Variant AAV and compositions, methods and uses for gene transfer to cells, organs and tissues

Abstract

To provide an AAV serotype with high targeting specificity for the liver and low seropositivity within the human body.SOLUTION: The invention relates to adeno-associated virus (AAV) serotype AAV-Rh74 and related AAV vectors, and AAV-Rh74 and related AAV vector mediated gene transfer methods and uses. In particular, AAV-Rh74 and related AAV vectors target polynucleotides to cells, tissues or organs for expression (transcription) of genes encoding therapeutic proteins and peptides, and the polynucleotides function as or are transcribed into inhibitory nucleic acid sequences.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 61 / 985,365, filed April 28, 2014. No. 61 / 857,161, filed July 22, 2013. This application claims priority to US Pat. No. 6,399,343, the entire contents of which are incorporated herein by reference. Can be put in. preface

[0002] Absence or defect (loss of function) in a desirable gene, or an undesirable or Genetic disorders caused by the expression of defective genes (gain of function) are An example of a genetic disease caused by loss of function is hemophilia, which Clotting factor VIII (FVIII, hemophilia A) or clotting factor IX (FIX, hemophilia B) It is an inherited bleeding disorder caused by a deficiency of the β-lactamase inhibitor β-lactamase. Examples include Huntington's disease, which is caused by a condition that encodes a mutant protein called HT by the "T" gene (which codes for the huntingtin protein) This is a disease caused by accumulation of mutant proteins, particularly in the basal ganglia and cerebral cortex. This gradually leads to the destruction of nerve cells.

[0003] Current treatment for hemophilia involves administering hemophilia as needed or prophylactically when bleeding occurs. Recombinant clotting factors are administered intravenously. However, this method of treatment has several drawbacks. For example, the need for repeated infusions, the cost of treatment, and the risk of developing an anti-treatment factor immune response. There is a risk of injury and potentially fatal bleeding. This has prompted the development of gene-based therapies for hemophilia. In this study, gene transfer-based therapy is suitable for hemophilia because: ) The therapeutic window is very wide. At levels just above 1% of normal, the phenotype was already shifted from severe to moderate. 100% free of any side effects; 2) tissue-specific treatment; and 3) there is no strict requirement for target gene expression, and Furthermore, the hepatic expression of coagulation factors has led to the development of It is known that immune tolerance to the factor itself can be induced, making it difficult to identify potentially harmful clotting factors. This reduces the likelihood of an immune response in the child.

[0004] Currently, adeno-associated virus (AAV) vectors are the gene transfer vector of choice. It is recognized that this is due to the excellent safety and efficacy profile of gene delivery in vivo. Among the AAV serotypes isolated so far, A AV2 and AAV8 have been used to target the liver in patients with severe hemophilia B. Both vectors are known to be effective, and in the case of AAV8, the therapeutic transgene can be delivered for a long time. Recent data in humans suggest that AAV vectors are able to express Targeting the liver with FIX results in long-term therapeutic expression of the FIX transgene It has been shown that

[0005] These data suggest that these serotypes are promising for the future, but they are known to have high toxicity to the liver. It has a high tropism and a low seroprevalence in humans. To identify new AAV serotypes (natural hosts of wild-type AAV) that possess is important to achieve two things: 1) to elicit an anti-AAV capsid immune response; To reduce the risk, the lowest possible vector dose was used to induce therapeutic levels in the liver. 2) to achieve expression of the transgene; and 3) to have pre-existing humoral immunity to AAV. For patient populations in which AAV gene transfer is not possible, we have developed an alternative AAV with unique seroprevalence. The present invention addresses the above needs and provides further advantages. bring about. Summary of the Invention

[0006] The present invention relates to an adeno-associated virus (AAV) serotype AAV-Rh74 vector and related The present invention provides AAV vectors and viral particles that can be used to infect various types of cells. Among the variants, AAV-Rh74 targets hepatocytes in the liver. Polynucleotide sequence As a vector for delivering Rh74, AAV-Rh74 and related AAV vectors have been shown to mediate the expression of Rh74 in cells. It activates the expression of nucleotides, such as proteins used in therapy. The encoding polynucleotide can be expressed at therapeutic levels following administration.

[0007] Exemplary AAV-Rh74 vectors and related AAV vectors include the AAV-Rh74 mutant The specific capsid mutants include the RH74 VP1 capsid sequence (SEQ ID NO:1 ) at any one of the 195th, 199th, 201st, or 202nd amino acid positions In certain embodiments, the residue comprises the RH74 VP 1. The 195th, 199th, 201st, or 202nd address of the capsid sequence (SEQ ID NO: 1) It corresponds to an A, V, P, or N amino acid at any one of the amino acid positions. In one embodiment, the capsid sequence is the RH74 VP1 capsid sequence (SEQ ID NO:1). an A residue at amino acid position 195, a V residue at amino acid position 199, A P residue at the 201st amino acid position or an N residue at the 202nd amino acid position In more particular embodiments, the capsid sequence comprises any two, three or more of the following: , or all four. They contain the RH74 VP1 capsid sequence (SEQ ID NO: 1) A residue at the 195th amino acid position and V residue at the 199th amino acid position residue at amino acid position 201, or a P residue at amino acid position 202 The N residue is

[0008] The recombinant AAV particles of the present invention include RHM4-1 (SEQ ID NO: 5), RHM15-1 ( Sequence ID number: 6), RHM15-2 (sequence ID number: 7), RHM15-3 / RHM1 5-5 (SEQ ID NO: 8), RHM15-4 (SEQ ID NO: 9), or RHM15- 6 (SEQ ID NO: 10). In certain embodiments, the recombinant AAV particle comprises a vector genome (e.g., an AAV vector genome). Such a combination of the present invention encapsidates or packages a viral vector (such as a viral vector). Recombinant AAV particles are prepared by injecting a viral (e.g., AAV) vector containing a heterologous polynucleotide sequence. - Includes the genome.

[0009] AAV-Rh74 and related AAV vectors have been studied in preclinical and clinical settings. The results of the polynucleotide transfection were significantly higher than those of several other serotypes. In particular, AAV-Rh74 regulates the expression levels of the hemophilia B protein in mice and dogs. In this study, AAV8, a representative example of liver transduction, was used The polynucleotides are targeted to the liver with efficiency comparable to or greater than that of the IL-1 ... See FIG. 2.) For example, the capsid mutant RHM4-1 (SEQ ID NO: 5) The V-Rh74 mutant showed comparable or superior efficacy to AAV8 in mice, and 4 - Targeting polynucleotides to the liver with greater efficiency than AAV (see e.g., Figure 5 ). Furthermore, data in non-human primates support the notion that AAV-Rh74 and Rh74 AAV-Rh74 mutants, such as HM4-1, mediate expression of liver-derived hFIX It has been shown to be approximately twice as potent as AAV8 (see, e.g., Figures 4 and 6).

[0010] Therefore, AAV-Rh74 and capsid mutants (e.g., RHM4-1) The AAV-Rh74 mutant is useful for delivering polynucleotides such as gene coding sequences. Expressing a protein that can be used and provide a desired or therapeutic benefit and inhibiting or reducing the expression of undesirable or defective genes. It can also be used for harmful nucleotides, making it possible to treat a variety of diseases. For example, AAV-Rh74 and AAV-Rh74 capsid mutants (e.g., RHM4-1 ) is a company that develops therapeutic genes (e.g., FIX, FVIII) for the treatment of hemophilia A, B, etc. They can be used as vectors for delivery to cells, tissues, and organs such as the liver. In addition, such AAV-Rh74 and AAV-Rh74 capsid mutants (e.g., Rh74 M4-1) vectors are used for a wide range of diseases, including other metabolic disorders or plasma protein deficiencies. They can be used to deliver genes to the body or for other therapeutic purposes. These can be used to deliver genes, for example, but not limited to, to the liver. The zinc finger nucleases used to carry out the editing and genes encoding alpha-amyloid nucleases for treating hepatitis virus infections. Local (hepatic) delivery or hepatic transduction of immunomodulatory drugs such as interferon Requires or requires the presence of a therapeutic transgene product in the bloodstream (expressed in the liver) Anything that requires targeting of the transgene for It can be used to treat diseases such as

[0011] In vitro, in vivo and ex vivo characterization of AAV-Rh74 vectors and capsid mutants ( For example, by related AAV vectors such as AAV-Rh74 variants (e.g., RHM4-1). In addition to efficient delivery of polynucleotides to cells, anti-AAV-Rh74 antibody responses in humans The prevalence of anti-AAV antibodies is lower than that of anti-AAV2 antibodies. 8 antibody prevalence (Table 1). - and related AAV-Rh74 (capsid) variants (e.g., RHM4-1). V vectors can be used to express, for example, other AAV serotypes (e.g., AAV1, AAV2, AAV3 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11) for use in many people, including gene transfer in humans who are serologically positive for In addition, AAV-Rh74 vectors and capsid mutants (e.g., RHM Related vectors, such as the AAV-Rh74 mutant, can produce high titers. Therefore, the AAV-Rh74 vector and Related AAV vectors can be produced in large quantities for a wider range of clinical diseases.

[0012] The present invention also includes AAV vector genomes (encapsidated or packaged). ), recombinant AAV-Rh74 vectors, and capsid mutant (e.g., RHM4-1) particles. In one embodiment, related AAV vectors, such as the AAV-Rh74 mutant, are provided. In another embodiment, the recombinant AAV vector comprises a heterologous polynucleotide sequence. The recombinant AAV vector genome may comprise the AAV-Rh74 capsid or a capsid mutant (e.g. Encapsidation by related AAVs, such as AAV-Rh74 mutants (e.g., RHM4-1) Or packaged.

[0013] AAV containing (encapsidating or packaging) a recombinant AAV vector genome -Rh74 vectors, and AAV-Rh74 (capsid) mutants (e.g., RHM4-1 In the recombinant AAV vectors of the invention, such as related AAV vectors, such as avian vesicles, heterologous polypeptides may be used. The nucleotide sequence is transcribed and then converted into a protein. The nucleic acid may itself be a functional or active nucleic acid (e.g., an inhibitory nucleic acid). It may also be transcribed as:

[0014] In various embodiments, the heterologous polynucleotide sequence encodes a protein having a therapeutic effect. In certain embodiments, the protein is a blood clotting factor (e.g., factor XIII). , factor IX, factor X, factor VIII, factor VIIa or protein C), cysts cystic fibrosis transmembrane regulator e regulator protein; CFTR), antibody, retinal pigment epithelium specific Target 65 kDa protein (retinal pigment epithelial m-specific 65 kDa protein (RPE65), erythropoietin Ethin (erythropoietin), LDL receptor, lipoprotein lipase, Ornithin tin transcarbamylase, beta globin, alpha globin, spectrin, alpha antithrombin Psin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7) , sulfamidase, enzymes involved in lysosomal storage diseases (AR SA), hypoxanthine-guanine phosphoribosyltransferase, β-25glutamine Glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, Branched-chain ketoacid dehydrogenase, growth factors (e.g., insulin-like growth factors 1 and 2, platelet-derived Neurotrophic factor, epidermal growth factor, nerve growth factor, neurotrophic factor 3 and 4, brain-derived neurotrophic factor cytokines (e.g., α, glial cell-derived growth factor, transforming growth factor α, β, etc.); Interferon, beta interferon, interferon gamma, interleukin 2, Interleukin 4, Interleukin 12, Granulocyte-macrophage colony-stimulating factor, amphotericin, etc.), suicide gene products (e.g., herpes simplex virus thymidine kinase, Cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine ases, tumor necrosis factors, etc.), drug resistance proteins (e.g., proteins that confer resistance to drugs used in cancer treatment), proteins that confer Von Hippel-Lindau (VHL), Adenomatous polyposis intestinalis (APC), peptides with immunomodulatory functions, tolerogenic or Immunogenic peptide or protein Tregitopes (regulatory T cell epitopes) , or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA -GUCY2D), Rab escort protein 1 (congenital choroideremia), LCA 5 ( LCA-Reversillin), Ornithine Ketoacid Aminotransferase (Gyratum Choroidal Atrophy) ), Retinoschisin 1 (X-linked retinoschisis) , USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR type retinitis pigmentosa), DFNB1 (connexin 26 refractory hearing), ACHM 2, 3 and 4 (color blindness), PKD-1 or PKD-2 (multiple cysts) cystic kidney), TPP1, CLN2, genetic defects causing lysosomal storage diseases (e.g., Sul phatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A , GM2-AP, NPC1, VPC2, sphingolipid activating protein, etc.), genome editor One or more zinc finger nucleases or genomic DNA fragments used for binding This is the donor sequence used as the repair template in mu-editing.

[0015] In an additional embodiment, the heterologous polynucleotide sequence encodes a therapeutic protein. As a result, unwanted or abnormal (dysfunctional) proteins present in the patient (endogenous In yet another embodiment, the heterologous polynucleotide inhibits the expression or function of a heterologous protein. The sequence is a polynucleotide that, when transcribed, is transcribed into an inhibitory nucleic acid (e.g., an inhibitory RNA). In yet another embodiment, the inhibitory nucleic acid is a single-stranded sequence or a double-stranded sequence or In yet another embodiment, the inhibitory nucleic acid is a microRNA (miRNA). A) siRNA, shRNA, trans-splicing RNA, antisense RNA, or triplex-forming RNA.

[0016] In yet another embodiment, the inhibitory nucleic acid is an inhibitor of the huntingtin (HTT) gene, dentatorubral pallidum gene, Genes associated with bulbar-luysian atrophy (e.g., atrophin 1 (ATN1)), spinal-bulbar muscular atrophy Ataxin 1,2, an androgen receptor on the X chromosome, in ataxia , 3 and 7, the voltage-gated calcium channel encoded by CACNA1A, Ca v 2 .1 P / Q, ataxin, also known as TATA-binding protein, ATXN8OS Inverse chain 8, 55 of serine-threonine protein phosphatase 2A in spinocerebellar ataxia kDa Regulatory Subunit B beta isoform (1, 2, 3, 6, 7, 8, 12, 17 type), FMR1 (Fragile X Mental Retardation 1) in fragile X syndrome, fragile X-associated tremor / movement disorder FMR1 (Fragile X Mental Retardation 1) in ataxia syndrome, F in fragile XE mental retardation MR1 (Fragile X Mental Retardation 2) or AF4 / FMR2 Family Member 2 (AF4 / FMR2 family member 2), myotonic dystrophy Myotonin-protein kinase (MT -PK), Frataxin in Friedreich's ataxia, Muscle atrophy Mutant superoxide dismutase 1 (SOD1) gene in endothelial lateral sclerosis; Apolipoprotein A, a gene involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease Protein B (APOB) and proprotein convertase subtilisin / kexin type Protease inhibitor 9 (PCSK9), hypercholesterolemia, and human immunodeficiency virus (HIV) infection The HIV transcriptional transactivator gene, HIV Tat, and human immunodeficiency virus (H IV) HIV transactivator response element genes, HIV TAR, and HIV CC chemokine receptor (CCR5) in infection and Rous sarcoma virus (RSV) infection RSV nucleocapsid protein in the liver and liver-specific in hepatitis C virus infection MicroRNA (miR-122), p53, acute kidney injury, delayed graft function, or It is a protein kinase inhibitor in renal injury, acute renal failure, advanced recurrent or metastatic solid malignancies. PKN3, LMP2 in metastatic melanoma, LMP2 is a proteasome inhibitor LMP in metastatic melanoma, also known as prothrombin beta type 9 (PSMB 9) 7. Proteasome subunit beta type 8 (PSMB 8), also known as metastatic Also known as proteasome subunit beta type 10 (PSMB 10) in melanoma MECL1 in solid tumors, vascular endothelial growth factor (VEGF) in solid tumors, Kinesin spindle proteins suppress apoptosis in chronic myeloid leukemia B-cells CLL / Lys ribonucleotide reductase M2 (RRM2) in lymphoma (BCL-2), solid tumors; Furin in solid tumors and polo-like kinase 1 (PKI) in liver cancer LK1), diacylglycerol acyltransferase 1 (D-LK1) in hepatitis C infection GAT1), beta-catenin in familial adenomatous polyposis, beta-adrenergic receptor agonist (BETA) in glaucoma Phosphoreceptor, DAN loss in diabetic macular edema (DME) or age-related macular degeneration RTP801 / Redd1, also known as wound-induced transcript 4 protein, is involved in the age-related yellowing of the skin. Vascular endothelial growth factor receptor I (VEGFR1) in macular degeneration or choroidal neovascularization Caspase 2 in arteritic ischemic optic neuropathy and keratin 6A in pachyonychia congenita N17K mutant protein, influenza A virus in influenza infection Genome and gene sequences of coronaviruses in severe acute respiratory syndrome (SARS) infections Genome and gene sequence of respiratory syncytial virus in respiratory syncytial virus infection ·Genetic sequence, genome of Ebola phylovirus in Ebola infection ·Genetic sequence, B and Genome and gene sequences of hepatitis B and C viruses in hepatitis B and C infections, simple hepatitis Genome and gene sequences of HSV virus in HSV infection, Coxsackie - Genome and gene sequence of Coxsackievirus B3 in virus B3 infection, primary myocardial infarction Silence of pathogenic alleles of torsinA (TOR1A)-like genes in ataxia Allele-specific silencing, pan-class I and HLA allele silencing in transplantation Gene-specific (silencing) in autosomal dominant retinitis pigmentosa (adRP) A mutant rhodopsin gene that binds to a transcript of any of the above genes or sequences. The inhibitory nucleic acid is inhibited from expressing the inhibitory nucleic acid.

[0017] The present invention includes (encapsidates or packages) a recombinant AAV vector genome. Recombinant AAV vectors of the above, as well as AAV-Rh74 vectors and capsid mutants (e.g. Related AAV vectors, such as AAV-Rh74 vector variants, such as AAV-Rh74 (RHM4-1) particles. In certain embodiments, the recombinant vector comprises additional elements that function in cis or trans. A recombinant virus that contains (encapsidates or packages) an AAV vector genome vectors (e.g., AAV), and / or AAV-Rh74 vectors or AAV- Related AAVs, such as Rh74(capsid) mutant (e.g., RHM4-1) particles, They also have a sequence adjacent to the 5' or 3' end of the heterologous polynucleotide sequence. One or more inverted terminal repeat (ITR) sequences, which act as promoters for driving the transcription of a heterologous polynucleotide sequence. The heterologous polynucleotide sequence may include a current control sequence (e.g., a constitutive or regulatable control element). promoter or enhancer that contributes to the transcription of the sequence, or tissue-specific expression control elements, A polyadenine sequence located 3' of the polynucleotide sequence, a selectable marker (e.g. (e.g., a protein that provides resistance to an antibiotic, such as kanamycin resistance), and / or The origin of replication.

[0018] Also includes (encapsidates or packages) recombinant AAV vector genomes The recombinant AAV vector of the present invention, and the AAV-Rh74 vector and capsid mutant ( For example, related AAV vectors such as the AAV-Rh74 mutant containing RHM4-1 particles In one embodiment, the recombinant vector genome comprises a heterologous polynucleotide. A filler polynucleotide sequence or stuffer (st In certain embodiments, the heterologous polynucleotide sequence In a more particular embodiment, the heterologous polynucleotide sequence has a length of less than about 4.7 Kb. The sequence is less than approximately 4.7 Kb in length and contains two ITR sequences from the adeno-associated virus (AAV). In a further particular embodiment, the heterologous polynucleotide sequence and the filtrate are located within the The total length of the rRNA or stuffer polynucleotide sequence is approximately 3.0-5. 5 Kb, or about 4.0 to 5.0 Kb, or about 4.3 to 4.8 Kb.

[0019] The filler or stuffer polynucleotide sequence does not impair the function or activity of the vector. The nucleic acid sequence may be located at any desired position within the vector sequence, provided that the nucleic acid sequence is not disrupted by the nucleic acid sequence. The filler or stuffer polynucleotide sequence is a complement of the heterologous polynucleotide sequence. The 5' and / or 3' sides adjacent to the outside of the 5' and / or 3' ends, respectively In another embodiment, the filler or stuffer is not disposed between the flanking ITR sequences. The heterologous polynucleotide sequence may be 5' and / or 3' Located between the 5' and / or 3' ITR sequences adjacent to the outer ends, respectively In yet another embodiment, the filler or stuffer polynucleotide sequence is , flanking the outside of the 5' and / or 3' ends of the heterologous polynucleotide sequence, respectively. The ITR sequence is preferably located adjacent to the 5' and / or 3' ITR sequences. In another embodiment, the filler or stuffer polynucleotide sequences are derived from, for example, genomic nucleic acids. It is intended to be positioned within a heterologous polynucleotide sequence such as an intron within a heterologous polynucleotide sequence.

[0020] Thus, in various embodiments, one filler or stuffer polynucleotide sequence The rows are flanked by AAV ITR sequences or by a single filler or stuffer polynucleotide. The nucleotide sequence is located inside the two ITR sequences of the adeno-associated virus (AAV). Or, one filler or stuffer polynucleotide sequence is an adeno-associated Located outside the two ITR sequences of the virus (AAV), or a first filler or stuffer polynucleotide sequence, The nucleotide sequence is located within two ITR sequences of the adeno-associated virus (AAV). The second filler or stuffer polynucleotide sequence is an adeno-associated virus (AA V) are located outside the two ITR sequences.

[0021] In a further particular embodiment, the filler or stuffer polynucleotide sequence is When it is located between two ITR sequences of the associated virus (AAV), it is The total length of the tid sequence and the filler or stuffer polynucleotide sequence is Approximately 3.0 to 5.5 Kb, approximately 4.0 to 5.0 Kb, or approximately 4.3 to 4.8 Kb. In another specific embodiment, the filler or stuffer polynucleotide sequence is When located outside the two ITR sequences of AAV, it is a filler or The length of the stuffer polynucleotide sequence shall be greater than 4.7 Kb and less than about 5.0 ~10.0 Kb or approximately 6.0-8.0 Kb.

[0022] Furthermore, in various embodiments, the filler or stuffer polynucleotide sequence , the length is about 1-10, 10-20, 20-30, 30-40, 40-50, 50-6 0, 60~75, 75~100, 100~150, 150~200, 200~250, 2 50~300, 300~400, 400~500, 500~750, 750~1,000 , 1,000~1,500, 1,500~2,000, 2,000~2,500, 2,5 00~3,000, 3,000~3,500, 3,500~4,000, 4,000~4 ,500, 4,500~5,000, 5,500~6,000, 6,000~7,000 , a sequence of 7,000 to 8,000 or 8,000 to 9,000 nucleotides.

[0023] Typically, the filler or stuffer polynucleotide sequences are inert or harmless. In various specific embodiments, the filler or stuffer polysaccharide is The nucleotide sequence is not a bacterial polynucleotide sequence, but a filler or stuffer sequence. The polynucleotide sequence is not a protein or peptide coding sequence, but a filler or The stuffer polynucleotide sequence is a heterologous polynucleotide sequence, an AAV inverted end repeat sequence, Interleaved repeat (ITR), expression control element, replication origin, selection marker or polyadenine (pol yA) sequences different from any of the sequences.

[0024] In various other embodiments, the filler or stuffer polynucleotide sequence is A heterologous polynucleotide sequence may or may not be an intron sequence. In embodiments, the intron sequence is located internally in the heterologous polynucleotide sequence. In embodiments, the intron sequence is an intron that encodes a protein and is within the genomic DNA that is also encoded by the heterologous polynucleotide sequence, Associated with a heterologous polynucleotide sequence.

[0025] Also includes (encapsidates or packages) recombinant AAV vector genomes The recombinant AAV vector of the present invention, and the AAV-Rh74 vector and capsid mutant ( For example, related AAV vectors such as the AAV-Rh74 mutant containing RHM4-1 particles In such an embodiment, the cells are lysed to release the virus ( AAV) particles (e.g., AAV-Rh74 vector or AAV-Rh74 capsid mutant helper cells that produce the relevant AAV vector (e.g., RHM4-1); or Includes target cells within which it is desired to express the heterologous polynucleotide sequence.

[0026] Also includes (encapsidates or packages) recombinant AAV vector genomes The recombinant AAV vector of the present invention, and the AAV-Rh74 vector and AAV-Rh74 Related AAV vectors, such as mutant (e.g., capsid mutants such as RHM4-1) particles, Such compositions may be comprised within a pharmaceutical composition. A recombinant vector (e.g., AA V), and AAV-Rh74 vectors and AAV-Rh74 mutants (e.g., RHM4 These vectors are useful in administering to patients related AAV vectors, such as capsid variants such as .ALPHA.-1. .

[0027] Also includes (encapsidates or packages) recombinant AAV vector genomes The recombinant AAV vector of the present invention, and the AAV-Rh74 vector and AAV-Rh74 Related AAV vectors, such as mutant (e.g., capsid mutants such as RHM4-1) particles, Thus, the present invention can be applied to a variety of methods and uses. Methods for delivering or introducing heterologous polynucleotide sequences into mammalian or mammalian cells and and usage.

[0028] In one embodiment, the method or use comprises a vector genome (encapsidating, (e.g., AAV-Rh74 vector, or AAV-Rh74 variant (e.g., For example, a heterologous polynucleoside (e.g., a capsid mutant such as RHM4-1) or a related AAV (e.g., a capsid mutant such as RHM4-1) may be used. Adeno-associated virus (AAV) vectors containing particles of the peptide sequence are then cultured under suitable conditions in mammalian cells. to a mammal or to a cell of a mammal, thereby delivering the heterologous polynucleotide sequence to the mammal. In one embodiment, the method or use comprises delivering or introducing into a mammalian cell. , allowing the introduction / delivery of heterologous polynucleotides into mammals and / or cells. In another aspect, the method includes introducing / delivering a heterologous polynucleotide into a mammal and / or cell. The heterologous polynucleotide is then transcribed to form a transcript. In an embodiment, the method allows for the introduction / delivery of a heterologous polynucleotide into a cell, followed by transfection. Transcription forms a transcript, which is then translated to produce the gene. More specifically, in the two embodiments described below, for example, The polynucleotide sequence is operable with expression control elements that confer transcription of the heterologous polynucleotide sequence. operably linked to, and optionally further to, translation of the transcript.

[0029] In additional embodiments, the method or use comprises administering a heterologous polynucleotide sequence to a subject (e.g. for delivery or transport to a subject (e.g., a mammal) or to a cell of a subject (e.g., a mammal) , one virus (e.g., AAV-Rh74, or an AAV-Rh74 capsid mutant ( For example, a related AAV) particle such as RHM4-1, a plurality of such viral (e.g., AAV) particles or one of the viruses (e.g., AAV-Rh74, or AAV-Rh74 capsid AAV particles or a plurality of such viruses (e.g., related AAV) such as a mutant (e.g., RHM4-1) The pharmaceutical composition comprising the AAV particles is administered to a subject (e.g., a mammal) or to a subject (e.g., a mammalian This involves administering the heterologous polynucleotide sequence to cells of the subject ( The delivery or transport may be to a subject (eg, a mammal) or to cells of a subject (eg, a mammal). In another embodiment, the method or use relates to a method for treating a subject having a deficiency in protein expression or function, or is required for the expression or function of a protein, or is an endogenous protein (e.g., Absent, abnormal, or dysfunctional protein) expression or function must be reduced. For treating a subject (e.g., a mammal), -Rh74, and AAV-Rh74 mutants (e.g., capsid mutants such as RHM4-1) (e.g., AAV) particles, a plurality of such viral (e.g., AAV) particles, or a single viral Rh74 and AAV-Rh74 mutants (e.g., RHM4-1 AAV) particles or a plurality of such viruses (e.g., AAV ) particles, and providing a pharmaceutical composition comprising one virus (e.g., AAV-Rh 74, and AAV-Rh74 mutants (e.g., capsid mutants such as RHM4-1), etc. A related AAV) particle, a plurality of said viral (e.g., AAV) particles, or a single viral (e.g., AAV) particle. For example, AAV-Rh74 and AAV-Rh74 mutants (e.g., RHM4-1, etc.) A related AAV) particle or a plurality of said viral (e.g., AAV) particles, such as capsid variants The method includes administering to a subject (e.g., a mammal) a pharmaceutical composition comprising the heterologous polynucleotide. The nucleotide sequence is expressed in a mammal. Alternatively, the heterologous polynucleotide sequence is The present invention relates to a method for detecting endogenous proteins (e.g., unwanted, abnormal, or Inhibitory sequences or proteins that reduce the expression or function of a protein (e.g., a dysfunctional protein) The quality of the data shall be coded.

[0030] The method of administration or delivery and the method of use may be in any manner suitable for the subject. In certain embodiments, a single virus (e.g., AAV-Rh74 or AAV-Rh74) is used. h74 capsid mutant (e.g., RHM4-1) or related AAV particles or multiple Viral (e.g., AAV) particles (e.g., AAV-Rh74 vectors, or AAV-Rh Related AAV vectors, such as 74 capsid mutants (e.g., RHM4-1), were administered intravenously , intra-arterial, intramuscular, subcutaneous, oral, through intubation, via catheter, through the skin, Administered or delivered intrathecally, by inhalation, intracavity, or mucosally.

[0031] Subjects include human and non-human (e.g., primate) mammals. In this embodiment, the subject benefits from or is able to express the heterologous polynucleotide sequence. is required.

[0032] In the present invention, recombinant vector (e.g., AAV) plasmids and recombinant vectors ( A virus (e.g., AAV) that contains (encapsidates or packages) AV-Rh74 and AAV-Rh74 mutants (e.g., capsid variants such as RHM4-1) Methods for producing related AAV particles, such as variants, are provided. In one embodiment, a recombinant Methods for producing viruses or AAV particles include the use of recombinant vector (e.g., AAV) plasmids. The vector is introduced into packaging helper cells to express the virus (e.g., AAV-Rh74, Related AAVs such as AAV-Rh74 mutants (e.g., capsid mutants such as RHM4-1) V) and recombinant viruses (e.g., AAV-Rh74, or AAV-Rh75, Produce AV-Rh74 capsid mutant (e.g., related AAV such as RHM4-1) particles In another embodiment, the method further comprises culturing the helper cells in an environment that allows the removal of contaminating nucleic acid. Recombinant viruses containing recombinant viral vectors (e.g., AAV-Rh74, and AAV-Rh74 variants (e.g., capsid variants such as RHM4-1). The method for producing recombinant virus or AAV particles having reduced amounts of recombinant AAV particles is described. The vector (e.g., AAV) plasmid is introduced into packaging helper cells to produce the virus. and infecting the recombinant virus (e.g., AAV-R h74, and AAV-Rh74 mutants (e.g., capsid mutants such as RHM4-1). The process involves culturing helper cells in an environment that allows production of AAV (and other related AAV) particles. Here, the produced recombinant viruses (e.g., AAV-Rh74 and AAV-Rh74 Mutants (e.g., capsid mutants such as RHM4-1) and other related AAV particles are contaminating A virus containing a recombinant viral vector genome in which nucleic acid is present (e.g., AAV-Rh7 4, and AAV-Rh74 mutants (e.g., capsid mutants such as RHM4-1). The number of AAV (associated) particles is determined by the presence of filler or stuffer polynucleotides in the recombinant viral vector. Viruses containing contaminating nucleic acids when produced in the absence of a nucleotide sequence (e.g. AAV-Rh74 and AAV-Rh74 mutants (e.g., capsids such as RHM4-1) In one embodiment, the number of contaminating nuclei is reduced relative to the number of related AAV particles, such as mutants. The acid may be a bacterial nucleic acid or a heterologous polynucleotide sequence, an ITR, a promoter, an enhancer, The sequence is other than the base sequence, origin of replication, polyA sequence or selection marker.

[0033] The helper cells include mammalian cells. In some embodiments, the helper cells are The nucleotide sequence is expressed as a sequence similar to that of the AAV-Rh74 vector, but not as a sequence similar to that of the AAV-Rh74 vector. Related AAV vectors, such as h74 mutants (e.g., capsid mutants such as RHM4-1) It provides helper (e.g., AAV) functions to package the vector into a specific AAV particle (e.g., AAV particle). In embodiments, the helper cells express the Rep and / or Cap proteins of AAV (e.g., Rep and / or Cap proteins of AAV). The helper cells provide the Rep and and / or a polynucleotide encoding the sequence of the Cap protein. or transiently transfected. Helper cells are transfected with Rep78 and / or R The polynucleotide encoding the sequence of the ep68 protein may be used to stably or transiently The cells are selectively transfected.

[0034] The recombinant vector (e.g., AAV) plasmids of the present invention can be used in any strain or in different blood groups. The serotype may be based on any serotype, including hybrids or chimeras of serotypes. The recombinant viral (e.g., AAV) particles of the present invention are typically AAV-Rh74, or AAV-Rh74. Related AAVs, such as AV-Rh74 mutants (e.g., capsid mutants such as RHM4-1) Representative AAV serotypes include hybrids or chimeras having different serotypes. The types are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8. including, but not limited to, AV8, AAV9, AAV10, AAV11, and Rh10. Therefore, the recombinant virus of the present invention containing a vector genome (e.g. AAV) particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AA VP1 of V7, AAV8, AAV9, AAV10, AAV11, or Rh10 serotypes; Different serotypes, mixtures of serotypes, or contains capsid proteins derived from hybrids or chimeras of different serotypes In addition, the recombinant vectors (e.g., AAV), sequences, plasmids, vectors of the present invention are The genome may represent any single serotype, a mixture of serotypes, or a hybrid of different serotypes. In various embodiments, the recombinant AAV may include elements derived from a recombinant AAV vector or chimera. Vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and AAV7. , AAV8, AAV9, AAV10, AAV11, Rh74 or Rh10 serotypes, or or mixed hybrids or chimeras comprising any of the above AAV serotypes. It contains the Cap, Rep and / or ITR from which it is derived. [Brief description of the drawings]

[0035] [Figure 1] Human factor IX (FIX) plasma concentrations in C57BL / 6 mice (n=5 per group) injected with AAV vector via the tail vein to express the FIX transgene under the control of a liver-specific promoter. Vector dose was 2.510 vector genomes per mouse. FIX transgene product (FIX protein) plasma concentrations were measured by ELISA at weeks 1, 2, and 4 after gene transfer. AAV-Rh74 provided high levels of FIX transgene expression.

[0036] [Diagram 2] Figure 1 shows canine FIX plasma concentrations in dogs with hemophilia B after delivery of 312 vector genomes per kilogram (kg) of body weight. AAV vectors were infused intravenously (IV) via the saphenous vein and FIX concentrations were monitored by ELISA. Expression of the therapeutic FIX transgene was driven by a liver-specific promoter. AAV8 and AAV-Rh74 vectors performed approximately equally in dogs with hemophilia B and outperformed AAV6.

[0037] [Figure 3A-3E] The AAV-Rh74 VP1, VP2 and VP3 amino acid sequences and, for VP1, the polynucleotide (DNA) sequence are shown (SEQ ID NOs: 1-4).

[0038] [Figure 4]Administration of AAV8 and AAVrh74 vectors expressing human factor IX (FIX) to rhesus monkeys and non-human primates (under the control of a liver-specific promoter), and expression of FIX in the animals. Animals administered the AAVrh74-FIX vector (at least two bars toward the right margin) expressed high levels of the FIX transgene, compared to other groups of animals administered the same amount.

[0039] [Diagram 5] FIG. 1 shows plasma human factor IX expression levels in animals administered AAV human factor IX expression vectors encapsidated by the indicated AAV (Rh74 mutants, such as the RHM4-1 mutant) compared to AAV human factor IX expression vectors encapsidated by Rh74 and AAV8.

[0040] [Figure 6] FIG. 1 shows administration of AAV8 and AAV-Rh74 mutant RHM4-1 vectors expressing human factor IX (under the control of a liver-specific promoter) to cynomolgus monkeys and non-human primates, and expression of FIX in the animals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention relates, at least in part, to an adeno-associated virus (AAV) serotype AAV-Rh7. 4 and AAV-Rh74 capsid mutants (e.g., RHM4-1, RHM15-1, RH M15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6) Related AAV variants such as HIV-1 and HIV-2 target hepatocytes, which are cells in the liver. Based on data showing that it has high specificity for polynucleotides (e.g., genes, AAV-Rh74 and AAV-Rh74 are used as vectors for the introduction / delivery of inhibitory nucleic acids into cells. and AAV-Rh74 mutants (e.g., RHM4-1, RHM15-1, RHM15-2, Capsids such as RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 Using relevant AAV mutants, such as the .ALPHA. mutant, therapeutic levels of expression in the liver after intravenous administration were In addition, the AAV-Rh74 vector and the AAV-Rh74 mutant can be provided. (For example, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM1 5-5, RHM15-4, and capsid variants such as RHM15-6) The mutants generated by gene transfer / delivery are significantly higher than those of some other serotypes. The expression levels of the proteins involved are regulated by the IL-11 receptor agonists (see, for example, Figures 1, 2, 4, 5, and 6). In particular, AAV-Rh74 vectors and related AAV-Rh74 capsid mutants (e.g., R HM4-1) in dogs with hemophilia B and / or in mice and / or cynomolgus monkeys In this study, the results were at least as good as, and usually superior to, AAV8, the gold standard for liver transduction. Thus, AAV-mediated targeting of genes delivered to the liver is possible with an efficiency comparable to that of Rh74 vectors and AAV-Rh74 capsid mutants (e.g., RHM4-1) The relevant AAV variants are characterized by the coding sequence (gene) of a protein that provides a desired or therapeutic benefit. ), and unwanted or defective (e.g., pathological Inhibitory (e.g., antisense) nucleotides that reduce or suppress expression of a target (targeted) gene. can be used to introduce / deliver a variety of diseases For example, recombinant vectors (e.g., For example, the AAV genome may be expressed as an AAV-Rh74 vector or an AAV-Rh74 capsid mutant. Body (e.g., RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM 15-5, RHM15-4, and RHM15-6). It can be encapsidated or encapsulated.

[0042] As mentioned above, adeno-associated virus (AAV) serotypes AAV-Rh74 and AAV- Rh74 capsid mutants (e.g., RHM4-1, RHM15-1, RHM15-2, R Related AAs such as HM15-3 / RHM15-5, RHM15-4, and RHM15-6 The V variants are capable of transfecting polynucleotide sequences into cells in vitro, in vitro and in vivo. Such polynucleotide sequences provide a means for the delivery of and encoding a protein such that the cells to be transfected express the encoded protein. For example, a recombinant AAV vector can be used to encode a desired protein or peptide. or an inhibitory sequence (e.g., RNA) that, when transcribed, e.g. For example, a heterologous polynucleotide containing a sequence that targets a gene whose expression is to be inhibited may be included. Thus, vector delivery and administration to a subject (e.g., a mammal) can be achieved in a time-dependent manner. The heterologous polynucleotides encoding the proteins and peptides as well as the expression or is a method to develop inhibitory nucleic acids that target genes to inhibit their function. This will provide the target audience with leic acids.

[0043] Thus, according to the present invention, there are provided polynucleotide sequences encoding peptides and proteins, and repressor nuclear factors that directly or transcribingly inhibit the expression or function of targeted genes. A vector genome (encapsidation vector) containing a heterologous polynucleotide sequence comprising an amino acid. AAV-Rh74 vectors and AAV-Rh74 variants (e.g. , RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, Related AAV vectors such as RHM15-4 and capsid variants such as RHM15-6 Variants are provided.

[0044] A recombinant "vector" or "AAV vector" is a recombinant vector that ... and uses molecular techniques to remove the wild-type genome from the virus (e.g., AAV). The nucleic acid sequence may be replaced with a foreign nucleic acid, such as a heterologous polynucleotide sequence (e.g., a therapeutic gene expression cassette). In the case of AAV, the inverted terminal repeats (ITRs) of the wild-type AAV genome are usually ) sequences are left in the AAV vector. For viral (e.g., AAV) genomic nucleic acid, the whole or part of the viral genome is foreign. Since the sequence of the recombinant viral vector (e.g., AAV) is replaced by the sequence of the Therefore, the genome of the vector (e.g., AAV) is distinct from the genome of the heterologous polynucleotide sequence. When a viral vector (e.g., AAV) incorporates a sequence derived from a human genome, the vector is considered to be “recombinant.” In the case of AAV, the "recombinant" vector is called a "rAAV vector." It is sometimes called "Kuta".

[0045] Thus, recombinant vector (e.g., AAV) sequences can be used to infect cells ex vivo, in vivo, Viruses (herein “particles”) are used to infect (transform) in vitro or in vivo. The recombinant vector sequence can be packaged into a recombinant vector vector (also called a "virion" or "cell"). When encapsidated or packaged within an AAV particle, the particle is referred to as a "rAAV" The virus particle or virion usually contains a vector genome in an encapsidated form. Examples include envelope proteins and AA In the case of V, this includes the capsid protein.

[0046] In certain embodiments, the recombinant vector (e.g., AAV) is a parvovirus. Parvoviruses are small viruses with a single-stranded DNA genome. Adeno-associated virus (AAV) belongs to the Parvoviridae family.

[0047] Parvoviruses, including AAV, secrete cellular components that ensure the nucleic acid and genetic material are stably maintained within the cell. Since it can enter cells and deliver nucleic acids and genetic material, it is a useful virus vector for gene therapy. Furthermore, these viruses secrete nucleic acid and genetic material at specific sites, e.g. AAV can be introduced into specific sites on chromosome 9. AAV is not involved in human pathogenic diseases, so AV vectors have been shown to be effective in transmitting AAV-associated pathogenicity or disease to patients without causing substantial AAV-associated pathogenesis or disease. Seed polynucleotide sequences (eg, therapeutic proteins and drugs) can be delivered.

[0048] AAV-Rh74 and related AAV variants such as AAV-Rh74 or AAV-Rh74 Related AAV serotypes (e.g., capsid mutants such as RHM4-1) and variants (e.g., capsid variants such as RHM4-2) For example, VP1, VP2 and / or VP3 sequences) can be used in, for example, AAV1-AAV11 or Rh 10, which may or may not be different from other AAV serotypes (e.g., AAV1-AA The VP1, VP2 and / or VP3 sequences are distinct from those of either the V11 or Rh10 serotypes. As used herein, the term "serotype" means Other AAV serotypes are used to refer to AAVs with serologically distinct capsids. Serological differentiation is the ability to detect differences between antibodies when comparing one AAV with another. Such differences in cross-reactivity are usually determined by the absence of cross-reactivity between capsids. Due to differences in protein sequence / antigenic determinants (e.g., V P1, VP2 and / or VP3 sequence differences). AAV-Rh74 variants, including capsid mutants. Although the variant may not be serologically distinct from Rh74 or other AAVs, These AAV-Rh74 mutants have at least one The nucleotide or amino acid residues differ.

[0049] Traditionally, a serotype is a group of all known serotypes that represent a virus of interest. A neutralizing activity test is performed on serotype-specific serum to determine whether antibodies that neutralize the target virus are present. This means that more isolates of viruses that exist in nature are being discovered and As mutant strains of C. serovars are produced, differences between existing serovars are not necessarily present. Therefore, it is possible that serological differences may be observed in new viruses (e.g., AAV). If this is not the case, the new virus (e.g., AAV) may be a subgroup or variant of the corresponding serotype. In many cases, mutant viruses with altered capsid sequences are classified as serotypes. Neutralizing activity to determine whether they belong to other serotypes based on the conventional definition of No serological tests have been performed for these strains. Therefore, for convenience and to avoid duplication, the term "serotype" is used. The term is used to refer to serologically distinct viruses (e.g., AAV) and viruses that are serologically indistinguishable. It refers broadly to both viruses that belong to a subgroup or variant of a given serotype (e.g., AAV).

[0050] Recombinant vector (e.g., AAV) plasmids, as well as methods and uses thereof, are described in detail below. Non-limiting examples include recombinant vectors (e.g., A AV) is, for example, AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, - Any AAV, such as AAV-10, -11, -rh74, -rh10, and AAV-2i8. The vectors may also be based on genomes. As a non-limiting example, one serotype of Recombinant vectors (e.g., AAV) that rely on genomes are plasmids that package the vectors. In addition, the capsid protein may be identical to one or more capsid proteins that are expressed by recombinant vectors (e.g. The plasmid can be based on an AAV (e.g., AAV2) serotype genome. The genome is distinct from one or more capsid proteins that package the vector. In this case, at least one of the three capsid proteins is, for example, AAV-Rh. 74 or related AAV variants such as AAV-Rh74 or AAV-Rh74 variants (e.g. , RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, and related AAVs such as capsid variants such as RHM15-4 and RHM15-6. Good too.

[0051] AAV-Rh74 shares the same gene / protein sequence as the sequence signature of AAV-Rh74. (See, for example, VP1, VP2, and VP3 in Figures 3A to 3E). The term "AAV vector related to AAV-Rh74" and its synonyms are used herein as used herein. AAV-Rh74 comprising one or more polynucleotide or polypeptide sequences and One or more AAV proteins (e.g., VP1, VP2, and Therefore, such AAV-Rh74-related AAV vectors The target vector may have one or more sequences that differ from AAV-Rh74, but is not a variant of AAV - exhibits a sequence substantially identical to one or more genes of Rh74, and / or have one or more functional characteristics of V-Rh74 (e.g., cell / tissue tropism). For example, the AAV-Rh74-related AAV variant RHM4-1 has a distinct capsid structure from the Rh74 capsid. Exemplary AAV-Rh74 and AAV-Rh75 capsids have a four amino acid sequence. 74 or AAV-Rh74 variants (e.g., RHM4-1, RH M15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and The sequences of related AAVs, such as capsid variants such as RHM15-6, are described herein. For example, VP1, VP2 and / or VP3 in Figures 3A to 3E. In one embodiment, the AAV-Rh74 related AAV vector is shown in Figures 3A to 3E. and one or more of the AAV-Rh74 VP1, VP2 and / or VP3 sequences. At least 80% or more than 80% (e.g., 85%, 90%, 95%, 96% , 97%, 98%, 99%, 99.5%, etc.) containing or consisting of the same sequence The nucleic acid sequence may comprise a polynucleotide, polypeptide or subsequence thereof.

[0052] According to the present invention, the method and use comprises the steps of: The partial sequence includes a standard AAV-Rh74 gene or a protein sequence thereof. Protein sequences (e.g., the VP1, VP2 and / or VP3 sequences shown in Figures 3A to 3E) Although the match rate is less than 100%, the AAV1-AAV11 and AAV-Rh10 genes or proteins, which are distinct and not identical to known AAV genes or proteins. In one embodiment, the AAV-Rh74 polypeptide or a subsequence thereof is selected from any standard AA V-Rh74 sequence or a subsequence thereof (e.g., VP1, VP2 and / or VP3 sequences) or more than 80% identical to up to 1 00% (e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to or consisting of such sequences. In some embodiments, the AAV-Rh74-related variant is AAV-Rh74 (e.g., RHM4 -1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15 -4, and capsid mutants such as RHM15-6) Have one, two, three or four of them.

[0053] AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 , AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8, and and recombinant vectors containing related hybrid and chimeric sequences of the mutants (e.g., V) one or more functional AAV ITR sequences are obtained by recombinant techniques known to those skilled in the art. Constructed to contain one or more adjacent heterologous polynucleotide sequences (transgenes) Such vectors can be used to express, for example, the rep gene and / or the cap gene. , having one or more of the wild-type AAV genes deleted in whole or in part, However, the recovery, replication and packaging of the recombinant vector into an AAV vector particle are not possible. At least one functional flanking ITR sequence (functional fl) required for binding Therefore, the AAV vector genome The ribosomal RNAi sequence is required in cis for replication and packaging (e.g., For example, ITR sequences.

[0054] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably. The term "deoxyribonucleic acid" is used interchangeably with "nucleic acid" All forms of nucleic acids, including DNA and ribonucleic acid (RNA), oligonucleotides The polynucleotides include genomic DNA, cDNA and antisense DNA, as well as Spliced ​​or unspliced ​​mRNA, rRNA, tRNA, and Inhibitory DNA or RNA (RNAi, e.g., short hairpin or short hairpin (sh)RNA, microRNA (miRNA), small molecule interferon Interfering or short interfering (si)RNA, trans-splicing RNA, or antisense oligonucleotides. Polynucleotides include naturally occurring, synthetic and intentionally modified. includes modified polynucleotides, as well as analogs and derivatives. , may be single, double or triple, or linear or circular, and may be of any length When discussing polynucleotides, it is important to remember that a particular polynucleotide may have a sequence or structure. The structure of the nucleic acid may be described, and by convention, the sequence is described here in the 5' to 3' direction. can be.

[0055] A "heterologous" polynucleotide is intended to be introduced or delivered into a cell via a vector. Heterologous polynucleotide refers to a polynucleotide inserted into a vector (e.g., AAV). The nucleotide sequence is usually distinct from the nucleic acid of the vector (e.g., AAV). Once introduced / delivered into a cell, the heterologous polypeptide is “foreign” to the nucleic acid of the host (e.g., AAV). The nucleotides are packaged in virions and can be expressed (e.g., transcribed and processed). Alternatively, the virion (e.g., AA The heterologous polynucleotide contained within V) need not be expressed. Although the term is not always used herein to refer to polynucleotides, When referring to an ochido, even if it lacks the modifier "heterogeneous," it is still considered heterogeneous despite the omission. It is intended to include species polynucleotides.

[0056] "Polypeptides," "proteins," and A "peptide" refers to a full-length native sequence, similar to a naturally occurring protein. e sequences), and functional subsequences, modified or altered sequences These functional parts are intended to be useful as long as they retain some of the functionality of the full-length native protein. In the methods and uses of the present invention, the polynucleotides may be Such polypeptides, proteins and peptides encoded by the leucine sequences are , which is deficient or poorly or incompletely expressed in the mammal to be treated. The protein may be identical to an endogenous protein such as This is not the case.

[0057] The adeno-associated virus (AAV) serotypes AAV-Rh74 and AAV-Rh74 of the present invention Mutants (e.g., RHM4-1, RHM15-1, RHM15-2, RHM15-3 / R HM15-5, RHM15-4, and capsid variants such as RHM15-6) AAV variants can be used to stably or transiently deliver polynucleotides into cells and to progeny cells. As used herein, a "transgene" can be used to introduce / deliver a The term "heterologous polynucleotide" is used for convenience to refer to the heterologous polynucleotide introduced into such a cell or organ. A transgene is a gene that encodes a polypeptide or protein. a gene that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed into an inhibitory polynucleotide A polynucleotide that does not contain expression control elements such as a promoter that activates transcription. The term includes any polynucleotide, such as a polynucleotide having a structure other than nucleotide sequence (unless otherwise specified).

[0058] For example, in cells containing a transgene, the transgene can be delivered via a vector (e.g., AAV). They are introduced or transferred into cells through "transformation." "Transform" or "Transfect" The term "transfected vector" refers to a method for introducing a molecule, such as a polynucleotide, into a cell or host organism. This refers to the following.

[0059] A cell that has a transgene introduced into it is called a "transformed cell." The term "transformed cell" or "transformant" is used to refer to a "transformed " or "transfected" cells (e.g., in mammals, A recombinant human cell (cell, tissue or organ cell) refers to the incorporation of an exogenous molecule, e.g., a polynucleotide or is generated in a cell following the introduction of a protein (e.g., a transgene) into the cell. It means a genetic change, hence the words "transgenic" or "transformed." The cells may be, for example, cells into which an exogenous molecule has been introduced or progeny thereof. The cells may be propagated and the introduced protein expressed or the nucleic acid transcribed. For the uses and methods, the transformed cells may be present within a subject.

[0060] The introduced polynucleotide may be integrated into the nucleic acid of a recipient cell or organism. If the introduced polynucleotide does not Once incorporated into the nucleic acid (genomic DNA) of an organism, it is stably maintained within the cell or organism. and can be inherited or passed on to progeny (descendants) of the recipient cell or organism. Finally, the introduced nucleic acid may only exist transiently inside a recipient cell or host organism. There is a gender.

[0061] The cells to be transformed may be of any origin (e.g. mesodermal, ectodermal or endodermal) and of any Non-limiting examples of cells include cells of tissue or organ types such as liver. (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., islet β cells), lung, brain (e.g., nerve, retinal or ependymal cells) or the central or peripheral nervous system, such as the spinal cord, kidney, eye (e.g. retinal cells), alveolar components), spleen, skin, thymus, testes, lungs, diaphragm, heart (heart cells), muscle or psoas muscle, or intestine (e.g. endocrine cells), adipose tissue (white, brown or beige adipocytes), muscle (e.g. fibroblasts), synovial cells, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells cells, nerve cells of the inner ear, or hematopoietic cells (e.g., blood cells or lymphocytes). Further examples include liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., pancreatic islet β cells), and central or peripheral, such as the alveoli, lungs, brain (e.g., neural, glial or ependymal cells) or spinal cord Nervous system, kidney, eye (e.g., retinal cellular components), spleen, skin, thymus, testes, lungs, diaphragm, heart viscera (cardiac cells), muscle or psoas muscle, or intestine (e.g. endocrine cells), adipose tissue (white, brown colored or beige adipocytes), muscle (e.g., fibroblasts), synovial cells, chondrocytes, osteoclasts cells, epithelial cells, endothelial cells, salivary gland cells, neural cells of the inner ear, or hematopoietic cells (e.g. blood Stem cells, such as pluripotent or multipotent progenitor cells, that develop or differentiate into cells (cells or lymphocytes) Examples include:

[0062] In certain embodiments, a "therapeutic molecule" refers to a molecule that inhibits the absence or proliferation of a protein in a cell or subject. are peptides or proteins that may alleviate or reduce symptoms resulting from dysfunction. or a "therapeutic" peptide or protein encoded by the transgene. The gene is one that provides a benefit to the subject, for example by repairing a genetic defect or altering a gene (expression or These compounds repair deficiencies in certain tissues (such as the brain, kidney, and liver) and confer anti-cancer effects.

[0063] Heterologous polynucleotides encoding gene products (e.g., therapeutic proteins) useful in the present invention Non-limiting examples of leutidine include cystic fibril transmembrane regulators (CFR 601.111 and 601.122. rosis transmembrane regulator protein, CF TR), including acquisition of functional blood clotting factors (e.g., factor XIII, factor IX, factor, factor X, factor VIII, factor VIIa or protein C), antibodies, retinal pigment Retinal pigment epithelial cell-specific 65 kDa protein (retinal pigment epithelial cell-specific 65 kDa protein thelium-specific 65 kDa protein, RPE65 Rhthropoietin (erythropoietin), LDL receptor, lipoprotein lipase, ol Nitin transcarbamylase, β-globin, α-globin, spectrin, α-antithrombin Lipsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7 ), sulfamidase, enzymes involved in lysosomal storage diseases (A RSA), hypoxanthine-guanine phosphoribosyltransferase, β-25 glucuronide Glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, Branched-chain ketoacid dehydrogenase, hormones, and growth factors (e.g., insulin-like growth factors 1 and 2) , platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factors 3 and 4, brain-derived Neurotrophic factors, glial cell-derived growth factors, transforming growth factors α and β, etc.), cytokines ( For example, alpha interferon, beta interferon, interferon gamma, interleukin-1 (IL-1), Interleukin 2, Interleukin 4, Interleukin 12, Granulocyte-macrophage colony-stimulating factors, lymphotoxins, etc.), suicide gene products (e.g., herpes simplex virus thymidine kinase Enzymes, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytosine kinases, tumor necrosis factors, etc.), drug resistance proteins (e.g., resistance-conferring proteins), tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL) , adenomatous polyposis coli (APC), peptides with immunomodulatory functions, tolerogenic or are immunogenic peptide or protein Tregitopes (regulatory T cell epitopes) ) [de Groot et al., Blood 2008 Oct 15;112(8):33 03], or hCDR1 [Sharabi et al., Proc Natl Acad Sc i US A. 2006 Jun 6;103(23):8810-5], Ins. Glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab S Coat protein 1 (congenital choroideremia), LCA5 (LCA-reversillin), Ornithionib Tinketoacid aminotransferase (gyral retinal and choroidal atrophy), retinoschisin (Retin oschisin)1 (X-linked retinoschisis), USH1C (Usher syndrome) 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR type 1 retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM2, 3 and 4 ( Color vision disorder), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, lysophotic kidney disease Genetic defects causing serosomal storage diseases (e.g., sulfatase, N-acetylglucosamine Amino acid-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC 2. Sphingolipid activating protein, etc.), one used in genome editing or Multiple zinc finger nucleases or genome editing to repair the template Genes that contain or encode donor sequences used as a template, but are not limited to It is not something that is done.

[0064] Heterologous polynucleotides encoding gene products (e.g., therapeutic proteins) useful in the present invention. Further non-limiting examples of nucleotides include those used to treat cystic fibrosis (or other diseases of the lungs), blood Hemophilia A, Hemophilia B, Thalassemia, Anemia and other blood disorders, AIDS, Alzheimer's Disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders Cancer, diabetes mellitus, muscular dystrophies (e.g. Duchenne ), Becker type, Gaucher's disease sease, Hurler's disease, adenosine deficiency of glycogen deaminase, glycogen storage disease and other metabolic disorders, retinal degenerative diseases (eye For the treatment of diseases and disorders, including diseases of the brain, liver, kidneys, and heart, as well as diseases of the liver and other solid organs. These include, but are not limited to, those that may be used.

[0065] All mammalian and non-mammalian organisms, including the non-limiting genes and proteins disclosed herein, Polynucleotides encoding gene products, whether known or unknown, in the form of mammalian cells Thus, the present invention encompasses non-mammals, non-human mammals, and humans. The genes and proteins include those from the human Non-Limiting Examples of Non-Mammalian Genes As an example, the Fok nuclease domain (Fok nuclease domain) derived from bacteria A non-limiting example of a FIX sequence from a mammal other than a human is the Yos Hitake et al., 1985, supra; Kurachi et al., 19 95, supra; Jallat et al., 1990, supra; Kurachi et al. al., 1982, Proc. Natl. Acad. Sci. USA 79: 6461-6464;Jaye et al., 1983, Nucl.Acids R es. 11:2325-2335;Anson et al., 1984, EMBO J.3:1053-1060;Wu et al., 1990, Gene 86:2 75-278; Evans et al., Proc Natl Acad Sci U SA 86:10095(1989); Blood 74:207-212; Pend urthi 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.

[0066] As described herein, a heterologous polynucleotide sequence (transgene) can be a suppressor or antagonist. These include inhibitory, antisense, siRNA (small interfering RNA), miRNA (microRNA), shRNA (short hairpin RNA), RNAi and and antisense oligonucleotides can regulate the expression of target genes. The present invention relates to a method for treating a disease by inhibiting the expression of a target gene involved in mediating a disease process, thereby preventing one or more symptoms of the disease. These include molecules that reduce, inhibit or ameliorate the condition.

[0067] Antisense is a single stranded molecule that binds RNA transcripts or DNA (e.g., genomic DNA). , double-stranded or triple-stranded polynucleotides and peptide nucleic acids (PNAs). An oligonucleotide derived from the transcription start site, e.g., between −10 and +10 from the start site Nucleotides are another example. Antisense can bind to double-stranded DNA and inhibit gene transcription. "RNAi" refers to the use of single- or double-stranded RNA sequences to inhibit the expression of genes. (See, e.g., Kennerdell et al., Cell 95:1017(1 998; and Fire et al., Nature, 391:806 (1998 )). Thus, in the methods and uses of the present invention, The double stranded RNA sequences may be used to inhibit or prevent gene expression / transcription. RNAi and RNAi are directed against target gene sequences (e.g., huntingtin or HTT) The nucleic acids may be based on nucleic acids encoding mammalian and human HTT. For example, a single-stranded or double-stranded nucleic acid (e.g., RNA) can be used to target an HTT transcript (e.g., mRNA). This can be a target.

[0068] "siRNA" stands for sequence-specific post-transcriptional gene silencing or gene knockdown siRNA refers to a therapeutic molecule involved in the RNA interference process for the treatment of cancer. (cognate) mRNA sequence that has homology with the mRNA. Small interfering RNA (siRNAs) ) can be synthesized in vitro or by synthesis of longer dsRNAs. It can be generated by cleavage with ribozyme III and is a mediator of sequence-specific mRNA degradation. The siRNA or other such nucleic acids of the present invention may be prepared by the synthesis of appropriately protected ribonucleosides. It can be chemically synthesized using phosphoramidites and a conventional DNA / RNA synthesizer. siRNA can be expressed as two separate, complementary RNA molecules or as a single RNA molecule having two complementary regions. Synthetic RNA molecules or synthesis reagents can be purchased from a number of vendors. , Applied Biosystems (Foster City, CA, USA) ), Proligo (Hamburg, Germany), Dharmacon Re search(Lafayette, Colo., USA), Pierce Che mical(part of Perbio Science, Rockford, Ill., USA), Glen Research(Sterling, Va., (USA), ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK) is a drug that inhibits the mRNA of target genes. The specific siRNA construct for this purpose may be 15 to 50 nucleotides in length, Typically, the length may be about 20 to 30 nucleotides. Such nucleic acid molecules can be prepared by those skilled in the art. The vectors can be readily incorporated into the viral vectors disclosed herein by conventional methods known to those skilled in the art. This can be done.

[0069] Genes (e.g., genomic DNA) that may be targeted using the inhibitory nucleic acid sequences of the present invention. A) or a transcript of a virulence gene (RNA or mRNA) includes, but is not limited to, These include, but are not limited to, the following: Genes associated with nucleotide repeat disease, dentatorubral-pallidoluysian atrophy (denta torubropallidolusyan atropy (e.g., atrophin ( Atrophin 1 (ATN1) androgen on the X chromosome in spinal and bulbar muscular atrophy Ataxin receptors, human ataxin-1, -2, -3 and -7, (CACNA 1A) v 2.1P / Q voltage-gated calcium channel, TATA Binding protein, ataxin 8 inverse chain, also known as ATXN8OS, in spinocerebellar ataxia (1, Serine / threonine protein phosphatases (types 2, 3, 6, 7, 8, 12, and 17) α-aspartase 2A 55 kDa regulatory subunit B beta isoform, fragile X syndrome FMR1 (Fragile X Mental Retardation 1), Fragile X-associated tremor / motor syndrome FMR1 (Fragile X Mental Retardation 1) in ataxia, FMR1 (Fragile XE Mental Retardation) in Weak X mental retardation 2) or AF4 / FMR2 family members; myotonic dystrophy Myotonin-protein kinase Frataxin in Friedreich's ataxia, muscle Mutations in the superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis ;Genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease;Apolipoproteins Proprotein convertase B (APOB) and proprotein convertase subtilisin (proprotein c Onvertase subtilisin / kexin type 9 (PCSK) 9) Hypercholesterolemia; HIV infection Human immunodeficiency virus transactivator of HIV Tat, a transcription factor-dependent transactivator); HIV TAR in HIV infection, human immunodeficiency Viral transactivator response element gene; CC gene in HIV infection Chemokine receptor 5 (CCR5); Rous sarcoma virus (RSV) infection RSV nucleocapsid protein in infection and liver in hepatitis C virus infection Intrinsic microRNA (miR-122); p53 in acute kidney injury or kidney transplant postoperative organ function acute renal failure due to impaired function or renal function; Protein kinase N3 (PKN3); LMP2, which is involved in the proteasome easome) subunit beta type 9 (PSMB9) also known as metastatic melanoma; LMP7, which is a proteasome subunit beta type 8 (PSMB8) in metastatic melanoma Also known as MECL1, which is a proteasome subunit beta type 10 (P SMB 10) Also known as metastatic melanoma; Vascular endothelial growth factor (VGF) in solid tumors EGF; kinesin spindle transcription in solid tumors Proteins inhibit apoptosis in chronic myeloid leukemia / B-cell lymphoma (CLL) L-2; Ribonucleotide Reductase M2 in Solid Tumors tide reductase M2 (RRM2); Furin in solid tumors in; polo-like kinase 1 (PL) in liver tumors K1), diacylglycerol acyltransferase (diaK1) in hepatitis C infection DGAT1 (cycloglycerol acyltransferase) 1 (familial) Beta-catenin in adenomatous polyposis coli; beta-2 adrenergic receptor; glaucoma; diabetic jaundice DA known as RTP801 / Redd1 in DME and age-related macular degeneration N-damage-induced transcript 4 protein; a potential risk factor for age-related macular degeneration or choroidal neovascularization Vascular endothelial growth factor receptor I in idal neivascularization (VEGFR1);Caspase 2 in non-arteritic ischemic optic neuropathy;In congenital pachyonychia Keratin 6AN17K mutant protein in influenza infection; Influenza A virus genome / gene sequence; a role in severe acute respiratory syndrome (SARS) infection Respiratory syncytial virus (RS) genome / gene sequence; Virus genome / gene sequence; Ebola virus genome / gene in Ebola infection Sequences: Hepatitis B and C virus genome / gene sequences in hepatitis B and hepatitis C infections ;Herpes simplex virus (HSV) genotype in HSV infection Virus / gene sequence, coxsackievirus B3 infection Coxsackievirus B3 genome / gene sequence in primary dystonia: a possible role for toxins in primary dystonia (torsin)A (TOR1A), pan-class I and transplant-specific HL Silencing of pathogenic alleles of genes such as the A allele or mutations in the gene autosomal dominant retinitis pigmentosa (adRP) Heterorhodopsin gene (RHO).

[0070] Polynucleotides, polypeptides and subsequences thereof include modified and mutant forms. As used herein, "modified" or "variant" The term "polynucleotide" and its synonyms refer to a polynucleotide, polypeptide or portion thereof. A modified sequence means a sequence that deviates from the standard sequence. The variant sequence may have approximately the same, more or less activity as the reference sequence. or function, but at least partially has the activity or function of the reference sequence.

[0071] The present invention also includes naturally occurring or non-naturally occurring variants. Such variants include A Such AAVs include AAV-Rh74 mutants, such as AAV-Rh74 capsid mutants. Specific examples of -Rh74 capsid mutants include RHM15-1, RHM15-2, RHM 15-3, RHM15-4, RHM15-5, RHM15-6, and RHM4-1 (e.g. For example, see Figure 5.

[0072] Such variants include gain-of-function and loss-of-function variants. For example, The wild-type human FIX DNA sequence, in which the protein variant or mutant retains activity, is In the methods and uses of the invention, therapeutically active or invariant human FIX and In a specific example, collagen IV has the same or greater therapeutic activity. When taken up into mammalian muscle tissue, a portion of FIX is transported to the muscle. Because it is retained in the interstitial space of muscle tissue, it is unable to participate in blood clotting. Mutations in the FIX sequence that result in proteins with reduced binding (e.g., loss of function) Such mutations are useful in the methods of the invention, for example, in the treatment of hemophilia. An example of a variant human FIX gene is a human FIX protein that is expressed at 5′-nucleotide positions from the start of the mature protein. The lysine at the th amino acid position codes for the amino acid alanine.

[0073] Non-limiting examples of modifications include substitutions of one or more nucleotides or amino acids in a reference sequence. Conversion (e.g. 1~3, 3~5, 5~10, 10~15, 15~20, 20~25, 25~3 0, 30-40, 40-50, 50-100, or more nucleotides or residues substitution of lysine residues with arginine (e.g., RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15- 6) substitution of one or more lysines with arginines; addition (e.g., insertion or 1~3, 3~5, 5~10, 10~15, 15~20, 20~25, 25~30, 30 ~40, 40-50, 50-100, or more nucleotides or residues), and and deletions (e.g., subsequences or fragments). In specific embodiments, modified sequences Alternatively, the variant sequence may retain at least part of the function or activity of the unaltered sequence. Such modifications and variants may, for example, alter the function or activity of the reference sequence as described below. It may have less than, the same or greater function or activity than the standard sequence, but at least the function of the standard sequence. or partially retains activity.

[0074] A variant may have one or more non-conservative or conservative amino acid sequence differences or modifications, or Conservative substitutions can be used to refer to a sequence that is more specific than a given sequence. "Titution" means the substitution of a single molecule by a biologically, chemically or structurally similar residue. The term "biologically similar" refers to the substitution of two amino acids to obtain a biologically similar "Structurally similar" means that the amino acids are of similar length and structure. It means having a chain of amino acids, e.g., alanine, glycine and serine or similar size. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Specific examples include isoleucine, valine, leucine, and the like. replacement of lysine with arginine, or Substitutions (e.g., RHM15-1, RHM15-2, RHM15-3 / RHM15-5, R HM15-4, and RHM15-6), replacement of aspartic acid with glutamic acid, Substitution of a polar residue such as a glutamine for a paragine or a threonine for a threonine. Concrete examples of conservative substitutions include substitution with isoleucine. or by a hydrophobic residue such as lysine, valine, leucine, or methionine. arginine, aspartic acid, glutamic acid, asparagine, glutamic acid, For example, conservative amino acid substitutions such as substitution of a polar residue with glutamine are included. Acid substitutions typically include substitutions within the following groups: glycine, aryl, arylsulfate, arylsulfates ... lanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine , glutamine; serine, threonine; lysine, arginine; and phenylalanine, Tyrosine. A "conservative substitution" also refers to the substitution of a substituted amino acid in place of an unsubstituted parent amino acid. This includes using

[0075] Thus, the present invention relates to a method for the treatment of a disease comprising administering to a subject a therapeutic agent capable of inhibiting one or more biological activities (e.g., a function in blood clotting). Gene and protein variants (e.g., polynucleotides as described below) that carry Such proteins or polypeptides include variants of the polypeptide encoding the polypeptide. A peptide variant is a protein or polypeptide that has altered or added properties. A protein or protein that has been or may be modified using recombinant DNA technology to polypeptide, e.g., enhanced protein stability in plasma or enhanced The variants include variants that confer a desired protein activity. The sequence may be different from a standard sequence, such as a protein or peptide.

[0076] At the nucleotide sequence level, naturally occurring and non-naturally occurring variant genes are typically A sequence having at least about 50%, more typically about 70%, and even more typically about 80% identity ( At the amino acid sequence level, natural and non-natural variants The body protein is typically at least about 70%, more typically at least about 70%, different from the standard protein. About 80%, more typically about 90% or more identical, but in non-conserved regions regions that are largely discrepant (e.g., 60%, 50%, or 40%) are permitted. In another embodiment, the sequence is at least 100% identical to the reference sequence. A match rate of 60%, 70%, 75% or more (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater concordance. Induction of nucleotide and amino acid changes in a nucleic acid, protein or polypeptide. The process of introducing the saccharide is well known to those skilled in the art (see, e.g., Sambrook et al., Molecular Biology, 1999, 144:131-132). cular Cloning: A Laboratory Manual (2007) reference).

[0077] "Identity", "homology" and their synonyms In the case of an "aligned" sequence, two or more subject entities This means that the referenced entities are the same. For example, Therefore, when two polypeptides are said to be identical, at least one region of the standard or protein is included. Two polynucleotides are said to be identical if they have the same amino acid sequence. , have the same polynucleotide sequence, at least within the standard region or protein. The identity may exist over a defined area (region or domain) of the sequence. A "region" or "area" refers to two or more of the same Thus, two protein or nucleic acid sequences are identical over one or more sequence areas or regions, the sequences are "Aligned" sequences are often , including corrections for missing or extra bases or amino acids (gaps) compared to the reference sequence. It refers to a sequence of multiple polynucleotides or proteins (amino acids).

[0078] There may be matches over the entire length of the sequences or only portions of the sequences. In certain embodiments, the length of the sequences sharing the percent identity is 2, 3, 4, 5 or more. More than 6, 7, 8, 9, 10 or more consecutive polynucleotides or amino acids. , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. consecutive amino acids In another particular embodiment, the length of the matching sequence is 20 or more than 20 consecutive Polynucleotides or amino acids, for example, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, etc. In yet another particular embodiment, the length of the matching sequence is 35 or more. Consecutive polynucleotides or amino acids, e.g., 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 45, 47, 48, 49, 50, etc. In yet another particular embodiment, the length of the matching sequence is 50 or more amino acids. More than 50-55, 55-60 consecutive polynucleotides or amino acids. , 60-65, 65-70, 75-80, 80-85, 85-90, 90-95, 95- 100, 100-110, etc., consecutive polynucleotides or amino acids.

[0079] The terms "homologous" and "homology" The term refers to a situation where two or more than two target entities are present at least once in a given area or part. "Area, region or sequence" refers to a sequence that is identical to or corresponds to a sequence of a A "domain" is a term used to describe a set of entities that are homologous or identical in parts. Therefore, two sequences are identical over one or more sequence regions. If there is a match, then the sequences share identity in that region. "Substantial homology" means a reference molecule or a target molecule having homology The structure or function (e.g. biological function) of the related / corresponding region or portion of the quasi-molecule A molecule has or has at least a partial structure or function of one or more of the following: means that the molecule is structurally or functionally conserved, as predicted by the .

[0080] The extent of identity between two sequences can be determined using computer programs and numerical algorithms. Such percent sequence identity (homology) can be ascertained using the The algorithm for calculating the sequence gaps and mismatches in the comparison region or area is typically For example, the BLAST (e.g., BLAST 2.0) search algorithm ( See, e.g., Altschul et al., J. Mol. Biol. 215:403 (199 0), publicly available from NCBI, suggests the following typical search parameters: Has: mismatch-2, gap open-5, gap extension For polypeptide sequence comparison, the BLASTP algorithm is commonly used. Rhythm can be set to PAM100, PAM 250, BLOSUM 62 or BLOSUM Use in combination with a scoring matrix such as 50 To quantify the degree of match, FASTA (e.g., FASTA2 and FASTA3) and the SSEARCH sequence comparison program have also been used (Pearson et al. ., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195 (1981) ) Protein structures are modeled using Delaunay-based topological mapping. Programs to quantify similarity have also been developed (Bostick et al., 2013). ochem Biophys Res Commun.304:320(2003)) .

[0081] Polynucleotides include additions and insertions, e.g., heterologous domains. For example, a heterologous domain) can be used to bind covalently or non-covalently to any type of molecular compound. Typically, additions and insertions (e.g., heterologous domains) are complementary or heterologous. The function or activity is imparted to the

[0082] Additions and insertions include one or more amino acid sequences that are not normally present in the reference native (wild-type) sequence. Chimeras, in which the molecule is a polynucleotide sequence or a protein sequence covalently linked to a sequence "Fusion" or "chimeric" The term " and its synonyms, when used in reference to a molecule, refers to a portion or part of the molecule. includes a distinct (heterogeneous) entity that is distinct from the molecule in question, The moieties do not normally occur together in nature, i.e., in fusion or chimeric The part contains or is composed of parts that do not normally coexist, Structurally distinct.

[0083] The term "vector" refers to a vector that is capable of manipulating by insertion or incorporation of a polynucleotide. can be a plasmid, virus (e.g., AAV vector), cosmid or other delivery vector Such vectors are used to introduce polynucleotides into cells. A gene that transfers / transfers the inserted polynucleotide into the cell and transcribes or translates the inserted polynucleotide The vector nucleic acid sequence is Generally, the heterologous polynucleotide contains at least one origin of replication for propagation in a cell, and optionally contains a heterologous polynucleotide. nucleotide sequences, expression control elements (e.g., promoters, enhancers), selection markers (e.g., These include additional elements such as a polyadenine sequence, which confer resistance to antibiotics, and a nucleic acid sequence that confer resistance to ribozymes.

[0084] As used herein, the term refers to a viral vector, such as a recombinant AAV vector. as modifiers of sequences such as recombinant polynucleotides and polypeptides The term "recombinant" refers to a composition (e.g., an AAV or sequence) that does not normally occur in nature. This means that the AAV vector has been engineered (i.e., modified) in a manner similar to that of the AAV vector. An example of a suitable recombinant vector is a vector that is normally present in the genome of a wild-type virus (e.g., AAV). For example, a non-existent polynucleotide may be inserted into the viral genome. An example of a recombinant polynucleotide is a heterologous polynucleotide that encodes a protein. A gene that is normally associated with a gene in the viral (e.g., AAV) genome. The vector may be cloned with or without the ', 3' and / or intron regions. The term "recombinant" as used herein refers to viruses such as AAV vectors. The term "protein vector" is used to refer to sequences such as vectors, polynucleotides and polypeptides. The present invention includes, but is not limited to, AAV, as well as polynucleotides and polypeptides. Recombinant forms of the sequences are expressly included even if omitted.

[0085] The recombinant vector "genome" (e.g., AAV vector genome) is then transformed into a cell ex vivo , for infecting (transducing or transforming) in vitro or in vivo Encapsidated or packaged into a virus (also referred to herein as a "particle" or "virion"). The recombinant AAV vector genome can be encapsidated or packaged into an AAV particle. When caged, the particle may be referred to as a "rAAV." The virion or vector normally contains proteins that encapsidate or package the vector genome. Examples include viral capsid and envelope proteins, and in the case of AAV Examples include AAV capsid proteins.

[0086] For recombinant plasmids, the vector "genome" is the final product of packaging or encapsidation. It refers to a portion of a recombinant plasmid sequence that is cloned to form a viral particle. When a recombinant vector is constructed or produced using a vector, the vector genome is The recombinant plasmid does not contain any portion of the "plasmid" that does not correspond to the vector genome sequence of the plasmid. The non-vector genomic portion of the plasmid is called the "plasmid backbone" and is responsible for propagation and recombination. It is important in the process of plasmid cloning and amplification required for virus production. However, it is not packaged or encapsidated within the viral (e.g., AAV) particle. Not done.

[0087] Thus, a vector "genome" is a vector that is packaged or expressed by a virus (e.g., AAV). It refers to the portion of a vector plasmid that is encapsidated and contains a heterologous polynucleotide sequence. The non-vector genomic portion of the recombinant plasmid is important for cloning and amplifying the plasmid. It contains a backbone, but is itself packaged or cloned by a virus (e.g., AAV). Not psidized.

[0088] Viral vectors are derived from one or more nucleic acid elements that make up the viral genome or Specific viral vectors include the adeno-associated virus (AAV) and the adenovirus-associated virus (AAV). These include parvovirus vectors such as AAV vectors.

[0089] Recombinant vector sequences are engineered by the insertion or integration of polynucleotides. A plasmid usually contains at least one origin of replication for propagation in a cell, and , which contains one or more expression control elements.

[0090] Vector sequences, including AAV vectors, can include one or more "expression control elements." Typically, expression control elements affect expression of an operably linked polynucleotide. The promoter and the nucleic acid sequence present in the vector are as described below. Regulatory elements, including expression control elements such as enhancers, are required to regulate proper transcription of the heterologous polynucleotide. and where possible, components that facilitate translation (e.g., promoters, enhancers, Intron splice signals, the correct splicing of genes that allows in-frame translation of mRNA (Maintaining the reading frame, stop codons, etc.) Such elements usually act in cis mode, but may also act in trans mode. This is also fine.

[0091] Expression regulation is effective at the levels of transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are located in the transcribed polynucleotide. Expression control elements are also located near the 5' end (i.e., "upstream") of the transcribed sequence. at the 3' end of the inserted sequence (i.e., "downstream") or within the transcript (e.g., within an intron). Expression control elements can be located at a fixed or long distance from the transcribed sequence. Separation, (e.g., from polynucleotides, 100 to 500, 500 to 1000, 2000 5,000 to 10,000 or more nucleotides apart) However, due to the length restriction of the AAV vector polynucleotide, Such expression control elements typically comprise from 1 to 1000 polynucleotides. The sequence is located within a range of nucleotides.

[0092] Functionally, expression of an operably linked heterologous polynucleotide is controlled by a regulatory factor (e.g., The gene can be controlled, at least in part, by a regulatory factor (e.g., a promoter) and thus They regulate the transcription of polynucleotides and, under appropriate conditions, the translation of the transcripts. A specific example of a control element is a promoter that is typically located 5' to the transcribed sequence. Another example of an expression control element is an enhancer, which regulates the transcription of a gene. The nucleic acid may be located 5', 3' to the sequence to be transcribed or within the sequence to be transcribed.

[0093] As used herein, the term "promoter" refers to a polynucleotide that encodes a recombinant product. A promoter may refer to a DNA sequence located adjacent to a nucleotide sequence. A promoter is a sequence that is operably linked to an adjacent sequence, such as a heterologous polynucleotide. Typically, the amount of expression from a heterologous polynucleotide is reduced by the amount expressed in the absence of a promoter. The amount of the gene expressed in the present invention is increased relative to the amount expressed in the

[0094] As used herein, the term "enhancer" refers to an enhancer that is located adjacent to a heterologous polynucleotide. Enhancers are usually located upstream of a promoter, but It can also be located downstream of or within a DNA sequence (e.g., a heterologous polynucleotide). Thus, an enhancer can be a 100 base pair, 200 base pair, or 100 bp sequence of a heterologous polynucleotide. Enhancers can be located 1 base pair, 300 base pairs, or further upstream or downstream. The expression of the heterologous polynucleotide is increased beyond the expression level increased by the promoter sequence. Add.

[0095] Expression control elements (e.g., promoters) are factors that are active in particular types of tissues or cells. and are referred to herein as "tissue-specific expression control elements / promoters." Tissue-specific Expression Regulatory elements usually target specific cells or tissues (e.g., liver, brain, central nervous system, spinal cord, eye, retina, etc. The expression control elements are active in certain by transcriptional activator proteins or other transcriptional regulators specific to a particular cell type, tissue, or organ. and thus typically has activity in such cells, tissues or organs.

[0096] For example, if expression in skeletal muscle is desired, a promoter active in muscle is used. Such promoters include skeletal α-actin, myosin light chain 2A promoters from genes encoding dystrophin, muscle creatine kinase, and , including synthetic muscle promoters that have higher activity than naturally occurring promoters. (e.g. Li, et al., Nat. Biotech. 17:241-245 (1999). As an example of a tissue-specific promoter, albumin promoter for liver is (Miyatake et al., J. Virol., 71:5124-32 (1997) ), Hepatitis B virus core promoter (Sandig et al., Gene Ther. 3: 1002-9 (1996)), alpha-fetoprotein (AFP) (Arbuthn ot et al., Hum. Gene. Ther., 7:1503-14 (1996)); For bone, osteocalcin (Stein et al., Mol. Biol. Rep., 2003). 4:185-96 (1997)), bone sialoprotein (Chen et al., J. Bone Miner. Res. 11:654-64 (1996)), for lymphocytes CD2 (Hansal et al., J. Immunol., 161:1063-8 (19 98)), immunoglobulin heavy chain, T cell receptor α chain, and nerve cell-specific enolate The NSE promoter (Andersen et al., Cell. Mol. Neur. obiol., 13:503-15 (1993)), neurofilament light chain genetic Child (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991), and the neuronal-specific vgf gene (Piccioli, et al., Neuron, 15:373-84 (1995)). can be.

[0097] In addition, the expression control element includes a ubiquitous or promiscuous promoter / enhancer, These can drive expression in many different cell types. The child was identified as having the cytomegalovirus (CMV) immediate early promoter / enhancer sequence, Routine Sarcoma virus (RSV) promoter / enhancer sequences and various types of mammalian Other viral promoter / enhancer sequences active in cells or those not found in nature Synthetic factors (e.g., Boshart et al., Cell, 41:521-530 (1985) ), SV40 promoter, dihydrofolate reductase promoter, cytoplasmic β-activator The promoters include the phosphoglycerol kinase (PGK) promoter, , but is not limited to these.

[0098] Expression control elements also can be regulatable, i.e., linked in a manner that allows them to be actuated by a signal or stimulus. and expressing the heterologous polynucleotide in a manner that increases or decreases expression of the heterologous polynucleotide. It is also possible to increase expression of an operably linked polynucleotide in response to a signal or stimulus. The adjustable element that is added is called an “inducible element” ( (i.e., signal-guided). Specific examples include, but are not limited to: However, promoters that are inducible by a signal or stimulus include hormone (e.g., steroid) inducible promoters. A regulatable element that, in response, decreases expression of an operably linked polynucleotide, Also called a "repressible element" (i.e., A signal reduces expression in such a way that expression is increased when the signal is removed or absent. Typically, the amount of increase or decrease due to such a factor is proportional to the amount of signal present. Or, the amount of expression is proportional to the amount of stimulation. As the amount of signal or stimulation increases, the amount of increase or decrease in expression also increases. A specific, non-limiting example is the zinc-inducible sheep metallothionein (MT) Promoter, steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter tator, T7 polymerase promoter system (WO 98 / 10088), tetracycline Culin inhibitory system (Gossen et al., Proc. Natl. Acad. Sc USA, 89:5547-5551 (1992), tetracycline-inducible System (Gossen et al., Science. 268:1766-1769 (199 5); Harvey et al., Curr. Opin. Chem. Biol. 2:51 2-518 (1998)), the RU486-inducible system (Wang et al., N at. Biotech. 15:239-243 (1997) and Wang et al. Gene Ther. 4:432-441 (1997)), and rapamycin induction Sexual system (Magari et al., J. Clin. Invest. 100:2865- 2872 (1997); Rivera et al., Nat. Medicine. 2:10 28-1032 (1996)). Certain other controllable factors are regulated by specific physiological conditions, such as temperature or acute phase. It is something that is under control.

[0099] Expression control elements also include the native factors for the heterologous polynucleotide. Elements (e.g., promoters) may be used to ensure that expression of the heterologous polynucleotide mimics the natural expression. Natural regulatory elements can be used when it is desired to regulate the expression of a heterologous polynucleotide. Regulating expression temporally or developmentally, tissue-specifically, or in response to specific transcriptional stimuli Introns, polyadenylation sites or Kozak Other naturally occurring regulatory elements, such as consensus sequences, may also be used.

[0100] As used herein, "operable linkage" means or "operably linked" means The physical or functional nature of the components described to enable them to function in the manner intended Operable linkage to a polynucleotide In the example of expression regulation in Specifically, for example, two operably linked DNA sequences are those in which the two DNAs are At least one of the two DNA sequences is capable of exerting a physiological effect on the other sequence. This means that the two are arranged in a relationship (cis or trans) such that

[0101] Vectors, including AAV vectors, can include one or more "expression control elements." These elements include, but are not limited to, AAV ITR sequences, promoter / Enhancer elements, transcription termination signals, 5' or 3' untranslated regions adjacent to the polynucleotide sequence The present invention also includes certain regions (eg, polyadenylation sequences) or all or part of intron I. Such elements also optionally include transcription termination signals. A specific example is the SV40 transcription termination signal.

[0102] As disclosed herein, AAV vectors typically contain between about 4 kb and about 5.2 kb or It is possible to insert DNA of a defined size range that is slightly larger than this. Therefore, for short sequences, the AAV vector packaged in the viral particle is permissive. The length of the viral genome sequence is adjusted to be at or near the normal size of the viral genome sequence. The insert comprises a fur or filler. In various embodiments, the filler / stuffer - A nucleic acid sequence is a non-translated (non-protein-coding) segment of a nucleic acid. In certain embodiments of the AAV vector, the heterologous polynucleotide sequence is 4. The filler or stuffer polynucleotide sequence is less than 7 Kb and is a heterologous polynucleotide. The total length of the nucleic acid sequence when combined with the nucleic acid sequence (e.g., inserted into a vector) is about 3.0 to 5.5 Kb, or 4.0 to 5.0 kb, or 4.3 to 4.8 kb.

[0103] Introns also allow AAV vectors to reach a length that allows them to be packaged into viral particles. can function as a filler or stuffer polynucleotide sequence for Introns and introns that act as filler or stuffer polynucleotide sequences Intron fragments (e.g., part of intron I of FIX) can also enhance expression. For example, the inclusion of an intron can improve expression compared to the absence of the intron. (Kurachi et al., 1995, supra).

[0104] The use of introns is not limited to the inclusion of intron I sequences of FIX, It may also contain other introns, and such introns may be expressed in the same gene (e.g., a heterologous polynucleotide). Introns that exist within the genomic sequence of FIX if the intron encodes FIX derived from a different gene or a different DNA sequence. Therefore, in the present invention, a homologous (related) gene (heterologous polynucleotide) may be used. A protein whose sequence is the same as or corresponds to a protein encoded by a genomic sequence. and non-cognate (unrelated) genes (where the heterologous polynucleotide sequence is (encoding a protein different from the protein encoded by the sequence) Other non-translated (non-protein-coding) sequences within the nucleic acid, such as introns present within the genome sequence, The i) regions also function as filler or stuffer polynucleotide sequences.

[0105] As used herein, the term "a portion of intron I" refers to a portion of intron I. FIX expression in the absence of plasmid or viral vector templates In the present study, the region that enhances FIX expression by 1.5-fold or more was approximately 0. It refers to a region of intron I having a length of 1 kb to about 1.7 kb of nucleotides. More specifically, a 1.3 kb portion of intron I.

[0106] As used herein, the term "oligonucleotide" refers to a group of two or more, usually four or more, ribonucleotides. A sequence defined as a nucleic acid molecule composed of nucleic acid or deoxyribonucleic acid, a primer The exact size of the oligonucleotide depends on various factors, the oligonucleotide Although it depends on the specific application and use of the oligonucleotide, typically, the oligonucleotide is about 5 to 1000 bp. It has a length of 50 nucleotides.

[0107] As used herein, the term "primer" refers to a single-stranded or double-stranded primer that is capable of binding to a biological system. They can be derived from a genome, generated by restriction enzyme digestion, or made synthetically, and have the appropriate cyclic nucleotides. Can function as an initiator of template-dependent nucleic acid synthesis under certain conditions Refers to a DNA oligonucleotide. A suitable nucleic acid template, suitable nucleosides of the nucleic acid Such as a triphosphate precursor, a polymerase enzyme, appropriate cofactors, and appropriate temperature and pH. Under appropriate environmental conditions, the action of a polymerase or similar activity will convert the 3′-terminated nucleotides of the primers into The primer can be extended by adding nucleotides to the ', resulting in a primer extension product. The length of the primer may vary depending on the specific environmental conditions and application requirements. For example, in diagnostic applications, the length of an oligonucleotide primer is typically 15 -30 nucleotides or more. Prime the synthesis of the desired extension product, i.e. Properly juxtaposed plates are available for initiation of synthesis by a polymerase or similar enzyme. to anneal with the desired template strand in a manner sufficient to provide a 3' hydroxyl group for the dimer. In order to be compatible with the desired template, the primer must have sufficient complementarity to the desired template. It is not necessary for the primer sequence to have perfect complementarity with the desired template. A complementary nucleotide sequence is linked to the 5' end of the primer, which is complementary to the other region. Alternatively, the primer sequence may be a match to the sequence of the desired template and the primer sequence for synthesis of the extension product. As long as there is sufficient complementarity to provide a functional template-primer complex, the non-reactive The complementary bases may be interspersed within the oligonucleotide primer sequence.

[0108] The polymerase chain reaction (PCR) is described in U.S. Pat. 195 and 4,965,188.

[0109] The term "specifically hybridize" is used as commonly in the art. The term "antibody" refers to the association of two single-stranded nucleic acid molecules having sufficient complementarity under previously defined conditions. In particular, the term is used to refer to fully complementary It refers to the hybridization of two polynucleotide sequences having complementary sequences, i.e., non-complementary sequences. Hybridization to double-stranded nucleic acid sequences is substantially excluded.

[0110] A "selectable marker gene" is a gene that, when expressed, causes transformed cells to produce an antibiotic (e.g., kanamycin). A "reporter" gene refers to a gene that confers a selectable phenotype, such as resistance to a gene that is selective for a particular gene. is a gene that provides a detectable signal. Non-limiting examples of reporter genes include , which contains the luciferase gene.

[0111] Polynucleotides and polypeptides, including modified forms, can be expressed in cells or in vitro. Produced using a variety of standard replication, translation and chemical synthesis techniques, as well as recombinant DNA technology The purity of the polynucleotide can be confirmed by sequencing, gel electrophoresis, etc. For example, nucleic acids can be determined by hybridization. n) or by using computer-based database screening techniques. Such techniques include, but are not limited to, the following: ) Genomic DNA or cDNA library that uses a probe to detect the homologous nucleotide sequence (2) hybridization of a common structure, for example, using an expression library (3) antibody screening to detect polynucleotides having a specific characteristic; For genomic DNA or cDMA using primers capable of annealing to the (4) Compilation of sequence databases of related sequences; (5) computer-assisted searching; and (6) differential screening of the subtracted nucleic acid library. .

[0112] Polynucleotides and polypeptides, including modified versions thereof, can be synthesized using, for example, automated synthesis equipment. Applied Biosystems, Foster City, CA They can also be produced by chemical synthesis using methods known to the skilled artisan, such as Peptides can be synthesized in whole or in part using chemical methods (e.g., Caruthers (1980). Nucleic Acids Res. Sym p. Ser.215; Horn (1980); and Banga, A.K., Therapeutic Peptides and Proteins, Formu lation, Processing and Delivery Systems( 1995) Technomic Publishing Co., Lancaste Peptides are synthesized by various solid-phase methods (e.g., Roberge Sc ience 269:202 (1995);Merrifield, Methods 289:3 (1997)) and can be produced by The peptide synthesis was carried out according to the manufacturer's instructions, for example, using an ABI 431A peptide synthesizer (Perkin Elektrol mer) may be used to achieve automated synthesis.

[0113] The term "isolated" used as a modifier of a composition means that the composition are completely different from those that are man-made or occur naturally in vivo. Generally, an isolated composition means a composition that is distinct from, or partially distinct from, the natural state of the organism. For example, one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes, etc. The term "isolated" means that the material is substantially free of one or more ingredients that are present in the material. For example, recombinant vector (e.g., AAV) sequences, or vector genomes produced by Viral particles that package or encapsidate the vector (e.g., AAV-Rh74 vector) or related AAV-Rh74 variants, such as capsid variants (e.g., RHM4-1). The term "isolated" does not exclude mixtures of AAV vectors with pharmaceutical preparations. The terms hybrid / chimera, multimer / oligomer, modified (e.g. phosphorylation) , glycosylated or lipidated), or other physical forms of the composition, such as derivatives, or Forms expressed in host cells engineered by hand are not excluded.

[0114] The methods and uses of the present invention include the introduction of heterologous polynucleotides (transgenes) into dividing and non-dividing cells. The present invention provides a means for delivery (transduction) of a wide range of host cells, including both endothelial and non-endothelial cells. Described recombinant vector (e.g., AAV) sequences, plasmids, vector genomes, recombinant viruses Capsid particles (e.g., AAV-Rh74 or capsid mutants (e.g., RHM4-1) Related AAVs, such as the AAV-Rh74 mutant, methods, uses and pharmaceutical formulations include Also useful as a method for administering peptides or nucleic acids to a subject in need thereof as a therapeutic agent. As a result, proteins, peptides or nucleic acids can be produced in the subject's body. The subject is deficient in, or has a genetic defect in, the protein, peptide or nucleic acid. The production of said proteins, peptides or nucleic acids is of therapeutic value as a method of treatment or otherwise. The protein, peptide or nucleic acid has the effect of inhibiting the production of or may be in need of treatment for a variety of conditions, such as neurodegenerative diseases, cancer or atherosclerosis. For example, in the treatment of atherosclerosis, the expression of target genes involved in the disease process must be increased in order to achieve therapeutic efficacy. Alternatively, it may be desirable to inhibit or reduce production.

[0115] Generally, recombinant vector (e.g., AAV) sequences, plasmids, vector genomes, recombinant Viral particles (e.g., AAV-Rh74 or capsid mutants (e.g., RHM4-1) The present invention relates to AAV-Rh74 variants and related AAVs, methods and uses thereof. The present invention relates to a method for treating or preventing one or more symptoms associated with any disease associated with undesired gene expression. It can be used to deliver any heterologous polynucleotide that has a biological effect to alleviate Recombinant vector (e.g., AAV) sequences, plasmids, vector genomes, recombinant The viral particles (e.g., AAV-Rh74 or capsid mutants (e.g., RHM4-1 The AAV-Rh74 variant and related AAVs, methods and uses are directed to treating a variety of disease conditions. The present invention may also be used to provide treatment for

[0116] There are many genetic diseases for which the defective genes are known and have been cloned. The above disease states are divided into two classes. The first are usually enzyme deficiency states, is a deficiency condition that is generally inherited recessively, while the other is a deficiency condition that is inherited dominantly and at least in part It is an imbalance state involving regulatory or structural proteins. Deficiency state disease In this paper, gene transfer is used to deliver normal genes to diseased tissues as replacement therapy, as well as for the treatment of cancer. Sense mutations can be used to generate animal models of disease. For certain diseases, gene transfer can be used to produce pathology in model systems. Therefore, the model system can be used in efforts to alleviate the pathology. Recombinant vector (e.g. AAV) sequences, plasmids, vector genomes, recombinant viral particles vectors (e.g., AAV-Rh74 vectors or capsid mutants (e.g., RHM4-1) The present invention relates to a method for the treatment of genetic diseases, comprising administering a therapeutic agent to the patient, the patient's family, or a combination thereof, including administering a therapeutic agent to the patient. As used herein, a pathology refers to a condition that causes or is associated with a disease. It is treated by partially or completely correcting the aggravating deficiency or imbalance. Site-specific integration of nucleic acid sequences is used to generate mutations or correct defects. It is also possible to use

[0117] Examples of conditions include cystic fibrosis (and other diseases of the lungs), hemophilia A, hemophilia B, and pulmonary Anemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Chinton's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes mellitus , muscular dystrophies (e.g. Duchenne, Becker er type), Gaucher's disease, Hurler's disease Hulererapos's disease, adenosine deaminase deficiency , glycogen storage diseases and other metabolic disorders, Pompe disease, congestive heart failure, retinal degenerative diseases (previously congenital choroideremia, Leber's congenital amaurosis and other diseases of the eye), and solid organs (brain, liver This includes, but is not limited to, diseases of the liver, kidneys, and heart.

[0118] Methods of treatment and use are provided herein, which include the use of the recombinant vector of the invention. vector genome, recombinant viral particles (e.g., AAV-Rh74 AAV-Rh74 mutants, such as vector or capsid mutants (e.g., RHM4-1) and any related AAV vectors), and viral particles of the invention comprising a vector genome (e.g. AAV-Rh7, such as AAV-Rh74 or a capsid variant (e.g., RHM4-1) The methods and uses of the present invention include, for example, gene transfer Introduction of a gene encoding a protein, or alteration of the expression or function of the gene, by addition or substitution is broadly applicable to diseases amenable to treatment by increasing or stimulating The methods and uses of the present invention also include, for example, gene knockout or reduction of gene expression (genetic By reducing or diminishing the expression or function of a gene, such as by gene knockdown It has broad applicability to treat a wide range of diseases.

[0119] Specific, non-limiting examples of diseases treatable by the present invention include those set forth herein, such as and lung diseases (e.g., cystic fibrosis), blood clotting disorders or bleeding disorders (e.g., inhibitors of Hemophilia A (with or without), Hemophilia B (with or without), Thalassemia, Blood disorders (e.g. anemia), Alzheimer's disease Hymer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), seizures lysosomal storage diseases, copper or iron storage diseases (e.g. Wilson's disease or Menkes disease), Lysosomal acid lipase deficiency, neurological or neurodegenerative disorders, cancer, type 1 or type 2 diabetes disease, Gaucher's disease, Hurler's disease s disease), adenosine deaminase deficiency, metabolic disorders (e.g., glycolysis) retinal degenerative diseases (e.g. RPE65 deficiency or insufficiency, congenital choroideremia, Other conditions, including, but not limited to, eye diseases, solid organ diseases (brain, liver, kidneys, heart), etc. It is not something that can be determined.

[0120] Furthermore, the recombinant vector (e.g., AAV) vector genome, recombinant viral particle, (e.g., AAV-Rh74 vector or capsid mutants (e.g., RHM4-1), etc. The present invention relates to a method for the treatment of cancer, infection, and related AAV vectors, such as the AAV-Rh74 variant of the To treat or alleviate symptoms associated with autoimmune diseases such as rheumatoid arthritis and Delivering nucleic acids encoding monoclonal antibodies or fragments thereof that provide beneficial biological effects. It can be utilized to achieve this.

[0121] In one embodiment, the method or use of the present invention comprises: (a) a virus comprising a vector genome; AAV-Rh74 or AAV-Rh74 capsid mutant (e.g., Rh providing a related AAV (e.g., M4-1), wherein the vector genome comprises a heterologous polypeptide; a nucleotide sequence (and optionally a filler / stuffer polynucleotide sequence), The sequence of the heterologous polynucleotide may include an expression regulator that confers transcription of the polynucleotide sequence. (b) providing said viral particle operably linked to a control element; and injecting said viral particles into said mammal so as to express a seed polypeptide in said mammal. and administering a predetermined amount.

[0122] In another embodiment, the method or use of the present invention comprises the step of administering the heterologous polynucleotide sequence to a mammal. This includes delivering or transferring heterologous polynucleotides into cells of an organism or mammal. A vector genome including a vector sequence (and optionally a filler / stuffer polynucleotide sequence) A viral (e.g., AAV) particle comprising a capsid (e.g., AAV-Rh74 or capsid). AAV-Rh variants (e.g., RHM4-1) or related AAVs (e.g., Rh74 variants) or multiple viral (e.g., AAV) particles (e.g., AAV-Rh74 or capsid variants In vivo, the AAV-Rh74 variant (e.g., AAV-Rh74 variant, such as RHM4-1) is transfected into mammalian cells. and administering the heterologous polynucleotide sequence to the mammal or cells of the mammal. By delivery or transfer into the cells of an animal.

[0123] In yet another embodiment, the method comprises treating a mammalian subject with a deficiency requiring expression or function of a protein. The method or use of the present invention for treating a subject comprising administering a heterologous polynucleotide sequence (and optionally a filtrate) to a subject A virus comprising a vector genome including a marker / stuffer polynucleotide sequence. (e.g., AAV) particles (e.g., AAV-Rh74 or capsid mutants (e.g., RHM 4-1) related AAVs such as AAV-Rh74 mutants) or multiple viruses (e.g. (e.g., AAV) particles (e.g., AAV-Rh74 or capsid mutants (e.g., RHM4-1 providing a relevant AAV, such as an AAV-Rh74 variant, to said viral particles; and delivering or transferring said viral particles or said plurality of viral particles into a mammal or into a cell of a mammal. wherein the heterologous polynucleotide sequence encodes a protein that is expressed in the mammal. or the heterologous polynucleotide sequence reduces the expression of an endogenous mammalian protein. The inhibitory sequence or protein that reduces the expression of the gene is encoded by the inhibitory sequence or protein.

[0124] In certain aspects of the methods and uses of the invention disclosed herein, the heterologous polynucleotide Expression of the optides may result in the production of proteins or inhibitory genes that confer a therapeutic effect on a mammal (e.g., a human). In a further particular embodiment, the nucleic acid encodes a filler / stuffer polynucleotide sequence. The sequence is contained in the vector sequence, and the total length together with the heterologous polynucleotide sequence is approximately 3. 0-5.5 Kb, or about 4.0-5.0 Kb, or about 4.3-4.8 Kb.

[0125] The methods and uses of the present invention include any therapeutic or beneficial treatment method. Various methods and uses of the invention further include, for example, shortening blood clotting time, replacing blood One or more adverse (e.g., catastrophic) effects resulting from or related to the disease, such as a reduction in the dosage of clotting factors inhibit, reduce or alleviate (e.g. physical) symptoms, disorders, ill-health, diseases or complications This includes:

[0126] Thus, the therapeutic or beneficial effect of a treatment may be any objectively or subjectively measurable effect. Therapeutic value is a detectable improvement or benefit to a particular subject. or beneficial effect means the alleviation or elimination of all or certain of the adverse symptoms, disorders, complications of a disease or illness. This does not necessarily mean complete removal, although in some cases complete removal is possible. Thus, a satisfactory clinical endpoint is one that is consistent with the outcome of a disease caused or associated with the disease. A gradual improvement in one or more adverse symptoms, disorders, illnesses, diseases, or complications of When there is an improvement or partial decrease in the (days, weeks, months, etc.) a period of time caused by or associated with a disease The worsening or progression of multiple adverse symptoms, disorders, diseases, conditions or complications is inhibited, reduced, or eliminated. It is achieved when it is reduced, diminished, inhibited, prevented, restricted, or controlled.

[0127] The vector genome of the present invention, a recombinant virus particle containing the vector genome (e.g., AAV AAV-Rh7, such as the Rh74 vector or a capsid mutant (e.g., RHM4-1) Compositions, such as related AAV vectors, such as 4 mutants, and methods and uses thereof, include those A sufficient or effective amount can be administered to a subject in need thereof. "sufficient amount" or "sufficient amount" means a single or multiple doses of the compound, either alone or in combination with one or more other compositions. A therapeutic agent such as a drug, procedure, protocol, or treatment regimen tic regimen) for any period (long or short) A measurable response is obtained or an expected or desired result or benefit is achieved or detectable over a period of time (e.g., minutes, hours, days, months, years, or cure). It refers to the amount that can be

[0128] A vector genome or viral particle (e.g., AAV, e.g., A AAV-R, such as the AV-Rh74 vector or a capsid mutant (e.g., RHM4-1) h74 mutant or other relevant AAV vector) dose, e.g., vector per kilogram of body weight The dose, expressed as the amount of target genome (vg / kg), will vary depending on the route of administration, the specificity of the different species required to exert a therapeutic effect, and the dosage. The expression level of the polynucleotide, the specific disease to be treated, and the host response to the viral vector. Primary immune response, the host immune response to the heterologous polynucleotide or expression product (protein) This varies based on several factors, including the amount of DNA fragmentation and the stability of the expressed protein. Those skilled in the art will appreciate that the present invention is not limited to the above. A range of doses of the virions for treating a patient with a disease or disorder can be readily determined. Generally, a therapeutically effective dose is at least 1×10 8 Vector genome / object per kilogram of body weight (vg / kg) or in the range of more than this, e.g., 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 Or beyond this range It is.

[0129] Take hemophilia as an example. In general, the disease phenotype is heavily influenced by the disease in order to achieve therapeutic benefit. To change from severe to moderate, blood clotting factors must have a concentration of 1% of that in a healthy individual. A severe phenotype is defined as joint damage and life-threatening bleeding. The conversion of mild phenotype to mild disease requires a high concentration of blood clotting factors. must exceed 5% of the healthy individual standard. In this regard, a typical dosage would be at least 100 mg of vector per kilogram of body weight of the subject. Genome (vg) is 1×10 10 (vg / kg) or to achieve the desired therapeutic effect. To reduce, about 1×10 10 From 1×10 11 vg / kg of body weight of the subject or approximately 1× 10 11 From 1×10 12 vg / kg of body weight of the subject, or approximately 1×10 12 From 1×1 0 13 The range is vg / kg of subject body weight.

[0130] For treatment (e.g. to cure or provide a therapeutic benefit or to bring about improvement) The term "effective amount" or "sufficient amount" refers to the administration of "Clinical amount" generally refers to any adverse symptom, consequence, or complication of a disease. to one, more than one, or all of, or caused by or associated with, a disease A measurable reduction in one or more adverse symptoms, illnesses, conditions, or complications associated with The amount is an amount effective to provide a response of a certain degree, and of course, to reduce, decrease, or eliminate the progression or worsening of the disease. It is said that the desired outcome of administration is to be able to inhibit, suppress, limit or control the can.

[0131] An effective or sufficient amount may, but does not necessarily, be provided in a single administration. A single administration may be required, and may be administered alone or in combination with another component (e.g., an agent), treatment. Administration may be in conjunction with a protocol or treatment regimen. The subject, type, status and severity of the disease or side effects of treatment (if any) In addition, the effective amount may be increased in proportion to the amount of the second composition (e.g., another drug or is an agent), one or more administrations in the absence of a treatment, protocol or therapeutic regimen When given in the form of a compound, an effective or sufficient amount may not necessarily be effective or sufficient. This is done by verifying the effectiveness and sufficiency of the above in a given subject. Additional doses, amounts or durations beyond those prescribed, or additional components (e.g., drugs or actions) This may include any substance, procedure, protocol or treatment regimen that is considered to be effective. The amount of the compound that can be administered may also be, for example, a compound for treating a coagulation disorder (e.g., hemophilia A or B). Reduced use of alternative treatments, treatment regimens or protocols, such as the administration of recombinant coagulation factor proteins. Contains a small amount that results in

[0132] An effective amount or a sufficient amount does not necessarily have to be effective in all and each and every subject being treated. It is not necessary that a treatment be effective in the majority of subjects in a given group or population. Efficacy or sufficient amount means effectiveness or sufficient in a group or the general population. It does not mean that a drug is effective or sufficient in a particular subject (patient), but rather that it is effective or sufficient in a particular subject (patient). As is typical in such methods, for a given method of treatment or use, a subject There are cases where one subject shows a large response, while another subject shows little or no response at all. Therefore, the appropriate amount depends on the condition to be treated, the desired therapeutic effect, and the individual. Individual subjects (e.g., subject bioavailability, gender, It depends on the age, etc.

[0133] The term "ameliorate" means to improve or ameliorate a disease or a symptom thereof in a subject. By detectable or measurable improvement in the underlying cellular response, we mean a detectable or measurable improvement in the underlying cellular response. A measurable improvement is a reduction in the incidence, frequency, severity, progression, or duration of a disease, or in the severity of a disease. A subjective or objective reduction or inhibition of complications associated with or caused by the disease. to harm, restrain, limit or control, or to affect the symptoms or underlying causes or consequences of disease This includes improvement in, or remission of, a disease.

[0134] Thus, the outcome of successful treatment may be determined by the incidence, frequency, severity, progression or duration of the disease. or one or more adverse symptoms or underlying causes or consequences of a disease in a subject. A "therapeutic effect" or "benefit" is the reduction, lowering, inhibiting, suppressing, limiting, controlling, or preventing As mentioned above, it may lead to one or more underlying causes of a disease or adverse symptoms. Treatment methods and uses that affect the causative factors of a disease or the adverse effects of a disease are considered to be beneficial. A reduction or reduction in exacerbations can also be the outcome of successful treatment, as can the stabilization of symptoms. .

[0135] Thus, a therapeutic benefit or improvement does not necessarily mean a complete elimination of a disease or a reduction in the severity of a disease. Eliminate one, most or all of the associated adverse symptoms, complications, consequences or underlying causes. Thus, a satisfactory clinical endpoint is one that demonstrates a gradual progression of the disease of interest. Improvement or relief of a disease over a short or long period (hours, days, weeks, months, etc.) Partially reduce, decrease, inhibit, suppress, limit, or control the occurrence, frequency, severity, progression, or duration or prevention or reversal (e.g., stabilization of one or more symptoms or complications) When a method or procedure, such as a treatment, that provides a potential therapeutic benefit or amelioration of a disease is found, The validity of a usage can be verified in a variety of ways.

[0136] The methods and uses of the present invention provide desirable therapeutically beneficial, additive, synergistic or complementary Any compound, agent, drug, treatment or other therapeutic regimen or agent that has an activity or effect. Examples of compositions and treatments that can be combined include These include pharmaceutical agents (proteins), agents and second active substances, such as drugs. The biologics (proteins), agents, drugs, treatments and therapies are not intended to be used in any other manner or method. or methods of the invention, such as, for example, therapeutic methods for treating a subject with a blood clotting disorder, The method of use may be administered or carried out substantially simultaneously with or after the method of use. do.

[0137] The compound, agent, drug, treatment or other treatment regimen or protocol may be a combination composition. As a single vector or separately, e.g., a vector genome or virus of the invention (e.g., AAV AAV-Rh7, such as the Rh74 vector or a capsid mutant (e.g., RHM4-1) 4) simultaneously, consecutively or sequentially with the delivery or administration of the relevant AAV vector (such as a variant) The present invention therefore relates to the methods and uses of the present invention. The method may be used in combination with any compound, agent, drug, therapeutic agent, or method described herein or known to one of skill in the art. combinations in combination with dimen, treatment protocols, processes, therapeutic agents or compositions. Compounds, agents, drugs, treatment plans, treatment protocols, processes, therapies, or The composition may comprise a vector genome or virus of the invention (e.g., AAV-Rh74 vector). AAV-Rh74 mutants, such as AAV-Rh74 tRNA or capsid mutants (e.g., RHM4-1) to a subject prior to, substantially simultaneously with, or following administration of a related AAV vector. Non-limiting examples of combination embodiments include the above or other compounds. any substance, agent, drug, therapeutic regimen, therapeutic protocol, process, therapeutic agent or composition. nothing.

[0138] The methods and uses of the present invention may involve, inter alia, the administration of other compounds, agents, drugs, therapeutic regimens, Methods and uses that result in reduced use or need for treatment protocols, processes or therapeutic agents For example, in the case of blood clotting disorders, the method or use of the present invention may include administering to a given subject A combination therapy to compensate for deficient or absent (abnormal or mutated) endogenous coagulation factors. Reducing the frequency or dosage of recombinant coagulation factor protein or eliminating administration altogether Therefore, according to the present invention, the use of another treatment or therapy has a therapeutic benefit, such as improving the Methods and uses that reduce the need or use of techniques are provided.

[0139] The present invention is useful in animals, including veterinary applications. Accordingly, suitable subjects include "Subject" refers to any mammal, including humans, and non-human mammals. The term "human" typically refers to humans and non-human primates (monkeys, gibbons, gorillas, chimpanzees, etc.) animals (e.g., tigers, orangutans, macaques), domestic animals (dogs and cats), domestic animals (e.g., chickens and This refers to mammals such as ducks, poultry, horses, cows, goats, sheep, and pigs, and also experimental Animals include mice, rats, rabbits, and guinea pigs. Human subjects include: Subjects include fetuses, neonates, infants, juveniles, and adults. Subjects include animal disease models, e.g. For example, mouse and other animal models of blood clotting disorders, and other animal models known to those of skill in the art. Includes disease models.

[0140] As described herein, vectors and viral particles comprising the vectors (e.g., A AAV-Rh74 or a capsid mutant thereof (e.g., RHM4-1) Related AAV variants (such as AAV variants) contain insufficient protein or no functional gene product (protein To provide proteins to subjects with deficiencies in proteins or to treat abnormalities that may cause disease. In subjects that produce normal, defective, or non-functional gene products (proteins), It can be used to provide inhibitory nucleic acids or proteins, and is therefore suitable for treatment. The subjects have insufficient or absent amounts of functional gene products (proteins) or Abnormal, defective, or non-functional gene products (proteins) that may cause disease or The subjects who are suitable for the treatment according to the present invention are those who produce or are at risk of producing a pulmonary edema (protein). Further indications include abnormal or defective (mutated) gene products (proteins) that lead to disease. This includes subjects that have or are at risk of developing abnormal or defective substances, By reducing the amount, expression or function of the (mutated) gene product (protein) Therapeutic agents that can treat or alleviate one or more symptoms of the disease and / or help relieve the disease Thus, the subject may be examined for the type, time of onset or severity of the disease. Subjects with such a deficiency, regardless of progression, severity, frequency, type, or duration of symptoms. Includes:

[0141] The term "Prophylaxis" and its synonyms refer to the prevention of exposure to, or administration of, a substance or substance to a subject. It means a method in which administration or in vivo delivery occurs prior to the onset of disease. Before the onset of adverse symptoms, conditions, complications, etc., caused by the For example, the present invention can be applied to the methods and uses of the present invention. Screening (e.g., genetic) can be performed to identify subjects, but the disease There are also subjects who do not show symptoms of the disease. Insufficient or absent functional gene product (protein) even if not present Abnormal, defective, or non-functional gene products that may cause disease (proteins) and abnormal or defective (mutated) genes that may cause disease Subjects found to be positive by screening for the gene product (protein) are included.

[0142] The methods and uses of the present invention may be administered systemically, locally or topically, or by any route. This includes delivery and administration via injection, infusion, oral (e.g., ingestion or inhalation) ) or topically (e.g., transdermally). Possible delivery and administration methods include intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intracavitary, intracranial, transdermal (localized), This includes oral administration, parenteral administration, e.g., via a mucosa or rectum. Routes of administration and delivery include intravenous (iv), intraperitoneal (ip), intraarterial, and intramuscular. , parenteral, subcutaneous, intrapleural, topical, dermal, intradermal, transdermal, parenteral, e.g., transmucosal, intracranial, Intrathecal, oral (digestion), mucosal, respiratory, intranasal, endotracheal intubation, intrapulmonary, intrapulmonary infusion, oral, sublingual , intravascular, subarachnoid, intracavity, iontophoresis, intraocular, eye drop, optical, intraglandular, intraorgan, intralymphatic Includes:

[0143] Whether the treatment is a preventative or therapeutic measure, the type, onset, progression, severity, and / or severity of the disease being treated; severity, frequency, duration or probability, desired clinical endpoint, previous or concurrent treatment, subjects The general health, age, sex, race, or immune competence of the individual, as well as any other factors that would be understood by one of ordinary skill in the art. The dosage will depend on and vary depending on the individual and other factors. The amount, number, frequency or duration of administration may vary depending on the individual. any adverse side effects, complications, or other risk factors of the treatment or therapy and the subject's condition. The amount of the therapeutically effective amount of the compound can be increased or decreased proportionately depending on the amount of the compound. This will affect the dose and timing required to deliver a sufficient amount to provide prophylactic benefit. Understand the factors that influence

[0144] The methods and uses of the invention disclosed herein include those in which a subject is identified as having a disease to be treated. after the subject has one or more symptoms of the disease, or after the subject has one or more symptoms of the disease. Patients who do not have multiple symptoms but test positive for the disease as described below Run within 1-2, 2-4, 4-12, 12-24, or 24-72 hours after the cutoff Of course, the methods and uses of the present invention can be used without first identifying a subject as having a disease to be treated. after the subject has one or more symptoms of the disease, or after the subject has been screened. 1-7, 7-14, 14-21, 21-48 days after testing positive for COVID-19 may be carried out beyond 48 days, even months or years later.

[0145] Recombinant vectors (e.g., AAV, e.g., AAV-Rh74 vectors or capsid mutants) (e.g., relevant AAV vectors such as AAV-Rh74 variants such as RHM4-1) , sequences, plasmids, vector genomes, recombinant viral particles (e.g., AAV, e.g., A AAV-R, such as the AV-Rh74 vector or a capsid mutant (e.g., RHM4-1) h74 mutant and related AAV vectors), as well as other compositions, agents, drugs, biological products, The agent (protein) can be incorporated into a pharmaceutical composition, e.g., a pharma- ceutically acceptable carrier or excipient. Such pharmaceutical compositions can be administered to a subject, inter alia, in vivo or ex vivo. The present invention is useful for administering and delivering

[0146] As used herein, "pharmaceutically acceptable" means "physiologically acceptable" and "physiologically acceptable" The term "fully acceptable" means that one or more A biologically acceptable formulation, gas, liquid or solid, suitable for multiple routes of administration. "Pharmaceutically acceptable" or "physiologically acceptable" components. A moiety is a substance that is not biologically or otherwise harmful, e.g., one that does not cause significant undesirable biological effects. Such pharmaceutical compositions are substances that can be administered to a subject without causing adverse effects. The article may be, for example, a viral vector, a viral particle (e.g., AAV-Rh74 or a capsid). mutants (e.g., AAV-Rh74 mutants, such as RHM4-1) or related AAVs (e.g., AAV-Rh74 mutants) can be used in administering transformed cells to a subject.

[0147] The composition may be in a solvent (aqueous or non-aqueous) suitable for administration or in vivo contact or delivery. ), solutions (aqueous or non-aqueous), emulsions (e.g. water-in-oil or oil-in-water), suspensions, capsules, elixirs, dispersion and suspension media, coatings, isotonic agents, absorption enhancers, or adsorbents Aqueous and non-aqueous solvents, solutions and suspensions may contain suspending agents and thickening agents. Such pharma- ceutically acceptable carriers include, but are not limited to, those containing a coating or non-coating agent. (coating) tablets, (hard or soft) capsules, microbeads, powders, granules and powders Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents and antifungal agents) may also be included. Agents) can also be incorporated into the composition.

[0148] As described below and as will be known to those skilled in the art, pharmaceutical compositions may be administered or delivered in a variety of ways, including, but not limited to, by administering or administering to a subject in need thereof. Thus, the pharmaceutical composition can be formulated to be compatible with various routes. The present invention includes a carrier, diluent or excipient suitable for administration by.

[0149] Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of the active compound. The preparation is usually sterile and may be isotonic with the blood of the intended subject. Non-limiting specific examples include water, saline, dextrose, fructose, ethanol, etc. The oils may be oils, animal oils, vegetable oils or synthetic oils.

[0150] For transmucosal or transdermal administration (e.g., topical contact), a penetrant may be included in the pharmaceutical composition. Penetrants are known in the art and can be used, for example, for transmucosal administration, such as with an interface. For transdermal administration, the active ingredient is a dermal formulation comprising a dermal extract or dermal gel, the active ingredient being a dermal gel or dermal gel. As an aerosol, spray, ointment, salve, gel or other liquid, as is commonly known in the art, For contact with the skin, the pharmaceutical composition typically contains Typically, the formulation may be an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers that can be used include petrolatum, lanolin, polyethylene glycol, and the like. These include lees, alcohol, transdermal enhancers, and combinations thereof.

[0151] Cosolvents and adjuvants are added to the formulation. Non-limiting examples of co-solvents include those that contain hydroxyl groups or other polar groups and are isopropyl. Alcohols such as propyl alcohol; propylene glycol, polyethylene glycol , polypropylene glycol, glycol ether and other glycols; glycerol; Contains polyoxyethylene alcohol and polyoxyethylene fatty acid ester. Examples of the surfactants include soy lecithin, oleic acid, and triolein. sorbitan esters, such as sorbitan acid; and polyvinylpyrrolidone.

[0152] The vector genome, viral particle (e.g., AAV-Rh74 vector or capsule) of the present invention Related AAV vectors, such as AAV-Rh74 mutants, such as rhm4-1 Drug delivery systems and pharmaceutical compositions suitable for the methods and uses are well known in the art. is known (see, for example, Remington: The Science and Pra ctice of Pharmacy (2003) 20th edition, Mack Publishing ishing Co., Easton, PA; Remington's Phar Maceutical Sciences (1990) 18th Edition, Mack Pu blishing Co., Easton, PA; The Merck Inde x (1996) 12th Edition, Merck Publishing Group, W hitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; A nsel and Stoklosa, Pharmaceutical Calcul ations (2001) 11th Edition, Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al. , Drug Delivery Systems (1980), RL Ju (see Liano, ed., Oxford, NY, pp. 253-315).

[0153] As used herein, the term "unit dosage form" refers to The term refers to physically discrete units suited as unitary dosages for subjects to be treated. Each unit may also contain a pharmaceutical carrier (excipient, diluent, vehicle or filler) as needed. and when administered in one or more doses, have a desired effect (e.g., a prophylactic or therapeutic effect). The unit dosage form may be a liquid composition or a lyophilized or The composition may be packaged in ampoules and vials, which may contain the composition in a lyophilized state, and the bio For example, a sterile liquid carrier can be added prior to administration or delivery into the body. Each of the given forms may be contained in a multi-dose kit or container. AAV) sequences, plasmids, vector genomes, recombinant viral particles (e.g., AA V-Rh74 vector or AAV-Rh74 capsid mutant (e.g., RHM4-1) and associated AAV vectors), and these pharmaceutical compositions have the advantages of ease of administration and uniformity of dosage. The compositions may be packaged in single or multiple unit dosage forms for administration.

[0154] The present invention relates to one or more components and a package in which the components are enclosed. The kit typically includes components for performing in vitro , instructions for use or component labeling for in vivo or in vitro use The kit may contain, for example, a vector (e.g., AAV), genome or package insert. The vector is a viral particle (e.g., AAV-Rh74 vector or a capsid mutant (e.g., RH AAV vectors, such as AAV-Rh74 variants, such as M4-1, and optionally other In the case where the compound includes a group of components such as a second active ingredient, such as a compound, agent, drug, or composition, There is a match.

[0155] A kit refers to a physical structure that contains one or more components of the kit. The materials used should be able to keep the components sterile and should be of materials commonly used for such purposes (e.g. For example, paper, cardboard, glass, plastic, aluminum foil, ampoules, vials, It consists of a tube, etc.

[0156] The label or insert may contain information identifying one or more components. For example, the clinical pharmacology, pharmacokinetics and pharmacokinetics of the active ingredient, including dose, mechanism of action, Labels or inserts may include information about the manufacturer, lot number, manufacturing date, The label or insert may include information identifying the location, date, and expiration date. It may include information identifying the manufacturer, lot number, place of manufacture and date. The label or insert contains information about the diseases for which the components of the kit can be used. The label or insert may include instructions for use, or a treatment protocol or A clinician or subject using one or more of the components of the kit in a treatment regimen Instructions for use may include information such as dosage, frequency or duration, and and the methods, uses, treatment protocols or prophylactic or therapeutic regimens described herein. The document may contain instructions on how to perform one of the following actions:

[0157] The label or insert shall notify the user of any benefit that the component may provide. Information, for example about prophylactic or therapeutic benefit, may be included on the label. or the insert may contain information about possible side effects, complications or reactions; For example, to alert a subject or clinician regarding situations in which it is not appropriate to use a particular composition. The composition may contain information to warn users of one or more of the following conditions that may be incompatible with the composition: The subject is taking, planning to take, or has multiple other medications, or the subject is currently are currently undergoing or undergoing another treatment protocol or regimen that is incompatible with the composition. If you are planning to or have had any adverse side effects or complications, please explain. The documentation may include information about such non-compliance.

[0158] Labels or inserts may be attached to components, kits, or packaging materials (e.g., boxes). Separate or attached to the ampoules, tubes or other containers that contain the components of the kit or attached to the vial, e.g., paper or cardboard "printed matter." The labels or inserts may also include barcode-printed labels, disks, CD-ROs, etc. M or DVD-ROM / RAM, optical disks such as DVDs, MP3s, magnetic tapes or The computer-readable medium may include a storage medium such as a digital storage medium. There are AM and ROM or mixtures of these, e.g. magnetic / optical storage media, FLA Examples of such cards include SH media and memory type cards.

[0159] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as defined above. Generally, it has the same meaning as understood by a person skilled in the art to which the present invention pertains. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Suitable methods and materials are described below.

[0160] All applications, publications, patents, and other references cited herein, including GenBank citations, are incorporated herein by reference. In the event of conflict, the present specification, including definitions, will control. Takes priority.

[0161] All of the features disclosed in this specification can be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Therefore, unless expressly stated otherwise, the disclosed features (e.g., recombinant Vector (e.g. AAV) sequences, plasmids, vector genomes or recombinant viral particles (e.g., AAV-Rh74 vector or capsid mutants (e.g., RHM4-1), etc. Related AAV vectors, such as the AAV-Rh74 variant of This is an example.

[0162] As used herein, the words "a," "an," and "the" refer to the singular. The words "," "the" and "the" include plurals unless expressly stated to the contrary. Thus, see, e.g., "a polynucleotide." In the process of producing such a polynucleotide, a plurality of such polynucleotides is included and referred to as a "vector." When referring to a virus, multiple such vectors are included, and "a virus" is used When referring to a "particle" or "a particle," it is important to remember that there are multiple such bills. Contains particles /

[0163] In this specification, all numerical values ​​or numerical ranges are expressly incorporated herein unless the context clearly indicates otherwise. This includes integer values ​​within such ranges and fractional parts of values ​​or integer values ​​within such ranges. For example, If you refer to 0% identity, then 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, etc., and 81.1% , 81.2%, 81.3%, 81.4%, 81.5%, etc., and also 82.1%, Includes 82.2%, 82.3%, 82.4%, 82.5%, etc.

[0164] A reference to more or less than an integer means that the number referenced is greater than or equal to the referenced number. This includes any number larger or smaller. So, for example, saying that something is less than 1000 The numbers are 999, 998, 997, ..., up to 1, and the numbers less than 100 are 9 Contains the numbers 9, 98, 97, …, 1.

[0165] In this specification, all numerical values ​​or numerical ranges are expressly defined as being within the meaning of the present invention, unless the context clearly indicates otherwise. Fractional parts of values ​​such as, and integer values ​​within such ranges, and Includes fractional parts of integer values. Thus, for example, referring to numerical ranges such as percentage ranges. When doing this, the range from 1 to 10 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 1.1. , 1.2, 1.3, 1.4, 1.5, etc. When referring to a range from 1 to 50, therefore , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, and 1.1, 1.2, 1.3, 1.4, 1.5 etc., 2.1, 2.2, 2.3, 2.4, 2.5 etc. include.

[0166] A set of ranges includes ranges that combine the boundary values ​​of several consecutive, different ranges. Therefore, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50 ~60, 60~75, 75~100, 100~150, 150~200, 200~250 , 250~300, 300~400, 400~500, 500~750, 750~1,0 00, 1,000~1,500, 1,500~2,000, 2,000~2,500, 2 ,500~3,000, 3,000~3,500, 3,500~4,000, 4,000 ~4,500, 4,500~5,000, 5,500~6,000, 6,000~7,0 When referring to 10, 7,000-8,000 or 8,000-9,000, 0, 50~100, 100~1,000, 1,000~3,000, 2,000~4,0 Includes 00 etc.

[0167] The present invention is described herein using categorical language to describe many embodiments and aspects. The invention also relates to substances or materials, method steps and conditions, protocols, This includes embodiments in which certain subject matter, such as procedures, are entirely or partially excluded. In certain embodiments or aspects of the invention, materials and / or method steps are excluded. Therefore, although the present invention is not expressly expressed in terms that the present invention does not include, Aspects not expressly included in the invention are disclosed.

[0168] Several embodiments of the present invention have been described. It is understood that those skilled in the art will be able to easily implement the present invention without departing from the spirit and scope of the present invention. Various modifications may be made to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. and modifications may be made. Accordingly, the examples set forth below are illustrative of the present invention. It is intended to be illustrative and not limiting of the scope of the invention as claimed. EXAMPLES

[0169] Example 1 The following examples include descriptions of various materials and methods. Mice: Male C57BL / 6J (WT) mice, 8–10 weeks old, n per experimental group = 5. Dogs have a deficiency in the FIX gene, a genetic mutation found at the University of North Carolina at Chapel Hill. The HB dog from the colony had the HBV mutation (Evans et al., Proc Natl Acad Sci USA 86:10095 (1989)).

[0170] AAV vector construction: In vivo studies in mice showed that ApoE-hAAT was associated with liver fixation. The study was carried out using a construct expressing human FIX under the control of a promoter. A nearly identical promoter and canine FIX transgene were used.

[0171] Gene transfer method: All vectors were delivered intravenously; via the tail vein in mice. The vector was delivered in PBS (200 microliters per mouse). In dogs, the vector was delivered via the saphenous vein.

[0172] FIX expression determination: ELISA was used to measure FIX levels. The FIX ELISA antibody pairs (capture and secondary antibodies) were purchased from Affinity Biosciences. In dogs, Haurigot et al. As described in Mol Ther 18:1318 (2010), Affin Antibody pairs from ity Biologicals were used.

[0173] Statistical analysis: Statistical analysis was performed using unpaired, two-tailed t-tests. p-values ​​< A value of 0.05 was considered statistically significant.

[0174] AAV antibody measurement: For antibody measurements, we used the method described by Manno et al., (Nat Med 1 2:342 (2006)) and Mingozzi et al. (Nat M We used the in vitro neutralization assay described in (ed 13:419 (2007)). Briefly, two AAV vector constructs were used in the assay, one of which is driven by the CMV promoter. A single-stranded AAV expressing beta-galactosidase under the control of (ssAAV-LacZ) Renilla simulans under the control of vector or chicken β-actin promoter (CBA) AAV-Rh7, a self-complementary vector expressing a Renilla reporter gene 4-CBA-Renilla was used. To increase efficiency, 2V6.11 cells (ATCC) were used to express the inducible promoter. The adenovirus gene E4 was expressed under the control of 1.25x10 cells per well. 4 Cellular Cells were seeded in 96-well plates and treated with 1:1000 dilution of ponasterone A (In Vitrogen) was added to the medium to induce E4 expression. On the day of measurement, heat inactivated Serial half-log dilutions of the 3.2-fold diluted test serum ) was mixed with the medium containing the virus. The virus concentration used in this study was approximately 1x10 for AAV2. 10 vg / ml and AA For V5, 6 or 8, it is about 5.5x10 10 The scAAV-Luc vector was In the case of reporters, the measured virus concentrations were about 50 to about 150 times lower. - Residual activity of the transgene was measured by colorimetric (ssAAV-LacZ) or luminometer ( scAAV-Luc) was used.

[0175] Anti-AAV capsid total IgG or Ig subclasses were assayed using capture assays. The ELISA plate was filled with 5 × 10 empty capsids of AAV. 10 The plates were coated with capsid particles / ml of 2% BSA in PBS for 2 hours at room temperature. After blocking with 0.05% Tween 20, serial dilutions of samples were loaded into the wells and incubated at 4°C. Incubated overnight. Biotin-conjugated anti-human IgG1, IgG2, IgG3, IgG4 Antibody or IgM antibody (Sigma) was used for antibody detection, and streptavidin-HRP was added for substrate detection. The concentrations of Ig were purified human IgG1, IgG2, and IgG 3. Standard curves prepared by serial dilution of IgG4 or IgM antibodies (Sigma) It was judged.

[0176] AAV generation: The process for vector production was described by Ayuso et al. Ther 17:503 (2010)). Example 2 This example is a study of human FIX transgenic animals (mice) and FIX expression after gene transfer. Includes an explanation of.

[0177] C57BL / 6 mice (n=5 per group) were transfected with a liver-specific promoter. Under (2.5 per mouse) 10 Vector genome) containing the factor IX (FIX) gene The AAV vector was injected via the tail vein. The plasma concentration of α-amyloid protein was measured by ELISA at the 1st, 2nd, and 4th weeks after gene transfer. The results are shown in Figure 1. AAV-Rh74 was the most potent agonist in animals. A high level of transgene expression was demonstrated. Example 3 This example includes the efficient preparation of AAV-Rh74 for therapeutic levels of FIX in dogs with hemophilia. The present application includes a description of animal studies and data demonstrating delivery by

[0178] Briefly, dogs with hemophilia B were administered 3 × 10 12 Vector The expression of the therapeutic FIX transgene was mediated by a liver-specific promoter. The levels of vector and FIX were measured by ELISA. The plasma concentrations of canine FIX were monitored at 100 mg / kg / day and 100 mg / kg / day, respectively. and AAV8 were equally effective in dogs with hemophilia B, and both were superior to AAV6. Ta. Example 4 This example includes a description of studies demonstrating the presence of anti-AAV neutralizing antibodies (NAbs) in humans.

[0179] Data in Table 1 show anti-AAV neutralizing antibodies (NAbs) measured in humans in vitro. Subjects with NAb titers below 1:1 were excluded from the study or were excluded from the study if they had anti-AAV antibodies. Low titers were defined as those with low gene transfer potential for that AAV serotype (highlighted in grey). Patients with titers between 1:1 and 1:3 will be considered as subjects with empty capsids as decoys. The antibody titers of the ELISA kits with titers higher than 1:3 are considered to be acceptable for AAV. pulls that are not considered permissive for AAV transduction following systemic injection are lightly shaded AAV-Rh74 has the highest anti-AAV Nab activity compared to AAV-2 and AAV-8. showed a low prevalence. [Table 1] [Table 2] Example 5 In this example, data showing the production abundance of various AAV serotypes, including AAV-Rh74, are presented. Included.

[0180] The data in Table 2 show the production yields of various AAV serotypes. The following data were collected: virus batch size in roller bottles, total vector production, and bottle The yield per unit is shown. All serotypes were expressed with the same expression cassette. AAV-Rh74 was packaged similarly to the other serotypes evaluated, i.e., AAV-8 , with production yields comparable to or exceeding those of AAV-dj and AAV-2. [Table 3] Example 6 In this example, human factor IX (FIX) is expressed under the control of a liver-specific promoter. When rhesus monkeys were administered the AAVrh74 vector containing the same amount of AAV8 vector, The data show that this resulted in higher levels of FIX production in animals than in mice. Includes details.

[0181] Briefly, animals were administered AAV8 or AAVrh74 at 2 x 1 kg body weight. ×10 12 The vector was administered at a dose of 1000 mg / kg in saline. or formulated in a mixture of vector and empty AAV capsids (designated EC).

[0182] Figure 4 shows human FIX in rhesus monkey plasma. The average (2-8 weeks) and standard error or mean histogram of X Animals that received the AAVrh74-FIX vector are shown in the right margin. The data are shown in the last two bars toward the end of the experiment. The animals (last two bars towards the right margin) are from another group of animals injected with the same dose. The FIX transgene was expressed at a higher level compared to the control (black and grey bars). Mean levels were assessed using unpaired, two-tailed Student's t-test. Compared.

[0183] One of the animals injected with AAV-RHM4-1-FIX was found to have a human factor IX transgene. This is a well-documented phenomenon and is consistent with the results of human FI Approximately 20% of macaques treated with the X vector developed this disease. Individuals expressed approximately twice the concentration of FIX compared to AAV8-treated macaques. Example 7 The Examples below include a description of several Rh74 capsid mutants.

[0184] In summary, various substitutions were introduced into the Rh74 capsid sequence to generate Rh74 capsid mutants. The different Rh74 capsid mutants and the amino acids substituted at each position are as follows: It was a street. [Table 4]

[0185] The RHM4-1 mutant contains alanine, leucine, proline, and asparagine substitutions. and amino acids 195, 199, 201, and 202 of the Rh74 VP1 capsid, respectively. The amino acid sequence of the RHM4-1 mutant VP1 capsid is as follows: (SEQ ID NO: 5). The substituted residues a, v, p, and n are underlined and in bold. As shown in. [Table 5]

[0186] The nucleic acid sequence of the RHM4-1 mutant VP1 capsid is as follows: The codons encoding a, b, c, and n are underlined and in bold (SEQ ID NO: 11). [Table 6]

[0187] RHM15-1, 15-2, 15-3, 15-4, 15-5, and 15-6 mutants also contained alanine, leucine, proline, and asparagine substitutions at Rh74, respectively. The VP1 capsid has amino acids 195, 199, 201, and 202 at positions Furthermore, these mutants had multiple lysines substituted for arginines at various positions. there was.

[0188] The amino acid sequence of the RHM15-1 mutant VP1 capsid is as follows (SEQ ID NO: No.:6). [Table 7]

[0189] The nucleic acid sequence of the RHM15-1 mutant VP1 capsid is as follows (SEQ ID NO: 12). [Table 8]

[0190] The amino acid sequence of the RHM15-2 mutant VP1 capsid is as follows (SEQ ID NO: No.:7). [Table 9]

[0191] The nucleic acid sequence of the RHM15-2 mutant VP1 capsid is as follows (SEQ ID NO: 13). [Table 10]

[0192] The amino acid sequences of the RHM15-3 / RHM15-5 mutant VP1 capsids are as follows: (Sequence ID number: 8). [Table 11]

[0193] The nucleic acid sequences of the RHM15-3 / RHM15-5 mutant VP1 capsids are as follows: (Sequence ID number: 14). [Table 12]

[0194] The amino acid sequence of the RHM15-4 mutant VP1 capsid is as follows (SEQ ID NO: No.:9). [Table 13]

[0195] The nucleic acid sequence of the RHM15-4 mutant VP1 capsid is as follows (SEQ ID NO: 15). [Table 14]

[0196] The amino acid sequence of the RHM15-6 mutant VP1 capsid is as follows (SEQ ID NO: No.:10). [Table 15]

[0197] The nucleic acid sequence of the RHM15-6 mutant VP1 capsid is as follows (SEQ ID NO: 16). [Table 16] Example 8 This example shows the use of the Rh74 capsid mutant to compare human factor IX with Rh74 and AAV8. Includes a description of factor expression studies.

[0198] In summary, the Rh74 capsid mutant was used to transfect AAV human factor IX expression vectors. The researchers then used AAV particles to infect mice and measure the expression levels of factor IX in the animals (blood The capsid mutants had the following amino acid substitutions at the indicated positions: Ta. [Table 17]

[0199] FIG. 5 shows plasma human factor IX expression levels in treated animals after two weeks. As shown, AAV human nucleotide sequence encapsidated by the RHM4-1 mutant capsid. The factor IX expression vector provided the highest level of expression and was encapsidated by Rh74 AAV human factor IX expression vector and AAV human factor IX encapsidated by AAV8 This was substantially greater expression than that produced by the factor IX expression vector. Example 9 In this example, human factor IX (FIX) is expressed under the control of a liver-specific promoter. The AAVrh74 mutant RHM4-1 vector was administered to cynomolgus monkeys (macaques), Production of FIX in animals at higher concentrations than when administered the same amount of AAV8 vector The report includes details of the data that shows the volume of

[0200] Cynomolgus monkeys were pre-selected for neutralizing AAV antibodies and had pre-treatment titers of less than 1:1. 100% were selected for successful transduction. These monkeys were then AAV8 vector expressing factor X transgene or AAV-Rh74 variant RHM4-1 Either vector, 3x10 12 The non-human primates were injected at a dose of 1000 mg / kg. The plasma concentration of the transgene product (protein) of human FIX in animals was The results are shown in FIG. 6.

[0201] One of the animals injected with AAV-RHM4-1-FIX was found to have a human factor IX transgene. This is a well-documented phenomenon that has been reported in humans and mice. Due to a slight difference in amino acids in the macaque protein, the human FIX vector It occurred in about 20% of macaques treated with RHM4-1. The lack of evidence was due to the generation of an antibody response to the human transgene. The second individual treated with -1 had approximately twice as much FIX compared to the AAV8-treated macaque. The concentration was expressed.

Claims

1. AV capsid sequence, said sequence being the RH74 VP1 capsid sequence (SEQ ID NO: No.: 1) any one of the 195th, 199th, 201st, or 202nd amino acid positions Amino acid substitutions or modifications to the RH74 VP1 capsid sequence (SEQ ID NO: 1) AAV capsid sequence with amino acid substitutions of arginine for srcDNA.

2. The residues are located at positions 195, 199 and 200 of the RH74 VP1 capsid sequence (SEQ ID NO:1). A, V, P, or N at any one of amino acid positions 1, 201, or 202 The AAV capsid sequence of claim 1, which corresponds to the amino acids.

3. The AAV capsid sequence is 19 of the RH74 VP1 capsid sequence (SEQ ID NO:1). An A residue at amino acid position 5, a V residue at amino acid position 199, A P residue at the first amino acid position or an N residue at the 202nd amino acid position 2. The AAV capsid sequence of claim 1 ,

4. The AAV capsid sequence is 19 of the RH74 VP1 capsid sequence (SEQ ID NO:1). An A residue at amino acid position 5, a V residue at amino acid position 199, A P residue at the 1st amino acid position or an N residue at the 202nd amino acid position The AAV capsid sequence of claim 1 having any two of the following:

5. The AAV capsid sequence is 19 of the RH74 VP1 capsid sequence (SEQ ID NO:1). An A residue at amino acid position 5, a V residue at amino acid position 199, A P residue at the 1st amino acid position or an N residue at the 202nd amino acid position The AAV capsid sequence of claim 1 having any three of the following:

6. The AAV capsid sequence is 19 of the RH74 VP1 capsid sequence (SEQ ID NO:1). An A residue at amino acid position 5, a V residue at amino acid position 199, A P residue at the 1st amino acid position and an N residue at the 202nd amino acid position 2. The AAV capsid sequence of claim 1 ,

7. The AAV capsid sequence according to any one of claims 1 to 6, or RHM4-1 (sequence ID number: 5), RHM15-1 (SEQ ID number: 6), RHM15-2 (SEQ ID number: : 7), RHM15-3 / RHM15-5 (SEQ ID NO: 8), RHM15-4 (SEQ ID NO: A recombinant AAV comprising the sequence ID number: 9) or RHM15-6 (SEQ ID NO: 10). particle.

8. The AAV capsid sequence according to any one of claims 1 to 6, or RHM4-1 (sequence ID number: 5), RHM15-1 (SEQ ID number: 6), RHM15-2 (SEQ ID number: : 7), RHM15-3 / RHM15-5 (SEQ ID NO: 8), RHM15-4 (SEQ ID NO: A recombinant AAV comprising the sequence ID number: 9) or RHM15-6 (SEQ ID NO: 10). A recombinant AAV particle, wherein the particle encapsidates a vector genome.

9. The AAV capsid sequence according to any one of claims 1 to 6, or RHM4-1 (sequence ID number: 5), RHM15-1 (SEQ ID number: 6), RHM15-2 (SEQ ID number: : 7), RHM15-3 / RHM15-5 (SEQ ID NO: 8), RHM15-4 (SEQ ID NO: A recombinant AAV comprising the sequence ID number: 9) or RHM15-6 (SEQ ID NO: 10). A recombinant AAV particle, wherein the particle encapsidates an AAV vector genome.

10. The AAV vector genome according to claim 8 or 9, comprising a heterologous polynucleotide sequence. Recombinant AAV particles.

11. Method for delivering or introducing heterologous polynucleotide sequences into a mammal or into a mammalian cell The method includes the step of: providing a recombinant AAV comprising a recombinant vector genome comprising a heterologous polynucleotide sequence; administering the particles to the mammal or to cells of the mammal to deliver the heterologous polynucleotide delivering or introducing a sequence to said mammal or to a cell of said mammal. Law.

12. The recombinant AAV particle comprises an AAV capsid sequence according to any one of claims 1 to 6. , or RHM4-1 (SEQ ID NO: 5), RHM15-1 (SEQ ID NO: 6), RH M15-2 (SEQ ID NO: 7), RHM15-3 / RHM15-5 (SEQ ID NO: 8 ), RHM15-4 (SEQ ID NO: 9), or RHM15-6 (SEQ ID NO: 1 0), or a recombinant AAV particle according to any one of claims 7 to 10.

2. The method according to claim 1.

13. The heterologous polynucleotide sequence may be an expression vector that confers transcription of the heterologous polynucleotide sequence.

13. The method of claim 11 or 12, operably linked to a current control element.

14. 1. A method of treating a mammal having a defect in protein expression or function, comprising: (a) administering to said mammal a Encodes a polypeptide capable of correcting the expression or function of a defective protein Recombinant AAV particles are provided that contain a recombinant vector genome that includes a heterologous polynucleotide sequence. wherein the heterologous polynucleotide sequence is a transcription factor of the heterologous polynucleotide sequence. The recombinant AAV particle is operably linked to an expression control element that confers transcription. (b) introducing into said mammal a gene encoding said polypeptide for expression in said mammal. administering a predetermined amount of the recombinant AAV particles.

15. The recombinant AAV particles have a sequence identity of 90% or more to the following sequence: MAADGYL PDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVL PGYKYLGPFNGLDKGEPVNAADAAAALEHDKAYDQQLQAGD NPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLE PLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQP AKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAG GGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRT WALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT QNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPP FPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLR TGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYY LSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYR QQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMA THKDDEERFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEE IKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGM VWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGGFGLKHPPP QILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEW ELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPR PIGTRYLTRNL, or the sequence has 1-50 amino acid substitutions, deletions or substitutions. The added VP1 sequence, The sequence has a 90% or higher identity to the following sequence: TAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQT GDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNE GADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHL YKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPR DWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIAN NLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQ YGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYN FEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGT AGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQ NNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFF PSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATE QYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQ GPIWAKIPHTDGNFHPSPLMGGGFGLKHPPPQILIKNTPVP ADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRW NPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRN L, or a VP2 sequence in which 1-50 amino acids have been substituted, deleted or added to said sequence. Columns, and / or MAAGGGAPMADNNEGADGVG, which has a sequence identity of 90% or more with the following sequence: SSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISN GTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTI QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTL NNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQL LFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNF AWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVL MFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVA DNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAK IPHTDGNFHPSPLMGGGFGLKHPPPQILIKNTPVPADPPTT FNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQY TSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL, or having a VP3 sequence in which 1-50 amino acids have been substituted, deleted or added to said sequence; 15. The method of claim 11, 12, or 14.

16. The recombinant AAV particles comprise the VP1 capsid sequence at positions 195, 196, and 197 of the VP1 capsid sequence (SEQ ID NO:1). An amino acid substitution at any one of the 9th, 201st, or 202nd amino acid positions, or 74 Amino acid substitution of lysine with arginine in the VP1 capsid sequence (SEQ ID NO: 1) The method of claim 15 , wherein

17. The residue is amino acid sequence 195, 199, 201, or 202 of the VP1 capsid sequence.

17. The method of claim 16, wherein the amino acid is an A, V, P, or N amino acid at any one of the amino acid positions. The method described.

18. The VP1 capsid sequence contains an A residue at amino acid position 195, an A residue at amino acid position 199, a V residue at amino acid position 201, a P residue at amino acid position 202, or The method of claim 15, having an N residue at the amino acid position of

19. The VP1 capsid sequence contains an A residue at amino acid position 195, an A residue at amino acid position 199, a V residue at amino acid position 201, a P residue at amino acid position 202, or The method of claim 15, wherein any two of the N residues at the amino acid positions are:

20. The VP1 capsid sequence contains an A residue at amino acid position 195, an A residue at amino acid position 199, a V residue at amino acid position 201, a P residue at amino acid position 202, or The method of claim 15, wherein any three of the N residues at the amino acid positions are:

21. The VP1 capsid sequence contains an A residue at amino acid position 195, an A residue at amino acid position 199, A V residue at amino acid position 102, a P residue at amino acid position 103, and a V residue at amino acid position 104. The method of claim 15, having an N residue at the amino acid position of

22. The recombinant AAV particles may comprise one or more of VP1, VP2, VP3, VP4, VP5, VP6, VP7, VP8, VP9, ​​VP10, VP11, VP12, VP13, VP14, VP15, VP16, VP17, VP18, VP19, VP119, VP111, VP112, VP113, VP154, 2, or VP3 sequence having a VP1, VP2, or VP3 sequence with a 95% or higher identity to the VP1, VP2, or VP3 sequence. The method of claim 11, 12, or 14, comprising an AAV that

23. The recombinant AAV particles may comprise one or more of VP1, VP2, VP3, VP4, VP5, VP6, VP7, VP8, VP9, ​​VP10, VP11, VP12, VP13, VP14, VP15, VP16, VP17, VP18, VP19, VP119, VP111, VP112, VP113, VP154, VP1, VP2, or VP3 sequences having 96% or more identity with the VP2, VP3, or VP4 sequences. The method of claim 11, 12, or 14.

24. The recombinant AAV particles may comprise one or more of VP1, VP2, VP3, VP4, VP5, VP6, VP7, VP8, VP9, ​​VP10, VP11, VP12, VP13, VP14, VP15, VP16, VP17, VP18, VP19, VP119, VP111, VP112, VP113, VP154, VP1, VP2, or VP3 sequences having 97% or more identity with the VP1, VP2, or VP3 sequences. The method of claim 11, 12, or 14.

25. The recombinant AAV particles may comprise one or more of VP1, VP2, VP3, VP4, VP5, VP6, VP7, VP8, VP9, ​​VP10, VP11, VP12, VP13, VP14, VP15, VP16, VP17, VP18, VP19, VP119, VP111, VP112, VP113, VP154, VP1, VP2, or VP3 sequences having 98% or greater identity with the VP1, VP2, or VP3 sequences. The method of claim 11, 12, or 14.

26. The recombinant AAV particles may comprise one or more of VP1, VP2, VP3, VP4, VP5, VP6, VP7, VP8, VP9, ​​VP10, VP11, VP12, VP13, VP14, VP15, VP16, VP17, VP18, VP19, VP119, VP111, VP112, VP113, VP154, VP1, VP2, or VP3 sequences having 99% or greater identity to the VP1, VP2, or VP3 sequences. The method of claim 11, 12, or 14.

27. The recombinant AAV particles may comprise one or more of VP1, VP2, VP3, VP4, VP5, VP6, VP7, VP8, VP9, ​​VP10, VP11, VP12, VP13, VP14, VP15, VP16, VP17, VP18, VP19, VP119, VP111, VP112, VP113, VP154, 2, or VP3 sequence having 99.5% or more identity with the VP1, VP2, or VP3 sequence 15. The method of claim 11, 12, or 14, comprising:

28. The recombinant AAV particles are VP1 of AAV-Rh74 as depicted in Figures 3A to 3E; VP2 or VP3 sequence, AAV vector related to AAV-Rh74 or AAV- Rh74, or RHM4-1 (SEQ ID NO: 5), RHM15-1 (SEQ ID NO: 6 ), RHM15-2 (SEQ ID NO: 7), RHM15-3 / RHM15-5 (SEQ ID NO: No.: 8), RHM15-4 (SEQ ID NO: 9), or RHM15-6 (SEQ ID NO: The method of claim 11, 12, or 14, comprising:

29. The heterologous polynucleotide sequence or the protein has a therapeutic effect on the mammal.

15. The method of claim 11, 12, or 14, wherein the gene is expressed at a level that has an effect.

30. The adeno-associated virus (AAV) vector may be Rh74, AAV2, or AAV8 serovar. The method according to claim 11, 12 or 14, which has a higher hepatocyte targeting activity than serum type. Law.

31. The heterologous polynucleotide sequence or the protein is administered to a cell, tissue or The method of claim 11, 12, or 14, wherein the gene is expressed in an organ.

32. The method of claim 31 , wherein the cells comprise secretory cells.

33. 32. The method of claim 31 , wherein the cell comprises an endocrine cell.

34. The cell may be a hepatocyte, a neuron, a glial cell, a retinal cell, an epithelial cell, a lung cell, or a whole cell.

32. The method of claim 31 , comprising a primary, pluripotent or multipotent stem cell.

35. The mammalian tissue or organ includes the liver, brain, central nervous system, spinal cord, eye, retina, or lung. The method of claim 31 .

36. The mammal has an insufficient amount of the protein, or a defective or abnormal protein.

15. The method of claim 11, 12, or 14, further comprising producing a quality.

37. The protein is the cystic fibrosis transmembrane conductance regulator protein (CFTR), a blood clotting regulator ( Blood clotting) factors (factor XIII, factor IX, factor X, factor VIII, factor VIIa) , protein C, etc.), acquisition of function of blood coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein Protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, Ornithine transcarbamylase, β-globin, α-globin, spectrin, α-anti Trypsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7 ), sulfamidase, enzymes related to lysosomal storage diseases (ARSA), hypoxanthine Guanine phosphoribosyltransferase, β25 glucocerebrosidase, Engylglycerinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones , growth factor, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor , nerve growth factor, neurotrophic factor 3 and 4, brain-derived neurotrophic factor, glial cell-derived growth factor Transforming growth factors alpha and beta, cytokines, alpha interferon, beta interferon Interferon gamma, interleukin 2, interleukin 4, interleukin 12. Granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, simple Herpes virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance protein, tumor suppressor Proteins (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lin dau (VHL), adenomatous polyposis colonica (APC), immunomodulatory, tolerogenic or immunogenic peptide or protein Tregitope or hCDR1, insulin, Glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort Protein 1 (choroideremia), LCA5 (LCA-reversillin), ornithine keto Acid aminotransferase (gyradophyseal atrophy), retinoschisis 1 (X-linked retinoschisis) syndrome), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XL RP), MERTK (AR type of RP: retinitis pigmentosa), DFNB1 (Connexin 26 deafness) ), ACHM2, 3 and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, gene products involved in lysosomal storage diseases (e.g., sulfatase N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activating protein), or a One or more zinc finger nucleases or repair templates for genome editing The method of claim 36, further comprising a donor sequence used as the rate.

38. The heterologous polynucleotide sequence may encode a polypeptide, peptide or protein. The method of claim 11, 12, or 14, comprising a gene that

39. The genes include the cystic fibrosis transmembrane conductance regulator protein (CFTR), blood coagulation (blood coagulation ) Factors (Factor XIII, Factor IX, Factor X, Factor VII, Factor VIIa, Factor Protein C, etc.), acquisition of blood coagulation factor functions, antibodies, 65 kDa protein specific to the retinal pigment epithelium Quality of life (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithin tin transcarbamylase, beta globin, alpha globin, spectrin, alpha antitriptyline adenosine deaminase (ADA), metal transporter (ATP7A or ATP7), Sulfamidase, enzyme associated with lysosomal storage disease (ARSA), hypoxanthine N-phosphoribosyltransferase, β25 glucocerebrosidase, sphingomyelinase Erinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth Factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve Growth factors, neurotrophic factors 3 and 4, brain-derived neurotrophic factor, glial cell-derived growth factor, Transforming growth factors alpha and beta, cytokines, alpha interferon, beta interferon, interferon Interferon gamma, interleukin 2, interleukin 4, interleukin 12, Granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, herpes simplex viral thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P4 50. Deoxycytidine kinase, tumor necrosis factor, drug resistance protein, tumor suppressor protein Proteins (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC), immunomodulatory, tolerogenic or Immunogenic peptide or protein Tregitope or hCDR1, insulin, glucose Enzyme, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein Choroideremia, LCA5 (LCA-reversilin), ornithine ketoacid amine notransferase (gyrate atrophy), retinoschisis 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP) , MERTK (AR type of RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), A CHM2, 3 and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP 1. CLN2, gene products related to lysosomal storage diseases (e.g., sulfatase, N -Acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC 1, VPC2, sphingolipid activating protein), or one or more or multiple zinc finger nucleases or repair templates for genome editing The method of claim 38, comprising or encoding a donor sequence for use as a donor sequence.

40. The heterologous polynucleotide sequence comprises an inhibitory nucleic acid. The method described.

41. The inhibitory nucleic acid may be a microRNA (miRNA), a siRNA, a shRNA, a trans 40. The nucleic acid sequence of claim 40, comprising a splicing RNA, an antisense RNA, or a triplex structure RNA. The method described above.

42. The inhibitory nucleic acid is a virulence gene, a transcript of a virulence gene, or a nucleotide repeat. Gene transcripts associated with Huntington's disease, Huntington (HTT) gene, dentatorubral-pallidoluysian corpus Atrophy-related genes (atrophin 1, ATN1), X-chromosome association in spinal-bulbar muscular atrophy Androgen receptors on chromosomes, human ataxin-1, -2, -3 and -7, (CACN Ca encoded by v 2.1P / Q voltage-dependent calcium channel, TAT A-binding protein, ataxin 8 inverse chain, also known as ATXN8OS, spinocerebellar ataxia (1 Serine / threonine protein phosphatases in the phosphodiesterase (types 2, 3, 6, 7, 8, 12, and 17) Transcription factor 2A 55 kDa regulatory subunit B beta isoform, FM in fragile X syndrome Fragile X Mental Retardation 1 (R1), Fragile X Mental Retardation 1 (FMR1) in Fragile X-associated Tremor / Ataxia FMR1 in fragile XE mental retardation (FMR2 in fragile XE mental retardation) or AF4 / FMR 2 family members; Myotonic protein kinase (PK) in myotonic dystrophy ( MT-PK); Frataxin in Friedreich's ataxia, Amyotrophic lateral sclerosis Mutations in the superoxide dismutase 1 (SOD1) gene that cause Parkinson's disease and / or or genes involved in the pathogenesis of Alzheimer's disease; apolipoprotein B (APOB) and Proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercholesterolemia Disease: Human immunodeficiency virus transduction of HIV Tat and transcription genes in HIV infection Activator; HIV TAR, human immunodeficiency virus transactivator in HIV infection CC chemokine receptor (CCCR) in HIV infection R5) Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection quality, liver-specific microRNA (miR-122) in hepatitis C virus infection; 53, acute kidney injury or delayed renal transplant function or acute renal failure due to kidney injury; progressive , Protein kinase N3 (PKN3) in recurrent or metastatic solid malignant tumors; Theasome subunit beta type 9 (PSMB9) Metastatic melanoma also known as LMP 2; Proteasome subunit beta type 8 (PSMB8) also known as metastatic melanoma LMP7; proteasome subunit beta type 10 (PSMB10) in metastatic melanoma Also known as MECL1; Vascular Endothelial Growth Factor (VEGF) in Solid Tumors; Solid Tumors Kinesin spindle protein in chronic myeloid leukemia and apoptosis inhibitor B -Cell CLL / lymphoma (BCL-2); Ribonucleotide Reductase in Solid Tumors rR-kinase M2 (RRM2); furin in solid tumors; polo-like kinase 1 (P LK1), diacylglycerol acyltransferase 1 (D GAT1), beta-catenin in familial adenomatous polyposis; beta-2 adrenergic receptor, Glaucoma; DNA damage-induced transcription in diabetic macular edema (DME) or age-related macular degeneration RTP801 / Redd1, also known as substance 4 protein; age-related macular degeneration or choroid Vascular endothelial growth factor receptor I (VEGFR1) in angiogenesis and the non-arteritic ischemic optic nerve Caspase 2 in onychomycosis; keratin 6AN17K mutant protein in pachyonychia congenita ; Influenza A virus genome / gene sequence in influenza infection; Severe acute SARS coronavirus genome / gene sequence in SARS infection; Respiratory syncytial virus (RSV) genome / gene sequences in respiratory syncytial virus infections; Ebola Ebola virus genome / gene sequence in infectious diseases; Hepatitis B and C infections Hepatitis B and C virus genome / gene sequences; Herpes simplex virus (HSV) Herpes simplex virus (HSV) genome / gene sequences in infectious diseases, Coxsackiewii Coxsackievirus B3 genome / gene sequences in Rhus B3 infection; primary dystonia Torsin A (TOR1A), pan-class I and specific HL in transplantation Silencing of pathogenic alleles of genes such as the A allele (targeting alleles) or mutations in the gene autosomal dominant retinitis pigmentosa (adRP) The method of claim 40, which binds to the heterorhodopsin gene (RHO).

43. The mammal may have a pulmonary disease (e.g., cystic fibrosis), a bleeding disorder (e.g., inhibitors of Hemophilia A or B with or without hemophilia, thalassemia, blood disorders (e.g., anemia) , Atzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Epilepsy, lysosomal storage disorders, copper or iron storage disorders (e.g., Wilson's disease or Menkes disease) Disease) Lysosomal acid lipase deficiency, neuropathy or neurodegenerative disease, cancer, type 1 or type 2 diabetes , Gaucher disease, Hurler disease, adenosine deaminase deficiency, metabolic disorders (e.g., glycoprotein retinal degenerative diseases (e.g., RPE65 deficiency, congenital choroideremia, and other eye diseases), solid organ diseases (e.g., brain, liver, kidney, heart), or infectious diseases Viral diseases (e.g., Hepatitis B and C, HIV, etc.), bacterial diseases, or fungal diseases 15. The method of claim 11, 12, or 14, having the following structure:

44. The expression control elements include constitutive or regulatable control elements.

15. The method according to claim 14.

45. The expression control element comprises a tissue-specific expression control element or a promoter.

15. The method according to claim 14, wherein

46. The AAV vector can be administered intravenously, intraarterially, intramuscularly, subcutaneously, orally, by intubation, or via a bolus of blood. By administering the drug via a catheter, percutaneously, intracranially, by inhalation, into a body cavity, or through a mucous membrane 15. The method of claim 11, 12, or 14, wherein the delivery is via a

47. 15. The method of claim 11, 12, or 14, wherein the mammal is a human.

48. the mammal is serologically positive for an AAV serotype other than AAV-Rh74; 15. The method of claim 11, 12, or 14.

49. The mammal is an AAV serotype 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, A Serologically tested for AV6, AAV7, AAV8, AAV9, AAV10, or AAV11 144. The method of claim 11, 12, or 143, which is positive.

50. the mammal is seronegative or seronegative for AAV-Rh74; 15. The method of claim 11, 12, or 14.

51. 15. The method of claim 11, 12, or 14, further comprising administering an empty capsid AAV. How to.

52. An empty capsid AAV-Rh74 or an AAV capsid according to any one of claims 1 to 6. pSid sequence, or RHM4-1 (SEQ ID NO: 5), RHM15-1 (SEQ ID NO: SEQ ID NO:6), RHM15-2 (SEQ ID NO:7), RHM15-3 / RHM15-5 (SEQ ID NO:8), SEQ ID NO: 8), RHM15-4 (SEQ ID NO: 9), or RHM15-6 (SEQ ID NO:

12. The method of claim 11, further comprising administering an empty capsid comprising:

15. The method according to claim 14,

53. The VP1, VP2, or VP3 sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative, non-conservative, or conservative and non-conservative amino acid substitutions.

52. The method according to claim 52.

54. The method of claim 11, 12, or 14, wherein the AAV particles comprise a pharmaceutical composition.

55. The method of claim 54, wherein the pharmaceutical composition comprises an empty capsid AAV.

56. The pharmaceutical composition comprises empty capsid AAV-Rh74 or any one of claims 1 to 6. or RHM4-1 (SEQ ID NO: 5), RHM1 5-1 (SEQ ID NO: 6), RHM15-2 (SEQ ID NO: 7), RHM15-3 / RHM15-5 (SEQ ID NO: 8), RHM15-4 (SEQ ID NO: 9), or 55. The method of claim 54, comprising administering to a mammalian subject the method of the present invention a method comprising administering to a mammalian subject the method of the present invention, comprising administering to a mammalian subject the method of the present invention method.

Images