CN114686448A

CN114686448A - Purified adeno-associated virus with liver specific targeting and application thereof

Info

Publication number: CN114686448A
Application number: CN202210331754.XA
Authority: CN
Inventors: 赵锴; 陈晨; 刘强; 章一品; 袁龙辉; 郑静; 肖啸; 杜增民; 蒋威; 吴博; 刘素丽
Original assignee: Shanghai Mianyi Biotechnology Co ltd
Current assignee: Shanghai Mianyi Biotechnology Co ltd
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-07-01

Abstract

The present invention relates to purified adeno-associated virus (AAV) and its use for the treatment of liver diseases or other diseases requiring the delivery of genes expressed in the liver. The purified AAV of the invention has excellent liver specific targeting property, lower neutralizing antibody level, lower immunogenicity and higher safety.

Description

Purified adeno-associated virus with liver specific targeting and application thereof

Technical Field

The present disclosure relates to purified adeno-associated virus (AAV) with liver-specific targeting and its use for the treatment of liver diseases or other diseases requiring the delivery of genes expressed in the liver.

Background

In recent years, the use of adeno-associated virus (AAV) vectors in gene therapy has attracted considerable attention. However, wild-type AAV serotypes (e.g., AAV2, AAV5, AAV8, and AAV9) can infect multiple tissues/organs in a mammal due to poor tissue specificity. To improve organ and tissue targeting of AAV, we have constructed a serotype mutant AAVz2 (FIGS. 1A and 1B, SEQ ID NO: 1, described in CN 113121652A) and purified it using affinity column chromatography plus one-step iodixanol ultracentrifugation, and have found that the thus purified AAVz2 has high affinity for retina and muscle.

Liver-targeted gene therapy using AAV vectors has enjoyed compelling success in a number of clinical trials for hemophilia a and hemophilia B (reference 1). In these cases, moderate doses of AAV administration are sufficient to safely restore blood clotting. However, broader application of AAV to other diseases may require more efficient hepatocyte gene delivery capability and less tissue/organ off-target transduction. Thus, the development of AAV serotypes with superior hepatocyte targeting is a promising direction.

Furthermore, pre-existing neutralizing antibodies (nabs) against AAV viruses are also one of the biggest obstacles to their use in a wider population. Circulating Nab can mask the receptor binding site exposed on the surface of capsid protein and block AAV from entering target cells or accelerate depletion of AAV by immune system, rendering the therapeutic effect of AAV vector ineffective. Although it has been reported that engineered AAV viruses have improved liver transduction in primary human hepatocytes and xenografted mouse models of human hepatocytes, pre-existing Nab significantly reduced the activity of these AAV serotypes (reference 2).

Reference:

1.Leebeek FWG,Miesbach W.Gene therapy for hemophilia:A review on clinical benefit,limitations,and remaining issues.Blood.2021；138:923-931

2.Perocheau DP,Cunningham S,Lee J,Antinao Diaz J,Waddington SN,Gilmour K,Eaglestone S,Lisowski L,Thrasher AJ,Alexander IE,Gissen P,Baruteau J.Age-related seroprevalence of antibodies against aav-lk03 in a uk population cohort.Human gene therapy.2019；30:79-87

disclosure of Invention

The inventors have unexpectedly found that the liver targeting of AAVz2(SEQ ID NO: 1) can be improved by a specific purification method, i.e.a two-step iodixanol density gradient ultracentrifugation.

Accordingly, in a first aspect, the present disclosure provides a purified adeno-associated virus (AAV), wherein the AAV is obtained by two-step iodixanol density gradient ultracentrifugation purification, the AAV is AAVz2, the amino acid sequence of which is as set forth in SEQ ID NO: 1 is shown.

In one embodiment, the iodixanol density gradient comprises: 15 w/v%, 25 w/v%, 40 w/v%, 60 w/v% iodixanol.

In one embodiment, purification of AAV does not include an affinity column chromatography step.

In one embodiment, the AAV is an AAV produced by a eukaryotic cell or prokaryotic cell package.

In one embodiment, the AAV is an AAV produced by three plasmid transfection or insect baculovirus method packaging.

The purified AAV of the present disclosure has improved liver targeting compared to AAV obtained by purification methods using affinity column chromatography plus one-step iodixanol ultracentrifugation.

In a second aspect, the present disclosure provides a method of purifying an adeno-associated virus (AAV), comprising: AAV was purified by two-step iodixanol density gradient ultracentrifugation.

In one embodiment, the AAV is AAVz 2.

In one embodiment, the method of the present disclosure comprises: (i) loading the solution containing AAV above iodixanol density gradient, and ultracentrifuging to obtain primary purified solution; and (ii) loading the primary purified solution obtained in the step (i) above the iodixanol density gradient, and performing ultracentrifugation to obtain the purified AAV.

In one embodiment, the methods of the present disclosure do not include an affinity column chromatography step.

The purification method disclosed by the invention improves the transduction efficiency of AAV on liver cells in vivo and in vitro, but does not obviously improve the transduction efficiency on other tissues and organs, so that the liver transduction specificity is obviously improved.

The AAV vector purified by the purification method disclosed by the invention has excellent liver specific targeting property, lower neutralizing antibody level, lower immunogenicity and higher safety. AAV vectors of the present disclosure with excellent liver-specific targeting can be applied for the prevention and treatment of liver-related diseases or other diseases that require drug delivery to the liver, and low off-target (non-target organ) effects and low immunogenicity enhance their safety potential.

In a third aspect, the present disclosure provides use of an AAV according to the first aspect in the preparation of a medicament for the treatment of a disease, wherein the disease is a liver disease or other disease requiring delivery of a gene for expression in the liver.

In a fourth aspect, the present disclosure provides a medicament comprising: an AAV according to the first aspect and an excipient.

In one embodiment, the excipient comprises a salt, an organic substance, and/or a surfactant.

In one embodiment, the medicament is for the prevention, diagnosis and treatment of liver diseases or other diseases requiring the delivery of genes expressed in the liver.

In one embodiment, the medicament is administered by a systemic route or a local route, preferably intravenous administration, oral administration, intranasal administration, intralobular administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, or intralesional administration; preferably, administration to the liver is by systemic or local routes.

In one embodiment, the liver disease comprises: primary or secondary liver cancer, cirrhosis, liver abscess, fatty liver, alcoholic liver disease, liver transplantation, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, autoimmune hepatitis, drug-toxic hepatitis, and other hepatitis.

In one embodiment, other diseases where delivery of genes to expression in the liver is desired include: hemophilia a and B, lysosomal storage disorders (e.g., MPS II and III), Fabry's disease, glycogen storage disease, bepier disease, Gaucher's disease, walman's disease, Wilson's disease, autoimmune diseases (e.g., multiple sclerosis, myasthenia gravis, rheumatic or rheumatoid arthritis, lupus erythematosus, autoimmune heart disease), cardiovascular or pulmonary diseases (e.g., hypertension, atherosclerosis, hypercholesterolemia, chronic obstructive pulmonary disease), hyperammonemia, diabetes, fisher's syndrome, comprehensive trauma, renal failure, anemia.

In a fifth aspect, the present disclosure provides a method of treating a disease comprising administering a therapeutically effective amount of a medicament according to the fourth aspect to a subject in need thereof, the subject having a liver disease or other disease requiring delivery of a gene to expression in the liver.

In a sixth aspect, the present disclosure provides a method of purifying an adeno-associated virus (AAV), comprising: (a) loading the solution containing AAV onto an affinity chromatography column; (b) eluting AAV from the affinity chromatography column with a first eluent comprising arginine and magnesium ions, and collecting the eluent containing AAV; and (c) ultracentrifuging the AAV-containing eluate obtained in step (b).

In one embodiment, the methods of the present disclosure increase the production of AAV by about 10%, about 20%, about 30%, about 40%, or even about 50% or more, as compared to a method in which the elution step is performed with an eluent that does not contain arginine and magnesium ions.

In one embodiment, the concentration of arginine in the first eluent is 200mM to 2000mM, preferably 400mM to 1000mM, more preferably 500mM to 800 mM; and/or the concentration of magnesium ions is 0.5mM to 3mM, preferably 1mM to 3mM, more preferably 1.5mM to 2.5 mM.

In one embodiment, the magnesium ion is formed from MgCl₂Or MgSO 2₄Provided is a method.

In one embodiment, the first eluent comprises: 0.1-2M AcOH, 200 + 2000mM arginine, 137 + 600mM NaCl or Na₂SO₄、0.5-3mM MgCl₂Or MgSO 2₄And 0.05% poloxamer 188.

In one embodiment, the first eluent has a pH of 2.5 to 3.2. When the pH of the first eluent is within this range, the purification effect can be more effectively achieved.

In one embodiment, the elution of AAV is also performed in step (b) using a second eluent comprising urea and magnesium ions.

In one embodiment, the second eluent comprises 2-6M urea and 0.5-3mM MgCl₂

In one embodiment, the ultracentrifugation is iodixanol density gradient ultracentrifugation or cesium chloride density gradient ultracentrifugation.

In one embodiment, the AAV is a wild-type AAV.

In one embodiment, the AAV is AAVz 2.

The purification method disclosed by the invention not only improves the yield of AAV, but also improves the transduction efficiency of AAV on liver cells in vivo and in vitro, but does not obviously improve the transduction efficiency of other tissues and organs, so that the liver transduction specificity is obviously improved.

In a seventh aspect, the present disclosure provides an AAV obtained by the method according to the fifth aspect.

In one embodiment, the AAV is a wild-type AAV.

In one embodiment, the AAV is AAVz 2.

In one embodiment, the AAV purified by the methods of the present disclosure has improved liver targeting compared to AAV obtained by performing the elution step with an eluent that does not contain arginine and magnesium ions.

The AAV of the present disclosure has excellent liver-specific targeting, can be applied to the prevention and treatment of liver-related diseases or other diseases requiring drug delivery to the liver, and low off-target (non-target organ) effects and low immunogenicity enhance its safety potential.

In an eighth aspect, the present disclosure provides use of an AAV according to the sixth aspect in the manufacture of a medicament for the treatment of a disease, wherein the disease is a liver disease or other disease requiring delivery of a gene for expression in the liver.

In a ninth aspect, the present disclosure provides a medicament comprising: an AAV according to the sixth aspect and an excipient.

In one embodiment, the medicament is administered by a systemic route or a local route, preferably intravenous administration, oral administration, intranasal administration, intralobular administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, or intralesional administration; preferably, administration to the liver is by systemic or local route.

In a tenth aspect, the present disclosure provides an affinity column eluate comprising: arginine and magnesium ions.

In one embodiment, the affinity column eluent comprises 200mM to 2000mM, preferably 400mM to 1000mM, more preferably 500mM to 800mM arginine and 0.5mM to 3mM, preferably 1mM to 3mM, more preferably 1.5mM to 2.5mM magnesium ions.

In one embodiment, the affinity column eluent comprises: 0.1-2M AcOH, 200 + 2000mM arginine, 137 + 600mM NaCl or Na₂SO₄、0.5-3mM MgCl₂Or MgSO 2₄And 0.05% poloxamer 188.

In one embodiment, the pH of the eluate from the affinity column is 2.5 to 3.2. When the pH of the elution solution of the affinity column of the present disclosure is within this range, the purification effect can be better achieved.

The affinity column eluate of the present disclosure significantly increases the elution yield of AAV by comprising arginine and magnesium ions, and also improves the efficiency of specific transduction of AAV to liver cells in vivo and in vitro.

Drawings

Figure 1A shows that the AAVz2 capsid protein was constructed by inserting the oligopeptide "fatpgp" after Q574 on AAV5 capsid protein variable region VIII.

FIG. 1B shows a PDB map of the 3D structure of VP1 protein of AAVz2 constructed by Chimera 1.15 (UCSF). The inserted oligopeptides are shown magnified in rectangular boxes.

FIG. 1C shows silver staining of individual AAV particles produced purified packaged GFP genes, with 3 parallel lanes per AAV serotype.

FIG. 1D shows the cross section from 2X 10⁸Production of each AAV produced in individual cells (medium + lysate) and obtained by the corresponding purification method. AAVz2-0 and wild type AAV: performing affinity column chromatography (the eluent does not contain arginine and magnesium ions) and one-step iodixanol ultracentrifugation purification to obtain the product; AAVz 2-1: two-step iodixanol ultracentrifugation purification; AAVz 2-2: performing affinity column chromatography (eluent contains arginine and magnesium ions) and one-step iodixanol ultracentrifugation purification to obtain the product. n is 3. vg: a vector genome; vp is as follows: number of virus particles.

Figure 2 shows the yield of wild type AAV5 purified by affinity column chromatography using different eluents. Elution peaks a and C1: and performing affinity column chromatography (eluent A + eluent C) and one-step iodixanol ultracentrifugation to purify AAV5, wherein ultraviolet absorption peaks at 260 nm and 280nm are taken as representatives. Elution peaks B and C2: affinity column chromatography (eluent B + eluent)C) One step of iodixanol ultracentrifugation is added to purify AAV 5. qPCR quantifies viral genome (vg), silver stain quantifies viral particle number (vp). Eluent A: 1M AcOH, 500mM NaCl, 0.05% poloxamer 188, pH 2.5. Eluent B: 1M AcOH, 500mM arginine, 2mM MgCl₂500mM NaCl, 0.05% poloxamer 188, pH 2.5; eluent C (C1 or C2): 6M Urea, 2mM MgCl₂。

FIG. 3A shows the infectivity of AAV by different purification methods on mouse liver. Scale bar 200 microns.

Figure 3B shows the relative quantification of the liver infectious capacity of AAV compared to the heart, lung, spleen and kidney infectious capacity based on the ratio of the number of GFP-positive hepatocytes to the number of GFP-positive heart, lung, spleen and kidney cells based on DAPI plaque count statistics. n-5 mice/group (3 males and 2 females). P <0.001, one-way analysis of variance.

FIG. 4 shows mRNA expression of GFP gene in liver, heart, lung, spleen, kidney and brain of mice injected tail vein with each AAV. n-4 mice/group. P <0.05, p <0.01, p <0.001, one-way anova.

FIG. 5A shows the infectious capacity of AAV obtained by different purification methods for various skeletal muscles. GA: gastrocnemius muscle; LO: the longest thoracic muscle; QU: quadriceps femoris; TR: the triceps brachii muscle; ST: sternocleidomastoid muscle; SO: the soleus muscle. Scale bar 200 microns.

Figure 5B shows the results of quantifying the muscle transduction efficiency of AAV based on the percentage of GFP-positive muscle cell area to total muscle cross-sectional area. n-5 mice/group (3 males 2 females), p <0.05, p <0.01, p < 0.001. And (4) one-way analysis of variance.

FIG. 6A shows that human hepatoma cells Huh7 and normal hepatocytes L02 were infected with the corresponding AAV vector packaging the GFP gene (MOI ═ 1X 10⁵vg/cell) at 48 hours. The upper diagram: a GFP signal; the following figures: bright field. Scale bar: 100 microns.

Fig. 6B shows the expression levels of GFP protein delivered by each AAV.

FIG. 6C shows statistics for the percentage of GFP positive Huh7 and L02 cells in FIG. 6A. The ratio of the area of GFP positive signals to the area of the entire bright field was calculated for Huh7 cells and L02 cells. P <0.05, p <0.01, p <0.001, n-6 well cells, one-way anova.

Fig. 6D shows statistics for GFP protein expression levels in the AAV-treated Huh7 and L02 cells of fig. 6B. P <0.05, p <0.001, n-4 well cells, one-way anova.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") in this context.

Herein, the term "treatment" includes: (1) inhibiting the condition, disease or disorder, i.e., arresting, reducing, or delaying the development of the disease or its recurrence or the development of at least one clinical or subclinical symptom thereof; or (2) ameliorating the disease, i.e., causing regression of at least one of the conditions, diseases or disorders or clinical or subclinical symptoms thereof.

Herein, the term "preventing" a condition, disease or disorder includes: preventing, delaying or reducing the incidence and/or likelihood of the occurrence of at least one clinical or subclinical symptom of a condition, disease or disorder developing in a subject who may be suffering from or susceptible to the condition, disease or disorder but who has not experienced or exhibited clinical or subclinical symptoms of the condition, disease or disorder.

Herein, the term "topical administration" or "topical route" refers to an administration having a local effect.

As used herein, the terms "transduction," "transfection," and "transformation" refer to the process of delivering an exogenous nucleic acid into a host cell, followed by transcription and translation of the polynucleotide product, which includes the introduction of an exogenous polynucleotide into the host cell using a recombinant virus.

As used herein, the term "gene delivery" refers to the introduction of an exogenous polynucleotide into a cell for gene delivery, including targeting, binding, uptake, transport, replicon integration, and expression.

As used herein, the term "gene expression" or "expression" refers to the process by which a gene is transcribed, translated, and post-translationally modified to produce the RNA or protein product of the gene.

As used herein, the term "infection" refers to the process by which a virus or viral particle comprising a polynucleotide component delivers a polynucleotide into a cell and produces its RNA and protein products, and may also refer to the process of replication of the virus in a host cell.

In this context, the terms "targeting" or "specificity" refer to the preferential entry of the virus into some cell or tissue, followed by the further expression of the viral genome or sequence carried by the recombinant transgene in the cell.

In this context, "recombinant" in relation to a polynucleotide means that the polynucleotide is a synthetic product that is different from the natural polynucleotide, constructed by multiple cloning steps. Recombinant viruses are viral particles comprising recombinant polynucleotides.

As used herein, the term "vector" refers to a macromolecule or series of macromolecules encapsulating a polynucleotide that facilitates delivery of the polynucleotide to a target cell in vitro or in vivo. Types of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The polynucleotides to be delivered are sometimes referred to as "transgenes," including, but not limited to, the coding sequences of certain proteins or synthetic polypeptides (which may enhance, inhibit, attenuate, protect, trigger, or prevent certain biological and physiological functions), the coding sequences of interest in vaccine development (e.g., polynucleotides that express proteins, polypeptides, or peptides suitable for eliciting an immune response in a mammal), RNAi components (e.g., shRNA, siRNA, snRNA, microRNA, ribozymes, antisense oligonucleotides, and antisense polynucleotides, which may knock down any endogenous gene activated in an aberrant manner or an exogenous gene that invades the host cell), or alternatively, biomarkers. Viral or bacterial polynucleotides are known in the art. The RNAi moiety typically has 60-100% identity in sequence to its target gene and results in at least a 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) reduction in the corresponding protein product.

As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, hybrid sequences thereof, and the like. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein interchangeably refers to a single-stranded or double-stranded molecule. Unless otherwise indicated, polynucleotides described herein include double-stranded forms and two complementary single strands that are known or predictable to make up the double-stranded form.

Herein, the terms "polypeptide" and "protein" are used synonymously herein to refer to polymers consisting of more than 20 amino acids. These terms also encompass synthetic or artificial amino acid polymers.

As used herein, "viral particle" refers to a functional viral unit formed by natural or synthetic viral genome packaged by viral capsid proteins, and functions include infecting or transducing tissue organs and cells, delivering viral genome into tissue organs and cells, and expressing the corresponding nucleic acid and protein products.

In this context, the term "Inverted Terminal Repeat (ITR)" includes any AAV viral terminal repeat or synthetic sequence that forms a hairpin structure and serves as a cis element to mediate viral replication, packaging and integration. The ITRs herein include, but are not limited to, terminal repeats from AAV types 1-11 (avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV). Furthermore, the AAV terminal repeat need not have a native terminal repeat, so long as the terminal repeat is available for viral replication, packaging, and integration.

In this context, the term "adeno-associated virus (AAV)" or "adeno-associated virus (AAV) serotype" includes native AAV (e.g., capsid proteins of native AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV) and other artificially engineered AAV. The genomic sequences, ITR sequences, Rep and Cap proteins of different AAV serotypes are known in the art. These sequences can be found in the literature or in public databases, for example GenBank databases such as GenBank (r) accession nos. NC 002077, NC 001401, NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC 001701, NC 001510, AF063497, U89790, AF043303, AF028705, AF028704, J02275, JO1901, J02275, XO1457, AF288061, AHO09962, AY028226, AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579, AY631965, AY 631966; the contents of which are incorporated herein by reference in their entirety; and, for example, Srivistava et al, J.Virol (1983)45: 555; chiorini et al, J.Virol (1998)71: 6823; chiorini et al, J.Virol (1999)73: 1309; Bantel-Schaal et al, J.Virol (1999)73: 939; xiao et al, J.Virol (1999)73: 3994; muramatsu et al, Virology (1996)221: 208; WO 00/28061; WO 99/61601; WO 98/11244; US 6156303.

The present inventors have surprisingly found that by using specific purification methods (two-step iodixanol or cesium chloride density gradient centrifugation) or specific chemical reagents (elution of affinity column containing arginine and magnesium ions), the production and/or liver cell targeting of existing AAV (e.g. AAVz2, described in CN 113121652A) can be improved, altering the distribution of AAV in systemic tissues.

In one embodiment, an AAV vector of the present disclosure may be loaded with an exogenous polynucleotide for delivery of the gene into a target cell. The AAV vectors of the present disclosure can be used to deliver nucleic acids to tissue organs and cells in vitro or in vivo. The AAV vectors of the present disclosure preferentially deliver liver-specific genes while off-target delivery to other organs (e.g., heart, lung, spleen, kidney, and brain) is relatively less, meaning that infection and potential adverse effects to off-target tissues are less frequent.

In one embodiment, the exogenous polynucleotide delivered by the AAV vector encodes a polypeptide that acts as a reporter (i.e., a reporter protein). The reporter protein is used to indicate cells successfully infected with AAV. These reporter proteins include, but are not limited to, Green Fluorescent Protein (GFP), β -galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase.

In some embodiments, the delivered nucleic acid comprises a nucleic acid encoding a native protein, which native protein is codon-optimized or non-codon-optimized, for therapeutic (e.g., medical or veterinary) use. The proteins are delivered to the liver by AAV vectors, expressed, secreted into the blood circulation and acted on other tissues and organs of the whole body to prevent and treat related diseases. Such proteins include, but are not limited to: cystic Fibrosis Transmembrane Regulator (CFTR), dystrophin (including truncated forms, known as mini-dystrophin or microdystrophin, see, e.g., Vincent et al, Nat Genet (1993) 5: 130; US 2003017131; Wang et al, Proc Natl Acad Sci USA (2000) 97: 13714-9; Harper et al, Nat Med (2002) 8: 253-61); mini-agglutinin, integrin beta 1, laminin beta 2, myoglycan alpha and beta, sarcomeric proteins, synuclein, gonadotropin, mini-lecithin, Lamin a/C, four half LIM domain protein 1(FHL1), follistatin, SOD1 or SOD2, full length or dominant negative myostatin; angiogenic factors (e.g., VEGF, angiopoietin 1 or 2) and angiogenesis inhibitors (e.g., endostatin and angiostatin); anti-inflammatory polypeptides (e.g., anti-inflammatory interleukins 4, 10, 11 and 13, full-length and dominant mutant Ikappa B, Pinch and ILK genes); coagulation factors (e.g., coagulation factor VIII, coagulation factor IX, coagulation factor X); spectrin, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, leptin, and leptin receptor; neurotrophic factors (e.g., BDNF, GDNF, NGF, semaphores, Slit1, 2 and 3, FGF7, 10 and 22) and corresponding neurotrophin receptors; LDL receptors, lipoprotein lipase, adrenergic receptors alpha and beta, B globulin, C globulin; insulin-like growth factors, such as IGF-1 and IGF-2; adenosine deaminase and the bone morphogenic protein superfamily (e.g., BMP1, 2, 4, 6, 7 and TGF- β, RANKL); BDNF, GDNF, NTF, APP, GRIA1 and 2, atrophin 1, SLC1A 1; SMN1, UBE1, DYNC1H 1; RPE65, RPGR, Bestrophin-1, CNGA3, CNGB3, retinochitin, retinal-specific phospholipid transport ATPase ABCA4 and retinal-specific ABC transporters.

In some embodiments, the polynucleotide packaged in the AAV viral particle may encode an artificially synthesized polypeptide or protein and deliver it to liver cells for expression, secretion, and systemic action with blood circulation. Such polypeptides or proteins include, but are not limited to: aflibercept (a recombinant VEGF soluble receptor with anti-angiogenic effect produced by renger Pharmaceuticals); recombinant interleukins 1, 18 and TNF-alpha antagonize soluble receptors; activin type II soluble receptors; antibodies or single chain antibodies, including but not limited to anti-VEGF antibodies (e.g., bevacizumab, ranibizumab and broucizumab), anti-sclerostin antibodies (e.g., Romosozumab and Blosozumab), anti-RANKL antibodies (e.g., Denosumab), anti-complement component C5 antibodies (e.g., Ravulizumab and Eculizumab), anti-PD-1 antibodies (e.g., Nivolumab, Pembrolizumab and cemimimab), PD-L1 antibodies (e.g., Avelumab and atezolimab), anti-CTLA-4 antibodies (e.g., Ipilimumab), anti-CGRP antibodies (e.g., freezumab, galanezumab and ereumumab), anti-HER 2 antibodies (e.g., Trastuzumab and Pertuzumab), anti-EGFR antibodies (e.g., Cetuximab, Panitumumab and neclizumab), pro-inflammatory antibodies against cytokines and their receptors (e.g., saakulab, cyanidumumab, basilizumab, adylizumab and adolizumab); modified enzymes such as Cethrin (neuroprotective drugs available from BioAxone BioSciences inc. for the treatment of spinal cord injury); antigens or antigenic fragments of the vaccine can be generated (e.g., spike proteins of coronavirus disease 2019(COVID 2019) or Severe Acute Respiratory Syndrome (SARS) coronavirus, envelope proteins of hepatitis a, b, c and Human Immunodeficiency Virus (HIV), various tumor cell immunogens such as MAGE antigens, HER2, ErbB2, mucin antigens and estrogen receptors). Vaccines can elicit a protective immune response to prevent the onset of certain diseases. Methods of delivering antibodies or vaccines into a subject, as typified by intramuscular injection, are known to those skilled in the art.

In some embodiments, the nucleic acid delivered by the AAV vector can encode relevant elements of gene editing (e.g., ZFNs, TALENs, and CRISPRs), including endonucleases or recombinases such as Cas9 and Cas13, as well as corresponding sgrnas.

In some embodiments, the deliverable heterologous polynucleotide comprises regulatory elements such as transcriptional/translational control signals, origins of replication, polyadenylation signals, Internal Ribosome Entry Sites (IRES) or 2A signals (e.g., P2A, T2A, F2A), promoters and enhancers (e.g., such as CMV promoter or other hybrid CMV promoters with vertebrate β -actin, β -globin or β -globin regulatory elements, EF1 promoter, ubiquitin promoter, T7 promoter, SV40 promoter, VP16 or VP64 promoter). The use of promoters and enhancers depends on their tissue-specific expression profile. Promoters and enhancers may be induced by chemicals or hormones (e.g., doxycycline or tamoxifen) to ensure gene expression at a specific time point. Furthermore, promoters and enhancers may be natural or artificial or chimeric sequences, i.e., prokaryotic or eukaryotic sequences.

In some embodiments, the inducible regulatory element for gene expression may be a tissue-specific or tissue-tropic promoter/enhancer element.

In some embodiments, the AAV vectors of the present disclosure are used in situations where it is desirable to label a particular cell (e.g., a liver cell), such as in research experiments.

In one embodiment, an AAV vector of the present disclosure comprises a reporter protein for indicating or labeling a cell successfully infected by a virus.

In some embodiments, AAV viral particles of the present disclosure are administered to a host cell in vitro, and the cell is then implanted into a subject. Thus, a heterologous nucleic acid packaged in an AAV is introduced into a subject by a cell for transcription and/or translation, resulting in a protein or RNA product that is secreted from the cell into the subject or that modulates the biological activity of the host.

One skilled in the art can use standard methods known to produce recombinant and synthetic polypeptides or proteins thereof, to produce antibodies or antigen-binding fragments, to alter nucleic acid sequences, to produce transformed cells, to construct recombinant AAV mutants, to engineer capsid proteins, to package vectors expressing AAV Rep and/or Cap sequences, and to transiently or stably transfect packaging cells. These techniques are known to those skilled in the art. See, e.g., MOLECULAR CLONING (MOLECULAR CLONING): a LABORATORY MANUAL (a LABORATORY MANUAL), second edition, (cold spring harbor, new york, 1989); AUSUBEL et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., N.Y.).

In one embodiment, the AAV viral particles of the present disclosure are formulated for administration to a human or other mammal in a medicament (e.g., injection, tablet, capsule, powder). The medicament may also comprise other ingredients such as pharmaceutical excipients, water-soluble or organic solvents (e.g. water, glycerol, ethanol, methanol, isopropanol, chloroform, phenol or polyethylene glycol), salts (e.g. sodium chloride, potassium chloride, phosphate, acetate, bicarbonate, Tris-HCl and Tris-acetate), dissolution retarding agents (e.g. paraffin), surfactants, antimicrobial agents, liposomes, lipid complexes, immunosuppressants (e.g. cortisone, prednisone, cyclosporine), microspheres of non-steroidal anti-inflammatory drugs (NSAIDs, e.g. aspirin, ibuprofen, paracetamol), rigid matrices, semi-solid carriers, nanospheres or nanoparticles. In addition, the drug may be delivered in a single dose or multiple doses by inhalation, systemic or local (e.g., intravenous, intramuscular, subcutaneous, oral, intraperitoneal, and intralesional) administration.

In one embodiment, the medicament of the present disclosure is at 10⁵-10¹⁴The titer of vg/mL comprises the AAV viral particles of the disclosure.

In one embodiment, the medicament of the present disclosure is used for preventing and/or treating diseases, such as liver diseases, including but not limited to primary or secondary liver cancer, liver cirrhosis, liver abscess, fatty liver, alcoholic liver disease, liver transplantation, hepatitis a, hepatitis b, hepatitis c, hepatitis d, hepatitis e, autoimmune hepatitis, drug toxicity hepatitis and other hepatitis; and other diseases indirectly associated with or requiring delivery of drugs to the liver, such as hemophilia a and B, lysosomal storage disorders (e.g., MPS II and III), Fabry's syndrome, glycogen storage disorders, beware disease (battlen disease), Gaucher's disease, walman's disease, Wilson's disease, autoimmune diseases (e.g., multiple sclerosis, myasthenia gravis, rheumatic or rheumatoid arthritis, lupus erythematosus, autoimmune heart disease), cardiovascular or pulmonary diseases (e.g., hypertension, atherosclerosis, hypercholesterolemia, chronic obstructive pulmonary disease), hyperammonemia, diabetes, sanfilippo syndrome, comprehensive trauma, renal failure, anemia.

In some embodiments, AAV is produced using a three-plasmid transfection method (plasmid 1: cis-element plasmid; plasmid 2: AAV Rep/Cap plasmid; plasmid 3: helper plasmid) well known to those skilled in the art.

In some embodiments, the disclosure also provides methods of producing AAV from a cell. These cells support efficient transfection of plasmids encoding AAV Rep/Cap proteins, helper genes, and viral vectors encoding native viral genomes or heterologous proteins.

The present disclosure is described in further detail below with reference to the accompanying drawings and examples. The following examples are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure. The experimental procedures, in which the specific conditions are not indicated in the examples, are carried out according to the conventional conditions known in the art or according to the conditions recommended by the manufacturer.

Examples

Example 1 purification of AAV

Wild-type AAV (e.g. AAV2, AAV5, AAV8 and AAV9) was purified by affinity column chromatography plus one-step iodixanol ultracentrifugation as described in CN 113121652 a (wild-type AAV adopts this purification method unless otherwise specified), as follows:

1) a pretreatment step: after 48 hours of transfection with three plasmids, cells and supernatant were collected, 0.1% Triton X-100 was added thereto, and the mixture was allowed to stand for 30 minutes to lyse the cells. Then, 5mM domiphen bromide (Demifene bromide) was added thereto, and the mixture was allowed to stand for 30 minutes to precipitate nucleic acid. Then, the conductivity was adjusted by adding 300mM NaCl. Centrifuge at 9000rpm for 30 minutes. The centrifuged supernatant was concentrated to 500 mL.

2) And (3) affinity column chromatography: the concentrated supernatant was loaded onto an affinity chromatography column (AAVX resin, Thermo Scientific) and rinsed with equilibration solution (20mM Tris-HCl, 500mM NaCl, 0.05% poloxamer 188, pH 7.2). After equilibration for 10 column volumes, virus samples were loaded. After all the virus samples are loaded, the balance liquid is used for post-balancing. After equilibration for 10 column volumes, the virus was eluted with eluent a (1M AcOH, 500mM NaCl, 0.05% poloxamer 188, pH 2.5), the eluent was collected and the pH was adjusted to about 7.

3) Iodixanol ultracentrifugation step: the solution from the previous step was loaded on top of a 15%, 25%, 40%, 60% iodixanol density gradient and centrifuged at 65000rpm for 2.5h and 6mL of virus solution was collected.

4) A concentration step: the solution from the previous step was concentrated to 3mL with a viral diluent (PBS +300mM NaCl + 0.05% poloxamer 188).

AAVz2 was purified by the same purification procedure as described above for wild type AAV, yielding AAVz 2-0.

AAVz2 was purified by the same purification procedure as for wild type AAV described above, to give AAVz2-1, except that the 2) affinity column chromatography step was replaced with the same procedure as the 3) iodixanol ultracentrifugation step.

AAVz2 was purified by the same purification procedure as for wild type AAV described above, to give AAVz2-2, except that eluent A used in 2) the affinity column chromatography step was replaced with eluent B (1M AcOH, 500mM arginine, 2mM MgCl₂500mM NaCl, 0.05% poloxamer 188, pH 2.5) + eluent C (6M urea, 2mM MgCl₂)。

Subsequently, viral yields of AAV obtained by different purification methods were quantified by qPCR and silver staining.

The results show that the number of viral genomes and viral particles of AAVz2-1 obtained by the two-step iodixanol ultracentrifugation method without affinity chromatography is comparable to wild-type AAV5, 8, 9 and AAVz2-0 and is much higher than wild-type AAV 2. Surprisingly, the yield of AAVz2-2 using a particular eluent was significantly increased (by about 50%) compared to other AAV (fig. 1C and fig. 1D).

In order to verify the influence of the addition of arginine and magnesium ions in the eluate on virus purification, wild-type AAV5 was purified by the same procedure as described above using eluate a + eluate C and eluate B + eluate C, respectively, to obtain AAV 5-2. The results show that the addition of arginine and magnesium ions in the eluate increased the yield of virus (fig. 2).

Furthermore, the inventors found that the arginine concentration in the eluate B was adjusted in the range of 200-2000mM or the MgCl concentration in the eluate B was adjusted in the range of 0.5-3mM₂At concentration, the obtained AAVz2 all had essentially the same properties as AAVz 2-2.

Example 2 transduction efficiency of purified AAV on mouse liver

To investigate the in vivo transduction tendencies of AAV obtained by different purification methods in example 1, C57BL/6 mice were injected by tail vein with 1X 10¹³The liver, heart, lung, spleen and kidney were immunofluorescent stained with GFP and DAPI at a vg/kg dose of various AAV serotype vectors packaged with GFP encoding genes 3 weeks after virus injection.

The results showed that up to 92.25% and 91.8% of hepatocytes in the AAVz2-1 and AAVz2-2 injection groups were GFP-positive, significantly better than the AAV5 injection group (only 22.64%), and better than the AAV8 (85.37%) and AAV9 (88.11%) injection groups (fig. 3A). Also, unlike AAV8 and AAV9 which also show higher transduction efficiency in the heart and mild to moderate infectivity of the lungs, spleen and kidneys, AAVz2-1 and AAVz2-2 hardly infect these tissue organs. The above results indicate that AAVz2-1 and AAVz2-2 have the ability to specifically transduce liver.

Quantification of GFP signal, liver transduction specificity of AAV was quantified by the ratio of GFP positive cells to other tissues/organs (heart, lung, spleen and kidney) in the liver. As shown in FIG. 3B, the relative specificity of the liver of AAVz2-1 and AAVz2-2 compared to the heart, lung, spleen and kidney is about 4-11 times higher than that of AAV5, AAV8, AAV9 and AAVz 2-0. These results again demonstrate the high liver targeting ability of AAVz2-1 and AAVz 2-2.

Next, GFP gene expression was tested in tissues and organs of mice injected tail vein with each AAV. C57BL/6 mice were injected tail vein with GFP transgenic for the corresponding AAV serotype at a dose of 1X 10¹³vg/kg. Homogenating liver, heart, lung, spleen, kidney and brain tissueRT-PCR was performed to detect expression of GFP gene mRNA delivered by AAV5, AAV8, AAV9 and AAVz2 purified by various methods, and results were presented as relative values of mRNA for each set of GFP compared to AAV 5.

The results showed that AAVz2-1 and AAVz2-2 delivered GFP at mRNA levels in the liver that were 14.7 ± 5.7 fold higher than AAV5, comparable to AAV8 and AAV9 (fig. 4). Also, the levels of GFP expression in heart, lung, spleen, kidney and brain of mice by AAVz2-1 and AAVz2-2 were similar to or lower than AAV5 and significantly lower than AAV8 and AAV 9. In addition, AAVz2-0 delivered mRNA levels of GFP in the liver, heart, lung, spleen, kidney, and brain of mice close to AAV 5.

The above results indicate that the purification methods of the present disclosure can improve the transduction specificity of AAV to the liver.

Example 3 transduction efficiency of purified AAV into mouse skeletal muscle

To investigate the ability of AAV obtained by the different purification methods in example 1 to infect skeletal muscle, a relatively high dose (5X 10) was used¹³vg/kg) of each AAV (packaged GFP gene) tail vein injection of C57BL/6 mice of both sexes. Muscles in various regions of the body, including hind limb (gastrocnemius, quadriceps, soleus), back (longissimus thoracis), forelimb (triceps brachii) and neck (sternocleidomastoid) were immunofluorescent stained with GFP 21 days after virus injection.

As shown in fig. 5A and fig. 5B, AAV9 showed the highest transduction efficiency to skeletal muscle: about 60% of the gastrocnemius, quadriceps and triceps cells, 30-40% of the soleus and pectoralis longissimus cells successfully expressed GFP; AAV8 is ranked second, with about 15-40% of gastrocnemius, quadriceps and longissimus cells being efficiently transduced; in the AAV 5-injected group, the GFP-positive quadriceps femoris cells also reached 19.19%. In contrast, AAVz2-1 and AAVz2-2 infected muscle fibers were negligible in number, indicating that they hardly transduced skeletal muscle cells in vivo. These results further confirm the liver transduction specificity of AAVz2-1 and AAVz2-2 under intravenous systemic dosing conditions.

Example 4 transduction efficiency of purified AAV on human liver cell lines

To further investigate the transduction efficiency of AAV obtained by the different purification methods in example 1 in human hepatocytes, the inventors selected human hepatocellular carcinoma cell line Huh7 and normal hepatocyte line L02, which are frequently used in studies of liver drug delivery.

The results showed that the multiplicity of infection (MOI) was 1X 10⁵In the case of vg/cell, the GFP-positive cell ratios (i.e., ratio of GFP signal area to total cell area) of AAVz2-1 and AAVz2-2 were significantly higher than AAVz2-0 and AAV5 (21.51 times for Huh7 cells and 20.83 times for L02 cells) and slightly higher than AAV8 and AAV9 (fig. 6A and 6C).

Western blot was used to detect expression of AAV-delivered GFP proteins in lysates of Huh7 and L02 cells, with tubulin as an internal control. Western blot experiments showed that the expression level of GFP protein was significantly higher in AAVz2-1 and AAVz2-2 than in AAVz2-0 and AAV5 (FIGS. 6B and 6D).

The above results indicate that the purification method of the present disclosure can effectively improve the infection efficiency of AAV on human liver cell lines.

Example 5 neutralizing antibody levels of AAV

Pre-existing neutralizing antibodies (nabs) in serum may lead to inactivation of AAV vectors for gene therapy. Among wild-type AAV, AAV5 has a significant advantage due to the lowest systemic Nab levels, with Nab positivity in some populations of only 3%.

Hemophilia is a representative disease that requires the delivery of a coagulation factor IX gene to the liver for treatment by AAV vectors. The inventors collected serum samples from 10 patients with hemophilia B, in a 1:2 and mixed with the specified AAV vector encoding luciferase, and then Huh7 cells were treated with a virus-serum mixture. The highest serum dilution at which luciferase activity was inhibited by at least 50% was defined as Nab titer.

As shown in table 1, patients of 4/10 had Nab titers against AAV8 and AAV9 that were greater than 1:4, which was defined as Nab-positive. However, none of the patients were positive for Nab of AAV5 and AAVz 2-1.

TABLE 1 neutralizing antibody levels of various AAV serotypes in serum of hemophilia B patients

In addition, 21 healthy rhesus monkeys were also examined for Nab-positivity. The results showed that 3/21 monkeys were positive for Nab of AAV5, 4/21 monkeys were positive for Nab of AAV8, 9/21 monkeys were positive for Nab of AAV9, and 1/21 monkeys were positive for Nab of AAVz2-1 (Table 2).

TABLE 2 neutralizing antibody levels of various AAV serotypes in rhesus monkey sera

Monkey ID	anti-AAV 5	anti-AAV 8	anti-AAV 9	anti-AAVz 2-1
					1	1:2	1:4	1:8	1:1
2	1:1	1:1	1:16	1:2
					3	1:8	1:32	1:32	1:8
4	<1:1	1:1	1:1	1:1
					5	<1:1	<1:1	<1:1	<1:1
6	<1:1	<1:1	1:2	<1:1
					7	1:4	1:16	1:32	1:4
8	1:8	1:4	1:8	1:4
					9	1:16	1:2	1:8	1:4
10	<1:1	1:1	1:2	<1:1
					11	1:1	1:2	1:1	<1:1
12	<1:1	<1:1	1:1	<1:1
					13	1:2	<1:1	<1:1	<1:1
14	<1:1	<1:1	1:1	<1:1
					15	<1:1	1:1	1:1	<1:1
16	1:4	1:1	1:2	1:1
					17	1:2	1:8	1:8	1:1
18	1:2	1:8	1:16	1:2
					19	1:1	1:1	1:8	<1:1
20	1:4	<1:1	1:2	<1:1
					21	1:1	<1:1	1:1	<1:1

The above results indicate that AAVz2-1 has a low Nab-positive rate.

All publications, patent applications, patents, nucleic acid and amino acid sequences, and other references mentioned in this disclosure are incorporated by reference herein in their entirety.

While the present disclosure has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing is a more detailed description of the disclosure than is possible with reference to the specific embodiments, and that no limitation to the specific embodiments of the disclosure is intended. Various changes in form and detail, including simple deductions or substitutions, may be made by those skilled in the art without departing from the spirit and scope of the present disclosure.

Sequence listing

<110> Shanghai baren Biotech Co., Ltd

<120> purified adeno-associated virus with liver specific targeting and application thereof

<130> PCNCNN221510G

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 731

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of AAVz2 capsid protein

<400> 1

Met Ser Phe Val Asp His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu

1 5 10 15

Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys

20 25 30

Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val

50 55 60

Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu

65 70 75 80

Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe

115 120 125

Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile

130 135 140

Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser

145 150 155 160

Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln

165 170 175

Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr

180 185 190

Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala

195 200 205

Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp

210 215 220

Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro

225 230 235 240

Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp

245 250 255

Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr

260 265 270

Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln

275 280 285

Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val

290 295 300

Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr

305 310 315 320

Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp

325 330 335

Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys

340 345 350

Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr

355 360 365

Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser

370 375 380

Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn

385 390 395 400

Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser

405 410 415

Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp

420 425 430

Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln

435 440 445

Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp

450 455 460

Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly

465 470 475 480

Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu

485 490 495

Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr

500 505 510

Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile

515 520 525

Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu

530 535 540

Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg

545 550 555 560

Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Phe Ala

565 570 575

Pro Thr Pro Gly Pro Ser Ser Thr Thr Ala Pro Ala Thr Gly Thr Tyr

580 585 590

Asn Leu Gln Glu Ile Val Pro Gly Ser Val Trp Met Glu Arg Asp Val

595 600 605

Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro Glu Thr Gly Ala His

610 615 620

Phe His Pro Ser Pro Ala Met Gly Gly Phe Gly Leu Lys His Pro Pro

625 630 635 640

Pro Met Met Leu Ile Lys Asn Thr Pro Val Pro Gly Asn Ile Thr Ser

645 650 655

Phe Ser Asp Val Pro Val Ser Ser Phe Ile Thr Gln Tyr Ser Thr Gly

660 665 670

Gln Val Thr Val Glu Met Glu Trp Glu Leu Lys Lys Glu Asn Ser Lys

675 680 685

Arg Trp Asn Pro Glu Ile Gln Tyr Thr Asn Asn Tyr Asn Asp Pro Gln

690 695 700

Phe Val Asp Phe Ala Pro Asp Ser Thr Gly Glu Tyr Arg Thr Thr Arg

705 710 715 720

Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730

<210> 2

<211> 2196

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleic acid sequence encoding AAVz2 capsid protein

<400> 2

atgtcttttg ttgatcaccc tccagattgg ttggaagaag ttggtgaagg tcttcgcgag 60

tttttgggcc ttgaagcggg cccaccgaaa ccaaaaccca atcagcagca tcaagatcaa 120

gcccgtggtc ttgtgctgcc tggttataac tatctcggac ccggaaacgg tctcgatcga 180

ggagagcctg tcaacagggc agacgaggtc gcgcgagagc acgacatctc gtacaacgag 240

cagcttgagg cgggagacaa cccctacctc aagtacaacc acgcggacgc cgagtttcag 300

gagaagctcg ccgacgacac atccttcggg ggaaacctcg gaaaggcagt ctttcaggcc 360

aagaaaaggg ttctcgaacc ttttggcctg gttgaagagg gtgctaagac ggcccctacc 420

ggaaagcgga tagacgacca ctttccaaaa agaaagaagg ctcggaccga agaggactcc 480

aagccttcca cctcgtcaga cgccgaagct ggacccagcg gatcccagca gctgcaaatc 540

ccagcccaac cagcctcaag tttgggagct gatacaatgt ctgcgggagg tggcggccca 600

ttgggcgaca ataaccaagg tgccgatgga gtgggcaatg cctcgggaga ttggcattgc 660

gattccacgt ggatggggga cagagtcgtc accaagtcca cccgaacctg ggtgctgccc 720

agctacaaca accaccagta ccgagagatc aaaagcggct ccgtcgacgg aagcaacgcc 780

aacgcctact ttggatacag caccccctgg gggtactttg actttaaccg cttccacagc 840

cactggagcc cccgagactg gcaaagactc atcaacaact actggggctt cagaccccgg 900

tccctcagag tcaaaatctt caacattcaa gtcaaagagg tcacggtgca ggactccacc 960

accaccatcg ccaacaacct cacctccacc gtccaagtgt ttacggacga cgactaccag 1020

ctgccctacg tcgtcggcaa cgggaccgag ggatgcctgc cggccttccc tccgcaggtc 1080

tttacgctgc cgcagtacgg ttacgcgacg ctgaaccgcg acaacacaga aaatcccacc 1140

gagaggagca gcttcttctg cctagagtac tttcccagca agatgctgag aacgggcaac 1200

aactttgagt ttacctacaa ctttgaggag gtgcccttcc actccagctt cgctcccagt 1260

cagaacctct tcaagctggc caacccgctg gtggaccagt acttgtaccg cttcgtgagc 1320

acaaataaca ctggcggagt ccagttcaac aagaacctgg ccgggagata cgccaacacc 1380

tacaaaaact ggttcccggg gcccatgggc cgaacccagg gctggaacct gggctccggg 1440

gtcaaccgcg ccagtgtcag cgccttcgcc acgaccaata ggatggagct cgagggcgcg 1500

agttaccagg tgcccccgca gccgaacggc atgaccaaca acctccaggg cagcaacacc 1560

tatgccctgg agaacactat gatcttcaac agccagccgg cgaacccggg caccaccgcc 1620

acgtacctcg agggcaacat gctcatcacc agcgagagcg agacgcagcc ggtgaaccgc 1680

gtggcgtaca acgtcggcgg gcagatggcc accaacaacc agttcgcacc aacaccggga 1740

ccaagctcca ccactgcccc cgcgaccggc acgtacaacc tccaggaaat cgtgcccggc 1800

agcgtgtgga tggagaggga cgtgtacctc caaggaccca tctgggccaa gatcccagag 1860

acgggggcgc actttcaccc ctctccggcc atgggcggat tcggactcaa acacccaccg 1920

cccatgatgc tcatcaagaa cacgcctgtg cccggaaata tcaccagctt ctcggacgtg 1980

cccgtcagca gcttcatcac ccagtacagc accgggcagg tcaccgtgga gatggagtgg 2040

gagctcaaga aggaaaactc caagaggtgg aacccagaga tccagtacac aaacaactac 2100

aacgaccccc agtttgtgga ctttgccccg gacagcaccg gggaatacag aaccaccaga 2160

cctatcggaa cccgatacct tacccgaccc ctttaa 2196

Claims

1.A purified adeno-associated virus (AAV), wherein said AAV is obtained by two-step iodixanol density gradient ultracentrifugation purification, and said AAV is AAVz2, and its amino acid sequence is as shown in SEQ ID NO: 1 is shown.

2. The AAV of claim 1, wherein the iodixanol density gradient comprises: 15 w/v%, 25 w/v%, 40 w/v%, 60 w/v% iodixanol.

3. The AAV of claims 1 or 2, wherein the AAV is an AAV produced by a eukaryotic cell or prokaryotic cell package.

4. The AAV of claims 1 or 2, wherein purification of the AAV does not comprise an affinity column chromatography step.

5. Use of an AAV according to any one of claims 1 to 4 in the preparation of a medicament for the treatment of a disease, wherein the disease is a liver disease or other disease requiring delivery of a gene for expression in the liver.

6. The use according to claim 5, wherein liver diseases include: primary or secondary liver cancer, cirrhosis, liver abscess, fatty liver, alcoholic liver disease, liver transplantation, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, autoimmune hepatitis, drug-toxic hepatitis, and other hepatitis.

7. The use according to claim 5 or 6, wherein other diseases where delivery of genes to expression in the liver is desired include: hemophilia a and B, lysosomal storage disorders (e.g., MPS II and III), fabry syndrome, glycogen storage disease, behcet's disease, gaucher's disease, walman's disease, wilson's disease, autoimmune diseases (e.g., multiple sclerosis, myasthenia gravis, rheumatic or rheumatoid arthritis, lupus erythematosus, autoimmune heart disease), cardiovascular or pulmonary diseases (e.g., hypertension, atherosclerosis, hypercholesterolemia, chronic obstructive pulmonary disease), hyperammonemia, diabetes, sanfilippo syndrome, comprehensive trauma, renal failure, anemia.

8. A medicament, comprising: the AAV and excipient of any one of claims 1 to 4.

9. The medicament according to claim 8, wherein the medicament is administered by systemic or topical route, preferably intravenous administration, oral administration, intranasal administration, intrapper-tilt administration, intramuscular administration, subcutaneous administration, intraperitoneal administration or intralesional administration; preferably, administration to the liver is by systemic or local route.

10. The medicament of claim 8 or 9, wherein the excipients comprise: salts, organics, and/or surfactants.