by 10 to show together the ALT and AST values.

[0058] Figure 23A shows the efficacy of AAV-FIX Padua mutant (FIX3) over a 50-day evaluation period. FIX activity (%) data from a fourth human hemophilia B patient who received a single injection of a vector containing 9 Indicates the data.

[0059] Figure 23B shows the efficacy of AAV-FIX Padua mutant (FIX3) over a 50-day evaluation period. Liver function tests (AL) of the fourth human hemophilia B patient who received a single injection of vector containing 9 The data shows the LDH values ​​(LD H1) is divided by 10 to show together the ALT and AST values.

[0060] Figure 24A shows the relative immunogenicity profiles of AAV-FIX39 Padua in human subjects. Shows the feel.

[0061] Figure 24B shows the correlation between AAV-FIX39 Padua and AAV8-FIX19 in human subjects. The immunogenicity profiles of the IgG antibodies are shown in Table 1.

[0062] The present invention provides modified nucleic acid sequences encoding proteins, such as the human FIX protein. In some embodiments, the modified The nucleic acid may encode factor IX, such as the native (wild-type) sequence encoding human factor IX. The number of CpG (cytosine-guanine) dinucleotides is increased compared to the reference nucleic acid sequence. In a further embodiment, a nucleic acid (e.g., encoding human Factor IX) is Reduced number of CpG dinucleotides compared to the reference factor IX coding sequence (natural sequence) Such modified nucleic acids may be incorporated into expression vectors (e.g., vector genomes) or plasmids. It is contained in mido.

[0063] The present invention further provides compositions, such as compositions, that include modified nucleic acid sequences encoding human FIX. In such compositions, the modified nucleic acid encodes human Factor IX. The number of CpG dinucleotides is reduced relative to the reference sequence, such as the natural (wild-type) sequence. The composition also includes an expression vector (e.g., a viral vector / vector genome), and and such modified FIX proteins encoding human FIX proteins with a reduced number of CpG dinucleotides. The plasmid contains the modified nucleic acid sequence.

[0064] In particular embodiments, the nucleic acid sequence encoding the human FIX protein encodes human factor IX. or a human nucleotide sequence I having 1 to 5 fewer CpG dinucleotides than the native sequence it encodes; It contains 5 to 10 fewer CpG dinucleotides than the natural (wild-type) sequence encoding factor X. or 10-15 fewer amino acids than the native (wild-type) sequence encoding human factor IX or the native (wild-type) sequence encoding human factor IX, or a human factor IX-encoding gene having 15 to 20 fewer CpG dinucleotides than the human factor IX-encoding gene; or having 20 to 25 fewer CpG dinucleotides than the native (wild-type) sequence; It contains 25 to 30 fewer CpG dinucleotides than the native (wild-type) sequence encoding human factor IX. or 30 to 40 amino acids from the natural (wild-type) sequence encoding human factor IX. or the native (wild-type) coding for human factor IX ) has 40 to 55 fewer CpG dinucleotides than the sequence of The nucleotide is completely missing.

[0065] Modified factor IX-encoding FIX, such as FIX with a reduced number of CpG dinucleotides, The nucleic acid may further comprise one or more additional cis elements. These include, but are not limited to, expression control elements, introns, ITRs, stop codons, po In certain embodiments, the polynucleotide comprises an lyA sequence and / or a filler polynucleotide sequence. Such cis-acting elements may be modified, for example, expression control elements, introns, cis-acting elements such as ITRs, polyA sequences and / or filler polynucleotide sequences The gene may have a reduced number of CpG dinucleotides. Current regulatory elements, introns, ITRs, polyA sequences and / or filler polynucleotide sequences In particular embodiments, one or more cis-acting elements such as In the present invention, expression control elements, introns, ITRs, polyA sequences and / or filler polynucleotides are One or more cis-acting elements, such as a nucleotide sequence, are 1 to 5 times larger than the reference cis-acting element. or 5 to 10 fewer CpG dinucleotides than the control cis-acting element or 10 to 10 less CpG dinucleotides than the control cis-acting element 5 fewer CpG dinucleotides; or 15-20 fewer than the control cis-acting element or 20-25 fewer CpG dinucleotides than the reference cis-acting element. or 25-30 fewer CpG dinucleotides than the control cis-acting element. pG dinucleotides; or 30-40 fewer CpGs than the reference cis-acting element dinucleotide; or 40 to 55 fewer CpG dinucleotides than the reference cis-element or lacking any CpG dinucleotides.

[0066] The present invention provides human FIX proteins, such as FIXs with a reduced number of CpG dinucleotides. In certain embodiments, the viral vector comprises a modified nucleic acid sequence encoding The vector may be an adenoviral vector, a lentiviral vector, or a perviral vector. In a more particular embodiment, the number of CpG dinucleotides is reduced. The modified nucleic acid sequence encoding a human FIX protein, such as a modified FIX, is then transfected into adenovirus-transfected cells. Included in an AAV vector.

[0067] In a more specific embodiment, the adeno-associated virus (AAV) vector is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 Capsids derived from AAV10, AAV11, Rh10, Rh74, and AAV-2i8 and their variants (e.g., capsid modifications such as amino acid insertions, additions, and substitutions). As will be appreciated by those skilled in the art, AAV capsids typically contain a VP1 tag. and two proteins called VP2 and VP3, which are essentially amino-terminal truncations of VP1. Depending on the capsid and other factors known to those skilled in the art, The three capsid proteins, VP1, VP2, and VP3, are typically expressed in a 1:1 ratio, respectively. VP3 is present in the capsid at a ratio approaching 10:10. However, this ratio, especially the ratio of VP3, may vary significantly and should not be construed as limiting in any way. Not possible.

[0068] AAV mutants include AAV-Rh74 mutants (e.g., Rh74 VP1 capsid sequence , SEQ ID NO: 1, AAV capsid mutants of FIG. 1). However, mutants 4-1, 15-1, 15-2, 15-3 / 15-5, shown in Table 1 The amino acid sequences of Rh74 VP2 and Rh74 VP3 are: These are provided in SEQ ID NO:2 (Figure 2) and SEQ ID NO:3 (Figure 3), respectively. [Table 1]

[0069] The 4-1 mutant (SEQ ID NO: 4) is located at amino acid positions 195 and 199 of the VP1 capsid. , 201 and 202 are alanine, valine, proline and asparagine, respectively. The substituted residues a, v, p, and n are underlined and bolded. The amino acid sequence of the 4-1 mutant VP1 capsid is shown in Figure 4 (SEQ ID NO: 4). For mutant 4-1, the VP2 sequence is SEQ ID NO: 27, and the VP3 sequence is SEQ ID NO: 28. The string consists of SEQ ID NO:3.

[0070] The 15-1, 15-2, 15-3, 15-4, 15-5 and 15-6 mutants also At amino acid positions 195, 199, 201, and 202 of the capsid, respectively, These mutants contained substitutions of thiamin, valine, proline, and asparagine. The 15-1 mutant VP had multiple lysine to arginine substitutions at various positions. The capsid amino acid sequence (SEQ ID NO: 5) of 15-1 is shown in Figure 5; The VP1 capsid amino acid sequence (SEQ ID NO: 6) is shown in Figure 6; The 15-5 mutant VP1 capsid amino acid sequence (SEQ ID NO: 7) is shown in FIG. The amino acid sequence of the 15-4 mutant VP1 capsid (SEQ ID NO: 8) is shown in FIG. The 15-6 mutant VP1 capsid amino acid sequence (SEQ ID NO: 9) is shown in Figure 1. 9. Examples of capsids that can be used herein include, but are not limited to: No. 6,392,235, including those disclosed in U.S. Patent Application Publication No. 2015 / 0023924.

[0071] Thus, lentiviral vectors and parvoviral vectors such as AAV vectors and human FIX proteins, such as FIX with a reduced number of CpG dinucleotides. The vector genome includes a modified nucleic acid sequence encoding (encapsidated, enveloped) AAV mutants (e.g., 4-1, 15-1, 15-2, 15-3 / 15-5, 15- Viral vector variants such as capsid variants such as 15-4 and 15-6 are provided. do.

[0072] In an exemplary study, AAV-Rh74 showed significantly higher genetic activity than several other serotypes. Mediated the protein expression levels produced by gene transduction / delivery. In particular, AAV-Rh74 Targeting gene for liver delivery of vectors and capsid variants (e.g., 4-1) The offspring are derived from dogs and / or mice with hemophilia B, the gold standard for liver gene transfer. It has an efficiency at least comparable to AAV8 in mice and / or macaques.

[0073] As disclosed herein, lentiviruses and vectors, including AAV serotypes and variants, Viral vectors, such as parvovirus vectors, can be used ex vivo, in vitro, and in vivo. The present invention provides a means for delivering polynucleotide sequences into cells in vivo, The nucleotide sequence can encode a protein and can be used to produce the encoded protein. For example, recombinant AAV vectors can express a desired protein or containing a heterologous polynucleotide encoding a peptide (e.g., Factor IX) Therefore, vector delivery or administration to a subject (e.g., a mammal) may be performed by Thus, the AAV serotype and capsid variants are identified and the resulting proteins and peptides are provided to the subject. lentiviral and parvoviral vectors, including variants such as 4-1 Viral vectors such as vectors have been used to deliver different types of antibodies to cells for expression and, optionally, for the treatment of various diseases. It can be used to transfer / deliver polynucleotides of species origin.

[0074] In certain embodiments, the recombinant vector (e.g., AAV) is a parvovirus vector. Parvoviruses are small viruses with a single-stranded DNA genome. Avian-associated viruses (AAVs) belong to the parvovirus family.

[0075] Parvoviruses, including AAV, are able to penetrate cells and transfer nucleic acid / genetic material. The nucleic acid / genetic material can be stably maintained in the cell, making it suitable for gene therapy vectors. Furthermore, these viruses are useful as vectors for the detection of viruses that are mutated on chromosome 19, for example. AAV can deliver nucleic acid / genetic material to specific sites, such as specific sites in AAV vectors are not essential for AAV pathogenesis, as they have not been associated with pathogenic disease in humans. or administering heterologous polynucleotide sequences (e.g., therapeutic) to a human patient without causing disease. It can deliver therapeutic proteins and agents.

[0076] Serotypes (e.g., capsid mutants such as 4-1) of AAV and AAV variants (e.g., For example, the VP1 sequence, VP2 sequence, and / or VP3 sequence) can be used in, for example, AAV1-AAV11. , Rh74, or Rh10 (e.g., AA V1-AAV11, VP1 sequence of either Rh74 or Rh10 serotype, VP2 sequence, and The sequence of the VP1 sequence may be different from the VP1 and / or VP2 sequences, or may not be different from the VP1 and / or VP2 sequences.

[0077] As used herein, the term "serotype" refers to a serotype that is distinct from other AAV serotypes. This distinction is used to describe AAVs with serologically distinct capsids. Compared to AAV, serological uniqueness is the lack of cross-reactivity between antibodies against one AAV Such cross-reactivity differences are usually determined based on the capsid protein sequence / Due to differences in antigenic determinants (e.g., VP1 sequence, VP2 sequence, and / or V P3 sequence differences). AAV mutants, including capsid mutants, were compared with control AAV or other AAVs. AAV mutations, despite the possibility that they may be serologically indistinguishable from AV serotypes The antibody has at least one nucleotide or amino acid sequence that is different from that of a control or other AAV serotype. The amino acid residues are different.

[0078] Under the conventional definition, serotypes are all existing serotypes differentiated with respect to neutralizing activity. The target virus is tested against a specific serum, and an antibody that neutralizes the target virus is identified. This means that no bodies were found. More natural virus isolates were discovered, and and / or, even if more capsid variants are generated, they will not be compatible with any of the currently existing serotypes. There may or may not be serological differences. Therefore, new viruses (e.g., A If the virus (e.g., AAV) does not have serological differences, this new virus (e.g., AAV) These may be subgroups or variants of the serotypes that contain capsid sequence modifications. For mutant viruses that are of a different serotype according to the conventional definition of serotype, Serological testing for neutralizing activity has not yet been performed to determine whether Therefore, for convenience and to avoid repetition, the term "serotype" is used to refer to a serological serologically distinct viruses (e.g., AAV) and serologically indistinguishable viruses (e.g., , AAV) that can belong to a subgroup or variant of a given serotype It broadly represents both.

[0079] Recombinant vector (e.g., AAV) plasmids, vector (e.g., AAV) genomes, And the methods and uses include any virus strain or serotype. Non-limiting examples include: Recombinant vector (e.g., AAV) plasmid or vector (e.g., AAV) genome , e.g., AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, - Based on any AAV genome, such as AAV-11, -rh74, -rh10, or AAV-2i8 Such vectors may be of the same strain or serotype (or subtype). The amino acid sequences may be based on a group or variants of the amino acid sequence, or may be different from each other. As a non-limiting example, a recombinant vector based on one serotype genome (e.g., AA V) The plasmid or vector (e.g., AAV) genome is a capsid that encases the vector. Furthermore, the recombinant vector (e.g., AAV) The genome of a plasmid or vector (e.g., AAV) is contained in a capsid that encases the vector. Based on a different AAV (e.g., AAV2) serotype genome than one or more of the serotype proteins In this case, at least one of the three capsid proteins may be, for example, AA V1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AA V9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8, or A AV-Rh74 mutants (e.g., 4-1, 15-1, 15-2, 15-3 / 15-5, 1 The capsid may be a mutant such as 15-4 or 15-6.

[0080] Therefore, AAV vectors contain genes identical to the gene / protein sequences characteristic of a particular serotype. As used herein, "AAV1 and related AAVs" refers to any AAV that is a member of a family of AAVs. A "vector" is a vector that contains one or more AAV proteins (e.g., VP1 sequence, VP2 sequence, and / or VP3 sequence), one or more polynucleotide sequences or polypeptides that constitute AAV1 Similarly, "AAV8" refers to a peptide sequence that has substantial sequence identity to the AAV8 peptide sequence. An "AAV vector" is an AAV vector that contains one or more AAV proteins (e.g., VP1 sequence, VP2 sequence, and / or VP3 sequence), one or more polynucleotide sequences constituting AAV8 or polypeptide sequence having substantial sequence identity to the AAV-Rh 74 and related AAV vectors contain one or more AAV proteins (e.g., VP1 sequence, VP2 sequence and / or VP3 sequence), one or more of the polypeptides constituting AAV-Rh74 and the like, which have substantial sequence identity to the nucleotide or polypeptide sequence. (See, for example, VP1, VP2, and VP3 in Figures 1 to 3.) AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8, etc. Such AAV vectors are related to the serotypes AAV1, AAV2, AAV3 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, Rh10, Rh74, and AAV-2i8. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 , AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i 8 that show substantial sequence identity to one or more genes and / or proteins and / or one or more of their functional properties (e.g., cell / tissue index) Exemplary AAV-R vectors, such as AAV-Rh74, may have a specific tropism. h74 or related AAV, or (e.g., 4-1, 15-1, 15-2, 15-3 / AAV-Rh74 (capsid variants such as 15-5, 15-4, and 15-6) The sequences of the mutants and related AAV mutants are, for example, VP1 as described in Figures 1 to 9. , VP2 and / or VP3.

[0081] In various exemplary embodiments, the AAV vector associated with the reference serotype is one More than AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AA V8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8 (e.g. For example, the AAV-Rh74 VP1 sequence, VP2 sequence, and At least 80% (e.g., 85%, 90%, 95% 96%, 97%, 98%, 99%, 99.5%, etc.) identical sequence or The polynucleotide, polypeptide, or subsequence thereof comprises:

[0082] The methods and uses of the present invention involve the use of AAV sequences (polypeptides and nucleotides), AAV-R h74 sequences (polypeptide and nucleotide) and subsequences thereof, V1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AA V9, AAV10, AAV11, Rh10 or AAV-2i8, e.g., AAV-Rh7 4 gene or protein sequence (e.g., VP1 sequence, VP2 sequence, etc., as shown in Figures 1 to 9) sequences and / or VP3 sequences) relative to a reference AAV serotype Although it shows identity, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AA V7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74AAV-2i 8 genes or proteins, and are distinct from and identical to known AAV genes or proteins. In one embodiment, the AAV polypeptide or subsequence thereof is 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV 9, any pair such as AAV10, AAV11, Rh10, Rh74 or AAV-2i8 A reference AAV sequence or a partial sequence thereof (e.g., the VP1 sequence shown in Figures 1 to 9, VP2 sequence and / or VP3 sequence) at least 80% or more (e.g., 85%, 8 5%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, 99.5% (i.e., up to 100%) of identical sequences In particular embodiments, the AAV variant comprises or consists of four amino acid substitutions One, two, three or four of these (e.g., capsid mutants 4-1, 15-1 , 15-2, 15-3 / 15-5, 15-4 and 15-6).

[0083] AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 , AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8, and , variants, related hybrid and chimeric sequences, including recombinant vectors (e.g., A AAV) consists of one or more heterologous polynucleotides flanked by one or more functional AAV ITR sequences. Recombination techniques known to those skilled in the art can be used to include the nucleotide sequence (genetic modification). Such vectors can be constructed using one or more of the wild-type AAV genes. Even if all or part of the gene (e.g., the Rep gene and / or the Cap gene) is deleted, Rescue, replication, and packaging of recombinant vectors into AAV vector particles Since it is necessary for the synthesis of the gene, at least one functional flanking ITR sequence may be retained. Thus, the AAV vector genome is required in cis for replication and packaging. The sequences will include sequences that are functional (e.g., functional ITR sequences).

[0084] The terms "polynucleotide" and "nucleic acid" refer to deoxyribonucleic acid (DNA). to represent all forms of nucleic acids, including oligonucleotides and ribonucleic acid (RNA) Polynucleotides are used interchangeably. They include genomic DNA, cDNA, and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA, tRNA, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering RNA (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides include natural, synthetic, and intentionally modified or altered (e.g., Polynucleotides include polynucleotides with reduced CpG dinucleotides. It may be a single strand, double strand, or triple strand, linear or circular, and may be of any length. When discussing polynucleotides, the sequence of a particular polynucleotide may be Sequences or structures are described herein according to the convention of providing sequences in the 5' to 3' direction. There are.

[0085] A "heterologous" polynucleotide is a polynucleotide that is introduced into a cell via a vector. Polynucleotides inserted into vectors (e.g., AAV) for transfer / delivery purposes The heterologous polynucleotide is typically distinct from the vector (e.g., AAV) nucleic acid. That is, the heterologous polynucleotide is not related to the nucleic acid of the virus (e.g., AAV). Once transferred / delivered into the cell, the heterologous origin contained in the vector The polynucleotide can be expressed (e.g., transcribed and translated, as appropriate). Alternatively, a heterologous gene contained in a vector and transferred or delivered into a cell The polynucleotide does not need to be expressed. The term "heterologous" is not necessarily used in reference to polynucleotides in this context. Reference to a polynucleotide without modification is not, despite its omission, a heterologous polynucleotide. An example of a heterologous sequence is a sequence that is different from a reference nucleic acid sequence. Nucleic acids encoding factor IX (e.g., nucleic acids with a reduced number of CpG dinucleotides) For example, a modified nucleic acid encoding Factor IX.

[0086] "Polypeptides," "proteins," and "Peptides" include not only full-length natural sequences like natural proteins, but also functional partial sequences. , modified forms or sequence variants that retain some functionality of the native full-length protein. In the methods and uses of the present invention, the present invention includes subsequences, modified forms or sequence variants having the same structure as the sequence of the present invention. Such polypeptides, proteins and The peptides and peptides are endogenous proteins that are defective or whose expression is impaired in the mammal being treated. It may be identical to the endogenous protein that is poorly expressed or is deficient, but it may be identical to the endogenous protein that is poorly expressed or deficient. It doesn't have to be.

[0087] In the present invention, adenoviral vectors, such as AAV1, AAV2, AA V3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, A such as AAV11, Rh10, Rh74 or AAV-2i8 and AAV-Rh74 variants Related AAV variants (e.g., 4-1, 15-1, 15-2, 15-3 / 15-5, 15- lentiviruses such as AAV vectors containing capsid variants such as 15-4 and 15-6 Viral and parvoviral vectors stably deliver polynucleotides to cells and their progeny. It can be used to introduce / deliver a substance continuously or temporarily. The term "transgene" refers to a gene that has been introduced or is intended to be introduced into a cell or organism. A transgene is used for convenience to refer to a heterologous polynucleotide that is Any polypeptide, such as a gene encoding a peptide or protein (e.g., Factor IX) Contains nucleotides.

[0088] For example, in cells carrying a transgene, the transgene may be delivered via a vector such as AAV, Introduction / transfer by "transduction" or "transfection" of cells. The terms "introduce" and "transfect" refer to the act of introducing a gene into a cell or host organism. This refers to the introduction of a molecule such as a polynucleotide of the present invention.

[0089] Cells into which a transgene has been introduced are called "transduced cells." The "cells" (e.g., those in mammals, such as cells or tissue or organ cells) are not infected with exogenous components. uptake of a gene (e.g., a polynucleotide or protein (e.g., a transgene) into a cell) This refers to the genetic change in a cell after transduction. Thus, a "transduced" cell can be For example, a cell into which an exogenous molecule has been introduced or its progeny. The introduced protein is expressed or the introduced nucleic acid is transcribed. For the uses and methods, the cells to be transduced may be present in a subject.

[0090] The introduced polynucleotide may be integrated into the nucleic acid of the recipient cell or organism. The introduced polynucleotide may or may not be integrated into the recipient cell or When integrated into the nucleic acid (genomic DNA) of an organism, the introduced polynucleotide: maintained in that cell or organism and further in the descendent cells or organisms of that recipient cell or organism. Finally, the introduced nucleic acid may be transferred to the recipient cells. It may be present only transiently in the cell or host organism.

[0091] Cells that can be transfected can be of any origin (e.g., mesodermal, ectodermal, or endodermal). Non-limiting examples of cells include cells of any tissue or organ type from the liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells), lung, brain (e.g., nerve, glia or ependymal cells) or the central or peripheral nervous system, such as the spinal cord, kidney, eye ( e.g., retinal, cellular components), spleen, skin, thymus, testes, lungs, diaphragm, heart (heart) , muscle or psoas muscle, intestine (e.g., endocrine glands), adipose tissue (white, brown, or beige), muscle Flesh (e.g., fibroblasts), synovial cells, chondrocytes, osteoclasts, epithelial cells, endothelial cells, saliva gland cells, inner ear nerve cells, or hematopoietic cells (e.g., blood or lymph). are found in the liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells), lung, and brain ( central nervous system or peripheral nervous system cells, such as nerve, glial, or ependymal cells, or the spinal cord Nervous system, kidney, eye (e.g., retinal, cellular components), spleen, skin, thymus, testes, lungs, thyroid Diaphragm, heart (cardiac), muscle or psoas, intestine (e.g., endocrine glands), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts), synoviocytes, chondrocytes, osteoclasts, epithelial cells alveolar cells, endothelial cells, salivary gland cells, inner ear nerve cells, or hematopoietic cells (e.g., blood or lymph) stem cells, such as pluripotent or multipotent progenitor cells, that can evolve or differentiate into .

[0092] In one embodiment, a "therapeutic molecule" refers to a molecule that is lacking or deficient in a protein in a cell or subject. or a peptide or protein capable of alleviating or reducing symptoms caused by The "therapeutic" peptide or protein encoded by the transgene may be, for example, Correcting a defect (expression or function) and providing a benefit to the subject, such as correcting a genetic defect It is something.

[0093] Heterologous vectors encoding useful gene products (e.g., therapeutic proteins) according to the present invention. Non-limiting examples of polynucleotides include those for diseases such as hemophilia A, hemophilia B, thalassemia, and anemia. and can be used in the treatment of diseases or disorders, including, but not limited to, blood clotting disorders. Includes things.

[0094] Encoding gene products, including but not limited to the genes and proteins disclosed herein All mammalian and non-mammalian forms of polynucleotides that are known or not known Thus, the present invention is directed to non-mammalian, non-human mammalian, and Human-derived genes and proteins, including the human genes and proteins described herein, This includes genes and proteins that function in a manner substantially similar to those of non-human mammals. Non-limiting examples of Factor IX sequences are described in Yoshitake et al., 1985, supra; Kurachi et al., 1995, supra. a; Jallat et al., 1990, supra; Kurachi et al., 1982, Proc. Natl. Acad. Sci. USA 79:6461-646 4; Jaye et al., 1983, Nucl. Acids Res. 11:2325-2335; Anson et al., 1984, EMBO J. 3: 1053-10 60; Wu et al., 1990, Gene 86:275-278; Evans et al., Proc Natl Acad Sci USA 86:10095 (1989), Blood 74:207-212; Pendurthi et al., 1992, Thromb.Res. 65:177-186; Sakar et al., 1990 , Genomics 1990, 6:133-143; and Katayama et al., 1979, Proc. Natl. Acad. Sci.USA 76 :4990-4994.

[0095] Polynucleotides, polypeptides and subsequences thereof include modified forms and variants. As used herein, the term "modify" or "variant" refers to a The terms and grammatical variations thereof refer to polynucleotides, polypeptides, or This means that the partial sequence deviates from the reference sequence. A variant sequence may have substantially the same, greater, or lesser activity or function as the reference sequence. It may have the same activity or function as the reference sequence, but may retain at least a partial activity or function of the reference sequence. In another embodiment, the modified nucleic acid encodes Factor IX and the control Factor IX Nucleic acid encoding a factor IX gene (e.g., a wild-type gene such as a human or other mammalian factor IX gene sequence) It has been modified to have fewer CpG dinucleotides compared to the normal factor IX sequence. It is being done.

[0096] Mutants also include gain- or loss-of-function mutants. For example, wild-type human factor IX factor DNA sequences, wherein mutants or variants of the protein retain activity, or Therapeutically effective or as therapeutically effective as or more effective than non-mutant human factor IX The methods and uses of the present invention include those active against naturally occurring human factor IX variants. In one non-limiting example, human factor IX, designated "Padua," contains the amino acid sequence at position 338. Padua FIX has L (leucine) instead of R (arginine). It has greater catalytic and clotting activity than human factor IX without the ua mutation. Changing residue 338 of human factor IX from arginine to alanine improved catalytic activity. (Chang et al., J. Biol. Chem., 273:12089-94 In another particular example, collagen IV plays a role in capturing factor IX. and when introduced into mammalian muscle tissue, is retained within the interstitial spaces in the muscle tissue. This means that some of the factor IX is not available to participate in blood clotting. Mutations in the sequence of factor IX (e.g., β-glucanase) that result in a protein with reduced binding to collagen IV Such mutations (e.g., loss of function) are useful for treating, for example, hemophilia. An example of a variant factor IX gene is a lysine at the fifth amino acid position from the beginning of the mature protein. It encodes a human FIX protein having the amino acid alanine instead of .

[0097] Non-limiting examples of modifications include replacing CpG with other dinucleotides in the transgene. (e.g., a gene encoding FIX with a reduced number of CpG dinucleotides) one or more nucleotides or amino acids (e.g., 1 to 2), such as the Factor IX-encoding gene 3 pieces, 3~5 pieces, 5~10 pieces, 10~15 pieces, 15~20 pieces, 20~25 pieces, 25~30 pieces 30-40, 40-50, or 50-100 or more nucleotides or residues) An example of an amino acid substitution is a conservative amino acid substitution in the capsid sequence. Another example of an acid substitution is an arginine (e.g., 4-1, 15-1, 15-2) in place of a lysine residue. -2, 15-3 / 15-5, 15-4 and / or 15-6 Further modifications can be additions to the reference sequence (e.g., , 1~3 pieces, 3~5 pieces, 5~10 pieces, 10~15 pieces, 15~20 pieces, 20~25 pieces, 25 ~30, 30-40, 40-50, or 50-100 or more nucleotides or residues insertions) and deletions (e.g., subsequences or fragments) of the reference sequence. In embodiments, the modified or variant sequence retains the function or Such modified forms and variants may be, for example, , the same as, less than, or greater than but less than the reference sequences described herein. Both may have partial function or activity.

[0098] As described herein, a variant may contain one or more non-conservative or conservative amino acids. The amino acid sequence may have differences or modifications, or both. Biological similarity is the replacement of one amino acid with a biologically, chemically, or structurally similar residue. means that the substitution does not destroy biological activity. have side chains of similar length, such as alanine, glycine, and serine, or Chemical similarity means that the residues have the same charge. Either or both are hydrophilic or hydrophobic. A particular example is isolo One hydrophobic residue, such as lysine, valine, leucine, or methionine, is substituted for another. Substitution of one group for another, or substitution of arginine for lysine , substitution of glutamic acid for aspartic acid, glutamine for asparagine Substitutions of one polar residue with another, such as a hydroxyl group for a threonine or a serine for a threonine, A particular example of a conservative substitution is isoleucine, valine, lysine, or thiamin. Substitution of lysine or methionine for one another, and arginine for lysine substitution of Glu for Aspartic acid; substitution of Glu for Asparagine One polar residue, such as a glutamine substitution, or a serine substitution for threonine, For example, conservative amino acid substitutions typically involve replacing one group with another. The following groups: glycine, alanine; valine, isoleucine, leucine; asparagine Acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine and phenylalanine, tyrosine, and the like. includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0099] Thus, the present invention provides a method for the preparation of a medicament ... (e.g., genetic mutations in the polynucleotides encoding the proteins described herein) Variants include polypeptides and protein variants. Variants are not intended to be derivatives of naturally occurring polynucleotides, proteins or peptides. It may differ from a reference sequence such as a polynucleotide, protein, or polypeptide. Such variants of the polypeptide are those in which the polynucleotide, protein or polypeptide is altered. modified or altered using recombinant DNA technology to have altered or additional properties The present invention includes proteins or polypeptides that can be decorated with PEG.

[0100] At the nucleotide sequence level, naturally occurring variant genes and non-naturally occurring variant genes are identical to the reference gene. For genes, typically at least about 50%, more typically about 70%, and even more typically Therefore, for example, FIX, which has a reduced number of CpG dinucleotides, is about 80% identical to The gene must be 80% or more identical to the wild-type FIX gene or For genes, 80%-85%, 85%-90%, 90%-95%, or higher (e.g., For example, 96%, 97%, 98%, 99% identity to the wild-type FIX gene. It may be possible.

[0101] At the amino acid sequence level, naturally occurring or non-naturally occurring variant proteins are Typically at least about 70% identical, more typically about 80% identical to the protein are identical, more typically about 90% or more identical. Essential sites are allowed to reside in non-conserved regions (e.g., less than 60%, less than 50%). or less than 70% identity, such as less than 40% identity). At least 60%, 70%, 75% or more identity to a reference sequence (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity )

[0102] "Identity," "homology," and their grammatical variations refer to the relationship between two or more mentioned components. are the same when they are "aligned" sequences. If two polypeptide sequences are identical, then the polypeptide sequences must be at least The two polynucleotides have the same amino acid sequence within the referenced region or portion. If the polynucleotide sequences are identical, then the polynucleotide sequences must be identical to at least the referenced region. The identity is determined by the sequence of the gene or region. It may be across an area (region or domain) of identity. A "region" represents a portion of two or more identical components that are referenced. If the peptide or nucleic acid sequences are identical over one or more areas or regions of the sequence, then the Protein or nucleic acid sequences share a region of identity. "Aligned" sequences are sequences that are It represents a sequence of polynucleotides or proteins (amino acids) of a number of sequences, often referred to as a reference sequence. It includes corrections for missing or added bases or amino acids (gaps) compared.

[0103] The identity may be over the entire length or a portion of the sequence. The length of sequences that share the identity percentage can be 2, 3, 4, 5 or more sequences. A polynucleotide or amino acid sequence (e.g., 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In a further specific embodiment, the identity is The length of the shared sequence is 21 or more consecutive polynucleotides or amino acids ( For example, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 pieces, 31 pieces, 32 pieces, 33 pieces, 34 pieces, 35 pieces, 36 pieces, 37 pieces, 38 pieces, 39 pieces, In a further specific embodiment, the polynucleotide sequence is a sequence of 40 consecutive polynucleotides or amino acids. The length of the sequence sharing identity is 41 or more contiguous polynucleotides or Amino acids (e.g., 42, 43, 44, 45, 45, 47, 48, 49 In a further specific embodiment, the polynucleotide sequence is a sequence of 50 or 50 consecutive polynucleotides or amino acids. In this case, the length of the sequence that shares identity is 50 or more consecutive polynucleotides. or amino acids (e.g., 50 to 55, 55 to 60, 60 to 65, 65 to 70 pieces, 70~75 pieces, 75~80 pieces, 80~85 pieces, 85~90 pieces, 90 pieces~ Contiguous polynucleotides such as 95, 95-100, 100-110, etc. is an amino acid).

[0104] The term "homologous" or "homology" refers to the relationship between two or more components being referred to. means sharing at least partial identity over a given region or portion. The same or identical "area, region, or domain" refers to two or more components This means that parts of the two sequences share homology or are identical. If two sequences are identical over more than one sequence region, then the two sequences are considered identical over those regions. "Substantial homology" means that one molecule shares identity with another molecule that shares homology. the structure of a reference molecule having a corresponding region or portion thereof; At least one or more partial structures or functions (e.g., biological functions or activities) of a compound. that the protein is structurally or functionally conserved to have or be expected to have at least means.

[0105] The degree of identity (homology) between two sequences can be determined using computer programs and / or mathematical algorithms. The percentage of sequence identity (homology) can be determined using the The algorithm generally calculates sequence gaps and fragments over the region or area of ​​comparison. Explain mismatches. For example, the BLAST (e.g., BLAST 2.0) search algorithm rhythm (e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), published by NCBI. Typical search parameters are: 2; gap open 5; gap extension 2. For polypeptide sequence comparison, the BLASTP algorithm typically uses scores such as PAM100, PAM250, BLOSUM62 or BLOSUM50 Used in conjunction with an alignment matrix. FASTA3 and SSEARCH sequence comparison programs also quantify the degree of identity. (Pearson et al., Proc. Natl. Acad. Sci . USA 85:2444 (1988); Pearson, Methods M ol Biol. 132:185 (2000); and Smith et al., J.M. ol. Biol. 147:195 (1981). Delaunay-based topology Programs have also been developed to quantify the similarity of protein structures using mapping (B ostick et al., Biochem Biophys Res Commun. 304: 320 (2003)).

[0106] The polynucleotide may contain additions and insertions (eg, one or more heterologous domains). Additions (e.g., heterologous domains) can be achieved by covalent attachment of any type of molecule to a synthetic or Typically, additions and insertions (e.g., heterologous domains) are Conferring complementary or distinct functions or activities.

[0107] Additions and insertions include chimeric or fusion sequences and may be modified from a reference native (wild-type) sequence. Polynucleotides that are not normally present and have one or more molecules covalently attached to the sequence The term "fusion" or "chimeric" and These grammatical variants, when used in reference to a molecule, refer to a portion or parts of that molecule. The moiety contains distinct (heterogeneous) components from the molecule, which are typically means that they do not occur together in nature, i.e., one of a fusion or chimera A moiety may comprise or consist of moieties that do not occur together in nature, and may be structurally It is something to be distinguished.

[0108] The term "vector" refers to a plasmid, virus (e.g., AAV vector), or other vector. a delivery vehicle that can be engineered by insertion or integration of a polynucleotide; Such vectors are used to introduce / transfer polynucleotides into cells and to transcribe or translate the inserted polynucleotide in the cell. The vector nucleic acid sequence can be used for a cloning vector. Generally, it contains at least an origin of replication for propagation in a cell and, optionally, a heterologous polynucleotide. Nucleotide sequences, expression control elements (e.g., promoters, enhancers), introns , ITR, selectable marker (e.g., antibiotic resistance), polyadenine (polyadenylation It contains additional elements such as a sequence (also called a transcription factor).

[0109] Viral vectors are derived from or contain one or more nucleic acid elements comprising the viral genome. A particular viral vector is the adeno-associated virus (AAV) ) vectors, including lentiviral and parvoviral vectors.

[0110] As used herein, the term "recombinant" refers to a recombinant lentivirus. or viral vectors, such as recombinant parvovirus (e.g., AAV) vectors. As modifiers, and as recombinant polynucleotides and recombinant polypeptides, After modification of the sequence, the construct (e.g., AAV or sequence) may be in a form that does not normally occur in nature. This means that the vector has been engineered (i.e., modified) in a similar manner. One particular example of a suitable recombinant vector is a vector containing a sequence normally contained in the genome of a wild-type virus (e.g., AAV). This may be the case when a non-existent polynucleotide is inserted into the viral genome. For example, an example of a recombinant polynucleotide is a heterologous polynucleotide that encodes a protein. When a nucleotide (e.g., a gene) is cloned into a vector, the 5', 3' The gene is usually present in the virus ( For example, the recombinant may be integrated into the genome of an AAV. is not necessarily used herein to refer to vectors such as viruses and AAV vectors, and It is used in reference to sequences such as, but not limited to, polynucleotides and polypeptides. , viruses, AAV, and recombinant sequences containing polynucleotides and polypeptides Forms are expressly included notwithstanding any such omissions.

[0111] A recombinant viral "vector" or "AAV vector" refers to a virus (e.g., an AA V) using molecular methods to remove the wild-type genome from the host and replace it with a non-naturally occurring nucleic acid. and heterologous polynucleotide sequences (e.g., CpG fragments) like AAV by Modified nucleic acid sequences encoding human FIX, such as FIX with a reduced number of nucleotides, are also available. For AAV, typically, the AA One or both inverted terminal repeat (ITR) sequences of the V genome are retained in the AAV vector. A "recombinant" viral vector (e.g., AAV) contains all of the viral genome. or a portion thereof, which is a heterologous polynucleotide sequence (e.g., a reduced number of CpG dinucleotides) a modified nucleic acid sequence encoding human FIX, such as a modified FIX a sequence that is non-native to the nucleic acid of the genome of a vector (e.g., AAV), It is distinct from the genome of a virus (e.g., AAV). Therefore, the incorporation of non-native sequences (e.g., a gene encoding human FIX, such as FIX with a reduced number of CpG dinucleotides) The modified nucleic acid sequence (which is a nucleic acid sequence that encodes a specific nucleotide sequence) can be used to make the viral vector (e.g., AAV) a "recombinant" vector. In the case of AAV, it can be referred to as a "rAAV vector."

[0112] Recombinant vector (e.g., lentivirus, parvovirus, AAV) sequences are then transferred to the host cells for subsequent ex vivo or in vivo transfection. For infection (gene transfer) of cells in vitro or in vivo, " The recombinant vector sequence may be encapsidated into an AAV particle. When the particle is encapsulated or enveloped, it can be referred to as a "rAAV." Such particles contain proteins that encapsidate or package the vector genome. Specific examples are , containing viral envelope proteins and, in the case of AAV, capsid proteins .

[0113] With respect to recombinant plasmids, the vector "genome" refers to the portion of the recombinant plasmid sequence that is which are ultimately enveloped or encapsidated to form viral (e.g., AAV) particles. Recombinant plasmids are used to construct or produce recombinant vectors. When a vector is used, the vector genome is the vector genome sequence of the recombinant plasmid. This non-vector genome of the recombinant plasmid does not contain any portion of the "plasmid" that does not correspond to the The genome portion is called the "plasmid backbone" and is required for propagation and recombinant virus production. It is important for the cloning and propagation of plasmids, a process that occurs in the is not packaged or encapsidated into a viral (eg, AAV) particle.

[0114] Thus, the vector "genome" is packaged or encoded by a virus (e.g., AAV). The capsid is encapsidated and expresses the portion of the vector plasmid containing the heterologous polynucleotide sequence. The non-vector genome portion of the recombinant plasmid is the "plasmid backbone" and The plasmid backbone is important for cloning and propagation of plasmids, e.g. It has a selectable marker such as mycobacterium typhimurium, but the non-vector genome of the recombinant plasmid The cytoplasmic portion is itself enveloped or encapsidated by the virus (e.g., AAV). I can't.

[0115] The amount of rAAV that encapsidates / encapsidates the vector genome can be determined, for example, by quantitative PCR. For example, this assay can be performed using real-time quantitative polymerase chain reaction assays. The chain reaction measures the physical number of packaged vector genomes, e.g., bulk A Can be performed at various stages of the manufacturing / purification process for AV vectors and final products .

[0116] Recombinant vector sequences are manipulated by the insertion or incorporation of polynucleotides. As disclosed herein, vector plasmids generally comprise a vector vector for propagation in a cell. The vector contains at least an origin of replication for the expression of the vector and one or more expression control elements.

[0117] The vector sequence, including the AAV vector, may contain one or more "expression control elements." Typically, expression control elements affect the expression of an operably linked polynucleotide. Nucleic acid sequences that affect the expression of a gene, such as promoters and enhancers, are also described herein. The regulatory elements present in the vector, including the expression regulatory elements, are expressed in a suitable heterologous polynucleotide. The promoters are included to facilitate transcription and, where appropriate, translation of the promoter (e.g., promoter Motors, enhancers, splicing signals for introns, mRNA introns Maintenance of the correct reading frame of the gene to allow in-frame translation, and Such factors typically act in cis and are referred to as "cis-acting" factors. It is called a transformer, but it may also act in a transformer.

[0118] Expression regulation is achieved at the levels of transcription, translation, splicing, and message stability. Typically, expression control elements that regulate transcription are involved in the transcription of the transcribed polynucleotide. Factor IX coding variants (e.g., FIX with a reduced number of CpG dinucleotides) The expression control gene is located near (i.e., "upstream") the 5' end of the modified nucleic acid that controls the expression. The regulatory factor may be located at the 3' end (i.e., "downstream") of the transcribed sequence, or may be located downstream of the transcriptional sequence. The expression control element may be located within a gene (e.g., in an intron). The polynucleotide may be spaced apart or spaced apart (e.g., 1 ~10, 10-25, 25-50, 50-100, 100-500, or However, the nucleotides (more than 10 nucleotides) may be spaced apart by a considerable distance. Due to the polynucleotide length constraints of certain vectors, such as AAV vectors, Such expression control elements typically contain 1 to 1000 nucleotides from the transcribed polynucleotide. It will be in the range of nucleotides.

[0119] Functionally, expression of an operably linked heterologous polynucleotide results in the expression of that factor (e.g., , promoter) regulates transcription of the polynucleotide and, optionally, translation of the transcript. The expression of the gene is at least partially controllable by the factor. A typical example is a promoter, which is usually located 5' to the transcribed sequence. Examples include enhancers, which can be located 5', 3', or within the transcribed sequence. - is.

[0120] As used herein, a "promoter" refers to a promoter that encodes a recombinant product. It can refer to a nucleic acid (e.g., DNA) sequence located adjacent to a nucleotide sequence. A promoter is typically linked to adjacent sequences (e.g., heterologous polynucleotides, The promoter is operably linked to a nucleic acid encoding a factor IX. The promoter typically increases the amount of heterologous expression compared to the amount expressed in the absence of the promoter. Increases the amount expressed from the source polynucleotide.

[0121] As used herein, an "enhancer" refers to a heterologous polynucleotide. Enhancers can refer to sequences located adjacent to a promoter. - located upstream of the element but downstream of the DNA sequence (e.g., heterologous polynucleotide) Alternatively, enhancers may be positioned within the DNA sequence and function. , 100 base pairs, 200 base pairs, or 300 base pairs or more upstream of the heterologous polynucleotide Enhancers can be located downstream or upstream of a promoter. Increasing the expressed heterologous polynucleotide beyond the increased expression provided by the To make.

[0122] Expression control elements (e.g., promoters) are those that are active in particular tissue or cell types. These are referred to herein as "tissue-specific expression regulators / promoters." Tissue-specific expression regulators typically express expression regulators that regulate specific cells or tissues (e.g., liver, brain, central nervous system, etc.). It is active in the nervous system, spinal cord, eyes, retina, bones, muscles, lungs, pancreas, heart, kidney cells, etc. Expression control factors are those that are unique to a particular cell type, tissue type, or organ type. These proteins are recognized by transcriptional activator proteins or other regulators of transcription, It is typically active in these cells, tissues or organs.

[0123] Examples of promoters active in skeletal muscle are skeletal α-actin, myosin light chain 2A, and dimer promoters derived from genes encoding strophin and muscle creatine kinase; and , including synthetic muscle promoters with higher activity than natural promoters (e.g., Li (See, e.g., Nat. Biotech., 17:241-245 (1999)). An example of a promoter that is tissue-specific is the human alpha-1 antitrypsin (hAAT) promoter. It is a motor; in particular, albumin, Miyatake, et al. J. Virol., 71:5124-32 (1997); hepatitis B v irus core promoter, Sandig, et al., Gene Ther. 3:1002-9 (1996); alpha-fetoprotein (AFP) , Arbuthnot, et al., Hum. Gene. Ther., 7:1503- 14 (1996)], bone (osteocalcin, Stein, et al., M ol. Biol. Rep., 24:185-96 (1997); ialoprotein, Chen, et al., J. Bone Miner. Res. 11 :654-64 (1996)), lymphocytes (CD2, H ansal, et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell rece ptor a chain), neuronal (neuron-specific enolase (NSE) promoter, Andersen, et al., Cel l. Mol. Neurobiol., 13:503-15 (1993); urofilament light-chain gene, Piccioli, et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991); the neuron-specific VGF gene, Pi See Ccioli, et al., Neuron, 15:373-84 (1995). An example of an enhancer active in this context is apolipoprotein E (apoE), HCR-1, and H CR-2 (Allan et al., J. Biol. Chem. 272:29113- 19 (1997).

[0124] Expression control elements drive the expression of polynucleotides in many different cell types. These include ubiquitous or promiscuous promoter / enhancers that can Cytomegalovirus (CMV) immediate-early promoter / enhancer sequence, Rous sarcoma virus Respiratory syncytial virus (RSV) promoter / enhancer sequences and their role in various mammalian cell types Other viral promoters / enhancers or synthetic elements not found in nature that are active (e.g., Boshart et al., Cell, 41:521-530 (1985)), SV4 0 promoter, dihydroleaf reductase promoter, cytoplasmic β-actin promoter , and phosphoglycerol kinase (PGK) promoters, but are not limited to these. It is not something that can be determined.

[0125] Expression control elements can confer expression in a controllable manner, i.e., signal or The stimulus increases or decreases expression of the operably linked heterologous polynucleotide. Regulation of increased expression of an operably linked polynucleotide in response to a signal or stimulus Factors that can be induced are called "inducible factors" (i.e., those that can be induced by a signal). Specific examples include hormone (e.g., steroid) inducible promoters, The present invention is not limited to the above. Regulatable factors that decrease the expression of a protein are called "inhibitory factors" (i.e., The signal regulates expression such that expression increases when the signal is removed or absent. Typically, the increase or decrease provided by such a factor is The greater the amount of signal or stimulus, the greater the increase in expression or A specific, non-limiting example is the zinc-induced ovine metallothionein (MT) ) promoter; steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter Motor; T7 polymerase promoter system (WO98 / 10088); Tet The lacycline repression system (Gossen et al., Proc. Natl. Acad. S ci. USA, 89:5547-5551 (1992)); tetracycline induction system; (Gossen et al., Science, 268:1766-1769 (1995); H Arvey et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); see also the RU486 inducible system (Wang et al., Nat. Biotech. 1 5:239-243 (1997), and Wang et al., Gene Ther. 4:43 2-441 (1997); and the rapamycin-inducible system (Magari et al., J. Cli n. Invest. 100:2865-2872(1997); Rivera et al., N at. Medicine. 2:1028-1032 (1996). In this context Other controllable factors that are useful in this context include specific physiological states (e.g., body temperature, acute phase, development). It is controlled by.

[0126] Expression control elements also include elements native to a heterologous polynucleotide. If it is desired that expression of the original polynucleotide mimics natural expression, the natural regulatory A promoter or other factor may be used to express a heterologous polynucleotide. Expression may be transient or developmental, or tissue-specific, or dependent on specific transcriptional stimuli. Natural factors can be used if they are to be controlled depending on the gene. , other natural expression control elements such as polyadenylation sites or Kozak consensus sequences can also be used.

[0127] As used herein, "operably linked" or "operably linked" The term "combined" refers to the physical or functional contiguous arrangement of components that allows them to function in an intended manner. It refers to something that allows them to function. In the example of a factor, the relationship is where the regulatory factor regulates the expression of the nucleic acid. For example, two operably linked DNA sequences are those that A relationship in which at least one of the DNA base sequences can have a physiological effect on the other sequences. This means that the molecules are arranged in a cis or trans relationship.

[0128] Therefore, modified nucleic acid sequences encoding human FIX proteins and AAV vectors Viral vectors, such as lentiviral vectors and parvoviral vectors, including In addition to vectors and plasmids containing the nucleic acid sequence, these compositions may also contain additional nucleic acid elements. These elements include, but are not limited to, AAV ITR sequences, expression control sequences, and the like. Regulatory (e.g., promoter / enhancer) elements, transcription termination signals, or stop codons , sequences flanking the polynucleotide, 5' or 3' untranslated regions (e.g., polyadenylation), or the introduction of genomic human factor IX (SEQ ID NO: 13). It may contain one or more copies of an intron, such as all or part of intron I.

[0129] Nucleic acid factors may be used, for example, to improve packing and reduce the presence of contaminating nucleic acids (e.g., to reduce the packing of the plasmid backbone), e.g., filler sequences or stuffer polypeptides As disclosed herein, AAV vectors can be used in a variety of applications, including the development of novel AAV vectors. Typically, a defined range, generally about 4 kb to about 5.2 kb or slightly larger Therefore, for shorter sequences, the virus The normal size of the viral genome sequence acceptable for AAV vector packaging into the vesicle In order to adjust the length to be at or near the size of the insert fragment, A stuffer or filler may be included. In some embodiments, the filler nucleic acid Sequence / Stuffer Nucleic acid sequences are sequences that are generated from untranslated (non-protein-coding) segments of nucleic acids. In certain embodiments of the AAV vector, the heterologous polynucleotide sequence has a length of less than 4.7 kb, and the filler or stuffer polynucleotide sequence is , when combined with the heterologous polynucleotide sequence (e.g., inserted into a vector) , approximately 3.0kb to 5.5kb, or approximately 4.0kb to 5.0kb, or approximately 4.3 It has a total length of 4.8 kb.

[0130] Introns are used to achieve the length of the AAV vector that can be packaged into viral particles. It can also function as a filler sequence or a stuffer polynucleotide sequence. Introns and and intron fragments (e.g., a portion of intron I of FIX) increase expression. The inclusion of an intron element can be used to express the gene in the absence of the intron element. expression can be increased compared to expression (Kurachi et al., 1995, sup ra).

[0131] The use of introns is not limited to the inclusion of Factor IX intron I sequences. , and may contain other introns, which may be associated with the same gene (e.g., For example, if the nucleic acid encodes factor IX, the introns are located in the genomic sequence of factor IX. (derived from an existing intron) or from an entirely different gene or other DNA sequence. and therefore, the cognate (related) gene (heterologous polynucleotide) The nucleic acid sequence encodes all or part of the same protein encoded by the genomic sequence. and non-cognate (unrelated) genes (where the heterologous polynucleotide sequence is (encoding proteins distinct from those encoded by the genome sequence) Other untranslated (protein-coding) regions of nucleic acid, such as introns found in genomic sequences The region (which does not encode a gene) may be a filler polynucleotide sequence or a stuffer polynucleotide sequence according to the present invention. It can also function as a polynucleotide sequence.

[0132] As used herein, a "portion of Intron I" refers to a region from about 0.1 kb to about A region of intron I having a nucleotide length of up to 1.7 kb, Typically, when compared to expression of FIX in the absence of a portion of the plasmid or increasing the expression of factor IX on a viral vector template by about 1.5 times or more The more specific portion is a 1.3 kb portion of intron I. A non-limiting example of a Factor IX intron I sequence is set forth in SEQ ID NO: 13. The intron is a chimera composed of the 5' and 3' portions of the first intron of FIX. It is A.

[0133] Expression control elements, ITRs, polyA sequences, filler sequences, or stuffer polynucleotides The code sequence may vary in length. In particular embodiments, the expression control elements, ITRs, p olyA, or a filler or stuffer polynucleotide sequence The rows are generally 1-10, 10-20, 20-30, 30-40, 40-50, and 50-60. , 60~75, 75~100, 100~150, 150~200, 200~250, 25 0~300, 300~400, 400~500, 500~750, 750~1000, 1 nucleotide length of 000-1,500, 1500-2000, or 2000-2500 It is an array of sizes.

[0134] According to one non-limiting embodiment, the AAV vector comprises a 4-1 capsid mutant VP1 tag. In transduced mammalian cells, AAV capsids containing the protein (SEQ ID NO: 4) and a genome for expressing a heterologous gene.

[0135] The capsid of this vector contains the VP2 protein and VP3 protein (SEQ ID NO:27 and SEQ ID NO:3, respectively). According to one embodiment, the VP1 protein and the VP2 protein are approximately 1:1 (or alternatively The VP3 protein is present in a stoichiometric ratio of 1:1, and the VP3 protein is present in either VP1 or VP2. For both VP1 and VP2, the ratios were roughly 5:1, 6:1, 7:1, 8: 1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1 , 17:1, 18:1, 19:1, 20:1, or other ratios. do.

[0136] In some embodiments, the 4-1 capsid mutant proteins (VP1, VP2, VP 3) AAV vector genomes, including but not limited to those having capsids containing The system contains a heterologous nucleic acid sequence encoding the human Factor IX (FIX) protein. In some embodiments, the FIX protein is wild-type, and in other embodiments, The FIX protein may contain substitutions or other mutations that alter the activity of the protein. In some embodiments, the mutations affect FIX catalytic activity and / or its ability to function as a procoagulant. In some embodiments, the FIX protein increases the activity of the protein. dua FIX protein, and the amino acid sequence corresponding to position 338 of the FIX protein In some embodiments, human FIX has an arginine to alanine substitution. The genes encoding FIX (including FIX Padua) contain, for example, CpG dinucleotides. Other types of codon optimization include codon optimization by shortening or removing codons. It is equally possible.

[0137] In some embodiments, the genome of the AAV vector comprises a left and right end of the genome (i.e., reverse orientations derived from AAV2, located at the 5' and 3' ends, respectively In some embodiments, the left ITR further comprises an intermediate terminal repeat (ITR). Nucleotides 1 to 141 from SEQ ID NO:12 (disclosed herein as SEQ ID NO:13) The right ITR comprises or consists of the nucleotides set forth in SEQ ID NO: 12. Nucleotides 4097 to 4204 (disclosed herein as SEQ ID NO:20) derived from Each ITR is made up of a nucleic acid sequence of variable length. Therefore, it may be separated from other elements in the vector genome.

[0138] In other embodiments, the genome of the AAV vector comprises expression control elements including a promoter. and optionally further comprising an enhancer. The AAV vector genome contains both a promoter and an enhancer, which are constitutively In some embodiments, the promoter may be inducible, inducible, or tissue-specific. The promoter, enhancer, or both are tissue-specific. Both the promoter and the promoter are selective in hepatocytes compared to certain other cell types. According to one embodiment, the enhancer is a human ApoE HCR-1 enhancer. The promoter is a human alpha-1 antitrypsin gene. In some embodiments, the promoter is the entire or a portion of the AA T promoter. The V vector genome is nucleotides 152 to 472 (herein) derived from SEQ ID NO: 12. Apo comprising or consisting of the nucleotides disclosed in SEQ ID NO: 14 E. Contains the HCR-1 enhancer and nucleotide 48 from SEQ ID NO:12 2-878 (disclosed herein as SEQ ID NO: 15) or In some embodiments, the AAT promoter comprises the ApoE HC The R-1 enhancer is located 5' of the AAT promoter, and its sequence is: They may be adjacent or separated by other nucleotide sequences. According to some embodiments, the enhancer and promoter are linked to a nucleic acid sequence encoding Factor IX. It is located 5' of the sequence and is adjacent to and linked to the first exon of the factor IX gene. or may contain 5' untranslated sequences (UTR) or Alternatively, it may be separated therefrom by some other sequence that functions as a spacer. In an exemplary, non-limiting embodiment, the 5'UTR sequence is a nucleic acid derived from SEQ ID NO:12. It comprises or consists of nucleotides 879 to 907.

[0139] In some embodiments, the gene encoding FIX, including the naturally occurring FIX Padua, The gene contains one or more introns present in the genomic sequence of human factor IX. In other embodiments, all introns may be omitted, for example, 10, and is also referred to herein as "FIX39." If present, introns are referred to as coding sequences for the As described above, it can act as a stuffer or filler sequence. Even if the entire gene is codon-optimized to reduce or eliminate CpG dinucleotides, good.

[0140] In one particular, non-limiting embodiment, the human Factor IX used in the AAV vector is The encoding gene contains nucleotides 908 to 3731 derived from SEQ ID NO: 12. or consisting of said nucleotides, which encode FIX Padua, It has been codon-optimized to remove CpG dinucleotides. 1 (nucleotides 908-995 from SEQ ID NO: 12), the first intron (sometimes Known as intron I; nucleotides 996-24 from SEQ ID NO:12 33), and exons 2 to 8 (nucleotides 2434-37 from SEQ ID NO: 12) 31).

[0141] In one embodiment, the gene encoding factor IX contains at its 3' end a human factor I The 3'UTR sequence from the Factor X gene (nucleotide 373 from SEQ ID NO: 12) 2-3779), and / or the Factor IX gene or A polyadenylate (polyA) sequence derived from another gene may be attached to the In one non-limiting example, the polyA sequence is derived from the bovine growth hormone (bGH) gene. and may comprise nucleotides 3820 to 4047 derived from SEQ ID NO: 12. In some embodiments, the nucleotide sequence may be 3 or 4. 'UTRs are variably spaced from the polyA sequence by intervening sequences of nucleotides It may be arranged.

[0142] In some embodiments, the above-described elements are present in a single AAV vector genome. According to a non-limiting example, the AAV vector may be In order, the left AAV ITR, the ApoE HCR-1 enhancer (or a portion thereof), hAAT promoter (or a part thereof), a part of the 5'UTR of human factor IX, human factor Nucleic acid encoding factor IX Padua (containing one or more introns, such as intron I) Optionally, a portion of the 3'UTR of human factor IX, a polyA sequence derived from bGH ( or a part thereof) and the genome including the AAV2 ITR. In some of these embodiments, the left AAV2 ITR comprises the nucleic acid sequence of SEQ ID NO:13. the ApoE HCR-1 enhancer has the nucleic acid sequence of SEQ ID NO: 14; The AAT promoter has the nucleic acid sequence of SEQ ID NO:15; the 5'UTR has the nucleic acid sequence of SEQ ID NO:1 6; the gene encoding FIX Padua (including intron I) , encoding the FIX protein encoded by the nucleic acid sequence of SEQ ID NO: 10; 3'UT R has the nucleic acid sequence of SEQ ID NO:18; the polyA region has the nucleic acid sequence of SEQ ID NO:19 and the right AAV2 ITR has the nucleic acid sequence of SEQ ID NO:20.

[0143] According to one embodiment, the genome of the AAV vector comprises a nucleotide sequence derived from SEQ ID NO:12. Nucleotides 1 to 4204, or at least 95%, 96%, or 97% of the nucleotides %, 98% or 99% identical to, or In some of these embodiments, the capsid comprises a 4-1 VP1 capsid. The VP2 and VP2 capsid proteins were prepared using the VP2 mutant (SEQ ID NO: 4) and the corresponding VP2 and VP2 capsid proteins. In one specific, non-limiting embodiment of the AAV vector, as used herein, "AAV The vector, called "-FIX39Padua," contains the 4-1 capsid mutant protein. (VP1, VP2, VP3) and a capsid derived from SEQ ID NO: 12 The genome contains a single strand containing a nucleic acid sequence corresponding to nucleotides 1 to 4204 of the target gene.

[0144] As used herein, an AAV "empty capsid" refers to an AAV vector genome. In contrast to the "capsid-containing genome" which contains the vector genome (and therefore The empty capsid contains the complete virus (including the AAV vector genome). The virus is a virus-like particle in that it reacts with one or more antibodies that react with the virus.

[0145] Empty capsids may be included in the AAV vector formulation. AV empty capsids may be added to AAV vector formulations or may be used as described herein. They may be administered separately to the subject according to the method of

[0146] Without intending to be bound by theory, AAV empty capsids are to bind or react with antibodies against the AAV vector, thereby reducing inactivation of the AAV vector Such decoys are believed to act as decoys for targeting AAV vectors. The antibody acts to absorb the AAV vector transgene, thereby Increase or improve transduction (transgene delivery) and then increase the transcript and / or the encoded Increases cellular expression of proteins.

[0147] Empty capsids can be produced and purified to a given quality, and their quantity can be determined. For example, concentration titration of empty capsids can be performed as described by (Sommer et al., Mol. Ther. 2003 Jan;7(1):122-8) Optical at a wavelength of 280 nm It can be measured by density-based absorbance measurement.

[0148] Empty AAV or empty capsids are sometimes found naturally in AAV vector preparations. Natural mixtures such as may be used in accordance with the present invention, or, if desired, It may be engineered to increase or decrease the amount of empty capsid and / or vector. For example, the amount of empty capsid is optionally used for vector-mediated gene transfer in a subject. It is expected to reduce the inhibitory effects of antibodies reactive with the AAV vectors that are intended to be used. The use of empty capsids is described in U.S. Publication No. 2014 / 0336245. It is written.

[0149] In some embodiments, the AAV empty capsid is formulated with the AAV vector. and / or administered to a subject. In particular embodiments, the AAV empty capsid is With a smaller or equal amount of vector (e.g., 1.5-fold to 1.5-fold greater than AAV empty capsid) 100-fold higher AAV vector or AAV empty capsid ratio In other specific embodiments, the AAV vector is formulated in excess of the AAV vector. capsid (e.g., 1x more AAV empty capsid relative to the AAV vector, e.g., A It is formulated with 1.5 to 100 times the amount of AAV empty capsid relative to the AV vector. Interestingly, subjects with low to negative titers of AAV neutralizing antibodies had smaller Amount of empty capsid (1 to 10 times the amount of AAV empty capsid relative to the AAV vector, 2-6 times more AAV empty capsids than the vector, or approximately 4-5 times more AAV empty capsids than the vector. 2 times more AAV empty capsids).

[0150] According to one embodiment, the pharmaceutical composition comprising the AAV vector comprises a 4-1 capsid in the composition. AAV vectors, including those containing mutant VP1, VP2, and VP3 proteins (i.e., those containing vector genomes) The ratio of empty capsids to V vectors was approximately 1.1, 1.2, 1.3, 1.4, and 1. .5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2 .5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3 .5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4 .5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5 .5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6 .5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7 .5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8 .5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9 It may be 0.5, 9.6, 9.7, 9.8, 9.9, 10 to 1, or another ratio. It may be a rate.

[0151] In some embodiments, the empty capsid is the same as that present in the AAV vector. VP1 capsid protein, VP2 capsid protein, and VP3 capsid protein In other embodiments, the empty capsid is different from that found in AAV vectors. This includes VP1 protein, VP2 protein, and VP3 protein, which have the amino acid sequence Typically, but not always, the capsid proteins of the empty capsid and the capsid proteins of the AAV vector are If the capsid proteins of the two serotypes are not sequence identical, they are of the same serotype. cormorant.

[0152] According to some embodiments, the composition comprises AAV-FIX39 Padua (or the equivalent). having the same capsid and at least 95%, 96%, 97%, 98%, or 99% identical genomic sequence) and optionally an excess of empty capsids containing the same capsid proteins, The ratio of empty capsids to capsids was approximately 1.1, 1.2, 1.3, 1.4, 1. 5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2. 5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3. 5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4. 5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5. 5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6. 5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7. 5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8. 5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9. 5, 9.6, 9.7, 9.8, 9.9, 10 to 1, or some other ratio. In an embodiment of the present invention, the AAV-FIX39 Padua to empty capsid in the composition In another embodiment, the ratio of AAV-FIX39 Padua to AAV-FIX39 is about 1:5. The composition comprising the empty capsid is useful for treating hemophilia, including severe, moderate, or mild hemophilia B. B.

[0153] A "selectable marker gene" that, when expressed, confers antibiotic resistance to the transfected cell. A reporter gene refers to a gene that confers a selectable phenotype, such as kanamycin. A reporter gene is one that provides a detectable signal. Non-limiting examples of reporter genes include: One good example is the luciferase gene.

[0154] Nucleic acids, polynucleotides, expression vectors (e.g., vector genomes), including modified forms Plasmids can be transformed into cells using a variety of standard cloning and recombinant DNA techniques. They can be produced by cell expression or by in vitro translation and chemical synthesis techniques. Polynucleotide purity can be measured by sequencing, gel electrophoresis, etc. For example, nucleic acids can be identified by hybridization or computer-based methods. Database screening techniques can be used to isolate These include, but are not limited to: (1) Genomic DNA or DNA with probes for detecting multiple homologous nucleotide sequences Hybridization of cDNA libraries (2) Identifying multiple polynucleotides with shared structural features, for example, using expression libraries. Antibody screening to detect peptides (3) Genomic DNA using primers that can anneal to the nucleic acid sequence of interest A or polymerase chain reaction (PCR) for cDNA (4) Computer search of sequence databases for related sequences (5) Differential screening of subtraction nucleic acid libraries

[0155] The term "isolated," when used as a modifier of a composition, means that the composition is isolated from humans. that it was made by hand or that it was made entirely or entirely from the in vivo environment in which it naturally occurs It means that the composition is at least partially separated. Generally, the separated composition is the composition that is at least partially separated. The composition may contain one or more substances with which it is normally associated in nature (e.g., one or more tags). Substantially free of proteins, nucleic acids, fats, carbohydrates, and cell membranes. may contain, for example, a recombinant vector sequence (e.g., rAAV) or a vector genome. or human-made particles, such as capsid-enveloped virus particles and pharmaceutical preparations. The term "isolated" does not exclude hybrid / chimeric, Multimers / oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or Alternative physical forms of the composition, such as derivative forms or forms made by the hand of man. This does not exclude forms expressed in different host cells.

[0156] The methods and uses of the present invention may involve the production of heterologous cells in host cells, including dividing and / or non-dividing cells. Providing a means for delivering (transfecting) the source polynucleotide (transgene) The recombinant vector (e.g., rAAV) sequences, vector genomes, recombinant viruses of the present invention The present invention relates to a method for the delivery of nucleic acids or proteins to a patient in need thereof as a therapeutic method. It is further useful in methods of delivering, administering or providing to a subject in need thereof. The nucleic acid can be transcribed and the protein produced in vivo in the subject. because the body has a nucleic acid or protein deficiency or because the nucleic acid or protein in the subject The production of pak may have some therapeutic effect, whether therapeutic or not. The subject may benefit from nucleic acids or proteins, or Or it may require protein.

[0157] Generally, recombinant lentiviral or parvoviral vectors (e.g., AAV) The sequences, vector genomes, recombinant viral particles, methods and uses are insufficient or undesirable and treating or ameliorating one or more symptoms associated with any disease associated with abnormal gene expression. To deliver any heterologous polynucleotide (transgene) that has a biological effect Recombinant lentiviral or parvoviral vectors (e.g., (e.g., AAV) sequences, plasmids, vector genomes, recombinant viral particles, methods and uses The use can be used to provide therapy for a variety of medical conditions.

[0158] The nucleic acids, vectors, recombinant vectors (e.g., rAAV), vector genomes and The recombinant viral particles, methods and uses allow the treatment of genetic diseases. The condition is usually a recessively inherited deficiency of an enzyme and, at least occasionally, a regulatory protein. They are divided into two classes: dominantly inherited imbalances and those involving steroid or structural proteins. For deficiency diseases, replacement therapy involves bringing normal genes into the affected tissue. and to create animal models for the disease using antisense mutations. For diseases of imbalance, model Gene transfer can be used to create a pathology in a system, which then counteracts the pathology. The model system can be used in efforts to treat deficiencies. Site-specific integration of nucleic acid sequences for the purpose of

[0159] Illustrative examples of conditions include congenital hemophilia such as hemophilia A, hemophilia B, thalassemia, and anemia. These include, but are not limited to, clotting factor disorders.

[0160] In accordance with the present invention, methods of treatment and uses are provided which comprise the use of nucleic acids, vectors, recombinant vectors and the like of the present invention. vectors (e.g., rAAV), vector genomes, and recombinant viral particles. The methods and uses of the present invention can be used to determine gene expression or function, e.g., gene addition. or replacement, or augmenting or enhancing.

[0161] In one embodiment, the method or use of the present invention comprises: (a) F, which has a reduced number of CpG dinucleotides in a vector or vector genome, etc. providing a modified nucleic acid encoding factor IX, such as factor IX; The modified nucleic acid sequence is operably linked to expression control elements that provide for transcription of the sequence. The steps and (b) administering a predetermined amount of the modified nucleic acid to a mammal so that Factor IX is expressed in the mammal. The method includes administering the compound to an animal.

[0162] In another embodiment, the method or use of the present invention comprises the step of transfecting a virus ( A viral (e.g., AAV) particle or multiple viral (e.g., AAV) particles (e.g., capsids By administering a mutant (e.g., 4-1) to a mammal or mammalian cells, Modifications encoding factor IX, such as FIX with fewer CpG dinucleotides delivering or transferring the nucleic acid sequence into a mammal or a mammalian cell, The vector genome contains a reduced number of CpG dinucleotides (and optionally ITRs, intracellular repeats, ron, polyA, filler polynucleotide sequence / stuffer polynucleotide sequence) and a modified nucleic acid encoding factor IX, such as FIX having the formula: Delivering or transferring modified nucleic acids encoding Factor IX into mammals or mammalian cells The method includes the steps of:

[0163] In particular aspects of the present invention, the methods and uses disclosed herein comprise the steps of: provides a therapeutic benefit to a mammal (e.g., a human). Expression of factor X is therapeutic in mammals (e.g., humans), such as mammals with hemophilia B. In various further specific embodiments, the number of CpG dinucleotides is reduced. The length of the modified nucleic acid encoding factor IX, such as factor IX, bound to the modified nucleic acid is approximately 3. 0Kb to 5.5Kb, or approximately 4.0Kb to 5.0Kb, or approximately 4.3Kb to 4. Filler polynucleotide sequence / stuffer polynucleotide sequence to have an overall length of 8 kb The primer sequence is contained within the vector sequence.

[0164] The methods and uses of the present invention include any therapeutic method that produces a therapeutic or beneficial effect. In various inventive methods and uses, one or more adverse (e.g., physical) symptoms, disorders, illnesses, diseases, or conditions associated with It also includes preventing, reducing or diminishing complications. In this case, the therapeutic or beneficial effects may include reduced scarring, reduced blood clotting time, reduced This includes, but is not limited to, bleeding episodes (duration, severity, frequency) observed. For example, reducing the duration, severity or frequency of joint or cerebral (brain) bleeding episodes. Hemophilia For bleeding disorders such as reduced amounts of coagulation factor proteins (e.g., factor IX protein) or supplementary coagulation factor proteins (e.g., Further, this includes, but is not limited to, the cessation of administration of anti-inflammatory drugs (e.g., factor IX protein). stomach.

[0165] Thus, the therapeutic or beneficial effect of a treatment may be any effect provided to a particular subject. A therapeutic effect or benefit is an objective or subjective measurable or detectable improvement or benefit. A beneficial effect may be the alleviation of all or any particular adverse symptom, disease, disorder, or complication of a disease. It may be, but need not be, a complete removal. The end point is the duration of the illness, whether short-term or long-term (hours, days, weeks, months, etc.). A gradual progression of adverse symptoms, illnesses, diseases, or complications caused or associated with a disease If there is a partial improvement or partial reduction, or if there is a minor improvement or partial reduction caused by the disease, or aggravation of one or more adverse symptoms, illnesses, diseases or complications associated with the disease. is achieved when there is a suppression, reduction, arrest, prevention, limitation or control of progression. .

[0166] Nucleic acids, vectors, recombinant vectors (e.g., rAAV), vector genomes, and vectors Compositions, methods, and uses of the invention, such as recombinant viral particles containing a vector genome can be administered in a sufficient or effective amount to a subject in need thereof. "An amount sufficient" or "amount sufficient" means a single dose or multiple doses, alone or in combination, one or more other compositions (therapeutic agents such as drugs), treatments, protocols or therapeutic modalities along with a detectable response of any duration (long or short term), expected or desired result, or any measurable or detectable effect or any duration (e.g., minutes, hours, days, months, years, or ) represents the amount that provides a benefit to a subject.

[0167] Those skilled in the art will be able to determine whether administration of a single rAAV / vector dose is sufficient or whether administration of multiple rAAV / vector doses is sufficient. For example, if FIX is administered in advance, it can be determined whether multiple doses of the drug are necessary. If the level is lower than the stated level (e.g., lower than the minimum that provides a therapeutic benefit), The investigator may decide to administer an additional dose of rAAV / vector, if appropriate. Cut.

[0168] The dose that achieves a therapeutic effect, e.g., the dose of vector genome per kilogram of body weight ( vg / kg) is determined based on the route of administration, the amount of heterologous polynucleotide required to achieve a therapeutic effect, and the level of nucleotide expression, the particular disease being treated, and any host immunity to the viral vector. response, host immune response to heterologous polynucleotides or expression products (proteins), and This is due to several factors, including but not limited to the stability of the expressed protein. Those skilled in the art will appreciate that the effectiveness of the method will vary depending on the particular disease or condition, based on the factors mentioned above and other factors. It is important to determine the dose range of rAAV / vector genomes for treating patients with this disease. Generally, the dose is about 100 mg / kg of subject weight to achieve a therapeutic effect. , at least 1 x 10 8 or more, e.g., 1 x 10 9 , 1×10 10 , 1×10 11 , 1 x10 12 , 1×10 13 or 1 x 10 14 or more vector genomes (vg / kg) Deaf.

[0169] In some embodiments, an AAV vector (e.g., AAV-FIX39Padu a, or the same capsid as that and at least 95%, 96%, 97%, 98% or 99% identical genomic sequence) is used in treating hemophilia B or other When administered to a subject (e.g., a human) with deficient factor IX activity, it can cause severe hemophilia. sufficient to convert B to mild or moderate hemophilia B or to render the patient apparently unaffected In other embodiments, the therapeutically effective amount of an AAV vector is an amount sufficient to treat a patient with hemophilia B. Human subjects may be able to forgo factor IX replacement therapy entirely or maintain adequate hemostasis. The amount is sufficient to allow the replacement FIX to be administered less frequently in order to prevent the occurrence of side effects. As understood by those with hemophilia B, factor replacement therapy is the current standard of care for hemophilia B. However, to compensate for the patient's inability to produce sufficient levels of functioning clotting factors, It requires frequent injections of recombinantly produced human factor IX.

[0170] Severe hemophilia B often occurs spontaneously (without preceding trauma) and affects the subject's muscles or joints. Characterized by frequent bleeding into the joints (e.g., at least once or twice a week) It is generally accepted that less than 1% of the FIX activity found in healthy humans is present in severe hematologic It is related to hemophilia B. People with moderate hemophilia B are more likely to develop hemophilia than people with severe hemophilia B. Bleeding occurs less frequently (e.g., about once per month) but not after surgery, trauma, or dental work It is generally accepted that women who have undergone procreation will bleed for a longer period than normal people. It is generally accepted that people with moderate disease do not spontaneously bleed frequently. FIX activity of 1% to 5% of normal is generally associated with moderate hemophilia B. Human subjects with moderate hemophilia B are less likely to undergo surgery or major trauma, if at all. The only result is excessive bleeding. Generally, mild hemophilia is 6% to 40% of normal. % of FIX activity. Generally, patients without symptoms of hemophilia B are considered healthy. The resulting individuals have FIX activity ranging from about 50% to 150% of normal. Information on this can be found in Fijnvandraat et al., Diagnosis and management ment of hemophilia, Br. Med. J., 344:36- It can be seen in 40 (2012).

[0171] Factor IX activity can be measured by a variety of methods known to those skilled in the art, for example One exemplary, non-limiting assay involves measuring FIX clotting activity in a plasma sample obtained from a subject. One-stage activated partial thromboplastin time (ACTP) for measuring Arterial thromboplastin time (APTT) analysis. X activity is often expressed in international units (IU). 1 IU is the amount of X-linked IgG obtained from a normal donor. It is defined as the FIX clotting activity present in 1 ml of pooled plasma. Using this convention, Severe hemophilia B is associated with FIX levels below 0.01 IU / ml, while moderate The disease is associated with FIX levels of 0.02-0.05 IU / ml, and mild disease is associated with FIX levels of 0.06-0.40 IU / ml and is disease-free. This is associated with FIX levels of 0.50-1.50 IU / ml.

[0172] As will be appreciated by those skilled in the art, naturally occurring FIX variants such as Padua variants may be used. Factor IX variants that have higher catalytic activity compared to wild-type human FIX , a given level of FIX activity at lower concentrations of active protein compared to "non-Padua" FIX. levels (e.g., 1 IU / ml).

[0173] In one embodiment, an AAV vector (e.g., AAV-FIX39 Padua, or has the same capsid and at least 95%, 96%, 97%, and 98% or 99% identical genomic sequence) is used to treat severe, moderate, or or mild hemophilia B, FIX activity is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or , 50% or more of the plasma FIX activity. In certain embodiments, a therapeutically effective amount is 1% or more of a therapeutically effective amount in subjects otherwise lacking such activity. or more (e.g., 1.5% to 10%, 10% to 15%, 15% of the FIX activity of the test sample) The amount is the amount that achieves FIX activity of 20% to 20%, 20% to 25%, 25% to 30% or more.

[0174] For the treatment of subjects with hemophilia B, AAV vectors (e.g., AAV-FIX3 9 Padua or the same capsid and at least 95%, 96% , 97%, 98% or 99% identical genomic sequence) is , at least 1 x per kilogram of subject weight to achieve the desired therapeutic effect 10 10 Vector genome (vg) (vg / kg), or approximately 1 x 10 of the weight of the subject 10 ~ 1×10 11 vg / kg or approximately 1 x 10 of the specimen weight 11 ~1×10 12 vg / kg( For example, roughly 1 x 1011 ~2×10 11 vg / kg, or approximately 2 x 10 11 ~3×10 11 v g / kg, or approximately 3 x 10 11 ~4×10 11 vg / kg, or approximately 4 x 10 11 ~5× 10 11 vg / kg, or approximately 5 x 10 11 ~6×10 11 vg / kg, or approximately 6 x 10 11 ~7×10 11 vg / kg, or approximately 7 x 10 11 ~8×10 11 vg / kg, approximately 8 x 1 0 11 ~9×10 11 vg / kg, or approximately 9 x 10 11 ~1×10 12 vg / kg), or Approximately 1 x 10 of the subject's weight 12 ~1×10 13 vg / kg. Further doses may be Approximately 100 mg / kg of subject weight is required to achieve the desired therapeutic effect. 5×10 10 ~1×10 10 Within the range of 1 vector genome (vg) or 1 kg of subject weight Approximately 1 x 10 per program 10 ~5×10 11 Within the range of 1000 mg / kg or the weight of the subject Approximately 5 x 10 per kilogram 11 ~1×10 12 vg / kg range or subject weight Approximately 1 x 10 per kilogram 12 ~5×10 13 It may be within the range of vg / kg. In this embodiment, the therapeutically effective amount of the AAV vector is approximately 2.0 x 10 11 vg / k g, 2.1 × 10 11vg / kg、2.2×10 11 vg / kg、2.3×10 11 vg / kg 、2.4×10 11 vg / kg、2.5×10 11 vg / kg、2.6×10 11 vg / kg、 2.7×10 11 vg / kg、2.8×10 11 vg / kg、2.9×10 11 vg / kg、3 .0×10 11 vg / kg、3.1×10 11 vg / kg、3.2×10 11 vg / kg、3. 3×10 11 vg / kg、3.4×10 11 vg / kg、3.5×10 11 vg / kg、3.6 ×10 11 vg / kg、3.7×10 11 vg / kg、3.8×10 11 vg / kg、3.9× 10 11 vg / kg、4.0×10 11 vg / kg、4.1×10 11 vg / kg、4.2×1 0 11 vg / kg、4.3×10 11 vg / kg 4.4×10 11 vg / kg、4.5×10 11 vg / kg、4.6×10 11 vg / kg、4.7×10 11 vg / kg、4.8×10 11 vg / kg、4.9×10 11 vg / kg、5.0×10 11 vg / kg、5.1×10 11 v g / kg、5.2×10 11 vg / kg、5.3×10 11 vg / kg、5.4×10 11 vg / kg、5.5×10 11vg / kg, 5.6 × 10 11 vg / kg, 5.7 × 10 11 vg / kg, 5.8 × 10 11 vg / kg, 5.9 × 10 11 vg / kg, 6.0 × 10 11 vg / k g, 6.1 × 10 11 vg / kg, 6.2 × 10 11 vg / kg, 6.3 × 10 11 vg / kg , 6.4×10 11 vg / kg, 6.5 × 10 11 vg / kg, 6.6 × 10 11 vg / kg, 6.7×10 11 vg / kg, 6.8 × 10 11 vg / kg, 6.9 × 10 11 vg / kg, 7 .0×10 11 vg / kg, 7.1 × 10 11 vg / kg, 7.2 × 10 11 vg / kg, 7. 3×10 11 vg / kg, 7.4 × 10 11 vg / kg, 7.5 × 10 11 vg / kg, 7.6 x10 11 vg / kg, 7.7 × 10 11 vg / kg, 7.8 × 10 11 vg / kg, 7.9× 10 11 vg / kg or 8.0 x 10 11 These doses are In any of the embodiments, the AAV vector is AAV-FIX39Padua, or , the same capsid and at least 95%, 96%, 97%, 98% or may have 99% identical genomic sequence and may be present in a pharmaceutically acceptable composition. may be administered to a subject alone or together with empty capsids of the same capsid species, The ratio of empty capsid to vector was approximately 2:1, 3:1, 4:1, 5:1, 6:1, It may be administered to a subject in a ratio of 7:1, 8:1, 9:1, 10:1 or other ratios.

[0175] Satisfactory results in diminishing, reducing, inhibiting, suppressing, limiting or controlling the progression or worsening of a disease. Although there are some indications that therapies are therapeutic (e.g., to alleviate or provide a therapeutic benefit or improvement), An "effective amount" or "sufficient amount" (to provide a therapeutic effect) typically refers to one, more, or all Any adverse symptoms, consequences or complications of the disease, e.g., caused by or A response to one or more adverse symptoms, illnesses, diseases, pathologies, or complications associated with the condition is effective in providing a measurable degree of

[0176] An effective or sufficient amount may, but need not, be provided in a single administration, and may be provided in multiple doses. Several doses may be required and may be administered alone or in combination with other compositions (e.g., drugs), treatments, or other therapeutic agents. may be administered in conjunction with any other treatment, protocol, or regimen, but does not necessarily have to be. For example, the amount may vary depending on the needs of the subject, the disease being treated, or the therapeutic effect (if any). May be increased proportionately as indicated by the type, condition, and severity of side effects of the treatment. Furthermore, because such a drug is considered effective or sufficient in a given subject, Further doses, amounts or durations of administration above the recommended dose, or further compositions (e.g. may include any drug, drug or agent), treatment, protocol or treatment plan; An effective or sufficient amount may be present if a second composition (e.g., another drug or agent), treatment, prophylaxis, or It is effective when given in a single dose or multiple doses without a protocol or treatment regimen. The amount considered effective is also determined based on the coagulation disorder (e.g., hemophilia A or hemophilia B). Other treatments and regimens, such as the administration of recombinant clotting factor proteins for the treatment of idiopathic ... or amounts that result in a reduction in the use of the protocol.

[0177] An effective or sufficient amount needs to be effective in every subject treated. and is effective in the majority of treated subjects in a given group or population. An effective amount or sufficient amount is not necessarily a measure of a group or the general population, but rather a measure of a particular subject. As is typical for such methods, Some subjects may respond more to a given method or use of treatment than others. Some subjects show a reaction or no reaction.

[0178] The term "ameliorate" refers to a detectable or measurable decrease in a disease or its symptoms in a subject. A detectable or measurable improvement in the disease or underlying cellular response is considered to be a significant improvement. A subjective or objective decrease in the incidence, frequency, severity, progression, or duration of undermine, inhibit, restrain, limit or control, or the disease caused or resulting from it complications related to the disease, or in the symptoms, underlying causes or consequences of the disease This includes improvement or recovery from the disease.

[0179] Thus, a successful therapeutic outcome is defined as the reduction in the disease or one or more of the symptoms of the disease in a subject. The occurrence, frequency, severity, progression or persistence of any adverse symptoms or underlying causes or consequences "Therapeutic effect" or "benefit" of decreasing, reducing, inhibiting, suppressing, limiting, controlling or preventing the duration of Therefore, it can be linked to one or more of the underlying causes or adverse symptoms of a disease. The methods and uses of treatments used are believed to be beneficial and may also stabilize the disease. Such a decrease or reduction in the deterioration or in the adverse symptoms thereof is considered a successful treatment. It is a success.

[0180] Thus, a therapeutic benefit or improvement may be achieved by reducing the adverse symptoms of or associated with the disease. Complete elimination of most or all of the symptoms, complications, consequences, or underlying causes Therefore, the gradual improvement in the subject's disease or the onset of the disease, Partially diminish, reduce, inhibit, suppress, limit, in frequency, severity, progression or duration; Control or prevention, or inhibition or reversal of a disease (e.g., one or more symptoms or complications) (stabilizing the environment) may exist for short or long periods (hours, days, weeks, months, etc.) A satisfactory endpoint is achieved when there is a potential therapeutic benefit or improvement in disease. The effectiveness of the method or use, such as a treatment, can be determined by various methods, such as clot formation time, etc. It can be confirmed.

[0181] According to some embodiments, a therapeutically effective amount of an AAV vector is administered to a person with hemophilia B. When administered to a subject, it induces a certain level of FIX activity over a certain period of time. In some of these embodiments, the effective amount of the AAV vector is an amount sufficient to At least 3, 4, 5, 6, 7, 8, 9, 10, 11 months months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 1.5 years, 2 years , 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years , 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years or longer duration , resulting in normal FIX activity in at least 1% of human subjects with hemophilia B. In embodiments, the effective amount of the AAV vector is administered for at least 3 months, 4 months, 5 months, 6 months, or month, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 1 5 months, 16 months, or 17 months, or at least 1.5 years, 2 years, 2.5 years, or 3 years , 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years , 8.5 years, 9 years, 9.5 years, 10 years or more duration In another embodiment, the effective amount of the AAV vector is: At least 3, 4, 5, 6, 7, 8, 9, 10, 11 months months, 12 months, 13 months, 14 months, 15 months, 16 months, or 17 months, or At least 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years , 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years or Provides at least 10% of normal FIX activity for a longer duration. In some embodiments, the effective amount of the AAV vector is administered for at least 3 months, 4 months, 5 months, 6 months, or more. , 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, or 17 months, or at least 1.5 years, 2 years, 2.5 years, or 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, At least 15 years for a duration of 8.5, 9, 9.5, 10 or more years In another embodiment, the effective amount of the AAV vector is: At least 3, 4, 5, 6, 7, 8, 9, 10, 11 months months, 12 months, 13 months, 14 months, 15 months, 16 months, or 17 months, or At least 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years , 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years or Provides at least 20% of normal FIX activity for a longer duration. In some embodiments, the effective amount of the AAV vector is administered for at least 3 months, 4 months, 5 months, 6 months, or more. , 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, or 17 months, or at least 1.5 years, 2 years, 2.5 years, or 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, At least 25 years for a duration of 8.5, 9, 9.5, 10 or more years In another embodiment, the effective amount of the AAV vector is: At least 3, 4, 5, 6, 7, 8, 9, 10, 11 months months, 12 months, 13 months, 14 months, 15 months, 16 months, or 17 months, or At least 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years , 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years or Provides at least 30% of normal FIX activity for a longer duration. In some embodiments, the effective amount of the AAV vector is administered for at least 3 months, 4 months, 5 months, 6 months, or more. , 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, or 17 months, or at least 1.5 years, 2 years, 2.5 years, or 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, At least 35 years for a duration of 8.5, 9, 9.5, 10 or more years In another embodiment, the effective amount of the AAV vector is: At least 3, 4, 5, 6, 7, 8, 9, 10, 11 months months, 12 months, 13 months, 14 months, 15 months, 16 months, or 17 months, or At least 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years , 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years or Provides at least 40% of normal FIX activity for a longer duration. In some embodiments, the effective amount of the AAV vector is administered for at least 3 months, 4 months, 5 months, 6 months, or more. , 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, or 17 months, or at least 1.5 years, 2 years, 2.5 years, or 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, At least 45 years for a duration of 8.5, 9, 9.5, 10 or more years In any of these embodiments, the AAV vector results in % of normal FIX activity. AAV-FIX39 Padua or the same capsid and a smaller have at least 95%, 96%, 97%, 98% or 99% identical genomic sequence or may be administered to a subject alone in a pharmaceutically acceptable composition, or The empty capsids of the same capsid species are also included, estimating the ratio of empty capsids to vector. 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 may be administered to the subject in other ratios.

[0182] According to another embodiment, the therapeutically effective amount of the AAV vector is administered to treat severe or moderate hemophilia. When administered to human subjects with B, the %, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32 %, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42 %, 43%, 44% or 45% FIX activity for a duration of at least 6 months. In some embodiments, the amount is sufficient to provide an AAV-FIX39 Padu a, or the same capsid as that and at least 95%, 96%, 97%, The dose of a gene with 98% or 99% identical genomic sequence is approximately 5.0 x 10 11 vg / kg and may be used alone or in combination with empty capsids of the same capsid species in a pharmaceutically acceptable composition. The ratio of empty capsid to vector was approximately 2:1, 3:1, 4:1, 5: 1, 6:1, 7:1, 8:1, 9:1, 10:1 or other ratios. good.

[0183] A therapeutically useful amount of an AAV vector (e.g., AAV-FIX39 Padua, or and a capsid that is at least 95%, 96%, 97%, 98% or In several human subjects who ingested In some cases, the FIX activity caused by the vector may persist for a long period of time (e.g., months or years). It will be appreciated that the amount of oxidative stress may decrease to a level that is no longer considered sufficient (e.g., The subject exhibits symptoms and / or FIX activity characteristic of moderate or severe hemophilia B. In such situations, it may be necessary to administer the same type of AAV vector to the subject as the initial treatment. In other embodiments, the subject may develop an immune response to the initial vector, among other things. When developed, it expresses FIX in target cells, but compared to the original AAV vector, designed to have capsids of different or variant serotypes that are less immunoreactive compared to The resulting AAV vector can then be given to the patient.

[0184] According to one embodiment, a therapeutically effective amount of an AAV vector is administered to a human subject with hemophilia B. A subject's recombinant human Factor IX to maintain proper hemostasis when administered to the body. An amount sufficient to reduce or eliminate the need for replacement therapy. In this study, a therapeutically effective dose of an AAV vector is considered to be moderate or The figure roughly estimates how often an average human subject with severe hemophilia B requires FIX replacement therapy. Right, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 10 In a related embodiment, the therapeutically effective amount of the AAV vector can be reduced by 0%. The average human subject with moderate or severe hemophilia B needs to maintain adequate hemostasis. The dose of recombinant human factor IX required by the body is roughly 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, These can be reduced by 75%, 80%, 85%, 90%, 95% or 100%. In any of the embodiments, the AAV vector is AAV-FIX39Padua, or The same capsid and at least 95%, 96%, 97%, 98% or or 99% identical to the genomic sequence of the target gene, and a pharmaceutically acceptable composition. Empty capsids for vectors alone or together with empty capsids of the same capsid species The ratio of the do is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 1 It may be administered to a subject in a ratio of 0:1 or other ratio.

[0185] In another embodiment, a therapeutically effective amount of an AAV vector is administered to a patient with severe hemophilia B. When administered to a human subject, the amount is sufficient to reduce or eliminate spontaneous bleeding into the joint. Thus, in some embodiments, a therapeutically effective amount of an AAV vector is The frequency of spontaneous bleeding into joints in human subjects with hemophilia B was compared with that in treated subjects with severe hemophilia B. Compared to the average untreated human subject, the results show roughly 5%, 10%, 15%, 20%, and 2% 5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 7 It can be reduced by 5%, 80%, 85%, 90%, 95% or 100%. Bleeding may be assessed by magnetic resonance imaging or ultrasound of the joint, or other methods well known to those skilled in the art. In any of these embodiments, the AAV vector can be detected using techniques. The target is AAV-FIX39 Padua or the same capsid and and a genomic sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the and may be present alone or in combination with other capsid species of the same type in a pharmaceutically acceptable composition. Empty capsids were also used, with the ratio of empty capsid to vector being approximately 2:1, 3:1, or 4:1. , 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or other ratios, may be administered to

[0186] Previous efforts to develop AAV vectors to treat hemophilia have been unsuccessful. , at least in part, due to robust immunity to the AAV capsid in previous clinical trials. This is thought to be due to the reaction (e.g., Nathwani et al., NEJM 2011 ; 365(25):2357-2365; and Manno et al., Nat Med 2 006; 12(3):342-347). Ongoing clinical trials are investigating the use of AAV vectors. One embodiment provides a method for administering an AAV vector to a human subject with severe hemophilia B. Six months after administration (Example 5), no or minimal immune response was observed. On the other hand, we have demonstrated that high levels of FIX activity can be produced. According to one embodiment, a therapeutically effective amount of an AAV vector is administered to a patient with severe or moderate hemophilia B. When administered to a subject, it does not produce an immune response or produces a minimal immune response over a meaningful period of time. at a concentration that produces an immune response while providing adequate FIX activity to maintain hemostasis. In certain embodiments, the immune response is an innate immune response, a humoral immune response, or It may be a cell-mediated immune response, or it may be all three types of immune response. In some embodiments, the immune response is directed against the capsid, vector genome, and / or may be directed to the Factor IX protein produced from the transduced cells.

[0187] According to one embodiment, a therapeutically effective amount of an AAV vector comprises the capsid, genome and / or or does not produce an immune response to the Factor IX protein produced by the transduced cells, or While eliciting a minimal immune response (i.e., antibodies), This provides adequate FIX activity to maintain hemostasis. The antibody response to such virus-like particles can be assessed by measuring the antibody titer in the serum or plasma of the subject. or using techniques well known to those skilled in the art of immunology. Any component of the AAV vector, such as the capsid protein, or factor IX Encoded by a vector genome such as Padua (or other FIX variants) The antibody titers against the gene products and the gene products produced in the transduced cells are Antibody titers can be measured using a variety of techniques, including those that detect the presence of antibodies. Before the antibody signal is no longer detectable in the particular assay being used The dilution factor is expressed as a ratio, e.g., 2x, 5x, 10x, or some other dilution factor. Different dilution ratios can be used, such as, for example, WO2015 / 006743 antibodies, such as ELISA, FACS or reporter gene assays as described in Any suitable assay for the presence of may be used, including but not limited to: Additionally, other assays may be used according to the knowledge of those skilled in the art. Measurements can be taken at different times after the first dose of the drug.

[0188] In one embodiment, a therapeutically effective amount of an AAV vector is a therapeutically effective amount of an AAV vector administered to a subject. 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks after administration After, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months After, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, 4 years, 5 years capsid, genome and / or transduced cells when measured after 2 or longer periods of time The antibody titer produced against the factor IX protein (FIX Padua, etc.) produced from 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1: 12, 1:13, 1:14, 1:15, 1:20, 1:30, 1:40, 1:50, 1: 60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:40 0, 1:500 or more, while in subjects with hemophilia B at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or more According to an exemplary, non-limiting embodiment, the AAV vector provides FIX activity. In both cases, capsid and / or transduced cells were identified 6 months after administration of the AAV vector. The antibody titer against factor IX produced by the antibody is 1:2, 1:3, or 1:4 or less. While inducing at least 20% FIX in subjects with severe hemophilia B, In any of these embodiments, the AAV vector is AAV-F IX39 Padua or the same capsid as it and at least 95% thereto; It may have 96%, 97%, 98% or 99% identity to the genome sequence. , alone or together with empty capsids of the same capsid species in a pharmaceutically acceptable composition. The ratio of empty capsid to vector was approximately 2:1, 3:1, 4:1, 5:1, 6:1, may be administered to a subject in a ratio of 7:1, 8:1, 9:1, 10:1, or other ratios. .

[0189] As mentioned above, previous attempts using AAV-mediated gene therapy for hemophilia B have There is no need for high doses of steroids, which cause immunosuppression, and the patient can self-report for a significant period of time. A limiting immune response prevented the treatment from being ineffective. This was thought to be a cellular immune response that eliminated hepatocytes transduced with the AAV vector. The results were consistent with elevated liver enzymes suggesting liver damage and capsid-specific The presence of target T cells could be detected by both.

[0190] In one embodiment, a therapeutically effective amount of an AAV vector comprises a capsid and / or a phenotype. Does not induce a cellular immune response to the factor IX protein produced by the transfected cells or Maintaining hemostasis in subjects with hemophilia B while generating a minimal cellular immune response The cellular immune response is mediated by the capsid protein or the first Analysis of factor IX-specific T cell activity and damage to liver cells The presence of elevated liver enzymes can be determined by at least two methods of testing. .

[0191] In some embodiments, the cellular immune response is directed against capsid proteins and / or genes. T cell activity specific to factor IX protein produced by the transfected hepatocytes Several different assays for T cell responses are available. In one exemplary, non-limiting embodiment, the T cell response is a T cell response in a patient with hemophilia B. Peripheral blood mononuclear cells (PBMCs) were collected from subjects previously treated with AAV vectors for treatment. The cells are then used in the vector peptides derived from the VP1 capsid protein and / or FIX Padua Incubation with factor IX protein produced by such transfected hepatocytes T cells that specifically recognize the capsid protein or factor IX protein are stimulated. stimulated to release cytokines such as interferon gamma or other cytokines, Afterwards, it is possible to measure the expression of the antibody using an ELISPOT assay or other assays well known to those skilled in the art. can be detected and quantified using (e.g., Manno et al., Nat Med 20 06; 12(3):342-347. A for treating hemophilia B Before and at different times after the subject receives a dose of the AV vector, e.g. T cell responses can be monitored weekly, monthly, or at other intervals. According to embodiments, the therapeutically effective amount of the AAV vector is administered weekly, monthly, or at other intervals. Or, 2 weeks, 1 month, 2 months, 3 months, 6 months after administration of the AAV vector The 10 analyzed were measured after 1 month, 9 months, 1 year, 2 years, or other time periods. 10, 20, 30, 40, 50, 100, 200, 300, per million PBMCs 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or Use the ELISPOT, a spot forming unit that does not exceed maintain hemostasis in subjects with hemophilia B while generating a T cell response measured by FIX activity (e.g., at least 1%, 5%, 10%, 20%, 30%) adequate to maintain In some of these embodiments, ELI SPOT analysis facilitated by peptides derived from AAV vector capsid proteins Enhanced interferon gamma (or other cytokine) production and transfected hepatocytes Factor IX protein (including FIXPadua or different variants) produced by the cells In any of these embodiments, the vector is designed to detect an AAV vector. AAV-FIX39 Padua or the same capsid and a smaller have at least 95%, 96%, 97%, 98% or 99% identical genomic sequence and may be present alone or in combination with other capsid species of the same capsid species in a pharmaceutically acceptable composition. The ratio of empty capsid to vector was approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or other ratios to the subject may be administered.

[0192] As a surrogate for the cellular immune response to the transfected hepatocytes, standard methods were used. The presence of higher than normal liver enzymes can be analyzed. Although not shown, specific AAV vectors, such as those used in previous clinical trials, may be used. T cells specific for the target gene can attack and kill the transfected hepatocytes. Transient release of liver enzymes into the circulation. Typical liver enzymes are alanine aminotransferase (ALT). ALT, aspartate aminotransferase (AST), and lactate dehydrogenase Includes LDH, but can also monitor other enzymes that indicate liver damage Normal levels of these enzymes in the circulation are typically elevated above which the enzyme levels , whereby normal is defined as the range having values ​​that are considered to be indicative of liver damage. The appropriate range will depend, in part, on the criteria used by the clinical laboratory performing the analysis. In one embodiment, a therapeutically effective amount of an AAV vector is administered to treat elevated levels of circulating liver enzymes. While it produces enzymes (such as ALT, AST, or LDH) in subjects with hemophilia B, Adequate FIX activity (e.g., at least 1%, 5%, 10%, 2%) is required to maintain hemostasis. 0%, 30% or higher FIX activity), resulting in the elevated levels of circulating liver enzymes. The mean values ​​for each range are 0%, 10%, 20%, 30%, 40%, and 60% of the upper limit of normal (ULN). 0%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 40 0%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000% or less, or at different time points after AAV vector administration (e.g., weekly or The highest measured in multiple samples extracted from the same subject under treatment (monthly intervals) Levels 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 9 0%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 80 0%, 900%, 1000%, 1500%, 2000% or less. In either case, the AAV vector is AAV-FIX39Padua or an equivalent thereof. The same capsid and at least 95%, 96%, 97%, 98% or 99% % identical genomic sequence and can be used alone in a pharmaceutically acceptable composition. or the ratio of empty capsids to vector along with empty capsids of the same capsid species Ratios are generally 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or or other ratios may be administered to the subject.

[0193] In a previous clinical trial using AAV vectors to treat hemophilia B, investigators The subject undergoing treatment undergoes an immune response to eliminate the transfected cells that produce the factor IX protein. To prevent the onset of a reaction, immunosuppressants such as steroids must be administered concomitantly. However, there have been cases in subjects receiving experimental treatment with certain AAV vectors. Due to the reduced immune response achieved, concomitant administration of immunosuppressants may not be necessary. Thus, in certain embodiments, a therapeutically effective amount of an AAV vector is The need for concomitant administration (before, simultaneously with, or after) of immunosuppressants (such as immunosuppressants of and is insufficient to maintain adequate hemostasis in subjects with severe or moderate hemophilia B. However, the immune response is not predictable in all subjects. The present method of treatment for hemophilia B includes administering AA in combination with an immunosuppressant. Before the AAV vector is administered to a subject with hemophilia B, the AAV vector is Concomitant administration of an immunosuppressant may occur simultaneously or after administration. In , immunosuppressants are administered after AAV vectors are administered to treat hemophilia B. They are administered to the subject over a period of days, weeks, or months. steroids (such as, but not limited to, prednisone or prednisolone), and and nonsteroidal immunosuppressants such as cyclosporine and rapamycin. The amount and duration of treatment required to achieve adequate immunosuppression will be determined for each subject receiving treatment. Dosage and duration of treatment will depend on factors specific to the individual body, but are within the skill of one of ordinary skill in the art. In some embodiments, the immunosuppressant may need to be administered more than once.

[0194] In one embodiment, a therapeutically effective amount of an AAV vector is administered to treat severe or moderate hemophilia. results in a consistent increase in FIX activity when administered to a population of human subjects with B Consistency can be measured using the mean and standard deviation (SD) or other statistical methods familiar to those skilled in the art. By calculating the variability of responses in a population of human subjects using statistical methods such as In some embodiments, a therapeutically effective amount of an AAV vector can be determined. When administered to a population of human subjects with severe or moderate hemophilia B, months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months or , Less than 15, Less than 14, Less than 13, Less than 12, Less than 11, Less than 10, Less than 9, Less than 8, 7 Less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 standard deviation of 1% to 5% Average FIX activity: less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9 , less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 standard deviation 0.5% to 7.5% mean FIX activity; <15, <14, <13, <12, <11 Full, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or Mean FIX activity of 5% to 10% with a standard deviation of less than 1; less than 15, less than 14, less than 13, and less than 1 Under 2, Under 11, Under 10, Under 9, Under 8, Under 7, Under 6, Under 5, Under 4, Under 3 Average FIX activity of 7.5% to 12.5% ​​with a standard deviation of more than, less than 2, or less than 1; less than 15; Under 14, Under 13, Under 12, Under 11, Under 10, Under 9, Under 8, Under 7, Under 6 , 10% to 15% average fix with a standard deviation of less than 5, less than 4, less than 3, less than 2, or less than 1 Activity: Less than 15, Less than 14, Less than 13, Less than 12, Less than 11, Less than 10, Less than 9, Less than 8 , less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 standard deviation of 12.5% ~17.5% mean FIX activity; <15, <14, <13, <12, <11 Less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 Average FIX activity of 15% to 20% with a standard deviation of less than 15, less than 14, less than 13, and less than 12 Less than, Less than 11, Less than 10, Less than 9, Less than 8, Less than 7, Less than 6, Less than 5, Less than 4, Less than 3 , mean FIX activity of 17.5% to 22.5% with a standard deviation of less than 2 or less than 1; less than 15; Under 14, Under 13, Under 12, Under 11, Under 10, Under 9, Under 8, Under 7, Under 6 , 20% to 25% average fix with a standard deviation of less than 5, less than 4, less than 3, less than 2, or less than 1 Activity: Less than 15, Less than 14, Less than 13, Less than 12, Less than 11, Less than 10, Less than 9, Less than 8 , less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 standard deviation 22.5% ~27.5% mean FIX activity; <15, <14, <13, <12, <11 Less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 Average FIX activity of 25% to 30% with a standard deviation of less than 15, less than 14, less than 13, and less than 12 Less than, Less than 11, Less than 10, Less than 9, Less than 8, Less than 7, Less than 6, Less than 5, Less than 4, Less than 3 , mean FIX activity of 27.5% to 32.5% with a standard deviation of less than 2 or less than 1; less than 15; Under 14, Under 13, Under 12, Under 11, Under 10, Under 9, Under 8, Under 7, Under 6 , 30% to 35% average fix with a standard deviation of less than 5, less than 4, less than 3, less than 2, or less than 1 Activity: Less than 15, Less than 14, Less than 13, Less than 12, Less than 11, Less than 10, Less than 9, Less than 8 , less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 standard deviation 32.5% ~37.5% mean FIX activity; <15, <14, <13, <12, <11 Less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 Mean FIX activity of 35% to 40% with a standard deviation of <15, <14, <13, <12 Less than, Less than 11, Less than 10, Less than 9, Less than 8, Less than 7, Less than 6, Less than 5, Less than 4, Less than 3 , mean FIX activity of 37.5% to 42.5% with a standard deviation of less than 2 or less than 1; less than 15; Under 14, Under 13, Under 12, Under 11, Under 10, Under 9, Under 8, Under 7, Under 6 , 40% to 45% average fix with less than 5, less than 4, less than 3, less than 2, or less than 1 standard deviation Activity: Less than 15, Less than 14, Less than 13, Less than 12, Less than 11, Less than 10, Less than 9, Less than 8 , 42.5% with a standard deviation of less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 ~47.5% mean FIX activity; or <15, <14, <13, <12, <11 Less than, Less than 10, Less than 9, Less than 8, Less than 7, Less than 6, Less than 5, Less than 4, Less than 3, Less than 2 or These embodiments provide an average FIX activity of 45% to 50% with a standard deviation of less than 1. In either case, the AAV vector is AAV-FIX39Padua or an equivalent thereof. The same capsid and at least 95%, 96%, 97%, 98% or 99% % identical genomic sequence and can be used alone in a pharmaceutically acceptable composition. or the ratio of empty capsids to vector along with empty capsids of the same capsid species Ratios are generally 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or or other ratios may be administered to the subject.

[0195] The methods and uses of the present invention may be used to achieve desired therapeutic, beneficial, additive, synergistic or complementary effects. any compound, agent, drug, treatment or other therapeutic regimen or having any activity or efficacy Exemplary combination compositions and treatments include biologics ( Proteins), agents, and second active ingredients such as pharmaceuticals. (proteins), agents, pharmaceuticals, treatments and therapies may be used in conjunction with any other method or use of the present invention. For example, a therapeutic method for treating a subject for a blood clotting disorder, They may be administered or performed simultaneously or subsequently.

[0196] The compound, agent, drug, treatment, or other treatment regimen or protocol may be administered in combination. It may be administered as a composition or as a nucleic acid, vector, recombinant vector (e.g., AAV), vector genome or recombinant viral particle delivery or administration (prior to They can be administered separately (before or after) simultaneously, sequentially or sequentially. Thus, the present invention relates to any method or use of the present invention that is disclosed herein or that is readily apparent to those skilled in the art. Any known compound, agent, drug, treatment regimen, treatment protocol, process, treatment The compounds, agents, drugs, A treatment regimen, treatment protocol, process, therapeutic agent or composition is administered to a subject using the core of the present invention. Acid, vector, recombinant vector (e.g., rAAV), vector genome or recombinant vector administration of the virus particles, or It can be executed.

[0197] In one embodiment, a combination composition comprises one or more immunosuppressants. In an embodiment, a method comprises administering one or more immunosuppressive agents to a mammal. In one embodiment, one combination composition comprises an AAV-F In one embodiment, a method comprises administering to a mammal a mammalian target of the immune system ..., the mammalian target of the immune system, and one or more immunosuppressants. Administering or delivering AAV-FIX particles to a subject and immunosuppressing a mammal Those skilled in the art will appreciate that such combinations with one or more immunosuppressive agents may be used. and the appropriate need or timing of administering the immunosuppressant to the mammal. The tag can be determined.

[0198] The methods and uses of the present invention may also be used in combination with other compounds, agents, drugs, treatment regimens, treatments, This includes methods and uses that reduce the need for or use of protocols, processes, or therapeutic agents, for example: For blood clotting diseases, the method or use of the present invention can be carried out by measuring the level of blood clotting in a given subject. Recombinant coagulation factors to supplement insufficient or defective (abnormal or mutated) endogenous coagulation factors Treatment involves less frequent or reduced doses or cessation of administration of coagulation factor proteins. Therefore, in accordance with the present invention, the need for or use of other treatments or therapies may be reduced. Methods and uses are provided.

[0199] The present invention is useful in human and animal, including veterinary, medical applications. Subjects include mammals, such as humans, and non-human mammals. , represents animals, typically mammals such as humans, non-human primates (apes, gibbons) , gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), livestock Animals (chickens, ducks, and other domestic birds, horses, cows, goats, sheep, pigs, etc.) and experiments Animals (mouse, rat, rabbit, guinea pig) are included. Human subjects include fetuses, newborns, Subjects include infants, juveniles, and adults. Subjects may be animals, animals, or animals that have undergone disease model testing (e.g., hematology). Mouse and other animal models of coagulation diseases, as well as other animal models known to those skilled in the art Includes:

[0200] Suitable subjects for treatment are those who produce insufficient amounts of functional gene product (protein). Those who have or are deficient in, or at risk of having, abnormal, partial A gene product (protein) that functions only in certain cases or does not function at all, and can lead to disease. Suitable subjects for treatment according to the present invention include those who have abnormalities associated with disease. or there is a risk of having or making defective (mutant) gene products (proteins), Decreasing the amount, expression or function of abnormal or defective (mutant) gene products (proteins) of that the administration of the drug leads to a cure of the disease or reduces one or more symptoms or Therefore, the target subjects include hemophilia patients ( For example, people who produce abnormal or insufficient blood clotting factors, such as those with hemophilia B, or who have a high blood This includes subjects who do not produce clotting factors.

[0201] Suitable subjects for treatment according to the present invention are those who have received supplemental proteins (e.g., to treat hemophilia) Those who have been or are currently being treated with recombinant blood clotting factors (e.g., FIX for Subjects suitable for treatment according to the present invention further include those with a substantive or phenotypic response to FIX protein. or have not developed a detectable immune response, or FIX-based gene therapy Further, those who have not developed an amount of inhibitory antibodies against the anti-FIX protein that interferes with or inhibits the include.

[0202] In other embodiments, a subject who has been determined (e.g., by genotyping) to have hemophilia B Pediatric human subjects who have been diagnosed with hemophilia B but who do not yet exhibit any of the symptoms of hemophilia B are treated in the first phase. or in other embodiments, to prevent any such symptoms from occurring. To prevent the condition from becoming as severe as it would otherwise be if left untreated Therefore, prophylactic treatment with AAV vectors is possible. Thus, human subjects treated prophylactically in this manner maintain hemostasis and, therefore, the progression of hemophilia B. To produce and maintain adequate FIX activity to prevent or reduce the severity of one or more symptoms. If AAV vectors are administered for this purpose, the patient should be monitored for at least 3, 4, 5, 6, or 7 months. months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months , 16 months, 17 months, 18 months or older. In either case, the AAV vector is AAV-FIX39Padua or an equivalent carrier. psid and at least 95%, 96%, 97%, 98% or 99% identical thereto and the genomic sequence of the gene, and may be administered alone or in a pharmaceutically acceptable composition. The ratio of empty capsids to vector was roughly calculated along with empty capsids of the same capsid species. 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or Other ratios may also be administered to the subject.

[0203] Administration or in vivo delivery to a subject is directed to a disease caused by or associated with the disease. It may be performed prior to the occurrence of any adverse Qi-related symptoms, conditions, complications, etc. For example: Selection (e.g., genetic) may be used to identify such subjects as potential candidates for the compositions, methods, and uses of the present invention. Such subjects may then be used to identify candidates for functional genes. Insufficient or absent amounts of the product (protein) or abnormal Positive feedback on the production of partially functional or non-functional gene products (proteins) This includes those selected to be active.

[0204] The methods and uses of the present invention may be administered systemically, locally or locally, or by any route ( Such delivery and administration includes parenteral (e.g., by injection or infusion) delivery and administration. Oral, e.g., intravascular, intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, or oral mucosal delivery; Typical routes of administration and delivery include intravenous (iv), intraperitoneal (ip) , intra-arterial, subcutaneous, intrapleural, intubated, intrapulmonary, intracavity, iontophoretic, intraorgan, intralymphatic administration This includes giving and delivering.

[0205] Alternatively or additionally, the AAV vector may be delivered to the liver via the portal vein. In another option, AAV vectors can be delivered to the liver via the hepatic artery by injecting them into the femoral artery. The introduced catheter can be used to deliver AAV vectors directly to the liver. non-surgical means, such as endoscopic retrograde cholangiopancreatography (ERCP), to It is also possible to avoid the bloodstream and AAV neutralizing antibodies. To deliver AAV vectors into subjects with developing or pre-existing anti-AAV antibodies Other delivery systems, such as the submandibular duct, can also be used as entry points.

[0206] The dosage may vary and depends on the type, onset, progression and severity of the disease that the treatment is intended to treat. frequency, duration or likelihood, desired clinical endpoint, previous or concurrent treatment The treatment, the subject's health status, age, sex, race or immunocompetence, and other factors may be taken into consideration by those skilled in the art. Any adverse side effects, complications, or other adverse effects of the treatment or therapy may depend on factors that are understood. The dose, number, and frequency of treatment should be proportionate to the risk factors for the condition of the subject. The intensity or duration of the treatment may be increased or decreased. This may affect the dose and timing required to provide a sufficient amount to provide benefit. You will understand the factors that may be causing this.

[0207] The methods and uses of the present invention may be used in conjunction with any of the following: Have been confirmed to have the disease in question or have one or more symptoms of the disease or the subject does not have one or more symptoms of the disease but is a patient who has a condition described herein. As shown, after screening and confirmation of a positive result, the test is continued for 1-2 hours, 2-4 hours, 4-1 It can be performed within 2 hours, 12 to 24 hours, or 24 to 72 hours. The methods and uses of the invention are carried out after the subject has been identified as having the disease for which the treatment is intended. or the subject has one or more symptoms of the disease, or the subject has one of the diseases Those who do not have the above symptoms but have been screened as positive as described herein After confirmation, 1-7 days, 7-14 days, 14-21 days, 21-48 days, or more This can be done within a period of days, months, or years.

[0208] The nucleic acids, vectors, recombinant vectors (e.g., rAAV), vector genomes and and recombinant viral particles, as well as other compositions, agents, pharmaceuticals, biologics (proteins, ) may be incorporated into a pharmaceutical composition (e.g., a pharmaceutically acceptable carrier, excipient). Such pharmaceutical compositions are particularly suitable for in vivo or ex vivo administration to a subject and It is useful for delivery.

[0209] As used herein, the terms "pharmaceutically acceptable" and "physiologically tolerable" are used interchangeably. The term "tolerable" refers to the biologically tolerable state of a gas, liquid, or solid, or mixture thereof. and the formulation is capable of being administered via one or more routes of administration for in vivo delivery or contact. "Pharmaceutically acceptable" or "physiologically acceptable" means that the compound is suitable for the intended use. The composition is a material that is not biologically or otherwise undesirable, e.g. The materials can be administered to a subject essentially without causing undesired biological effects. Thus, such pharmaceutical compositions may be used, for example, to deliver viral vectors or viral particles to a subject. It may also be used in administering

[0210] Such compositions may contain solvents (e.g., water) that are compatible with pharmaceutical administration or in vivo contact or delivery. solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions , syrups, elixirs, dispersions and suspensions, coatings, isotonic agents, and absorption Aqueous and non-aqueous solvents, solutions and suspensions contain suspending agents. Such pharmaceutically acceptable carriers may include tablets (coatings) and thickeners. coated or uncoated), capsules (hard or soft), microcapsules Includes beads, powders, granules and crystals. Supplementary active compounds (e.g., preservatives, antimicrobials, Antiviral and antifungal agents may also be incorporated into the composition.

[0211] Pharmaceutical compositions may be prepared in a variety of ways, including, but not limited to, those described herein or known to those skilled in the art. The pharmaceutical composition may be formulated to suit the route of administration or delivery of the drug. The present invention also includes a carrier, diluent or excipient suitable for administration by administration.

[0212] Compositions suitable for parenteral application include aqueous solutions, non-aqueous solutions, suspensions or emulsions of the active compound. The preparation is typically sterile and equivalent to the blood of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, sorbitol, and sorbitol. Contains ethanol, animal oil, vegetable oil, or synthetic oil.

[0213] Co-solvents and adjuvants may be added to the dosage form. Non-limiting examples of co-solvents include, for example, ibuprofen, cefolia, sorbitol, sorbitol, sorbitol-based ... Alcohols such as isopropyl alcohol, propylene glycol, polyethylene glycol Glycols such as ethanol, polypropylene glycol, glycol ethers, etc.; glycerols roll; polyoxyethylene alcohol, polyoxyethylene fatty acid ester, Contains hydroxyl or other polar groups. Adjuvants include, for example, surfactants such as soy lecithin and oleic acid; sorbitan triphosphate; sorbitan esters such as oleate; and polyvinylpyrrolidone.

[0214] Pharmaceutical compositions and delivery systems suitable for the compositions, methods and uses of the present invention are well known in the art. known (e.g., Remington: The Science and Pr actice of Pharmacy (2003) 20 th ed., Mack Publishing Co., Easton, PA; Remington’s Pharmaceutical Sciences (1990) 18 th ed., Mack Publishing Co., Easton, PA; The Mer ck Index (1996) 12 th ed., Merck Publishing g Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancast er, Pa.; Ansel and Stoklosa, Pharmaceuti cal Calculations (2001) 11 th ed., Lippinc ott Williams & Wilkins, Baltimore, MD; Poznansky et al. Drug Delivery Systems (198 0), R. L. Juliano, ed., Oxford, NY, pp. . See 253-315.

[0215] As used herein, the term "unit dosage form" refers to a dosage form containing a single dose of a compound to be administered to a subject to be treated. Represents physically discrete units suitable as single amounts for analytes. Each unit may optionally contain a drug carrier. When administered in one or more doses, In this case, a predetermined amount calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) is Unit dose forms may be present, for example, in ampoules and vials. The unit dosage form may comprise a liquid composition or may be freeze-dried or lyophilized. For example, the composition may be in a modified state prior to administration or delivery in vivo. A sterile liquid carrier may be added. Individual unit dosage forms may be provided in multi-dose kits or packaging containers. Recombinant vector (e.g., rAAV) sequences, recombinant viral particles, The compounds and their pharmaceutical compositions are preferably administered in a single unit for ease of administration and uniformity of dosage. The composition may be packaged in a single unit dose or multiple unit doses.

[0216] The present invention provides kits having packaging material and one or more components therein. Kits typically include instructions for the components or for the in vitro, in vivo, or ex vivo use of the components. The kit may contain, for example, nucleic acids, compositions, or a label or packaging insert containing instructions for use. Such vectors, such as recombinant vectors, viral (e.g., AAV) vectors or viral particles, A collection of components, optionally containing a second active ingredient, such as another compound, agent, drug, or composition. It may also contain a hydroxyl group.

[0217] A kit refers to the physical structure that contains one or more components of the kit. is capable of maintaining its components sterile and is commonly used for such purposes. It may be made of any material (e.g., paper, corrugated fiber, glass, plastic, packs, foils, ampoules, vials, tubes, etc.

[0218] The label or insert may contain information about one or more ingredients, dosage, mechanism of action, pharmacokinetics, and pharmacodynamics. The label or insert may also contain information identifying the clinical pharmacology of the active ingredient(s). Contains information identifying the manufacturer, lot number, manufacturer's location and date, and expiration date. The label or insert must identify the manufacturer, lot number, manufacturer's location, and date. The label or insert may contain information specifying how the kit components can be used. The label or insert may contain information about the method, use, or treatment of the and a clinician for using the kit components in one or more of the following treatment protocols or regimens. or instructions for the subject, such as dosage, frequency or duration, and The uses, treatment protocols, or prophylactic or therapeutic regimens described herein It may include instructions for doing either.

[0219] The label or insert may include information about the prophylactic or therapeutic benefit the ingredient may provide. The label or insert may include information about any benefits of the particular composition. potential risks, such as warnings to subjects or clinicians about situations in which it is not appropriate to use the The information may include information about potential adverse side effects, complications, or reactions. have, will have, or currently have one or more other drugs that may be incompatible with the composition If the subject is currently taking other therapeutic protocols or treatments that are incompatible with the composition If you are currently receiving or have plans to receive any of the following medications, you may experience side effects or complications. may occur and the instructions therefore contain information about such incompatibilities. Good too.

[0220] A label or insert is a "printed material," e.g., paper, cardboard, or label that identifies a component, kit, or Separate from or attached to the packaging material (e.g., box) or containing the kit components Labels or inserts are not included in ampoules, tubes, or vials. -coded printed label, disc, CD- or DVD-ROM / RA M, optical disks such as DVD, MP3, magnetic tape, or electrical storage such as RAM, ROM Media, or hybrids such as magnetic / optical storage media, flash media or memory cards The present invention may further include computer-readable media such as a computer readable medium.

[0221] Unless otherwise specified, all technical and scientific terms used herein are and have the same meaning as commonly understood by a person skilled in the art to which this invention pertains. Methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. Any suitable method and material can be used; suitable methods and materials are described herein.

[0222] All patents, patent applications, publications and other references cited herein, including but not limited to, Gen. Bank citations and ATCC citations are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.

[0223] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may have alternatives serving the same, equivalent, or similar purpose. Therefore, unless expressly stated otherwise, the disclosed features may be substituted. (e.g., nucleic acid variants, vector plasmids, recombinant vectors (e.g., rAAV) sequences) A recombinant virus particle (e.g., a recombinant virus particle) is an example of a genera of equivalent or similar characteristics.

[0224] As used herein, unless expressly stated otherwise, "a" (a) and the singular form "the" includes plural referents. Thus, for example, "nucleic acid A reference to a "nucleic acid" includes a plurality of such nucleic acids. A reference to a "vector" includes a plurality of such vectors. A reference to a virus or a particle refers to a plurality of such viruses. Contains viruses / particles.

[0225] As used herein, unless expressly stated otherwise, a numerical value or numerical range includes all such ranges and all values ​​or fractions of integers within the ranges. For example, a reference to 80% or more identity is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% etc. , as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1 %, 82.2%, 82.3%, 82.4%, 82.5%, etc.

[0226] References to integers with "greater than" or "less than" mean values ​​greater than the referenced value. This includes any number greater than or less than the reference value, respectively. For example, therefore, "100 References to "less than" include 99, 98, 97, etc., and all numbers on the way to the number 1 (1). Also, "less than 10" includes all numbers on the way to number 1 (1), such as 9, 8, 7, etc. include.

[0227] As used herein, unless expressly stated otherwise, all numerical or Ranges include values ​​and integer sub-values ​​within such ranges, as well as sub-values ​​of integers within such ranges. Thus, for purposes of explanation, reference to a numerical range such as 1-10 includes 1, 2, 3, 4, and so on. , 5, 6, 7, 8, 9, 10, etc., and 1.1, 1.2, 1.3, 1.4, 1.5, etc. Thus, a reference to a range of 1-50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to a maximum of 50, or 1, 50, also 1.1, 1.2, 1.3, 1.4, 1.5, 2.1, 2.2, Includes 2.3, 2.4, 2.5, etc.

[0228] A reference to a continuous range is a range that combines the values ​​of the different range boundaries in that series. Thus, to illustrate a reference to a continuous range, for example, 1-10, 10-2 0, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 1 00-150, 150-200, 200-250, 250-300, 300-400, 4 00-500, 500-750, 750-850 are 1-20, 1-30, 1-40, 1 -50, 1-60, 10-30, 10-40, 10-50, 10-60, 10-70, 1 0-80, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90 , 50-75, 50-100, 50-150, 50-200, 50-250, 100-2 00, 100-250, 100-300, 100-350, 100-400, 100-5 00, 150-250, 150-300, 150-350, 150-400, 150-4 Ranges include 50, 150-500, etc.

[0229] The present invention is set forth herein to describe a number of embodiments and aspects. The invention is generally disclosed using generic terms. The invention also includes materials, process steps, and the like. Certain subjects, such as plans and conditions, protocols or procedures, may be excluded in whole or in part. For example, in certain embodiments or aspects of the invention, materials and / or Therefore, in terms of what the invention does not include, Even if the present invention is not generally expressed herein, it is not expressly excluded in the present invention. Unspecified embodiments are nevertheless disclosed herein.

[0230] Many embodiments of the present invention have been described. However, those skilled in the art will appreciate that the spirit and scope of the present invention are readily apparent. Various modifications may be made to the present invention to adapt it to various uses and situations without departing from the spirit and scope of the present invention. Further modifications and variations are possible. Accordingly, the following examples are intended to be illustrative. and is not intended to limit the scope of the claimed invention in any way. There is no.

[0231] Accordingly, the following examples illustrate the scope of the invention, which is set forth in the appended claims. It is not intended to limit the scope of the invention. Example 1 Vector Design / Preparation

[0232] 338L Padua mutation (Simioni P et al., N Engl J Med 20 9, 361:1671) encodes a human factor IX protein with high specific activity. A novel Factor IX nucleic acid was designed ("FIX39-Padua", SEQ ID NO: 10, Figure 1). 10) FIX39Padua contains CpG dinucleotides in the FIX coding sequence and intron sequence. It is completely deficient in nucleotides. For comparative testing, FIX19 (Mingozzi (Sci. Transl Med. 2013) was prepared and placed in FIX Padua. To eliminate any potentially disruptive results, FIX Padua is included. ("FIX19-Padua", SEQ ID NO: 11, Figure 11).

[0233] Plasmid ("pAAV-ApoE_hAAT-FIX39", 11,125 base pairs, sequence The FIX39 Padua expression plasmid (column 12, Figure 12A) was synthesized and used to express the FIX39 Padua gene. pAAV-ApoE_hAAT- contained the cassette and factors listed in Table 2. A map of FIX39 is shown in Figure 13. [Table 2]

[0234] The FIX39Padua coding sequence and the sequence of intron A are set forth in SEQ ID NO:25. It is described in (Figure 15). In addition, FIX19PaduaCDS and the same regulatory factors, Adeno-associated inverted terminal repeats (ITRs) and pAAV-ApoE_hAATFIX3 A plasmid containing the same liver-specific ApoE / hAAT promoter as in 9 was also synthesized.

[0235] FIX39Padua ("AAV-FIX39Padua") transgene and FIX1 For the 9Padua ("AAV-FIX19Padua") transgene, triple transfection was performed. The transfection process and subsequent double cesium chloride gradient centrifugation (Ayu so E, Met et al., Gene Ther 2010, 17:503) using 4 An AAV vector containing the -1 capsid mutation (SEQ ID NO: 4) was prepared. The linearized plasmid was used to titer the vector by quantitative PCR. For the study described in Example 3, 200 μL per mouse for tail vein injection Dilute the base with PBS, 5% sorbitol, 0.001% F68 to a final volume of 100 ml. The culprit was diluted. Example 2 In vitro gene transfer with AAV mutant 4-1

[0236] Four different multiplicities of infection ranging from 500 to 62,500 vector genomes per cell The 4-1 mutant capsid (SEQ ID NO: 4) expressing luciferase at a multiplicity of infection (MOI) of 100 was used. Primary hepatocytes from cynomolgus monkeys and humans were transduced with the α-glucan-1-phosphate dehydrogenase (GD)-1 ... Luciferase expression was then analyzed. As shown in Figure 16, The ratio of human hepatocytes to non-human primate hepatocytes ranged from 0 to 10, depending on the MOI used. These in vitro generated data were in the range of 0.8 to 1.5. Previous in vivo observations were made when comparing the expression of coagulation factor IX in rat and human subjects. Seems to be consistent with the observations. Example 3 Efficacy Study

[0237] Efficacy of AAV-FIX39Padua and AAV-FIX19Padua in mice A study was conducted to compare the effects of 10-week-old mice on the 1st and 2nd groups of mice. x10 11 vg / kg or 1 × 10 12 vg / kg of either AAV-FIX39 Padu a and AAV-FIX19Padua. After vector administration, heparinized Blood was collected by retro-orbital bleeding using a capillary tube. Plasma was stored at 4°C. Separate by centrifugation at 9000 rpm for 10 minutes and store frozen at -80°C until analysis. It remained.

[0238] The collected plasma was used to assess the expression of the hFIX transgene. Kit (Affinity Biologicals, Ancaster, ON, Canada) ) was used to measure human FIX levels in plasma.

[0239] The activity level of human FIX was determined by activated partial thromboplastin time (aPTT) assay. The plasma samples were prepared from human FIX-deficient plasma (George King Bio and aPTT reagent (Trinity Biotech) at a ratio of 1:1:1. by volume, followed by incubation at 37°C for 180 seconds. The aPTT assay was performed. Coagulation was initiated by adding 25 mM calcium chloride. Clot formation was assessed using a STart4 coagulation device (Diagnostica Stago). The reaction time was measured. Starting with 120 μl followed by 1:2 serial dilutions (120 μl + 120 μl), A standard curve was generated using normal pooled plasma from the 17th week after vector administration. The human standard curve was used to calculate the activity of each sample. Activity was also measured in untreated mice. The average value was then calculated as the additional (i.e., human) activity attributed to the FIX Padua protein. To calculate sex, the genotype was subtracted from the treated sample.

[0240] As shown in Figure 17, AAV-FIX39 Padua and AAV-FIX19 Padua appears to express substantially equivalent levels of FIX.

[0241] 1 × 10 12 Mice treated with a vector dose of 1000 vg / kg The activity of human FIX was measured in the presence of antigen. The activity ratio to antigen ranged from 5.2 to 7.5. Within the scope of both the FIX19 Padua Group and the FIX39 Padua Group The average was 6.4 (Table 3). [Table 3]

[0242] These results suggest that the potencies of both expression cassettes are substantially similar. However, the two constructs were also analyzed in a plasmid-based tail vein injection setting. The rationale for assessing FIX levels resulting from in vivo administration of NA is AAV titration, Both expression cassettes were compared without potential interference from differences in vector production, etc. This was to

[0243] As shown in Figure 18, both naked expression cassettes were able to express FIX. The data obtained in the AAV setting are comparable in efficacy to those in the These results confirmed that FIX19Padua and FIX39Padua expression It is shown that the cassettes have similar efficacy. Example 4 AAV-FIX39 Padua gene therapy

[0244] To determine the safety and kinetics of a single intravenous infusion of AAV-FIX39 Padua The AAV4-1 capsid mutants used have shown excellent efficacy in preclinical studies. Safety and efficacy (1 × 10 12 maximal survival in NHPs 3 months after vector injection of 2000 vg / kg The ability to achieve a retained FIX activity level of up to 35% was demonstrated. Neutralizing antibodies (Abs) cross-reacting with the V4-1 capsid mutant were more abundant than those with AAV8. The design of this study is provided in Table 4. [Table 4] TIFF0007818558000005.tif65170 Example 5 Clinical Results

[0245] Four subjects with hemophilia B were injected with the AAV-FIX39Padua vector alone. The first two subjects, aged 23 and 18, were given intravenous injections of the drug. The third subject was 47 years old and had a history of HCV infection. All four subjects had a response to the novel AAV capsid. The neutralizing antibodies were selected and found to be negative.

[0246] Subjects were given 5 x 10 11 vg / kg AAV-FIX39Pa The dua vector was injected intravenously. All AAV-FIX39Padua vectors administered to each subject were shown in Figures 20 to 23. and are summed with the amount of AAV empty capsid shown.

[0247] Figures 19 to 23 show the results of the study. Administered on day 1 of study. This was reflected by increased FIX activity throughout the entire study evaluation period. Thus, the results show increased Factor IX production in all four subjects.

[0248] The initial increase in FIX activity, approximately from day 0 to day 3, was observed after 100 IU / kg of Alp rolix™ or BeneFIX™, which It is a recombinant FIX-Fc fusion protein with an approximate half-life of 82 hours. Factor IX activity caused by the X39Padua vector was approximately 6 minutes after AAV vector injection. It begins after the 8th day.

[0249] As summarized in FIG. 19, and in FIGS. 20, 21A, 22A and 23A, As shown for individual subjects in the study, Factor IX was detected in all four subjects. The activity gradually increased over the evaluation periods of 183 days, 102 days, 69 days, and 50 days. These data are considered to be stable at 5×10 11 vg / kg AAV-FIX A single infusion of the Padua vector provides sufficient and sustained factor IX immunity in patients with hemophilia B. The production and activity of factors that result in meaningful and beneficial blood clotting activity to provide hemostasis. It indicates that you provide

[0250] As shown in Figures 19, 20A, 21A, 22A, and 23A, the factor IX activity levels Bell was measured at 183, 102, 69, and 50 days after injection for subjects 1-4, respectively. These were 28%, 41%, 26%, and 33% of the normal values, respectively. Subject #3, who had an extended product, presented with a suspected ankle hemorrhage 2 days after vector infusion and was subsequently admitted to hospital. The patient himself was treated, but otherwise no factor was infused and no bleeding occurred during the evaluation period.

[0251] Immunosuppressants (steroids) were not administered to any of the subjects. There is no persistent elevation of transaminases above the upper limit of normal, and no adverse effects of treatment No adverse effects were observed (Figures 20B, 21B, 22B, and 23).

[0252] T cell responses to AAV and to FIX were monitored in all four subjects. ELISPOT was used to measure the response of the IgG to the IgG. The response was absent or very low. Notably, the time course for factor IX levels to rise to a stable level was remarkably consistent over the number of days. Assuming an 8-fold increase in specific activity of the Padua variant of factor IX, Thus, moderate variations in antigen levels result in larger changes in activity levels.

[0253] Published data (Nathwani et al., N Engl J Med. 371( 21):1994-2004(2014)) was developed using AAV8 expressing wild-type factor IX. They have shown long-term expression of factor IX in vector-infused hemophilia B patients. While the level of expression was significantly higher at the smallest dose (2 x 10 11 vector genome [vg] / kg body weight) The normal values ​​were 1.4% to 2.2% at the highest dose (2 × 1012 vg / kg) Furthermore, 4 / 6 subjects who received the highest dose had a mean ... , transaminases increased relative to the highest dose (but 2 × 10 11 or 6 x 10 11 The immunosuppressant (prednisolone) was administered to reduce the risk of pulmonary embolism (which was not observed at lower doses of 200 mg / kg). Data from natural history studies of hemophilia patients suggest that A blood concentration of ~12% of FIX is required to reduce the annual number of joint bleeds to zero. (den Uijl et al., Haemophilia 17(1):4 1-4 (2011)).

[0254] These are bioengineered factor IX transgenes expressing high specific activity. These are the first clinical results using the novel AAV capsid. The observed factor IX activity levels were 28%, 41%, 26%, and 33% of normal. Substantially higher circulation than found in previous studies based on published data Factor IX levels and to reduce the number of spontaneous joint bleeds per year to zero. exceed the circulating factor IX levels required for

[0255] Furthermore, the intrinsic factor IX activity levels observed in this study were significantly higher after vector infusion. without the use of recombinant factor IX and without the use of immunosuppressants (steroids) These results demonstrate the feasibility of liver-directed therapy, which consequently does not require immunosuppression. Low-dose AAV vector administration enables an important goal for targeted gene therapy. We demonstrate the development of AAV-FIX vectors capable of directing high levels of coagulation factor expression. The factor IX activity levels observed in this study were maintained over the course of the study. It was held. Example 6 Reduced immunogenicity of the AAV-FIX39 Padua vector

[0256] For the current Phase I / II study, validated interferon-gamma (IF Ng) Enzyme-linked immunospot (ELISPOT) assay Regarding potential immune responses to AV vectors, 5 × 11 vg / kg AAV-FIX Four subjects receiving 39 Padua were monitored. Purified erythrocytes isolated from weekly blood samples were The isolated PBMCs were examined in an interferon-γ ELISPOT assay. Six AAV capsid peptide pools, each containing a different peptide, were transfected into 2e5 cells. T cell responses were assessed by incubating the cells with a biotinylated antibody against IFNg. Subsequent colorimetric development yields spots per million cells detected using The highest response at each time point was reported as single-forming units (SFU). The pools are shown as SFU / million cells. The critical point is over 50 SFUs and three times the media control (blue line). Samples 840-003-001, 840-001-002, 840-001-004 and 8 40-001-005 (shown in black) is available at 26 weeks, 14 weeks, 11 weeks, and 8 weeks. The ELISPOT was performed on two subjects, CP-16 and PT1. 7 received 1e12vg / kg of AAV8-FIX19 vector, and 1 subject from a previous trial in which 2e12vg / kg of AAV8-FIX19 was administered. Shown in red.

[0257] The historically accepted criterion for a positive T-cell response is an S >50. FU and 3x values ​​of background (media) controls were used to measure the FU and 3x values ​​of background (media) controls 2 days after injection. After six weeks, there was little or no response in three subjects. (Figure 24A). This was done by using the codon 1441 to deliver the FIX transgene cassette to three subjects. Using optimized AAV8 vectors, robust IFNg T cell responses were observed as early as week 2 A response was observed in a previous unpublished study by our group (Figure 24B). In stark contrast, AAV-2 (Manno et al., 2006 Nat Med) Other previously published studies and the AAV-8 self-complementary vector (Nathwa Other previously published studies using AA We show evidence of early T cell responses to the V capsid. Importantly, the transgene No reaction to the product has been observed in this trial.

[0258] Immunity against transfected hepatocytes presenting AAV capsid T cell epitopes Activation of T cells via the IL-1 response is a test subject in which transgene expression is short-lived and eventually lost. It is hypothesized that AAV-FIX may play a role in the body. The reduced immunogenicity profile of the 39Padua vector holds promise for overall efficacy This means significant improvement. Rh74 VP1 amino acid sequence (SEQ ID NO: 1) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQD NGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYD QQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGI GKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLG SGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV ITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYST PWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFN IQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSA HQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEY FPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPL IDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNW LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLV NPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSV MLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNS QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQ VSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTE GTYSEPRPIGTRYLTRNL Rh74 VP2 amino acid (SEQ ID NO:2) TAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQT GDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNE GADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHL YKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPR DWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIAN NLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQ YGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYN FEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGT AGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQ NNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFF PSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATE QYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQ GPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVP ADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRW NPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRN L Rh74 VP3 amino acid (SEQ ID NO:3) MAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITT STRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWG YFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQV KEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQ YLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPG PCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPG VAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLT SEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGA LPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLK HPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSV EIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTY SEPRPIGTRYLTRNL 4-1 Mutant VP1 capsid amino acid sequence (SEQ ID NO: 4) 1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQ QKQDNGRGLVLPGYKYLGPFNGLD 61 KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHA DAEFQERLQEDTSFGGNLGRAVFQ 121 AKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDS STGIGKKGQQPAKKRLNFGQTGDS 181 ESVPDPQPIGEPPA A PSG V G PN TMAAGGGAPMADNN EGADGVGSSSGNWHCDSTWLGDRV 241 ITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYF GYSTPWGYFDFNRFHCHFSPRDWQ 301 RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIA NNLTSTIQVFTDSEYQLPYVLGSA 361 HQGCLPPFPADVFMIPQYGYLTLNNGSQAV GRSSF YCLEYFPSQMLRTGNNFEFSYNFED 421 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGG TAGTQQLLFSQAGPNNMSAQAKNW 481 LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGR DSLVNPGVAMATHKDDEERFFPSS 541 GVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVAT EQYGVVADNLQQQNAAPIVGAVNS 601 QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPL MGGFGLKHPPPQILIKNTPVPADP 661 PTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTNVDFAVNTE 721 GTYSEPRPIGTRYLTRNL 4-1 Mutant VP2 capsid amino acid sequence (SEQ ID NO: 27) TAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQT GDSESVPDPQPIGEPPA A PSG V G PN T MAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITT STRTWALPTYNNHLYKQISNGTS GGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNN WGFRPKRLNFKLFNIQVKEVTQNEG TKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPAD VFMIPQYGYLTLNNGSQAVGRSSFYCL EYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMN PLIDQYLYYLSRTQSTGGTAGTQQLLF SQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAW TGATKYHLNGRDSLVNPGVAMAT HKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEI KTTNPVATEQYGVVADNLQQQNAAP IVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHP SPLMGGFGLKHPPPQILIKNTPVPADPP TTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEI QYTSNYYKSTNVDFAVNTEGTYSEPRPI GTRYLTRNL 4-1 Mutant VP3 capsid amino acid sequence (SEQ ID NO: 3) MAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITT STRTWALPTYNNHLYKQISNGTS GGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNN WGFRPKRLNFKLFNIQVKEVTQNEG TKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPAD VFMIPQYGYLTLNNGSQAVGRSSFYCL EYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMN PLIDQYLYYLSRTQSTGGTAGTQQLLF SQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAW TGATKYHLNGRDSLVNPGVAMAT HKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEI KTTNPVATEQYGVVADNLQQQNAAP IVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHP SPLMGGFGLKHPPPQILIKNTPVPADPP TTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEI QYTSNYYKSTNVDFAVNTEGTYSEPRPI GTRYLTRNL Amino acid sequence of mutant VP1 capsid of 15-1 (SEQ ID NO: 5) 1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQ QRQDNGRGLVLPGYRYLGPFNGLD 61 KGEPVNAADAAALEHDRAYDQQLQAGDNPYLRYNHA DAEFQERLQEDTSFGGNLGRAVFQ 121 AKKRVLEPLGLVESPVRTAPGKKRPVEPSPQRSPDS STGIGKKGQQPARKRLNFGQTGDS 181 ESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNN EGADGVGSSSGNWHCDSTWLGDRV 241 ITTSTRTWALPTYNNHLYRQISNGTSGGSTNDNTYF GYSTPWGYFDFNRFHCHFSPRDWQ 301 RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTRTIA NNLTSTIQVFTDSEYQLPYVLGSA 361 HQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFY CLEYFPSQMLRTGNNFEFSYNFED 421 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGG TAGTQQLLFSQAGPNNMSAQAKNW 481 LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGR DSLVNPGVAMATHRDDEERFFPSS 541 GVLMFGRQGAGRDNVDYSSVMLTSEEEIRTTNPVAT EQYGVVADNLQQQN AAPIVGAVNS 601 QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPL MGGFGLKHPPPQILIKNTPVPADP 661 PTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSTNVDFAVNTE 721 GTYSEPRPIGTRYLTRNL 15-2 mutant VP1 capsid amino acid sequence (SEQ ID NO: 6) 1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQ QRQD NGRGLVLPGY RYLGPFNGLD 61 KGEPVNAADAAALEHDRAYDQQLQAGDNPYLRYNHA DAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPLGLVESPVRTAPGKKRPVEPSPQRSPDS STGI GKRGQQPARK RLNFGQTGDS 181 ESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNN EGAD GVGSSSGNWH CDSTWLGDRV 241 ITTSTRTWALPTYNNHLYRQISNGTSGGSTNDNTYF GYST PWGYFDFNRF HCHFSPRDWQ 301 RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTRTIA NNLT STIQVFTDSE YQLPYVLGSA 361 HQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFY CLEY FPSQMLRTGN NFEFSYNFED 421 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGG TAGT QQLLFSQAGP NNMSAQAKNW 481 LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGR DSLV NPGVAMATHR DDEERFFPSS 541 GVLMFGKQGAGRDNVDYSSVMLTSEEEIRTTNPVAT EQYG VVADNLQQQN AAPIVGAVNS 601 QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPL MGGF GLKHPPPQIL IKNTPVPADP 661 PTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKR WNPE IQYTSNYYKS TNVDFAVNTE 721 GTYSEPRPIGTRYLTRNL 15-3 / 15-5 mutant VP1 capsid amino acid sequence (SEQ ID NO: 7) 1 MAADGYLPDWLEDNLSEGIR EWWDLKPGAPKPKAN QQRQD NGRGLVLPGY RYLGPFNGLD 61 KGEPVNAADAAALEHDRAYDQQLQAGDNPYLRYNHA DAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPLGLVESPVRTAPGKKRPVEPSPQRSPDS STGI GKRGQQPAKK RLNFGQTGDS 181 ESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNN EGAD GVGSSSGNWH CDSTWLGDRV 241 ITTSTRTWALPTYNNHLYRQISNGTSGGSTNDNTYF GYST PWGYFDFNRF HCHFSPRDWQ 301 RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTRTIA NNLT STIQVFTDSE YQLPYVLGSA 361 HQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFY CLEY FPSQMLRTGN NFEFSYNFED 421 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGG TAGT QQLLFSQAGP NNMSAQAKNW 481 LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGR DSLV NPGVAMATHR DDEERFFPSS 541 GVLMFGRQGAGRDNVDYSSVMLTSEEEIRTTNPVAT EQYG VVADNLQQQN AAPIVGAVNS 601 QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPL MGGF GLKHPPPQIL IKNTPVPADP 661 PTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKR WNPE IQYTSNYYKS TNVDFAVNTE 721 GTYSEPRPIGTRYLTRNL 15-4 mutant VP1 capsid amino acid sequence (SEQ ID NO: 8) 1 MAADGYLPDWLEDNLSEGIREWWDLKPGAP KPKAN QQRQD NGRGLVLPGY RYLGPFNGLD 61 KGEPVNAADAAALEHDRAYDQQLQAGDNPY LRYNH ADAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPLGLVESPVRTAPGKKRPVEPSP QRSPD SSTGI GKRGQQPAKK RLNFGQTGDS 181 ESVPDPQPIGEPPAAPSGVGPNTMAAGGGA PMADN NEGAD GVGSSSGNWH CDSTWLGDRV 241 ITTSTRTWALPTYNNHLYRQISNGTSGGST NDNTY FGYST PWGYFDFNRF HCHFSPRDWQ 301 RLINNNWGFRPKRLNFKLFNIQVKEVTQNE GTRTI ANNLT STIQVFTDSE YQLPYVLGSA 361 HQGCLPPFPADVFMIPQYGYLTLNNGSQAV GRSSF YCLEY FPSQMLRTGN NFEFSYNFED 421 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSR TQSTG GTAGT QQLLFSQAGP NNMSAQAKNW 481 LPGPCYRQQRVSTTLSQNNNSNFAWTGATK YHLNG RDSLV NPGVAMATHR DDEERFFPSS 541 GVLMFGKQGAGRDNVDYSSVMLTSEEEIRT TNPVA TEQYG VVADNLQQQN AAPIVGAVNS 601 QGALPGMVWQNRDVYLQGPIWAKIPHTDGN FHPSP LMGGF GLKHPPPQIL IKNTPVPADP 661 PTTFNQARLASFITQYSTGQVSVEIEWELQ KENSK RWNPE IQYTSNYYKS TNVDFAVNTE 721 GTYSEPRPIGTRYLTRNL 15-6 Mutant VP1 Capsid Amino Acid Sequence (SEQ ID NO: 9) 1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQ QRQDNGRGLVLPGY RYLGPFNGLD 61 KGEPVNAADAAALEHDRAYDQQLQAGDNPYLRYNHA DAEFQERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPLGLVESPVRTAPGKKRPVEPSPQRSPDS STGIGKKGQQPAKK RLNFGQTGDS 181 ESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNN EGADGVGSSSGNWH CDSTWLGDRV 241 ITTSTRTWALPTYNNHLYRQISNGTSGGSTNDNTYF GYSTPWGYFDFNRF HCHFSPRDWQ 301 RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTRTIA NNLTSTIQVFTDSE YQLPYVLGSA 361 HQGCLPPFADVFMIPQYGYLTLNNGSQAVGRSSFY CLEYFPSQMLRTGN NFEFSYNFED 421 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGG TAGTQQLLFSQAGP NNMSAQAKNW 481 LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGR DSLVNPGVAMATHR DDEERFFPSS 541 GVLMFGRQGAGRDNVDYSSVMLTSEEEIRTTNPVAT EQYGVVADNLQQQN AAPIVGAVNS 601 QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPL MGGFGLKHPPPQIL IKNTPVPADP 661 PTTFNQARLASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYYKS TNVDFAVNTE 721 GTYSEPRPIGTRYLTRNL FIX39 nucleic acid sequence (sequence number: 10) ATGCAGAGGGTGAACATGATCATGGCTGAGAGCCCTGGCC TGATCACCATCTGCCTGCTGGGCTACCTGCTGTCTGCTGA ATGTACAGTTTTTCTTGATCATGAAAATGCCAACAAAAATT CTGAATAGACCAAAGAGGTATAACTCTGGCAAGCTTGAAG AGTTTGTACAGGGAATCTGGAGAGAGAGTGTATGGAAGA GAAGTGCAGCTTTGAGGAAGCCAGAGAGGTTTGAAAAT ACAGAGAGAACAACTGAATTTTGGAAGCAGTATGTGGATG GTGATCAATGTGAGAGCAATCCCTGCTTGAATGGGGGGAG CTGTAAAGATGATATCAACAGCTATGAATGTTGGTGTCCC TTTGGATTTGAGGGGAAAAACTGTGAGCTTGATGTGACCT GTAATATCAAGAATGGCAGGTGTGAGCAATTTTGCAAGAA TTCTGCTGATAACAAAGTGGTCTGTAGCTGCACTGAGGGA TATAGGCTGGCTGAAAACCAGAAGAGCTGTGAACCTGCAG TGCCTTTTCCCTGTGGGAGAGTGTCTGTGAGCCAAACCAG CAAGCTGACTAGGGCTGAAGCAGTCTTTCCTGATGTAGAT TATGTGAATAGCACTGAGGCTGAGACAATCCTTGACAATA TCACTCAGAGCACACAGAGCTTCAATGACTTCACCAGGGT GGTAGGAGGGGAGGATGCCAAGCCTGGGCAGTTCCCCTGG CAGGTAGTGCTCAATGGAAAAGTGGATGCCTTTTGTGGAG GTTCAATTGTAAATGAGAAGTGGATTGTGACTGCAGCCCA CTGTGTGGAAACTGGAGTCAAGATTACTGTGGTGGCTGGA GAGCACAATATTGAGGAAACTGAGCACACTGAGCAGAAGA GGAATGTGATCAGGATTATCCCCCACCACAACTACAATGC TGCTATCAACAAGTACAACCATGACATTGCCCTCCTGGAA CTGGATGAACCCCTGGTCTTGAACAGCTATGTGACACCCA TCTGTATTGCTGATAAAGAGTACACCAACATCTTCTTGAA ATTTGGGTCTGGATATGTGTCTGGGCTGGGGCAGGGTGTTC CATAAAGGCAGGTCTGCCCTGGTATTGCAGTATTTGAGGG TGCCTCTGGTGGATAGAGCAACCTGCTTGCTGAGCACCAA GTTTACAATCTACAACAATATGTTCTGTGCAGGGTTCCAT GAAGGTGGTAGAGACAGCTGCCAGGGAGATTCTGGGGGTC CCCATGTGACTGAGGTGGAGGGAACCAGCTTCCTGACTGG GATTATCAGCTGGGGTGAGGAGTGTGCTATGAAGGGAAAG TATGGGATCTACACAAAAGTATCCAGATATGTGAACTGGA TTAAGGAGAAAACCAAGCTGACTTGA FIX19 nucleic acid sequence(sequence number:11) ATGCAGCGCGTGAACATGATCATGGCCGAGAGCCCTGGCC TGATTACCATCTGCCTGTTAGGATATCTACTCAGTGCTGA ATGTACAGTTTTTCTTGATCATGAAAACGCCAACAAAATC CTGAACCGGCCCAAGCGGTACAACTCAGGCAAGCTGGAAG AGTTCGTGCAGGGCAACCTGGAACGGGAGTGCATGGAAGA GAAGTGCAGCTTCGAGGAAGCCCGGGAGGTGTTCGAGAAC ACCGAGCGGACCACCGAGTTCTGGAAGCAGTACGTGGACG GCGACCAGTGCGAGTCAAACCCCTGCCTGAACGGCGGCAG CTGCAAGGACGATATCAACAGCTACGAGTGCTGGTGCCCC TTCGGCTTCGAGGGCAAGAACTGCGAGCTGGACGTGACCT GCAACATCAAGAACGGCCGCTGCGAGCAGTTCTGCAAGAA CAGCGCCGACAACAAGGTGGTGTGCTCATGCACTGAGGGC TACCGGCTGGCCGAGAACCAGAAGAGCTGCGAGCCCGCCG TGCCCTTCCCCTGCGGCAGAGTGTCCGTGAGCCAGACCAG CAAGCTGACCAGGGCCGAGGCCGTGTTCCCTGACGTGGAC TACGTGAACTCAACCGAGGCCGAGACAATCCTGGACAACA TCACCCAGAGCACCCAGTCCTTCAACGACTTCACCCGGGT GGTGGGCGGCGAGGACGCCAAGCCCGGCCAGTTCCCTTGG CAGGTGGTGCTGAACGGCAAGGTGGACGCCTTCTGCGGCG GCTCAATCGTGAACGAGAAGTGGATCGTGACAGCCGCCCA CTGCGTGGAGACAGGCGTGAAGATCACCGTGGTGGCCGGC GAACACAATATCGAGGAAACCGAGCACACCGAGCAGAAAC GGAACGTGATCCGGATTATCCCCCACCACAACTACAACGC CGCCATCAACAAGTACAACCACGATATCGCCCTGCTGGAA CTGGACGAGCCTCTGGTGCTGAATTCATACGTGACCCCCA TCTGTATCGCCGACAAAGAGTACACCAACATCTTTCTGAA GTTCGGCAGCGGCTACGTGTCCGGCTGGGGCAGGGTGTTC CACAAGGGCCGCAGCGCCCTGGTGCTGCAGTACCTGCGGG TGCCCCTGGTGGACAGAGCCACCTGCCTGCGGTCAACCAA GTTCACCATCTACAACAACATGTTCTGCGCCGGCTTCCAC GAGGGCGGCAGGGACAGCTGCCAGGGCGACAGCGGCGGAC CCCACGTGACCGAGGTGGAGGGCACCAGCTTTCTGACCGG CATCATCTCATGGGGCGAGGAATGCGCCATGAAGGGCAAG TACGGAATCTACACTAAGGTGTCAAGATACGTGAACTGGA TCAAAGAGAAAACCAAGCTGACCTGA pAAV-ApoE_hAAT-FIX39 (SEQ ID NO: 12) LOCUS FIX39 11125 bp DNA circular UNA DEFINITION AKA FIX39 Step 4. ACCESSION urn.local...e-3um3omk VERSION urn.local...e-3um3omk KEYWORDS. SOURCE ORGANISM . FEATURES Location / Qualifiers repeat_region 1..141 / Imported_from="<a " href=""http: / / wishar t.biology.ualberta.ca / PlasMapper"">Pla " sMapper" / Transferred_From=" LAAV-2_ITR" / Transferred_Simila rity="100.00%" / modified_by="User" / label="AAV2 ITR" enhancer 152..472 / created_by="User" / modified_by="User" / Transferred_From=" ApoE HCR-1" / Transferred_Simila rity="100.00%" / label="ApoE HCR-1" promoter 482..878 / vntifkey=21 / ApEinfo_fwdcolor= "#ffff00" / ApEinfo_revcolor=" #0080ff" / Transferred_From=" hAAT Promoter" / Transferred_Simila rity="100.00%" / modified_by="User" / label="hAAT Promot er" 5'UTR 879..907 / created_by="User" / label="FIX 5'UTR" CDS order(908..995,2434 ..3731) / created_by="User" / Transferred_From=" hFIX CDS" / Transferred_Simila rity="79.22%" / modified_by="User" / label="hFIX CDS" intron 996..2433 / created_by="User" / Transferred_From=" hFIX Intron" / Transferred_Simila rity="100.00%" / modified_by="User" / label="hFIX Intron" 3'UTR 3732..3779 / created_by="User" / label="hF9 3' UTR" Terminator 3820..4047 / Imported_from="<a href=""http: / / wishar t.biology.ualberta.ca / PlasMapper"">Pla sMapper" / Transferred_From=" bGH_PA term" / Transferred_Simila rity="100.00%" / label="bGH_PA term" repeat_region complement(4097..42 04) / modified_by="User" / label="AAV2 ITR" misc_feature 4219..8579 / modified_by="User" / label="Lambda Stuf fer" Gene complement(8491..85 80) / Imported_from="<a href=""http: / / wishar t.biology.ualberta.ca / PlasMapper"">Pla sMapper" / Transferred_From=" cosN" / Transferred_Simila rity="100.00%" / label="cosN" rep_origin 8680..8986 / modified_by="User" / label="F1 Ori" misc._marker 9284..10096 / modified_by="User" / label="Kan R" rep_origin complement(10453..1 1120) / modified_by="User" / label="pUC Ori" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgct ca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagt ga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggcct ag taggctcaga ggcacacagg agtttctggg 181 ctcaccctgc ccccttccaa cccctcag tt cccatcctcc agcagctgtt tgtgtgctgc 241 ctctgaagtc cacactgaac aaacttca gc ctactcatgt ccctaaaatg ggcaaacatt 301 gcaagcagca aacagcaaac acacagcc ct ccctgcctgc tgaccttgga gctggggcag 361 aggtcagaga cctctctggg cccatgcc ac ctccaacatc cactcgaccc cttggaattt 421 cggtggagag gagcagaggt tgtcctgg cg tggtttaggt agtgtgagag gggtacccgg 481 ggatcttgct accagtggaa cagccact aa ggattctgca gtgagagcag agggccagct 541 aagtggtact ctcccagaga ctgtctga ct cacgccaccc cctccacctt ggacacagga 601 cgctgtggtt tctgagccag gtacaatg ac tcctttcggt aagtgcagtg gaagctgtac 661 actgcccagg caaagcgtcc gggcagcg ta ggcgggcgac tcagatccca gccagtggac 721 ttagcccctg tttgctcctc cgataact gg ggtgaccttg gttaatattc accagcagcc 781 tcccccgttg cccctctgga tccactgc tt aaatacggac gaggacaggg ccctgtctcc 841 cagctcag gcaccaccac tgaggtgg ga cagtgaatac cactttcaca atctgctagc 901 aaggttatg cagaggtga acatgatc at ggctgagagc cctggcctga tcaccatctg 961 cctgctgggc tacctgctgt ctgctgaa tg tacaggtttg tttcctttt tataatacat 1021 tgagtatgct tgccttttag atatagaa at atctgattct gtctcttca ctaaatttg 1081 attacks slowly slowly ag tctaacagcc agcaccagg ttggtaagta 1141 ctggttctt gttagcttagg ttttctc tt cttcactttt aaaactaaat nagatgacaa 1201 tgcttatgat gcaataaggt tttaaa ca ctgttcagtt cagtatttgg tcatgtaatt 1261 cctgttaaa aacagtcatc tccttggt tt aaaaaatta aagtgggaaaaaaaaah 1321 taggaata taggaaaaaaacc and agtatttg tttggactta ccactttgaa 1381 Atcaattgg Gaaaaaag Storm gt gggccttatt acacaaaag tctgatttta 1441 Agatatgtga Cattcagg Ttcaga gt atgtaggag gtgtgtctct aattttaa 1501 attatatc ttcaatttaa agttttag tt aaaactaaa gattacctt tcattagcaa 1561 gctgttagtt atcaccaag ctttcat gg attaggaaaaatcattg tctcatctc 1621 aacatcttg gagttgatat tgggga you will see gttgagttcc ctaggggaga 1681 aaagct windows wind gt aggagttag ctattgcac atattacact 1741 ttgttttttc acactacag tgacttta she is screaming very quickly 1801 attackacca tttggacaa cagcatgt tc tcacagtaag cacttatcac acttacttgt 1861 Hurricane Gatcaatc Tagtagct ga cagtaccagg atcaggggtg caccctaa 1921 GCACCAG AAGCTGACT GCCCTGT gg ttcccactcc agacatgatg tcagctgtga 1981 atcaccc cctggaccat attackaggc tt ctgttcttca ggagacattt gttcaagtc 2041 atttgggca ccatattctg aaaacagc cc agccagggtg atggatcact ttgcaaagat 2101 cctcaatgag ctattttcaa gtgatgac aa agtgtgaagt taagggctca tttgagaact 2161 ttctttttca tccaaagtaa attcaaat at gattagaaat ctgacctttt attactggaa 2221 ttctcttgac taaaagtaaa attgaatt tt aattcctaaa tctccatgtg tatacagtac 2281 tgtgggaaca tcacagattt tggctcca tg ccctaaagag aaattggctt tcagattatt 2341 tggattaaaa acaaagactt tcttaaga ga tgtaaaattt tcatgatgtt ttcttttttg 2401 ctaaaactaa agaattattc ttttacat tt cagtttttct tgatcatgaa aatgccaaca 2461 aaattctgaa tagaccaaag aggtataa ct ctggcaagct tgaagagttt gtacagggga 2521 atctggagag agagtgtatg gaagagaa gt gcagctttga ggaagccaga gaagtgtttg 2581 aaaatacaga gagaacaact gaattttg ga agcagtatgt ggatggtgat caatgtgaga 2641 gcaatccctg cttgaatggg gggagctg ta aagatgatat caacagctat gaatgttggt 2701 gtccctttgg atttgagggg aaaaactg tg agcttgatgt gacctgtaat atcaagaatg 2761 gcaggtgtga gcaattttgc aagaattc tg ctgataacaa agtggtctgt agctgcactg 2821 agggatatag gctggctgaa aaccagaa ga gctgtgaacc tgcagtgcct tttccctgtg 2881 ggagagtgtc tgtgagccaa accagcaa gc tgactagggc tgaagcagtc tttcctgatg 2941 tagattatgt gaatagcact gaggctga ga caatccttga caatatcact cagagcacac 3001 agagcttcaa tgacttcacc agggtggt ag gaggggagga tgccaagcct gggcagttcc 3061 cctggcaggt agtgctcaat ggaaaagt gg atgccttttg tggaggttca attgtaaatg 3121 agaagtggat tgtgactgca gcccactg tg tggaaactgg agtcaagatt actgtggtgg 3181 ctggagagca caatattgag gaaactga gc acactgagca gaagaggaat gtgatcagga 3241 ttatccccca ccacaactac aatgctgc you tcaaagta caaccatgac attgccctcc 3301 tggaactgga tgaacccctg gtcttgaa ca gctatgtgac acccatctgt attgctgata 3361 aagagtacac caacatcttc ttgaaatt tg ggtctggata tgtgtctggc tggggcaggg 3421 tgttccataa aggcaggtct gccctggt at tgcagtattt gagggtgcct ctggtggata 3481 gagcaacctg cttgctgagc accaagtt ta caatctacaa caatatgttc tgtgcagggt 3541 tccatgaagg tggtagagac agctgcca gg gagattctgg gggtccccat gtgactgagg 3601 tggaggaac cagcttcctg actgggat ta tcagctgggg tgaggagtgt gctatgaagg 3661 gaaagtatgg gatctacaca aaagtatc ca gatatgtgaa ctggattaag gagaaaacca 3721 agctgacttg atgaaagatg gatttcca ag gttaattcat tggaattgaa attaacaga 3781 gatctagagc tgaattcctg cagccagg gg gatcagcctc tactgtgcct tctagttgcc 3841 agccatctgt tgtttgcccc tccccctt gc cttccttgac cctggaaggt gccactccca 3901 ctgtcctttc ctaataaaat gaggaaat tg catcacattg tctgagtagg tgtcattcta 3961 ttctgggggg tggggtgggg caggacag ca agggggagga ttgggaagac aatagcaggc 4021 atgctgggga tgcagtgggc tctatggc tt ctgaggcaga aagaaccagc tggggctcga 4081 gatccactag ggccgcagga acccctag tg atggagtgg ccactccctc tctgcgcgct 4141 cgctcgctca ctgaggcgc cggggctt tg cccggcggc ctcagtgagc gagcgacgc 4201 gcagctgcct gcaggggcag cttgaagg aa atactaaggc aaaggtactg caagtgctcg 4261 caacattcgc tttgcggat tattgccg ta gtgccgcgac gccgggggca agatgcagag 4321 attgccatgg tacaggccgt gcggttga ta ttgccaaaac agagctgtgg gggagagttg 4381 tcgagaaaga gtgcggaaga tgcaaagg cg tcggctattc aaggatgcca gcaagcgcag 4441 catatcgcgc tgtgacgatg ctaatccc aa accttaccca acccacctgg tcacgcactg 4501 ttaagccgct gtatgacgct ctggtggt gc aatgccacaa agaagagtca atcgcagaca 4561 acattttgaa tgcggtcaca cgttagca gc atgattgcca cggatggca catattacg 4621 gcatgatatt gacttattga father gg gtaaatttga ctcaacgatg ggttaattcg 4681 ctcgttgtgg tagtgagatg aaaagagg cg gcgcttacta ccgattccgc ctagttggtc 4741 acttcgacgt atcgtctgga actccaac ca tcgcaggcag agaggtctgc aaaatgcaat 4801 cccgaaacag ttcgcaggta attack ag cctgcataac ggtttcgggga tttttatat 4861 ctgcacaaca ggtaagagca ttgagtcg to aatcgtgaag agtcggcgag cctggttagc 4921 cagtgctctt tccgttgtgc tgattaa gc gathering agcagaaccg gathering 4981 tgcgtacagg cgtcatcgcc gcccagca ac agcacaaccc aaactgagcc gtagccactg 5041 tctgtcctga attcattagt aatagtta cg ctgcggcctt ttacacatga ccttcgtgaa 5101 agcgggtggc agcggtcgc gctaacaa cc tcctgccgtt ttgcccgtgc atatcggtca 5161 cgaacaaatc tgattactaa acacagta gc ctggattgt tctatcagta atcgacctta 5221 ttcctaatta atagagcaa atcccctt at tgggggtaag acatgaagat gccagaaaaa 5281 catgacctgt tggccgccat tctcgcgg ca aaggaacaag gcatcggggc aatccttgcg 5341 tttgcaatgg cgtaccttcg cggcagat at aatggcggtg cgtttacaaa aacagtaatc 5401 gacgcaacga tgtgcgccat tatcgcct ag ttcattcgtg accttctcga cttcgccgga 5461 ctaagtagca atctcgctta tataacga gc gtgttatcg gctacatcgg tactgactcg 5521 attggttcgc tttcaaacg cttcgctg ct aaaaaagccg gagtagaaga tggtagaaat 5581 caataatcaa cgtaaggcgt tcctcgat at gctggcgtgg tcggagggaa ctgataacgg 5641 acgtcagaaa accagaaatc atggttat ga cgtcattgta ggcggagagc tatttactga 5701 ttactccgat caccctcgca aacttgtc ac gctaaaccca aaactcaaat caacaggcgc 5761 cggacgctac cagcttcttt cccgttgg tg ggatgcctac cgcaagcagc ttggcctgaa 5821 agacttctct ccgaaaagtc aggacgct gt ggcattgcag cagattaagg agcgtggcgc 5881 tttacctatg attgatcgtg gtgatatc cg tcaggcaatc gaccgttgca gcaatatctg 5941 ggcttcactg ccgggcgctg gttatggt ca gttcgagcat aaggctgaca gcctgattgc 6001 aaaattcaaa gaagcgggcg gaacggtc ag agagattgat gtatgagcag agtcaccgcg 6061 attatctccg ctctggttat ctgcatca tc gtctgcctgt catgggctgt taatcattac 6121 cgtgataacg cattaccta caaagccc ag cgcgacaaaa atgccagaga actgaagctg 6181 gcgaacgcgg caattactga catgcaga tg cgtcagcgtg atgttgctgc gctcgatgca 6241 aaatacacga aggagttagc tgatgcta aa gctgaaaatg atgctctgcg tgatgatgtt 6301 gccgctggtc gtcgtcggtt gcacatca aa gcagtctgtc agtcagtgcg tgaagccacc 6361 accgcctccg gcgtggataa tgcagcct cc cccgactgg cagacaccgc tgaacgggat 6421 tatttcaccc tcagagagag gctgatca ct atgcaaaaac aactggaagg aacccagaag 6481 tatattaatg agcagtgcag atagagtt gc ccatatcgat gggcaactca tgcaattatt 6541 gtgagcaata cacacgcgct tccagcgg ag tataaatgcc taaagtaata aaaccgagca 6601 atccattac gaatgtttgc tgggtttc tg ttttaacaac atttctgcg ccgccacaaa 6661 ttttggctgc atcgacagtt ttcttctg cc caattccaga aacgaagaaa tgatgggtga 6721 tgtttcctt tggtgctact gctgccgg tt tgttttgaac agtaaacgtc tgttgagcac 6781 atcctgtaat aagcagggcc agcgcagt ag cgagtagcat ttttttcatg gtgttattcc 6841 cgatgcttt tgaagttcgc agaatcgt and gtgtagaaaa ttaaacaaac cctaaacaat 6901 gagttgaaat ttcatattgt tatattt at taatgtatgt caggtgcgat gaatcgtcat 6961 tgtattcccg gattaactat gtccacag cc ctgacgggga acttctctgc gggagtgtcc 7021 gggaataatt aaaacgatgc acacaggg tt tagcgcgtac acgtattgca ttatgccaac 7081 gccccggtgc tgacacggaa gaaaccgg ac gttatgattt agcgtggaaa gatttgtgta 7141 gtgttctgaa tgctctcagt aatagta at gaattatcaa aggtatagta atatctttta 7201 tgttcatgga tatttgtaac ccatcgga aa actcctgctt tagcaagatt ttccctgtat 7261 tgctgaaatg tgatttctt tgatttca ac ctatcatagg acgtttctat aagatgcgtg 7321 tttcttgaga attaacatt tacaacct tt ttaagtcctt ttattaacac ggtgttatcg 7381 ttttctaaca cgatgtgaat attatctg tg gctagatagt aaatataatg tgagacgttg 7441 tgacgtttta gttcagaata aaacaatt ca cagtctaaat cttttcgcac ttgatcgaat 7501 atttctttaa aaatggcaac ctgagcca tt ggtaaaacct tccatgtgat acgagggcgc 7561 gtagttgca ttcgtttt tatcgttt ca atctggtctg acctccttgt gttttgttga 7621 tgattatgt caaatattag gaatgttt tc acttaatagt attggttgcg taacaaagtg 7681 cggtcctgct ggcattctgg agggaaat ac aaccgacaga tgtatgtaag gccaacgtgc 7741 tcaaatcttc atacagaaag atttgaag ta atattttaac cgctagatga agagcaagcg 7801 catggagcga caaaatgaat aaagaaca at ctgctgatga tccctccgtg gatctgattc 7861 gtgtaaaaaa tatgcttaat agcaccat tt ctatgagtta ccctgatgtt gtaattgcat 7921 gtatagaaca taaggtgtct ctggaagc at tcagagcaat tgaggcagcg ttggtgaagc 7981 acgataataa tatgaaggat tattccct gg tggttgactg atcaccataa ctgctaatca 8041 ttcaaactat tagtctgtg acagagcc aa cacgcagtct gtcactgtca ggaaagtggt 8101 aaaactgcaa ctcaattact gcaatgcc ct cgtaattaag tgaatttaca atacgtcct 8161 gttcggaggg aagaacgcgg gatgttca tt cttcatcact tttaattgat gtatatgctc 8221 tcttttctga cgttagtctc cgacggca gg cttcaatgac ccaggctgag aaattcccgg 8281 accctttttg ctcaagagcg atgttaat tt gttcaatcat ttggttagga aagcggatgt 8341 tgcgggttgt tgttctgcgg gttctgtt ct tcgttgacat gaggttgccc cgtattcagt 8401 gtcgctgatt tgtattgtct gaagttgt tt ttacgttaag ttgatgcaga tcaattaata 8461 cgatacctgc gtcataattg attatttg and gtggtttgat ggcctccacg cacgttgtga 8521 tatgtagatg ataatcatta tcacttta cg ggtcctttcc ggtgatccga caggttacgg 8581 ggcggcgacc tgcctgatgc ggtatttt ct ccttacgcat ctgtgcggta tttcacaccg 8641 catacgtcaa agcaaccata gtacgcgc cc tgtagcggcg cattaagcgc ggcgggtgtg 8701 gtggttacgc gcagcgtgac cgctacac tt gccagcgcct tagcgcccgc tcctttcgct 8761 ttcttccctt cctttctcgc cacgttcg cc ggctttcccc gtcaagctct aaatcgggg 8821 ctccctttag ggttccgatt tagtgctt ta cggcacctcg accccaaaaa acttgatttg 8881 ggtgatggtt cacgtagtgg gccatcgc cc tgatagacgg ttttcgcc tttgacgttg 8941 gagtccacgt tctttaatag tggactct tg ttccaaactg gaacaacact caactctatc 9001 tcgggctatt cttttgatt agacctgc ag gcatgcaagc ttggcactgg ccgtcgtttt 9061 acaacgtcgt gactgggaaa acctggc gt tacccaactt aatcgccttg cagcacatcc 9121 cccttcgcc agctggcgta atagcgaa ga ggcccgcacc gatcgccctt cccaacagtt 9181 gcgcagcctg aatggcgaat gcgattta tt caacaaagcc gccgtcccgt caagtcagcg 9241 taatgctctg ccagtgttac aaccaatt aa ccaattctga ttagaaaaac tcatcgagca 9301 tcaaatgaaa ctgcaattta ttcatatc ag gattatcaat accatatttt tgaaaaagcc 9361 gttctgtaa tgaaggagaa aactcacc ga ggcagttcca taggatggca agatcctggt 9421 atcggtctgc gattccgact cgtccaac at the caterpillar tattatttc ccctcgtca 9481 aaataaggtt atcaagtgag aaatcacc that gagtgacgac tgaatccggt gagaatggca 9541 aaagcttatg catttctttc cagacttg tt cacaggcca gccattacgc tcgtcatca 9601 aatcactcgc atcaccaaa ccgttatt ca ttcgtgattg cgcctgagcg agacgaaata 9661 cgcgatcgct gttaaaagga catch aa caggatcga atgcaaccgg cgcaggaca 9721 ctgccagcgc atcacaat ttttcacc tg aatcaggata ttcttctaat acctggaatg 9781 ctgttttccc ggggatcgca gtggtgag that accatgcatc atcaggagta cggataaaat 9841 gcttgatggt cggagaggc ataattc cg tcagccagtt tagtctgacc atctcatctg 9901 taacatcatt ggcaacgcta cctttgcc to gtttcagaaa caactctggc gcatcgggct 9961 tcccataca tcgatagatt gtcgcacc tg attgcccgac attatcgcga gcccatttat 10021 acccatata atcagcatcc atgttgga to ttaatcgcgg cttcgagcaa gacgtttccc 10081 gttgaatatg gctcataaca ccccttgt at tactgttat gtaagcagac agttttattg 10141 ttcatgatga tatatttta tcttgtgc aa tgtaacatca gagattttga gacacaacgt 10201 ggctttgttg aataaatcga acttttgc tg agttgaagga tcagatcacg catcttcccg 10261 acaacgcaga ccgttccgtg gcaaagca aa agttcaaaat caccaactgg tccacctaca 10321 acaaagctct catcaaccgt ggctccct ca cttctggct ggatgatggg gcgattcagg 10381 cctggtatga gtcagcaaca cttcttc ac gaggcagacc tctcgacgga gttccactga 10441 gcgtcagacc ccgtagaaaa gatcaaag ga tcttttgag atcctttttt tctgcgcgta 10501 atctgctgct tgcaaacaaa aaaaccac cg ctaccagcgg tggttgttt gccggatcaa 10561 gagctaccaa ctctttttcc gaaggtaa ct ggcttcagca gagcgcagat accaaatact 10621 gttcttctag tgtagccgta gttaggcc ac cacttcaaga actctgtagc accgcctaca 10681 tacctcgctc tgctaatcct gttaccag tg gctgctgcca gtggcgataa gtcgtgtctt 10741 accgggttgg actcaagacg atagttac cg gataaggcgc agcggtcggg ctgaacgggg 10801 ggttcgtgca cacagcccag cttggagc ga acgacctaca ccgaactgag atacctacag 10861 cgtgagctat gagaaagcgc cacgcttc cc gaagggagaa aggcggacag gtatccggta 10921 agcggcaggg tcggaacagg agagcgca ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ / label="ApoE HCR-1" promoter 482..878 / label="hAAT Promot er" CDS order(908..995,2434 ..3731) / label="hFIX CDS" intron 996..2433 / label="hFIX Intron" 3'UTR 3732..3779 / label="hF9 3' UTR" Terminator 3820..4047 / label="bGH_PA term" repeat_region complement(4097..42 37) / label="AAV2 ITR" misc_feature 4248..8713 / label="Eukaryotic Stuffer" rep_origin 8754..9060 / label="F1 Ori" CDS complement(9355..10 170) / label="Kanamycin r esistance" rep_origin complement(10527..1 1194) / label="pUC Ori" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgct ca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagt ga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggcct ag taggctcaga ggcacacagg agttctggg 181 ctcaccctgc ccccttccaa cccctcag tt cccatcctcc agcagctgtt tgtgtgctgc 241 ctctgaagtc cacactgaac aaacttca gc ctactcatgt ccctaaaatg ggcaaacatt 301 gcaagcagca aacagcaaac acacagcc ct cctgcctgc tgaccttgga gctggggcag 361 aggtcagaga cctctctggg cccatgcc ac ctccaacatc cactcgaccc cttggaattt 421 cggtggagag gagcagaggt tgtcctgg cg tggttaggt agtgtgagag gggtacccgg 481 ggatcttgct accagtggaa cagccact aa ggattctgca gtgagagcag agggccagct 541 aagtggtact ctcccagaga ctgtctga ct cacgccaccc cctccacctt ggacacagga 601 cgctgtggtt tctgagccag gtacaatg ac tccttcggt aagtgcagtg gaagctgtac 661 actgcccagg caaagcgtcc gggcagcg ta ggcgggcgac tcagatccca gccagtggac 721 ttagcccctg ttgctcctc cgataact gg ggtgaccttg gttaatattc accagcagcc 781 tccccgttg cccctctgga tccactgc tt aaatacggac gaggacagggg cctgtctcc 841 cagctcag gcaccaccac tgaggtgg ga cagtgaatac cactttcaca atctgctagc 901 aaggttatg cagaggtga acatgatc at ggctgagagc cctggcctga tcaccatctg 961 cctgctgggc tacctgctgt ctgctgaa tg tacaggtttg tttcctttt tataatacat 1021 tgagtatgct tgccttttag atatagaa at atctgattct gtctcttca ctaaatttg 1081 attacks slowly slowly ag tctaacagcc agcaccagg ttggtaagta 1141 ctggttctt gttagcttagg ttttctc tt cttcactttt aaaactaaat nagatgacaa 1201 tgcttatgat gcaataaggt tttaaa ca ctgttcagtt cagtatttgg tcatgtaatt 1261 cctgttaaa aacagtcatc tccttggt tt aaaaaatta aagtgggaaaaaaaaah 1321 taggaata taggaaaaaaacc ac agtatttttg tttggactta ccactttgaa 1381 atcaaattgg cacaaaag cacaaaca gt ggccttattt acacaaaag tctgatttta 1441 agatatgtga caattcaagg tttcaga gt atgtaaggag gtgtgtctct aattttttaa 1501 attached ttcaatttaa agttttag tt aaaacataaa throwcctt tcattagcaa 2101 cctcaatgag ctattttcaa gtgatgac aa agtgtgaagt you are tttgagaact 2161 ttctttttca tccaaagtaa attcaaat at gattagaat ctgacctttt attack 2221 ttctcttgac taaaagtaaa attgaatt tt aattcctaaa tctccatgtg fathercagtac 2281 tgtgggaaca tcacagattt tggctcca tg ccctaaagag aaattggctt tcagattatt 2341 tggattaaaa acaaagactt tcttaaga ga tgtaaaattt tcatgatgtt ttctttttg 2401 ctaaaacta agaattattc ttttacat tt cagtttttct tgatcatga aatgccaaca 2461 aaattctgaa tagaccaaag aggtata ct ctggcaagct tgaagagttt gtacagggga 2521 atctggagag agagtgtatg gaagagaa gt gcagctttga ggaagccaga gaagtgtttg 2581 aaaatacaga gagaacaact gaattttg ga agcagtatgt ggatggtgat caatgtgaga 2641 gcaatccctg cttgaatggg gggagctg ta aagatgatat caacagctat gaatgttggt 2701 gtccctttgg atttgagggg aaaaactg tg agcttgatgt gacctgtaat atcaagaatg 2761 gcaggtgtga gcaattttgc aagaattc tg ctgataacaa agtggtctgt agctgcactg 2821 agggatatag gctggctgaa aaccagaa ga gctgtgaacc tgcagtgcct tttccctgtg 2881 ggagagtgtc tgtgagccaa accagcaa gc tgactagggc tgaagcagtc tttcctgatg 2941 tagattatgt gaatagcact gaggctga ga caatccttga caatatcact cagagcacac 3001 agagcttcaa tgacttcacc agggtggt ag gaggggagga tgccaagcct gggcagttcc 3061 cctggcaggt agtgctcaat ggaaaagt gg atgccttttg tggaggttca attgtaaatg 3121 agaagtggat tgtgactgca gcccactg tg tggaaactgg agtcaagatt actgtggtgg 3181 ctggagagca caatattgag gaaactga gc acactgagca gaagaggaat gtgatcagga 3241 ttatccccca ccacaactac aatgctgc you tcaaagta caaccatgac attgccctcc 3301 tggaactgga tgaacccctg gtcttgaa ca gctatgtgac acccatctgt attgctgata 3361 aagagtacac caacatcttc ttgaaatt tg ggtctggata tgtgtctggc tggggcaggg 3421 tgttccataa aggcaggtct gccctggt at tgcagtattt gagggtgcct ctggtggata 3481 gagcaacctg cttgctgagc accaagtt ta caatctacaa caatatgttc tgtgcagggt 3541 tccatgaagg tggtagagac agctgcca gg gagattctgg gggtccccat gtgactgagg 3601 tggaggaac cagcttcctg actgggat ta tcagctgggg tgaggagtgt gctatgaagg 3661 gaaagtatgg gatctacaca aaagtatc ca gatatgtgaa ctggattaag gagaaaacca 3721 agctgacttg atgaaagatg gatttcca ag gttaattcat tggaattgaa attaacaga 3781 gatctagagc tgaattcctg cagccagg gg gatcagcctc tactgtgcct tctagttgcc 3841 agccatctgt tgtttgcccc tccccct gc cttccttgac cctggaaggt gccactccca 3901 ctgtcctttc ctaataaaat gaggaaat tg catcacattg tctgagtagg tgtcattcta 3961 ttctgggggg tggggtgggg caggacag ca agggggagga ttgggaagac aatagcaggc 4021 atgctgggga tgcagtgggc tctatggc tt ctgaggcaga aagaaccagc tggggctcga 4081 gatccactag ggccgcagga acccctag tg atggagtgg ccactccctc tctgcgcgct 4141 cgctcgctca ctgaggccgg gcgaccaa ag gtcgcccgac gccgggctt tgcccgggcg 4201 gcctcagtga gcgagcgagc gcgcagct gc ctgcaggggc ccatgggcag atgcaccacc 4261 tgtctcagtg caaagccctg cctaagta gg ctggtcataa gaccatgtgt ctggctgtaa 4321 ctccaattga ttgtcagcat caataaaa ct tggccaacac tgttatatac tggtattgat 4381 agttacaact gacatattt gtttaagc aa ttggatta gattcacat gcaatgatat 4441 cagggtcctt ctcctctggt tagtgtat tg gggggaatt ggacatctct cagctcagta 4501 ggctagttag gccaggatgg atgacatc ca cagcccctgg gcagagagat tatgatgtag 4561 ctagtctgac tcctgacaaa gacttgct tc ctggagcttc tactactttc tggtggatgg 4621 ctaagaaata tggttgtgtt cttttaag tc tgaagagcat tatttttgcc aacccctgac 4681 caacatcct tgccaagga aaggccta aa attackattgc attack attack 4741 acttggtttt ggaatgtttg gcctttca gg atcatagcta tcaaaatatt agctatttgg 4801 ggtatgagat gtctgcttgg tcaaggac aa gttcttaag acatcatgtt ggggaataat 4861 ggggaaaatg ggaaggctta tgctctga gt aagacatctg to attack tgtcaaacat 4921 ttttgttagt catagtctaa tggggagcc tg ttttccctct ttaatataca ttcacatctg 4981 aatttatgct cttcattgac aatgccag cc cagaacaaca gctcttaccc tttggttttc 5041 ttcctaacct ttaactccaa tgtaacca tt acctgccatt tcagtaaaac cattattctc 5101 cctacttacc cacccaagtt gtacaata aa gagtgtttgc tctcactcat atacaaagca 5161 aattcatttg tttgtgatgt acagcttg ct atgcccacag atgtggtttg cctagtcctt 5221 tgctctaggt catttgactg ggaacaga tg ggatgctcac tttggtttt aatggttaac 5281 tagtcattga aatgcatttc atcaaata at cttagaggat aattgtttaa atgtctgtcc 5341 agactagctt tgtagagcca ggtgccat ta cacatgtcac cttcttattt ctcttaattg 5401 aatttttatc atctgagata ggaataat ag agggcttttt caagtgaaga tattactata 5461 gtctaaagac cttagtgtaa catcctgg cc cctaaggaaa aacaagttct ggttcataca 5521 tataataact ttgcatgtta tctgccac tg agatgtgtcc taatccaaca gaaaggattg 5581 aatctctgta gctaggtgta caggcaa ga gctgtacagg gaacctttaa agatagcttc 5641 agccaaagc tgaggaagt ggatgag and tgggaaat gctaagacat tttaagatt 5701 Tctttaggt CAAAAATAGA ATTAAAAAA ta gaccattcc ctggacattt tctgtaggtt 5761 atactgtta actattggta atgcata tg ctacaactta atatgtctgc ttgtgagtt 5821 tagcattgtc tccttgtcat tccagaaa tg aaatggcaa tacattaa tcagaacata 5881 aagggaac agggtataaa ggctcaat tt agtcacatca ttcccttc cacccaccc 5941 cctttaaacc agatgtttgc caatgcat this acatgcaga tgtttcctga aagaaagtttt 6001 agtaactca gcagacacct tattttct tt tcagcagaa agactatga gatggtggtt 6061 gtggttgttc tgggaggag agatata aa tgatacacat tattcaat cattcatga 6121 cctcactgca cacttatagt tattgtac ct gttgtcttt tgctgtcaag cctagctaag 6181 atcatttgga atgttcaaga tcactcat ac atgcatgtgc acacatacac atgcacatat 6241 gttcactccc tatttcatcc acatgaac ta agattactga tgtgtacaga ttcaaagcac 6301 ttttattcttt ttccaaaggc aagaagct ga gctactttcc agaatagttg tgaaagaccc 6361 tgtcatactt ctgcattgtt tcctccac ac cacctccatc cagttcctta tgaatggtta 6421 ctggttttca aaaatatgag ataaattg ag tgtataaaag tcatttttag acaaaatgaa 6481 acaggaaatg aaagaaacca gaatctct cc tcatttgtgg atgggccagc tccaccatgt 6541 catggttaat ctgcagggag gaaatact ag atttgattgc agatcagact gcagcaaacc 6601 tgctgtgact aaggcatcaa gagaaagc aa gcaacagctg gggcttcagt ggtgaaaaca 6661 ttatatatct agctttgaaa tatgaaat ac tgtttagcag tgtcacctag aaaagagtgt 6721 ttcaaaatgc tgatgcttca taagaacc tt tctcttcaga gttgtttct ttatctttca 6781 aattagccag ggtggggaaat aaagtgat ca cttggtgaag aaatctcaca aagaagaaca 6841 tagagagttc actttcatct ggagtaat ga acagattgaa caaactagaa atggttagtc 6901 tgttaaagaa aaggtgtagg tgagctgt tt gcaagagcca caagggaaag gggaagacaa 6961 cttctttgtg gacttaaggg tgaaagtt gc aagcaggcaa gaccattctg acctccatta 7021 agaaagccct ttccaaccaa caaccact gg gttggttact caggttgggc agcattggga 7081 gcaaatgttg attgaacaaa tgtttgtc ag aattgttgac ttaaagagct gttctgtcac 7141 tggggacagc agcagctaga tagcccca tt cagggagagg gcatttgttc acctggccag 7201 agatcagagc aggctaaggg actgctgg ga tcctgtccag ctttgagacc ctacagagcc 7261 atgttcacct agcaggtatc ccttctga gg tcactctcat ttcttacctt attccagggc 7321 tttcacctca gcttgccagg ctggagcc aa gggccaaggc agcctcacct tgttggctat 7381 ggtagcttcc caggagcccc ctatggtt ca ggaacagctc tgcctgcccc atcctgtttg 7441 ctacctccta aagccaaagg cactggtg gg ccaggccagc ttctaaagtc acacaaggtt 7501 agaaggttcc tgacaggaag ggcttgag gc caatggaagg aggtacttca gtttccctcc 7561 agatgcccag tgatgggctc agagctcc tt gagaacttgg gaaaggaagc agggtctctg 7621 aagaaatact tcaggagtag aaagagga ag ctagagggtt aaatgcacta cacaggaaca 7681 gaaatgagtt tttcttagg ttagtata tg tctagaggtg tagtaaacta aaacaagtct 7741 tgaattgcat acagccactt agggaaga aa tgaaaacctt tgaatattag tgaaaaaagg 7801 gaaactgcaa cccctgtatt actagata gc tttcatcaac agctcaaaac agacagattt 7861 ttataggttt actgtgtgca ctttaata ca agggcagtgg ttcagaacta gtcaggtcct 7921 gaaaaggatt taccaaatgt tgagtgtg cc ctctagtgtt cacacttccc agctttcttc 7981 ctataaaggt ggatcaaggc acttgctt ac aactggaact gaaatcctcc aagtggaact 8041 agacattgag atggagaaaa tattcatt gt ccactgtaat tatgcaagga atatccagtt 8101 gagataatgg acttgcctct tatctaat aa tacccaggct caatgggtca ctgctttgtc 8161 cactttgcc aaaattcaag cacagcta ag ttgatatttt aggacaaagg cagcttacta 8221 tccagccaga ggggagtaga atatggtt aa gagagagtgg aaagaatgaa tgagccctgc 8281 tattcctcac tgcctggatg gctataag like cagcccttat ggaggcctta ggtcttgctt 8341 cataatattc cagtttgaaa agggtttg aa aagacctcct agaaaaatca gtagttttc 8401 tcttttgagt aacatgtagc aaaaaaaa tt tcatcatgta ggtacagggga acaccctaat 8461 aactattaat ctcaaggagt caagccag tg tgttcctaa tgtatctgct gtatccccat 8521 gaagcaaatt ttgccatcag agaaactg ac tcatggggaa aaaatccaag gacctcaaat 8581 caccaaaaga agccattcct cagatttg cc tagcttaag cttccctgtc tctcattgtg 8641 tgttgctttc aatgcagtta cataaatg gc ttttttgttt atgcaccaaa aacactaatt 8701 catctgcaaa gctataggtc aaagcaac ca tagtatgcac cctgctagct ggcgcattaa 8761 gcgcggcggg tgtggtggtt acgcgcag cg tgaccgctac acttgccagc gccttagcgc 8821 ccgctccttt cgctttcttc cttcctt tcgccacgtt cgccggcttt ccccgtcaag 8881 ctctaaatcg ggggctccct ttagggtt cc gatttagtgc tttacggcac ctcgacccca 8941 aaaaacttga tttgggtgat ggttcacg ta gtgggccatc gccctgatag acggtttttc 9001 gccctttgac gttggagtcc acgttctt ta atagtggact cttgttccaa actggaacaa 9061 cactcaactc tatctcgggc tattcttt tg atttagacct gcaggcatgc aagcttggca 9121 ctggccgtcg tttacaacg tcgtgact gg gaaaaccctg gcgttaccca acttaatcgc 9181 cttgcagcac atcccccttt cgccagct gg cgtaatacg aagaggcccg caccgatcgc 9241 ccttcccaac agttgcgcag cctgaatg gc gaatgcgatt tattcaacaa agccgccgtc 9301 ccgtcaagtc agcgtaatgc tctgccag tg ttacaaccaa ttaaccaatt ctgattagaa 9361 aaactcatcg agcatcaaat gaaactgc aa tttattcata tcaggattat caataccata 9421 ttttgaaaa agccgtttct gtaatgaa gg agaaaactca ccgaggcagt tccataggat 9481 ggcaagatcc tggtatcggt ctgcgatt cc gactcgtcca acatcaatac aacctattaa 9541 tttcccctcg tcaaaaataa ggttatca ag tgagaaatca ccatgagtga cgactgaatc 9601 cggtgagaat ggcaaaagct tatgcatt tc tttccagact tgttcaacag gccagccatt 9661 acgctcgtca tcaaaatcac tcgcatca ac caaaccgtta ttcattcgtg attgcgcctg 9721 agcgagacga aatacgcgat cgctgtta aa aggacaatta caaacaggaa tcgaatgcaa 9781 ccggcgcagg aacactgcca gcgcatca ac aatattttca cctgaatcag gatattcttc 9841 tatacctgg aatgctgttt tcccgggg and cgcagtggtg agtaaccatg catcatcagg 9901 agtacggata aaatgcttga tggtcgga ag aggcataaat tccgtcagcc agtttagtct 9961 gaccatctca tctgtaacat cattggca ac gctaccttg ccatgtttca gaaacaactc 10021 tggcgcatcg ggcttcccat acaatcga ta gattgtcgca cctgattgcc cgacattatc 10081 gcgagcccat ttatacccat ataaatca gc atccatgttg gaatttaatc gcggcttcga 10141 gcaagacgtt tccgttgaa tatggctc at aacacccctt gtattactgt ttatgtaagc 10201 agacagtttt attgttcatg atgatata tt tttatcttgt gcaatgtaac atcagagatt 10261 ttgagacaca acgtggcttt gttgaata aa tcgaactttt gctgagttga aggatcagat 10321 cacgcatctt cccgacaacg cagaccgt tc cgtggcaaag caaaagttca aaatcaccaa 10381 ctggtccacc tacaacaaag ctctcatc aa ccgtggctcc ctcactttct ggctggatga 10441 tggggcgatt caggcctggt atgagtca gc aacaccttct tcacgaggca gacctcga 10501 cggagttcca ctgagcgtca gaccccgt ag aaaagatcaa aggatcttct tgagatcctt 10561 ttttctgcg cgtaatctgc tgcttgca aa caaaaaaacc accgctacca gcggtggttt 10621 gtttgccgga tcaagagcta ccaactct tt ttccgaaggt aactggcttc agcagagcgc 10681 agataccaaa tactgttctt ctagtgta gc cgtagttagg ccaccacttc aagaactctg 10741 tagcaccgcc tacatacctc gctctgct aa tcctgttacc agtggctgct gccagtggcg 10801 ataagtcgtg tcttaccggg ttggactc aa gacgatagtt accggataag gcgcagcggt 10861 cgggctgaac ggggggttcg tgcacaca gc ccagcttgga gcgaacgacc tacaccgaac 10921 tgagatacct acagcgtgag ctatgaga aa gcgccacgct tcccgaaggg agaaaggcgg 10981 acaggtatcc ggtaagcggc agggtcgg aa caggagagcg cacgagggag cttccagggg 11041 gaaacgcctg gtatctttat agtcctgt cg ggtttcgcca cctctgactt gagcgtcgat 11101 ttttgtgatg ctcgtcaggg gggcggag cc tatggaaaaa cgccagcaac gcggcctttt 11161 tacggttcct ggccttttgc tggccttt tg ctcacatgt / / INTRON A nucleic acid sequence(SEQ ID NO:17) GTTTGTTTCCTTTTTATAATACATTGAGTATGCTTGCCT TTTAGATAGAAATATCTGATTCTGTCTTCTTCACTAAA TTTTGATTACATGATTTGACAGCAATATTGAAGAGTCTAA CAGCCAGCACCCAGGTTGGTAAGTACTGGTTCTTTGTTAG CTAGGTTTTCTTCTTCTTCACTTTTAAAACTAAATATAGTG GACAATGCTTATGATGCAATAAGGTTTAATAAACACTGTT CAGTTCAGTATTTGGTCATGTAATTCCTGTTAAAAAAACAG TCATCTCCTTGGTTTAAAAAAAATTAAAAGTGGGAAAAACAA AGAAATAGCAGAATATAGTGAAAAAAATAACCACAGTAT TTTTGTTTGGACTTACCACTTTGAAATCAAATTGGGAAAC AAAAGCACAACAGTGGCCTTATTTACACAAAAAGTCTGA TTTTAAGATATGTGATCAAGGTTTCAPAGATGTA AGGAGGTGTGTCTCTAATTTTTTAAATTATATATCTTCAA TTTAAAGTTTGTTACTTAAAAAAAAGTAACCTTTHELP AGCAAGCTGTTAGTTATCACCAAAAGCTTTTCATGGATTAG GAAAAAATCATTTTGTCTCTATCTCAAACATCTTGGAGTT GATTTGGGGAAACAATACTCAGTTGAGTTCCCTAGG GGAGAAAAGCTTAAGAATTGACAAAGAGTTA GTTAGCTATTGCAACATATATCACTTTGTTTTTTCACAAC TACAGTGACTTTATTTATTTCCCAGAGGAAGGCATACAGG GAAGAAATTATCCCATTTGGAAACAGCATGTTCTCACA GTAAGCACTTATCACACTTACTTGTCAACTTTCTAGAATC AAATCTAGTAGCTGACAGTACCAGGATCAGGGGTGCCAAC CCTAAGCACCCCCAGAAAGCTGACTGGCCCTGTGGTTCCC ACTCCAGACATGATGTCAGCTGTGAATCCACCTCCCTGG ACCATAATTAGGCTTCTGTTCTTCAGGAGACATTTGTTCA AAGTCATTTGGGCAACCATATTCTGAAAACAGCCCAGCCA GGGTGATGGATCACTTTGCAAAGTCCTCAATGAGCTATT TTCAAGTGATGACAAAGTGTGAGTTAAGGGCTCATTTGA BACKTTCTTTTTCATCATCHATTATTCAAATTACKGROUP GAAATCTGACCTTTTATTACTGGAATTCTCTTGACTAAAA GTAAAATTGAATTTTAATTCCTAAATCTCCATGTGTATAC AGTACTGTGGGAACATCACAGATTTTGGCTCCATGCCCTA AAGAGAAATTGGCTTTCAGATTATTTGGATTAAAAACAAA GACTTTCTTAAGAGATGTAAAATTTTCATGATGTTTTCTT TTTTGCTAAAACTAAAGAATTATTCTTTTACATTTCAG Figure 15 FIX39 nucleic acid sequence including intron A (intron A is underlined) (SEQ ID NO: 25) ATGCAGAGGGTGAACATGATCATGGCTGAGAGCCCTGGCC TGATCACCATCTGCCTGCTGGGCTACCTGCTGTCTGCTGA ATGTACAG GTTTGTTTCCTTTTTTATAATACATTGAGTAT GCTTGCCTTTTAGATATAGAAATATCTGATTCTGTCTTCT TCACTAAATTTTGATTACATGATTTGACAGCAATATTGAA GAGTCTAACAGCCAGCACCCAGGTTGGTAAGTACTGGTTC TTTGTTAGCTAGGTTTTCTTCTTCTTCACTTTTAAAACTA AATAGATGGACAATGCTTATGATGCAATAAGGTTTAATAA ACACTGTTCAGTTCAGTATTTGGTCATGTAATTCCTGTTA AAAAACAGTCATCTCCTTGGTTTAAAAAAATTAAAAGTGG GAAAACAAAGAAATAGCAGAATATAGTGAAAAAAAATAAC CACAGTATTTTTGTTTGGACTTACCACTTTGAAATCAAAT TGGGAAACAAAAGCACAAACAGTGGCCTTATTTACACAAA AAGTCTGATTTTAAGATATGTGACAATTCAAGGTTTCAGA AGTATGTAAGGAGGTGTGTCTCTAATTTTTTAAATTATAT ATCTTCAATTTAAAGTTTTAGTTAAAACATAAAGATTAAC CTTTCATTAGCAAGCTGTTAGTTATCACCAAAGCTTTTCA TGGATTAGGAAAAAATCATTTTGTCTCTATCTCAAACATC TTGGAGTTGATATTTGGGGAAACACAATACTCAGTTGAGT TCCCTAGGGGAGAAAAGCAAGCTTAAGAATTGACACAAAG AGTAGGAAGTTAGCTATTGCAACATATATCACTTTGTTTT TTCACAACTACAGTGACTTTATTTATTTCCCAGAGGAAGG CATACAGGGAAGAAATTATCCCATTTGGACAAAACAGCATG TTCTCACAGTAAGCACTTATCACACTTACTTGTCAACTTT CTAGAATCAAATCTAGTAGCTGACAGTACCAGGATCAGGG GTGCCAACCCTAAGCACCCCCAGAAAGCTGACTGGCCCTG TGGTTCCCACTCCAGACATGATGTCAGCTGTGAAATCCAC CTCCCTGGACCATAATTAGGCTTCTGTTCTTCAGGAGACA TTTGTTCAAAGTCATTTGGGCAACCATATTCTGAAAACAG CCCAGCCAGGGTGATGGATCACTTTGCAAAAGATCCTCAAT GAGCTATTTTCAAGTGATGACAAAGTGTGAAGTTAAGGGC TCATTTGAGAACTTTCTTTTTCATCCAAAGTAAATTCAAA TATGATTAGAAATCTGACCTTTTATTACTGGAATTCTCTT GACTAAAAAGTAAAATTGAATTTTAATTCCTAAATCTCCAT GTGTATACAGTACTGTGGGAACATCACAGATTTTGGCTCC ATGCCCTAAAGAGAAATTGGCTTTCAGATTATTTGGATTA AAAACAAAGACTTTCTTAAGAGATGTAAAATTTTCATGAT GTTTTCTTTTTTGCTAAAACTAAAGAATTATTCTTTTACA TTTCAG TTTTTCTTGATCATGAAATGCCAACAAAATTCT GAATAGACCAAAGAGGTATAACTCTGGCAAGCTTGAAGAG TTTGTACAGGGGAATCTGGAGAGAGAGTGTATGGAAGAGA AGTGCAGCTTTGAGGAAGCCAGAGAAGTGTTTGAAAATAC AGAGAGAACAACTGAATTTTGGAAGCAGTATGTGGATGGT GATCAATGTGAGAGCAATCCCTGCTTGAATGGGGGGAGCT GTAAAGATGATATCAACAGCTATGAATGTTGGTGTCCCTT TGGATTTGAGGGGAAAAACTGTGAGCTTGATGTGACCTGT AATATCAAGAATGGCAGGTGTGAGCAATTTTGCAAGAATT CTGCTGATAACAAAGTGGTCTGTAGCTGCACTGAGGGATA TAGGCTGGCTGAAAACCAGAAGAGCTGTGAACCTGCAGTG CCTTTTCCCTGTGGGAGAGTGTCTGTGAGCCAAACCAGCA AGCTGACTAGGGCTGAAGCAGTCTTTCCTGATGTAGATTA TGTGAATAGCACTGAGGCTGAGACAATCCTTGACAATATC ACTCAGAGCACACAGAGCTTCAATGACTTCACCAGGGTGG TAGGAGGGGAGGATGCCAAGCCTGGGCAGTTCCCCTGGCA GGTAGTGCTCAATGGAAAAGTGGATGCCTTTTGTGGAGGT TCAATTGTAAATGAGAAGTGGATTGTGACTGCAGCCCACT GTGTGGAAACTGGAGTCAAGATTACTGTGGTGGCTGGAGA GCACAATATTGAGGAAACTGAGCACACTGAGCAGAAGAGG AATGTGATCAGGATTATCCCCCACCACAACTACAATGCTG CTATCAACAAGTACAACCATGACATTGCCCTCCTGGAACT GGATGAACCCCTGGTCTTGAACAGCTATGTGACACCCATC TGTATTGCTGATAAAGAGTACACCAACATCTTCTTGAAAT TTGGGTCTGGATATGTGTCTGGCTGGGGCAGGGTGTTCCA TAAAGGCAGGTCTGCCCTGGTATTGCAGTATTTGAGGGTG CCTCTGGTGGATAGAGCAACCTGCTTGCTGAGCACCAAGT TTACAATCTACAACAATATGTTCTGTGCAGGGTTCCATGA AGGTGGTAGAGACAGCTGCCAGGGAGATTCTGGGGGTCCC CATGTGACTGAGGTGGAGGGAACCAGCTTCCTGACTGGGA TTATCAGCTGGGGTGAGGAGTGTGCTATGAAGGGAAAGTA TGGGATCTACACAAAAGTATCCAGATATGTGAACTGGATT AAGGAGAAAACCAAGCTGACTTGA

Claims

1. 1. A recombinant adeno-associated virus (rAAV) vector comprising a capsid and a genome, wherein the capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO: 4, and the genome comprises a nucleic acid sequence encoding a human factor IX (FIX) protein, wherein the coding sequence is not naturally occurring and is at least 70% identical to SEQ ID NO: 10, encodes a human FIX protein identical to the human FIX protein encoded by SEQ ID NO: 10, has a reduced number of CpG dinucleotides compared to a wild-type nucleic acid sequence encoding the human FIX protein, and is optionally separated by an intron.

2. 2. The rAAV vector of claim 1, wherein the genome further comprises an additional sequence selected from the group consisting of an intron, an expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs), and a filler polynucleotide sequence, and the additional sequence is optionally modified to have a reduced number of CpG dinucleotides compared to the unmodified sequence.

3. The rAAV vector of claim 2, wherein the expression control element comprises an enhancer comprising the sequence set forth in SEQ ID NO:

14.

4. The rAAV vector of claim 2, wherein the expression control element comprises a promoter comprising the sequence set forth in SEQ ID NO:

15.

5. The rAAV vector of claim 2 , wherein the expression control element confers expression in the liver.

6. 3. The rAAV vector of claim 2, wherein the intron is present in the sequence encoding a human FIX protein, or the expression control element is operably linked to the sequence encoding a human FIX protein, or the AAV ITR is located at the 5' or 3' end of the genome, or the filler polynucleotide sequence is adjacent to the 5' or 3' end of the sequence encoding a human FIX protein.

7. The rAAV vector of claim 6, wherein the expression control element comprises a human alpha-1-antitrypsin (hAAT) gene promoter or a portion thereof, or an apolipoprotein E (ApoE) HCR-1 or HCR-2 enhancer or a portion thereof.

8. 2. The rAAV vector of claim 1, wherein the sequence encoding the human FIX protein has 1 to 55 fewer CpG dinucleotides than the native sequence encoding human FIX.

9. 2. The rAAV vector of claim 1, wherein the sequence encoding human FIX protein does not contain any CpG dinucleotides.

10. 2. The rAAV vector of claim 1, wherein the sequence encoding the human FIX protein is interrupted by an intron.

11. 2. The rAAV vector of claim 1, wherein the sequence encoding human FIX protein comprises a sequence having 80% or more identity to SEQ ID NO:

10.

12. 1. A recombinant adeno-associated virus (rAAV) vector comprising a capsid and a genome, wherein the capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:4, and the genome comprises a nucleic acid sequence encoding a human factor IX (FIX) protein, wherein the coding sequence is not naturally occurring and is at least 70% identical to SEQ ID NO:10, and encodes a human FIX protein identical to the human FIX protein encoded by SEQ ID NO:10, and is optionally separated by an intron.

13. 13. The rAAV vector of claim 1 or 12, wherein the intron comprises the sequence set forth in SEQ ID NO:

17.

14. 13. The rAAV vector of claim 1 or 12, further comprising a polyadenylation sequence located 3' to the sequence encoding human FIX.

15. 15. The rAAV vector of claim 14, wherein the polyadenylation sequence is depleted of all CpG dinucleotides.

16. 15. The rAAV vector of claim 1 or 14, wherein the genome further comprises an ITR sequence of any of the AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, or Rh74.

17. A pharmaceutical composition comprising the rAAV vector of claim 1 or 16.

18. 18. The pharmaceutical composition of claim 17, further comprising an empty capsid AAV.

19. 19. The pharmaceutical composition of claim 18, wherein the empty capsid AAV is selected from AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, or is an AAV capsid comprising a VP1 protein comprising the amino acid sequence of SEQ ID NO:

4.

20. 19. Use of the rAAV vector of claim 1 or 12 in the manufacture of a medicament for treating hemophilia B.

21. 13. The rAAV vector of claim 1 or 12, wherein the nucleic acid coding sequence is at least 75% identical to SEQ ID NO:

10.

22. 13. The rAAV vector of claim 1 or 12, wherein the nucleic acid coding sequence is at least 80% identical to SEQ ID NO:

10.

23. 13. The rAAV vector of claim 1 or 12, wherein the nucleic acid coding sequence is at least 85% identical to SEQ ID NO:

10.

24. 13. The rAAV vector of claim 1 or 12, wherein the nucleic acid coding sequence is at least 90% identical to SEQ ID NO:

10.

25. 13. The rAAV vector of claim 1 or 12, wherein the nucleic acid coding sequence is at least 95% identical to SEQ ID NO:

10.

26. 13. The rAAV vector of claim 1 or 12, wherein the nucleic acid coding sequence is at least 96% identical to SEQ ID NO:

10.

27. 13. The rAAV vector of claim 1 or 12, wherein the nucleic acid coding sequence is at least 97% identical to SEQ ID NO:

10.

28. 13. The rAAV vector of claim 1 or 12, wherein the nucleic acid coding sequence is at least 98% identical to SEQ ID NO:

10.

29. 13. The rAAV vector of claim 1 or 12, wherein the nucleic acid coding sequence is at least 99% identical to SEQ ID NO:

10.

30. A recombinant adeno-associated virus (rAAV) vector for treating hemophilia B, the rAAV vector comprising a capsid and a genome; the capsid comprises a VP1 protein having the amino acid sequence of SEQ ID NO:4; The genome is single-stranded and is ordered 5' to 3' (a) a first adeno-associated virus (AAV) inverted terminal repeat (ITR); and (b) an apolipoprotein E (ApoE) HCR-1 enhancer or a portion thereof; (c) an α1-antitrypsin (AAT) gene promoter or a portion thereof; (d) a nucleic acid comprising a sequence encoding a human Factor IX (FIX) Padua variant, wherein the coding sequence of said nucleic acid is not naturally occurring and is at least 70% identical to SEQ ID NO:10, and encodes the same human FIX protein as that encoded by SEQ ID NO:10, and optionally is separated by an intron; (e) a polyadenylation sequence; and (f) A recombinant adeno-associated virus (rAAV) vector containing elements of the second AAV ITR.

31. 31. Use of a therapeutically effective amount of the rAAV vector of claim 30 in the manufacture of a medicament for treating a human patient with hemophilia B.

32. 32. The use of claim 31, wherein the human patient has severe hemophilia B symptoms and the medicament is effective in reducing the symptoms from severe to moderate or mild hemophilia B symptoms.

33. 1. A packaging cell comprising: (i) a nucleic acid comprising a sequence encoding a human Factor IX (FIX) protein, wherein the coding sequence of said nucleic acid is not naturally occurring and is at least 70% identical to SEQ ID NO:10, encoding the same human FIX protein as that encoded by SEQ ID NO:10, optionally separated by an intron; (ii) an adeno-associated virus (AAV) Rep protein; (iii) an AAV capsid protein comprising the amino acid sequence of SEQ ID NO:4; and (iv) adenovirus helper proteins.

34. A recombinant AAV (rAAV) vector produced by incubating the packaging cell of claim 33 under conditions that allow packaging of a nucleic acid comprising the sequence of nucleotides 142 to 4096 of SEQ ID NO: 12 into an AAV particle.

35. 35. An rAAV vector produced by the method of claim 34, wherein the rAAV vector is isolated or purified.

36. 1. A recombinant adeno-associated virus (rAAV) vector comprising a capsid and a genome, wherein the capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:4, and the genome comprises at least one AAV inverted terminal repeat (ITR) and a nucleic acid comprising a sequence encoding a human factor IX (FIX) protein, wherein the coding sequence of the nucleic acid is not naturally occurring and is at least 90% identical to SEQ ID NO:10, and encodes a human FIX protein identical to the human FIX protein encoded by SEQ ID NO:10, and is optionally separated by an intron.

37. 37. The rAAV vector of claim 36, wherein the genome further comprises an expression control element operably linked to the sequence encoding a human FIX protein.

38. 38. The rAAV vector of claim 37, wherein the expression control elements comprise a promoter and, optionally, an enhancer.

39. 39. The rAAV vector of claim 38, wherein the expression control element is tissue-specific.

40. 40. The rAAV vector of claim 39, wherein the expression control element is liver-specific.

41. 39. The rAAV vector of claim 38, wherein the genome further comprises a polynucleotide stuffer.

42. 39. The rAAV vector of claim 38, wherein the genome further comprises a transcription terminator.

43. 1. A recombinant adeno-associated virus (rAAV) vector comprising a capsid and a genome, wherein the capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:4, and the genome comprises AAV inverted terminal repeats (ITRs) and a liver-specific enhancer and promoter operably linked to a nucleic acid comprising a sequence encoding a human factor IX (FIX) protein, wherein the coding sequence of the nucleic acid is not naturally occurring and is at least 70% identical to SEQ ID NO:10 and encodes a human FIX protein identical to the human FIX protein encoded by SEQ ID NO:

10.

44. 44. The rAAV vector of claim 43, wherein the first AAV ITR is located at the 5' or 3' end of the genome, and the second AAV ITR, if present, is located at the opposite end of the genome.

45. 45. The rAAV vector of claim 44, wherein the genome further comprises an intron or polynucleotide stuffer.

46. 46. ​​The rAAV vector of claim 45, wherein the genome is single-stranded DNA.

47. 46. ​​The rAAV vector of claim 45, wherein the intron comprises the nucleic acid sequence set forth in SEQ ID NO:

17.

48. 46. ​​The rAAV vector of claim 45, wherein the sequence and intron of the nucleic acid encoding a human FIX protein comprises the nucleic acid sequence of SEQ ID NO:

25.

49. The rAAV vector of claim 43, wherein the promoter is a human alpha-1-antitrypsin (AAT) promoter or a portion thereof, and the enhancer is an apolipoprotein E (ApoE) HCR-1 or HCR-2 enhancer or a portion thereof.

50. 50. The rAAV vector of claim 49, wherein the enhancer comprises the nucleic acid sequence of SEQ ID NO:

14.

51. 50. The rAAV vector of claim 49, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO:

15.

52. 44. The rAAV vector of claim 43, wherein the AAV ITRs are AAV2 ITRs.

53. A recombinant adeno-associated virus (rAAV) vector for treating hemophilia B, the rAAV vector comprising a capsid and a genome; the capsid comprises a VP1 protein having the amino acid sequence of SEQ ID NO:4; The genome (a) a first AAV inverted terminal repeat (ITR); (b) a liver-specific enhancer and promoter; (c) a nucleic acid sequence encoding a human Factor IX (FIX) Padua variant, the coding sequence of which is not naturally occurring, has at least 70% identity to SEQ ID NO:10, encodes the same human FIX protein as that encoded by SEQ ID NO:10, and is separated by an intron; (d) a polyadenylation sequence; and (e) A recombinant adeno-associated virus (rAAV) vector containing elements of the second AAV ITR.

54. 54. The rAAV vector of claim 53, wherein the enhancer is an apolipoprotein E (ApoE) HCR-1 enhancer or a portion thereof, the promoter is a human alpha 1-antitrypsin (hAAT) promoter or a portion thereof, the intron is hFIX intron A or a portion thereof, and the polyadenylation sequence is a bGH polyA sequence or a portion thereof.

55. (a) the nucleic acid sequence of the first AAV ITR comprises nucleotides 12 to 141 of SEQ ID NO:26; (b) the nucleic acid sequence of the enhancer comprises nucleotides 152 to 472 of SEQ ID NO: 12; (c) the nucleic acid sequence of the promoter comprises nucleotides 482 to 878 of SEQ ID NO: 12; (d) the nucleic acid sequence encoding human FIX consists of nucleotides 908-995 and 2434-3731 of SEQ ID NO: 12; (e) the nucleic acid sequence of the intron comprises nucleotides 996 to 2433 of SEQ ID NO:12; (f) the nucleic acid sequence of the polyadenylation sequence comprises nucleotides 3820 to 4047 of SEQ ID NO: 12; (g) the nucleic acid sequence of the second AAV ITR comprises nucleotides 3557 to 3686 of SEQ ID NO: 26; 54. The rAAV vector of claim 53.

56. 54. The rAAV vector of claim 53, wherein the genome further comprises a 5'UTR located after the promoter and before the coding sequence, and a 3'UTR located after the coding sequence and before the polyadenylation sequence.

57. 57. The rAAV vector of claim 56, wherein the nucleic acid sequence of the 5'UTR comprises nucleotides 879 to 907 of SEQ ID NO: 12 and the 3'UTR comprises nucleotides 3732 to 3779 of SEQ ID NO:

12.

58. 58. The rAAV vector of claim 57, wherein the genome is single-stranded DNA and the nucleic acid sequence comprises nucleotides 142 to 4096 of SEQ ID NO: 12.

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