Mutant AAV, and compositions, methods, and uses for gene transfer into 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 of related applications This application is based on U.S. Provisional Patent Application No. 61 / 985,365, filed on April 28, 2014. The patent number, and U.S. Provisional Patent Application No. 61 / 857,161 filed on July 22, 2013. These applications claim priority, and the entire contents of these applications are incorporated herein by reference. It can be inserted. preface

[0002] Desirable gene deficiency or defect (loss of function), or undesirable or Genetic disorders caused by the expression (gain of function) of defective genes include a variety of diseases. It is related to the concept of "ki" (vital energy). An example of a genetic disorder caused by loss of function is hemophilia, which is... Clotting factor VIII (FVIII, hemophilia A) or clotting factor IX (FIX, hemophilia B) It is a hereditary hemorrhagic disorder caused by a deficiency of [substance name]. An example of a hereditary disorder resulting from gain-of-function. One example is Huntington's disease, which is a condition in which a mutated protein called "HT" is encoded. The T" gene (which codes for the huntingtin protein) It is a disease caused by the accumulation of mutated proteins, particularly in the basal ganglia and cerebral cortex. This gradually leads to the destruction of nerve cells.

[0003] Current treatments for hemophilia involve combining the treatments as needed or preventively when bleeding occurs. Alternative coagulation factors are administered intravenously. However, this treatment method has several drawbacks. For example, the need for repeated intravenous infusions, the cost of treatment, and the expression of an immune response due to anti-treatment factors. There is a risk of bleeding that can be fatal. Therefore, the development of gene-based therapies for hemophilia has progressed. In this context, gene transfer-based therapies are suitable for hemophilia, and the reasons for this are, firstly, 1 One of the advantages is the very wide therapeutic window. At a level slightly exceeding 1% of the normal range, the phenotype has already shifted from severe to moderate. 1) No side effects at all, at a 100% level, and furthermore, 2) Tissue-specific treatment The expression of the introduced gene is not strictly required, and 3) the endpoint of the therapeutic effect is not measured. One of the reasons is that there is a considerable amount of experience in performing the procedure. Furthermore, due to the hepatic expression of coagulation factors, coagulation It is known that immune tolerance to the factor itself is induced, potentially leading to harmful coagulation factors. This reduces the likelihood of an immune response in the offspring.

[0004] Currently, adeno-associated virus (AAV) vectors are the optimal gene transfer vectors. It is recognized that this is due to the superior safety and effectiveness of gene delivery in vivo. This is because it has a rofile. Of the AAV serotypes isolated so far, A AV2 and AAV8 have been used as targets for the liver of patients with severe hemophilia B. This vector is also known to work effectively, and in the case of AAV8, the therapeutic transgene is long It has been reported that it is expressed periodically. Recent data from humans suggests that AAV vectors By targeting the liver using this method, therapeutic levels of FIX transgenes can be expressed over the long term. It has been indicated that this should be done.

[0005] Based on this data, the above serotypes are considered promising for the future, but they have high efficacy against the liver. It has tropism and a low serological positivity rate in the human body. Identifying a new AAV serotype (natural host of wild-type AAV) having such valence is important for achieving the following two points. 1) To induce a therapeutic level of expression of the transgene in the liver with the lowest possible vector dose to reduce the risk of causing an anti-AAV capsid immune response and, 2) To enable treatment of patient groups with existing humoral immunity to AAV for whom AAV gene transfer is not possible with alternative AAV serotypes having a specific seropositivity rate. The present invention addresses the above-mentioned desires and provides further advantages. SUMMARY OF THE INVENTION

[0006] The present invention provides an adeno-associated virus (AAV) serotype AAV-Rh74 vector, and related AAV vectors and virus particles. Such vectors include AAV-Rh74 that targets hepatocytes in the liver among various cell types. As vectors for delivering polynucleotide sequences, AAV-Rh74 and related AAV vectors activate the expression of polynucleotides in cells. A polynucleotide encoding a protein such as a protein used in treatment can be expressed at a therapeutic level after administration.

[0007] Exemplary AAV-Rh74 vectors and related AAV vectors include AAV-Rh74 variants. Certain capsid variants include a Rh74 capsid sequence in which any one of the amino acid positions 195, 199, 201, or 202 of the RH74 VP1 capsid sequence (SEQ ID NO: 1) is amino acid substituted In certain embodiments, the residue is at amino acid position 195, 199, 201, or 202 of the RH74 VP1 capsid sequence (SEQ ID NO: 1) ​​​​​​​​​​​​ Corresponds to amino acids A, V, P, or N at any one of the amino acid positions. Further specification In this embodiment, the capsid sequence is the RH74 VP1 capsid sequence (sequence ID number: 1) A residue at the 195th amino acid position, V residue at the 199th amino acid position, A P residue at the 201st amino acid position, or an N residue at the 202nd amino acid position. It has a group. Furthermore, in a particular embodiment, the capsid sequence is any two or three of the following: They have either all four or the RH74 VP1 capsid sequence (sequence ID number: 1) A residue at the 195th amino acid position, V at the 199th amino acid position A residue, a P residue at the 201st amino acid position, or at the 202nd amino acid position It is an N residue.

[0008] The recombinant AAV particles of the present invention are RHM4-1 (sequence ID number: 5), RHM15-1 ( Sequence ID number: 6), RHM15-2 (Sequence ID number: 7), RHM15-3 / RHM1 5-5 (Sequence ID number: 8), RHM15-4 (Sequence ID number: 9), or RHM15- Includes AAV capsid sequences of any capsid variant, such as 6 (sequence ID number: 10). In one embodiment, recombinant AAV particles are vector genomes (e.g., AAV vector genomes). Capsidizing or packaging a viral vector (such as NM). Alternate AAV particles are viral (e.g., AAV) vectors containing heterologous polynucleotide sequences. - Includes the genome.

[0009] AAV-Rh74 and related AAV vectors have been studied in preclinical and clinical settings. The introduction of polynucleotides produced was significantly higher than in some other serotypes. It modulates protein expression levels. In particular, AAV-Rh74 is used in mice and dogs with hemophilia B. In this regard, AAV8, a representative example of hepatic transduction, is less common. The liver is targeted with polynucleotides with efficiency equal to or exceeding that of other targets. (Figure 1 and See Figure 2.) For example, AA such as the capsid mutant RHM4-1 (sequence ID number: 5) The V-Rh74 mutant is equivalent to or better than AAV8 in mice, and Rh7 It targets the liver with polynucleotides with superior efficiency compared to 4-AAV (see, for example, Figure 5). Furthermore, data from non-human primates has shown that AAV-Rh74 and R AAV-Rh74 variants such as HM4-1 mediate the expression of liver-derived hFIX. It has been shown to be approximately twice as effective as AAV8 (see, for example, Figures 4 and 6).

[0010] Therefore, AAV-Rh74 and capsid variants (e.g., RHM4-1) The AAV-Rh74 mutant delivers polynucleotides such as gene coding sequences. To express proteins that can be used and provide desired or therapeutic benefits. This can be achieved, and it can also reduce or suppress the expression of undesirable or defective genes. Because it can also be used for harmful nucleotides, it can treat a variety of diseases. For example... For example, AAV-Rh74 and AAV-Rh74 capsid variants (e.g., RHM4-1 ) is a method of treating hemophilia A, B, etc. by introducing therapeutic genes (e.g., FIX, FVIII) into cells. It can be used as a vector for delivery to tissues and organs such as the liver. Furthermore, such AAV-Rh74 and AAV-Rh74 capsid variants (for example, RH M4-1) Vectors are used for a wide range of diseases, including other metabolic disorders or plasma protein deficiencies. It can be used to deliver genes to [unclear]. Or, it can be used for other therapeutic purposes. It can be used to deliver genes, for example, but is not limited to the genome in the liver. Zinc finger nuclease is used to perform the editing. Genes encoding nucleases, ALF for treating hepatitis virus infections Local (liver) delivery of immunomodulatory drugs such as interferon, or hepatic transduction. To provide therapeutic transgene products in the bloodstream or to ensure their presence (expression in the liver) This can be achieved by targeting the introduced gene for any purpose that requires It can be used to treat diseases.

[0011] In vitro, in vivo and in vitro AAV-Rh74 vectors and capsid variants ( For example, related AAV vectors such as AAV-Rh74 variants like RHM4-1) In addition to efficient delivery of polynucleotides to cells, it also has anti-AAV-Rh74 properties in humans. The prevalence of the body is lower than the prevalence of anti-AAV2 antibodies, and anti-AAV The prevalence differs from that of the 8 antibodies (Table 1). Due to the low serum prevalence, the AAV-Rh74 vector... - and related AAs such as AAV-Rh74 (capsid) variants (e.g., RHM4-1) V vectors can be used with other AAV serotypes, for example (e.g., AAV1, AAV2, AAV3) , AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV This includes gene transfer into humans who are serologically positive for (11, etc.), and is used by many people. It is possible. In addition, the AAV-Rh74 vector and capsid variants (e.g., RHM) are also possible. Related vectors, including the AAV-Rh74 mutant (4-1), exhibit high titer. It can be efficiently generated using (Table 2). Therefore, the AAV-Rh74 vector and Related AAV vectors can be generated in large quantities for a wider range of clinical diseases.

[0012] The present invention also includes AAV vector genomes (capsidized or packaged). ), recombinant AAV-Rh74 vector, and capsid variant (e.g., RHM4-1) granules This invention provides related AAV vectors, such as AAV-Rh74 variants. In one embodiment, The recombinant AAV vector contains a heterogeneous polynucleotide sequence. In another embodiment, Recombinant AAV vector genomes are AAV-Rh74 capsid or capsid variants (e.g.) Capsidization occurs due to related AAVs such as AAV-Rh74 variants (RHM4-1), etc. Or it is packaged.

[0013] AAV containing recombinant AAV vector genome (capsidized or packaged) -Rh74 vector and AAV-Rh74 (capsid) variant (e.g., RHM4-1 ) In the recombinant AAV vector of the present invention, such as related AAV vectors such as particles, heterogeneous The nucleotide sequence is transcribed and then converted into a protein. Alternatively, heterologous polynucleotides Rheotides themselves are used to create transcripts that have function or activity (e.g., as inhibitory nucleic acids). It may also be transcribed as follows.

[0014] In various embodiments, heterogeneous polynucleotide sequences can coat proteins that have therapeutic effects. In certain embodiments, the protein is a blood coagulation factor (e.g., factor XIII). Factor IX, Factor X, Factor VIII, Factor VIIa, or Protein C), cyst cystic fibrosis transmembrane regulator e regulator protein; CFTR), antibody, retinal pigment epithelium specific 65 kDa protein (retinal pigment epithelial m-specific 65 kDa protein (RPE65), erythropoiesis Ethin (erythropoiesis-promoting factor), LDL receptor, lipoprotein lipase, orni Tin transcarbamylase, β-globin, α-globin, spectrin, α-antitriol Psin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7) Sulfamidase, an enzyme involved in lysosomal storage diseases (AR SA), Hypoxanthine guanine phosphoribosyltransferase, β-25 Lucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, Branched-chain keto acid dehydrogenases, growth factors (e.g., insulin-like growth factors 1 and 2, platelet-derived) Next growth factor, epidermal growth factor, nerve growth factor, neurotrophic factors 3 and 4, brain-derived neurotrophic factor Child cells, glial cell-derived growth factors, transforming growth factors α, β, etc.), cytokines (e.g., α Interferon, β-interferon, interferon-γ, interleukin-2, Interleukin-4, Interleukin-12, Granulocyte-macrophage colony-stimulating factor, (e.g., phenotoxins), suicide gene products (e.g., herpes simplex virus thymidine kinase, Cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidinequinate (e.g., tumor necrosis factor), drug resistance proteins (for example, resistance to drugs used in cancer treatment) Proteins to be conferred), tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), Intestinal adenomatous polyposis (APC), immunomodulatory peptides, tolerogenic or Immunogenic peptides or proteins: Tregitopes (regulatory T cell epitopes) , or hCDR1, insulin, glucokinase, granyl cyclase 2D (LCA -GUCY2D), Rab escort protein 1 (congenital choroidal absence), LCA 5 ( LCA-level cylin), ornithine keto acid aminotransferase (cerebral gyroretinoid choroidal atrophy) ), Retinoschisin 1 (X-linked retinoschiosis) USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR-type retinitis pigmentosa), DFNB1 (connexin 26-26 deficiency) Hearing impairment, ACHM 2, 3 and 4 (color blindness), PKD-1 or PKD-2 (multiple sacral vesicles) (Splenic kidney disease), TPP1, CLN2, genetic defects that cause lysosomal storage disorders (e.g., Sul Fatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A (GM2-AP, NPC1, VPC2, sphingolipid-activating proteins, etc.), genome ed One or more zinc finger nucleases, or geno, used in the fitting process. This is a donor sequence used as a repair template in muediting.

[0015] In an additional aspect, heterogeneous polynucleotide sequences encode therapeutic proteins, and As a result, the patient has undesirable or abnormal (dysfunctional) proteins (endogenous) Inhibits the expression or function of proteins. In another embodiment, heterogeneous polynucleotides. The sequence is a polynucleotide that, when transcribed, is transcribed into an inhibitory nucleic acid (e.g., inhibitory RNA). In another embodiment, the inhibitory nucleic acid may be a single-stranded sequence or a double-stranded sequence. These form a triple-stranded sequence. In another embodiment, the inhibitory nucleic acid is a microRNA (miRN). A) siRNA, shRNA, transsplicing RNA, antisense RNA, Alternatively, it is triple-stranded RNA.

[0016] In other embodiments, the inhibitory nucleic acid is the huntingtin (HTT) gene, dentate rubra pallidum. Genes associated with bulbuluysian atrophy (e.g., atrophine 1 (ATN1)), bulbar spinal muscular atrophy Androgen receptors on the X chromosome in dyslexia, human attaxin 1,2 ,3 and 7, voltage-gated calcium channel Ca encoded in CACNA1A v 2 .1 P / Q, also known as TATA-binding protein or ATXN8OS, is ataxin. 8 reverse chain, serine / threonine protein phosphatase 2A in spinocerebellar ataxia 55 kDa adjustment subunit B beta isoform (1,2,3,6,7,8,12,17 Type), FMR1 (Vulnerability X Mental Retardation 1) in Vulnerability X Syndrome, Vulnerability X-related earthquake / luck FMR1 (Vulnerability X Intellectual Disability 1) in Ataxia Syndrome, F in Vulnerability XE Intellectual Disability MR1 (Vulnerability X Intellectual Disability 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 lateral sclerosis (LSC) Apolipo Protein B (APOB) and precursor protein-converting enzyme subtilisin / kexinty PCSK9, hypercholesterolemia, and human immunodeficiency virus (HIV) infection The HIV transcriptional transactivator gene, HIV Tat, human immunodeficiency virus (H) IV) HIV Transactivator Response Array Genes, HIV TARs, HIV CC chemokine receptor (CCR5) in infection, Roussarcoma virus (RSV) infection RSV nucleocapsid protein in hepatitis C virus infection, liver-specific MicroRNA (miR-122), p53, acute kidney injury or delayed development of transplanted kidney function This is a protein-related condition in renal injury, acute renal failure, and advanced, recurrent, or metastatic solid malignancies. Nase N3 (PKN3), LMP2 in metastatic melanoma, LMP2 is proteasome LMP in metastatic melanoma, also known as bunit beta 9 (PSMB 9). 7. Also known as proteasome subunit beta-8 (PSMB 8), metastatic Also known as proteasome subunit beta-10 (PSMB 10) in melanoma. MECL1, vascular endothelial growth factor (VEGF) in solid tumors, in solid tumors Kinesin spindle protein, apoptosis suppression in chronic myeloid leukemia (CLL) B cells BCL-2 cystoma, ribonucleotide reductase M2 (RRM2) in solid tumors, Furin in solid tumors, polo-like kinase 1 (P) in liver cancer LK1), diacylglycerol acyltransferase 1 (D) in hepatitis C infection GAT1), betacatenin in familial adenomatous polyposis, betaadrenaline in glaucoma Phosphate receptors, DAN loss in diabetic macular edema (DME), or age-related macular degeneration. RTP801 / Redd1, also known as the wound-inducing transcript 4 protein, is associated with aging and yellowing. Vascular endothelial growth factor receptor I (VEGFR1) in plaque degeneration or choroidal angiogenesis, non Caspase 2 in arteritis-induced ischemic optic neuropathy, keratin 6A in congenital onychoplasty N17K mutant protein, influenza A virus in influenza infection The genome / gene sequence of the coronavirus in severe acute respiratory syndrome (SARS) infection. Genome and gene sequences, genome of respiratory syncytial virus in respiratory syncytial virus infection • Genetic sequence, genome of Ebola filovirus in Ebola infection • Genetic sequence, type B The genome and gene sequences of hepatitis B and C viruses in hepatitis C infection, and simplex hepatitis Genome and gene sequences of the HSV virus in pesvirus (HSV) infection, Coxsacki - Genome and gene sequence of Coxsackievirus B3 in virus B3 infection, primary muscle Silencing of pathogenic alleles of genes like torsin A (TOR1A) in ataxia Allele-specific silencing, pan-class I and HLA alleles in transplantation In gene-specific (silencing) autosomal dominant retinitis pigmentosa (adRP) A mutant rhodopsin gene, or a transcript of any of the aforementioned genes or sequences, binds to the mutant rhodopsin gene. It inhibits the expression of inhibitory nucleic acids.

[0017] This invention includes (capsidizes or packages) a recombinant AAV vector genome. Recombinant AAV vectors, AAV-Rh74 vectors, and capsid variants (e.g.) Related AAV vectors such as AAV-Rh74 vector variants, including RHM4-1 particles This includes additional elements that function in sys or trans. In certain embodiments, recombinant Recombinant viruses containing (capsidizing or packaging) AAV vector genomes AAV (for example) vectors, and / or AAV-Rh74 vectors or AAV- Related AAVs such as Rh74 (capsid) variant (e.g., RHM4-1) particles are as follows: They also have a single nucleotide adjacent to the 5' or 3' end of a heterogeneous polynucleotide sequence. Multiple reverse terminal repeat (ITR) sequences, which drive the transcription of heterogeneous polynucleotide sequences. Current control sequences (e.g., constitutive or adjustable control elements, such as heterogeneous polynucleotide combinations) Promoters or enhancers that contribute to the transcription of a sequence, or tissue-specific expression regulatory elements), heterogeneous A polyadenine sequence located at the 3' position of a polynucleotide sequence, and selectable markers (for example) (For example, proteins that provide resistance to antibiotics such as kanamycin resistance), and / or This is the replication base point.

[0018] Also, recombinant AAV vectors containing genomes (capsidized or packaged) The recombinant AAV vector, AAV-Rh74 vector, and capsid variant of the present invention ( For example, related AAV vectors such as the AAV-Rh74 variant containing RHM4-1) particles , may include additional elements. In one embodiment, the recombinant vector genome is a heterologous polynucleo The nucleotide sequence and the filler polynucleotide sequence or stuffer (st (uffer) Contains polynucleotide sequences. In certain embodiments, heterogeneous polynucleotide sequences It has a length of less than approximately 4.7 Kb. Furthermore, in certain embodiments, heterogeneous polynucleotides are used. The sequence is less than approximately 4.7 KB in length and contains two ITR sequences of adeno-associated virus (AAV). It shall be located inside. Furthermore, in certain embodiments, heterogeneous polynucleotide sequences and phi The combined length of the Lar or Stuffer polynucleotide sequences is approximately 3.0–5. The file size should be 5Kb, or approximately 4.0-5.0Kb, or approximately 4.3-4.8Kb.

[0019] Filler or stuffer polynucleotide sequences impair the function or activity of the vector. Unless otherwise specified, it can be placed at any desired position within the vector array. In one embodiment The filler or stuffer polynucleotide sequence is a heterogeneous polynucleotide sequence. The 5' and / or 3' ends adjacent to the outside of the 5' and / or 3' ends, respectively. It shall not be placed between the lateral ITR sequences. In another embodiment, fillers or stuffing The polynucleotide sequence is the 5' and / or 3' ends of the heterogeneous polynucleotide sequence. It is located between the adjacent 5' and / or 3' ITR sequences on the outer side of the terminal. In yet another embodiment, the filler or stuffer polynucleotide sequence is , adjacent to the outside of the 5' and / or 3' ends of heterogeneous polynucleotide sequences, respectively It shall be positioned adjacent to the 5' and / or 3' side ITR sequences. In another embodiment, the filler or stuffer polynucleotide sequence is, for example, a genomic nucleic acid. It is assumed that the nucleotides are positioned within heterogeneous polynucleotide sequences, similar to introns.

[0020] Therefore, in various embodiments, one filler or stuffer polynucleotide mixture The columns are adjacent to the ITR sequence of the AAV, or to a single filler or stuffer poly The nucleotide sequence is located inside the two ITR sequences of adeno-associated virus (AAV). Alternatively, one filler or stuffer polynucleotide sequence is adeno-associated. Located outside the two ITR sequences of the virus (AAV), or two fillers Alternatively, there is a stuffer polynucleotide sequence, and the first filler or stuffer poly The nucleotide sequence is located inside the two ITR sequences of adeno-associated virus (AAV). The second filler or stuffer polynucleotide sequence is adeno-associated virus (AA) This would mean that it would be located outside the two ITR sequences in V).

[0021] Furthermore, in certain embodiments, the filler or stuffer polynucleotide sequence is adeno-linked If located between two ITR sequences of the companion virus (AAV), then a heterologous polynucleo The total length of the combined nucleotide sequence and filler or stuffer polynucleotide sequence is: Approximately 3.0-5.5Kb, or approximately 4.0-5.0Kb, or approximately 4.3-4.8Kb. In another specific embodiment, the filler or stuffer polynucleotide sequence is ad If located outside the two ITR sequences of the associated virus (AAV), the filler... Alternatively, the length of the stuffer polynucleotide sequence shall be greater than 4.7 Kb, approximately 5.0 The file size should be approximately 10.0 KB or about 6.0-8.0 KB.

[0022] Furthermore, in various embodiments, the filler or stuffer polynucleotide sequence is The lengths are approximately 1-10, 10-20, 20-30, 30-40, 40-50, and 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 The sequence should consist of 7,000-8,000 or 8,000-9,000 nucleotides.

[0023] Typically, filler or stuffer polynucleotide sequences are inactive or harmless. , has no function or activity. In various specific embodiments, filler or stuffer polymer The nucleotide sequence is not a bacterial polynucleotide sequence, but rather a filler or stuffer. Polynucleotide sequences are not sequences that code for proteins or peptides, but rather fillers or The stuffer polynucleotide sequence is a heterogeneous polynucleotide sequence, with the AAV reversed end. Re-sequence (ITR), expression regulatory element, origin of replication, selection marker or polyadenine (pol yA) This sequence is different from any of the sequences.

[0024] In other various embodiments, the filler or stuffer polynucleotide sequence is This refers to intron sequences that are related to or unrelated to heterogeneous polynucleotide sequences. In one embodiment, the intron sequence is located inside a heterogeneous polynucleotide sequence. In this embodiment, the intron sequence is such that the intron codes for a protein, and that protein Because it is located within genomic DNA that is also encoded by heterogeneous polynucleotide sequences, It is associated with heterogeneous polynucleotide sequences.

[0025] Also, recombinant AAV vectors containing genomes (capsidized or packaged) The recombinant AAV vector, AAV-Rh74 vector, and capsid variant of the present invention ( For example, related AAV vectors such as the AAV-Rh74 variant containing RHM4-1) particles , can be contained within cells. In such embodiments, cells are lysed and the virus ( AAV) particles (e.g., AAV-Rh74 vector or AAV-Rh74 capsid variant) Helper cells that produce related AAV vectors (for example, RHM4-1), or It contains target cells in which heterogeneous polynucleotide sequences are desirable to be expressed internally.

[0026] Also, recombinant AAV vectors containing genomes (capsidized or packaged) The Recombinant AAV Vector, and AAV-Rh74 Vector and AAV-Rh74 of the present invention Related AAV vectors such as mutant particles (e.g., capsid mutants like RHM4-1) such compositions may contain recombinant vectors (e.g., AAVs). Recombinant vectors containing (capsidizing or packaging) genomes (e.g., AA V), and AAV-Rh74 vectors and AAV-Rh74 variants (e.g., RHM4 This is useful when administering related AAV vectors (such as capsid variants like -1) to patients. .

[0027] Also, recombinant AAV vectors containing genomes (capsidized or packaged) The Recombinant AAV Vector, and AAV-Rh74 Vector and AAV-Rh74 of the present invention Related AAV vectors such as mutant particles (e.g., capsid mutants like RHM4-1) It can be used in various methods and applications. Therefore, it can be applied to living organisms or cells, for example, A method for delivering or introducing heterologous polynucleotide sequences into the cells of a dairy or mammal, and And provide instructions on how to use it.

[0028] In one embodiment, the method or use includes a vector genome (capsidizing, pa (Package) (For example, AAV-Rh74 vector, or AAV-Rh74 variant (example) For example, heteropolynucleo (caused by related AAVs such as capsid variants like RHM4-1) Adeno-associated virus (AAV) vectors containing cytoplasmic nucleotide sequence particles are subjected to suitable conditions. Administer to mammalian cells and introduce the heterologous polynucleotide sequence into the mammalian cells. or includes the step of delivering or introducing into mammalian cells. In one embodiment, the method or use is This enables the introduction / delivery of heterologous polynucleotides into mammals and / or cells. In another embodiment, the method involves introducing / transporting heterologous polynucleotides into mammals and / or cells. This enables the formation of a transcript through the transcription of heterologous polynucleotides. In one embodiment, the method enables the introduction / delivery of heterologous polynucleotides into cells, and then, By copying, a transcript is formed, and then translation is performed to produce genes. It forms substances (proteins). More specifically, for example, in the two embodiments described below, heterogeneous Polynucleotide sequences are expression regulatory elements that confer transcription of heterologous polynucleotide sequences and manipulation They are combined as possible, and if necessary, the transcript is further translated.

[0029] In additional embodiments, the method or use involves introducing a heterogeneous polynucleotide sequence into a subject (e.g., It is intended for delivery or transport to the cells of a mammal or subject (e.g., a mammal). , one virus (for example, AAV-Rh74, or AAV-Rh74 capsid variant) For example, related AAV particles such as RHM4-1), multiple such virus (e.g., AAV) particles The offspring, or one of the viruses (e.g., AAV-Rh74, or AAV-Rh74 capsule) Related AAV particles such as variants (e.g., RHM4-1) or multiple such viruses (e.g., For example, a pharmaceutical composition containing AAV particles is used in a test subject (e.g., a mammal) or a test subject (e.g., a milkmammal). This includes administering a heterologous polynucleotide sequence to cells of the same type. It is used to deliver or transport cells to a subject (e.g., a mammal) or to the cells of a test subject (e.g., a mammal). In another embodiment, the method or use is for those with a deficiency in protein expression or function, It requires the expression or function of a protein, or an endogenous protein (for example, preferably) The expression or function of a protein (which is absent, abnormal, or dysfunctional) needs to be reduced. It is intended to treat a subject (e.g., a mammal) and one virus (e.g., AAV). -Rh74 and AAV-Rh74 variants (e.g., capsid variants such as RHM4-1) ) related AAV particles, multiple such virus (e.g., AAV) particles, or one virus Rus (e.g., AAV-Rh74, and AAV-Rh74 variants (e.g., RHM4-1) Related AAV particles (such as capsid variants) or multiple AAV (e.g., AAV) To provide a pharmaceutical composition containing particles of a single virus (e.g., AAV-Rh 74, and AAV-Rh74 variants (e.g., capsid variants such as RHM4-1), etc. A related AAV particle, multiple such virus (e.g., AAV) particles, or one virus ( For example, AAV-Rh74 and AAV-Rh74 variants (e.g., RHM4-1, etc.) Associated AAV particles (such as capsid variants) or multiple such virus (e.g., AAV) particles The method includes administering a pharmaceutical composition containing a heterogeneous polynucleus to a subject (e.g., a mammal). The creotide sequence is expressed in mammals. Or, here, the heterologous polynucleotide sequence is In the subject (e.g., mammals), endogenous proteins (e.g., undesirable, abnormal, and This refers to repressive sequences or proteins that reduce the expression or function of dysfunctional proteins. It shall be considered to represent the quality.

[0030] The method and use for administration or delivery may be in any form suitable for the subject. i. In certain embodiments, one virus (for example, AAV-Rh74, or AAV-Rh74) may be used. Related AAV) particles such as h74 capsid variants (e.g., RHM4-1) or multiple such Virus (e.g., AAV) particles (e.g., AAV-Rh74 vector, or AAV-Rh Related AAV vectors such as 74-capsid variants (e.g., RHM4-1) are administered intravenously. , into the artery, into the muscle, subcutaneously, into the oral cavity, via intubation, through a catheter, into the skin, head It is administered or delivered into the lid, by inhalation, into the lumen, or onto the mucous membrane.

[0031] The subjects include humans and non-human mammals (e.g., primates). In this state, the subject benefits from the expression of heterologous polynucleotide sequences or their expression It requires.

[0032] In the present invention, recombinant vector (e.g., AAV) plasmid and recombinant vector ( Viruses (e.g., AAV) that are capsidized or packaged (e.g., A AV-Rh74 and AAV-Rh74 variants (e.g., capsid variants such as RHM4-1) A method is provided for producing related AAV particles such as (alternative) particles. In one embodiment, recombinant Methods for producing viruses or AAV particles include recombinant vectors (e.g., AAV) plasmids The virus (e.g., AAV-Rh74) is introduced into packaging helper cells. Related AAs such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1) V) to propagate and infect with recombinant viruses (e.g., AAV-Rh74, or A It produces related AAV particles such as AV-Rh74 capsid variants (e.g., RHM4-1). This includes culturing helper cells under conditions that allow for this process. In other embodiments, contaminating nucleic acids Recombinant viruses containing recombinant viral vectors (e.g., AAV-Rh74) and related AAV-Rh74 variants (e.g., capsid variants such as RHM4-1) A method for producing recombinant viruses or AAV particles with reduced amounts of AAV particles is recombinant The vector (e.g., AAV) plasmid is introduced into packaging helper cells and the virus Propagating and infecting with viruses (e.g., AAV), and recombinant viruses (e.g., AAV-R h74 and AAV-Rh74 variants (e.g., capsid variants such as RHM4-1) This includes culturing helper cells in an environment capable of producing any relevant AAV particles. Here, the recombinant viruses produced (e.g., AAV-Rh74, and AAV-Rh74) Related AAV particles, such as mutants (e.g., capsid mutants like RHM4-1), are contaminating the image. Viruses containing recombinant viral vector genomes with nucleic acids (e.g., AAV-Rh7) 4, and AAV-Rh74 variants (e.g., capsid variants such as RHM4-1), etc. The number of related AAV particles in the recombinant viral vector is due to filler or stuffer polymer. Viruses containing contaminating nucleic acids when produced under conditions without a nucleotide sequence (for example, AAV-Rh74 and AAV-Rh74 variants (e.g., capsids such as RHM4-1) The number of related AAV particles (such as mutants) is reduced. In one embodiment, the number of contaminating nuclei Acids are bacterial nucleic acids, or heterologous polynucleotide sequences, ITRs, promoters, enhancers - The starting point for replication is a sequence other than the poly(A) sequence or the selected marker.

[0033] Helper cells include mammalian cells. In one embodiment, helper cells are heterologous poly(IQS). The nucleotide sequence is used in viral particles (e.g., AAV-Rh74 vector and AAV-Rh74 vector). Related AAV vectors such as h74 variants (e.g., capsid variants such as RHM4-1) It provides a helper (e.g., AAV) function to package AAV particles (such as AAV particles). In the application morphology, helper cells use AAV's Rep and / or Cap proteins (e.g., R Provides ep78 and / or Rep68 proteins. Helper cells provide Rep The polynucleotide encoding the sequence of the Cap protein provides stability. Alternatively, they are temporarily transfected. Helper cells are Rep78 and / or R The ep68 protein sequence is encoded by a polynucleotide, which is either stable or transient. It is transfected.

[0034] The recombinant vector (e.g., AAV) plasmid of the present invention can be used with any strain or different blood. It may be based on any serotype, including hybrids or chimeras consisting of clear forms. The recombinant virus (e.g., AAV) particles of the present invention are typically AAV-Rh74, or A Related AAVs such as AV-Rh74 variants (e.g., capsid variants such as RHM4-1) Based on, but includes hybrids or chimeras with different serotypes. Representative AAV blood The clear types are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, A This includes, but is not limited to, AV8, AAV9, AAV10, AAV11, and Rh10. It is not the case that the recombinant virus of the present invention, including the vector genome (for example) AAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AA VP1 of V7, AAV8, AAV9, AAV10, AAV11, or Rh10 serotype, Like VP2 or VP3 capsid proteins, different serotypes, mixtures of serotypes, It contains capsid proteins derived from hybrids or chimeric forms consisting of different serotypes. Furthermore, the recombinant vector (e.g., AAV), sequence, plasmid, vector of the present invention. A genome can be any single serotype, a mixture of serotypes, or a hybrid of different serotypes. It may contain elements derived from a rhizome or chimera. In various embodiments, recombinant AAV The vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and AAV7. , AAV8, AAV9, AAV10, AAV11, Rh74 or Rh10 serotype, or a mixed hybrid or chimera comprising any of the above AAV serotypes Includes derived Cap, Rep, and / or ITR. [Brief explanation of the drawing]

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

[0036] [Figure 2] This shows the canine FIX plasma concentrations in dogs with hemophilia B after delivery of 312 vector genomes per kilogram (kg) of body weight. AAV vectors were administered intravenously (IV) via the saphenous vein, and FIX concentrations were monitored by ELISA. Therapeutic FIX transgene expression was activated by a liver-specific promoter. AAV8 and AAV-Rh74 vectors functioned nearly equally well in dogs with hemophilia B, outperforming AAV6.

[0037] [Figure 3] The amino acid sequences of AAV-Rh74 VP1, VP2, and VP3 are shown, and for VP1, the polynucleotide (DNA) sequence (sequence ID numbers: 1-4) is also shown.

[0038] [Figure 4]This figure shows the effects of administering AAV8 and AAVrh74 vectors expressing human factor IX (FIX) to rhesus monkeys and non-human primates (under the control of liver-specific promoters), and the expression of FIX in the animals. Compared to other groups of animals administered the same amount, animals administered with the AAVrh74-FIX vector (at least two bars towards the right margin) expressed higher levels of the FIX transgene.

[0039] [Figure 5] The plasma expression levels of human factor IX in animals administered with an AAV human factor IX expression vector capsidized by a specified AAV (such as the RHM4-1 variant or other Rh74 variant) are shown, compared to those of AAV human factor IX expression vectors capsidized by Rh74 and AAV8.

[0040] [Figure 6] This figure shows the effects of administering AAV8 and AAV-Rh74 mutant RHM4-1 vectors expressing human factor IX to cynomolgus monkeys and non-human primates (under the control of liver-specific promoters), and the expression of FIX in these animals. [Modes for carrying out the invention]

[0041] This invention relates at least in part to adeno-associated virus (AAV) serotype AAV-Rh7 4 and AAV-Rh74 capsid variants (e.g., RHM4-1, RHM15-1, RH (M15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6) Related AAV mutants, such as those mentioned above, affect hepatocytes, which are cells of the liver. Based on data showing high directivity. Polynucleotides (e.g., genes, AAV-Rh74, and 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 related AAV variants such as (mutants), therapeutic levels of expression in the liver after intravenous administration were investigated. Furthermore, we can provide AAV-Rh74 vectors and AAV-Rh74 variants. (For example, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM1 Related AAVs (capsid variants such as 5-5, RHM15-4, and RHM15-6) The mutants are generated via gene transfer / delivery at a significantly higher rate than several other serotypes. It regulates the expression levels of the proteins (see, for example, Figures 1, 2, 4, 5, and 6). In particular, AAV-Rh74 vectors and related AAV-Rh74 capsid variants (e.g., R HM4-1) is found in dogs with hemophilia B, and / or in mice and / or cynomolgus monkeys. Furthermore, it is at least as good as, and usually surpasses, AAV8, a representative example of hepatic transduction. With high efficiency, it is possible to target genes delivered to the liver. Therefore, AAV- Rh74 vectors and AAV-Rh74 capsid variants (e.g., RHM4-1), etc. Related AAV variants are used to code for proteins that provide desired or therapeutic benefits (genes). Heterogeneous polynucleotides such as ) and undesirable or defective (e.g., pathological Inhibitors (e.g., antisense) nucleotides that reduce or suppress the expression of a target gene. Because it can be used for introduction / delivery, it can be used to treat a variety of diseases. For example, recombinant vectors are used to introduce / deliver heterologous polynucleotides into cells. For example, the AAV genome is modified using an AAV-Rh74 vector or an AAV-Rh74 capsid mutation. Body (for example, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM) Within related AAV variants such as 15-5, RHM15-4, and RHM15-6, the packaging It can be di- or capsidized.

[0042] As mentioned above, adeno-associated virus (AAV) serotypes AAV-Rh74 and AAV- Rh74 capsid variants (e.g., RHM4-1, RHM15-1, RHM15-2, R Related AAs such as HM15-3 / RHM15-5, RHM15-4, and RHM15-6) The V mutant has been shown to be able to transfer polynucleotide sequences into cells in vitro, in vitro, and in vivo. It provides a means of delivery. Such polynucleotide sequences allow polynucleotides to be delivered. The cells that are being coded will express the encoded protein, thus encoding the protein. This is possible. For example, recombinant AAV vectors can encode the desired protein or peptide. A heterologous polynucleotide that inhibits a sequence (e.g., RNA) when transcribed, for example. For example, a heterogeneous polynucleotide containing a sequence that targets a gene whose expression is inhibited. Therefore, vector delivery and administration to the target (e.g., mammals) is possible. Not only heterologous polynucleotides encoding proteins and peptides, but also expression in the target Inhibitory nucleic acids (NUCs) target genes to inhibit their function. It will be provided for use with leic acid.

[0043] Therefore, according to the present invention, polynucleotide sequences encoding peptides and proteins, and repressive nuclei that directly or transcribably inhibit the expression or function of targeted genes. A vector genome containing a heterogeneous polynucleotide sequence containing an acid (capsidized) (or package) AAV-Rh74 vector and AAV-Rh74 variant (for example) , RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, Related AAV vectors (such as capsid variants RHM15-4 and RHM15-6) A variant is provided.

[0044] Recombinant "vectors" or "AAV vectors" are wild-type genomes of viruses such as AAV. It originates from and uses molecular techniques to remove the wild-type genome from viruses (e.g., AAV). , using foreign nucleic acids such as heterologous polynucleotide sequences (e.g., therapeutic gene expression cassettes) It is being replaced. Typically, in the case of AAV, it is the reverse end repeat (ITR) of the wild-type AAV genome. One or both of the sequences remain in the AAV vector. Regarding the genomic nucleic acid of a virus (e.g., AAV), all or part of the viral genome is foreign. Because it is substituted with the sequence, recombinant viral vectors (e.g., AAV) are viral It is distinct from the AAV genome (for example). Therefore, it is distinct from other genomes such as heterogeneous polynucleotide sequences. If the original sequence is incorporated, the viral vector (e.g., AAV) is called "recombined." It is defined as an "e" vector, and in the case of AAV, the "recombinant" vector is called "rAAV vector". They are sometimes referred to as "Kuta".

[0045] Therefore, recombinant vector sequences (e.g., AAV) are used to ex vivo and in vi cells. To infect (transform) a virus (here, "particles") in vivo or in vivo The recombinant vector sequence can be packaged within (also referred to as "billion"). If an AAV particle is capsidized or packaged within it, that particle is called "rAAV". It may be referred to as such. The virus particle or virion typically capsulates the vector genome. Includes proteins that are encapsulated or packaged. Examples include envelope proteins and AA In the case of V, a capsid protein is an example.

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

[0047] Parvoviruses, including AAV, have a mechanism to ensure that their nucleic acids and genetic material are stably maintained within cells. Because it can enter cells and introduce nucleic acids and genetic material, it is a useful vector for gene therapy. Furthermore, these viruses use nucleic acids and genetic material at specific sites, for example, the first It can be introduced into specific sites on chromosome 9. AAV is not involved in human pathogenic diseases, so A AV vectors do not cause substantial AAV-related pathogenicity or disease in patients. It can deliver various polynucleotide sequences (e.g., therapeutic proteins and drugs).

[0048] AAV-Rh74 and related AAV variants such as AAV-Rh74 or AAV-Rh74 Related AAV serotypes (e.g., capsid variants such as RHM4-1) For example, the VP1, VP2 and / or VP3 sequences are AAV1-AAV11 or Rh Different from or not different from other AAV serotypes, including 10 (e.g., AAV1-AA) The VP1, VP2 and / or VP3 sequences are different from those of either the V11 or Rh10 serotype. (This is correct). The term “serotype” as used in this specification means Other AAV serotypes are used to refer to AAVs that have serologically different capsids. The serological distinction is made when comparing one AAV with another, between antibodies. The determination is based on the absence of cross-reactivity. Such differences in cross-reactivity are usually due to the capsid. This is due to differences in protein sequence / antigenic determinants (for example, AAV serotype V). Differences in P1, VP2, and / or VP3 sequences. AAV-Rh74 variants including capsid variants. Even though the variant may not be serologically different from Rh74 or other AAVs, These AAV-Rh74 variants have at least one characteristic compared to Rh74 or other AAVs. The nucleotides or amino acid residues are different.

[0049] In the traditional definition, a serotype is a group of existing serotypes of a virus of interest whose properties are known. A neutralization activity test was performed on serotype-specific serum to determine the presence of antibodies that neutralize the target virus. It means not to. More isolated strains of viruses that exist in nature have been discovered, and caps As mutants of Sid are created, differences from existing serotypes do not necessarily exist. It is possible that there are no such differences. Therefore, if serological differences are observed in a new virus (e.g., AAV), If not, this new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. In many cases, for mutant viruses with altered capsid sequences, the serotype Neutralizing activity to determine whether they belong to other serotypes based on the conventional definition. Serological testing has not been performed. Therefore, for convenience and to avoid duplication, we will use the term "serotype". The term refers to viruses that are serologically different (e.g., AAV) and viruses that are serologically identical. This broadly refers to both viruses belonging to a subgroup or variant of a particular serotype (e.g., AAV).

[0050] Recombinant vectors (e.g., AAV) plasmids, as well as their methods and uses, are as follows: This includes virus strains or serotypes. Non-limiting examples include recombinant vectors (e.g., A AV) For example, AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, - Any AAV such as 10, -11, -rh74, -rh10, and AAV-2i8 The genome can also be relied upon. The vector may be from the same or different strains or serum. It may depend on the type (or subgroup or variant). A non-limiting example is a single serotype. Recombinant vectors (e.g., AAV) plasmids that rely on NM package the vector It may be identical to one or more capsid proteins. Furthermore, the recombinant vector (for example) AAV plasmids can rely on AAV (e.g., AAV2) serotype genomes. 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 Related AAV variants such as 74 or AAV-Rh74 or AAV-Rh74 variants (for example) , RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, Associated AAVs such as capsid variants (RHM15-4 and RHM15-6) That's good too.

[0051] AAV-Rh74 has a gene / protein sequence identical to the sequence features of AAV-Rh74. (See, for example, VP1, VP2, and VP3 in Figures 3A to 3E). The phrase "AAV vectors related to AAV-Rh74" and its synonyms are, One or more polynucleotide or polypeptide sequences containing AAV-Rh74, and a large portion One or more AAV proteins having the same sequence (e.g., VP1, VP2 and This refers to the AAV vector (or VP3 sequence) related to AAV-Rh74. The ter may have one or more sequences different from AAV-Rh74, but AAV - Shows a sequence that is substantially identical to one or more genes of Rh74, and / or AA Possesses one or more functional characteristics of V-Rh74 (e.g., cell / tissue orientation). Example For example, the AAV variant RHM4-1, which is related to AAV-Rh74, is different from the Rh74 capsid. It has a capsid with four amino acids. Exemplary AAV-Rh74 and AAV-Rh Related AAV variants such as 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 like RHM15-6, are described herein. For example, including VP1, VP2 and / or VP3 in Figures 3A to 3E. In one example of the application method, the AAV vector related to AAV-Rh74 is shown in Figures 3A to 3E. One or more of the AAV-Rh74 VP1, VP2, and / or VP3 sequences are used. At least 80% or more than 80% (for example, 85%, 90%, 95%, 96%) (e.g., 97%, 98%, 99%, 99.5%) Contains or consists of the same sequence It has a polynucleotide, polypeptide, or partial sequence.

[0052] According to the present invention, the method and method of use are provided for the AAV-Rh74 sequence (polypeptide and nucleotide) The leotide (and its partial sequence) is a standard AAV-Rh74 gene or a tan. Protein sequences (for example, the VP1, VP2, and / or VP3 sequences shown in Figures 3A to 3E) Although it shows a concordance rate of less than 100%, the genes for AAV1-AAV11 and AAV-Rh10 Or it is different from and not identical to a well-known AAV gene or protein such as a protein. In one embodiment, the AAV-Rh74 polypeptide or a partial sequence thereof is any standard AA V-Rh74 sequence or a subsequence thereof (for example, VP1 shown in Figures 3A to 3E) (VP2 and / or VP3 sequences) match at least 80% or more than 80% and up to 1 Up to 00% (for example, 85%, 85%, 87%, 88%, 89%, 90%) 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Contains or consists of sequences that match 99% or 99.5% of the original sequence. In this embodiment, AAV-Rh74-related mutants are AAV-Rh74 (e.g., RHM4) -1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15 The four amino acid substitutions mentioned in -4 and capsid variants such as RHM15-6 It has 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 Recombinant vectors containing the relevant hybrid and chimeric sequences of the mutants (e.g., AA) V) Using recombinant techniques known to those skilled in the art, one or more functional AAV ITR sequences are obtained The process involves constructing a sequence containing one or more adjacent heterologous polynucleotide sequences (transgenes). Yes, it is possible. Such vectors can be, for example, rep genes and / or cap genes. , having one or more wild-type AAV genes that are either completely or partially missing It is possible to recover recombinant vectors, replicate them, and package them into AAV vector particles. At least one functional adjacent ITR sequence (functional fl) is required for aging. It holds the anking ITR sequence. Therefore, the AAV vector genome This refers to the sequence required on the same molecule (in cis) for replication and packaging (for example) (Includes ITR sequences).

[0054] In this specification, “polynucleotide” and “nucleic acid” The term "nucleotide (nucleotide)" is used interchangeably, and it is deoxyribonucleic acid. All forms of nucleic acids and oligonucleotides, including DNA and ribonucleic acid (RNA). It refers to polynucleotides, including genomic DNA, cDNA, and antisense DNA, Spliced ​​or unspliced ​​mRNA, rRNA, tRNA, and, Restrictive DNA or RNA (RNAi, e.g., low molecular weight hairpin type) Alternatively, short hairpin (sh) RNA, microRNA (miRNA), low molecular weight RNA Interfering or short interfering (SiO) RNA, trans-splicing RNA, or anti-interfering RNA It contains (nucleotide RNA). Polynucleotides are naturally occurring, synthetic, and intentionally modified. This includes modified polynucleotides, as well as their analogs and derivatives. Polynucleotides are They may exist as a single element, twice or three times their original size, or in a linear or circular form, and of any length. It may have. When discussing polynucleotides, the sequence or structure of a particular polynucleotide may be considered. The structure may be described, and here, following convention, its arrangement is written from 5' to 3'. It can be done.

[0055] "Heterogeneous" polynucleotides are intended to be introduced and delivered into cells via vectors. This refers to polynucleotides inserted into a vector (e.g., AAV). Otide is usually different from the nucleic acid of the vector (e.g., AAV). That is, the virus (e.g.) For example, it is "foreign" to the nucleic acid of AAV. Once introduced / delivered into a cell, the heterologous polymer Nucleotides are housed in virions and are expressible (e.g., transcription and adaptation). (Translation in urgent cases) Or, virions (e.g., AA) introduced and delivered into the cell. The heterogeneous polynucleotides held within V) do not need to be expressed. The term is not always used here when referring to polynucleotides, but polynucleotide When referring to Ochido, even if the modifier "different species" is omitted, it still means "different species" despite the omission. It is intended to contain species polynucleotides.

[0056] Polynucleotide sequences encode polypeptides, proteins, and Peptides, like naturally occurring proteins, have a full-length, native-type sequence (nativ (e sequences) and also include functional subsequences, modified or altered sequences As long as these functional parts retain some degree of the function of the full-length native protein, This includes separate sequences, modified or diverse sequences. In the methods and uses of the present invention, polynucle Such polypeptides, proteins, and peptides encoded by rheotide sequences are , deficiency, or insufficient or incomplete expression in the target mammal. It may be identical to an endogenous protein, but being identical is a necessary condition. That's not the case.

[0057] The present invention relates to adeno-associated virus (AAV) serotypes AAV-Rh74 and AAV-Rh74. Mutants (e.g., RHM4-1, RHM15-1, RHM15-2, RHM15-3 / R) Related to capsid variants such as HM15-5, RHM15-4, and RHM15-6. AAV mutants can deliver polynucleotides stably or transiently to cells and then to progeny cells. Can be used for input / delivery. In this specification, "transgene" refers to a transgene. The term "ene)" is, for convenience, used to refer to a heterologous polynucleotide introduced into such a cell or organ. Used to refer to an ocide. An introduced gene is a polypeptide or protein that codes for a polypeptide. A gene that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed. polynucleotides (for example, those containing expression regulatory elements such as promoters that activate transcription) This includes all polynucleotides, such as those that do not exist.

[0058] For example, in cells containing a transgene, the transgene is transmitted via a vector (e.g., AAV). It is introduced and transferred through the "transformation" of cells. "to transform" or "to transfect" The term "ct)" refers to the introduction of molecules such as polynucleotides into cells or host organisms. This refers to the matter.

[0059] Cells into which an introduced gene has been introduced are called "transformed cells (transformed cells)." It is called a "transformed cell" or "transformed organism". "or "transfected" cells (for example, in mammals, Cells, tissues, or organ cells) are those that incorporate exogenous molecules, such as polynucleotides or This occurs in cells following the introduction of a protein (e.g., a transgene) into the cell. It means a genetic change. Therefore, it is called "genetically modified" or "transformed." The cells are, for example, cells into which exogenous molecules have been introduced, or their progeny. These cells are, The gene may be amplified, and the introduced protein will be expressed or the nucleic acid will be transcribed. Gene therapy For use and methods, transformed cells may be present in the subject.

[0060] The introduced polynucleotides may be incorporated into the nucleic acids of receptor cells or organisms. Or, it may not be incorporated. If the introduced polynucleotide is incorporated into the receptor cell or When incorporated into the nucleic acids (genomic DNA) of an organism, it is stably maintained within the cell or organism. It can be inherited or passed down to receptor cells or offspring (offspring) of an organism. Finally, the introduced nucleic acids may only exist transiently within the receptor cell or host organism. It has a sex.

[0061] What is the origin (e.g., mesoderm, ectoderm, or endoderm) of the cells that are transformed? These may be cells of a tissue or organ type. A non-limiting example of cells is the liver. (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., islet β-cells), lungs, brain (e.g., nerve cells, g Central or peripheral nervous system (such as retinal or ependymal cells) or spinal cord, kidneys, eyes (e.g., retinal cells) Cellular components), spleen, skin, thymus, testes, lungs, diaphragm, heart (cardiac cells), muscles or psoas muscles, Or the intestines (e.g., endocrine cells), adipose tissue (white, brown, or beige fat cells), muscle (For example, fibroblasts), synovial cells, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells Examples include cells, nerve cells of the inner ear, or hematopoietic cells (e.g., blood cells or lymphocytes). Further examples include the liver (e.g., hepatocytes, sinusoidal endothelial cells) and the pancreas (e.g., islet β cells). Central or peripheral cells such as cells, lungs, brain (e.g., nerves, glial or ependymal cells), or spinal cord. The nervous system, kidneys, eyes (e.g., retinal cell components), spleen, skin, thymus, testicles, lungs, diaphragm, heart Organs (heart cells), muscles or psoas muscles, or intestines (e.g., endocrine cells), adipose tissue (white, brown) Colored or beige fat cells), muscle (e.g., fibroblasts), synovial cells, chondrocytes, bone clastic cells. Cells, epithelial cells, endothelial cells, salivary gland cells, nerve cells of the inner ear, or hematopoietic cells (e.g., blood cells) Stem cells, such as pluripotent or pluripotent progenitor cells, that develop or differentiate into cells or lymphocytes. These are some examples.

[0062] In one embodiment, the “therapeutic molecule” is the absence of a protein in a cell or subject. These are peptides or proteins that may alleviate or reduce symptoms resulting from dysfunction. It is a quality. Or, a “therapeutic” peptide or protein encoded by a transgene. This is something that benefits the subject, for example, by repairing genetic defects or gene expression It repairs deficiencies in (or functions) or imparts anti-cancer effects.

[0063] heterogeneous polynucleations encoding useful gene products (e.g., therapeutic proteins) in the present invention A non-limiting example of leotide is cystic fibrin transmembrane regulator (cystic fibrin). rosis transmembrane regulator protein, CF TR), including acquisition of functional blood coagulation factors (e.g., factor XIII, factor IX), blood coagulation factors. Factors (Factor X, Factor VIII, Factor VIIa or Protein C), antibodies, retinal color Retinal pigment epithelial cell-specific 65 kDa protein (retinal pigment epithelial cell-specific 65 kDa protein thelium-specific 65 kDa protein (RPE65), e Rislopoietin (erythropoiesis-promoting factor), LDL receptor, lipoprotein lipase, ol Nittine transcarbamylase, β-globin, α-globin, spectrin, α-antitoxin Lipsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7) ), sulfamidase, enzyme involved in lysosomal storage diseases (A RSA), hypoxanthine guanine phosphoribosyltransferase, β-25 Lucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, Branched-chain keto acid dehydrogenases, hormones, 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, β, etc.), cytokines ( For example, α-interferon, β-interferon, interferon-γ, interleuk Interleukin 2, Interleukin 4, Interleukin 12, Granulocyte-Macrophage Colony Stimulation Factors, lymphotoxins, etc.), suicide gene products (e.g., herpes simplex virus thymidine) Cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytoxin (e.g., din kinase, tumor necrosis factor), drug resistance proteins (for example, drugs used in cancer treatment) Proteins that confer resistance), tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL) adenomatous polyposis (APC), peptides with immunomodulatory functions, tolerogenicity and These are immunogenic peptides or proteins called 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], Insulin Glucokinase, granyl cyclase 2D (LCA-GUCY2D), Rabs Coat protein 1 (congenital choroidal absence), LCA5 (LCA-level cylin), orni Tin keto acid aminotransferase (cerebral gyroretinal choroidal atrophy), retinosin (Retin Usher syndrome (USH1C) is a condition characterized by X-linked retinal schiosis (oschisin)1 and USH1C (usher syndrome). 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR (Type 1 retinitis pigmentosa), DFNB1 (connexin 26 hearing loss), ACHM2, 3 and 4 ( Color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, lyso Genetic defects that cause somma storage disorders (e.g., sulfatase, N-acetylglucosa) Min-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC 2. Sphingolipid-activating proteins, etc.), one or more zinc finger nucleases, or a donor sequence used as a repair template in genome editing, or a gene containing or encoding the same, but not limited thereto.

[0064] Another non-limiting example of a heterologous polynucleotide encoding a useful gene product (e.g., a therapeutic protein) in the present invention is cystic fibrosis (or other diseases of the lung), blood diseases such as 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, (true) diabetes, muscular dystrophy (e.g., Duchenne type (Duchenne ), Becker type (Becker)), Gaucher's disease (Gaucher's di sease), Hurler's disease, adenosine deaminase deficiency, glycogen storage disease and other metabolic disorders, retinal degenerative diseases (other diseases of the eye), and diseases of solid organs (brain, liver, kidney, heart), including those that may be used for the treatment of diseases and disorders, but not limited thereto. All mammalian and non-mammalian forms of polynucleotide-encoding gene products, including the non-limiting genes and proteins disclosed in the specification, are included, whether known or unknown. Therefore, the present invention includes genes and proteins from non-mammals, non-human mammals and humans, and the genes and proteins function substantially similarly to the human

[0065] genes and proteins described below. Non-limiting examples of non-mammalian genes are included, whether known or unknown. Explicitly included. Therefore, the present invention includes genes and proteins from non-mammals, non-human mammals and humans, and the genes and proteins function substantially similarly to the human genes and proteins described below. Non-limiting examples of non-mammalian genes include those from non-mammals, non-human mammals and humans, and the genes and proteins function substantially similarly to the human genes and proteins described below. As for the Fok nuclease domain, which is derived from bacteria, Examples include omain. Non-restrictive examples of FIX sequences in non-human mammals include Yos hitake et al., 1985, cited above; Kurachi et al., 19 1995, cited above; Jallat et al., 1990, cited above; 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. It is described in Acad.Sci.USA 76:4990-4994.

[0066] As described herein, heterologous polynucleotide sequences (transgenes) are repressive and anti- Contains antisense nucleic acid sequences. Inhibitory, antisense, siRNA (small interfering RNA), miRNA (microRNA), shRNA (small hairpin RNA), RNAi Antisense oligonucleotides can regulate the expression of target genes. Such molecules This involves inhibiting the expression of target genes involved in mediating the course of the disease, thereby reducing the symptoms of one or more diseases. It contains molecules that reduce, inhibit, or alleviate the condition.

[0067] Antisense is a single strand that binds RNA transcripts or DNA (e.g., genomic DNA). It includes double-stranded or triple-stranded polynucleotides and peptide nucleic acids (PNAs). The oligo obtained from the transcription start site, for example, a position between -10 and +10 from the start site. Nucleotides are another specific example. Triple-stranded structure (triplex forming) Antisense can bind to double-stranded DNA and inhibit gene transcription. "RNAi" is the use of single-stranded or double-stranded RNA sequences to inhibit gene expression. (For example, Kennerdell et al., Cell 95:1017(1 1998); and Fire et al., Nature, 391:806 (1998) (See reference). Therefore, in the method and use of the present invention, two from the target gene coding region This stranded RNA sequence may be used to inhibit or prevent gene expression / transcription. RNAi and RNAi encode target gene sequences (e.g., huntingtin or HTT). It can be created based on nucleic acids, for example, nucleic acids that encode mammalian and human HTT. If a single-stranded or double-stranded nucleic acid (e.g., RNA) is labeled with HTT transcription (e.g., mRNA), It can be used as a target.

[0068] siRNA is used for sequence-specific post-transcriptional gene silencing or gene knockdown. This refers to therapeutic molecules involved in the RNA interference process for target genes. siRNA is a cognate of the target gene. (Cognate) mRNA sequences are homologous. Small interfering RNAs (siRNAs) ) can be prepared by in vitro synthesis or by cleavage of longer double-stranded RNAs with ribonuclease III and is a mediator of sequence-specific mRNA degradation. The siRNAs or other such nucleic acids of the invention can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA / RNA synthesizers. siRNAs can be synthesized as two separate complementary RNA molecules or as a single RNA molecule having two complementary regions. Vendors of synthetic RNA molecules or synthetic reagents include 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). Specific siRNA constructs for inhibiting the mRNA of a target gene can be 15-50 nucleotides in length and more typically about 20-30 nucleotides in length. Such nucleic acid molecules can be readily incorporated into the viral vectors disclosed herein by conventional methods known to those of skill in the art.

[0069] Non-limiting examples of genes (e.g., genomic DNA A) or pathogenic gene (RNA or mRNA) transcripts that may be targeted using the inhibitory nucleic acid sequences according to the invention include, but are not limited to, Examples, though not limited to them, include the Huntington's (HTT) gene. Genes associated with nucleotide repeat diseases, dentatorubralpallidoluysian atrophy (denta torubropallidolusyan atropy) (for example, atropine ( atrophin)1, ATN1); androgens on the X chromosome in spinal and bulbar muscular atrophy Ataxin receptors, human ataxin-1, -2, -3 and -7, (CACNA) Ca encoded by 1A) v 2.1 P / Q voltage-gated calcium channels, TATA The binding protein, also known as ATXN8OS, is the reverse chain of ataxin 8, and is associated with spinocerebellar ataxia (1, Serine / threonine protein phosphata in types 2, 3, 6, 7, 8, 12, and 17 -ze2A 55 kDa regulatory subunit B beta-isoform, fragile X disease FMR1 (Fragile X Intellectual Disability 1), Fragile X-related tremor / motion FMR1 in ataxia (Vulnerable X Intellectual Disability 1), FMR1 in fragile XE intellectual disability (Brittle) Mild intellectual disability (2) or AF4 / FMR2 family member; myotonic dystrophy Myotonin-protein kinase :MT-PK); Frataxin in Friedreich's ataxia, muscle Mutations of the superoxide dismutase 1 (SOD1) gene in atrophic lateral sclerosis (ALS) ;Genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein C (APOB) and proprotein convertase subtilisin (proprotein c onvertase subtilisin) / kexin type 9 (PCSK 9) hypercholesterolemia; HIV infection HIV Tat in infections, the transcription gene of human immunodeficiency virus transactivator ( transactivator; HIV TAR in HIV infection, human immunodeficiency virus Viral transactivation activator response element gene; CC gene in HIV infection Mokine receptor (CCR5); Roussarcoma virus (RSV) RSV nucleocapsid protein in infectious diseases, liver in hepatitis C virus infection Unique microRNA (miR-122); p53, acute kidney injury or organ function after kidney transplantation Acute renal failure due to impaired function or kidney damage; in progressive, recurrent, or metastatic solid malignancies Protein kinase N3 (PKN3); LMP2, which is the proteasome (prot Also known as easome subunit beta type 9 (PSMB9) metastatic melanoma; LMP7 is a metastatic melanoma caused by proteasome subunit beta-8 (PSMB8). Also known as MECL1, this is a proteasome subunit beta-type 10(P SMB 10) Also known as metastatic melanoma; vascular endothelial growth factor (V) in solid tumors EGF; Kinesin spindle in solid tumors Protein, apoptosis suppression in chronic myeloid leukemia (CLL) / lymphoma (BC 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 in hepatitis C infection (dia cylglycerol acyltransferase 1 (DGAT1), familial Beta-catenin in adenomatous polyposis; beta-2 adrenergic receptor; glaucoma; diabetic jaundice DA, known as RTP801 / Redd1 in varicose edema (DME) and age-related macular degeneration. N-damage-induced transcript 4 protein; age-related macular degeneration or choroidal neovascularization (choro Vascular endothelial growth factor receptor I in idal neivascularization (VEGFR1); caspase 2 in non-arteritic ischemic optic neuropathy; congenital onychoplasty Keratin 6AN17K mutant protein in influenza infection The genome / gene sequence of the virus A; the role of the virus in severe acute respiratory syndrome (SARS) infection. Ronavirus genome / gene sequence; RSV in respiratory syncytial virus (RS) infection Virus genome / gene sequence; Ebola filovirus genome / gene in Ebola infection Sequence; Hepatitis B and C virus genomes / gene sequences in hepatitis B and hepatitis C infections ;Herpes simplex virus (HSV) genotype in herpes simplex virus (HSV) infection Mu / genetic sequence, Coxsackievirus B3 infection Coxsackievirus B3 genome / gene sequence; tosin in primary dystonia (torsin)A (TOR1A), pan-class I, and specific HL in transplantation Silencing of pathogenic alleles of genes like the A allele (targeting alleles) Silencing; or mutations in autosomal dominant retinitis pigmentosa (adRP). Heterorhodopsin genes (RHO).

[0070] Polynucleotides, polypeptides, and their subsequences include modified and mutant forms. The terms “modified” or “variant” as used herein refer to The words ) and their synonyms refer to polynucleotides, polypeptides or their parts This means that the sequence is a sequence that deviates from the standard sequence. Therefore, it is a modified and Variant sequences are nearly identical to standard sequences, or exhibit more or less activity. Alternatively, it may have the function of the standard sequence, but at least partially it has the activity or function of the standard sequence.

[0071] Furthermore, the present invention includes natural or unnatural manifolds. Such manifolds include A This includes AAV-Rh74 variants such as AV-Rh74 capsid variants. -Specific examples of Rh74 capsid variants include RHM15-1, RHM15-2, RHM 15-3, RHM15-4, RHM15-5, RHM15-6, and RHM4-1 (for example) (See Figure 5)

[0072] Furthermore, such manifolds include both function-gaining and function-lossing manifolds. For example, The wild-type human FIX DNA sequence in which protein variants or mutants retain activity is this In the method and use of the invention, a therapeutically effective or non-mutant human FIX and It has equivalent or greater therapeutic activity. For example, Collagen IV has: It plays a role in capturing FIX, and once incorporated into mammalian muscle tissue, some of the FIX becomes muscle Because it is retained in the interstitial space of muscle tissue, it cannot participate in blood coagulation. Mutations in the FIX sequence that result in a protein with reduced binding (e.g., loss of function) For example, in the treatment of hemophilia, this is a useful mutation in the method of invention. An example of atypical human FIX gene is human FIX protein from the start of the mature protein to 5 The lysine at the second amino acid position codes to the amino acid alanine.

[0073] An example of non-restrictive modification is the positioning of one or more nucleotides or amino acids in the reference sequence. Exchange (for example, 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-3) 0, 30-40, 40-50, 50-100, or even more nucleic acids or residues (base), for example, lysine residues are substituted with arginine (e.g., RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15- 6) Substitution of lysine with one or more 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 even more nucleotides or residues), This includes and deletions (e.g., subsequences or fragments). In specific embodiments, the modified sequence Alternatively, the variant sequence retains at least partially the function or activity of the unmodified sequence. Such variations and manifolds may have the functions or features of the standard sequence, such as those described below. It may have less, the same, or greater function or activity than the standard sequence, but at least the function of the standard sequence. Alternatively, it partially retains activity.

[0074] A manifold is a difference or modification of one or more non-conservative or conserved amino acid sequences, or It can have both. "Titation" refers to a combination of biologically, chemically, or structurally similar residues. This involves substituting one amino acid. "Biologically similar" means that the substitution results in a biological similarity. This means that the activity is not lost. "Structurally similar" means that the amino acids are of similar length. Having a chain, for example, alanine, glycine, and serine or similar sizes. Chemically similar means that the residues have the same charge, or are both hydrophilic or hydrophobic. This means having [a certain characteristic]. Specific examples include isoleucine, valine, and leucine. Substitution by a single hydrophobic residue such as methyl phosphate or methionine, or by arginine substitution of lysine. Substitution (e.g., RHM15-1, RHM15-2, RHM15-3 / RHM15-5, R HM15-4 and RHM15-6), substitution of aspartic acid with glutamic acid, as Substitutions by a single polar residue, such as the substitution of paragine with glutamine, or threonine This includes substitution with serine, etc. Specific examples of conservative substitution include, for example, isoleucine. Substitution with a single hydrophobic residue such as n, valine, leucine, or methionine, or lysine Substitution of arginine, substitution of aspartic acid with glutamic acid, substitution of asparagine This includes substitutions by a single polar residue, such as substitution by lutamine. For example, conservative lutamine Acid substitutions typically include substitutions within the following groups: glycine, a Lanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine , glutamine; serine, threonine; lysine, arginine; and phenylalanine, Tyrosine. "Conservative substitution" also involves using a substituted amino acid in place of an unsubstituted parent amino acid. This includes using it.

[0075] Thus, the present invention relates to one or more biological activities (for example, functions in blood coagulation). Genes and protein variants (e.g., polynucleotides such as those described below) that carry such genes and protein variants This includes a variant of an ocidal encoding protein. Plitter variants have altered or added properties to proteins or polypeptides. Proteins that have been modified or may be modified using DNA recombination technology or Contains polypeptides, for example, enhanced protein stability in plasma, or enhanced It contains manifolds that confer the protein activity. The manifolds are native polynucleotides. The sequence may differ from a standard sequence such as that of a protein or peptide.

[0076] At the nucleotide sequence level, native and non-natural variant genes are typically standard. The sequence matches at least approximately 50%, more typically approximately 70%, and even more typically approximately 80%. (90% or more identical). At the amino acid sequence level, there is natural and unnatural diversity. Body proteins are typically at least about 70% of standard proteins, more typically While the agreement rate is approximately 80%, and more typically around 90% or higher, in the non-conservative region... Areas where there is a large difference are acceptable (e.g., 60%, 50%, or 40%). (For example, a match rate of less than 70%). In another embodiment, compared to the standard sequence, at least Also, agreement rates of 60%, 70%, 75% or higher (for example, 80%, 85%, 90%) It has an agreement rate of 95%, 96%, 97%, 98%, 99%, or higher. Induction of nucleotide and amino acid changes within rheotides, proteins, or polypeptides. The input process is well known to those skilled in the art (e.g., Sambrook et al, Mole cular Cloning: A Laboratory Manual (2007) reference).

[0077] "Identity," "homology," and their synonyms are, In the case of an "aligned" array, two or more target entities This means that the referenced entities are the same. Therefore, if two polypeptides are identical, at least a standard region or intraprotein region is required. In this case, they have the same amino acid sequence. They have the same polynucleotide sequence, at least within a standard region or within the protein. Identity may exist beyond the defined area (region or domain) of the sequence. An “area” or “region” refers to two identical or more than two identical areas. It refers to a portion of the target entity. Therefore, two protein sequences or nucleic acid sequences. However, if a match occurs across one or more sequence areas or regions, these sequences are considered to be within that region. They have identity within the region. “Aligned” sequences are often This includes corrections to missing or extra bases or amino acids (gaps) compared to the standard sequence. This refers to a sequence of multiple polynucleotides or proteins (amino acids).

[0078] The sequences may match along their entire length, or they may match in only a portion of their length. In certain embodiments, the lengths of sequences sharing a percentage agreement rate are 2, 3, 4, 5 or so. A continuous polynucleotide or amino acid exceeding 10, for example, 6, 7, 8, 9, 10 , consecutive amino acids such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 In another specific embodiment, the length of the matching sequence is 20 or more consecutive sequences. These are polynucleotides or amino acids, for example, 20, 21, 22, 23, 24, 25 , consecutive amino acids such as 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 In yet another specific embodiment, the length of the matching array is 35 or greater than 35. These are consecutive polynucleotides or amino acids, for example, 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 45, 47, 48, 49, 50 etc consecutive A It is a mino acid. In yet another specific embodiment, the length of the matching sequence is 50 or 50. A series of polynucleotides or amino acids exceeding 50-55, 55-60 , 60-65, 65-70, 75-80, 80-85, 85-90, 90-95, 95- These are consecutive polynucleotides or amino acids, such as 100, 100-110, etc.

[0079] The terms "homologous" and "homology" The word is a term used when two or more target entities are less than one in a given area or part. This also means that they partially coincide. "Homologous or identical" means "area, region or A "domain" is defined as a group of two or more entities that are homologous or identical in some respect. This means that the two arrays span one or more array regions. In this case, these sequences have identity within their respective regions. "Substantial homology ( "Substantial homology" means a standard molecule or a standard molecule having homology. The structure or function (e.g., biological function) of the related / corresponding region or part of a quasi-molecule. At least one of the structures or functions of the molecule (or activity) is a partial structure or function of the molecule. This means that the molecule is structurally or functionally preserved, as is expected. .

[0080] The range of agreement (homology) between two sequences is determined by computer programs and numerical calculations. This can be verified using gorism. Such sequence percentage agreement (homology) The algorithm for calculating this typically involves array gaps and discrepancies in the comparison region or area. Calculate the search algorithm of BLAST (e.g., BLAST 2.0). For example, Altschul et al., J. Mol. Biol. 215:403 (199 0) See reference (publicly available from NCBI), which shows typical search parameters such as the following: Possesses: Mismatch -2, Gap interval (gap open) 5, Gap extension (gap extension)2. For comparing polypeptide sequences, the BLASTP algorithm is generally used. Set the rhythm to PAM100, PAM 250, BLOSUM 62 or BLOSUM It is used in combination with a scoring matrix like the one shown in 50. To quantify the degree of agreement, use FASTA (e.g., FASTA2 and FASTA3). The SSEARCH sequence comparison program is also 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) Using Delaunay-based phase mapping, the protein structure Programs for quantifying similarity have also been developed (Bostick et al., Bi ochem Biophys Res Commun.304:320(2003)) .

[0081] Polynucleotides include, for example, adducts and inserts, which are heterogeneous domains. For example, heterogeneous domains can form covalent or non-covalent bonds to a compound of any type of molecule. This may also be the case. Typically, adducts and inserts (e.g., heterogeneous domains) are complementary or heterogeneous. To impart a certain function or activity.

[0082] Adducts and inserts include one or more that are not normally present in the standard natural (wild) sequence. A chimeric sequence is a polynucleotide sequence or protein sequence in which molecules are covalently bonded to each other. Includes sequences and fusion sequences. "Fusion" or "chimeric" The word "and its synonyms" when used in reference to a molecule refers to a part or portion of the molecule. However, this means that it includes a different entity (of a different type) that is distinct from the molecule in question. Those parts do not normally exist together in a natural state. That is, they are fused or chimeras. A part includes or is composed of parts that do not normally coexist. They are structurally distinct.

[0083] The term "vector" refers to a device that manipulates polynucleotides through insertion or incorporation. Plasmids, viruses (e.g., AAV vectors), cosmids, or other transportable materials. This refers to a vehicle. Such vectors guide polynucleotides into cells. Genes that are imported / transported, and that transcribe or translate the inserted polynucleotides into cells. It is used for the operation (i.e., “cloning vector”). The vector nucleic acid sequence is Generally, it contains at least one origin of replication for proliferation within the cell, and optionally, heterogeneous polynucleotides. Rheotide sequence, expression regulatory elements (e.g., promoter, enhancer), selection marker (e.g.) For example, antibiotic resistance, and additional factors such as polyadenine sequences.

[0084] As used here, it is a modifier for viral vectors such as recombinant AAV vectors. as a modifier of sequences such as recombinant polynucleotides and polypeptides The term "recombinant" refers to a composition (e.g., AAV or sequence) that does not normally occur in nature. This means that it has been manipulated (i.e., modified) in a specific style. Like an AAV vector. One example of a recombinant vector is one that is normally present in the genome of wild-type viruses (e.g., AAV). One example is when a polynucleotide that is not present is inserted into the viral genome. An example of recombinant polynucleotides is a heterologous polynucleotide that codes for a protein. A gene (for example) is usually associated with a virus (for example, AAV) genome. Cronin within the vector with or without ',3' and / or intron regions This is one example of a case where it is modified. Here, the term "recombinant" refers to a type of vector such as AAV vectors. The term is always used to refer to sequences such as vectors, polynucleotides, and polypeptides. It is not something that can be found, but includes AAV, as well as polynucleotides and polypeptides. Recombination types in arrays are to be explicitly included, even if they are omitted.

[0085] Recombinant vectors "genomes" (e.g., AAV vector genomes) allow cells to ex vivo To infect (transduce or transform) in vitro or in vivo Viruses (also referred to here as "particles" or "virions") undergo capsidization or packaging within the virus. It can be caged. Recombinant AAV vector genomes can be capsidized or packed into AAV particles. When caged, these particles are sometimes referred to as "rAAV". Alternatively, virions typically produce proteins that capsidize or package vector genomes. Includes, for example, viral capsids and envelope proteins, and in the case of AAV. AAV capsid protein is one example.

[0086] For recombinant plasmids, the vector "genome" is ultimately packaged or capsulated. This refers to a portion of the recombinant plasmid sequence that is converted into a virus particle. When recombinant vectors are constructed or manufactured using the recombinant gene, the vector genome is the recombinant gene. It does not contain any "plasmid" portion that does not correspond to the vector genome sequence of Mido. The non-vector genome portion of Rasmid is called the "plasmid backbone," and is involved in replication and recombination. It is important in the plasmid cloning and amplification processes necessary for virus production. However, the virus itself is not packaged or capsidized within the virus (e.g., AAV) particle. It will not be done.

[0087] Therefore, the vector "genome" is packaged by a virus (e.g., AAV) or This refers to a portion of a vector plasmid that has been capsidized and contains heterologous polynucleotide sequences. The non-vector genome portion of the replacement plasmid is important for plasmid cloning and amplification. It includes the backbone, but the package or data itself can be infected by a virus (e.g., AAV). It is not psidified.

[0088] Viral vectors are derived from one or more nucleic acid elements that make up the viral genome, or Or they are created based on them. Certain viral vectors include adeno-associated viruses ( This includes parvovirus vectors such as AAV vectors.

[0089] Recombinant vector sequences are manipulated by the insertion or incorporation of polynucleotides. A tar plasmid typically has at least one replication origin for growth within a cell, and , comprising one or more expression regulatory elements.

[0090] A vector sequence containing an AAV vector can contain one or more "expression regulatory elements". Typically, expression regulators affect the expression of manipulably bound polynucleotides. This is a nucleic acid sequence that gives the following promoter and Regulatory elements, including expression regulatory elements such as enhancers, are responsible for appropriate heterologous polynucleotide transcription. and, where possible, include translation (e.g., promoter, enhancer, Intron splice signals enable the correct gene translation of mRNA. Maintaining the reading frame (stop codon, etc.). While eel elements usually act in the cis state, they can also act in the trans state. That's fine.

[0091] Expression regulation is effective at levels such as transcription, translation, splicing, and message stability. It is possible. Typically, expression regulatory elements that regulate transcription are transcribed polynucleotides. It is juxtaposed near the 5' end (i.e., “upstream”). The expression regulatory element is also transcribed. At the terminal 3' of the sequence (i.e., “downstream”) or within the transcript (e.g., within an intron) It can be positioned at a certain distance from the transcribed sequence, or at a long distance. Decomposition, (for example, from polynucleotides, 100-500, 500-1000, 2000) (Positioned at intervals of ~5000, 5000~10,000 or more nucleotides) This is possible. However, due to the limitations on the length of the polynucleotides in AAV vectors... These expression regulatory elements typically consist of 1 to 1000 polynucleotides. It is located within the range of nucleotides.

[0092] Functionally, the expression of heterologously linked polynucleotides acts as a regulatory factor (e.g.) For example, it can be controlled at least partially by promoters, and as a result, regulatory factors It regulates the transcription of polynucleotides and, under appropriate conditions, also regulates the translation of transcripts. A specific example of your element is typically a promoter located at 5' of the sequence being transcribed. One example is an enhancer, which is a transcriptional regulator. They may be located at the 5', 3' of the sequence being transcribed, or within the sequence being transcribed.

[0093] The term "promoter" used here refers to a polynucleotide that codes for recombinant products. It can refer to a DNA sequence located near the ocidal sequence. A promoter is usually, for example, It is linked to nearby sequences, such as heterologous polynucleotides, in a way that allows it to function. - Normally, the amount expressed from a heterologous polynucleotide is expressed in the absence of a promoter. Increase the amount expressed compared to the amount normally expressed.

[0094] The term "enhancer" used here refers to a substance located in the vicinity of a heterologous polynucleotide. It may refer to a sequence that does this. Enhancers are usually located upstream of promoters, It can also be located downstream of or within a DNA sequence (e.g., heterologous polynucleotides). Yes, it works. Therefore, the enhancer is 100 base pairs of heterogeneous polynucleotides, 200 salts. Enhancers can be located at the base pair, 300 base pairs, or further upstream or downstream. The expression of heterologous polynucleotides is increased beyond the expression level increased by the promoter sequence. To add.

[0095] Expression regulators (e.g., promoters) are factors that are active in specific types of tissues or cells. This includes, and here we refer to it as "tissue-specific expression regulatory elements / promoters". The control element is usually a specific cell or tissue (e.g., liver, brain, central nervous system, spinal cord, eye, retina). It is active in cells (such as bone, muscle, lung, pancreas, heart, and kidney cells). The expression regulators are specific By transcription-activating proteins or other transcriptional regulators specific to a particular type of cell, tissue, or organ. Because it is recognized in this way, it usually has activity within such cells, tissues, or organs.

[0096] For example, if you want expression within skeletal muscle, you would use a promoter that is active in muscle. Such promoters include the α-actin skeleton and myosin light chain 2A. , dystrophin, promoter from the gene encoding muscle creatine kinase, and It includes synthetic muscle promoters that have higher activity than naturally occurring promoters. (e.g. Li, et al., Nat. Biotech. 17:241-245 (See 1999). As an example of a tissue-specific promoter, for the liver, albumin (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), Regarding bone, osteocalcin (Stein et al., Mol. Biol. Rep., 2 4:185-96 (1997), bone sialoprotein (Chen et al., J. Bone Miner. Res. 11:654-64 (1996), on lymphocytes CD2 (Hansal et al., J. Immunol., 161:1063-8 (19 98)) Immunoglobulin heavy chain, T cell receptor α chain, and for nerves, nerve cell-specific ene NSE promoter (Andersen et al., Cell. Mol. Neur obiol., 13:503-15 (1993), Neurofilament Light Chain Genetics Child (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991), neuronal cell-specific VGF gene (Piccioli, et al., Neuron, 15:373-84 (1995)) is particularly noteworthy. It can be done.

[0097] Furthermore, the expression regulatory elements include ubiquitous or indiscriminate promoters / enhancers, These can drive expression in numerous different cell types. The child is the earliest promoter / enhancer sequence of cytomegalovirus (CMV), Rouss Sarcoma virus (RSV) promoter / enhancer sequences, and various types of mammals Other viral promoter / enhancer sequences that are active in cells, or that do not exist in nature Synthetic factors (e.g., Boshart et al., Cell, 41:521-530 (1985) (See reference), SV40 promoter, dihydrofolate reductase promoter, cytoplasmic β-actin This includes the tin promoter and the phosphoglycerol kinase (PGK) promoter. However, this is not limited to these.

[0098] The expression regulatory elements are also linked in a way that makes them controllable, i.e., activatable by signals or stimuli. To express heterologous polynucleotides in a manner that increases or decreases their expression. It can also increase the expression of operably bound polynucleotides in response to a signal or stimulus. The adjustable elements that are added are called "inducible elements". It is also called (guided by signals). Specific examples are limited to this. However, hormone-inducible promoters (e.g., steroids) are included. In response, a moduloable element that reduces the expression of a operably bound polynucleotide is It is also called a “repressible element” (that is, trust The signal is reduced in a manner such that the expression increases when the number is removed or absent. Typically, the amount that increases or decreases due to such factors is the signal present. Alternatively, it is proportional to the amount of stimulation; as the amount of signal or stimulation increases, the increase and decrease in expression also increase. It becomes effective. A specific non-specific example is zinc-derived sheep metallothionein (MT). Promoter, steroid hormone-induced mouse mammary tumor virus (MMTV) promoter T7 polymerase promoter system (WO 98 / 10088), Tetrasyl Clin-inhibiting system (Gossen et al., Proc. Natl. Acad. Sc) i. 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 See also 2-518 (1998), RU486 inductive 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 Systems (Magari et al., J. Clin. Invest. 100:2865- 2872 (1997); Rivera et al., Nat. Medicine. 2:10 28-1032 (1996)) is cited as an example. In this context, the potential usefulness Other controllable regulators include, for example, temperature or certain physiological conditions such as the acute phase. It is subject to control.

[0099] Expression regulatory elements also include natural factors for heterologous polynucleotides. Factors (e.g., promoters) allow the expression of heterologous polynucleotides to mimic native expression. It can be used when desired. Natural regulators are used in the development of heterologous polynucleotides. Controlling the present state transiently or developmentally, in a tissue-specific manner, or in response to specific transcriptional stimuli. It may be used at times. Introns, polyadenylation sites or Kozak Other natural regulators, such as consensus sequences, may also be used.

[0100] The term "operable linkage" as used in this specification Or the phrase “operably linked” means The physical or functional aspects of the components as described so that the components can function in the intended manner. This refers to a juxtaposition, a manipulative binding with polynucleotides. In examples of expression regulation, the regulatory elements have a relationship that adjusts the expression of nucleic acids. Specifically, for example, two manipulably linked DNA sequences are two DNA sequences that are... At least one of the two DNA sequences exerts a physiological effect on the other sequence. This means they are arranged in a relationship (cis or trans) that allows for such a relationship to occur.

[0101] A vector containing an AAV vector may contain one or more “expression regulatory elements”. These elements include, but are not limited to, AAV ITR array, promoter / Enhancer elements, transcription termination signals, 5' or 3' untranslated regions adjacent to polynucleotide sequences This includes a region (e.g., a polyadenylated sequence), or all or part of intron I. Such elements also include, if necessary, transcription termination signals. Unrestricted transcription termination signals A specific example is the SV40 transcription termination signal.

[0102] As disclosed herein, AAV vectors typically range from about 4kb to about 5.2kb or It is possible to insert DNA that is slightly larger than this, within a defined size range. Therefore, For short sequences, they are acceptable as AAV vectors packaged in viral particles. To adjust the length of the viral genome sequence to the normal size or a length close to it, the staff The insert contains fur or filler. In various embodiments, filler / stuff Nucleic acid sequences are untranslated (non-protein-coding) segments of nucleic acids. In certain embodiments of the AAV vector, the heterologous polynucleotide sequence has a length of 4. Less than 7Kb, the filler or stuffer polynucleotide sequence is heterogeneous polynucleotide The total length when combined with the rheotide sequence (for example, inserted into a vector) is approximately 3.0 to 5.5. It is in KB, or 4.0-5.0kb, or 4.3-4.8kb.

[0103] Introns also help to achieve the length necessary to package the AAV vector into a viral particle. It can function as a filler or stuffer polynucleotide sequence. Introns and i, which function as filler or stuffer polynucleotide sequences. Intron fragments (for example, part of intron I of FIX) can also improve expression. For example, including introns can improve gene expression compared to when introns are absent. This could potentially cause it (see Kurachi et al., 1995, above).

[0104] The use of introns is not limited to the inclusion of intron I sequences in FIX. This includes other introns, and such introns are the same gene (e.g., heterologous polynucleotides). When a character codes for FIX, the intron is an intron that exists within the genome sequence of FIX. (derived from) or related to a completely different gene or another DNA sequence This may also be the case. Therefore, in the present invention, related genes (heterogeneous polynucleotides) The sequence is the whole or a part of the same protein as the protein encoded by the genome sequence. (encodes), and non-homogeneous (unrelated) genes (heterogeneous polynucleotide sequences in the genome) (The sequence codes for a protein different from the protein that is encoded by it.) Other untranslated elements within nucleic acids, such as introns present in the NM sequence (protein coding) (i) The region also functions as a filler or stuffer polynucleotide sequence.

[0105] The term “part of intron I” as used herein refers to a part of intron I. Compared to FIX expression in the absence of plasmid or viral vector template In this region, approximately 0.5 times or more enhances FIX expression. This refers to an intron I region with nucleotides ranging in length from 1 kb to approximately 1.7 kb. Specifically, it is the 1.3kb portion of intron I.

[0106] The term "oligonucleotide" used here refers to two or more, usually four or more, ribonucleotides. A sequence defined as a nucleic acid molecule composed of nucleic acids or deoxyribonucleic acid, primer and probes. The exact size of oligonucleotides depends on various factors, oligonucleotides Depending on the specific use and application of the rheotide, typically oligonucleotides are about 5- It has a length of 50 nucleotides.

[0107] The term "primer" used here refers to a single-stranded or double-stranded biological system It is derived from mu, produced by restriction enzyme digestion, or synthesized, and is suitable for use in a ring. Under certain conditions, it can function as an initiator for template-dependent nucleic acid synthesis. This refers to DNA oligonucleotides. Appropriate nucleic acid templates, appropriate nucleosides of nucleic acids. Triphosphate precursor, polymerase enzyme, appropriate cofactor, and appropriate temperature and pH, etc. Under appropriate environmental conditions, the polymerase or similar activity can cause the primer to 3 Nucleotides are added to the primer to extend it, and the primer extension product can be obtained. It is possible. The length of the primer may vary depending on specific environmental conditions and application requirements. For example, in diagnostic applications, the length of oligonucleotide primers is typically 15 ~30 nucleotides or more. This initiates the synthesis of the desired extension product, i.e., Appropriately juxtaposed plastics are provided for the initiation of synthesis by polymerase or similar enzymes. To anneal with the desired template chain in a manner sufficient to give the 3' hydroxyl group of the imer Therefore, the primer must have sufficient complementarity to the desired template. The primer sequence does not need to be perfectly complementary to the desired template. For example, non The complementary nucleotide sequence is linked to the 5' end of the complementary primer in other regions. Good. Alternatively, the primer sequence may match the desired template sequence for the synthesis of the extension product. Insofar as it has sufficient complementarity to functionally provide a template-primer complex, non-phase Complementary bases may be scattered within the oligonucleotide primer sequence.

[0108] Polymerase chain reaction (PCR) is U.S. Patent No. 4,683,195, 4,800, It is described in issues 195 and 4,965,188.

[0109] The expression "specifically forms hybrids" is not commonly used in this field. Under predetermined conditions, two single-stranded nucleic acid molecules with sufficient complementarity associate. It means (sometimes expressed as "fully complementary"). In particular, this term means fully complementary This refers to the hybrid formation of two polynucleotide sequences having a non-complementary sequence. Hybridization with the main strand nucleic acid sequence is effectively ruled out.

[0110] When a "selection marker gene" is expressed, it can lead to the delivery of antibiotics (e.g., Kanamycin) to transformed cells. This refers to a gene that confers a selectable phenotype, such as resistance. A "reporter" gene. A reporter gene is a gene that provides a detectable signal. A non-limiting example of a reporter gene is... It has a luciferase gene.

[0111] Polynucleotides and polypeptides, including modified forms, may be used in cell expression or in vitro. Produced using various standard replication and recombinant DNA technologies via translation and chemosynthesis techniques. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, etc. It is determinable. For example, nucleic acids undergo hybridization. n) Isolating using computer-based database screening technology. This is possible. Such technologies include, but are not limited to, the following: (1 ) Using probes to detect homogeneous nucleotide sequences in genomic DNA or cDNA (2) Hybridization of the expression library; for example, using an expression library to create a common structure Antibody screening to detect polynucleotides having specific characteristics; (3) Target nucleic acid sequence Using primers capable of annealing, for genomic DNA or cDMA polymerase chain reaction (PCR); (4) Compilation of sequence databases of related sequences Search by computer; and (5) difference screening of the subtracted nucleic acid library. .

[0112] Polynucleotides and polypeptides, including their modified forms, are synthesized, for example, by automated synthesis machines. Location (e.g., Applied Biosystems, Foster City, CA) Skilled technicians like (see reference) can produce it by chemical synthesis using known methods. Peptides can be synthesized whole or partially using chemical methods (for example) Caruthers (1980). Nucleic Acids Res. Sym p. Ser.215; Horn (1980); and Banga, AK, Therapeutic Peptides and Proteins, Formu lation, Processing and Delivery Systems( 1995) Technomic Publishing Co., Lancaste (See r, PA). Peptide synthesis is carried out using various solid-phase methods (e.g., Roberge Sc) ience 269:202 (1995);Merrifield, Methods This can be done using Enzymol. 289:3 (see 1997), and also manufactured Follow the instructions of the person in charge, for example, the ABI 431A peptide synthesizer (Perkin El Automated synthesis can be achieved using mer.

[0113] The word “isolated” used as a modifier for composition refers to composition It is completely different from something that is produced by human hands or occurs naturally in the biological environment. Or it means that it is partially different. Generally, isolated compositions are in their natural state. For example, one or more proteins, nucleic acids, lipids, carbohydrates, and cell membranes are associated with them. Substantially contains no one or more materials that are present. The term "isolated" is used when referring to materials that are not made by human hands. For example, recombinant vector (e.g., AAV) sequences, or vector genes, produced by Virus particles that package or capsidize the virus (e.g., AAV-Rh74 vector) Or related AAV-Rh74 variants such as capsid variants (e.g., RHM4-1). This does not exclude mixtures of AAV vectors and pharmaceutical formulations. The terms hybrid / chimera, multimer / oligomer, and modified (e.g., phosphorylation) are used to describe hybrid / chimera, multimer / oligomer, and modified (e.g., phosphorylation) , glycosylation or lipid addition, or another physical form of the composition such as derivatives, or human This does not exclude morphologies expressed in host cells created manually.

[0114] The method and use of the present invention involves introducing heterologous polynucleotides (transgenes) into dividing cells and non-dividing cells. This invention provides a means for delivering (transducing) a wide range of host cells, including both types of fissure cells. Recombinant vector (e.g., AAV) sequences, plasmids, vector genomes, recombinant viruses S particles (e.g., AAV-Rh74 or capsid variants (e.g., RHM4-1)) Related AAVs such as AAV-Rh74 variants, methods, uses, and pharmaceutical formulations are proteins, pe It is also useful as a method of administering butylene or nucleic acid to subjects who require it as a therapeutic agent. As a result, proteins, peptides, or nucleic acids may be produced within the subject's body. The subject is deficient in the protein, peptide, or nucleic acid, or has the protein, peptide, or nucleic acid within the subject. The production of such proteins, peptides, or nucleic acids is a therapeutic method or other method that provides therapeutic effects. Therefore, one may benefit from or have these effects of the protein, peptide or nucleic acid. They may need it. Or, for example, neurodegenerative diseases, cancer or atherosclerosis. In the treatment of pulsile sclerosis, for therapeutic effects to be obtained, the expression of target genes involved in the course of the disease is important. Alternatively, it may be desirable to inhibit or reduce its generation.

[0115] Generally, recombinant vector (e.g., AAV) sequence, plasmid, vector genome, recombinant Viral particles (e.g., AAV-Rh74 or capsid variants (e.g., RHM4-1)) Related AAVs such as AAV-Rh74 variants, methods and uses are insufficient. Treating one or more symptoms related to any disease involving the undesirable expression of genes or To alleviate the effects, use to deliver any heterologous polynucleotide with biological effects It can be used. Recombinant vector (e.g., AAV) sequence, plasmid, vector genome, recombinant Viral particles (e.g., AAV-Rh74) or capsid variants (e.g., RHM4-1) (AAVs such as AAV-Rh74 variants, etc., related AAVs), methods and usage are applicable to various pathological conditions. It may also be used to provide a treatment for the opposite condition.

[0116] There are many genetic disorders for which the defective gene is known and has been cloned. Generally speaking, The above disease conditions are classified into two classes. One is usually an enzyme deficiency state, One is a deficiency that is generally inherited recessively, while the other is inherited dominantly, and at least some of them An imbalance involving regulatory or structural proteins. A disease characterized by a deficiency. Therefore, gene transfer is used as a replacement therapy to deliver normal genes to the affected tissue, as well as anti- Sense mutations can be used to create animal models of diseases. In disease states, gene transfer can be used to create disease conditions in model systems. Therefore, this model system can be used in efforts to resolve the pathological condition. Recombinant vector (e.g., AAV) sequence, plasmid, vector genome, recombinant viral particle Offspring (e.g., AAV-Rh74 vector or capsid variant (e.g., RHM4-1)) Which AAV-Rh74 variant or other related AAV vector, methods and uses are for genetic diseases To enable the treatment of the patient. As used here, a pathological condition is the cause of the disease, or Treatment involves partially or completely correcting the deficient or imbalance that is causing the problem. Site-specific integration of acid sequences is used to induce mutations or correct defects. It can also be used.

[0117] Examples of pathological conditions include cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, and Anemia, anemia and other blood disorders, AID, Alzheimer's disease, Parkinson's disease, Pentington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, (true) diabetes mellitus. , muscular dystrophy (for example, Duchenne type, Becker type) (er) type, Gaucher's disease, Haller's disease Hurlerapos's disease, adenosine deaminase deficiency , glycogen storage disorders and other metabolic disorders, Pompe disease, congestive heart failure, retinal degenerative diseases (previous Congenital choroidal absence, Leber congenital amaurosis, and other eye disorders), and solid organs (brain, liver). This includes, but is not limited to, diseases of the organs (kidneys, heart, etc.).

[0118] In the present invention, a treatment method and a method of use are provided, and these include recombinant vectors of the present invention Vector genome (e.g., AAV), recombinant virus particles (e.g., AAV-Rh74) AAV-Rh74 variants such as vector or capsid variants (e.g., RHM4-1) The virus particles of the present invention (e.g., any related AAV vector) and vector genome. AAV-Rh74 or capsid variants such as RHM4-1 This includes related AAVs such as 4 mutants. The methods and uses of the present invention are, for example, gene-attached Introduction of a protein-coding gene by addition or substitution, or gene expression or function It can be widely applied to diseases that are susceptible to treatment by increasing or stimulating certain factors. The methods and uses of the present invention also include, for example, gene knockout or reduction of gene expression ( By reducing or decreasing gene expression or function (such as gene knockdown), It can be widely applied to treatable diseases.

[0119] Non-limiting specific examples of diseases treatable by the present invention include those shown herein, Furthermore, lung diseases (e.g., cystic fibrosis), blood coagulation disorders or hemorrhagic disorders (e.g., inhibitors) Hemophilia A, hemophilia B (whether present or absent), Mediterranean anemia, blood disorders (e.g., anemia), Alzheimer's disease. Heimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), cerebrospinal fluid Hmm, lysosomal storage disorders, copper or iron storage disorders (e.g., Wilson's disease or Menkes disease), Lysosomal acid lipase deficiency, neuropathy or neurodegenerative disease, cancer, type 1 or type 2 diabetes Diseases, Gaucher's disease, Hurler's disease (s disease), adenosine deaminase deficiency, metabolic disorders (e.g., glycogen (Gene storage disorders), retinal degenerative diseases (e.g., RPE65 deficiency or insufficiency, congenital choroidal absence, etc.) This includes, but is not limited to, other eye diseases and diseases of solid organs (brain, liver, kidneys, heart), etc. It is not something that can be determined.

[0120] Furthermore, the recombinant vector (e.g., AAV) vector genome and recombinant viral particles of the present invention. (For example, AAV-Rh74 vector or capsid variant (e.g., RHM4-1), etc.) Related AAV vectors such as the AAV-Rh74 variant, methods and uses are for cancer, infection To treat or alleviate symptoms associated with diseases and autoimmune diseases such as rheumatoid arthritis. Sending a nucleic acid encoding a monoclonal antibody or a fragment thereof that provides beneficial biological effects. It can be used to achieve a goal.

[0121] In one embodiment, the method or use of the present invention involves (a) a virus containing a vector genome Rh74 particles (e.g., AAV-Rh74 or AAV-Rh74 capsid variants (e.g., RH) The step involves providing related AAVs such as M4-1), wherein the vector genome is heterologous poly(AAV). It includes a nucleotide sequence (and optionally a filler / stuffer polynucleotide sequence), The sequence of the aforementioned heterologous polynucleotide is an expression that confers transcription of the polynucleotide sequence. (b) the steps of providing the virus particles which are operablely bound to the element, and (b) the different In order to express the species polypeptide in the mammal, the virus particles are introduced into the mammal. The system includes a step of administering a predetermined amount.

[0122] In another embodiment, the method or use of the present invention involves transferring heterologous polynucleotide sequences to mammals This includes delivery or transfer into the cells of an object or mammal, which is a heterologous polynucleo Vectors containing nucleotide sequences (and optionally filler / stuffer polynucleotide sequences) Virus particles (e.g., AAV) containing nom (e.g., AAV-Rh74 or capsicum) AAV variants such as RHM4-1 and related AAV variants such as Rh74 variants or multiple virus (e.g., AAV) particles (e.g., AAV-Rh74 or capsid mutation) Related AAVs (such as AAV-Rh74 variants, e.g., RHM4-1) are used in mammalian feeding To administer to a substance or mammalian cells, thereby introducing heterologous polynucleotide sequences into mammals or mammals. By delivery or transfer into animal cells.

[0123] In yet another embodiment, a deficient mammal that requires protein expression or function is used to treat a protein-decreasing mammal. The present invention provides a method or use for the treatment of heterogeneous polynucleotide sequences (and optionally a FI A virus containing a vector genome (including a Lar / Stuffer polynucleotide sequence) (e.g., AAV) particles (e.g., AAV-Rh74 or capsid variants (e.g., RHM) 4-1) AAV-Rh74 variants and other related AAVs or multiple viruses (for example) AAV) particles (e.g., AAV-Rh74 or capsid variants (e.g., RHM4-1) The steps include providing related AAVs such as AAV-Rh74 variants, and the viral particles The child or the plurality of virus particles are delivered or transferred into a mammal or a mammalian cell. This includes a step where heterologous polynucleotide sequences are used to create proteins expressed within mammals. Either the heterologous polynucleotide sequence reduces the expression of endogenous mammalian proteins. It encodes an inhibitory sequence or protein that induces depression.

[0124] In certain aspects of the methods and uses of the present invention disclosed herein, different polynucleotides Otide expression is a protein that has therapeutic or inhibitory effects on mammals (e.g., humans). It encodes nucleic acids. In more specific embodiments, filler / stuffer polynucleotides The column is included in the vector sequence, and the total length when combined with the heterogeneous polynucleotide sequence is approximately 3. The file size is 0-5.5Kb, or approximately 4.0-5.0Kb, or approximately 4.3-4.8Kb.

[0125] The methods and uses of the present invention do not constitute any therapeutic or beneficial treatment method. Includes. Various methods and uses of the present invention further include, for example, shortening blood coagulation time, blood replacement. One or more adverse effects caused by or related to the disease, such as a reduction in the dosage of liquid coagulation factors (e.g.) For example, to inhibit, reduce, or alleviate physical symptoms, disorders, ill health conditions, diseases, or complications. This includes the following.

[0126] Therefore, the therapeutic or beneficial effects of treatment are not subject to any objective or subjective measurability. A value, or a detectable improvement or benefit given to a specific subject. Therapeutic Or, beneficial effects include all or specific adverse symptoms, disorders, diseases, or complications of illness. This doesn't necessarily mean complete removal, but it can be done completely in some cases. Therefore, a satisfactory clinical endpoint is one in which the disease or related conditions are resolved. The gradual improvement of one or more harmful symptoms, disorders, illnesses, diseases or complications When an improvement or partial reduction is observed, or over a short period or over a long period (several hours, (Several days, weeks, months, etc.) One or a disease caused by or related to the disease The exacerbation or progression of multiple adverse symptoms, disorders, illnesses, diseases, or complications is inhibited or reduced. It is achieved when it is reduced, decreased, suppressed, prevented, limited, or controlled.

[0127] The vector genome of the present invention, recombinant virus particles containing the vector genome (e.g., AAV) -AAV-Rh7 vector or capsid variant (e.g., RHM4-1) Compositions such as related AAV vectors (including 4 variants), methods, and uses are these A sufficient or effective dose can be administered to subjects who require it. "tive amount" or "sufficient amount" This refers to a single or multiple doses containing, alone or one or more other components. A substance (such as a drug or other therapeutic agent), procedure, protocol, or treatment regimen (therapeutic regimen) When combined with an anti-tic regimen, it can detect the reaction for any period (long-term or short-term). A response can be obtained, or the expected or desired result or benefit can be measured in the object. Or, a detectable period (e.g., minutes, hours, days, months, years, or healing) is given. It refers to the amount that is used.

[0128] A vector genome or viral particle that exerts therapeutic effects (e.g., AAV, e.g., A AV-Rh74 vector or AAV-Rh74 vector or capsid variant (e.g., RHM4-1) The dose of related AAV vectors (such as the h74 variant), for example, the vector per kilogram of body weight. The dosage expressed in terms of the amount of the target genome (vg / kg) varies based on several factors such as the route of administration, the expression level of the heterologous polynucleotide required to exert a therapeutic effect, the specific disease to be treated, the host immune response to the viral vector, the host immune response to the heterologous polynucleotide or the expressed product (protein), and the stability of the expressed protein, etc. However, the factors are not limited to these. Those skilled in the art can easily determine the dosage range of virions for treating patients with specific diseases or disorders based on the above-mentioned factors and other factors. Generally, the dosage that can achieve a therapeutic effect is at least 1×10 vector genomes per kilogram of the subject's body weight (vg / kg) or more than this. For example, 1×10 8 vector genome / subject per kilogram of body weight, or in a range exceeding this, such as 1×10 9 1×10 10 1×10 11 1×10 12 1×10 13 1×10 14 or a range exceeding this is.

[0129] Taking hemophilia as an example, generally, in order to obtain a therapeutic effect, to change the phenotype of the disease from severe to moderate, the blood coagulation factor needs to exceed 1% of the concentration based on healthy people. A severe phenotype is characterized by joint damage and life-threatening bleeding . To change a disease with a moderate phenotype to a mild one, the concentration of the blood coagulation factor needs to exceed 5% of the concentration based on healthy people. Regarding the treatment of subjects suffering from hemophilia, a typical dosage is at least 1×10 vector genomes per kilogram of the subject's body weight (vg / kg), or, to achieve the desired therapeutic effect 1×10 10 (vg / kg), or ​​To reduce it, approximately 1 x 10 10 From 1 x 10 11 Within the range of vg / kg, for the amount corresponding to the target body weight, or approximately 1 × 10 11 From 1 x 10 12 Within the range of vg / kg, for the amount corresponding to the target body weight, or approximately 1 x 10⁻⁶ 12 from 1x1 0 13 This is the amount corresponding to the subject's body weight within the range of vg / kg.

[0130] For the purpose of treatment (e.g., to restore, or to provide therapeutic benefits or improvement) The “effective amount” or “sufficient amount” of administration "Critical amount" generally refers to one of the adverse symptoms, consequences, or complications of a disease. For multiple or all of the above, or caused by or associated with the disease. A measurable degree of one or more harmful symptoms, diseases, conditions, or complications This is an effective amount to provide a certain degree of response, and of course, it reduces the progression or worsening of the disease. Desirable outcomes of administration include being able to inhibit, suppress, limit, or control the effect. El.

[0131] An effective or sufficient amount may be provided in a single dose, but is not necessarily required. Multiple doses may be required, and they may also be administered alone or with other components (e.g., the active ingredient) or treatments. It may be administered in combination with a protocol or treatment regimen. For example, The target, type, status and severity of the disease, or (if any) the need for side effects of treatment. The effective amount may be increased in proportion to this. In addition, a second composition (for example, another drug or (The active substance), procedure, protocol, or treatment regimen is administered once or multiple times without the presence of such a substance. If given by means of, an effective or sufficient amount does not necessarily have to be effective or sufficient. This is to ensure that it is effective and sufficient for the specified subject, Additional doses, amounts, or durations exceeding the prescribed dosage, or further components (e.g., drugs or effects) This is because it may include substances, procedures, protocols, or treatment regimens. The amount that can be used is, for example, a combination for the treatment of coagulation disorders (e.g., hemophilia A or B). Reduced use of alternative therapies, treatment regimens, or protocols, such as administration of replacement coagulation factor proteins. It contains an amount that brings about a small result.

[0132] An effective or sufficient dose is not necessarily effective in all and each of the subjects being treated. Furthermore, it does not need to be effective in the majority of those being treated within a given group or population. Effective dose or sufficient dose means effective or sufficient for a group or general population. This does not mean that it is effective, but rather that it is effective or sufficient for a specific subject (patient). While this is typical in such methods, for a given treatment method or use, a certain subject In some cases, one object may show a strong reaction, while another object shows little to no reaction. Yes. Therefore, the appropriate amount depends on the condition to be treated, the desired therapeutic effect, and the individual. Each subject (for example, the bioavailability of the subject, sex, It depends on age, etc.

[0133] The word "ameliorate" refers to the disease or its symptoms, or This means a detectable or measurable improvement in the underlying cellular response. Measurable improvement refers to the onset, frequency, severity, progression, or duration of the disease, or the disease itself. Subjective or objective reduction or inhibition of complications related to or caused by the disease. Including harm, suppression, limitation, or control, or the symptoms, underlying causes, or consequences of a disease. This includes improvement in the condition or recovery from the disease.

[0134] Therefore, the outcome of successful treatment is related to the onset, frequency, severity, progression, or duration of the disease. Or, one or more adverse symptoms, underlying causes, or consequences of the disease in the subject. , “therapeutic effect” or “benefit” of reducing, lowering, inhibiting, suppressing, limiting, controlling or preventing This can lead to: As mentioned above, the underlying cause of one or more of the disease or adverse symptoms Treatment methods and uses that affect the cause are considered beneficial. Just as symptoms stabilize, a reduction or mitigation of worsening symptoms can also be considered a result of successful treatment. .

[0135] Thus, therapeutic benefit or improvement does not necessarily mean the complete elimination of the disease, or the reduction of the disease. Eliminating one, most or all of the associated harmful symptoms, complications, consequences, or underlying causes. It does not require. Therefore, a satisfactory clinical endpoint is a progressive outcome in the disease in question. Improvement, or improvement of the disease, either in a short period or over a long period (several hours, several days, several weeks, several months, etc.) Partial reduction, decrease, inhibition, suppression, limitation, or control of occurrence, frequency, severity, progression, or duration. Or prevention or reversal (for example, stabilization of one or more symptoms or complications) It is when it is observed. Methods or treatments that bring about potential therapeutic benefits or improvement of the disease. The effectiveness of its use can be verified by various methods.

[0136] The methods and uses of the present invention provide desirable therapeutic advantages, additional, synergistic or complementary Any compound, active substance, drug, treatment or other therapeutic regimen that has activity or effect This may be combined with the protocol. Examples of compositions and treatments to be combined include biological It contains a second active substance, such as a formulation (protein), an active agent, and a drug. Biological products (proteins), active substances, drugs, treatments and therapies, other methods or uses , or, for example, a therapeutic method for treating a subject with a blood coagulation disorder, and the method and It may be administered or carried out substantially simultaneously with, or after, the use of the product. ru.

[0137] Compounds, active substances, drugs, treatments or other treatment plans or protocols are complex compositions. As, or separately, for example, the vector genome or virus of the present invention (e.g., AAV) -AAV-Rh7 vector or capsid variant (e.g., RHM4-1) Simultaneously with the delivery or administration of related AAV vectors (such as 4 variants), either sequentially or sequentially. It can be administered (before or after). Therefore, the present invention relates to the method and use of the present invention. The law applies to any compound, active substance, drug, therapeutic agent described herein or known to those skilled in the art. Combinations of Dimen, treatment protocols, processes, therapeutic agents, or compositions We provide: compounds, active ingredients, drugs, treatment plans, treatment protocols, processes, therapies, and more. The composition may be a vector genome or virus of the present invention (e.g., AAV-Rh74 vector). AAV-Rh74 variants such as tar or capsid variants (e.g., RHM4-1) Administer to the subject substantially simultaneously with, or after, the administration of the related AAV vector. This is possible. Non-limiting specific examples of combination embodiments include the above or other combinations. Includes substances, active ingredients, drugs, treatment regimens, treatment protocols, processes, therapeutic agents, or compositions. nothing.

[0138] The methods and uses of the present invention are, in particular, related to other compounds, active substances, drugs, therapeutic regimens, Methods and uses that result in reducing the use or need of treatment protocols, processes, or therapeutic drugs. This includes the method of use. For example, in the case of blood coagulation disorders, the method or method of use of the present invention applies to a specified target. Combinations performed to supplement intrinsic coagulation factors that are deficient or lacking (abnormal or mutated) Reduce the frequency, reduce the dosage, or discontinue the administration of replacement coagulation factor proteins. It has therapeutic benefits, such as improvement. Therefore, according to the present invention, another treatment or therapy Methods and uses that reduce the need for or use of the law are provided.

[0139] The present invention is useful in animals, including for veterinary applications. Therefore, suitable subjects are This includes mammals like humans and non-human mammals. The term typically refers to humans, non-human primates (monkeys, gibbons, gorillas, chimpanzees) (Little geese, orangutans, macaques), livestock (dogs and cats), livestock (for example, chickens and This refers to domestic poultry such as ducks, and mammals such as horses, cows, goats, sheep, and pigs, and furthermore, experiments This refers to animals, including mice, rats, rabbits, and guinea pigs. Human subjects include: Includes fetuses, newborns, infants, young children, and adults. The subjects include animal disease models, for example. For example, mouse and other animal models of blood coagulation disorders, and other animals well known to those skilled in the art. Includes disease models.

[0140] As described herein, a vector and a virus particle containing the vector (for example, A AV-Rh74 or AAV-Rh74 variants such as capsid variants (e.g., RHM4-1) Related AAVs (such as allogenes) have insufficient protein quantity or functional gene products (tan). (Protein) provides protein to subjects who are deficient in it, or can cause disease. In subjects that produce gene products (proteins) that are normally incomplete or non-functional It can be used to provide inhibitory nucleic acids or proteins. Therefore, it is suitable for treatment. The subjects had insufficient or missing amounts of functional gene products (proteins), or abnormal, incomplete, or non-functional gene products (Tan) that can cause disease A subject that produces (protein) or is at risk of producing it. A subject suitable for treatment according to the present invention Furthermore, as an elephant, an abnormal or defective (mutated) gene product (protein) that leads to disease The subject includes objects that have or are at risk of generating such abnormal or defective properties. By reducing the amount, expression, or function of the (mutated) gene product (protein), This treatment leads to the cure of the disease, or alleviates one or more of the symptoms of the disease, and helps to recover from the disease. It can be cured. Therefore, the subject of the study must have a specific type of disease, a specific time of onset, or a specific severity. Subjects with such defects, regardless of progression, severity, frequency, type of symptoms, or duration. It includes.

[0141] The word “prophylaxis” and its synonyms refer to contact with an object, This refers to a method in which administration or intra vivo delivery is performed before the onset of disease. Before any adverse symptoms, conditions, or complications that may result from this occur, administer the drug to the target or use it in the following manner: Internal delivery is possible. For example, as a candidate to which the method and use of the present invention should be applied Screening (for example, genetic screening) can be performed to identify the target, but disease There are also subjects in whom the symptoms are not apparent. Therefore, even if such subjects show symptoms of the disease... Even if not, the amount of functional gene products (proteins) is insufficient, or missing. Damaged, disease-causing, abnormal, incomplete, or non-functional gene products (Proteins) that produce abnormalities or defects (mutations) that can cause disease This includes subjects who tested positive through screening for genetic products (proteins).

[0142] The methods and uses of the present invention are systemic, local, or localized, or via any route. This includes delivery and administration, for example, by injection, intravenous drip, or orally (for example, by oral ingestion or inhalation). This includes delivery and administration by means of or locally (e.g., transdermally). Delivery and administration methods include intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intracavitary, intracranial, and percutaneous (local) delivery. This includes administration by means of the mucous membrane or rectum, and parenteral administration, for example. The administration and delivery routes include intravenous (IV), intraperitoneal (IP), intraarterial, and intramuscular. Extraintestinal, subcutaneous, intrapleural, local, dermal, intradermal, percutaneous, parenteral, for example, transmucosal, intracranial, Intrathecal, oral (digestive), mucosal, respiratory, nasal, endotracheal intubation, intrapulmonary, intrapulmonary infusion, oral, sublingual Intravascular, subarachnoid, intracavitary, iontophoresis, intraocular, eye drops, optical, intraglandular, intraorganic, intralymphatic It includes.

[0143] Whether the treatment is preventive or therapeutic, the type of disease being treated, onset, progression, and severity. Severity, frequency, duration or probability, desired clinical endpoint, previous or concurrent treatments, subject The overall health condition, age, sex, race or immune capacity, and the ability to understand the skills of a person skilled in the art. The dose depends on and changes with other factors. The amount, number, frequency or duration of administration may vary depending on the circumstances. Adverse side effects, complications, or other risk factors for the procedure or treatment, and the condition of the subject. Depending on the circumstances, it can be increased or decreased proportionally. Those skilled in the art will know that therapeutic or This casts a shadow on the dosage and timing required to deliver a sufficient amount to provide preventative benefits. We can understand the factors that influence the outcome.

[0144] The methods and uses of the present invention disclosed herein are provided for subjects who have a disease of treatment. After that, after the subject has one or more symptoms of the disease, or after the subject has one or more symptoms of the disease Although they do not have multiple symptoms, they were found to be positive through screening as described below. It must be carried out within 1-2, 2-4, 4-12, 12-24, or 24-72 hours after rejection. It is possible. Of course, the method and use of the present invention are applicable when the subject has a disease to be treated. After the subject has one or more symptoms of the disease, or the subject has a screen After being diagnosed as positive by the test, 1-7, 7-14, 14-21, 21-48 days later It can also be done more than 48 days later, several months or even years later.

[0145] Recombinant vectors (e.g., AAV, e.g., AAV-Rh74 vector or capsid mutation) (e.g., AAV-Rh74 variants such as RHM4-1, etc., and related AAV vectors) Sequences, plasmids, vector genomes, recombinant viral particles (e.g., AAV, e.g., A AV-Rh74 vector or AAV-Rh74 vector or capsid variant (e.g., RHM4-1) Related AAV vectors (such as the h74 variant), and other compositions, active substances, drugs, and biological products The agent (protein) is incorporated into a pharmaceutical composition, for example, as a carrier or excipient acceptable as a pharmaceutical. Such pharmaceutical compositions can be incorporated into a target organism, either in vivo or in vitro. It is useful for giving and serving.

[0146] The term "pharmaceutically acceptable" as used herein "tolerant" and "physiologically acceptable" The phrase "ly acceptable)" means that in intra vivo delivery or contact, one or A bioacceptable formulation, gas, liquid or solid or any of the above, suitable for multiple routes of administration. It means a combination. "Acceptable as a pharmaceutical" or "Physiologically acceptable" The substance is not harmful in any biological or other way, for example, a significantly undesirable biological substance. It is a substance that can be administered to a subject without causing any adverse effects. Therefore, such a pharmaceutical composition The objects include, for example, viral vectors, viral particles (e.g., AAV-Rh74 or capsids) AAV variants (e.g., RHM4-1) and related AAVs such as Rh74 variants, as well as This can be used when administering transformed cells to a subject.

[0147] The composition is a solvent (aqueous or non-aqueous) suitable for drug administration or in vivo contact or delivery. ), solutions (aqueous or non-aqueous), emulsions (e.g., water-in-oil or oil-in-water), suspensions, silo Toppings, elixirs, dispersions and suspensions, coatings, isotonic agents, absorption enhancers, or absorbents. Contains yield retarders, etc. Aqueous and non-aqueous solvents, solutions and suspensions contain suspending agents and thickeners. That is also acceptable. Such pharmaceutically acceptable carriers include (coated or non-coated carriers). (Coated) tablets, (hard or soft) capsules, microbeads, powders, granules and crystals Crystals are included. Auxiliary active compounds (e.g., preservatives, antibacterial agents, antiviral agents, and antifungal agents) are also present. The agent can also be incorporated into the composition.

[0148] As described below or as is well known to those skilled in the art, pharmaceutical compositions specify the administration or delivery. It can be formulated to suit the pathway. Therefore, the pharmaceutical composition can be formulated to suit various pathways. Contains a carrier, diluent, or excipient suitable for administration by means of [method].

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

[0150] For transmucosal or transdermal administration (e.g., local contact), a penetrating agent is included in the pharmaceutical composition. It can be done. Penetrating agents are well known in the field, and for example, in the case of transmucosal administration, at the interface. It contains activators, bile salts, and fusidic acid derivatives. When administered transdermally, the active ingredient is... Aerosols, sprays, ointments, patches, gels or It can be formulated as a cream. For skin contact, as a pharmaceutical composition, In terms of type, it can be an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Includes. Suitable carriers include petrolatum, lanolin, polyethylene, etc. Examples include recalls, alcohol, transdermal promoters, and combinations thereof.

[0151] The cosolvent and adjuvants are added to the formulation. It may also be used. Non-restrictive examples of cosolvents include those containing a hydroxyl group or other polar group, and isopropyl alcohol. Alcohols such as polypropyl alcohol; propylene glycol, polyethylene glycol Glycols such as polypropylene glycol and glycol ether; glycerol; Contains polyoxyethylene alcohol and polyoxyethylene fatty acid ester. Examples of surfactants include soy lecithin, oleic acid, and triolein. This includes sorbitan esters such as sorbitan acid, and polyvinylpyrrolidone.

[0152] The vector genome, viral particles (e.g., AAV-Rh74 vector or capsulphur) of the present invention Related AAV variants such as AAV-Rh74 variants (e.g., RHM4-1) Pharmaceutical compositions and delivery systems suitable for the method and use of the pharmacopoeia (pharmacopoeia), in the art, This is known (e.g., Remington: The Science and Pra Case of Pharmacy (2003), 20th edition, Mack Publ 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), R.L. Ju See Liano (ed.), Oxford, NY, pp. 253–315.

[0153] The term “unit dosage form” as used herein refers to The term refers to a physically separated unit suitable as a unit dose for the subject being treated. Each unit may include a pharmaceutical carrier (excipient, diluent, vehicle, or filler) as needed. When administered in one or more doses, the desired effect (for example, preventive or therapeutic effect) can be achieved. Contains a predetermined amount calculated to produce the fruit. The unit dosage form is a liquid composition or lyophilized or The composition may be contained in a freeze-dried state and may be enclosed in ampoules and vials, and may be biological Before internal administration or delivery, for example, a sterile liquid carrier may be added. Each of the given forms can be contained in a multi-dose kit or container. Recombinant vector ( For example, AAV sequences, plasmids, vector genomes, recombinant viral particles (e.g., AA V-Rh74 vector or AAV-Rh74 capsid variant (e.g., RHM4-1) Which related AAV vectors, and these pharmaceutical compositions, offer ease of administration and dose uniformity. For this purpose, it can be packaged in single or multiple unit dose forms.

[0154] The present invention relates to one or more components and a package in which the components are enclosed. The kit includes the materials for the in vitro application. The kit typically includes the components in an in vitro application. , labels including instructions for use or component instructions for use in vivo or in vitro. The kit includes, for example, a vector (e.g., AAV) genome or These are viral particles (e.g., AAV-Rh74 vector or capsid variant (e.g., RH) Related AAV vectors such as AAV-Rh74 variants (M4-1), and other vectors as needed. A field containing a group of components such as a second active substance, compound, active substance, drug, or composition. There is a match.

[0155] A kit refers to a physical structure that contains one or more components of a kit. Materials that can keep their components sterile, and are commonly used for such purposes. For example, paper, cardboard, glass, plastic, aluminum foil, ampoules, vials, etc. It consists of (tubes, etc.).

[0156] Labels or inserts may include information that identifies one or more components. For example, the clinical pharmacology of the active ingredient, including the dose, mechanism of action, pharmacokinetics, and drug Mechanical information can be included. The label or insert should include the manufacturer, lot number, and manufacturing date. It may include information that identifies the location, date, and expiration date. The label or insert may include: This may include information identifying the manufacturer, lot number, place of manufacture, and date of manufacture. The bell or insert contains information about the disease in which the kit components can be used. It may include: the label or insert may include the method, use or treatment protocol or Clinicians or patients using one or more components of the kit in a treatment regimen Instructions for use may be included. The instructions for use may include the dosage, frequency or duration, and The methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein Instructions for carrying out any of the following can be included.

[0157] The label or insert should state that the component may offer any benefit whatsoever. Information may include, for example, information about preventive or therapeutic benefits. (Label) Alternatively, the implant may include information about possible side effects, complications, or reactions. For example, regarding situations where the use of a particular composition is inappropriate, the subject or clinician should be warned. Information that may be disclosed may be included. The person is taking, plans to take, or has multiple other medications, or the subject is currently Currently, you are receiving or undergoing another treatment protocol or regimen that is incompatible with the composition. If you have or are planning to have this done, please be aware that harmful side effects or complications may occur. The documentation may include information regarding such nonconformities.

[0158] Labels or inserts can be components, kits, or packaging materials (e.g., boxes). Ampoules, tubes, etc., that are either attached separately or attached to the kit components. Alternatively, it could include "printed material," such as paper or cardboard, attached to the vial. Labels or inserts may also include labels, discs, and CD-ROS with barcodes printed on them. Optical discs such as M or DVD-ROM / RAM, DVDs, MP3s, magnetic tapes, or digital media. Computer-readable media such as mechanical memory media may be included, and as an electrical memory medium, R AM and ROM or mixtures thereof exist, for example, magnetic / optical storage media, FLA Examples include SH media or memory-type cards.

[0159] Unless otherwise defined, all technical and scientific terms used in this specification are defined as: Generally, it has the same meaning as understood by those skilled in the art in the field to which this invention belongs. Using methods and materials similar to or equivalent to those described herein in carrying out or testing the present invention. This can be done, but preferred methods and materials are described below.

[0160] All applications, publications, patents and other references cited herein, as well as GenBank references, are subject to the same conditions. All references to the ATCC are invoked in their entirety. In case of any inconsistency, this specification, including its definitions, shall prevail. It takes priority.

[0161] All features disclosed herein can be combined in any combination. Each feature disclosed in the specification is replaced by an alternative feature that serves the same, equivalent, or similar purpose. It can be replaced. Therefore, unless otherwise stated, the disclosed characteristics (e.g., recombinant) Vector (e.g., AAV) sequence, plasmid, vector genome, or recombinant viral particle (For example, AAV-Rh74 vector or capsid variant (e.g., RHM4-1), etc.) Related AAV vectors such as the AAV-Rh74 variant are of the same or similar genus. This is an example.

[0162] The singular forms "a" and "and" used in this specification indicate singularity. The words "the" and "that" include multiple things unless explicitly stated otherwise. Therefore, see, for example, "polynucleotide". When doing so, multiple such polynucleotides are included, and a "vector" is created. When referring to ")", multiple such vectors are included, and "virus (a virus) When referring to "a particle" or "a particle", use multiple such billios. It contains particles.

[0163] In this specification, unless otherwise explicitly stated in the context, all numbers or numerical ranges are this This includes integer values ​​within a range and values ​​within that range or fractional parts of integer values. For example, at least 8 When referring to 0% identity, use 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%, This includes percentages such as 82.2%, 82.3%, 82.4%, 82.5%, etc.

[0164] When referring to an integer that is greater than or less than a given number, it implies that the number referred to is greater than or less than the given number. This includes any number that is larger or smaller than 1000. Therefore, for example, the statement that it is less than 1000 is also true. The reference to numbers smaller than 100 is 999, 998, 997, ..., up to 1. Includes numbers from 9, 98, 97, ... up to 1.

[0165] In this specification, unless otherwise explicitly stated in the context, all numbers or ranges of numbers are... The fractional part of such a value, and integer values ​​within such a range, and within such a range This includes the fractional part of an integer value. Therefore, it refers to numerical ranges such as percentage ranges, for example. When doing so, the range from 1 to 10 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 1.1 This includes 1.2, 1.3, 1.4, 1.5, etc. When referring to the range of 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 50 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] When we refer to a series of ranges, it includes a range that combines the boundary values ​​of multiple consecutive and different ranges. Hmm. 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 00, 7,000-8,000, or 8,000-9,000, use 10-5 0, 50-100, 100-1,000, 1,000-3,000, 2,000-4,0 Includes 00, etc.

[0167] This invention uses definitive language to describe many embodiments and aspects. The present invention also discloses substances or materials, methods, steps and conditions, and protocols. This includes embodiments in which certain subjects, such as procedures, are completely or partially excluded. In certain embodiments or aspects of the specification, the steps of materials and / or methods are excluded. However However, this invention is not explicitly stated in terms of what is not included in this invention. Embodiments not expressly included in the invention are disclosed.

[0168] Several embodiments of the present invention have been described. Those skilled in the art will understand the spirit and scope of the present invention. Without departing from the present invention, various modifications may be made to adapt it to various uses and conditions. And modifications can be made. Therefore, the embodiments described below illustrate the present invention. This is a matter that does not limit the scope of the claimed invention. [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 miscellaneous form of the FIX gene at the University of Chapel Hill, North Carolina. These are HB dogs from colonies carrying the S mutation (Evans et al., Proc). Natl Acad Sci USA 86:10095 (1989)).

[0170] AAV vector construction: In vivo studies using mice have shown that ApoE-hAAT liver stabilization The study was conducted using a construct that expresses human FIX under the control of a promoter. The same study was also performed on dogs. The experiment was conducted using nearly identical promoters and canine FIX transgenes.

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

[0172] FIX expression determination: ELISA was used to measure FIX levels. In mice, FIX ELISA antibody pairs (supplementary antibody and secondary antibody) are manufactured by Affinity Biol. This is from ogicals. 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 a two-tailed t-test for unpaired samples. p-value < A value of 0.05 was considered statistically significant.

[0174] AAV antibody measurement: For antibody measurement, see Manno et al., (Nat Med 1 2:342 (2006)) and Mingozzi et al. (Nat M Using the in vitro neutralization assay described in ed 13:419 (2007) In short, two AAV vector constructs were used in the measurement, and the CMV promoter Beta-galactosidase expression under the control of (ssAAV-LacZ) - Single-stranded Under the control of a vector or chicken β-actin promoter (CBA), sea urchins AAV-Rh7, a self-complementary vector expressing the Take (Renilla) reporter gene. 4-CBA-Renilla was used. In vitro transduction of AAV vectors To improve efficiency, 2V6.11 cells (ATCC) were used, and the inducible promoter was controlled. Adenovirus gene E4 was expressed in the following: 1.25 x 10⁶ per well. 4 cells Cells were seeded in a 96-well plate at the specified concentration, and ponasterone A (In) was added in a 1:1000 dilution. E4 expression was induced by adding a ( vitrogen) to the culture medium. On the day of measurement, the thermoactive 3.2-fold dilution series of serum for chemical testing (serial half-log dilutions) ) was mixed with a culture medium containing the virus. For the ssAAV-LacZ vector, measurement was performed. The virus concentration used was approximately 1x10⁻¹⁶ in the case of AAV2. 10 vg / ml, and AA For V5, 6, or 8, it is approximately 5.5 x 10 10 The result was vg / ml. In Kurt's case, the virus concentration in the measurement was approximately 50 to 150 times lower. - The residual activity of the transgene can be determined by colorimetric analysis (ssAAV-LacZ) or luminometer ( Measured using scAAV-Luc).

[0175] Anti-AAV capsid whole IgG or Ig subclasses can be detected using a capture assay (capture a Measurement was performed using ELISA. The ELISA plate was filled with 5 × 10⁶ empty capsids of AAV. 10 The plates were coated with capsid particles / ml. The plates were left at room temperature for 2 hours in PBS with 2% BSA. Block with 0.05% Tween20, fill the wells with serial dilutions of the sample, and leave at 4°C. Incubated overnight. Biotin-conjugated anti-human IgG1, IgG2, IgG3, IgG4 Antibodies or IgM antibodies (Sigma) are used for antibody detection, and streptavidin-HRP It was added for substrate detection. The concentrations of Ig were: purified human IgG1, IgG2, IgG 3. For standard curves created by serial dilutions with IgG4 or IgM antibody (Sigma) The determination was made.

[0176] AAV generation: The process for vector production is described by Ayuso et al. (Gene This is described in detail in Ther 17:503 (2010). Example 2 This example involves research on human FIX gene-transformed animals (mice) and FIX expression after gene transfer. This includes an explanation.

[0177] Control of liver-specific promoters in C57BL / 6 mice (n=5 per group) Below (2.5 per mouse) 10 The vector genome contains the factor IX (FIX) gene. The AAV vector was injected via the tail vein. The human FIX transgene product (T) was injected into the mouse. Plasma concentrations of the protein were measured using ELISA at weeks 1, 2, and 4 after gene transfer. Therefore, it was measured, and the results are shown in Figure 1. AAV-Rh74 is the most important factor in animals. It showed high levels of transgene expression. Example 3 This example demonstrates the effective adjustment of AAV-Rh74 levels for therapeutic FIX in dogs with hemophilia. This includes descriptions of animal experiments and data demonstrating delivery by this method.

[0178] In general terms, in dogs with hemophilia B, 3 × 10⁶ per kg of body weight is administered via the saphenous vein. 12 Vector —The genome was infused intravenously (IV). The expression of the therapeutic FIX transgene was a liver-specific progenitor. Activated by a motor. The vector level and FIX level were determined by ELISA. The plasma concentrations of canine FIX were monitored. AAV-Rh74 and Both AAV8 and AAV6 are equally effective in dogs with hemophilia B, and both are superior to AAV6. Ta. Example 4 This example includes a description of a study demonstrating the existence of anti-AAV neutralizing antibodies (NAbs) in humans.

[0179] The data in Table 1 shows anti-AAV neutralizing antibodies (NAb) measured in humans in vitro. Subjects with NAb titers of 1:1 or less are subjected to experiments using or anti-AAV antibodies that have not been used in the experiment. It is defined as having a low titer, and the gene transfer of that AAV serotype (highlighted in gray) It is assumed that the patient is a target. Patients with titers between 1:1 and 1:3 will be given an empty capsid as a decoy. As long as it is used in this way, AAV is considered acceptable. Sun Pull is thought not to tolerate AAV transduction after whole-body injection, and is therefore lightly shaded. AAV-Rh74 is the most effective anti-AAV Nab compared to AAV-2 and AAV-8. It showed a low prevalence rate. [Table 1] [Table 2] Example 5 In this example, the explanation of the data showing the production levels of various AAV serotypes, including AAV-Rh74, is provided. It is included.

[0180] Table 2 shows the production levels of various AAV serotypes. Reported data This includes the virus batch size in the roller bottle, the total vector generation amount, and the bottle. The yield per unit is shown. All serotypes are expressed using the same expression cassette. It was packaged. AAV-Rh74 was evaluated against other serotypes, namely AAV-8. It has a production volume comparable to or exceeding that 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 AAVrh74 vector was administered to rhesus monkeys, and the same amount of AAV8 vector was administered to them... Data showing that it led to higher levels of FIX production in animals than combined Details are included.

[0181] Simply put, animals contain either AAV8 or AAVrh74 at a rate of 2 per kg of body weight. ×10 12 The vector genome (vg) was administered at a dose of 1 kg / kg. The vector was in physiological saline. Alternatively, it was formulated in a mixture of the vector and an empty AAV capsid (denoted as EC).

[0182] Figure 4 shows human FIX measured by detection ELISA in rhesus monkey plasma. The average of X (2-8 weeks) and the histogram of the standard error or arithmetic mean. This is a togram plot. Animals that received the AAVrh74-FIX vector are shown in the right margin. It is displayed in the last two bars. The data receives an AAVrh74 vector. The animals (the last two bars towards the right margin) are the same as the other animals injected with the same dose. This indicates that the FIX transgene was expressed at a higher level compared to the black and gray bars. The mean level is calculated using a two-tailed Student t-test, where the sample is not paired. I compared them.

[0183] One of the individuals injected with AAV-RHM4-1-FIX developed a human factor IX transgenic gene. It produced inhibitors to the offspring, but this is a well-established phenomenon, and human FI It occurs in approximately 20% of macaque monkeys treated with X vector. The individuals expressed approximately twice the FIX concentration compared to macaque monkeys treated with AAV8. Example 7 The following examples include descriptions of several Rh74 capsid variants.

[0184] In summary, various substitutions are introduced into the Rh74 capsid sequence to create Rh74 capsid mutants. The following were generated. Different Rh74 capsid variants and the amino acids after substitution at each position are as follows: It was a street. [Table 4]

[0185] The RHM4-1 mutant has alanine, leucine, proline, and asparagine substitutions. The 195th, 199th, 201st, and 202nd amino acids of the Rh74 VP1 capsid, respectively. It was located at the following position. The amino acid sequence of the RHM4-1 mutant VP1 capsid is as follows: Yes (sequence ID number: 5). Note that the substituted residues a, v, p, and n are underlined and in bold. This is shown. [Table 5]

[0186] The nucleic acid sequence of the RHM4-1 mutant VP1 capsid is as follows. Note that a, v, p The codons encoding , and n are underlined and shown in bold (sequence ID number: 11). [Table 6]

[0187] RHM15-1, 15-2, 15-3, 15-4, 15-5, and 15-6 mutants Furthermore, alanine, leucine, proline, and asparagine substitutions are used in Rh74. The VP1 capsid has amino acids at positions 195, 199, 201, and 202. Furthermore, these mutants possess multiple lysines in which arginine is substituted at various positions. there was.

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

[0189] The nucleic acid sequence of the RHM15-1 mutant VP1 capsid is as follows (sequence ID number: 12). [Table 8]

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

[0191] The nucleic acid sequence of the RHM15-2 mutant VP1 capsid is as follows (sequence ID number: 13). [Table 10]

[0192] The amino acid sequence of the RHM15-3 / RHM15-5 mutant VP1 capsid is as follows: Yes (array ID number: 8). [Table 11]

[0193] The nucleic acid sequence of the RHM15-3 / RHM15-5 mutant VP1 capsid is as follows: (Array ID number: 14). [Table 12]

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

[0195] The nucleic acid sequence of the RHM15-4 mutant VP1 capsid is as follows (sequence ID number: 15). [Table 14]

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

[0197] The nucleic acid sequence of the RHM15-6 mutant VP1 capsid is as follows (sequence ID number: 16). [Table 16] Example 8 This example compares human IX with Rh74 and AAV8 using the Rh74 capsid mutant. Includes an explanation of factor expression studies.

[0198] In summary, the Rh74 capsid variant was used to create an AAV human factor IX expression vector. The mice were packaged and infected with AAV particles, and the expression level of factor IX was measured in the animals (blood). The determination was made within the plasma. The capsid mutant has the following amino acid substitutions at the indicated position. Ta. [Table 17]

[0199] Figure 5 shows the expression levels of human factor IX in plasma two weeks after treatment in treated animals. As shown in the figure, AAV human 2177 is capsidized with the RHM4-1 mutant capsid. The factor IX expression vector provides the highest level of expression and is capsidized by Rh74. AAV Human Factor IX Expression Vector and AAV8 Capsidated AAV Human Factor IX The expression level was substantially higher than that generated 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 (macaque monkeys), FIX production in animals at higher concentrations than when the same amount of AAV8 vector is administered. This includes detailed data showing that it led to a certain volume.

[0200] Cynomolgus monkeys were pre-selected to neutralize AAV antibodies, and their pre-treatment titers were 1:1. Individuals with full potential were selected to ensure successful trait introduction. These monkeys were subsequently introduced into human I. AAV8 vector expressing factor X transgene or AAV-Rh74 mutant RMH4-1 Choose one of the vectors, 3x10 12 The dose was administered by infusion at a rate of vg / kg. (For non-human primates) The plasma concentration of the human FIX transgene product (protein) in animals was observed throughout the observation period. The results were measured weekly by ELISA and are shown in Figure 6.

[0201] One of the individuals injected with AAV-RHM4-1-FIX developed a human factor IX transgenic gene. It produced inhibitory factors for the offspring, but this is a well-established phenomenon, and in humans and ma Due to slight differences in amino acids in the civet monkey protein, human FIX vector It occurs in approximately 20% of macaque monkeys treated with -. It occurred in one monkey treated with RHM4-1. The reason there was no visible result was because an antibody reaction occurred against the human transgene. RHM4 The second individual treated with -1 showed approximately twice the FIX compared to the macaque monkey treated with AAV8. The concentration was expressed.

Claims

1. Recombinant AAV (rAAV) particles comprising a VP1 capsid protein having one amino acid sequence of sequence ID number: 5, sequence ID number: 6, sequence ID number: 7, sequence ID number: 8, or sequence ID number: 10, wherein the genome of the recombinant AAV particle comprises an AAV reverse terminal repeat (ITR) and a heterologous polynucleotide sequence encoding a heterologous polynucleotide to be transcribed into a polypeptide, peptide, protein, or inhibitory nucleic acid, wherein the polynucleotide is operably linked to an expression regulatory element, and the heterologous polynucleotide sequence is adjacent to a 5' or 3' AAV reverse terminal repeat (ITR).

2. The recombinant AAV particle according to claim 1, wherein the expression control element includes a constitutive or controllable control element.

3. The recombinant AAV particle according to claim 1, wherein the expression control element comprises a tissue-specific expression control element or a promoter.

4. The recombinant AAV particle according to claim 3, wherein the tissue-specific expression regulatory element or promoter is liver-specific.

5. The recombinant AAV particle according to claim 1, wherein the expression regulatory element includes a liver-specific promoter.

6. The recombinant AAV particle according to claim 1, wherein the expression regulatory element is the ApoE-hAAT liver-specific promoter.

7. Recombinant AAV particles according to any one of claims 1 to 6, wherein the heterogeneous polynucleotide sequence is linked to the polyA sequence.

8. The recombinant AAV particle according to any one of claims 1 to 7, wherein the genome of the recombinant AAV particle further comprises one or more untranslated regions.

9. The recombinant AAV particle according to any one of claims 1 to 7, wherein the heterogeneous polynucleotide sequence is adjacent to 5' and 3' AAV reverse terminal repeats (ITRs).

10. Recombinant AAV particle according to any one of claims 1 to 9, wherein the AAV reverse terminal repeat (ITR) is derived from AAV2.

11. Recombinant AAV particles containing a VP1 capsid protein having one amino acid sequence of sequence ID number: 5, sequence ID number: 6, sequence ID number: 7, sequence ID number: 8, or sequence ID number: 10, wherein the genome of the recombinant AAV particle is arranged in the order from 5' to 3', (a) The first AAV reverse terminal repeat (ITR), (b) Liver-specific promoter and (c) A heterogeneous polynucleotide sequence encoding a heterogeneous polynucleotide that is operably linked to a polypeptide, peptide, protein, or the liver-specific promoter and transcribed into an inhibitory nucleic acid, (d) Poly-A sequence and (e) Second AAV reverse terminal repeat (ITR) and Recombinant AAV particles, including those mentioned above.

12. The recombinant AAV particle according to claim 11, wherein the first and second AAV reverse end repeats (ITRs) are derived from AAV2.

13. The recombinant AAV particle according to claim 11 or 12, wherein the liver-specific promoter is the ApoE-hAAT liver-specific promoter.

14. The recombinant AAV particle according to any one of claims 11 to 13, wherein the genome of the recombinant AAV particle further comprises one or more untranslated regions.

15. The aforementioned proteins include cystic fibrous transmembrane regulatory factor proteins (CFTR), blood coagulation (coagulation) factors including functional blood coagulation factors (e.g., factor XIII, factor X, factor VIII, factor VIIa, protein C), antibodies, erythropoietin, LDL receptors, lipoprotein lipases, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidases, hypoxanthine guanine phosphoribosyltransferases, β25-glucocerebrosidases, sphingomyelinases, lysosomal hexosaminidases, branched-chain keto acid dehydrogenases, and hormones. Mon, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factors 3 and 4, brain-derived neurotrophic factor, glial cell-derived growth factors, transforming growth factors α and β, cytokines, α-interferon, β-interferon, interferon γ, interleukin 2, interleukin 4, interleukin 12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance protein, tumor suppressor protein, p53, Rb, Wt-1, NF1, Von Recombinant AAV particles according to any one of claims 1 to 12, comprising Hippel-Lindau (VHL), adenomatous polyposis (APC), immunomodulatory peptides, immunotolerogenic or immunogenic peptides or regulatory T cell epitopes or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), DFNB1 (connexin 26 hearing loss), ACHM2, 3 and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid-activating protein), or zinc finger nuclease for genome editing, or a donor sequence used as a repair template for genome editing.

16. The recombinant AAV particle according to any one of claims 1 to 12, wherein the inhibitory nucleic acid comprises microRNA (miRNA), siRNA, shRNA, trans-splicing RNA, antisense RNA, or triple-stranded RNA.

17. A pharmaceutical composition comprising recombinant AAV particles according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17, further comprising empty capsid AAV.

19. The pharmaceutical composition according to claim 17, further comprising an empty capsid AAV-Rh74, or an empty capsid containing an AAV capsid sequence of sequence ID number: 5, sequence ID number: 6, sequence ID number: 7, sequence ID number: 8, or sequence ID number:

10.

20. A method for producing recombinant AAV particles according to any one of claims 1 to 16, A method comprising the step of culturing helper cells comprising a recombinant plasmid containing the genome of a recombinant AAV particle according to any one of claims 1 to 16, and a recombinant plasmid containing a nucleic acid encoding any one of sequence ID number: 5, sequence ID number: 6, sequence ID number: 7, sequence ID number: 8, or sequence ID number: 10.

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