BR112016001210B1 - RECOMBINATED AAV PARTICLES, AND PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

RECOMBINATED AAV PARTICLES, THEIR USES AND PHARMACEUTICAL COMPOSITIONS. The present 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 polynucleotides that function as or are transcribed into inhibitory nucleic acid sequences.

Description

CROSS REFERENCE TO RELATED ORDERS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 61/985,365, filed April 28, 2014, and U.S. Provisional Patent Application No. 61/857,161, filed July 22, 2013, all the orders incorporated herein by reference in their entirety.

Introduction

[0002] Genetic disorders, caused by the absence of or a defect in a desirable gene (loss of function) or expression of an undesirable or defective gene or (gain of function), lead to a variety of diseases. An example of a genetic loss-of-function disorder is hemophilia, an inherited bleeding disorder caused by deficiency of either clotting factor VIII (FVIII, hemophilia A) or factor IX (FIX, hemophilia B). An example of a gain-of-function genetic disorder is Huntington's disease, a disease caused by a pathological “HTT” gene (encoding the huntingtin protein) that encodes a mutated protein that accumulates and leads to the gradual destruction of neurons, particularly in ganglia. basal cells and in the cerebral cortex.

[0003] Current treatment for hemophilia consists of intravenous administration of recombinant clotting factor or on demand, in case bleeding occurs, or prophylactically. However, this therapeutic approach has several drawbacks such as the need for repeated infusions, the cost of treatment, the risk of developing immune responses to antitherapeutic factors and the risk of potentially fatal bleeding. These limitations have prompted the development of gene-based therapies for hemophilia. For this purpose, hemophilia is ideal for therapy based on gene transfer since 1) the therapeutic window is very wide, as levels just above 1% of normal can already result in a change in phenotype from severe to moderate and 100% levels are not associated with any side effects; 2) tissue-specific expression of the therapeutic transgene is not strictly required; and 3) there is considerable experience in measuring therapeutic efficacy endpoints. Furthermore, expression in the liver of clotting factor has been shown to induce immunological tolerance to the clotting factor itself, reducing the likelihood of potentially harmful immune responses against clotting factor.

[0004] Currently, adeno-associated virus (AAV) vectors are recognized as the gene transfer vectors of choice since they have the best safety and efficacy profile for gene delivery in vivo. Of the AAV serotypes isolated to date, AAV2 and AAV8 have been used to target the livers of humans affected by severe hemophilia B. Both vectors worked efficiently and in the case of AAV8 long-term expression of the therapeutic transgene was documented. Recent data in humans have shown that targeting the liver with an AAV vector achieves long-term expression of the FIX transgene at therapeutic levels.

[0005] Although these data are promising, the identification of AAV serotypes with high liver tropism and low seroprevalence in humans (a natural host for wild-type AAV) is critical to 1) achieving therapeutic levels of liver transgene expression in lowest possible vector dose to decrease the risk of triggering immune responses to the anti-AVV capsid; and 2) alternating AAV serotypes with unique seroprevalence will allow treating those patient populations otherwise ineligible for AAV gene transfer due to preexisting humoral immunity to AAVs. The invention addresses these needs and provides additional benefits.

summary

[0006] The invention provides adeno-associated virus (AAV) serotype AAV-Rh74 vector and related AAV vectors and viral particles. Such vectors include AAV-Rh74, which targets liver cells, hepatocytes, among other cell types. As a vector for polynucleotide sequence delivery, AAV-Rh74 and related AAV vectors direct polynucleotide expression in cells. Polynucleotides that encode proteins, such as proteins for therapeutic applications, are capable of being expressed at therapeutic levels upon administration.

[0007] Exemplary AAV-Rh74 and related AAV vectors include AAV-Rh74 variants. Particular capsid variants include a Rh74 capsid sequence with an amino acid substitution at any one of amino acid position 195, 199, 201 or 202 of the VP1 RH74 capsid sequence (SEQ ID NO:1). In particular aspects, the residues correspond to an A, V, P or N amino acid at any one of amino acid position 195, 199, 201 or 202 of the VP1 RH74 capsid sequence (SEQ ID NO:1). In more particular aspects, the capsid sequence has an A residue at amino acid position 195; a V residue at amino acid position 199, a P residue at amino acid position 201, or an N residue at amino acid position 202 of the VP1 RH74 capsid sequence (SEQ ID NO:1). In further more particular aspects, the capsid sequence has any two, three or all of the following: an A residue at amino acid position 195; a V residue at amino acid position 199, a P residue at amino acid position 201, or an N residue at amino acid position 202 of the VP1 RH74 capsid sequence (SEQ ID NO:1).

[0008] Recombinant AAV particles of the invention include an AVV capsid sequence of any capsid variant such as RHM4-1 (SEQ ID NO:5), RHM15-1 (SEQ ID NO:6), RHM15-2 (SEQ ID NO:7), RHM15-3/RHM15-5 (SEQ ID NO:8), RHM15-4 (SEQ ID NO:9) or RHM15-6 (SEQ ID NO:10). In particular embodiments, a recombinant AAV particle encapsulates or packages a vector genome (e.g., viral vector such as an AAV vector genome). Such recombinant AAV particles of the invention include a viral vector (e.g., AAV) genome that also includes a heterologous polynucleotide sequence.

[0009] Vector-mediated polynucleotide transfer of AAV-Rh74 and related AAV produced protein expression levels that were significantly higher than several other serotypes currently studied in preclinical and clinical settings. In particular, AAV-Rh74 targets polynucleotides to the liver with efficiency at least comparable to or greater than the gold standard for liver transduction, AAV8, both in mice and in dogs with hemophilia B. (See, for example, Figures 1 and 2). AAV-Rh74 variants such as, for example, capsid variant RHM4-1 (SEQ ID NO:5) target polynucleotides to the liver with efficiency comparable to or greater than AAV8 and greater than AAV Rh74 in mice (see, e.g., Figure 5). Furthermore, data in non-human primates show that AAV-Rh74 and variants of AAV-Rh74 such as RHM4-1 are approximately twice as potent as AAV8 in measuring liver-derived expression of hFIX (see, for example, Figures 4 and 6).

[0010] In this way, AAV-Rh74 and AAV-Rh74 variants such as capsid variants (e.g., RHM4-1) can be used to deliver polynucleotides, such as gene coding sequences, to express proteins that provide a benefit desirable or therapeutic, as well as for inhibitory nucleotides that reduce or inhibit the expression of an undesirable or defective gene, thereby treating a variety of diseases. For example, AAV-Rh74 and AAV-Rh74 capsid variants (e.g., RHM4-1) can be used as vectors to deliver to cells, tissues, and organs such as liver therapeutic genes (e.g., FIX, FVIII ) to treat hemophilia A, B, etc. Such AAV-Rh74 and AAV-Rh74 capsid variant vectors (e.g., RHM4-1) can also be used to deliver genes for a wide range of other metabolic or plasma protein deficiencies or for other therapeutic purposes such as, but not limited to, genes encoding zinc finger nucleases to perform genome editing in the liver and for local (liver) administration of immunomodulatory agents such as alpha-interferon to treat hepatitis virus infections or treat virtually any disease that requires or transduction in the liver or presence of the therapeutic transgene product in the bloodstream (which can be achieved by targeting the transgene for expression in the liver).

[0011] In addition to the efficient delivery of polynucleotides by AAV-Rh74 and related AAV vectors such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1) to cells in vitro, ex vivo and in vivo, The prevalence of anti-AAV-Rh74 antibodies in humans is lower than that of anti-AAV2 antibodies, and differs from that of anti-AAV8 antibodies (Table 1). Due to low seroprevalence, AAV and related AAV-Rh74 vectors such as AAV-Rh74 (capsid) variants (e.g., RHM4-1) can be used in a larger percentage of humans who would otherwise not be eligible for transfer. gene, for example, humans who may be seropositive for other AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, etc.). Furthermore, AAV-Rh74 and related vectors such as AAV-Rh74 variants including capsid variants (e.g., RHM4-1) can be efficiently produced at high titers (Table 2). In this way, AAV-Rh74 and related AAV vectors can be produced in large quantities for more prevalent clinical diseases.

[0012] According to the invention, there are provided recombinant AAV-Rh74 vectors and related AAV vectors such as AAV-Rh74 variants such as capsid variant particles (e.g., RHM4-1) that include (encapsulate, package) AAV vector genomes. In one embodiment, a recombinant AAV vector includes a heterologous polynucleotide sequence. In another embodiment, a recombinant AAV vector genome is encapsulated or packaged by an AAV-Rh74 capsid or related AAV such as AAV-Rh74 variants such as a capsid variant (e.g., RHM4-1).

[0013] In the invention recombinant AAV vectors, such as AAV-Rh74 vectors and related AAV vectors such as AAV-Rh74 (capsid) variant particles (e.g., RHM4-1) that include (encapsulate, package) genomes of recombinant AAV vector, the heterologous polynucleotide sequence can be transcribed and subsequently translated into a protein. Alternatively, the heterologous polynucleotide may be transcribed into a transcript that alone has a function or activity (e.g., as an inhibitory nucleic acid).

[0014] In various aspects, the heterologous polynucleotide sequence encodes a therapeutic protein. In particular aspects, the protein is a blood clotting factor (e.g., Factor XIII, Factor IX, Factor X, Factor VIII, Factor VIIa or protein C), CFTR (cystic fibrosis transmembrane regulatory protein), an antibody, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, β-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, a hormone, a growth factor ( for example, insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor 3 and 4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor α and β, etc.), a cytokine, α-interferon, β-interferon, interferon-Y, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony stimulating factor, lymphotoxin, etc.), a suicide gene product (e.g., herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, etc.), a drug resistance protein (e.g. , which provides resistance to a drug used in cancer therapy), a tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (Choroideremia), LCA 5 (LCA-Lebercillin), ornithine ketoacid aminotransferase (Atrophy do Girato), Retinoschisin1 (X-linked Retinoschisis), USH1C (Usher Syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis pigmentosa), DFNB1 (Connexin 26 deafness), ACHM 2, 3 and 4 (Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1, CLN2, gene deficiencies causing lysosomal storage diseases (e.g. sulfatases, N-acetylglucosamine-1-phosphate transferase , cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activating proteins) or one or more zinc finger nucleases for genome editing or donor sequences used as repair templates for genome editing.

[0015] In further aspects, the heterologous polynucleotide sequence encodes a therapeutic protein that in turn inhibits expression or function of an undesirable or aberrant (dysfunctional) protein present (endogenous) in an individual. In further aspects, the heterologous polynucleotide sequence is a polynucleotide that, when transcribed, is transcribed into an inhibitory nucleic acid (e.g., inhibitory RNA). In more particular aspects, an inhibitory nucleic acid is a single-stranded sequence or forms a double- or triple-stranded sequence. In further more particular aspects, an inhibitory nucleic acid is a micro-RNA (miRNA), siRNA, shRNA, trans-binding RNA, antisense RNA, or triplex-forming RNA.

[0016] In additional more particular aspects, an inhibitory nucleic acid inhibits expression of: huntingtin gene (HTT), a gene associated with dentatorubropalidolusian atrophy (e.g., atrophin 1, ATN1); androgen receptor on the opposite of Ataxin 8, also known as ATXN8OS, beta isoform of the 55 kDa regulatory B subunit of serine/threonine protein phosphatase 2A in spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12, 17), FMR1 ( fragile X mental retardation 1) in fragile X syndrome, FMR1 (fragile X mental retardation 1) in fragile X-associated tremor/ataxia syndrome, FMR1 (fragile in mental retardation due to fragile XE; Myotonin-protein kinase (MT-PK) in myotonic dystrophy; Frataxin in Friedreich's ataxia; a superoxide dismutase 1 (SOD1) gene mutant in amyotrophic lateral sclerosis; a gene involved in the pathogenesis of Parkinson's disease and/or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin/chexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, human immunodeficiency virus transactivator of transcription gene, in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transactivator response element gene, in HIV infection; C-C chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis C virus infection; acute kidney injury or kidney transplantation with delayed graft function or acute kidney failure due to damage to the kidney; protein kinase N3 (PKN3) in advanced recurrent or metastatic solid diseases; LMP2, LMP2 also known as proteasome subunit beta type 9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta type 8 (PSMB 8), metastatic melanoma; MECL1 also known as proteasome subunit beta type 10 (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, B cell CLL suppressor of apoptosis/lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; Furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta2 adrenergic receptor, glaucoma; RTP801/Redd1 also known as DAN damage-inducible transcript 4 protein, in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in non-arteritic ischemic optic neuropathy; Keratin 6A N17K mutant protein in pachyonychia congenita; influenza A virus genome/gene sequences in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome/gene sequences in SARS infection; respiratory syncytial virus genome/gene sequences in respiratory syncytial virus infection; Ebola filovirus genome/gene sequence in Ebola infection; genome/gene sequences of hepatitis B and C viruses in hepatitis B and C infection; herpes simplex virus (HSV) genome/gene sequences in HSV infection, coxsackievirus B2 genome/gene sequences in coxsackievirus B3 infection; silencing of a pathogenic allele of a gene (allele-specific silencing) such as torsin A (TOR1A) in primary dystonia, pan-class I allele-specific and HLA in transplantation; mutant rhodopsin gene (RHO) in autosomal dominantly inherited retinitis pigmentosa (adRP); or the inhibitory nucleic acid binds to a transcript of any of the above genes or sequences.

[0017] The recombinant AAV vectors of the invention and AAV-Rh74 and related AAV vectors such as AAV-Rh74 vector variants such as capsid variant particles (e.g., RHM4-1) that include (encapsulate, package) genome of recombinant AAV vector include additional elements that function in cis or in trans. In particular embodiments, a recombinant viral vector (e.g., AAV) and/or AAV-Rh74 vector or related AAV vector such as an AAV-Rh74 (capsid) variant particle (e.g., RHM4-1) that includes (encapsulates, packages) recombinant AAV vector genome also has: one or more Inverted Terminal Repeat (ITR) sequences flanking the 5' or 3' terminus of the heterologous polynucleotide sequence; an expression control sequence that directs transcription of the heterologous polynucleotide sequence (e.g., a promoter or enhancer that contributes to transcription of the heterologous polynucleotide sequence, such as a constitutive or regulatable control element or specific expression control element tissue); a poly-Adenine sequence located 3' of the heterologous polynucleotide sequence; a selectable marker (e.g., a protein that provides antibiotic resistance, such as kanamycin resistance); and/or an origin of replication.

[0018] Recombinant AAV vectors of the invention and AAV-Rh74 vectors and related AAV vectors such as AAV-Rh74 variants including capsid variant particles (e.g. RHM4-1) that include (encapsulate, package) vector genome of recombinant AAV may also include additional elements. In one embodiment, a recombinant vector genome includes a heterologous polynucleotide sequence and a filler or stuffer sequence. In particular aspects, a heterologous polynucleotide sequence has a length of less than about 4.7 Kb. In further particular aspects, a heterologous polynucleotide sequence has a length of less than 4.7 Kb and is positioned within two adeno-associated virus (AVV) ITR sequences. In further particular aspects, a filler or stuffer sequence of a length that when combined with the heterologous polynucleotide sequence the total combined length of the heterologous polynucleotide sequence and filler or stuffer sequence is between about 3.0-5.5 Kb or between about 4.0-5.0 Kb or between about 4.3-4.8 Kb.

[0019] Cargo or stuffer polynucleotide sequences can be located in the vector sequence at any desired position so that they do not prevent a function or activity of the vector. In one aspect, a filler or stuffer sequence is not positioned between a 5' and/or 3' ITR flanking the respective 5' and/or 3' termini of a heterologous polynucleotide sequence. In another aspect, a filler or stuffer sequence is positioned within a 5' and/or 3' ITR that flanks the respective 5' and/or 3' termini of a heterologous polynucleotide sequence. In a further aspect, a filler or stuffer sequence is positioned adjacent to the 5' and/or 3' ITR flanking the respective 5' and/or 3' termini of a heterologous polynucleotide sequence. In a further aspect, a filler or stuffer sequence is positioned within a heterologous polynucleotide sequence, e.g., analogous to an intron within a genomic nucleic acid.

[0020] Thus, in various embodiments, the filler or stuffer sequence is positioned adjacent to an AVV ITR sequence; positioned within two adeno-associated virus (AVV) ITR sequences; positioned outside two adeno-associated virus (AVV) ITR sequences; or there are two loading or stuffer polynucleotide sequences, a first filler or stuffer sequence positioned within two adeno-associated virus (AAV) ITR sequences and a second filler or stuffer sequence positioned outside two adeno-associated virus (AAV) ITR sequences. associated (AAV).

[0021] In more particular aspects, a filler or stuffer sequence has a length that when combined with said heterologous polynucleotide sequence the total combined length of the heterologous polynucleotide sequence and filler or stuffer sequence is between about 3.0- 5.5 Kb, between about 4.0-5.0 Kb or between about 4.3-4.8 Kb, when positioned within two adeno-associated virus (AAV) ITR sequences. In other more particular aspects, a filler or stuffer sequence has a length greater than 4.7 Kb, between about 5.0-10.0 Kb, or between 6.0-8.0 Kb, when positioned outside the two adeno-associated virus (AAV) ITR sequences.

[0022] In various further aspects, a filler or stuffer sequence is a sequence between about 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, 750-1000, 1000-1500, 15002000, 2000-2500, 2.50 0-3,000, 3,000-3,500, 3,500-4,000, 4,000 4,500, 4,500-5,000, 5,5000-6,000, 6,000-7,000, 7,000-8,000, or 8,0009,000 nucleotides in length.

[0023] Typically, a filler or stuffer sequence is inert or innocuous and has no function or activity. In several particular respects, a filler or stuffer sequence is not a bacterial polynucleotide sequence, a filler or stuffer sequence is not a sequence encoding a protein or peptide, a filler or stuffer sequence is a sequence distinct from either: the heterologous polynucleotide sequence, an AAV inverted terminal repeat (ITR) sequence, an expression control element, an origin of replication, a selectable marker, or a poly-Adenine (poly-A) sequence.

[0024] In several additional particular aspects, a filler or stuffer sequence is an intron sequence that is related to or unrelated to the heterologous polynucleotide sequence. In particular aspects, the intron sequence is positioned within the heterologous polynucleotide sequence. In other particular aspects, the intron sequence is related to the heterologous polynucleotide sequence since the intron is in genomic DNA, such as genomic DNA that encodes a protein, which protein is also encoded by the heterologous polynucleotide sequence.

[0025] The recombinant AAV vectors of the invention and AAV-Rh74 and related AAV vectors such as AAV-Rh74 variants such as capsid variant particles (e.g., RHM4-1) that include (encapsulate, package) genome of Recombinant AAV vector can be included in the cells. In such embodiments, the cells may comprise helper cells lysed to produce virus (AAV) particles (e.g., AAV-Rh74 vectors or related AAV vectors such as AAV-Rh74 capsid variants (e.g., RHM4-1) ) or target cells where it is desirable to express the heterologous polynucleotide sequence.

[0026] Recombinant AAV vectors and AAV-Rh74 vectors and related AAV vectors such as AAV-Rh74 variant particles (e.g., capsid variants such as RHM4-1) of the invention that include (encapsulate, package) genome of recombinant AAV vector can be included in pharmaceutical compositions. Such compositions are useful for administering recombinant vector (e.g., AAV) and virus particles such as AAV-Rh74 vectors and related AAV vectors such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1 ) that include (encapsulate, package) recombinant vector genomes (e.g., AAV) to an individual.

[0027] Recombinant AAV vectors and AAV-Rh74 vectors and related AAV vectors such as AAV-Rh74 variant particles (e.g., capsid variants such as RHM4-1) of the invention that include (encapsulate, package) genome of recombinant AAV vector can be employed in various methods and uses. In this way, methods and uses are provided for administering or transferring a heterologous polynucleotide sequence to an organism or cell, such as a mammal or a cell of a mammal.

[0028] In one embodiment, a method or use includes administering an adeno-associated virus (AAV) vector that includes a heterologous polynucleotide sequence (e.g., via an AAV-Rh74 or related AAV vector such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1)) that include (encapsulate, package) the vector genome to a mammal or a mammalian cell under conditions suitable for administering or transferring the polynucleotide sequence heterologous to the mammal or cell of a mammal. In one aspect, the method or use allows transfer/administration of the heterologous polynucleotide to the mammal and/or cell. In another aspect, the method allows transfer/administration of the heterologous polynucleotide to the mammal and/or cell and subsequent transcription of the heterologous polynucleotide in this manner forming a transcript. In a further aspect, the method allows transfer/administration of the heterologous polynucleotide to the cell, subsequent transcription to form a transcript and subsequent translation to form a gene product (protein). In particular, for example, in the latter two aspects a heterologous polynucleotide sequence is operably linked to an expression control element conferring transcription of the heterologous polynucleotide sequence and optionally subsequent translation of the transcript.

[0029] In additional embodiments, a method or use is for administering or transferring a heterologous polynucleotide sequence to an individual (e.g., mammal) or a cell of an individual (e.g., mammal) and includes administering a viral particle (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 capsid variant particles (e.g., RHM4-1), a plurality of such viral particles (e.g., AAV) or a pharmaceutical composition of such viral particle (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 capsid variant particles (e.g., RHM4-1)) or plurality of such viral particles (e.g., AAV) to an individual (e.g., mammal ) or a cell of the individual (e.g., mammal), thereby administering or transferring a heterologous polynucleotide sequence to the individual (e.g., mammal) or cell of the individual (e.g., mammal). In another embodiment, a method or use is for treating an individual (e.g., mammal) deficient in or in need of protein expression or function or in need of expression or function of an endogenous protein (e.g., an undesirable, aberrant, or dysfunctional protein), which includes provision of a viral particle (e.g., AAV-Rh47 and related AAV such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1)), a plurality of such viral particles (e.g., AAV) or a pharmaceutical composition of a viral particle (e.g., AAV-Rh74 and related AAV such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1)) or plurality of such viral particles (e.g., AAV); and administering the viral particle (e.g., AAV-Rh74 and related AAV such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1)), plurality of such viral particles (e.g., AAV) or composition pharmaceutical viral particle (e.g., AAV-Rh74 and related AAV such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1)) or a plurality of such viral particles (e.g., AAV) to the individual (e.g., mammalian), where the heterologous polynucleotide sequence is expressed in the mammal or where the heterologous polynucleotide sequence encodes an inhibitory sequence or protein that reduces expression or function of an endogenous protein (e.g., an undesirable, aberrant, or dysfunctional protein ) in the individual (e.g. mammal).

[0030] Methods and uses for administration or application include any mode compatible with an individual. In particular embodiments, a viral particle (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 capsid variant (e.g., RHM4-1)) or plurality of such viral particles (e.g., AAV) (e.g. , AAV-Rh74 vectors or related AAV vectors such as AAV-Rh74 capsid variants (e.g., RHM4-1)) is administered or applied intravenously, intra-arterially, intramuscularly, subcutaneously, orally, via intubation, through catheter, dermally, intracranially, via inhalation, intracavity or mucosally.

[0031] Individuals include mammals, such as humans and non-humans (e.g., primates). In particular embodiments, an individual would benefit from or have a need for expression of a heterologous polynucleotide sequence.

[0032] According to the invention, methods of producing plasmid particles and recombinant vector (e.g., AAV) viruses (e.g., AAV-Rh74 and related AAV such as variants of AAV-Rh74 (e.g., variants of capsid such as RHM4-1)) that include recombinant (encapsulated, packaged) vector (e.g., AAV) are provided. In one embodiment, a method of producing recombinant viral or AAV particles includes introducing into a packaging helper cell a recombinant vector plasmid (e.g., AAV) to produce a productive viral infection (e.g., AAV-Rh74 and AAV related such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1)); and culturing the helper cells under conditions to produce recombinant viral particles (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 capsid variants (e.g., RHM4-1)). In another embodiment, a method of producing recombinant viral or AAV particles with reduced amounts of recombinant viral particles (e.g., AAV-Rh74 and related AAV such as AAV-Rh74 variants (e.g., capsid variants such as RHM4- 1)) where the recombinant viral vector includes contaminating nucleic acid, includes introduction into a packaging helper cell of a recombinant vector plasmid (e.g., AAV); and culturing the helper cells under conditions to produce recombinant viral particles (e.g., AAV-Rh74 and related AAV such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1)), wherein the recombinant viral particles ( for example, AAV-Rh74 and related AAV such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1)) produced have reduced numbers of viral particles (e.g., AAV-Rh74 and related AAV such as variants of AAV-Rh74 (e.g., capsid variants such as RHM4-1)) with recombinant vector genomes that contain contaminating nucleic acid compared to viral particle numbers (e.g., AAV-Rh74 and related AAV such as AAV variants - Rh74 (e.g., capsid variants such as RHM4-1)) that contain contaminating nucleic acid produced under conditions where the filler or stuffer sequence is absent from the recombinant viral vector. In particular aspects, the contaminating nucleic acid is bacterial nucleic acid; or a sequence other than the heterologous polynucleotide sequence, or ITR, promoter, enhancer, origin of replication, poly-Adenine sequence or selectable marker.

[0033] Helper cells include mammalian cells. In particular embodiments, a helper cell provides helper functions (e.g., AAV) to package the heterologous polynucleotide sequence into a viral particle (e.g., AAV particles such as AAV-Rh74 vectors and related AAV such as AAV-Rh74 variant). Rh74 (e.g. capsid variants such as RHM4-1)). In particular aspects, a helper cell provides AAV Rep and/or Cap proteins (e.g., Rep78 and/or Rep68 proteins); a helper cell is stably or transiently transfected with polynucleotide(s) encoding Rep and/or Cap protein sequence(s); a helper cell is stably or transiently transfected with sequence(s) encoding Rep78 and/or Rep68 protein polynucleotide.

[0034] Recombinant vector plasmids of the invention (e.g., AAV) can be based on any strain or serotype, including hybrids or chimeras of different serotypes. The recombinant viral particles of the invention (e.g., AAV) are typically based on AAV-Rh74 or related AAV such as AAV-Rh74 and related AAV such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1). , but also include hybrids or chimeras with different serotypes. Representative AAV serotypes include, without limitation, serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and Rh10. In this way, the recombinant viral particles of the invention (e.g., AAV) comprising vector genomes may include a capsid protein of a different serotype, a mixture of serotypes or hybrids or chimeras of different serotypes such as a VP1, VP2 capsid protein. or VP3 of serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and Rh10. Furthermore, the recombinant vectors of the invention (e.g., AAV), sequences, plasmids, vector genomes may include elements of any serotype, a mixture of serotypes, or hybrids or chimeras of different serotypes. In various embodiments, a recombinant AAV vector includes a Cap, Rep and/or ITR sequence derived from serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh74 or Rh10 or a mixture, hybrid or chimera of any of the above AAV serotypes.

Description of the Drawings

[0035] Figure 1 shows plasma levels of human factor IX (FIX) in C57BL/6 mice (n=5 per group) injected via the tail vein with AAV vectors expressing the FIX transgene under the control of a specific promoter of liver. Vector dose 2,510 vector genomes per mouse. Plasma levels of FIX transgene product (FIX protein) were measured by ELISA at weeks 1, 2, and 4 post-gene transfer. AAV-Rh74 conferred the highest levels of FIX transgene expression.

[0036] Figure 2 shows plasma levels of canine FIX in dogs with hemophilia B after administration of 312 genome vectors per kilogram (kg) of weight. AAV vectors were infused intravenously (IV) via the saphenous vein and FIX levels were monitored via ELISA. Expression of the therapeutic FIX transgene was driven by a liver-specific promoter. The AAV8 and AAV-Rh74 vectors performed more or less equally in dogs with hemophilia B and were both superior to AAV6.

[0037] Figure 3 shows amino acid sequences of VP1, VP2 and VP3 of AAV-Rh74 and, for VP1, polynucleotide (DNA) sequence (SEQ ID NOs: 1-4).

[0038] Figure 4 shows administration of AAV8 and AAV-Rh74 vector expressing human factor IX (FIX) (under the control of a liver-specific promoter) to rhesus macaques, a non-human primate, and expression of FIX in the animals. Animals receiving the AAV-Rh74-FIX vectors (the last two bars towards the right margin) expressed the FIX transgene at higher levels compared to the other groups of animals injected with the same dose.

[0039] Figure 5 shows expression levels of human Factor IX in plasma in animals administered with AAV human Factor IX expression vector encapsulated by the indicated AAV (Rh74 variants, such as RHM4-1 variant), compared with vector of expression of AAV human Factor IX encapsulated from Rh74 and AAV8.

[0040] Figure 6 shows vector administration of AAV8 and AAV-Rh74 variant RHM4-1 expressing Factor IX (under the control of a liver-specific promoter) to Cynomolgus macaques, a non-human primate, and expression of FIX in the animals .

Detailed Description

[0041] The invention is based, at least in part, on data indicating that the adeno-associated virus (AVV) serotype AAV-Rh74 and related AAV variants such as AAV-Rh74 capsid variants (e.g., RHM4- 1, RHM15-1, RHM15-2, RHM15-3/RHM15- 5, RHM15-4 and RHM15-6) have a high tropism for hepatocytes, which are liver cells. As a vector for transfer/administration of polynucleotide (e.g., genes, inhibitory nucleic acid, etc.) to cells, AAV-Rh74 and related AAV variants such as AAV-Rh74 variants (e.g., capsid variants such as RHM4- 1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4 and RHM15-6) can be used to provide therapeutic levels of expression in liver after intravenous administration. Furthermore, AAV-RH74 vectors and related AAV variants such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15- 4 and RHM15-6) mediate protein expression levels produced by gene transfer/administration that were significantly higher than several other serotypes (see, for example, Figures 1, 2, 4, 5 and 6). In particular, AAV-Rh74 vector and related AAV-Rh74 capsid variants (e.g., RHM4-1) target genes for delivery to the liver with efficiency at least comparable to and typically superior to the gold standard for liver transduction. AAV8, in dogs with hemophilia B and/or in mice and/or monkeys. In this way, AAV-Rh74 vectors and related AAV variants such as AAV-Rh74 capsid variants (e.g., RHM4-1) can be used to transfer/deliver heterologous polynucleotides, such as coding sequences (genes) for proteins that provide a desirable or therapeutic benefit, as well as inhibitory nucleic acid (e.g., antisense) that reduces or inhibits expression of an undesirable or defective (e.g., pathological) gene, thereby treating a variety of diseases. For example, a recombinant vector genome (e.g., AAV) can be packaged or encapsulated within the AAV-Rh74 vector or related AAV variants such as AAV-Rh74 capsid variants (e.g., RHM4-1, RHM15- 1, RHM15-2, RHM15-3/RHM15-5, RHM15-4 and RHM15-6) in order to transfer/administer a heterologous polynucleotide to a cell.

[0042] As shown here, adeno-associated virus (AAV) serotype AAV-Rh47 and related AAV variants such as AAV-Rh74 capsid variants (e.g., RHM4-1, RHM15-1, RHM15-2, RHM15 -3/RHM15-5, RHM15-4 and RHM15-6) provide a means for delivering polynucleotide sequences into cells ex vivo, in vitro and in vivo. Such polynucleotide sequences may encode proteins such that cells into which the polynucleotides are administered express the encoded proteins. For example, a recombinant AAV vector may include heterologous polynucleotides encoding a desired protein or peptide, or a heterologous polynucleotide that when transcribed comprises an inhibitory sequence (e.g., RNA), e.g., a sequence that targets a gene for inhibition of expression. . Application or administration of a vector to an individual (e.g., mammal) in this manner provides not only heterologous polynucleotides encoding proteins and peptides to the individual, but also inhibitory nucleic acids that target genes for inhibition of expression or function in the individual.

[0043] Thus, according to the invention AAV-Rh74 vectors and related AAV vector variants such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1, RHM15-1, RHM15-2 , RHM15-3/RHM15-5, RHM15-4 and RHM15-6) that include (encapsulate or package) vector genome including polynucleotide sequences encoding peptides and proteins, as well as polynucleotide sequences that directly or when transcribed comprise inhibitory nucleic acids that target genes for inhibition of expression or function, are provided.

[0044] A “recombinant vector” or “AAV vector” is derived from the wild-type genome of a virus, such as AAV, through the use of molecular methods to remove the wild-type genome of the virus (e.g., AAV) , and replacing with a non-native nucleic acid, such as a heterologous polynucleotide sequence (e.g., a therapeutic gene expression cassette). Typically, for AAV one or both inverted terminal repeat (ITR) sequences from the wild-type AAV genome are retained in the AAV vector. A recombinant viral vector (e.g., AAV) is distinguished from a viral genome (e.g., AAV) in that all or a portion of the viral genome has been replaced with a non-native sequence with respect to the viral genomic nucleic acid (e.g. , AAV) such as a heterologous polynucleotide sequence. Incorporation of a non-native sequence such as a heterologous polynucleotide thereof defines the viral vector (e.g., AAV) as a “recombinant” vector, which in the case of AAV may be referred to as an “rAAV vector.”

[0045] A recombinant vector sequence (e.g., AAV) can be packaged into a virus (also referred to herein as a “particle” or “virion”) for subsequent infection (transformation) of a cell, ex vivo, in vitro or in vivo. Where a recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle may be referred to as an “rAAV”. Such particles or virions will typically include proteins that encapsulate or package the vector genome. Particular examples include viral envelope proteins and, in the case of AAV, capsid proteins.

[0046] In particular embodiments, a recombinant vector (e.g., AAV) is a parvovirus vector. Parvoviruses are small viruses with a single-stranded DNA genome. “Adeno-associated viruses” (AAV) are in the parvovirus family.

[0047] Parvoviruses including AAV are useful viruses as gene therapy vectors since they can penetrate cells and introduce nucleic acid/genetic material so that the nucleic acid/genetic material can be stably maintained in cells. Furthermore, these viruses can introduce nucleic acid/genetic material at specific sites, for example, such as a specific site on chromosome 19. Because AAVs are not associated with pathogenic disease in humans, AAV vectors are capable of delivering sequences of heterologous polynucleotide (e.g., proteins and therapeutic agents) to human patients without causing substantial AAV pathogenesis or disease.

[0048] AAV-Rh74 and related AAV variants such as AAV-Rh74 or related AAV such as AAV-Rh74 variant serotypes (e.g., capsid variants such as RHM4-1) (e.g., VP1, VP2 sequences and/or VP3) may or may not be distinct from other AAV serotypes including, for example, AAV1-AAV11 or Rh10 (e.g., distinct from VP1, VP2 and/or VP3 sequences of any of the AAV1-AAV11 or Rh10 serotypes). ). As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological distinction is determined based on the range of cross-reactivity between antibodies to one AAV compared to another AAV. Such differences in cross-reactivity are generally due to differences in capsid protein sequences/antigenic determinants (e.g., due to differences in sequence of VP1, VP2 and/or VP3 of AAV serotypes). Despite the possibility that AAV-Rh74 variants including capsid variants may not be serologically distinct from Rh74 or another AAV, they differ in at least one nucleotide or amino acid residue compared to Rh74 or another AAV.

[0049] Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing serotypes and characterized for neutralizing activity and any antibodies that neutralize the virus of interest have been found. As more naturally occurring virus isolates are found and/or capsid mutants generated, they may or may not have serological differences with any of the currently existing serotypes. Thus, in cases where the new virus (e.g., AAV) has no serological difference, this new virus (e.g., AAV) would be a subgroup or variant of the corresponding serotype. In many cases, serology testing for neutralization activity still has to be performed on mutated viruses with capsid sequence modifications to determine whether they are of another serotype according to the traditional definition of serotype. Therefore, for the sake of convenience and to avoid repetition, the term “serotype” refers broadly to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are not serologically distinct that they may be within a subgroup or a variant of a given serotype.

[0050] Recombinant vector plasmids (e.g., AAV), as well as methods and uses thereof, include any viral strain or serotype. As a non-limiting example, a recombinant vector plasmid (e.g., AAV) can be based on any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -rh74, -rh10 or AAV-2i8, for example. Such vectors may be based on the same lineage or serotype (or subgroup or variant) or be different from each other. As a non-limiting example, a serotype genome-based recombinant vector (e.g., AAV) plasmid may be identical to one or more of the capsid proteins that package the vector. Further, a recombinant vector plasmid (e.g., AAV) may be based on an AAV serotype genome (e.g., AAV2) distinct from one or more of the capsid proteins packaging the vector, in which case at least one of the three Capsid proteins could be an AAV-Rh74 or related AAV variant such as AAV-Rh74 or related AAV such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4 and RHM15-6), for example.

[0051] AAV-Rh74 has gene/protein sequences identical to the characteristic sequences for AAV-Rh74 (see, for example, VP1, VP2, VP3 of Figure 3). As used herein, an “AAV-Rh74-related AAV vector” and grammatical variations thereof refers to one or more AAV proteins (e.g., VP1, VP2, and/or VP3 sequences) that have substantial sequence identity with one or more polynucleotide or polypeptide sequences comprising AAV-Rh74. Such AAV-Rh74 related AAV vectors may then have one or more sequences distinct from AAV-Rh74, but may exhibit substantial sequence identity with one or more genes and/or have one or more functional features of AAV-Rh74 (e.g. , such as cell/tissue tropism). For example, AAV-Rh74 related AAV variant RHM4-1 has a capsid with four amino acids different from the Rh74 capsid. AAV-Rh74 and exemplary related AAV variants such as AAV-Rh74 or related AAV such as AAV-Rh74 variant sequences (e.g., capsid variants such as RHM4-1, RHM15-1, RHM15-2, RHM15-3 /RHM15-5, RHM15-4 and RHM15-6) include VP1, VP2 and/or VP3 shown here, for example, in Figure 3. In an exemplary non-limiting embodiment, an AAV vector related to AAV-Rh74 has a polynucleotide , polypeptide or subsequence thereof that includes or consists of a sequence at least 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc. ) identical to one or more AAV-Rh74 VP1, VP2 and/or VP3 sequences shown in Figure 3.

[0052] According to the invention, methods and uses include AAV-Rh74 sequences (polypeptides and nucleotides) and subsequences thereof that exhibit less than 100% sequence identity with an AAV-Rh74 gene or protein sequence of reference (e.g., VP1, VP2, and/or VP3 sequences shown in Figure 3), but are distinct from and not identical to known AAV genes or proteins, such as AAV1-AAV11, AAV-Rh10, etc. genes or proteins . In one embodiment, an AAV-Rh74 polypeptide or subsequence thereof includes or consists of a sequence that is at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical to any reference AVV-Rh74 sequence or subsequence thereof (e.g., VP1, VP2 and/or VP3 sequences shown in Figure 3). In particular aspects, an AAV-Rh74-related variant has one, two, three, or four of the four amino acid substitutions shown in AAV-Rh74 (e.g., capsid variant RHM4-1, RHM15-1, RHM15-2, RHM15- 3/RHM15-5, RHM15-4 and RHM15-6).

[0053] Recombinant vectors (e.g., AAV), including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 and variant, related, hybrid and chimeric, can be constructed using recombinant techniques that are known to those skilled in the art, to include one or more heterologous polynucleotide sequences (transgenes) flanked with one or more functional AAV ITR sequences. Such vectors may have one or more of the wild-type AAV genes deleted in whole or in part, e.g., a rep and/or cap gene, but retain at least one functional flanking ITR sequence as necessary for rescue. replication and packaging of the recombinant vector into an AAV vector particle. An AAV vector genome would then include sequences required in cis for replication and packaging (e.g., functional ITR sequences).

[0054] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA and antisense DNA, and mRNA, rRNA, bound or unbound tRNA, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), ( si)Small or short interfering RNA, trans-binding RNA or antisense RNA). Polynucleotides include naturally occurring, synthetic and intentionally altered or modified polynucleotides as well as analogs and derivatives. Polynucleotides can be single, double or triplex, linear or circular and can be of any length. In polynucleotides under discussion, a sequence or structure of a particular polynucleotide may be described herein in accordance with the convention of providing the sequence in the 5' to 3' direction.

[0055] A “heterologous” polynucleotide refers to a polynucleotide inserted into a vector (e.g., AAV) for the purposes of vector-mediated transfer/administration of the polynucleotide to a cell. Heterologous polynucleotides are typically distinct from the vector nucleic acid (e.g., AAV), that is, they are non-native with respect to the viral nucleic acid (e.g., AAV). Once transferred/administered to a cell, a heterologous polynucleotide, contained within the virion, can be expressed (e.g., transcribed and translated if appropriate). Alternatively, a heterologous polynucleotide transferred/administered to a cell, contained within the virion, does not need to be expressed. Although the term “heterologous” is not always used herein in reference to polynucleotides, reference to a polynucleotide even in the absence of the “heterologous” modifier is intended to include heterologous polynucleotides despite the omission.

[0056] The “polypeptides”, “proteins” and “peptides” encoded by the “polynucleotide sequences” include native full-length sequences, as with naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants as long as the subsequence, modified form or variant retains some degree of functionality of the native full-length protein. In methods and uses of the invention, such polypeptides, proteins and peptides encoded by the polynucleotide sequences may be, but need not be, identical to the endogenous protein that is defective, or whose expression is insufficient, or deficient in the treated mammal.

[0057] AAV-Rh74 serotype of the adeno-associated virus (AAV) of the invention and related AAV variants such as AAV-Rh74 variants (e.g., capsid variants such as RHM4-1, RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4 and RHM15-6) can be used to introduce/deliver polynucleotides stably or transiently into cells and their progeny. The term “transgene” is used here to conveniently refer to such a heterologous polynucleotide that has been introduced into a cell or organism. Transgene includes any polynucleotide, such as a gene that encodes a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (e.g., does not have an expression control element, such as a promoter that directs transcription).

[0058] For example, in a cell having a transgene, the transgene was introduced/transferred by means of a vector, such as AAV, “transforming” the cell. The terms “transform” and “transfect” refer to the introduction of a molecule such as a polynucleotide into a host cell or organism.

[0059] A cell into which the transgene has been introduced is referred to as a “transformed cell” or “transformant”. In this way, a “transformed” or “transfected” cell (e.g., in a mammal, such as a cell or tissue or organ cell), means a genetic change in a cell following incorporation of an exogenous molecule, e.g., a polynucleotide or protein (e.g., a transgene) in the cell. In this way, a “transfected” or “transformed” cell is a cell into which, or a progeny thereof, an exogenous molecule has been introduced, for example. The cell(s) can be propagated and the introduced protein expressed or nucleic acid transcribed. For gene therapy uses and methods, a transformed cell may be in an individual.

[0060] The introduced polynucleotide may or may not be integrated into the nucleic acid of the recipient cell or organism. If an introduced polynucleotide integrates into the nucleic acid (genomic DNA) of the recipient cell or organism it can be stably maintained in that cell or organism and further passed on to or inherited by the progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist in the recipient host cell or organism only transiently.

[0061] Cells that can be transformed include a cell from any type of tissue or organ, of any origin (for example, mesoderm, ectoderm or endoderm). Non-limiting cell examples include cells of the liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., islet beta cells), lung, central or peripheral nervous system, such as brain (e.g., neural cells, glial or ependymal) or spine, kidney, eye (e.g., retinal, cell components), spleen, skin, thymus, testicles, lung, diaphragm, heart (cardiac), muscle or psoas, or intestine (e.g., endocrine), adipose tissue (white, brown, or beige), muscle (e.g., fibroblasts), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nerve cells, or hematopoietic cells (e.g., blood or lymph) . Additional examples include stem cells, such as pluripotent or multipotent progenitor cells that develop or differentiate into cells of the liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., islet beta cells), lung, central nervous system or peripheral, such as brain (e.g., neural, glial, or ependymal cells) or spine, kidney, eye (retinal, cellular components), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle, or psoas , or intestine (e.g., endocrine), adipose tissue (white, brown, or beige), muscle (e.g., fibroblasts), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nerve cells or hematopoietic (e.g. blood or lymph).

[0062] A “therapeutic molecule” in one embodiment is a peptide or protein that can alleviate or reduce symptoms that result from an absence or defect in a protein in a cell or individual. Alternatively, a “therapeutic” peptide or protein encoded by a transgene is one that confers a benefit to an individual, e.g., correcting a genetic defect, correcting a gene deficiency (expression or functional), or an anticancer effect.

[0063] Particular non-limiting examples of heterologous polynucleotides encoding gene products (e.g., therapeutic proteins) that are useful according to the invention include, but are not limited to: genes comprising or encoding CFTR (fibrosis transmembrane regulatory protein cystic cyst), a blood clotting factor (Factor XIII, Factor IX, Factor X, Factor VIII, Factor VIIa, protein C, etc.) including gain-of-function blood clotting factor, an antibody, a 65 kDa retinal pigment epithelium-specific (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, β-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, a hormone, a growth factor (e.g. , insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor 3 and 4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth α and β, etc.), a cytokine, α-interferon, β-interferon, interferon-Y, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony stimulating factor, lymphotoxin, etc.) , a suicide gene product (e.g., herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, etc.), a drug resistance protein (e.g., that provides resistance to a drug used in cancer therapy), a tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein Tregitope [de Groot et al., Blood 2008 Oct 15;112(8):3303] or hCDR1 [Sharabi et al., Proc. Natl. academic Sci. USA 6 June 2006;103(23):8810-5], insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort 1 protein (Choroideremia), LCA 5 (LCA-Lebercillin), ornithine ketoacid aminotransferase (Gyrate Atrophy), Retinoschisin 1 (X-linked Retinoschisis), USH1C (Usher Syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis pigmentosa), DFNB1 (Connexin deafness 26), ACHM 2, 3 and 4 (Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1, CLN2, gene deficiencies causing lysosomal storage diseases (e.g. sulfatases, N-acetylglucosamine- 1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, a sphingolipid activating protein, etc.), one or more zinc finger nucleases for genome editing or donor sequences used as repair templates for genome editing .

[0064] Additional non-limiting examples of heterologous polynucleotides encoding gene products (e.g., therapeutic proteins) that are useful according to the invention include those that can be used in the treatment of a disease or disorder including, but not limited to, fibrosis cystic (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (e.g., Duchenne, Becker), Gaucher disease, Hurler disease, adenosine deaminase deficiency, glycogen storage diseases and other metabolic defects, degenerative retinal diseases (and other diseases of the eye), and diseases of solid organs (e.g. brain, liver, kidney, heart).

[0065] All mammalian and non-mammalian forms of polynucleotides encoding gene products, including the non-limiting genes and proteins disclosed herein, are expressly included, either known or unknown. Thus, the invention includes genes and proteins from non-mammalian, non-human mammals and humans, which genes and proteins function in a manner substantially similar to the human genes and proteins described herein. A non-limiting example of a non-mammalian gene is a Fok nuclease domain, which is of bacterial origin. Non-limiting examples of mammalian non-human FIX sequences are described in Yoshitake et al., 1985, supra; Kurachi et al., 1995, supra; Jallat et al., 1990, supra; Kurachi et al., 1982, Proc. Natl. academic Sci. USA 79:6461-6464; Jaye et al., 1983, Nucl. Acids Res 11:2325-2335; Anson et al., 1984, EMBO J. 3:1053-1060; Wu et al., 1990, Gene 86:275-278; Evans et al., Proc. Natl. academic Sci. USA 86:10095 (1989), Blood 74:207-212; Pendurthi et al., 1992, Thromb. Res. 65:177-186; Sakar et al., 1990, Genomics 1990, 6:133-143; and Katayama et al., 1979, Proc. Natl. academic Sci USA 76:4990-4994.

[0066] As shown here, heterologous polynucleotide sequences (transgene) include inhibitory and antisense nucleic acid sequences. Inhibitory, antisense, siRNA (small interfering RNA), miRNA (micro RNA), shRNA (small hairpin RNA), RNAi and antisense oligonucleotides can modulate expression of a target gene. Such molecules include those capable of inhibiting expression of a target gene involved in mediating a disease process, thereby reducing, inhibiting or alleviating one or more symptoms of a disease.

[0067] Antisense includes single-, double- or triple-stranded polynucleotides and peptide nucleic acids (PNAs) that bind to RNA or DNA transcripts (e.g., genomic DNA). Oligonucleotides derived from the transcription start site of a target gene, for example, between positions -10 and +10 of the start site, are another particular example. Triplex-forming antisense can bind to double-stranded DNA in this manner inhibiting gene transcription. “RNAi” is the use of single- or double-stranded RNA sequences for inhibition of gene expression (see, for example, Kennerdell et al., Cell 95:1017 (1998); and Fire et al., Nature, 391:806 ( 1998)). Double-stranded RNA sequences from a target gene coding region can then be used to inhibit or prevent gene expression/transcription in accordance with the methods and uses of the invention. Antisense and RNAi can be produced based on nucleic acids encoding target gene sequences (e.g., huntingtin or HTT), such as nucleic acid encoding mammalian and human HTT. For example, a single- or double-stranded nucleic acid (e.g., RNA) can target the HTT transcript (e.g., mRNA).

[0068] An “siRNA” refers to a therapeutic molecule involved in the process of RNA interference for sequence-specific post-transcriptional gene silencing or gene inactivation. siRNAs have homology to the cognate mRNA sequence of the targeted gene. Small interfering RNAs (siRNAs) can be synthesized in vitro or generated through ribonuclease III cleavage of longer dsRNA and mediate sequence-specific mRNA degradation. siRNA or other nucleic acids of the invention can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. siRNA can be synthesized as two separate, complementary RNA molecules or as a single RNA molecule with two complementary regions. Commercial suppliers of synthetic RNA molecules or synthesis reagents include Applied Biosystems (Foster City, CA, USA), Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford , III, USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass, USA) and Cruachem (Glasgow, UK). Specific siRNA constructs for RNA inhibition of a target gene can be between 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 using conventional methods known to one of skill in the art.

[0069] Particular non-limiting examples of genes (e.g., genomic DNA) or transcript of a pathogenic gene (e.g., RNA or mRNA) that can be targeted with inhibitory nucleic acid sequences in accordance with the invention include, but are not limited to: genes associated with polynucleotide repeat diseases such as huntingtin gene (HTT), a gene associated with dentatorubropalidolusian atrophy (e.g., atrophin 1, ATN1); androgen receptor on the 8, also known as ATXN8OS, beta isoform of the 55 kDa regulatory subunit B of serine/threonine protein phosphatase 2A in spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12, 17), FMR1 (mental retardation by Fragile X 1) in fragile X syndrome, FMR1 (fragile X mental retardation 1) in fragile X-associated tremor/ataxia syndrome, FMR1 (fragile by fragile XE; Myotonin-protein kinase (MT-PK) in myotonic dystrophy; Frataxin in Friedreich's ataxia; a mutant superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; a gene involved in the pathogenesis of Parkinson's disease and/or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin/chexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, human immunodeficiency virus transactivator of transcription gene, in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transactivator response element gene, in HIV infection; C-C chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis C virus infection; acute kidney injury or kidney transplantation with delayed graft function or acute kidney failure due to damage to the kidney; protein kinase N3 (PKN3) in advanced recurrent or metastatic solid diseases; LMP2, LMP2 also known as proteasome subunit beta type 9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta type 8 (PSMB 8), metastatic melanoma; MECL1 also known as proteasome subunit beta type 10 (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, B cell CLL suppressor of apoptosis/lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; Furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta2 adrenergic receptor, glaucoma; RTP801/Redd1 also known as DAN damage-inducible transcript 4 protein, in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in non-arteritic ischemic optic neuropathy; Keratin 6A N17K mutant protein in pachyonychia congenita; influenza A virus genome/gene sequences in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome/gene sequences in SARS infection; respiratory syncytial virus genome/gene sequences in respiratory syncytial virus infection; Ebola filovirus genome/gene sequence in Ebola infection; genome/gene sequences of hepatitis B and C viruses in hepatitis B and C infection; herpes simplex virus (HSV) genome/gene sequences in HSV infection, coxsackievirus B3 genome/gene sequences in coxsackievirus B3 infection; silencing of a pathogenic allele of a gene (allele-specific silencing) such as torsin A (TOR1A) in primary dystonia, pan-class I allele-specific and HLA in transplantation; or mutant rhodopsin gene (RHO) in autosomal dominantly inherited retinitis pigmentosa (adRP).

[0070] Polynucleotides, polypeptides and subsequences thereof include modified and variant forms. As used herein, the term “modify” or “variant” and grammatical variations thereof means that a polynucleotide, polypeptide or subsequence thereof deviates from a reference sequence. Modified sequences and variants may then have substantially the same, greater or lesser activity or function than a reference sequence, but at least retain partial activity or function of the reference sequence.

[0071] In this way, the invention also includes naturally occurring and non-naturally occurring variants. Such variants include AAV-Rh74 variants such as AAV-Rh74 capsid variants. Particular examples of such AAV-Rh74 capsid variants include RHM15-1, RHM15-2, RHM15-3, RHM15-4, RHM15-5, RHM15-6 and RHM4-1 (see, for example, Figure 5).

[0072] Such variants also include gain and loss of function variants. For example, wild-type human FIX DNA sequences, whose protein variants or mutants retain activity, or are therapeutically effective or are comparably or even more therapeutically active than invariant human FIX in the methods and uses of the invention. In one particular example, collagen IV serves to trap FX, meaning that when introduced into the muscle tissue of a mammal some of the FIX is not available to participate in blood clotting because it is trapped in the interstitial spaces in the muscle tissue. A mutation in the FIX sequence that results in a protein with reduced binding to collagen IV (e.g., loss of function) is a useful mutant in the methods of the invention, for example, for treating hemophilia. An example of such a mutant human FIX encodes a human FIX protein with the amino acid alanine in place of lysine at the fifth amino acid position from the beginning of the mature protein.

[0073] Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (e.g., 1-3, 35, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40 , 40-50, 50-100 or more nucleotides or residues), such as a lysine residue for an arginine (e.g., one or more arginine substitution of a lysine as shown in any of RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4 and RHM15-6), additions (e.g. insertions or 1-3, 3-5, 5-10, 10-15, 15-20, 20- 25, 25-30, 30-40, 40-50, 50-100 or more nucleotides or residues) and deletions (e.g., subsequences or fragments) from a reference sequence. In particular embodiments, a modified or variant sequence retains at least part of a function or activity of the unmodified sequence. Such modified forms and variants may have less than, the same, or greater than, but at least a part of, a function or activity of a reference sequence, for example, as described herein.

[0074] A variant may have one or more non-conservative or conservative amino acid sequence differences or modifications or both. A “conservative substitution” is the replacement of an amino acid with a biologically, chemically or structurally similar residue. Biologically similar means that the substitution does not destroy a biological activity. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine and serine, or a similar size. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Particular examples include replacing the hydrophobic residue, such as isoleucine, valine, leucine or methionine, with another or replacing one polar residue with another, such as replacing lysine with arginine (e.g., RHM15-1, RHM15-2 , RHM15-3/RHM15-5, RHM15-4 and RHM15-6), aspartic acids for glutamic or asparagine for glutamine, threonine for serine and the like. Particular examples of conservative substitutions include replacing one hydrophobic residue such as isoleucine, valine, leucine or methionine with another, replacing one polar residue with another, such as replacing lysine with arginine, aspartic acid with glutamic acid, or asparagine with glutamine. It's similar. For example, conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. A “conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0075] In this way, the invention includes gene and protein variants (for example, of polynucleotides encoding proteins described here) that retain one or more biological activities (for example, function in blood clotting, etc.). Such protein or polypeptide variants include proteins or polypeptides that have been or can be modified using recombinant DNA technology such that the protein or polypeptide has altered or additional properties, for example, variants conferring increased protein stability in plasma or increased protein activity. . Variants may differ from a reference sequence, such as naturally occurring polynucleotides, proteins, or peptides.

[0076] At the nucleotide sequence level, a naturally occurring or non-naturally occurring variant gene will typically be at least about 50% identical, more typically about 70% identical, even more typically about 80% identical (90% or more identity) to the reference gene. At the amino acid sequence level, a naturally occurring and non-naturally occurring variant protein will typically be at least 70% identical, more typically about 80% identical, even more typically about 90% or more identity with the reference protein, although substantial regions of non-identity are allowed in non-conserved regions (e.g., less than 70% identical, such as less than 60%, 50%, or even 40%). In other embodiments, the sequences have at least 60%, 70%, 75% or more identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity) with a reference sequence. Procedures for introducing nucleotide and amino acid changes into a polynucleotide, protein or polypeptide are known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2007)).

[0077] The terms “identity”, “homology” and grammatical variations thereof mean that two or more reference entities are the same, when they are “aligned” sequences. Thus, by way of example, when two polypeptide sequences are identical, they have the same amino acid sequence, at least within the region or portion referred to. Where two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within the region or portion referred to. The identity can be in a defined area (region or domain) of the sequence. An “area” or “region” of identity refers to a portion of two or more named entities that are the same. In this way, where two protein or nucleic acid sequences are identical in one or more sequence areas or regions they share identity within that region. An “aligned” sequence refers to multiple polynucleotide or protein (amino acid) sequences, often containing corrections for missing or additional bases or amino acids (gaps) compared to a reference sequence.

[0078] The identity may extend over the entire length of the sequence or a portion of the sequence. In particular aspects, the length of the sequence sharing percent identity is 2, 3, 4, 5 or more contiguous polynucleotides or amino acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., contiguous amino acids. In further particular aspects, the length of sequences sharing identity is 20 or more contiguous polynucleotide or amino acids, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, etc., contiguous amino acids. In further particular aspects, the length of the sequence sharing identity is 35 or more contiguous polynucleotide or amino acids, e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49 , 50, etc., contiguous amino acids. In further particular aspects, the length of the sequence sharing identity is 50 or more contiguous polynucleotides or amino acids, e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85 , 85-90, 90-95, 95-100, 100-110, etc., contiguous polynucleotides or amino acids.

[0079] The terms “homologous” and “homology” mean that two or more entities referred to share at least partial identity in a given region or portion. “Areas, regions or domains” of homology or identity mean that a portion of two or more entities referred to share homology or are the same. Thus, where two sequences are identical in one or more sequence regions they share identity in those regions. “Substantial homology” means that a molecule is structurally or functionally conserved such that it has or is predicted to have at least partial structure or function of one or more of the structures or functions (e.g., biological function or activity) of the reference molecule or relevant/corresponding region or portion of the reference molecule with which it shares homology.

[0080] The degree of identity (homology) between two sequences can be determined using a computer program and mathematical algorithm. Such algorithms can calculate percentage sequence identity (homology) generally accounting for sequence gaps and mismatches in the region or area of comparison. For example, a BLAST search algorithm (e.g., BLAST 2.0) (see, e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI) has exemplary search parameters such as follows: Mismatch -2; gap opening 5; gap extension 2. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a classification matrix, such as PAM100, PAM250, BLOSUM 62, or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparisons are also used to quantify the degree of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith and others, J. Mol. Biol. 147:195 (1981)). Programs for quantifying protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem. Biophys. Res. Commun. 304:320 (2003)).

[0081] Polynucleotides include additions and insertions, for example, heterologous domains. An addition (e.g., heterologous domain) can be a covalent or non-covalent attachment of any type of molecule to a composition. Typical additions and insertions (e.g., a heterologous domain) confer a complementary or distinct function or activity.

[0082] Additions and insertions include chimeric and fusion sequences, which is a polynucleotide or protein sequence having one or more molecules not normally present in a native reference sequence (wild type) covalently linked to the sequence. The terms “fusion” and “chimeric” and grammatical variations thereof, when used in reference to a molecule, mean that a portion or part of the molecule contains a different entity distinct (heterologous) from the molecule since they do not typically exist together in nature. That is, for example, a portion of the fusion or chimera includes or consists of a portion that does not exist together in nature, and is structurally distinct.

[0083] The term “vector” refers to a plasmid, virus (e.g., AAV vector), cosmid or other vehicle that can be manipulated through insertion or incorporation of a polynucleotide. Such vectors can be used for genetic manipulation (i.e., “cloning vectors”) to introduce/transfer polynucleotides to cells, and transcribe or translate the inserted polynucleotide into cells. A vector nucleic acid sequence generally contains at least one origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g. antibiotic resistance), poly-Adenine sequence.

[0084] As used herein, the term “recombinant”, as a viral vector modifier, such as recombinant AAV vectors, as well as a sequence modifier such as recombinant polynucleotides and polypeptides, means that the compositions (e.g., AAV or sequences) have been manipulated (i.e., engineered) in a way that does not generally occur in nature. A particular example of a recombinant vector, such as an AAV vector, would be where a polynucleotide that is not normally present in the wild-type (e.g., AAV) viral genome is inserted into the viral genome. For example, an example of a recombinant polynucleotide would be where a heterologous polynucleotide (e.g. gene) encoding a protein is cloned into a vector, with or without 5', 3' and/or intron regions that the gene is normally associated within. of the viral genome (e.g. AAV). Although the term “recombinant” is not always used herein in reference to viral vectors, such as AAV vectors, as well as sequences such as polynucleotides and polypeptides, recombinant forms of AAV, and sequences including polynucleotides and polypeptides, are expressly included notwithstanding any type omission.

[0085] A recombinant vector “genome” (e.g., an AAV vector genome) can be encapsulated or packaged into a virus (also referred to herein as a “particle” or “virion”) for subsequent infection (transduction or transformation) of a cell, ex vivo, in vitro or in vivo. Where a recombinant AAV vector genome is encapsulated or packaged into an AAV particle, the particle may be referred to as an “rAAV”. Such particles or virions will typically include proteins that encapsulate or package the vector genome. Particular examples include viral capsid and envelope proteins, and in the case of AAV, AAV capsid proteins.

[0086] For a recombinant plasmid, a vector “genome” refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsulated to form a viral particle. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, the vector genome does not include the portion of the “plasmid” that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is referred to as the 'plasmid backbone', which is important for cloning and amplification of the plasmid, a process that is necessary for propagation and production of recombinant viruses, but is not itself packaged. or encapsulated in virus particles (e.g. AAV).

[0087] In this way, a vector “genome” refers to the portion of the vector plasmid that is packaged or encapsulated by viruses (e.g., AAV) and that contains a heterologous polynucleotide sequence. The non-vector genome portion of the recombinant plasmid includes the backbone that is important for cloning and amplification of the plasmid, but is itself not packaged or encapsulated by viruses (e.g., AAV).

[0088] A viral vector is derived from or based on one or more nucleic acid elements that comprise a viral genome. Particular viral vectors include parvovirus vectors, such as adeno-associated virus (AAV) vectors.

[0089] Recombinant vector sequences are manipulated through insertion or incorporation of a polynucleotide. As disclosed herein, a vector plasmid generally contains at least one origin of replication for propagation in a cell and one or more expression control elements.

[0090] Vector sequences including AAV vectors may include one or more “expression control elements”. Typically, expression control elements are nucleic acid sequence(s) that influence expression of an operably linked polynucleotide. Control elements, including expression control elements as shown herein such as promoters and enhancers, present within a vector are included to facilitate appropriate heterologous polynucleotide transcription and if appropriate translation (e.g., a promoter, enhancer, splicing signal for introns, maintenance of the correct reading frame of the gene to allow translation into mRNA frame and stop codons, etc.). Such elements typically act in cis, but can also act in trans.

[0091] Expression control can be affected at the level of transcription, translation, splicing, message stability, etc. Typically, an expression control element that modulates transcription is juxtaposed near the 5' end of the transcribed polynucleotide (i.e., “upstream”). Expression control elements can also be located at the 3' end of the transcribed sequence (i.e., “downstream”) or within the transcript (e.g., in an intron). Expression control elements can be located at a distance from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, 5000 to 10,000 or more nucleotides from the polynucleotide), even at considerable distances. However, due to nucleotide length limitations, for AAV vectors, such expression control elements will typically be within 1 to 1000 nucleotides from the polynucleotide.

[0092] Functionally, expression of operably linked heterologous polynucleotide is at least in part controllable by the element (e.g., promoter) such that the element modulates transcription of the polynucleotide and, as appropriate, translation of the transcript. A specific example of an expression control element is a promoter, which is generally located 5' to the transcribed sequence. Another example of an expression control element is an enhancer, which may be located 5', 3' of the transcribed sequence, or within the transcribed sequence.

[0093] A “promoter” as used herein may refer to a DNA sequence that is located adjacent to a polynucleotide sequence that encodes a recombinant product. A promoter is typically operably linked to an adjacent sequence, e.g., heterologous polynucleotide. A promoter typically increases an amount expressed from a heterologous polynucleotide compared to an amount expressed when no promoter exists.

[0094] An “enhancer” as used herein may refer to a sequence that is located adjacent to the heterologous polynucleotide. Enhancer elements are typically located upstream of a promoter element, but also function and can be located downstream of or within a DNA sequence (e.g., a heterologous polynucleotide). In this way, an enhancer element can be located 100 base pairs, 200 base pairs or 300 base pairs upstream or downstream of a heterologous polynucleotide. Enhancer elements typically increase the expression of a heterologous polynucleotide above an increased expression provided by a promoter element.

[0095] Expression control elements (e.g., promoters) include those active in a particular tissue or cell type, referred to herein as “tissue-specific expression control elements/promoters”. Tissue-specific expression control elements are typically active in a specific cell or tissue (e.g., liver, brain, central nervous system, spinal cord, eye, retina, bone, muscle, lung, pancreas, heart, kidney, cell, etc. ). Expression control elements are typically active in these cells, tissues, or organs because they are recognized by transcriptional activator proteins, or other transcription regulators, that are unique to a cell type, tissue, or organ.

[0096] For example, if expression in skeletal muscle is desired, a muscle-active promoter can be used. These include promoters of genes encoding skeletal α-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, as well as synthetic muscle promoters with activities greater than naturally occurring promoters (see, e.g., Li et al., Nat Biotech. 17:241-245 (1999)). Examples of promoters that are tissue specific for liver are albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B core promoter, Sandig et al., Gene Ther. 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)], bone (osteocalcin, Stein et al., Mol. Biol. Rep. 24:185-96 (1997); bone sialoprotein, Chen et al., J. Bone Miner. Res. 11:654-64 (1996)), lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin; T cell receptor a chain), neuronal promoter (Neuron-Specific Enolase (NSE)), Andersen et al., Cell Mol. Neurobiol., 13:503-15 (1993); neurofilament light chain gene, Piccioli et al., Proc. Natl. academic Sci. USA, 88:5611-5 (1991); the neuron-specific vgf gene, Piccioli et al., Neuron., 15:373-84 (1995)]; among others.

[0097] Expression control elements also include ubiquitous or promiscuous promoters/enhancers that are capable of directing expression of a polynucleotide in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter/enhancer sequences, the Rous sarcoma virus (RSV) promoter/enhancer sequences, and the other active viral promoters/enhancers in a variety of mammalian cell types, or synthetic elements that are not present in nature (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter and the phosphoglycerol kinase (PGK) promoter (Phosphoglycerol kinase).

[0098] Expression control elements can also confer expression in a manner that is regulatable, that is, a signal or stimulus increases or decreases expression of the operably linked heterologous polynucleotide. A regulatable element that increases expression of the operably linked polynucleotide in response to a signal or stimulus is also referred to as an “inducible element” (i.e., is induced by a signal). Particular examples include, but are not limited to, a hormone (e.g., steroid) inducible promoter. A regulatable element that decreases expression of the operably linked polynucleotide in response to a signal or stimulus is referred to as a “repressible element” (i.e., the signal decreases expression such that when the signal is removed or is absent, expression is increased ). Typically, the amount of increase or decrease imparted by such elements is proportional to the amount of signal or stimulus present; the greater the amount of signal or stimulus, the greater the increase or decrease in expression. Particular non-limiting examples include zinc-inducible sheep metallothionein (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (WO 98/10088); the tetracycline repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); the RU486-inducible system (Wang et al., Nat. Biotech. 15:239-243 (1997) and Wang et al., Gene Ther. 4:432-441 (1997)]; and the rapamycin-inducible system (Magari et al. , J. Clin. Invest. 100:2865-2872 (1997); Rivera et al., Nat. Medicine. 2:1028-1032 (1996)). Other adjustable control elements that may be useful in this context are those that are regulated by a specific physiological state, e.g. temperature, acute phase.

[0099] Expression control elements also include the native element(s) for the heterologous polynucleotide. A native control element (e.g., promoter) can be used when it is desirable that expression of the heterologous polynucleotide mimics native expression. The native element can be used when expression of the heterologous polynucleotide must be regulated temporally or developmentally or in a tissue-specific manner or in response to specific transcriptional stimuli. Other native expression control elements, such as introns, polyadenylation sites or Kozak consensus sequences, can also be used.

[00100] As used herein, the term “operable link” or “operably linked” refers to a physical or functional juxtaposition of the components so described in a manner that allows them to function in their intended manner. In the example of an expression control element in operable binding with a polynucleotide, the reaction is such that the control element modulates expression of the nucleic acid. More specifically, for example, the two operably linked DNA sequences means that the two DNAs are arranged (cis or trans) in a relationship such that at least one of the DNA sequences is capable of exerting a physiological effect on the other sequence.

[00101] Vectors including AAV vectors may further include additional nucleic acid elements. Such elements include, without limitation, one or more copies of an AAV ITR sequence, a promoter/enhancer element, a transcription termination signal, 5' or 3' untranslated regions (e.g., polyadenylation sequences) flanking a polynucleotide sequence or all or a portion of intron I. Such elements optionally also include a transcription termination signal. A particular non-limiting example of a transcription termination signal is the SV40 transcription termination signal.

[00102] As disclosed herein, AAV vectors typically accept DNA inserts having a defined size range that is generally about 4 kb to about 5.2 kb or slightly more. Thus, for shorter sequences, inclusion of a stuffer or payload in the insertion fragment in order to adjust the length to close to or to the normal size of the virus genomic sequence acceptable for packaging the AAV vector into a virus particle. In various embodiments, a cargo/stuffer nucleic acid sequence is an untranslated (non-protein coding) segment of nucleic acid. In particular embodiments of an AAV vector, a heterologous polynucleotide sequence has a length of less than 4.7 Kb and the filler or stuffer sequence has a length that when combined (e.g., inserted into a vector) with a sequence of heterologous polynucleotide has a total length of between about 3.0-5.5 Kb or between about 4.0-5.0 Kb or between about 4.3-4.8 Kb.

[00103] An intron can also function as a filler or stuffer sequence in order to obtain a length for packaging the AAV vector into a virus particle. Introns and intron fragments (e.g., intron I portion of FIX) that function as a filler or stuffer sequence can also increase expression. For example, inclusion of an intron element can increase expression compared to expression in the absence of the intron element (Kurachi et al., 1995, supra).

[00104] The use of introns is not limited to the inclusion of FIX intron I sequences, but also includes other introns, introns that may be associated with the same gene (for example, where the heterologous polynucleotide encodes FIX, the intron is derived of an intron present in the FIX genomic sequence) or associated with a completely different gene or other DNA sequence. In this way, other untranslated (non-protein coding) regions of nucleic acid, such as introns found in genomic sequences of cognate (related) genes (the heterologous polynucleotide sequence encodes all or a portion of the same protein encoded by the genomic sequence) and Non-cognate (unrelated) genes (the heterologous polynucleotide sequence encodes a protein that is distinct from the protein encoded by the genomic sequence) may also function as cargo or stuffer polynucleotide sequences according to the invention.

[00105] An “intron I portion” as used herein means intron I region having a nucleotide length of from about 0.1 kb to about 1.7 kb, a region that increases FIX expression, typically by about 1.5-fold or more in a plasmid or viral vector model when compared to FIX expression in the absence of a portion of intron I. A more specific portion is a 1.3 kb portion of intron 1.

[00106] The term “oligonucleotide” as used herein refers to sequences, primers and probes defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, typically more than three. The exact size of the oligonucleotide will depend on several factors and the particular application and use of the oligonucleotide, but typically an oligonucleotide is between about 5-50 nucleotides in length.

[00107] The term “primer” as used herein refers to a DNA oligonucleotide, either single or double stranded, or derived from a biological system, generated by restriction enzyme digestion or produced synthetically that, when placed in the appropriate environment , is capable of functionally acting as a template-dependent nucleic acid synthesis initiator. When present with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors, and conditions such as suitable temperature and pH, the primer can be extended at its 3' terminus by the addition of nucleotides. by the action of polymerase or similar activity to provide a primer extension product. The primer may vary in length depending on the particular conditions and application needs. For example, in diagnostic applications, the oligonucleotide primer is typically 15-30 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to initiate synthesis of the desired extension product, i.e., anneal to the desired template strand in a manner sufficient to provide the 3' hydroxyl portion of the primer in appropriate juxtaposition for use in the initiation of synthesis by an enzyme or polymerase or similar. It is not necessary for the primer sequence to represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be affixed to the 5' end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the desired template strand sequence to functionally provide a template-primer complex for synthesis of the extension product.

[00108] Polymerase chain reaction (PCR) was described in U.S. Patent Nos. 4,683,195, 4,800,195 and 4,965,188.

[00109] The expression “specifically hybridizes” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit hybridization under predetermined conditions generally used in the art (sometimes called “substantially complementary”). In particular, the term refers to the hybridization of two polynucleotide sequences with substantially complementary sequences, to the substantial exclusion of hybridization with other non-complementary single-stranded nucleic acid sequences.

[00110] A “selectable marker gene” refers to a gene that when expressed confers a selectable phenotype, such as antibiotic resistance (e.g., kanamycin), in a transformed cell. A “reporter” gene is one that provides a detectable signal. A non-limiting example of a reporter gene is the luciferase gene.

[00111] Polynucleotides and polypeptides including modified forms can be made using various standard cloning, recombinant DNA technology, through cellular expression or in vitro translation and chemical synthesis techniques. The purity of polynucleotides can be determined through sequencing, gel electrophoresis and the like. For example, nucleic acids can be isolated using hybridization techniques or computer-based database evaluation. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody evaluation to detect polypeptides having shared structural features, for example, using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to a nucleic acid sequence of interest; (4) computer searching of sequence databases for related sequences; and (5) different evaluation of a subtracted nucleic acid library.

[00112] Polynucleotides and polypeptides including modified forms can also be produced through chemical synthesis using methods of skill in the art, for example, an automatic synthesis apparatus (see, for example, Applied Biosystems, Foster City, CA). Peptides can be synthesized, in whole or in part, using chemical methods (see, for example, Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, A.K. Therapeutic Peptides and Proteins, Formulations , Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be carried out using various solid phase techniques (see, e.g., Roberge, Science 269:202 (1995); Merrifield, Methods Enzymol. 289:3 (1997)) and automated synthesis can be achieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer) according to the manufacturer's instructions.

[00113] The term “isolated”, when used as a modifier of a composition, means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. In general, the isolated compositions are substantially free of one or more materials with which they are normally associated in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane. The term “isolate” does not exclude man-made combinations, e.g., a recombinant vector sequence (e.g., AAV) or virus particle (e.g., AAV-Rh74 vector or related AAV vector such as variants of AAV-Rh74 such as capsid variants (e.g., RHM4-1)) that packages or encapsulates a vector genome and a pharmaceutical formulation. The term “isolated” also does not exclude alternative physical forms of the compositions, such as hybrids/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms or forms expressed in human-made host cells.

[00114] Methods and uses of the invention provide a means for administering (transduction) heterologous polynucleotides (transgenes) into a wide range of host cells, including both dividing and non-dividing cells. Recombinant vector sequences (e.g., AAV), plasmids, vector genomes, recombinant virus particles (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 variants such as capsid variants (e.g., RHM4- 1)), methods, uses and pharmaceutical formulations of the invention are additionally useful in a method of administering a protein, peptide or nucleic acid to an individual in need thereof, as a method of treatment. In this way, the protein, peptide or nucleic acid can then be produced in vivo in an individual. The individual may benefit from or have a need for the protein, peptide, or nucleic acid because the individual has a deficiency of the protein, peptide, or nucleic acid or because production of the protein, peptide, or nucleic acid in the individual may cause some therapeutic effect, such as a treatment method or otherwise. Alternatively, it may be desirable to inhibit or reduce expression or production of a target gene involved in a disease process, for example, for the treatment of a neurodegenerative disease, cancer or atherosclerosis, for example, to obtain a therapeutic effect.

[00115] In general, recombinant vector sequences (e.g., AAV), plasmids, vector genomes, recombinant virus particles (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 variants such as capsid variants ( for example, RHM4-1)), methods and uses can be used to administer any heterologous polynucleotide (transgene) with a biological effect to treat or improve one or more symptoms associated with any disorder related to insufficient or undesirable gene expression. Recombinant vector sequences (e.g., AAV), plasmids, vector genomes, recombinant virus particles (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 variants such as capsid variant particles (e.g., RHM4- 1)), Methods and uses can be used to provide therapy for various disease states.

[00116] There are several inherited diseases where defective genes are known and have been cloned. In general, the above disease states fall into two classes: deficiency states, usually of enzymes, which are usually inherited in a recessive manner, and unbalanced states, at least sometimes involving regulatory or structural proteins, which are inherited in a recessive manner. dominant way. For deficiency state diseases, gene transfer could be used to bring a normal gene into affected tissues for replacement therapy, as well as create animal models for the disease using antisense mutations. For unbalanced disease states, gene transfer could be used to create a disease state in a model system, which could then be used in efforts to combat the disease state. In this way, recombinant vector sequences (e.g., AAV), plasmids, vector genomes, recombinant virus particles (e.g., AAV-Rh74 vectors or related AAV vectors such as AAV-Rh74 variants such as capsid variants (e.g. RHM4-1)), methods and uses allow for the treatment of genetic diseases. As used herein, a disease state is treated by partially or completely remedying the deficiency or imbalance that causes the disease or makes it more severe. The use of site-specific integration of nucleic acid sequences to cause mutations or correct defects is also possible.

[00117] Illustrative disease states include, but are not limited to: cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood clotting disorders, AIDS, Alzheimer's disease, Parkinson's, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (e.g., Duchenne, Becker), Gaucher disease, Hurler disease, adenosine deaminase deficiency, glycogen storage diseases and other metabolic defects, Pompe disease, congestive heart failure, degenerative retinal diseases (choroideremia, Leber congenital amaurosis and other diseases of the eye), diseases of solid organs (e.g. brain, kidney, heart) and the like.

[00118] According to the invention, treatment methods and uses are provided, which include recombinant vectors of the invention (e.g., AAV), vector genomes, recombinant virus particles (e.g., AAV-Rh74 vectors or vectors of related AAV such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) and viral particles of the invention (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 variants such as capsid (e.g. RHM4-1)) including vector genomes. Methods and uses of the invention are broadly applicable to diseases subject to treatment by introducing a gene encoding a protein or increasing or stimulating gene expression or function, for example, gene addition or replacement. Methods and uses of the invention are also broadly applicable to diseases subject to treatment by reducing or decreasing gene expression or function, for example, gene inactivation or reduction of gene expression (gene inactivation).

[00119] Particular non-limiting examples of diseases treatable in accordance with the invention include those shown here as well as a lung disease (e.g., cystic fibrosis), a blood clotting or hemorrhagic disorder (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, a blood disorder (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases, an accumulation disorder copper or iron (e.g., Wilson or Menkes disease), lysosomal acid lipase deficiency, a neurological or neurodegenerative disorder, cancer, type 1 or type 2 diabetes, Gaucher disease, Hurler disease, adenosine deaminase deficiency, a defect metabolic disease (e.g., glycogen storage diseases), a degenerative retinal disease (such as RPE65 deficiency or defect, choroideremia, and other diseases of the eye), and a disease of a solid organ (e.g., brain, liver, kidney, heart ).

[00120] Furthermore, recombinant vectors of the invention (e.g., AAV), vector genomes, recombinant virus particles (e.g., AAV-Rh74 vectors or related AAV vectors such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)), methods and uses may be employed to administer nucleic acids encoding monoclonal antibodies or fragments thereof to provide beneficial biological effects to treat or ameliorate symptoms associated with cancers, infectious diseases and autoimmune diseases such as arthritis rheumatoid.

[00121] In one embodiment, a method or use of the invention includes: (a) providing a viral particle (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 capsid variants (e.g., RHM4-1) ) comprising a vector genome, the vector genome comprising a heterologous polynucleotide sequence (and optionally a filler/stuffer sequence), wherein the heterologous polynucleotide sequence is operably linked to an expression control element conferring transcription of said sequence. polynucleotide; and (b) administering an amount of the viral particle to the mammal such that said heterologous polynucleotide is expressed in the mammal.

[00122] In another embodiment, a method or use of the invention includes administering or transferring a heterologous polynucleotide sequence to a mammal or a mammalian cell, through the administration of a viral (e.g., AAV) particle (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) or plurality of viral particles (e.g., AAV) (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) comprising a vector genome, the vector genome comprising the heterologous polynucleotide sequence (and optionally a filler/stuffer sequence) to a mammal or a cell of a mammal, thereby administering or transferring the heterologous polynucleotide sequence to the mammal or mammalian cell.

[00123] In a further embodiment, a method or use of the invention for treating a deficient mammal in need of protein expression or function includes provision of a viral (e.g., AAV) particle (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) or plurality of viral particles (e.g., AAV) (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) comprising a vector genome, the vector genome comprising a heterologous polynucleotide sequence (and optionally filler/stuffer sequence); and administering the viral particle or plurality of viral particles to the mammal, wherein the heterologous polynucleotide sequence encodes a protein expressed in the mammal, or where the heterologous polynucleotide sequence encodes an inhibitory sequence or protein that reduces expression of an endogenous protein in the mammal.

[00124] In particular aspects of the methods and uses of the invention disclosed herein, expression of the heterologous polynucleotide encodes an inhibitory protein or nucleic acid that provides a therapeutic benefit to the mammal (e.g., human). In additional particular aspects, a filler/stuffer sequence is included in the vector sequence such that the combined length of the heterologous polynucleotide sequence has a total length of between about 3.0-5.5 Kb or between about 4. 0-5.0 Kb or between about 4.3-4.8 Kb.

[00125] Methods and uses of the invention include treatment methods, which result in any therapeutic or beneficial effect. In various methods and uses of the invention, there are further included inhibiting, lessening, or reducing one or more adverse (e.g., physical) symptoms, disorders, illnesses, diseases, or complications caused by or associated with the disease, such as blood clotting time. reduced blood, reduced administration dosage of supplemental clotting factor protein.

[00126] A therapeutic or beneficial effect of treatment is then any measurable or detectable objective or subjective improvement or benefit provided to a particular individual. A therapeutic or beneficial effect may be, but need not be, complete ablation of all or any adverse symptom, disorder, illness or complication of a disease. In this way, a satisfactory clinical end point is reached when there is an incremental improvement or partial reduction in an adverse symptom, disorder, illness or complication caused by or associated with a disease or an inhibition, diminution, reduction, suppression, prevention, limit or control of worsening or progression of one or more adverse symptoms, disorders, illness or complications caused by or associated with the disease, over a short or long duration (hours, days, weeks, months, etc.).

[00127] Compositions, such as vector genomes, recombinant virus particles (e.g., AAV-Rh74 vectors or related AAV vectors such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) including vector genomes, and methods and uses of the invention can be administered in a sufficient or effective amount to an individual in need thereof. An “effective amount” or “sufficient amount” refers to an amount that provides, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic agents such as a drug), treatments, protocols or agents of therapeutic regimens, a detectable response of any duration of time (long or short duration), an expected or desired result in or a benefit to an individual of any measurable or detectable degree or for any duration of time (e.g., for minutes, hours , days, months, years or cured).

[00128] The dose of vector genome or virus particle (e.g., such as AAV-Rh74 vector or related AAV vector such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) To obtain a therapeutic effect, for example, the dose in vector genomes/per kilogram of body weight (vg/kg), will vary based on several factors including, but not limited to: route of administration, the level of expression of heterologous polynucleotide required to obtain a therapeutic effect, the specific disease treated, any host immune response to the viral vector, a host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the expressed protein. One skilled in the art can readily determine a virion dose range for treating a patient having a particular disease or disorder based on the factors mentioned above, as well as other factors. In general, doses will range from at least 1x108 or more, e.g., 1x109, 1x1010, 1x1011, 1x1012, 1x1013, or 1x1014, or more, vector genomes per kilogram (vg/kg of individual weight, to obtain a therapeutic effect.

[00129] Using hemophilia as an example, generally speaking, it is believed that in order to obtain a therapeutic effect, the concentration of a blood clotting factor that is greater than the 1% factor concentration found in a normal individual is necessary to change a severe disease phenotype to a moderate one. A severe phenotype is characterized by joint damage and life-threatening hemorrhages. To convert a moderate disease phenotype into a mild one, it is believed that a blood clotting factor concentration greater than 5% of normal is required. With respect to treatment such as a hemophiliac individual, a typical dose is at least 1x1010 vector genomes (vg) per kilogram (vg/kg) of the individual's body weight or between about 1x1010 to 1x1011 vg/kg of the individual's body weight or between about 1x1011 to 1x1012 vg/kg of the subject's weight or between about 1x1012 to 1x1013 vg/kg of the subject's weight, to obtain a desired therapeutic effect.

[00130] Doses of “an effective amount” or “sufficient amount” for treatment (e.g., to improve or to provide a therapeutic benefit or improvement) typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, disorders, illnesses, pathologies or complications, for example, caused by or associated with the disease, to a measurable degree, although diminishing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the disease is a satisfactory outcome.

[00131] An effective amount or a sufficient amount may, but need not, be provided in a single administration, may require multiple administrations, and may, but need not be, administered alone or in combination with another composition (e.g., agent), treatment, protocol or therapeutic regimen. For example, the amount may be proportionally increased as indicated by the individual's need, type, state and severity of the disease treated or side effects (if any) of the treatment. Further, an effective amount or a sufficient amount need not be effective or sufficient if given in single or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol, or therapeutic regimen, since additional doses, amounts or duration above and below such doses, or additional compositions (e.g., drugs or agents), treatments, protocols or therapeutic regimens may be included so that they are considered effective or sufficient in a given individual. Amounts considered effective also include amounts that result in a reduction in the use of another treatment, regimen, or therapeutic protocol, such as administration of recombinant clotting factor protein for treatment of a clotting disorder (e.g., hemophilia A or B).

[00132] An effective amount or a sufficient amount does not need to be effective in each and every individual treated, nor the majority of individuals treated in a given group or population. An effective amount or a sufficient amount means efficacy or sufficiency in a particular individual, not a group or general population. As is typical for such methods, some individuals will exhibit a greater or lesser response or no response to a given method of treatment or use. Thus, appropriate amounts will depend on the condition treated, the desired therapeutic effect, as well as the particular individual (e.g., bioavailability within the individual, gender, age, etc.).

[00133] The term “improvement” means a detectable or measurable improvement in an individual's disease or symptom thereof or a basic cellular response. A detectable or measurable improvement includes a subjective or objective diminution, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression or duration of the disease or complication caused by or associated with the disease or an improvement in a symptom or an underlying cause or a consequence of the disease or a reversal of the disease.

[00134] In this way, a successful treatment result can lead to a “therapeutic effect” or “benefit” of lessening, reducing, inhibiting, suppressing, limiting, controlling or preventing the occurrence, frequency, severity, progression or duration of a disease or one or more adverse symptoms or underlying causes or consequences of disease in an individual. Treatment methods and uses that affect one or more underlying causes of illness or adverse symptoms are then considered beneficial. A decrease or reduction in worsening, such as stabilization of the disease, or an adverse symptom thereof, is also a result of successful treatment.

[00135] A therapeutic benefit or improvement therefore need not be complete ablation of the disease or any, most or all adverse symptoms, complications, consequences or underlying causes associated with the disease. In this way, a satisfactory end point is reached when there is an increasing improvement in an individual's disease or a diminution, reduction, inhibition, suppression, limit, control or partial prevention in the occurrence, frequency, severity, progression or duration or inhibition or reversal of the disease. illness (e.g., stabilization of one or more symptoms or complications), over a short or long duration of time (hours, days, weeks, months, etc.). The effectiveness of a method or use, such as a treatment that provides a therapeutic benefit or potential improvement of a disease, can be determined through various methods.

[00136] The methods and uses of the invention can be combined with any compound, agent, drug, treatment or other therapeutic regimen or protocol having a therapeutic, beneficial, additive, synergistic or complementary activity or effect. Exemplary combination compositions and treatments include second additives such as biological agents (proteins) and drugs. Such biological agents (proteins), drugs, treatments and therapies may be administered or performed prior to, substantially contemporaneously with or following any other method or use of the invention, e.g., a therapeutic method of treating an individual for a blood clotting disease. .

[00137] The compound, agent, drug, treatment or other therapeutic regimen or protocol can be administered as a combination composition or administered separately, such as concomitantly or serially or sequentially (prior to or following) with the application or administration of a vector or virus genome (e.g., AAV-Rh74 vector or related AAV vector such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) of the invention. The invention then provides combinations where a method or use of the invention is in combination with any other compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition shown herein or known to one skilled in the art. The compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition may be administered or performed prior to, substantially contemporaneously with or following administration of a vector or virus genome (e.g., AAV-Rh74 vector or of related AAV such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) of the invention to an individual. Specific non-limiting examples of combination embodiments then include the above compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition or others.

[00138] Methods and uses of the invention also include, among other things, methods and uses that result in a need for or reduced use of another compound, agent, drug, therapeutic regimen, treatment protocol, process or remedy. For example, for a blood clotting disease, a method or use of the invention has a therapeutic benefit if in a given individual a less frequent or reduced dose or elimination of administration of a recombinant clotting factor protein to supplement the clotting factor deficient or defective endogenous (abnormal or mutant) in the individual is achieved. Thus, according to the invention, methods and uses of reducing the need or use of other treatment or therapy are provided.

[00139] The invention is useful in animals including veterinary medical applications. Suitable individuals then include mammals, such as humans, as well as non-human mammals. The term “individual” refers to an animal, typically a mammal, such as a human, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal ( poultry such as chickens and ducks, horses, cows, goats, sheep, pigs) and experimental animals (mouse, rat, rabbit, guinea pigs). Human individuals include fetal, neonatal, infant, juvenile, and adult individuals. Subjects include animal disease models, for example, mouse and other animal models of blood clotting diseases, and others known to those of skill in the art.

[00140] As shown herein, vectors and virus particles (e.g., AAV-Rh74 and related AAV such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) comprising such vectors can be used to providing a protein to an individual where there is an insufficient amount of the protein or a deficiency in a functional gene product (protein) or providing an inhibitory nucleic acid or protein to an individual who produces an aberrant, partially functional or non-functional gene product ( protein) that can lead to disease. In this way, individuals suitable for treatment include those having or at risk of producing an insufficient amount of or having a deficiency in a functional gene product (protein) or producing an aberrant, partially functional or non-functional gene product (protein), which may lead to disease. Individuals suitable for treatment in accordance with the invention also include those having or at risk of producing an aberrant or defective (mutant) gene product (protein) that leads to a disease in a manner that reduces amounts, expression or function of the gene product. (protein) aberrant or defective (mutant) would lead to treatment of the disease or reduce one or more symptoms or improve the disease. Target individuals then include individuals who have such defects regardless of the type of disease, timing or degree of onset, progression, severity, frequency, or type or duration of symptoms.

[00141] “Prophylaxis” and grammatical variations thereof mean a method where in vivo contact, administration or application to an individual is prior to illness. In vivo administration or application to an individual may be carried out prior to the development of an adverse symptom, condition, complication, etc. caused by or associated with the disease. For example, an evaluation (e.g., genetic) may be used to identify such individuals as candidates for methods and uses of the invention, but the individual may not manifest the disease. Such individuals then include those screened positive for an insufficient amount of or a deficiency in a functional gene product (protein) or who produce an aberrant, partially functional or non-functional gene product (protein) that can lead to disease; and individuals who have been screened positive for an aberrant or defective (mutant) gene product (protein) that leads to the disease, although such individuals do not manifest symptoms of the disease.

[00142] Methods and uses of the invention include application and administration systemically, regionally or locally, or through any route, for example, through injection, infusion, orally (e.g., ingestion or inhalation) or topically (e.g., transdermally) . Such application and administration include intravenously, intramuscularly, intraperitoneally, intradermally, subcutaneously, intracavity, intracranially, transdermally (topically), parenterally, for example, transmucosally or rectally. Exemplary routes of administration and application include intravenous (i.v.), intraperitoneal (i.p.), intra-arterial, intramuscular, parenteral, subcutaneous, intrapleural, topical, dermal, intradermal, transdermal, parenterally, e.g., transmucosal, intracranial, intraspinal, oral ( alimentary), mucosal, respiratory, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, intralymphatic.

[00143] Doses may vary and depend on whether the treatment is prophylactic or therapeutic, the type, onset, progression, severity, frequency, duration or probability of the disease to which treatment is directed, the desired clinical end point, previous or simultaneous treatments , the general health, age, gender, race or immunological competence of the individual and other factors that will be understood by one skilled in the art. The dose amount, number, frequency or duration may be proportionately increased or reduced as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy and the condition of the individual. One skilled in the art will understand the factors that may influence the dosage and time required to provide a sufficient amount to provide a therapeutic or prophylactic benefit.

[00144] Methods and uses of the invention as disclosed herein can be practiced within 1-2, 2-4, 4-12, 12-24 or 24-72 hours after an individual has been identified as having the disease targeted for treatment, have one or more symptoms of the disease or have been evaluated and are identified as positive as shown here even though the individual does not have one or more symptoms of the disease. Of course, the methods and uses of the invention can be practiced 1-7, 7-14, 14-21, 21-48 or more days, months or years after an individual has been identified as having the disease targeted for treatment, has a or more symptoms of the disease or have been evaluated and are identified as positive as shown here.

[00145] Recombinant vector (e.g., AAV, such as AAV-Rh74 vector or related AAV vector such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)), sequences, plasmids, genomes vector, recombinant virus particles (e.g., AAV, such as AAV-Rh74 vector or related AAV vector such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) and other compositions, agents, drugs, biologics (proteins) can be incorporated into pharmaceutical compositions, for example, a pharmaceutically acceptable carrier or excipient. Such pharmaceutical compositions are useful for, among other things, administration and application to an individual in vivo or ex vivo.

[00146] As used herein the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, that is suitable for one or more routes of administration, administration or contact in vivo . A “pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, for example, the material can be administered to an individual without causing substantial undesirable biological effects. In this way, such a pharmaceutical composition can be used, for example, in the administration of a viral vector or viral particle (e.g., AAV-Rh74 or related AAV such as AAV-Rh74 variants such as capsid variants (e.g., RHM4- 1)) or transformed cell to an individual.

[00147] Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion media and suspension, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or contact or in vivo administration. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules and crystals. Supplementary additive compounds (e.g., preservative, antibacterial, antiviral and antifungal agents) may also be incorporated into the compositions.

[00148] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or application, as shown here or known to one skilled in the art. Thus, pharmaceutical compositions include carriers, diluents or excipients suitable for administration via various routes.

[00149] Compositions suitable for parenteral administration comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, preparations that are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.

[00150] For transmucosal or transdermal administration (e.g., topical contact), penetrants may be included in the pharmaceutical composition. Penetrants are known in the art and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. For transdermal administration, the active ingredient may be formulated into aerosols, sprays, ointments, pastes, gels or creams as generally known in the art. For skin contact, pharmaceutical compositions typically include ointments, creams, lotions, pastes, gels, sprays, aerosols or oils. Carriers that can be used include petroleum jelly, lanolin, polyethylene glycols, alcohols, transdermal enhancers, and combinations thereof.

[00151] Co-solvents and adjuvants can be added to the formulation. Non-limiting examples of co-solvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants such as soy lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[00152] Pharmaceutical compositions and delivery systems suitable for vector genomes, virus particles (e.g., AAV-Rh74 vector or related AAV vector such as AAV-Rh74 variants such as capsid variants (e.g., RHM4- 1)) and methods and uses of the invention are known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA: The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, PA.: Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, N.Y., pp. 253 -315).

[00153] A “unit dosage form” as used herein refers to physically separate units suitable as unit dosages for the individual to be treated; each unit containing a predetermined amount optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or loading agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect) . Unit dosage forms may be contained in, for example, ampoules and vials, which may include a liquid composition or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, may be added prior to administration or in vivo application. Individual unit dosage forms may be included in multidose cases or containers. Recombinant vector sequences (e.g., AAV), plasmids, vector genomes, recombinant virus particles (e.g., AAV-Rh74 vector or related AAV vector such as AAV-Rh74 capsid variants (e.g., RHM4- 1)) and pharmaceutical compositions thereof may be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.

[00154] The invention provides cases with packaging material and one or more components therein. A kit typically includes a label or leaflet including a description of the components or instructions for in vitro, in vivo or ex vivo use of the components therein. A kit may contain a set of such components, for example, a vector genome (e.g., AAV) or virus particle (e.g., AAV-Rh74 vector or related AAV vector such as AAV-Rh74 variants such as capsid variants (e.g., RHM4-1)) and optionally a second active, such as another compound, agent, drug or composition.

[00155] A case refers to a physical structure housing one or more case components. The packaging material can hold the compounds sterilely and can be made from material generally used for such purposes (e.g. paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tube, etc.).

[00156] Labels or leaflets may include information identifying one or more components present, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels and leaflets may include information identifying the manufacturer, batch number, place and date of manufacture, expiration dates. Labels and leaflets may include information identifying manufacturer information, lot numbers, manufacturer location and date. Labels or leaflets may include information about a disease for which a kit component may be used. Labels or package inserts may include instructions for the physician or individual to use one or more of the components of the kit in a treatment method, use, or protocol or therapeutic regimen. Instructions may include dosage amounts, frequency and duration and instructions for practicing any of the methods, uses and treatment protocols or prophylactic or therapeutic regimens described therein.

[00157] Labels or leaflets may include information about any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or package inserts may include information about adverse side effects, complications or reactions, such as risks to the individual or physician in relation to situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could occur when the individual has taken, will take or is currently taking one or more other medications that may be incompatible with the composition or the individual has been, will be or is currently on another treatment protocol or therapeutic regimen that would be incompatible with the composition and then instructions could include information regarding such incompatibilities.

[00158] Labels or leaflets include "printed", for example, paper or cardboard, or separated from or affixed to a component, a case or packaging material (e.g. a box) or attached to an ampoule, tube or vial containing a case component. Labels or leaflets may also include a computer-readable medium, such as a printed label with a bar code, a disc, an optical disc such as a CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape or an electrical storage medium. such as RAM or ROM or hybrids thereof such as magnetic/optical storage media, flash memory or memory cards.

[00159] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by one of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, suitable methods and materials are described herein.

[00160] All applications, publications, patents and other references, citations to GenBank and citations to the ATCC mentioned here are incorporated by reference in their entirety. In case of conflict, the report, including definitions, will prevail.

[00161] All features disclosed here can be combined in any combination. Each characteristic revealed in the report can be replaced by an alternative characteristic serving the same, equivalent or similar purpose. In this manner, unless expressly stated otherwise, the disclosed features (e.g., a recombinant vector sequence (e.g., AAV), plasmid, vector genome, or recombinant virus particle (e.g., AAV-RH74 vector or related AAV vector such as AAV-Rh74 capsid variants (e.g., RHM4-1)) are an example of a genus of equivalent or similar characteristics.

[00162] As used herein, the singular forms “a, an”, “and” and “the, a” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a “polynucleotide” includes a plurality of such polynucleotides, reference to a “vector” includes a plurality of such vectors, and reference to a “virus” or “particle” includes a plurality of such virions/particles. .

[00163] As used herein, all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to at least 80% identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, etc. as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc, 82.1%, 82.2%, 82.3%, 82.4%, 82 .5%, etc and so on.

[00164] Reference to an integer with more (greater) or less includes any number greater or less than the reference number, respectively. Thus, for example, a reference to less than 1,000 includes 999, 998, 997, etc., always one number (1) downwards; and less than 100 includes 99, 98, 97, etc., always one number (1) down.

[00165] As used herein, all numeric values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as a percentage range, such as 1-10, includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 as well as 1.1 , 1.2, 1.3, 1.4, 1.5, etc and so on. Reference to a range of 1-50 then includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc, up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc, 2.1, 2.2, 2.3, 2.4, 2.5 , etc., and so on.

[00166] Reference to a series of ranges includes ranges that combine the limit values of different ranges within the series. In this way, to illustrate reference to a series of ranges of 11-10, 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, 750-1,000, 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, 6,000-7,000, 7,000-8,000, or 8,000-9,000 includes ranges of 10-50, 50-100, 100-1,000, 1,000-3,000, 2. 000-4,000, etc.

[00167] The invention is generally disclosed herein using affirmative language to describe the various embodiments and aspects. The invention also specifically includes embodiments where particular subject matter is excluded, in whole or in part, such as substances or materials, method steps and conditions, protocols or procedures. For example, in certain embodiments or aspects of the invention, materials and/or method steps are excluded. Thus, although the invention is not generally expressed herein in terms of what the invention does not include, aspects that are not expressly excluded in the invention are nevertheless disclosed herein.

[00168] Various embodiments of the invention have been described. Nevertheless, one skilled in the art, without departing from the spirit and scope of the invention, may make various changes and modifications of the invention to adapt it to various uses and conditions. Therefore, the examples that follow are intended to illustrate, but not limit, the scope of the claimed invention.

Examples Example 1 This example includes a description of various materials and methods.

[00169] Mice: Male C57BL/6J mice (WT) 8-10 weeks old, n=5 per experimental group. The dog is an HB dog from the University of North Carolina Chapel Hill colony carrying a nonsense mutation in the FIX gene (Evans et al., Proc. Natl. Acad. Sci. USA 86:10095 (1989)).

[00170] AAV Vector Constructs: In vivo studies in mice were performed using a construct expressing human FIX under the control of the ApoE-hAAT liver-specific promoter. The dog study used an almost identical promoter and the canine FIX transgene.

[00171] Gene Transfer Methodology: All vectors were administered intravenously. In mice via the tail vein (a volume of 200 microliters per mouse was administered, the vector was diluted in PBS). In dogs, the vector was administered through the saphenous vein.

[00172] Determination of FIX Expression: ELISA was used to measure FIX levels. In mice, the human FIX ELISA antibody pair (capture and secondary) is from Affinity Biologicals. In dogs, an antibody pair also from Affinity Biologicals was used as described in Haurigot et al. (Mol. Ther. 18:1318 (2010)).

[00173] Statistical analysis: Statistical analysis was performed with a two-variable, unpaired t-test. P values <0.05 were considered statistically significant.

[00174] AAV Antibody Measurements: An in vitro neutralization assay described in Manno et al. (Nat. Med. 12:342 (2006) and Mingozzi et al. (Nat. Med. 13:419 (2007)) was used to antibody measurement. In brief, two AAV vector constructs were used in the assay, a single-stranded vector expressing β-galactosidase under the control of the CMV promoter (ssAV-LacZ) or a self-complementary vector expressing the Renilla reporter gene, AAV - Rh74-CBA-Renilla, under the control of the chicken β-actin (CBA) promoter To increase the transduction efficiency of AAV vectors in vitro, 2V6.11 cells (ATCC) were used, which expressed the gene adenoviral E4 under the control of an inducible promoter. Cells were seeded in a 96-well plate at a density of 1.25x104 cells/well and a 1:1000 dilution of ponasterone A (Invitrogen) was added to the medium to induce expression of E4. On the day of the assay, serial semi-logarithmic dilutions of heat-inactivated test serum were mixed with virus-containing medium. For the ssAAV-LacZ vector, virus concentration used in the assay was ~1x1010 vg/ml for AAV2 and ~5.5x1010 vg/ml for AAV5, 6, or 8. For the ssAAV-Luc vector, the virus concentration in assay was between ~50 and 150 times smaller. Residual activity of the reporter transgene was measured using either a colorimetric assay (ssAAV-LacZ) or a luminometer (scAAV-Luc).

[00175] Anti-AAV capsid IgG or total Ig subclasses were measured with a capture assay; ELISA plates were coated with 5x1010 capsid particles/ml empty AAV capsids. Plates were blocked with 2% BSA, 0.05% Tween 20 in PBS for 2 hours at room temperature and serial dilutions of samples were loaded into the wells and incubated overnight at 4° C. Antibodies IgG1, IgG2, Biotin-conjugated anti-human IgG3, IgG4, or IgM (Sigma) were used as detection antibodies; streptavidin-HRP was added for substrate detection. Ig concentration was determined against standard curves made with serial dilution of purified human IgG1, IgG2, IgG3, IgG4, or IgM (Sigma).

[00176] AAV Production: The process for vector production is described in detail in Ayuso et al. (Gene Ther. 17:503 (2010)).

Example 2 This example includes a description of animal (mouse) studies of human FIX gene transfer and FIX expression after gene transfer.

[00177] C57BL/6 mice (n=5 per group) were injected via the tail vein with AAV vectors carrying the Factor IX (FIX) gene (2,510 vector genomes per mouse) under the control of a factor IX (FIX)-specific promoter. liver. Plasma levels of human FIX transgene product (protein) in mice were determined by ELISA at weeks 1, 2, and 4 post-gene transfer and are illustrated in Figure 1. AAV-Rh74 showed the highest level of expression of transgene in animals.

Example 3 This example includes a description of animal studies and data demonstrating efficient AAV-Rh74-mediated delivery of FIX at therapeutic levels in dogs with hemophilia.

[00178] In short, dogs with hemophilia B were infused intravenously (IV) through the saphenous vein with 3x102 vector genomes per kg of weight. Expression of the therapeutic FIX transgene was driven by a liver-specific promoter. FIX vectors and levels were monitored using ELISA. Plasma levels of canine FIX are shown in Figure 2. AAV-Rh74 and AAV8 performed almost equally in dogs with hemophilia B and both were superior to AAV6.

Example 4 This example includes a description of studies showing the presence of anti-AAV neutralizing antibodies (Nab) in humans

[00179] The data in Table 1 shows anti-AAV neutralizing antibodies (NAb) measured in humans with an in vitro assay. Individuals with a NAb titer of less than or equal to 1:2 are defined as pure or low-titer anti-AAV antibodies and are eligible for gene transfer for this AAV serotype (highlighted in gray). Patients with titers between 1:1 and 1:3 are considered permissive for AAV as long as empty capsids are used as bait. Samples with titers greater than 1:3 are considered nonpermissive for AAV transduction after systemic injection and are filled with light gray. AAV-Rh74 exhibited the lowest prevalence of anti-AAV Nab compared with AAV-2 and AAV-8. Table 1

Example 5 This example includes a description of data showing production amounts of different AAV serotypes including AAV-Rh74

[00180] The data in Table 2 show production yield of different AAV serotypes. Virus batch size in round bottles, total vector yield, and yield per bottle are reported. All serotypes were packaged with the same expression cassette. AAV-Rh74 has a yield comparable to or greater than the other serotypes evaluated, namely AAV-8, AAV-dj and AAV-2. Table 2

Example 6 This example includes a description of data showing that the AAVrh74 vector expressing human Factor IX (FIX) under the control of a liver-specific promoter administered to rhesus macaques led to amounts of FIX production in animals and at greater levels than the vector of AAV8 administered in the same amount

[00181] In short, animals were administered with either AAV8 or AAVrh74 at a dose of 2x1012 vector genomes (vg)/kg of weight. Vectors were formulated either in saline or in a mixture of vector and empty AAV capsid (called EC).

[00182] Figure 4 is a histogram plot of the mean (weeks 2 to 8) and standard errors or the mean of human FIX measured via an ELISA that specifically detects human FIX in rhesus macaque plasma. Animals receiving the AAVrh74-FIX vector are shown in the last two bars toward the right margin. The data show that animals receiving the AAVrh74 vectors (the last two bars towards the right margin) expressed the FIX transgene at higher levels compared to the other groups of animals injected at the same dose (black and gray bars). Mean levels were compared using two-variable, unpaired Student's t-test.

[00183] One of the animals receiving AAV-RHM4-1-FIX developed an inhibitor against the human Factor IX transgene product, which is a well-documented phenomenon that occurs in approximately 20% of monkeys treated with a human FIX vector. The second RHM4-1-treated animal expressed approximately 2-fold higher FIX levels compared to the AAV8-treated monkeys.

Example 7 This example includes a description of several Rh74 capsid variants

[00184] In short, several substitutions were introduced into the Rh74 capsid sequence to produce Rh74 capsid variant. The different Rh74 capsid variants, and amino acids substituted at each position, were as follows: Table 3: Variant Amino Acid Substitutions and Indicated Positions in VP1 Capsid RHM4_1 G195A-L199V-S201P-G202N RHM15_1 G195A-L199V-S201P-G202N K (137/259/333/530/552/569/38/51/77/169/547)R RHM15_2 G195A-L199V- S201P-G202N K(137/259/333/530/552/569/38/51/ 77/163/169)R RHM15_3 G195A-L199V- S201P-G202N 137/259/333/530/552/569/38/51/77/163/668)R RHM15_5 G195A-L199V- S201P-G202N K(137/259/333/530/552/569/38/51/77 /547/163)R RHM15_6 G195A-L199V- S201P-G202N K(137/259/333/530/552/569/38/51/77/547/688)R

[00185] The RHM4-1 variant had a substitution of an alanine, a leucine, a proline and an asparagine at amino acid positions 195, 199, 201 and 202, respectively, of the Rh74 VP1 capsid. The amino acid sequence of the VP1 capsid of the RHM4-1 variant, with residues a, v, p and n substituted, underlined and in bold, is as follows (SEQ ID NO:5): erlqedtsfggnlgravfq 121 akkrvleplglvespvktapgkkrpvepspqrspdsstgigkkgqqpakkrlnfgqtgds 181 esvpdpqpigeppaapsgvgpntmaagggapmadnnegadgvgsssgnwhcdstwlgdrv 241 ittstrtwalptynnhly kqisngtsggstndntyfgystpwgyfdfnrfhchfsprdwq 301 rlinnnwgfrpkrlnfklfniqvkevtqnegtktiannltstiqvftdseyqlpyvlgsa 361 hqgclppfpadvfmipqygyltlnngsqav grssfycleyfpsqmlrtgnnfefsynfed 421 vpfhssyahsqsldrlmnplidqylyylsrtqstggtagtqqllfs qagpnnmsaqaknw 481 lpgpcyrqqrvsttlsqnnnsnfawtgatkyhlngrdslvnpgvamathkddeerffpss 541 gvlmfgkqgagkdnvdyssvmltseeeikttnpvateqygvvadnlqqqnaapivgavns 601 qgalpgmvwqnrdvylqgpiwa kiphtdgnfhpsplmggfglkhpppqilikntpvpadp 661 pttfnqaklasfitqystgqvsveiewelqkenskrwnpeiqytsnyykstnvdfavnte 721 gtyseprpigtryltrnl

[00186] The nucleic acid sequence of the VP1 capsid of the RHM4-1 variant, with codons encoding a, v, p and n, underlined and in bold, is as follows (SEQ ID NO: 11): ccaaagccaaccagcaaaagcaggac 121 aacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgac 181 aagggggagcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgac 241 cagcagctccaagcgggtgacaatccgtacctgcggtataatcacgccgacgccgagttt 301 caggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgcgcagtctt ccag 361 gccaaaaagcgggttctcgaacctctgggcctggttgaatcgccggttaagacggctcct 421 ggaaagaagagaccggtagagccatcaccccagcgctctccagactcctctacgggcatc 481 ggcaagaaaggccagcagcccgcaaaaagagactcaattttgggcagactggcgactca 541 gag tcagtccccgaccctcaaccaatcggagaaccaccagcagccccctctggtgtggga 601 cctaatacaatggctgcaggcggtggcgctccaatggcagacaataacgaaggcgccgac 661 ggagtgggtagttcctcaggaaattggcattgcgattccacatggctgggcgacagagtc 721 atcaccaccagcacc cgcacctgggccctgcccacctacaacaaccacctctacaagcaa 781 atctccaacgggacctcgggaggaagcaccaacgacaacacctacttcggctacagcacc 841 ccctgggggtattttgacttcaacagattccactgccacttttcaccacgtgactggcag 901 cgactcatcaacaacaactggggattccggcccaaga ggctcaacttcaagctcttcaac 961 atccaagtcaaggaggtcacgcagaatgaaggcaccaagaccatcgccaataaccttacc 1021 agcacgattcaggtctttacggactcggaataccagctcccgtacgtgctcggctcggcg 1081 caccagggctgcctgcctccgttcccggcggacgtcttcatgat tcctcagtacgggtac 1141 ctgactctgaacaatggcagtcaggctgtgggccggtcgtccttctactgcctggagtac 1201 tttccttctcaaatgctgagaacgggcaacaactttgaattcagctacaacttcgaggac 1261 gtgcccttccacagcagctacgcgcacagccagagcctgg accggctgatgaaccctctc 1321 atcgaccagtacttgtactacctgtcccggactcaaagcacgggcggtactgcaggaact 1381 cagcagttgctattttctcaggccgggcctaacaacatgtcggctcaggccaagaactgg 1441 ctacccggtccctgctaccggcagcaacgcgtctccacgacact gtcgcagaacaacaac 1501 agcaactttgcctggacgggtgccaccaagtatcatctgaatggcagagactctctggtg 1561 aatcctggcgttgccatggctacccacaaggacgacgaagagcgattttttccatccagc 1621 ggagtcttaatgtttgggaaacagggagctggaaaagacaacg tggactatagcagcgtg 1681 atgctaaccagcgaggaagaaataaagaccaccaacccagtggccacagaacagtacggc 1741 gtggtggccgataacctgcaacagcaaaacgccgctcctattgtaggggccgtcaatagt 1801 caaggagccttacctggcatggtgtggcagaaccgggacgtgtacctgcagggtcccatc 1861 tg ggccaagattcctcatacggacggcaactttcatccctcgccgctgatgggaggcttt 1921 ggactgaagcatccgcctcctcagatcctgattaaaaacacacctgttcccgcggatcct 1981 ccgaccaccttcaatcaggccaagctggcttctttcatcacgcagtacagtaccggccag 2041 gtcagcg tggagatcgagtgggagctgcagaaggagaacagcaaacgctggaacccagag 2101 attcagtacacttccaactactacaaatctacaaatgtggactttgctgtcaatactgag 2161 ggtacttattccgagcctcgccccattggcacccgttacctcacccgtaatctg

[00187] Variants RHM15-1, 15-2, 15-3, 15-4, 1-5 and 15-6 also had substitutions of an alanine, a leucine, a proline and an asparagine at amino acid positions 195, 199 , 201 and 202, respectively, of the Rh74 VP1 capsid. Furthermore, these variants had multiple lysine to arginine substitutions at various positions.

[00188] The amino acid sequence of the VP1 capsid of the RHM15-1 variant is as follows (SEQ ID NO:6): 20 fdfnrfhchfsprdwq 301 rlinnnwgfrpkrlnfklfniqvkevtqnegtrtiannltstiqvftdseyqlpyvlgsa 361 hqgclppfpadvfmipqygyltlnngsqavgrssfycleyfpsqmlrtgnnfefsynfed 421 vpfhssyahsqsldrlmnplidq ylyylsrtqstggtagtqqllfsqagpnnmsaqaknw 481 lpgpcyrqqrvsttlsqnnnsnfawtgatkyhlngrdslvnpgvamathrddeerffpss 541 gvlmfgrqgagrdnvdyssvmltseeeirttnpvateqygvvadnlqqqn aapivgavns 601 qgalpg mvwqnrdvylqgpiwakiphtdgnfhpsplmggfglkhpppqilikntpvpadp 661 pttfnqaklasfitqystgqvsveiewelqkenskrwnpeiqytsnyykstnvdfavnte 721 gtyseprpigtryltrnl

[00189] The nucleic acid sequence of the VP1 capsid of the RHM15-1 variant is as follows (SEQ ID NO:12): ggggtctggtgcttcctggctacaggtacctcggacccttcaacggactcgac 181 aagggggagcccgtcaacgcggcggacgcagcggccctcgagcacgacagggcctacgac 241 cagcagctccaagcgggtgacaatccgtacctgcggtataatcacgccgacgccgagttt 301 caggagc gtctgcaagaagatacgtcttttgggggcaacctcgggcgcgcagtcttccag 361 gccaaaaagcgggttctcgaacctctgggcctggttgaatcgccggttaggacggctcct 421 ggaaagaagagaccggtagagccatcaccccagcgctctccagactcctctacgggcatc 481 ggcaagaaa ggccagcagcccgcaagaaagagactcaattttgggcagactggcgactca 541 gagtcagtccccgaccctcaaccaatcggagaaccaccagcagccccctctggtgtggga 601 cctaatacaatggctgcaggcggtggcgctccaatggcagacaataacgaaggcgccgac 660 ggtattttgacttcaacagattccactgccacttttcaccacgtgactggcag 901 cgactcatcaacaacaactggggattccggcccaagaggctcaacttcaagctcttcaac 961 atccaagtcaaggaggtcacgcagaatgaaggcaccagga ccatcgccaa taaccttacc 1021 agcacgattcaggtctttacggact cggaataccagctcc cgtacgtgct cggctcggcg 1081 caccagggctgcctgcctccgttcccggcggacgtcttca tgattcctca gtacgggtac 1141 ctgactctgaacaatggcagtcaggctgtgggccggtcgt ccttctactg cctggagtac 1201 tttccttctca aatgctgagaacgggcaacaactttgaat tcagctacaa cttcgaggac 1261 gtgcccttccacagcagctacgcgcacagccagagcctgg accggctgat gaaccctctc 1321 atcgaccagtacttgtactacctgtcccggactcaaagca cgggcggtac tgcaggaact 1381 cagcagtt gctattttctcaggccgggcctaacaacatgt cggctcaggc caagaactgg 1441 ctacccggtccctgctaccggcagcaacgcgtctccacga cactgtcgca gaacaacaac 1501 agcaactttgcctggacgggtgccaccaagtatcatctga atggcagaga ctctctggtg 1561 aatcctggcgttgccatggctacccacagggacgacgaag agcgattttt tccatccagc 1621 ggagtcttaatgtttgggagacagggagctggaagagaca acgtggacta tagcagcgtg 1681 atgctaaccagcgaggaagaaataaggaccaccaacccag tggccacaga acagtac ggc 1741 gtggtggccgataacctgcaacagcaaaacgccgctccta ttgtaggggc cgtcaatagt 1801 caaggagccttacctggcatggtgtggcagaaccgggacg tgtacctgca gggtcccatc 1861 tgggccaagattcctcatacggacggcaactttcatccct cgccgctgat gggaggct TT g 2101 attcagtacacttccaactactacaaatctacaaatgtgg actttgctgt caatactgag 2161 ggtacttattccgagcctcgccccattggcacccgttacc tcacccgtaa tctgtaa

[00190] The amino acid sequence of the VP1 capsid of the RHM15-2 variant is as follows (SEQ ID NO:7): FQ 121 fgyst pwgyfdfnrf hchfsprdwq 301 rlinnnwgfrpkrlnfklfniqvkevtqnegtrtiannlt stiqvftdse yqlpyvlgsa 361 hqgclppfpadvfmipqygyltlnngsqavgrssfycley fpsqmlrtgn nfefsynfed 421 vpfhssyahsqsldrlmnplidqylyylsrtqstggtagt qqllfsqagp nnmsaqaknw 481 lpgpcyrqqrvsttlsqnnnsn fawtgatkyhlngrdslv npgvamathr ddeerffpss 541 gvlmfgkqgagrdnvdyssvmltseeeirttnpvateqyg vvadnlqqqn aapivgavns 601 qgalpgmvwqnrdvylqgpiwakiphtdgnfhpsplmggf glkhpppqil ikntpvpadp 661 pttfnqaklasfitqystgqvsveiewelqkenskrwnpe iqytsnyyks tnvdfavnte 721 gtyseprpigtryltrnl

[00191] The nucleic acid sequence of the VP1 capsid of the RHM15-2 variant is as follows (SEQ ID NO:13): ccggggtctggtgcttcctggctacaggtacctcg gacccttcaa cggactcgac 181 aagggggagcccgtcaacgcggcggacgcagcggccctcg agcacgacag ggcctacgac 241 cagcagctccaagcgggtgacaatccgtacctgcggtata atcacgccga cgccgagttt 301 caggagcgtctgcaagaagatacgtcttttgggggcaacc tcgggcgcgc agtcttccag 361 gccaaaaagcgggttctcgaacctctgggcctggttgaat cgccggttag gacggctcct 421 ggaaagaagagaccggtagagccatcaccccagcgctctc cagactcctc tacgggcatc 481 ggcaagagaggccagcagcccgcaagaaagagactcaat t ttgggcagac tggcgactca 541 gagtcagtccccgaccctcaaccaatcggagaaccaccag cagccccctc tggtgtggga 601 cctaatacaatggctgcaggcggtggcgctccaatggcag acaataacga aggcgccgac 661 ggagtgggtagttcctcaggaaattggcattgcgattcca cat ggctggg cgacagagtc 721 atcaccaccagcacccgcacctgggccctgcccacctaca acaaccacct ctacaggcaa 781 atctccaacgggacctcgggaggaagcaccaacgacaaca cctacttcgg ctacagcacc 841 ccctgggggtattttgacttcaacagattccactgccact tttcaccacg tgact ggcag 901 cgactcatcaacaacaactggggattccggcccaagaggc tcaacttcaa gctcttcaac 961 atccaagtcaaggaggtcacgcagaatgaaggcaccagga ccatcgccaa taaccttacc 1021 agcacgattcaggtctttacggactcggaataccagctcc cgtacgtgct cggctcggcg 1080 cttcgaggac 1261 gtgcccttccacagcagctacgcgcacagccagagcctgg accggctgat gaaccctctc 1321 atcgaccagtacttgtactacctgtcccggactcaaagca cgggcggtac tgcaggaact 1381 cagcagttgctattttctcaggccgggcctaacaacatgt cggct caggc caagaactgg 1441 ctacccggtccctgctaccggcagcaacgcgtctccacga cactgtcgca gaacaacaac 1501 agcaactttgcctggacgggtgccaccaagtatcatctga atggcagaga ctctctggtg 1561 aatcctggcgttgccatggctggctacccacagggacgacgaag agcgatt ttt tccatccagc 1621 ggagtcttaatgtttgggaaacagggagctggaagagaca acgtggacta tagcagcgtg 1681 atgctaaccagcgaggaagaaataaggaccaccaacccag tggccacaga acagtacggc 1741 gtggtggccgataacctgcaacagcaaaacgccgctccta ttgtagggg c cgtcaatagt 1801 caaggagccttacctggcatggtgtggcagaaccgggacg tgtacctgca gggtcccatc 1861 tgggccaagattcctcatacggacggcaactttcatccct cgccgctgat gggaggcttt 1921 ggactgaagcatccgcctcctcagatcctgattaaaaaca cacctgttcc cgcggatcct 1981 ccgaccaccttcaatcaggccaagctggcttctttcatca c gcagtacag taccggccag 2041 gtcagcgtggagatcgagtgggagctgcagaaggagaaca gcaaacgctg gaacccagag 2101 attcagtacacttccaactactacaaatctacaaatgtgg actttgctgt caatactgag 2161 ggtacttattccgagcctcgccccattggcacccgttacc tca cccgtaa tctgtaa

[00192] The amino acid sequence of the VP1 capsid of the RHM15-3/RHM15-5 variant is as follows (SEQ ID NO:8). 1 maadgylpdwlednlsegir ewwdlkpgapkpkanqqrqd ngrglvlpgy rylgpfngld 61 kgepvnaadaaalehdraydqqlqagdnpylrynhadaef qerlqedtsf ggnlgravfq 121 akkrvleplglvespvrtapgkkrpvepspqrspdsstgi gkrgqqpa kk rlnfgqtgds181 niqvkevtqnegtrtiannlt stiqvftdse yqlpyvlgsa 361 hqgclppfpadvfmipqygyltlnngsqavgrssfycley fpsqmlrtgn nfefsynfed 421 vpfhssyahsqsldrlmnplidqylyylsrtqstggtagt qqllfsqagp nnmsaqaknw 4 81 lpgpcyrqqrvsttlsqnnnsnfawtgatkyhlngrdslv npgvamathr ddeerffpss 541 gvlmfgrqgagrdnvdyssvmltseeeirttnpvateqyg vvadnlqqqn aapivgavns 601 qgalpgmvwqnrdvylqgpiwakiphtdgnfhpsplmggf g lkhpppqil ikntpvpadp 661 pttfnqaklasfitqystgqvsveiewelqkenskrwnpe iqytsnyyks tnvdfavnte 721 gtyseprpigtryltrnl

[00193] The nucleic acid sequence of the VP1 capsid of the RHM15-3/RHM15-5 variant is as follows (SEQ ID NO:14) 1 atggctgccgatggttatcttccagattggctcgaggaca acctctctga gggcattcgc 61 gagtggtgggacctgaaacctggagccccgaaacccaaag ccaaccagca aaggcagg ac 121 aacggccggggtctggtgcttcctggctacaggtacctcg gacccttcaa cggactcgac 181 aagggggagcccgtcaacgcggcggacgcagcggccctcg agcacgacag ggcctacgac 241 cagcagctccaagcgggtgacaatccgtacctgcggtata atcacgccga cgccgagttt 301 caggagcgtctgcaagaagatacgtcttttgggggcaacc tcgggcgcgc agtcttccag 361 gccaaaaagcgggttctcgaacctctgggcctggttgaat cgccggttag gacggctcct 421 ggaaagaagagaccggtagagccatcaccccagcgctctc ca gactcctc tacgggcatc 481 ggcaagagaggccagcagcccgcaaaaaagagactcaatt ttgggcagac tggcgactca 541 gagtcagtccccgaccctcaaccaatcggagaaccaccag cagccccctc tggtgtggga 601 cctaatacaatggctgcaggcggtggcgctccaatggcag acaataacga agg cgccgac 661 ggagtgggtagttcctcaggaaattggcattgcgattcca catggctggg cgacagagtc 721 atcaccaccagcacccgcacctgggccctgcccacctaca acaaccacct ctacaggcaa 781 atctccaacgggacctcgggaggaagcaccaacgacaaca cctacttcgg ctacagcacc 8 41 ccctgggggtattttgacttcaacagattccactgccact tttcaccacg tgactggcag 901 cgactcatcaacaacaactggggattccggcccaagaggc tcaacttcaa gctcttcaac 961 atccaagtcaaggaggtcacgcagaatgaaggcaccagga ccatcgccaa taaccttacc 1021 agcacgattcaggtctttacggactcggaataccagctcc cgtacgtgct cggctcggcg 1081 caccagggctgcctgcctccgttcccggcggacgtcttca tgattcctca gtacgggtac 1141 ctgactctgaacaatggcagtcaggctgtgggccggtcgt ccttctactg cctgg agtac 1201 tttccttctcaaatgctgagaacgggcaacaactttgaat tcagctacaa cttcgaggac 1261 gtgcccttccacagcagctacgcgcacagccagagcctgg accggctgat gaaccctctc 1321 atcgaccagtacttgtactacctgtcccggactcaaagca cgggcggtac t gcaggaact 1381 cagcagttgctattttctcaggccgggcctaacaacatgt cggctcaggc caagaactgg 1441 ctacccggtccctgctaccggcagcaacgcgtctccacga cactgtcgca gaacaacaac 1501 agcaactttgcctggacgggtgccaccaagtatcatctga atggcagaga ctctct ggtg 1561 aatcctggcgttgccatggctacccacagggacgacgaag agcgattttt tccatccagc 1621 ggagtcttaatgtttgggagacagggagctggaagagaca acgtggacta tagcagcgtg 1681 atgctaaccagcgaggaagaaataaggaccaccaacccag tggccacaga acag tacggc 1741 gtggtggccgataacctgcaacagcaaaacgccgctccta ttgtaggggc cgtcaatagt 1801 caaggagccttacctggcatggtgtggcagaaccgggacg tgtacctgca gggtcccatc 1861 tgggccaagattcctcatacggacggcaactttcatccct cgccgctgat gggaggcttt 1921 ggactgaagcatccgcctcctcagatcctgattaaaaaca cacctgttcc cgcggatcct 1981 ccgaccaccttcaatcaggccaagctggcttctttcatca cgcagtacag taccggccag 2041 gtcagcgtggagatcgagtgggagctgcagaaggagaaca gcaaacgctg gaacccagag 2101 attcagtacacttccaactactacaaatctacaaatgtgg actttgctgt caat actgag 2161 ggtacttattccgagcctcgccccattggcacccgttacc tcacccgtaa tctgtaa

[00194] The sequence of the amino acid of VP1 capsid of the RHM15-4 variant is how it follows (seq ID no: 9): 1 maadgylpdwlednlsegirewdlkpgapggggddddddddddddlpgy rylgpfngld 61 kgepvnaadeaalehdraydqq EF QerlqedSF GGGLGRAVFQ 121 AKKRVLEPLGLvesPVTAPGKRPVEPSP QRSPDDSSSGI GKRGQQPAKK RLNFGQTGDS 181 ESVPDPDPQPigePPTMAAGGGGA PMADNNEGAD GVGSSGSGSGSGSGSGSGSGSSGSSGSSGSSGSSGSSGSSGSSGSSGSSGSSGSSGSSGSGSSGSGSGSSGSSGSSGSGSGSGSGSGSGS lyrqisngtsggst ndntyfgyst pwgyfdfnrf hchfsprdwq 301 rlinnnwgfrpkrlnfklfniqvkevtqne gtrtiannlt stiqvftdse yqlpyvlgsa 361 hqgclppfpadvfmipqygyltlnngsqav grssfycley fpsqmlrtgn nfefsynfed 421 vp fhssyahsqsldrlmnplidqylyylsr tqstggtagt qqllfsqagp nnmsaqaknw 481 lpgpcyrqqrvsttlsqnnnsnfawtgatk yhlngrdslv npgvamathr ddeerffpss 541 gvlmfgkqgagrdnvdyssvmltseeeirt tnpvateqyg vvad NLQQQN AAPIVGAVNS 601

[00195] The nucleic acid sequence of the VP1 capsid of the RHM15-4 variant is as follows (SEQ ID NO:15): ccggggtctggtgcttcctggctacaggtacctcg gacccttcaa cggactcgac 181 aagggggagcccgtcaacgcggcggacgcagcggccctcg agcacgacag ggcctacgac 241 cagcagctccaagcgggtgacaatccgtacctgcggtata atcacgccga cgccgagttt 301 caggagcgtctgcaagaagatacgtcttttgggggcaacc tcgggcgcgc agtcttccag 361 gccaaaaagcgggttctcgaacctctgggcctggttgaat cgccggttag gacggctcct 421 ggaaagaagagaccggtagagccatcaccccagcgctctc cagactcctc tacgggcatc 481 ggcaagagaggccagcagcccgcaaaaaagagactcaatt t tgggcagac tggcgactca 541 gagtcagtccccgaccctcaaccaatcggagaaccaccag cagccccctc tggtgtggga 601 cctaatacaatggctgcaggcggtggcgctccaatggcag acaataacga aggcgccgac 661 ggagtgggtagttcctcaggaaattggcattgcgattcca catggct ggg cgacagagtc 721 atcaccaccagcacccgcacctgggccctgcccacctaca acaaccacct ctacaggcaa 781 atctccaacgggacctcgggaggaagcaccaacgacaaca cctacttcgg ctacagcacc 841 ccctgggggtattttgacttcaacagattccactgccact tttcaccacg tgactggca g 901 cgactcatcaacaacaactggggattccggcccaagaggc tcaacttcaa gctcttcaac 961 atccaagtcaaggaggtcacgcagaatgaaggcaccagga ccatcgccaa taaccttacc 1021 agcacgattcaggtctttacggactcggaataccagctcc cgtacgtgct cggctcggcg 1080 cttcgaggac 1261 gtgcccttccacagcagctacgcgcacagccagagcctgg accggctgat gaaccctctc 1321 atcgaccagtacttgtactacctgtcccggactcaaagca cgggcggtac tgcaggaact 1381 cagcagttgctattttctcaggccgggcctaacaacatgt cggct caggc caagaactgg 1441 ctacccggtccctgctaccggcagcaacgcgtctccacga cactgtcgca gaacaacaac 1501 agcaactttgcctggacgggtgccaccaagtatcatctga atggcagaga ctctctggtg 1561 aatcctggcgttgccatggctggctacccacagggacgacgaag agcgatt ttt tccatccagc 1621 ggagtcttaatgtttgggaaacagggagctggaagagaca acgtggacta tagcagcgtg 1681 atgctaaccagcgaggaagaaataaggaccaccaacccag tggccacaga acagtacggc 1741 gtggtggccgataacctgcaacagcaaaacgccgctccta ttgtagggg c cgtcaatagt 1801 caaggagccttacctggcatggtgtggcagaaccgggacg tgtacctgca gggtcccatc 1861 tgggccaagattcctcatacggacggcaactttcatccct cgccgctgat gggaggcttt 1921 ggactgaagcatccgcctcctcagatcctgattaaaaaca cacctgttcc cgcggatcct 1981 ccgaccaccttcaatcaggccaggctggcttctttcatca c gcagtacag taccggccag 2041 gtcagcgtggagatcgagtgggagctgcagaaggagaaca gcaaacgctg gaacccagag 2101 attcagtacacttccaactactacaaatctacaaatgtgg actttgctgt caatactgag 2161 ggtacttattccgagcctcgccccattggcacccgttacc tca cccgtaa tctgtaa

[00196] The amino acid sequence of the VP1 capsid of the RHM15-6 variant is as follows (SEQ ID NO:10): 121 gystpwgyfdfnrf hchfsprdwq 301 rlinnnwgfrpkrlnfklfniqvkevtqnegtrtiannltstiqvftdse yqlpyvlgsa 361 hqgclppfpadvfmipqygyltlnngsqavgrssfycleyfpsqmlrtgn nfefsynfed 421 vpfhssyahsqs ldrlmnplidqylyylsrtqstggtagtqqllfsqagp nnmsaqaknw 481 lpgpcyrqqrvsttlsqnnnsnfawtgatkyhlngrdslvnpgvamathr ddeerffpss ggfglkhpppqil ikntpvpadp 661 pttfnqarlasfitqystgqvsveiewelqkenskrwnpeiqytsnyyks tnvdfavnte 721 gtyseprpigtryltrnl

[00197] The nucleic acid sequence of the VP1 capsid of the RHM15-6 variant is as follows (SEQ ID NO:16): ccggggtctggtgcttcctggctacaggtacctcg gacccttcaa cggactcgac 181 aagggggagcccgtcaacgcggcggacgcagcggccctcg agcacgacag ggcctacgac 241 cagcagctccaagcgggtgacaatccgtacctgcggtata atcacgccga cgccgagttt 301 caggagcgtctgcaagaagatacgtcttttgggggcaacc tcgggcgcgc agtcttccag 361 gccaaaaagcgggttctcgaacctctgggcctggttgaat cgccggttag gacggctcct 421 ggaaagaagagaccggtagagccatcaccccagcgctctc cagactcctc tacgggcatc 481 ggcaagaaaggccagcagcccgcaaaaaagagactcaatt ttgggcagac tggcgactca 541 gagtcagtccccgaccctcaaccaatcggagaaccaccag cagccccctc tggtgtggga 601 cctaatacaatggctgcaggcggtggcgctccaatggcag acaataacga aggcgccgac 661 ggagtgggtagttcctcaggaaattggcattgcgattcca catgg ctggg cgacagagtc 721 atcaccaccagcacccgcacctgggccctgcccacctaca acaaccacct ctacaggcaa 781 atctccaacgggacctcgggaggaagcaccaacgacaaca cctacttcgg ctacagcacc 841 ccctgggggtattttgacttcaacagattccactgccact tttcaccacg tgactgg Cag 901 1080 cttcgaggac 1261 gtgcccttccacagcagctacgcgcacagccagagcctgg accggctgat gaaccctctc 1321 atcgaccagtacttgtactacctgtcccggactcaaagca cgggcggtac tgcaggaact 1381 cagcagttgctattttctcaggccgggcctaacaacatgt cggct caggc caagaactgg 1441 ctacccggtccctgctaccggcagcaacgcgtctccacga cactgtcgca gaacaacaac 1501 agcaactttgcctggacgggtgccaccaagtatcatctga atggcagaga ctctctggtg 1561 aatcctggcgttgccatggctggctacccacagggacgacgaag agcgatt ttt tccatccagc 1621 ggagtcttaatgtttgggagacagggagctggaagagaca acgtggacta tagcagcgtg 1681 atgctaaccagcgaggaagaaataaggaccaccaacccag tggccacaga acagtacggc 1741 gtggtggccgataacctgcaacagcaaaacgccgctccta ttgtagggg c cgtcaatagt 1801 caaggagccttacctggcatggtgtggcagaaccgggacg tgtacctgca gggtcccatc 1861 tgggccaagattcctcatacggacggcaactttcatccct cgccgctgat gggaggcttt 1921 ggactgaagcatccgcctcctcagatcctgattaaaaaca cacctgttcc cgcggatcct 1981 ccgaccaccttcaatcaggccaggctggcttctttcatca c gcagtacag taccggccag 2041 gtcagcgtggagatcgagtgggagctgcagaaggagaaca gcaaacgctg gaacccagag 2101 attcagtacacttccaactactacaaatctacaaatgtgg actttgctgt caatactgag 2161 ggtacttattccgagcctcgccccattggcacccgttacc tca cccgtaa tctgtaa

Example 8 This example includes a description of human Factor IX expression studies using the RH74 capsid variants compared to Rh74 and AAV8

[00198] In brief, RH74 capsid variants were used to package an AAV human Factor IX expression vector, the AAV particles used to infect mice, and Factor IX expression levels determined in the animals (plasma). The capsid variants had the following amino acid substitutions at the indicated positions: /77/169/547)R RHM15_2 G195A-L199V- S201P-G202N (137/259/333/530/552/569/38/51/77/163/547)R RHM15_4 G195A-L199V- S201P-G202N K(137/259/333/530/552/569/38/51/ 77/163/668)R RHM15_5 G195A-L199V- S201P-G202N 137/259/333/530/552/569/38/51/77/547/688)R

[00199] Figure 5 shows expression levels of human Factor IX in plasma in treated animals after 2 weeks. As illustrated, capsid-encapsidated AAV human Factor IX expression vector of the RHM4-1 variant provided the highest level of expression and was substantially greater than the expression produced by the Rh74-encapsulated AAV human Factor IX expression vector and AAV8 encapsulated AAV human Factor IX expression vector.

Example 9 This example includes a description of data showing that the AAVrh74 variant RHM4-1 vector expressing human Factor IX (FIX) under the control of a liver-specific promoter administered to cynomolgus macaques led to the production of amounts of FIX in animals and at levels higher than the AAV8 vector administered in the same amount

[00200] Cynomolgus monkeys were pre-screened for antibodies to neutralizing AVV and animals with pretreatment titers <1:1 were selected to ensure that transduction was successful. Monkeys were then infused with either AAV8 or AAV-Rh74 variant RHM4-1 vectors expressing a human Factor IX transgene at a dose of 3x102 vg/kg. Plasma levels of human FIX transgene product (protein) in non-human primates were determined by ELISA weekly during the study and are illustrated in Figure 6.

[00201] One of the animals receiving AAV-RHM4-1-FIX developed an inhibitor against the human Factor IX transgene product, which is a well-documented phenomenon that occurs in approximately 20% of monkeys treated with a human FIX vector due to small amino acid differences between human and monkey proteins. The loss of expression in an animal treated with RHM4-1 was due to the development of an antibody response against the human transgene. The second RHM4-1-treated animal expressed approximately 2-fold higher FIX levels compared to the AAV8-treated monkeys.

Claims (18)

1. Recombinant AAV particle, characterized by the fact that it comprises an AAV capsid comprising an AAV VP1 capsid protein, wherein the AAV particle encapsulates a vector genome comprising a heterologous polynucleotide sequence encoding the Factor IX protein , and wherein the amino acid sequence of the AAV VP1 capsid protein is shown as SEQ ID NO: 5. 2. Recombinant AAV particle, according to claim 1, characterized by the fact that the vector genome further comprises an expression control element conferring transcription of said heterologous polynucleotide sequence encoding the Factor IX protein. 3. Recombinant AAV particle according to claim 2, characterized in that the expression control element comprises a constitutive or regulatable control element. 4. Recombinant AAV particle according to claim 3, characterized in that the expression control element comprises a tissue-specific expression control element or promoter. 5. Recombinant AAV particle according to any one of claims 1 to 3, characterized by the fact that one or more inverted terminal repeat (ITR) sequences flank the 5' or 3' terminus of the heterologous polynucleotide sequence encoding the protein of Factor IX. 6. Recombinant AAV particle according to any one of claims 1 to 3, characterized in that the vector genome further comprises a filler or stuffer sequence. 7. Recombinant AAV particle, characterized in that it comprises an AAV capsid encapsulating a vector genome comprising a heterologous polynucleotide sequence, wherein the AAV capsid comprises a protein comprising the amino acid sequence of SEQ ID NO:5, wherein the heterologous polynucleotide sequence encodes a human Factor IX protein, and wherein the vector genome further comprises a liver-specific promoter or enhancer, and AAV inverted terminal repeats flanking the 5' and 3' termini of the polynucleotide sequence heterologous. 8. Recombinant AAV particle according to any one of claims 1 to 3 or 7, characterized in that the heterologous polynucleotide sequence further comprises at least a portion of intron I of human Factor IX. 9. Recombinant AAV particle according to claim 8, characterized by the fact that the vector genome further comprises a filler or stuffer sequence. 10. Pharmaceutical composition, characterized by the fact that it comprises the recombinant AAV particle, as defined in any one of claims 1 to 9. 11. Recombinant AAV particle according to any one of claims 5 to 9, characterized by the fact that the AAV inverted terminal repeat sequences are from AAV2. 12. Recombinant AAV particle according to any one of claims 1 to 9 or 11, characterized by the fact that said polynucleotide sequence is flanked by 5' and 3' untranslated regions. 13. Recombinant AAV particle according to claims 1 to 9, 11 or 12, characterized in that the heterologous polynucleotide sequence further comprises a poly A sequence. 14. Recombinant AAV particle according to any one of claims 1 to 9 or 11 to 13, characterized in that the heterologous polynucleotide sequence further comprises at least a portion of an intron. 15. Recombinant AAV particle according to any one of claims 1 to 9 or 11 to 14, characterized in that the heterologous polynucleotide sequence further comprises at least a portion of intron I of the human Factor IX gene. 16. Recombinant AAV particle according to claim 15, characterized by the fact that said portion of intron I of the human Factor IX gene has a nucleotide length of 0.1 kb to 1.7 kb. 17. The recombinant AAV particle of claim 7, wherein said liver-specific enhancer and promoter is an ApoE-hAAT enhancer and promoter. 18. Recombinant AAV particle according to any one of claims 1 to 9 or 11 to 18, characterized in that the Factor IX protein is a human gain-of-function variant that is more active than human Factor IX type wild.