Recombinant adeno-associated viruses with enhanced hepatotropism and uses thereof

Abstract

The present invention describes a recombinant adeno-associated virus. The recombinant adeno-associated virus may contain a capsid protein with enhanced hepatocyte tropism. The recombinant adeno-associated virus exhibits low immunogenicity in humans. The recombinant adeno-associated virus may contain an expression cassette containing a polynucleotide sequence encoding a therapeutic agent for the treatment of liver disease. The present invention also provides a packaging system for the recombinant adeno-associated virus, a method for producing the recombinant adeno-associated virus, a pharmaceutical composition containing the recombinant adeno-associated virus, and the use of the composition in the treatment of liver diseases such as Fabry disease and hepatitis B.

Description

[Technical field]

[0001] Sequence Listing

[0002] The sequence listing included in this application is submitted as an ASCII text file and is incorporated herein by reference in its entirety. The ASCII document has a creation date of November 6, 2020, a text name of SEQLIST.TXT, and a size of 26KB.

[0003] The present invention relates to recombinant adeno-associated viruses comprising an adeno-associated virus capsid protein with enhanced liver tropism and an expression cassette comprising a polynucleotide sequence encoding a therapeutic agent useful for gene therapy of liver disease. In some embodiments, the therapeutic agent encoded by the nucleic acid sequence comprises alpha-galactosidase A (GLA / Gla) or a short hairpin RNA targeting the genome of Hepatitis B virus. The present invention also relates to compositions comprising these recombinant adeno-associated viruses and to the use of these recombinant adeno-associated viruses to treat patients with liver disease (e.g., Fabry disease or Hepatitis B). [Background technology]

[0004] Adeno-associated viruses (AAV) are small, non-enveloped, replication-defective viruses that contain a linear, single-stranded DNA genome. The genome of AAV contains inverted terminal repeats (ITRs) located at the ends of the DNA sequence and open reading frames (ORFs) that code for replication proteins (Rep) and capsid proteins (Cap). AAV has generally proven to be safe, and no studies have shown a significant association with the pathogenesis of tumors or other diseases (Guylene (2005) Arch Ophthalmal 123:500-506). In addition to safety, AAV has other advantages such as high infection efficiency, wide infectious range, and long-term expression (David (2007) BMC Bio 7:75), making it more suitable as a vector for gene therapy. Therefore, AAV is widely used to treat many different diseases, including cancer, retinal diseases, arthritis, acquired immune deficiency syndrome (AIDS), liver diseases, and neurological diseases.

[0005] Fabry disease, a type of liver disease, is a rare genetic disease belonging to the lysosomal storage disorders caused by mutations in the α-galactosidase A (gla) gene. Deficiency of GLA can lead to the accumulation of glycolipids in blood vessels, other tissues and organs, impairing their function. Symptoms include pain, kidney disease, skin lesions, fatigue, nausea and neurological disorders. Treatment includes enzyme replacement therapy with recombinant GLA. Hepatitis B is a liver disease caused by the hepatitis B virus (HBV), which is transmitted to healthy people through bodily fluids such as blood and semen of infected individuals, and symptoms include fatigue, loss of appetite, stomach pain, nausea and jaundice. Approximately 25% of patients with chronic hepatitis B develop cirrhosis and liver cancer. According to the World Health Organization (WHO), in 2015, of the approximately 257 million people with chronic hepatitis B worldwide, an estimated 887,000 people are estimated to have died from the disease. Therefore, it is crucial to develop safe and effective treatments for such widespread liver diseases. AAV can be used as a gene therapy vector to deliver therapeutic agents useful in liver diseases such as Fabry disease and Hepatitis B. Treatment of Hepatitis B involves gene therapy that involves the use of short hairpin RNA (shRNA) that targets the genome of the Hepatitis B virus. When using AAV to deliver therapeutic agents to treat liver diseases, it is desirable for AAV to have enhanced hepatotropism.

[0006] AAV is divided into many variants called serotypes, and is listed in the literature as AAV1-AAV12 (Gao et al. (2004) J Virol 78: 6381-6388; Mori et al. (2004) Virology 330: 375-383; Schmidt et al. (2008) J Virol 82: 1399-1406). AAV usually has a human or primate host, and AAV1-6 are isolated from humans, and therefore may induce a clear immune response in humans. AAV7 and AAV8 were isolated from the heart tissue of rhesus monkeys (Gao et al. (2002) PNAS 99: 11854-11859), and AAV9-12 were isolated from humans and cynomolgus monkeys. All serotypes of AAV have a typical icosahedral structure, but due to differences in their capsid protein sequences and surface topologies, different serotypes of AAV have different binding abilities and tropisms for different types of cell surface receptors (Timpe (2005) Curr Gene Ther 5:273-284). For example, it is known that AAV2 has tropism for many types of cells, especially for neurons, AAV1 and AAV7 have enhanced tropism for skeletal muscle, AAV3 has enhanced tropism for megakaryocytes, AAV5 and AAV6 have enhanced tropism for airway epithelial cells, while AAV8 has enhanced tropism for hepatocytes.

[0007] Natural AAV has limited tropism, and therapeutic agents containing different AAV capsid proteins have significantly different efficacies against their respective target cells. In addition, humans and other primates usually produce neutralizing antibodies against natural AAV variants, resulting in a short half-life and loss of efficacy after administration of AAV. Thus, a great deal of research has been conducted on genetic engineering of AAV capsid proteins to enhance their tropism for specific cell types and reduce their immunogenicity. Engineered AAVs have been applied in clinical practice. For example, AAV2.5 contains a chimeric capsid protein obtained by adding five amino acids that determine skeletal muscle tropism in the AAV1 capsid protein to the capsid protein of AAV2. AAV2.5 carrying a minidystrophin gene has been used to treat Duchenne muscular dystrophy (Bowles et al. (2012) Mol Ther 20:443-455), and its phase I clinical trial has already been completed. The safety of these engineered AAVs was also evaluated. The results showed that AAV2.5 has stronger skeletal muscle tropism and lower immunogenicity than native AAV2.

[0008] Currently, there remains a need for engineered AAVs that exhibit stronger hepatotropism and lower immunogenicity in humans, which would be valuable and improved tools for delivering therapeutic agents to treat liver diseases (e.g., Fabry disease or hepatitis B). Summary of the Invention

[0009] Some aspects of the present specification relate to recombinant AAV (rAAV). In some embodiments, the rAAV has improved properties such as higher packaging yields, enhanced gene expression levels, lower immunogenicity and / or enhanced hepatocyte tropism. In these embodiments, the rAAV comprises an expression cassette comprising a polynucleotide sequence encoding a therapeutic agent for treating liver disease. In some embodiments, the therapeutic agent is shRNA or GLA. In certain embodiments, the liver disease is Hepatitis B or Fabry disease. In one aspect, the specification relates to a vector comprising an shRNA or GLA expression cassette, e.g., a plasmid comprising the expression cassette. In yet another aspect, the specification relates to a packaging system for producing the rAAV of the present invention. In yet another aspect, the specification relates to a plasmid system for packaging the rAAV of the present invention. In yet another aspect, the specification relates to a cell comprising a plasmid system for packaging the rAAV of the present invention, including an isolated engineered cell comprising the packaged rAAV. In yet another aspect, the specification relates to a method for packaging the rAAV of the present invention. In yet another aspect, the present specification relates to compositions comprising the rAAV. In yet another aspect, the present specification relates to the use of compositions comprising the rAAV of the present invention in the preparation of medicaments for the prevention and treatment of liver diseases, such as Hepatitis B and Fabry disease, and in methods of treating liver diseases, such as Hepatitis B and Fabry disease.

[0010] Additional features and advantages of the disclosed embodiments will be set forth in part in the description that follows, and in part will be obvious from the description, or may be obtained by practice of the disclosed embodiments. The features and advantages of the disclosed embodiments will be realized and obtained by means of the elements and combinations pointed out in the appended claims.

[0011] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the embodiments disclosed in the claims.

[0012] The drawings constitute a part of this specification. The drawings illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the disclosed embodiments as recited in the appended claims. [Brief description of the drawings]

[0013] The above, as well as other objects, features and advantages, will become apparent from the following description of specific disclosed embodiments of the invention, as illustrated in the drawings, which are not necessarily to scale or comprehensive, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

[0014] [Figure 1A] Figures 1A-1I show plasmid maps. Specifically, Figures 1A-1D show various plasmid maps containing different promoters and nucleotide sequences encoding Gluc. Figures 1E-1I show various plasmid maps containing different promoters and nucleotide sequences encoding GLA. [Figure 1B] Figures 1A-1I show plasmid maps. Specifically, Figures 1A-1D show various plasmid maps containing different promoters and nucleotide sequences encoding Gluc. Figures 1E-1I show various plasmid maps containing different promoters and nucleotide sequences encoding GLA. [Figure 1C] Figures 1A-1I show plasmid maps. Specifically, Figures 1A-1D show various plasmid maps containing different promoters and nucleotide sequences encoding Gluc. Figures 1E-1I show various plasmid maps containing different promoters and nucleotide sequences encoding GLA. [Figure 1D] Figures 1A-1I show plasmid maps. Specifically, Figures 1A-1D show various plasmid maps containing different promoters and nucleotide sequences encoding Gluc. Figures 1E-1I show various plasmid maps containing different promoters and nucleotide sequences encoding GLA. [Figure 1E]Figures 1A-1I show plasmid maps. Specifically, Figures 1A-1D show various plasmid maps containing different promoters and nucleotide sequences encoding Gluc. Figures 1E-1I show various plasmid maps containing different promoters and nucleotide sequences encoding GLA. [Figure 1F] Figures 1A-1I show plasmid maps. Specifically, Figures 1A-1D show various plasmid maps containing different promoters and nucleotide sequences encoding Gluc. Figures 1E-1I show various plasmid maps containing different promoters and nucleotide sequences encoding GLA. [Figure 1G] Figures 1A-1I show plasmid maps. Specifically, Figures 1A-1D show various plasmid maps containing different promoters and nucleotide sequences encoding Gluc. Figures 1E-1I show various plasmid maps containing different promoters and nucleotide sequences encoding GLA. [Figure 1H] Figures 1A-1I show plasmid maps. Specifically, Figures 1A-1D show various plasmid maps containing different promoters and nucleotide sequences encoding Gluc. Figures 1E-1I show various plasmid maps containing different promoters and nucleotide sequences encoding GLA. [Figure 1I] Figures 1A-1I show plasmid maps. Specifically, Figures 1A-1D show various plasmid maps containing different promoters and nucleotide sequences encoding Gluc. Figures 1E-1I show various plasmid maps containing different promoters and nucleotide sequences encoding GLA. [Figure 2A] 2A-B show plasmid maps containing the nucleotide sequence encoding the shRNA targeting the HBV genome. [Figure 2B] 2A-B show plasmid maps containing the nucleotide sequence encoding the shRNA targeting the HBV genome. [Figure 3A]3A-3F are graphs showing the tropism of AAV2 / 8, AAV2 / 3B, AAV2 / 7 and AAV2 / 9 for various hepatic cell lines. [Figure 3B] 3A-3F are graphs showing the tropism of AAV2 / 8, AAV2 / 3B, AAV2 / 7 and AAV2 / 9 for various hepatic cell lines. [Figure 3C] 3A-3F are graphs showing the tropism of AAV2 / 8, AAV2 / 3B, AAV2 / 7 and AAV2 / 9 for various hepatic cell lines. [Figure 3D] 3A-3F are graphs showing the tropism of AAV2 / 8, AAV2 / 3B, AAV2 / 7 and AAV2 / 9 for various hepatic cell lines. [Figure 3E] 3A-3F are graphs showing the tropism of AAV2 / 8, AAV2 / 3B, AAV2 / 7 and AAV2 / 9 for various hepatic cell lines. [Figure 3F] 3A-3F are graphs showing the tropism of AAV2 / 8, AAV2 / 3B, AAV2 / 7 and AAV2 / 9 for various hepatic cell lines. [Figure 4A] 4A to 4J are graphs showing the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines. [Figure 4B] 4A to 4J are graphs showing the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines. [Figure 4C] 4A to 4J are graphs showing the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines. [Figure 4D] 4A to 4J are graphs showing the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines. [Figure 4E] 4A to 4J are graphs showing the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines. [Figure 4F] 4A to 4J are graphs showing the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines. [Figure 4G] 4A to 4J are graphs showing the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines. [Figure 4H] 4A to 4J are graphs showing the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines. [Figure 4I] 4A to 4J are graphs showing the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines. [Figure 4J] 4A to 4J are graphs showing the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines. [Figure 5A] 5A to 5J are graphs showing the tropism of AAV2 / 8 and AAV2 / X to human primary cultured hepatoma cells. [Figure 5B] 5A to 5J are graphs showing the tropism of AAV2 / 8 and AAV2 / X to human primary cultured hepatoma cells. [Figure 5C] 5A to 5J are graphs showing the tropism of AAV2 / 8 and AAV2 / X to human primary cultured hepatoma cells. [Figure 5D] 5A to 5J are graphs showing the tropism of AAV2 / 8 and AAV2 / X to human primary cultured hepatoma cells. [Figure 5E] 5A to 5J are graphs showing the tropism of AAV2 / 8 and AAV2 / X to human primary cultured hepatoma cells. [Figure 5F] 5A to 5J are graphs showing the tropism of AAV2 / 8 and AAV2 / X to human primary cultured hepatoma cells. [Figure 5G] 5A to 5J are graphs showing the tropism of AAV2 / 8 and AAV2 / X to human primary cultured hepatoma cells. [Figure 5H] 5A to 5J are graphs showing the tropism of AAV2 / 8 and AAV2 / X to human primary cultured hepatoma cells. [Figure 5I] 5A to 5J are graphs showing the tropism of AAV2 / 8 and AAV2 / X to human primary cultured hepatoma cells. [Figure 5J] 5A to 5J are graphs showing the tropism of AAV2 / 8 and AAV2 / X to human primary cultured hepatoma cells. [Figure 6]Figures 6A to 6N are fluorescent images showing in vivo EGFP expression in various tissues of cynomolgus monkeys administered the AAV2 / X-CMV-EGFP vector, namely, the heart (Figure 6A), lung (Figure 6B), liver (Figures 6C to 6G), brain (Figure 6H), testis (Figure 6I), biceps femoris (Figure 6J), stomach (Figure 6K), jejunum (Figure 6L), kidney (Figure 6M), and spleen (Figure 6N). [Figure 7] Figures 7A to 7N are fluorescent images showing in vivo EGFP expression in various tissues of cynomolgus monkeys administered the AAV2 / 8-CMV-EGFP vector, namely, the heart (Figure 7A), lung (Figure 7B), liver (Figures 7C to 7G), brain (Figure 7H), testis (Figure 7I), biceps femoris (Figure 7J), stomach (Figure 7K), jejunum (Figure 7L), kidney (Figure 7M), and spleen (Figure 7N). [Figure 8] FIG. 8 is a graph showing the levels of neutralizing antibodies to AAV2 / 8 or AAV2 / X in pooled human serum. [Figure 9A] 9A-9B are schematic diagrams showing expression cassettes comprising different promoters and polynucleotides encoding different transgenes. [Figure 9B] 9A-9B are schematic diagrams showing expression cassettes comprising different promoters and polynucleotides encoding different transgenes. [Figure 10A] 10A-10B are graphs showing the expression levels of Gluc and GLA under different promoters. Fig. 10A is a graph showing the expression levels of Gluc under DC172 promoter, DC190 promoter, or CMV promoter. Fig. 10B is a graph showing the expression levels of GLA under DC172 promoter or LP1 promoter. [Figure 10B] 10A-10B are graphs showing the expression levels of Gluc and GLA under different promoters. Fig. 10A is a graph showing the expression levels of Gluc under DC172 promoter, DC190 promoter, or CMV promoter. Fig. 10B is a graph showing the expression levels of GLA under DC172 promoter or LP1 promoter. [Figure 11] FIG. 11 is a schematic diagram showing an expression cassette containing a WPRE sequence. [Figure 12A] 12A to 12B are graphs showing a comparison of GLA activity in normal mice using AAV2 / X having a DC172 promoter or an LP1 promoter, with or without the addition of a WPRE sequence. [Figure 12B] 12A to 12B are graphs showing a comparison of GLA activity in normal mice using AAV2 / X having a DC172 promoter or an LP1 promoter, with or without the addition of a WPRE sequence. [Figure 13A] 13A to 13D are graphs showing GLA activity levels in various organs of model mice administered AAV2 / X or AAV2 / 8. [Figure 13B] 13A to 13D are graphs showing GLA activity levels in various organs of model mice administered AAV2 / X or AAV2 / 8. [Figure 13C] 13A to 13D are graphs showing GLA activity levels in various organs of model mice administered AAV2 / X or AAV2 / 8. [Figure 13D] 13A to 13D are graphs showing GLA activity levels in various organs of model mice administered AAV2 / X or AAV2 / 8. [Figure 14A] 14A to 14D are graphs showing GLA activity levels in various organs of model mice administered AAV2 / X at different MOIs. [Figure 14B] 14A to 14D are graphs showing GLA activity levels in various organs of model mice administered AAV2 / X at different MOIs. [Figure 14C] 14A to 14D are graphs showing GLA activity levels in various organs of model mice administered AAV2 / X at different MOIs. [Figure 14D]14A to 14D are graphs showing GLA activity levels in various organs of model mice administered AAV2 / X at different MOIs. [Figure 15A] 15A-15B are graphs showing the reduction of hepatitis B surface antigen (HBsAg) levels by shRNA encoded by a nucleotide sequence contained in AAV2 / X or AAV2 / 8. [Figure 15B] 15A-15B are graphs showing the reduction of hepatitis B surface antigen (HBsAg) levels by shRNA encoded by a nucleotide sequence contained in AAV2 / X or AAV2 / 8. [Figure 16A] 16A-16B are graphs showing the reduction of hepatitis B E antigen (HBeAg) levels by shRNA encoded by a nucleotide sequence contained in AAV2 / X or AAV2 / 8. [Figure 16B] 16A-16B are graphs showing the reduction of hepatitis B E antigen (HBeAg) levels by shRNA encoded by a nucleotide sequence contained in AAV2 / X or AAV2 / 8. [Figure 17A] 17A-17B are graphs showing the reduction of HBV DNA levels by shRNA encoded by nucleotide sequences contained in AAV2 / X or AAV2 / 8. [Figure 17B] 17A-17B are graphs showing the reduction of HBV DNA levels by shRNA encoded by nucleotide sequences contained in AAV2 / X or AAV2 / 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Although examples and features of the disclosed principles have been described herein, modifications, variations, and other implementations may be made without departing from the spirit and scope of the disclosed embodiments. The words "comprising," "having," "containing," and "including," as well as other similar forms, are equivalent and open-ended in meaning, and the item or items introduced by these words do not imply an exhaustive list of the item or items, or are limited to only the illustrated item or items. It should also be noted that the terms "a," "an," and "said" as used in this specification and the appended claims include the plural unless the context clearly dictates otherwise. Unless otherwise defined, 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 invention belongs. The terms used herein to describe the present invention are intended to describe specific embodiments only, and are not intended to limit the present invention. Unless otherwise indicated, standard methods known to those of skill in the art can be used for producing recombinant or synthetic polypeptides, manipulating nucleic acid sequences, producing transformed cells, constructing recombinant AAV, modifying capsid proteins, constructing vectors to express AAV Rep and / or Cap, and transient or stable transfection of packaging cells. Recombinant AAV

[0016] Some aspects of the present specification relate to recombinant AAV (rAAV). As used herein, the term "recombinant AAV" generally refers to an infectious, replication-defective AAV virus that has been modified to have specific properties and / or contain a desired therapeutic nucleic acid. In some embodiments, the virus may contain wild-type AAV capsid proteins and a modified genome. In some embodiments, the virus contains modified AAV capsid proteins. The term "wild-type AAV genome" refers to an unmodified linear ssDNA molecule that contains ITRs at both ends. The ITRs provide an origin of replication for the viral genome. The wild-type AAV genome also contains an open reading frame that encodes a capsid protein (Cap) and a replication protein (Rep). The Cap protein is a structural protein that forms the shell structure that contains the AAV genome. The Rep protein is a non-structural protein that functions in AAV replication and packaging.

[0017] Cap protein is encoded by the cap gene. AAV capsid proteins play an important role in determining the tropism of the virus. As used herein, the term "tropism" generally refers to the tendency of a virus to invade a particular type of cell or tissue and / or to interact with a particular cell surface that contributes to the invasion of such cells or tissues. As used herein, the term "tropism property" refers to the pattern of transduction of a virus into one or more target cells, tissues and / or organs. For example, some AAV capsid proteins may show effective transduction of neurons but not cardiac tissue. The tropism property of AAV can be altered by modifying its capsid protein. Various methods for modifying AAV capsid proteins are known in the art. For example, in US Patent US9186419, two or more different AAV capsid protein sequences are scrambled and shuffled to integrate portions of two or more capsid protein sequences to obtain AAV capsid proteins with modified tropism properties. rAAVs containing scrambled capsid proteins are known as chimeric or mosaic AAVs.

[0018] In some embodiments, the rAAV of the present invention is a mosaic AAV that comprises a capsid protein that has stronger hepatocyte tropism in an organism than the corresponding AAV of other serotypes. In some embodiments, the organism refers to a mammal. In certain embodiments, the organism is a human. As will be understood by those skilled in the art, AAV8 is a known serotype that has stronger hepatocyte tropism. In some embodiments, the rAAV described herein comprises an AAV capsid protein that has superior hepatocyte tropism compared to rAAV that comprises an AAV8 capsid protein. In one embodiment, the rAAV is a mosaic AAV that comprises a capsid protein (AAVX) having an amino acid sequence as shown in SEQ ID NO:1. In certain embodiments, the rAAV comprises a capsid protein (AAVX) having an amino acid sequence as shown in SEQ ID NO:1 and at least one ITR from the AAV2 serotype, and is designated as AAV2 / X.

[0019] AAVs are known to elicit immune responses in humans. Consistent with the disclosed embodiments, rAAVs containing a mosaic capsid protein are less immunogenic in an organism than corresponding rAAVs containing a different serotype capsid protein. In some embodiments, the organism is a mammal. In certain embodiments, the organism is a human. As used herein, the term "immunogenicity" generally refers to the strength of an immune response elicited by an antigen. The term "immune response" generally refers to the process by which an organism recognizes and resists bacteria, viruses, and other biotic and non-biotic substances that may be foreign or harmful. These substances are commonly referred to as "antigens." Two types of immune responses are found in humans: innate and adaptive. Innate immune responses are not specific to a particular antigen, whereas adaptive immune responses arise following antigen exposure. Host immune responses during rAAV administration may adversely affect long-term expression of transgenes in humans, reduce the efficacy of delivered therapeutic agents, and / or cause undesirable side effects. Over 90% of the population exposed to wild-type AAV may induce a pre-existing immune response that inhibits clinical efficacy following administration of certain serotypes of rAAV. For example, approximately 70% of the world's population has neutralizing antibodies (NAbs) against AAV1 and AAV2 in the circulatory system. As will be understood by those skilled in the art, the term "neutralizing antibodies" generally refers to antibodies produced by the adaptive immune response that specifically bind to surface structures of infectious particles and prevent them from interacting with host cells, thereby allowing cells to defend against pathogens and infectious particles. In some embodiments, the rAAVs described herein exhibit less immunogenicity than corresponding rAAVs that include capsid proteins from other serotypes. In certain embodiments, the rAAVs described herein exhibit less immunogenicity than corresponding rAAVs that include AAV8 capsid proteins. In some embodiments, the rAAVs include a capsid protein having an amino acid sequence as set forth in SEQ ID NO:1. In some embodiments, the capsid protein having the amino acid sequence set forth in SEQ ID NO:1 is encoded by a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence similarity to SEQ ID NO:10.In some embodiments, the capsid protein having the amino acid sequence set forth in SEQ ID NO:1 is encoded by the polynucleotide sequence set forth in SEQ ID NO: 10. In certain embodiments, the rAAV is AAV2 / X (comprising a capsid protein having the amino acid sequence set forth in SEQ ID NO:1 and the ITRs from AAV2).

[0020] In some aspects, rAAVs comprising an expression cassette are included herein. In some embodiments, the expression cassette may comprise at least a portion of the genome of a wild-type AAV. In certain embodiments, the expression cassette may comprise at least one polynucleotide sequence encoding a therapeutic agent. The term "expression cassette" generally refers to a nucleic acid sequence comprising at least one polynucleotide sequence encoding a therapeutic agent, components required for expression of the therapeutic agent, and other nucleic acid sequences. The therapeutic agent is useful for treating illnesses or diseases, including liver disease. Non-limiting examples of liver disease include Fabry disease, hepatitis B, hemophilia A, hemophilia B, Crigler-Najjar, Wilson disease, OTC deficiency (ornithine carbamoylase deficiency), glycogen storage disease type Ia (GSDIa), citrullinemia type I, methylmalonic acidemia, and other diseases. In certain embodiments, the liver disease is hepatitis B. In another embodiment, the liver disease is Fabry disease.

[0021] For example, the therapeutic agent may comprise a polypeptide, peptide, or nucleic acid. In some embodiments, the therapeutic agent is an antibody or antigen-binding fragment thereof, a therapeutic peptide, or shRNA. In some embodiments, the therapeutic agent is an shRNA that targets the HBV genome. In some embodiments, the shRNA has the sequence set forth in SEQ ID NO:3. In another embodiment, the therapeutic agent is GLA, which is useful for treating Fabry disease. In some embodiments, GLA has the amino acid sequence set forth in SEQ ID NO:2.

[0022] RNA interference (RNAi) is a biological process that is ubiquitous in animals, plants, fungi, and other organisms. RNAi controls gene expression in organisms by regulating messenger RNA (mRNA). During this process, double-stranded RNA (dsRNA) is cleaved into small fragments of approximately 20-25 nucleotides in length by an enzyme called Dicer. These fragments, called small interfering RNAs (siRNAs), sometimes also called short interfering RNAs or silencing RNAs, are double-stranded RNAs that are assembled into the RNA-induced silencing complex (RISC), a protein complex that belongs to a family of proteins that includes Argonaute. After binding in this way, one strand of the dsRNA is removed, allowing the remaining strand to bind to the mRNA sequence via Watson-Crick base pairing. This binding allows the Argonaute protein to cleave or destroy the target mRNA or to recruit other mRNA regulators. Short hairpin RNA (shRNA) is an artificial RNA molecule that contains a stem region in which the sense and antisense strands are complementary paired and can form a hairpin structure connected by a loop structure formed by unpaired nucleotides. shRNA also contains two inverted repeat sequences. After shRNA enters a cell, it is rewound into sense and antisense strands by the host cell's RNA helicase. The antisense RNA strand binds to RISC, which recognizes and interacts with mRNA that contains a sequence complementary to the antisense strand. Subsequently, cleavage and degradation of the mRNA caused by RISC results in downregulation of the target gene. Due to the characteristics such as specificity, efficacy and heritability of the gene sequence, shRNA is widely used in scientific research and clinical practice. There are various commonly used methods for introducing shRNA into cells, such as direct delivery of plasmids and introduction by viral or bacterial vectors. In vivo expression of shRNA via plasmid or viral vector is superior to direct synthesis of siRNA.After cloning the dsRNA sequence corresponding to the shRNA into a plasmid or viral vector containing a suitable promoter, cells are transfected with the plasmid or infected with the virus, and the shRNA of interest is transcribed under the control of the promoter. AAV is a virus commonly used to deliver shRNA. Although AAV is commonly used as a vector to deliver shRNA to cells, various aspects of AAV vectors for shRNA delivery need to be optimized. For example, there is still a need to develop AAV with good hepatocyte tropism and / or low immunogenicity in humans. In addition, short shRNA sequences generally result in low packaging yields in vitro and low transgene expression levels in the host organism. Therefore, there is a need to optimize packaging efficiency and transgene expression levels.

[0023] According to some embodiments, the expression cassette may also include at least one filler sequence. As used herein, the term "filler sequence" generally refers to a nucleic acid sequence other than at least one polynucleotide encoding a therapeutic agent and components necessary for transcription and expression of said therapeutic agent. In some embodiments, the length of the filler sequence is selected such that the length of the expression cassette approximates the length of the wild-type AAV genome. As used herein, the term "approximate" is equivalent to the term "substantially similar." In some embodiments, the length of the expression cassette is about 3.2 kb to about 5.2 kb. In another embodiment, the length of the expression cassette is about 1.6 kb to about 2.6 kb. In certain embodiments, the length of the expression cassette may be greater than 5.2 kb or less than about 1.6 kb, depending on the characteristics of the polynucleotide sequence encoding the therapeutic agent or the application of the rAAV. For example, the filler sequence may include a non-coding sequence. The term "non-coding" generally refers to a nucleic acid sequence that does not encode a protein or other biologically active molecule. For example, the non-coding sequence may be an intron or a gene regulatory element. In certain embodiments, the non-coding sequence is a human non-coding sequence. Optionally, the human non-coding sequence is inactive or harmless, i.e. has no function or activity. Non-limiting examples of human non-coding sequences include a fragment or a combination of sequence fragments selected from the blood clotting factor IX intron sequence, or a portion of the sequence of human cosmid C346, or the HPRT-intron sequence. For example, the filling sequence may comprise the HPRT-intron 2 sequence shown in SEQ ID NO: 4. The filling sequence can be located upstream or downstream of at least one polynucleotide encoding a therapeutic agent. In a preferred embodiment, at least one polynucleotide encoding a therapeutic agent is located upstream of at least one filling sequence. For example, at least one polynucleotide encoding a therapeutic agent can encode an shRNA, and at least one filling sequence is located downstream of the shRNA.

[0024] Consistent with embodiments of the present invention, the expression cassette may further comprise a promoter located upstream of at least one polynucleotide sequence encoding a therapeutic agent and at least one filler sequence (if present). As will be appreciated by those skilled in the art, the promoter may be any type of promoter, such as a constitutive promoter or an inducible promoter, depending on the application of the rAAV employed. In some embodiments, the promoter is an RNA polymerase type II promoter or an RNA polymerase type III promoter. Examples of promoters include, but are not limited to, LP1 promoter, ApoE / hAAT promoter, DC172 promoter, DC190 promoter, ApoA-I promoter, TBG promoter, LSP1 promoter, 7SK promoter, H1 promoter, U6 promoter, and HDIFN promoter. In a particular embodiment, the promoter is an H1 promoter comprising the sequence set forth in SEQ ID NO:9.

[0025] According to one embodiment of the present invention, the expression cassette may further comprise at least one AAV ITR. As will be understood by those skilled in the art, the term "ITR" refers to a sequence of about 145 nucleotides in length derived from the end of the wild-type AAV genome. The ITR sequence may be required for AAV replication and packaging. The at least one ITR in the rAAV disclosed herein may be of any serotype derived from AAV, such as clade AF, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or hybrid / chimeric forms thereof. In a particular embodiment, the ITR is derived from AAV2.

[0026] In some disclosed embodiments, the rAAV comprises a single-stranded expression cassette. In another embodiment, the rAAV comprises a double-stranded or self-complementary (scAAV) expression cassette. The scAAV vector genome comprises DNA strands that form double-stranded DNA by intramolecular annealing after the virus is uncoated in the target cell. By skipping second strand synthesis, the scAAV is rapidly expressed in the target cell. In some embodiments, the rAAV comprises a single-stranded expression cassette with a length of about 3.2 kb to about 5.2 kb. In another embodiment, the rAAV comprises a double-stranded expression cassette with a length of about 1.6 kb to about 2.6 kb. In some embodiments herein, the expression cassette further comprises at least one reporter sequence located downstream of the promoter sequence. Examples of reporters include Gaussia luciferase and fluorescent proteins such as EGFP.

[0027] Some aspects of the present specification relate to rAAV that increases expression of therapeutic agents in host cells or organisms compared to the corresponding rAAV of another serotype. Alternatively or additionally, the rAAV of the present invention shows higher packaging yield than the corresponding rAAV of another serotype. In some embodiments, the corresponding rAAV is AAV8 serotype. Methods for detecting gene expression are well known in the art. Exemplary methods include, but are not limited to, quantitative polymerase chain reaction (qPCR) using reporter genes, Western blot, Northern blot, and fluorescence microscopy.

[0028] In one embodiment, the rAAV comprises a capsid protein comprising the amino acid sequence set forth in SEQ ID NO:1, and an expression cassette comprising two ITRs from the AAV2 serotype, a promoter from 5' to 3', a polynucleotide encoding the shRNA, and a human non-coding filling sequence. In some embodiments, the shRNA targets the Hepatitis B Virus (HBV) genome. In a particular embodiment, the polynucleotide encoding the shRNA comprises the sequence set forth in SEQ ID NO:3. In some embodiments, the filling sequence comprises the sequence set forth in SEQ ID NO:4. In a particular embodiment, the promoter comprises the sequence set forth in SEQ ID NO:9. In some embodiments, the expression cassette comprises the sequence set forth in SEQ ID NO:5. In another particular embodiment, the rAAV comprises a capsid protein comprising the amino acid sequence set forth in SEQ ID NO:1, and an expression cassette comprising two ITRs from the AAV2 serotype, a promoter from 5' to 3', and a GLA. In another embodiment, the amino acid sequence of the GLA comprises SEQ ID NO:2. In one embodiment, the expression cassette comprises the sequence set forth in SEQ ID NO:6-7. Virus packaging

[0029] Aspects of the present specification include rAAV plasmid systems, cells for packaging rAAV disclosed herein, and methods for packaging rAAV disclosed herein. As used herein, the terms "virus packaging" and "virus production" are used interchangeably. Various methods for producing rAAV are known in the art. The term "packaging system" generally includes a plasmid containing an expression cassette that includes a polynucleotide encoding a gene of interest, a plasmid containing polynucleotides encoding AAV structural and nonstructural proteins, and other components that aid in rAAV production. In some embodiments, the packaging system includes a helper virus. rAAV is a replication-defective virus, i.e., it is incapable of replicating itself. The helper virus enables the replication of rAAV by providing components that support the replication of rAAV. Examples of helper viruses include, but are not limited to, adenovirus (Ad) and herpes simplex virus (HSV). For example, a plasmid containing a polynucleotide encoding a gene of interest, the rep and cap genes of AAV can be transfected into cells that already contain Ad. The cells are then maintained for rAAV production before obtaining rAAV. In some embodiments, the packaging system may comprise a two-plasmid AAV packaging system. For example, the expression cassette can be cloned into one plasmid, and the rep, cap and helper virus genes can be cloned into a second plasmid. As used herein, the term "helper virus genes" refers to all the DNA sequences of the helper virus necessary for AAV production. This plasmid is transfected into cells suitable for rAAV production. In another embodiment, the packaging system may comprise a three-plasmid AAV packaging system.For example, an expression cassette can be cloned into a first plasmid, the AAV rep and cap genes can be cloned into a second plasmid, and the helper virus genes can be cloned into a third plasmid. The three plasmids are transfected into a cell expression system for the production of rAAV. In other embodiments, packaging systems including baculovirus are also encompassed. Baculoviruses are pathogens that infect insects and other arthropods. For example, the expression cassette, the AAV rep and cap genes can be cloned into a baculovirus plasmid. These plasmids are then transfected into insect cells, such as Sf9 cells, where baculovirus is produced. Baculovirus is then used to infect insect cells, such as Sf9 cells, where rAAV is produced. Baculovirus packaging systems have many advantages, including ease of scale-up and the ability to grow insect cells in serum-free medium.

[0030] Each aspect of the present specification includes a cell that includes the AAV packaging plasmid system described herein. As will be appreciated by those skilled in the art, the AAV packaging plasmid system can be used to transfect any cell line suitable for the production of rAAV. In some embodiments, the cell includes bacterial cells, mammalian cells, yeast cells, and insect cells. The cell can be a suspension cell or an adherent cell. Exemplary cells include, but are not limited to, E. coli cells, HEK293 cells, HEK293T cells, HEK293A cells, HEK293S cells, HEK293FT cells, HEK293F cells, HEK293H cells, HEK293H cells, HeLa cells, SF9 cells, SF21 cells, SF900 cells, and BHK cells.

[0031] The disclosed embodiments also include methods of producing rAAV as described herein. In some embodiments, the methods may include introducing a packaging plasmid system into a cell suitable for rAAV production, culturing the cell under appropriate conditions, obtaining the produced rAAV, and optionally purifying the rAAV. Methods of purifying rAAV are well known in the art. For example, rAAV can be purified using chromatography. As used herein, "chromatography" refers to various methods known in the art for specifically separating one or more components from a mixture. These methods include, but are not limited to, affinity chromatography, ion exchange chromatography, and size exclusion chromatography, which are known in the art.

[0032] Aspects of the invention also include isolated and engineered cells that contain the rAAV disclosed herein. In some embodiments, the engineered cells are animal cells. In some embodiments, the engineered cells are mammalian cells. In one embodiment, the engineered cells are human cells. Compositions and Disease Treatment

[0033] Aspects of the invention include compositions comprising the rAAV disclosed herein. The terms "composition" and "formulation" are used interchangeably herein. In some embodiments, the composition is a therapeutic composition. In some embodiments, the composition comprising the rAAV may further comprise one or more additional therapeutic agents. In certain embodiments, the composition comprising the rAAV may further comprise one or more pharma- ceutically acceptable excipients and / or diluents. Although the compositions provided herein primarily refer to compositions suitable for administration to humans, one of skill in the art will appreciate that such compositions are generally suitable for administration to any other animal. Formulations of the invention may include, but are not limited to, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, rAAV-infected cells, and combinations thereof.

[0034] The compositions disclosed herein can be prepared using any method known in the art. In some embodiments, the compositions of the invention are aqueous formulations (i.e., formulations that include water). In certain embodiments, the formulations of the invention include water, sterile water, or water for injection (WFI). In some embodiments, the rAAV may be formulated in PBS. In certain embodiments, the rAAV formulation may include a buffer system. Illustrative examples of buffer systems include, but are not limited to, buffers that include phosphate, Tris, and / or histidine.

[0035] According to some embodiments herein, the compositions disclosed herein may include one or more excipients and / or diluents. As will be appreciated by those skilled in the art, the presence of an excipient and / or diluent may provide certain advantages, such as (1) increased stability, (2) increased transfection or transduction of cells, (3) sustained or delayed release of the therapeutic agent encoded by the transgene, (4) altered biodistribution (e.g., targeting the virus to specific tissues or cell types), (5) increased translation of the protein encoded by the transgene, (6) altered release profile of the protein encoded by the transgene, and / or (7) regulatable expression of the transgene herein. Excipients used herein include, but are not limited to, any and / or all solvents, dispersion media or other liquid vehicles, dispersions or suspensions, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, suitable for the particular dosage form desired. In some embodiments, the compositions may include surfactants, including anionic, zwitterionic, or nonionic surfactants. Surfactants may aid in controlling shear forces in suspension cultures.

[0036] Additionally, some aspects of the invention include compositions that include various concentrations of rAAV, which can be optimized depending on the characteristics of the formulation and its application. For example, the concentration of rAAV particles can be about 1×10 6 VG (vector genome) / mL ~ approx. 1 x 10 18In certain embodiments, the concentration of rAAV particles in the formulation can be between about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×10 11 , 2.1×10 11 , 2.2×10 11 , 2.3×10 11 , 2.4×10 11 , 2.5×10 11 , 2.6×1011 、2.7×10 11 、2.8×10 11 、2.9×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、7.1×10 11 、7.2×10 11 、7.3×10 11 、7.4×10 11 、7.5×10 11 、7.6×10 11 、7.7×10 11 、7.8×10 11 、7.9×10 11 、8×10 11 、9×10 11 、1×10 12 、1.1×10 12 、1.2×10 12 、1.3×10 12 、1.4×10 12 、1.5×10 12 、1.6×10 12 、1.7×10 12 、1.8×10 12 、1.9×10 12 、2×10 12 、2.1×10 12 、2.2×10 12 、2.3×10 12 、2.4×10 12 、2.5×10 12 、2.6×10 12 、2.7×10 12 、2.8×10 12 、2.9×10 12 、3×10 12 、4×10 12 、4.1×10 12 、4.2×10 12 、4.3×10 12 、4.4×10 12 、4.5×10 12 、4.6×10 12 、4.7×10 12 、4.8×10 12 、4.9×10 12 、5×1012 、6×10 12 、7×10 12 、7.1×10 12 、7.2×10 12 、7.3×10 12 、7.4×10 12 、7.5×10 12 、7.6×10 12 、7.7×10 12 、7.8×10 12 、7.9×10 12 、8×10 12 、8.1×10 12 、8.2×10 12 、8.3×10 12 、8.4×10 12 、8.5×10 12 、8.6×10 12 、8.7×10 12 、8.8 ×10 12 、8.9×10 12 、9×10 12 、1×10 13 、1.1×10 13 、1.2×10 13 、1.3×10 13 、1.4×10 13 、1.5×10 13 、1.6×10 13 、1.7×10 13 、1.8×10 13 、1.9×10 13 、2×10 13 、2.1×10 13 、2.2×10 13 、2.3×10 13 、2.4×10 13 、2.5×10 13 、2.6×10 13 、2.7×10 13 、2.8×10 13 、2.9×10 13 、3×10 13 、3.1×10 13 、3.2×10 13 、3.3×10 13 、3.4×10 13 、3.5×10 13 、3.6×10 13 、3.7×10 13, 3.8×10 13 , 3.9×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 6.7×10 13 , 7×10 13 , 8×10 13 , 9×10 13 , 1×10 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , 9×10 14 , 1×10 15 , 2×10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , 9×10 15 , 1×10 16 , 2×10 16 , 3×10 16 , 4×10 16 , 5×10 16 , 6×10 16 , 7×10 16 , 8×10 16 , 9×10 16 , 1×10 17 , 2×10 17 , 3×10 17 , 4×10 17 , 5×10 17 , 6×10 17 , 7×10 17 , 8×10 17 , 9×10 17 , or 1 × 10 18 VG / mL is fine.

[0037] In some embodiments, the concentration of rAAV in the composition is about 1×10 6 VG / mL ~ approx. 1×10 18 In certain embodiments, the concentration of the composition in the delivery agent may be between about 1×106 、2×10 6 、3×10 6 、4×10 6 、5×10 6 、6×10 6 、7×10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、1.1×10 12、1.2×10 12 、1.3×10 12 、1.4×10 12 、1.5×10 12 、1.6×10 12 、1.7×10 12 、1.8×10 12 、1.9×10 12 、2×10 12 、2.1×10 12 、2.2×10 12 、2.3×10 12 、2.4×10 12 、2.5×10 12 、2.6×10 12 、2.7×10 12 、2.8×10 12 、2.9×10 12 、3×10 12 、3.1×10 12 、3.2×10 12 、3.3×10 12 、3.4×10 12 、3.5×10 12 、3.6×10 12 、3.7×10 12 、3.8×10 12 、3.9×10 12 、4×10 12 、4.1×10 12 、4.2×10 12 、4.3×10 12 、4.4×10 12 、4.5×10 12 、4.6×10 12 、4.7×10 12 、4.8×10 12 、4.9×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12 、1×10 13 、2×10 13 、2.1×10 13 、2.2×10 13 、2.3×10 13 、2.4×10 13 、2.5×10 13 、2.6×10 13, 2.7×10 13 , 2.8×10 13 , 2.9×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 6.7×10 13 , 7×10 13 , 8×10 13 , 9×10 13 , 1×10 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , 9×10 14 , 1×10 15 , 2×10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , 9×10 15 , 1×10 16 , 2×10 16 , 3×10 16 , 4×10 16 , 5×10 16 , 6×10 16 , 7×10 16 , 8×10 16 , 9×10 16 , 1×10 17 , 2×10 17 , 3×10 17 , 4×10 17 , 5×10 17 , 6×10 17 , 7×10 17 , 8×10 17 , 9×10 17 , or 1 × 10 18 Total VG / mL is fine.

[0038] Another aspect of the present disclosure is the total dose of rAAV in a composition, e.g., in a bottle of product or formulation for administration to a patient. In some embodiments, the total dose of rAAV in a composition is about 1×10 6 VG ~ approx. 1×10 18 In one embodiment, the total dose of rAAV in the formulation can be between about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×1011 、2.1×10 11 、2.2×10 11 、2.3×10 11 、2.4×10 11 、2.5×10 11 、2.6×10 11 、2.7×10 11 、2.8×10 11 、2.9×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、7.1×10 11 、7.2×10 11 、7.3×10 11 、7.4×10 11 、7.5×10 11 、7.6×10 11 、7.7×10 11 、7.8×10 11 、7.9×10 11 、8×10 11 、9×10 11 、1×10 12 、1.1×10 12 、1.2×10 12 、1.3×10 12 、1.4×10 12 、1.5×10 12 、1.6×10 12 、1.7×10 12 、1.8×10 12 、1.9×10 12 、2×10 12 、2.1×10 12 、2.2×10 12 、2.3×10 12 、2.4×10 12 、2.5×10 12 、2.6×10 12 、2.7×10 12 、2.8×10 12 、2.9×10 12 、3×10 12 、4×10 12 、4.1×10 12 、4.2×10 12 、4.3×10 12 、4.4×1012 、4.5×10 12 、4.6×10 12 、4.7×10 12 、4.8×10 12 、4.9×10 12 、5×10 12 、6×10 12 、7×10 12 、7.1×10 12 、7.2×10 12 、7.3×10 12 、7.4×10 12 、7.5×10 12 、7.6×10 12 、7.7×10 12 、7.8×10 12 、7.9×10 12 、8×10 12 、8.1×10 12 、8.2×10 12 、8.3×10 12 、8.4×10 12 、8.5×10 12 、8.6×10 12 、8.7×10 12 、8.8×10 12 、8.9×10 12 、9×10 12 、1×10 13 、1.1×10 13 、1.2×10 13 、1.3×10 13 、1.4×10 13 、1.5×10 13 、1.6×10 13 、1.7×10 13 、1.8×10 13 、1.9×10 13 、2×10 13 、2.1×10 13 、2.2×10 13 、2.3×10 13 、2.4×10 13 、2.5×10 13 、2.6×10 13 、2.7×10 13 、2.8×10 13 、2.9×10 13 、3×10 13 、3.1×10 13, 3.2×10 13 , 3.3×10 13 , 3.4×10 13 , 3.5×10 13 , 3.6×10 13 , 3.7×10 13 , 3.8×10 13 , 3.9×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 6.7×10 13 , 7×10 13 , 8×10 13 , 9×10 13 , 1×10 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , 9×10 14 , 1×10 15 , 2×10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , 9×10 15 , 1×10 16 , 2×10 16 , 3×10 16 , 4×10 16 , 5×10 16 , 6×10 16 , 7×10 16 , 8×10 16 , 9×10 16 , 1×10 17 , 2×10 17 , 3×10 17 , 4×10 17 , 5×10 17 , 6×10 17 , 7×10 17 , 8×10 17 , 9×10 17 , or 1 × 10 18 VG is fine.

[0039] Consistent with the embodiments herein, methods of treating a disease in a patient in need thereof are also included, comprising administering to the patient a therapeutically effective amount of a rAAV or composition thereof disclosed herein. In some embodiments, the methods relate to gene therapy. As used herein, the term "gene therapy" generally refers to the delivery of a therapeutic nucleic acid to cells of a subject to treat the disease. In other embodiments, the methods relate to the use of a rAAV or composition disclosed herein in the preparation of a medicament for treating the disease in a patient. As used herein, the term "patient" may refer to a subject suffering from a disease or other illness. The patient may be a human or any other animal. In some embodiments, the disease is a liver disease. In some embodiments, the liver disease is Hepatitis B or Fabry disease.

[0040] For example, the rAAV herein can include an expression cassette comprising a polynucleotide sequence encoding an shRNA useful for treating Hepatitis B or GLA useful for treating Fabry disease. In some embodiments, administration of an rAAV comprising GLA results in higher levels of GLA expression in tissues compared to administration of a corresponding rAAV comprising AAV8 capsid protein. In other embodiments, administration of an rAAV comprising an shRNA for treating Hepatitis B results in higher inhibition of Hepatitis B surface S antigen (HBsAg), Hepatitis B E antigen (HBeAg) and / or HBV DNA compared to administration of a corresponding rAAV comprising AAV8 capsid protein. As used herein, the term "corresponding rAAV" refers to an rAAV that differs from the subject rAAV only in the capsid protein.

[0041] The rAAV or composition disclosed herein may be administered by any method known in the art. In some embodiments, the rAAV or composition may be administered by intravenous administration. In some embodiments, the rAAV or composition may be administered by infusion or injection. In some embodiments, the rAAV or composition may be administered by parenteral injection. Other methods of administration include, but are not limited to, subcutaneous injection, intravenous injection, intraperitoneal injection, and intramuscular injection. In a specific embodiment, the rAAV or composition is administered by intravenous injection. In some embodiments, the rAAV or composition may be administered in a single dose. In another embodiment, the rAAV or composition may be administered in multiple doses.

[0042] Consistent with embodiments of the present invention, a method of treating a disease in a patient includes administering a second active agent in addition to the rAAV or composition disclosed herein. In some embodiments, the second active agents may be administered simultaneously. In other embodiments, the second active agents may be administered sequentially. For example, a method of treating a patient with Hepatitis B may include additional administration of lamivudine and / or entecavir in addition to administering a pharmaceutical composition disclosed herein. Without being limited thereto, the present invention includes the following aspects. [Aspect 1] (a) an adeno-associated virus (AAV) capsid protein having the amino acid sequence set forth in SEQ ID NO:1, and (b) A recombinant adeno-associated virus (rAAV) comprising an expression cassette comprising a polynucleotide sequence encoding a therapeutic agent useful for gene therapy of liver disease. [Aspect 2] The rAAV of embodiment 1, wherein the therapeutic agent encoded by the polynucleotide sequence is an shRNA targeting alpha-galactosidase A (GLA) or the genome of Hepatitis B virus (HBV). [Aspect 3] The rAAV of aspect 1 or 2, wherein the expression cassette further comprises a promoter located upstream of the polynucleotide sequence, and / or a human non-coding filling sequence located downstream of the polynucleotide sequence. [Aspect 4] The rAAV of aspect 3, wherein the promoter is an RNA polymerase II promoter or an RNA polymerase III promoter. [Aspect 5] The rAAV of aspect 3 or 4, wherein the promoter is an LP1 promoter, an ApoE / hAAT promoter, a DC172 promoter, a DC190 promoter, an ApoA-I promoter, a TBG promoter, an LSP1 promoter, a 7SK promoter, an H1 promoter, a U6 promoter, or an HD-IFN promoter. [Aspect 6] The promoter is (i) for a polynucleotide sequence encoding GLA, the LP1 or DC172 promoter is employed; or (ii) The rAAV of any one of aspects 3 to 5, wherein an H1 promoter is employed for a polynucleotide sequence encoding an shRNA. [Aspect 7] The rAAV of any one of aspects 1 to 6, wherein the expression cassette further comprises a first AAV inverted terminal repeat (ITRs) and a second AAV ITR. [Aspect 8] The rAAV of aspect 7, wherein the first and / or second ITRs are derived from any serotype of AAV selected from the group consisting of clades A-F, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and any hybrid / chimeric forms thereof. [Aspect 9] The rAAV of aspect 7 or 8, wherein the first and / or second ITRs are derived from an AAV2 serotype. [Aspect 10] (i) GLA comprises the sequence shown in SEQ ID NO:2; or (ii) The rAAV according to any one of aspects 2 to 9, wherein a polynucleotide sequence encoding the shRNA comprises the sequence shown in SEQ ID NO:3. [Aspect 11] 11. The rAAV of any one of aspects 3 to 10, wherein the human non-coding filling sequence is a human factor IX intron sequence, a human cosmid C346 sequence, an HPRT-intron sequence, or a combination thereof. [Aspect 12] The rAAV of embodiment 11, wherein the human non-coding filling sequence is an HPRT-intron sequence comprising the sequence set forth in SEQ ID NO:4. [Aspect 13] The rAAV of embodiment 1, wherein the expression cassette comprises the sequence set forth in SEQ ID NO:5. [Aspect 14] The rAAV of embodiment 1, wherein the expression cassette comprises a sequence as set forth in SEQ ID NO: 6 or 7. [Aspect 15] The rAAV of embodiment 13 or 14, wherein the expression cassette further comprises the sequence set forth in SEQ ID NO:8. [Aspect 16] A composition comprising an rAAV according to any one of aspects 1 to 15. [Aspect 17] 17. The composition according to embodiment 16, further comprising a pharma- ceutically acceptable excipient and / or diluent. [Aspect 18] A method for treating liver disease in a patient, the method comprising administering to the patient a therapeutically effective amount of an rAAV according to any one of aspects 1 to 15, or a composition according to aspect 16 or 17. [Aspect 19] 19. The method of embodiment 18, wherein the liver disease is Fabry disease or Hepatitis B. [Aspect 20] 20. The method of aspect 18 or 19, wherein the rAAV or composition is administered intravenously. [Aspect 21] The method of any one of embodiments 18 to 20, further comprising administering a second therapeutic agent. [Aspect 22] By administration of the rAAV or composition, (i) increased levels of GLA expression in liver tissue compared to a corresponding rAAV containing an AAV2 / 8 serotype capsid protein; or (ii) The method according to any one of aspects 18 to 21, wherein the inhibitory effect against Hepatitis B surface antigen (HBsAg), Hepatitis B E antigen (HBeAg) or HBV DNA is enhanced compared to a corresponding rAAV comprising an AAV2 / 8 serotype capsid protein. [Aspect 23] The therapeutic efficacy of the rAAV is approximately 1×10 6 VG ~ approx. 1×10 18 The method of any one of embodiments 18 to 22, comprising VG. [Aspect 24] 18. Use of an rAAV according to any one of aspects 1 to 15, or a composition according to aspect 16 or 17, in the preparation of a medicament for treating liver disease in a patient. [Aspect 25] 25. The use according to aspect 24, wherein the liver disease is Fabry disease or Hepatitis B. [Aspect 26] 26. The use according to aspect 24 or 25, wherein the rAAV or composition thereof is suitable for intravenous administration. [Aspect 27] By administration of the rAAV or composition, (i) increased levels of GLA expression in liver tissue compared to a corresponding rAAV containing an AAV2 / 8 serotype capsid protein; or (ii) The use according to any one of aspects 24 to 26, which has an enhanced inhibitory effect against Hepatitis B surface antigen (HBsAg), Hepatitis B E antigen (HBeAg) or HBV DNA compared to a corresponding rAAV comprising an AAV2 / 8 serotype capsid protein. [Aspect 28] The therapeutic efficacy of the rAAV is approximately 1×10 6 VG ~ approx. 1×10 18 28. The use according to any one of embodiments 24 to 27, comprising VG. [Aspect 29] 18. The rAAV according to any one of aspects 1 to 15 or the composition according to aspect 16 or 17 for use in treating liver disease in a patient. [Aspect 30] 30. The rAAV or composition for use according to aspect 29, wherein the liver disease is Fabry disease or Hepatitis B. [Aspect 31] The rAAV or composition for use according to aspect 29 or 30, wherein the rAAV or composition is suitable for intravenous administration or intravenous injection. [Aspect 32] By administration of the rAAV or the composition, (i) increased levels of GLA expression in liver tissue compared to a corresponding rAAV containing an AAV2 / 8 serotype capsid protein; or (ii) enhanced inhibition of Hepatitis B surface antigen (HBsAg), Hepatitis B E antigen (HBeAg) or HBV DNA compared to a corresponding rAAV containing an AAV2 / 8 serotype capsid protein; 32. An rAAV or composition for use according to any one of aspects 29 to 31. [Aspect 33] The therapeutic efficacy of the rAAV is approximately 1×10 6 VG ~ approx. 1×10 18 33. A composition for use according to any one of embodiments 29 to 32, comprising VG. EXAMPLES

[0043] Example 1: Plasmid construction

[0044] Construction of pSC-DC172-Gluc, pSC-DC190-Gluc and pSC-CMV-Gluc plasmids Three sequence fragments including the DC172 promoter sequence (SEQ ID NO: 20), the DC190 promoter sequence (SEQ ID NO: 22) and the CMV promoter sequence with Xho I and Not I restriction sites at both ends were synthesized and digested with Xho I and Not I restriction enzymes separately. A Gaussia luciferase (Gluc) sequence with Not I and Xba I restriction sites at both ends was synthesized and digested with Not I and Xba I restriction enzymes. The pSC-CMV-EGFP plasmid (Figure 1A) was digested with Xho I and Xba I, and then ligated with the digested DC172 and Gluc fragments to generate the plasmid pSC-DC172-Gluc (Figure 1B). The pSC-CMV-EGFP plasmid (Figure 1A) was digested with Xho I and Xba I restriction enzymes, and then ligated with the digested DC190 and Gluc fragments to generate the plasmid pSC-DC190-Gluc (Figure 1C). The pSC-CMV-EGFP plasmid (Figure 1A) was digested with XhoI and XbaI restriction enzymes and then ligated with the digested CMV and Gluc fragments to generate the plasmid pSC-CMV-Gluc (Figure 1D). These plasmids were used to generate self-complementary double-stranded AAV viral vectors (scAAVs).

[0045] Construction of pSNAV2.0-DC172-GLA, pSNAV2.0-DC172-GLA-wpre, pSNAV2.0-LP1-GLA and pSNAV2.0-LP1-GLA-wpre plasmids Three sequence fragments containing LP1 promoter (SEQ ID NO: 21) with Xho I and Not I restriction sites at both ends, α-galactosidase (GLA, GeneBank NM_000169.2) with Not I and Sal I restriction sites at both ends, and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE / wpre, SEQ ID NO: 8) with Sal I restriction sites at both ends were synthesized and then digested with the respective restriction enzymes. The pSNAV2.0-EGFP plasmid (Figure 1E) was digested with Xho I and Sal I, and then ligated with DC172 digested with Xho I and Not I and GLA sequence fragment digested with Not I and Sal I to generate pSNAV2.0-DC172-GLA plasmid (Figure 1F). pSNAV2.0-DC172-GLA was digested with Sal I and then ligated with the digested WPRE fragment to generate the pSNAV2.0-DC172-GLA-wpre plasmid (Figure 1G). pSNAV2.0-EGFP was digested with Xho I and Sal I and then ligated with the Xho I and Not I digested LP1 and the Not I and Sal I digested GLA fragment to generate the plasmid pSNAV2.0-LP1-GLA (Figure 1H). pSNAV2.0-LP1-GLA was digested with Sal I and ligated with the digested WPRE fragment to generate the plasmid pSNAV2.0-LP1-GLA-wpre (Figure 1I). These plasmids were used to generate single-stranded AAV viral vectors (ssAAVs).

[0046] Construction of pSC-H1-shRNA-intron2 plasmid We constructed the plasmid pSC-CMV-EGFP, a shuttle vector used for the production of self-complementary double-stranded AAV. The plasmid preserves the ITRs from AAV2. This vector was digested with Bgl II and Xho I restriction enzymes, and then ligated with a DNA sequence containing the H1 promoter (SEQ ID NO: 9) and a polynucleotide sequence encoding an shRNA targeting HBV (SEQ ID NO: 3), which was digested with Bgl II and Xho I, to replace the CMV-EGFP sequence in this vector, forming a new plasmid, pSC-H1-shRNA (Figure 2A). Intron2 from HPRT-intron (GenBank: M26434.1, positions 1846-3487) was synthesized, digested with Bgl II and Hind III restriction enzymes, and inserted into the pSC-H1-shRNA vector, which was also digested with Bgl II and Hind III restriction enzymes, to generate the pSC-H1-shRNA-intron2 plasmid (Figure 2B).

[0047] Example 2: Virus production and virus titer detection The recombinant AAV virus vectors used in this experiment were produced in HEK293 cells (obtained from ATCC) by a conventional three-plasmid packaging system (Xiao et al., Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol. 72(3): 2224(1998)). An appropriate amount of purified virus sample was added to the digestion reaction solution described in Table 1 and incubated at 37°C for 30 minutes, and then incubated at 75°C for another 10 minutes to inactivate DNase I. The treated AAV was diluted and analyzed by qPCR, and the reaction system and procedure are shown in Table 2.

[0048] [Table 1] [Table 2]

[0049] The primers used in qPCR are shown in Table 3. Viral titers measured by qPCR are shown in Table 4. [Table 3] [Table 4]

[0050] Example 3: Selection of AAV vectors 3.1 Comparison of the tropism of AAV2 / 8, AAV2 / 3B, AAV2 / 7, and AAV2 / 9 for various hepatic cell lines in vitro Six different hepatocyte cell lines (HepG2, Huh-7, 7402, 7721, Huh-6 and L-02) were infected with scAAV2 / 8-CMV-EGFP, scAAV2 / 3B-CMV-EGFP, scAAV2 / 7-CMV-EGFP and scAAV2 / 9-CMV-EGFP, respectively, and the infection efficiency was analyzed and compared by flow cytometry. As a result, as shown in Figure 3A-F, AAV2 / 3B showed the same infection efficiency as AAV2 / 8 in Hep G2 at different MOIs. At different MOIs, AAV2 / 3B had a higher infection efficiency in the Huh-7 cell line than AAV2 / 8. In the other four cell lines, AAV2 / 8 had a higher infection efficiency than AAV2 / 3B at different MOIs.

[0051] 3.2 Comparison of the tropism of AAV2 / 8 and AAV2 / X for various hepatic cell lines in vitro According to the experimental results of Example 3.1, AAV2 / 8 had the strongest tropism for hepatocytes. AAVX is a recombinant AAV containing a capsid protein generated using DNA shuffling. The tropism of AAV2 / 8 and AAV2 / X for hepatocytes was compared. scAAV2 / 8-CMV-EGFP or scAAV2 / X-CMV-EGFP was infected into five hepatocyte cell lines (Huh-6, 7402, Huh-7, HepG2 and 7721), respectively, and the infection efficiency was analyzed and compared by flow cytometry. The evaluation method adopted is to compare the difference in MOI required when two AAVs have the same infection efficiency in the same cell line.

[0052] In various hepatic cell lines, the infection efficiency of scAAV2 / X-CMV-EGFP was significantly higher than that of scAAV2 / 8-CMV-EGFP. In Huh-6 cells, the MOI of AAV2 / 8-CMV-EGFP was approximately 3 times that of AAV2 / X-CMV-EGFP to achieve the same infection efficiency. In 7402 cells, the MOI of AAV2 / 8-CMV-EGFP was approximately 10 times that of AAV2 / X-CMV-EGFP to achieve the same infection efficiency. In Huh-7 cells, the MOI of AAV2 / 8-CMV-EGFP was approximately 100-300 times that of AAV2 / X-CMV-EGFP to achieve the same infection efficiency. In HepG2 cells, the MOI of AAV2 / 8-CMV-EGFP was approximately 30-100 times that of AAV2 / X-CMV-EGFP to reach the same infection efficiency. In 7721 cells, the MOI of AAV2 / 8-CMV-EGFP was approximately 30-100 times that of AAV2 / X-CMV-EGFP to reach the same infection efficiency. The results are shown in Figure 4A-Figure 4J. These results consistently demonstrated that the infection efficiency of AAV2 / X was significantly improved compared to AAV2 / 8, and also demonstrated that the tropism of AAV2 / X for various hepatic cell lines was better than that of AAV2 / 8.

[0053] 3.3 Comparison of the tropism of AAV2 / 8 and AAV2 / X for human primary hepatocytes The tropism of AAV2 / 8 and AAV2 / X for primary hepatocytes from human HBV patients was further evaluated by infection assay. Primary hepatocytes were isolated from five hepatocellular carcinoma patients (HCC307N1, HCC061A2, HCC213F1, HCC893D1, and HCC554A4, shown in Table 5). The cells were cultured and infected with scAAV2 / 8-CMV-EGFP or scAAV2 / X-CMV-EGFP. For HCC307N1 cells, scAAV2 / 8-CMV-EGFP or scAAV2 / X-CMV-EGFP was used at an MOI of 5000, 15000, 50000, 150000, or 500000; for HCC061A2 cells, scAAV2 / 8-CMV-EGFP was used at an MOI of 5000, 15000, 50000, 150000, or 500000; For C061A2 cells, MOIs of 500, 1500, 5000, 15000, or 50000 were used for scAAV2 / X-CMV-EGFP; for the remaining three cell lines, MOIs of 500, 1500, 5000, 15000, 50000, 150000, or 500000 were used for AAV2 / 8-CMV-EGFP or AAV2 / X-CMV-EGFP. 48 hours after infection, the infected cells were photographed under a fluorescent microscope, and the GFP positive rate and fluorescence intensity were detected by flow cytometry.

[0054] As a result, as shown in Figures 5A-5J, under the same MOI conditions, the percentage of GFP-positive cells obtained by scAAV2 / X-CMV-EGFP infection was higher and the fluorescence intensity was stronger than that obtained by scAAV2 / 8-CMV-EGFP infection. These results in primary cultured hepatocytes were consistent with the experimental results in hepatic cell lines, indicating that the tropism of AAV2 / X for primary cultured hepatocytes is superior to that of AAV2 / 8.

[0055] [Table 5]

[0056] 3.4 Comparison of the tropism of AAV2 / 8 and AAV2 / X in cynomolgus monkeys in vivo Six cynomolgus monkeys (three males and three females) were divided into two groups of three. One group of animals was intravenously injected with scAAV2 / 8-CMV-EGFP at a single dose of 1E+12vg / kg, and the other group was intravenously injected with scAAV2 / X-CMV-EGFP at a single dose of 1E+12vg / kg. All animals were euthanized 7 days after administration, and the tissues of the heart, lung, liver, brain, testis, ovary, biceps femoris, stomach, jejunum, kidney and spleen were collected, and the expression of GFP protein in each tissue was observed by fluorescence microscopy.

[0057] GFP protein expression was observed in the liver tissues of the test animals. The results are shown in Figures 6A-6N, 7A-7N and Table 6. The average number of GFP-positive cells in the liver tissues of three animals administered scAAV2 / 8-CMV-EGFP was 15.00±4.47, 8.20±2.39, and 8.00±5.83 cells / field, respectively, whereas the average number of GFP-positive cells in the liver tissues of three animals administered scAAV2 / X-CMV-EGFP was 123.40±8.02, 79.80±23.06, and 54.40±28.01 cells / field, respectively. No significant difference in GFP protein expression was observed between liver tissues from the same individual. These results showed that the number of GFP protein-positive cells was statistically significantly higher in the liver tissues of cynomolgus monkeys administered scAAV2 / X-CMV-EGFP than in the liver tissues of cynomolgus monkeys administered scAAV2 / 8-CMV-EGFP.

[0058] The results of the above in vitro and in vivo hepatotropism experiments fully demonstrated that AAV2 / X has superior hepatotropism to AAV2 / 8 both in vitro and in vivo.

[0059] [Table 6]

[0060] Example 4: Comparison of neutralizing antibody levels against different AAV serotypes in serum 4.1 Comparison of neutralizing antibodies against AAV2 / 8, AAV2 / 3B, and AAV2 / 2 in human sera In this experiment, the MOI of the virus was kept constant and the serum was serially diluted. The MOI of viral infection was 10,000. The serially diluted serum and virus were mixed in a 1:1 ratio and incubated at 37°C for 1 hour. HepG2 cells were cultured in a 24-well plate for 24 hours and then infected with Ad5 adenovirus. After 2 hours, the virus-containing medium was removed and the cells were washed with DPBS. The diluted serum and virus mixture or virus alone (with the same MOI) was then added to the cells and incubated for 48 hours. After incubation, the cells were harvested and analyzed by flow cytometry. The level of neutralizing antibodies was calculated by taking the reciprocal of the serum dilution selected from the serial dilution that achieved a cell infection efficiency equivalent to 50% of that achieved with serum-free rAAV (Lochrie MA et al. (2006) Virology 353: 68-82; Mori S et al. (2006) Jpn J Infect Dis 59: 285-293).

[0061] The experimental results are shown in Table 7. Table 7 shows the neutralizing antibody levels in human serum. In the serum samples of 13 people, the neutralizing antibody level of AAV2 / 8 was the lowest. The neutralizing antibody level of AAV2 / 3B was about 10 times higher than that of AAV2 / 8. In the serum samples of 11 people, the neutralizing antibody level of AAV2 / 2 was equal to or lower than that of AAV2 / 3B. In the serum samples of 2 people, the neutralizing antibody level of AAV2 / 2 was higher than that of AAV2 / 3B. The results showed that the neutralizing antibody level of AAV2 / 8 was significantly lower (more than 10 times lower than that of AAV2 / 3B). These results showed that AAV2 / 8 is more suitable as a gene therapy vector than AAV2 / 3B, and that AAV2 / 8 has lower immunogenicity in humans than other serotypes such as AAV2 / 3B or AAV2 / 2.

[0062] [Table 7]

[0063] 4.2 Comparison of neutralizing antibody levels against AAV2 / 8 and AAV2 / X in cynomolgus monkey sera Using the above protocol, neutralizing antibody levels against AAV2 / X and AAV2 / 8 were measured in the serum of 12 cynomolgus monkeys. The virus MOI was 2000. Serially diluted cynomolgus monkey serum was mixed with scAAV2 / X-CMV-EGFP or scAAV2 / 8-CMV-EGFP in a 1:1 ratio and incubated at 37°C for 1 h. 7402 cell line was cultured in 24-well plates. The diluted cynomolgus monkey serum and virus mixture or virus alone (with the same MOI) was then added to the cells and incubated for 48 h. After incubation, the cells were harvested and analyzed by flow cytometry.

[0064] Serum samples were diluted in four steps with a dilution range of 5-100 times. Neutralizing antibody levels less than 5 were considered negative. Twelve samples were numbered 1# to 12#, respectively. As a result, as shown in Table 8, samples 1#, 2#, and 4# had negative neutralizing antibody levels against the two AAV serotypes. Sample 7# had low neutralizing antibody levels against both serotypes. Samples 3# and 12# had high neutralizing antibody levels against scAAV2 / 8, but were negative for neutralizing antibody against scAAV2 / X. Samples 5#, 6#, 8#, 9#, 10#, and 11# had significantly lower neutralizing antibody levels against scAAV2 / X than those against scAAV2 / 8. These results indicated that the neutralizing antibody levels against AAV2 / X in cynomolgus monkeys were significantly lower than those against AAV2 / 8. These results indicated that AAV2 / X has lower immunogenicity and can serve as a more preferable drug delivery vector.

[0065] [Table 8]

[0066] 4.3 Comparison of neutralizing antibody levels against AAV2 / 8 and AAV2 / X in human serum In these experiments, cells were infected with rAAV at a fixed MOI, and then neutralizing antibody levels against AAV2 / 8 and AAV2 / X in sera from healthy humans were detected and compared. The level of neutralizing antibodies was calculated by taking the reciprocal of the serum dilution factor selected from the serial dilution that achieved 50% of the cell infection efficiency achieved by rAAV without serum. Sera from 20 normal individuals were serially diluted using the protocol described above and analyzed for neutralizing antibody levels against AAV2 / X and AAV2 / 8. 7402 cell line was cultured in 24-well plates. Human serum samples were serially diluted. scAAV2 / 8-CMV-EGFP or scAAV2 / X-CMV-EGFP at MOI 2000 was added to the serially diluted sera at a 1:1 ratio and incubated at 37°C for 1 h. Next, this mixture or scAAV2 / 8-CMV-EGFP or scAAV2 / X-CMV-EGFP at an MOI of 2000 was added to the cultured cells and incubated for 48 hours, after which the cells were harvested and analyzed by flow cytometry.

[0067] As a result, as shown in Figure 8 and Table 9, it was found that in nine serum samples (2#, 5#, 6#, 7#, 9#, 15#, 16#, 17#, 20#), the neutralizing antibody levels against AAV2 / 8 were higher than those against AAV2 / X. In four serum samples (4#, 13#, 14#, 18#), the neutralizing antibody levels against AAV2 / X were higher than those against AAV2 / 8. In three samples (1#, 3#, 8#), the neutralizing antibody levels against AAV2 / 8 were equivalent to those against AAV2 / X. In four serum samples (10#, 11#, 12#, 19#), the neutralizing antibody levels against both AAV2 / X and AAV2 / 8 were negative. These in vitro infection experiments compared AAV2 / 8 and AAV2 / X neutralizing antibody levels in 20 randomly selected human samples, and the results are representative of results from a larger population.

[0068] These results demonstrated that AAV2 / X has superior hepatotropism and lower immunogenicity in human hepatocytes than AAV2 / 8, making it more suitable as a vector for delivering therapeutic agents.

[0069] [Table 9]

[0070] Example 5: Pharmacodynamics Experiments Example 5.1: AAV2 / X for the treatment of Fabry disease Promoter Selection 129 normal mice were divided into 4 groups, including 3 dosing groups with 3 mice in each group and 1 negative control group. The animals in each dosing group were intravenously injected with scAAV2 / X-CMV-Gluc, scAAV2 / X-DC172-Gluc, or scAAV2 / X-DC190-Gluc at a dose of 3E+10vg / animal, respectively (Figure 9A). The negative control group was intravenously injected with PBS. After intravenous injection, the tail was cut and blood was collected 1, 2, 3, and 4 weeks, respectively, to analyze the expression of Gluc. For specific methods, refer to the kit instructions (such as Gaussia Luciferase Detection Kit from GaiNing BioPharmaceuticals).

[0071] The results showed that there was no expression of Gluc in the PBS negative control group. The experimental results at each detection point showed that the expression level of Gluc was the highest after infection of normal mice with rAAV carrying the DC172 promoter, followed by the expression level of Gluc by rAAV carrying the DC190 promoter or CMV promoter (Figure 10A).

[0072] Next, pSNAV2.0-DC172-GLA was constructed using the DC172 promoter, and pSNAV2.0-LP1-GLA was constructed using the LP1 promoter (Figure 9B), generating recombinant ssAAV2 / X-DC172-GLA or ssAAV2 / X-LP1-GLA. Three groups of normal mice were administered the recombinant viruses ssAAV2 / X-DC172-GLA, ssAAV2 / X-LP1-GLA, or PBS, respectively, using the above protocol. The dose was 1E+15vg / animal. The tails were snipped and blood was collected 2, 3, 4, 5, 6, 7, and 8 weeks after injection, respectively, and GLA activity was analyzed by substrate fluorescence. Specifically, 10 μL of serum test sample was added to a 96-well fluorescent plate, 40 μL of substrate (5 mM 4-methylumbelliferone-α-D-galactoside (ACROS, 337162500) and 100 mM N-acetyl-D-galactosamine (Sigma, A2795)) was added and mixed well, and the mixture was incubated at 37°C in the dark for 1 h, after which the reaction was stopped with 0.3 M glycine-NaOH. Using different molar concentrations of 4-MU (Sigma, M1381) as a standard, the expression level was calculated from the degree of fluorescence. As a result, as shown in Figure 10B, at each time point, the GLA expression level of rAAV with the LP1 promoter in normal mice was higher than that of rAAV with the DC172 promoter in normal mice. Based on these results, the LP1 promoter was selected as the promoter to be used in the next stage of detection.

[0073] Analysis of woodchuck hepatitis virus posttranscriptional regulatory elements in gene expression. The effect of expression regulatory elements such as WPRE was studied. Recombinant AAV viruses ssAAV2 / X-DC172-GLA, ssAAV2 / X-DC172-GLA-wpre, ssAAV2 / X-LP1-GLA or ssAAV2 / X-LP1-GLA-wpre (Figure 11) were injected into four groups of normal mice, respectively. There were three animals in each experimental group, and the rAAV vector was injected into the tail vein at a dose of 3E+10vg / animal. Another three experimental animals were taken and administered PBS as a negative control group. After 1, 2, 3, 4, 5, 6, 7 and 8 weeks after injection, the tails were cut, blood was collected, and the activity of GLA was measured.

[0074] As a result, it was found that the addition of WPRE to ssAAV2 / X-DC172-GLA or ssAAV2 / X-LP1-GLA increased the expression of the foreign gene. As shown in Figures 12A to 12B, the GLA expression level of ssAAV2 / X-DC172-GLA-wpre or ssAAV2 / X-LP1-GLA-wpre was only 1.5 to 2 times that of the viral vector without WPRE.

[0075] Detection of enzyme activity in various tissues of mice infected with AAV2 / X-LP1-GLA and AAV2 / 8-LP1-GLA The relationship between administration of different rAAVs and GLA expression levels was measured. GLA-deficient model mice (Ohshima T et al. (1997) Proc Natl Acad Sci USA 94(6):2540-2544) were divided into two groups, and each group was administered ssAAV2 / X-LP1-GLA or ssAAV2 / 8-LP1-GLA at a dose of 1E+10vg / animal. Each group contained six animals, including three female mice and three male mice. Two control groups were set up in the experiment: a wild-type mouse control group and a blank model mouse (Gla- / -) control group. All the animals in the groups were sacrificed 7 days after intravenous tail injection, and serum, liver tissue, heart tissue and kidney tissue were collected using known techniques, and the samples were homogenized and centrifuged at 4°C for 30 minutes. The supernatant was collected and centrifuged again at 4°C for 10 minutes. After the final centrifugation, the supernatant was collected and the protein concentration was measured by BCA assay. GLA activity was measured by substrate fluorometric method.

[0076] The results showed that the GLA enzyme activity in serum, liver, kidney, and heart tissues of animal tissues infected with ssAAV2 / X-LP1-GLA or ssAAV2 / 8-LP1-GLA was higher than that in wild-type control mice. Furthermore, in animals infected with ssAAV2 / X-LP1-GLA, the GLA enzyme activity in the measured tissues was higher than that in animal tissues infected with sAAV2 / 8-LP1-GLA. After systemic administration of rAAV, the GLA enzyme activity in serum and liver was significantly higher than that in heart and kidney. Thus, although the amount of rAAV reaching the kidney and heart via the blood circulation was relatively small, the enzyme activity of GLA was still higher than that in wild-type. These results showed that ssAAV2 / X-LP1-GLA achieved better efficacy compared to ssAAV2 / 8-LP1-GLA. The results are shown in Figures 13A to 13D.

[0077] Analysis of the effect of AAV2 / X viral titer on GLA activity in different tissues The relationship between rAAV administration dose and GLA enzyme activity was measured by in vivo experiments. GLA-deficient model mice were divided into three groups and administered ssAAV2 / X-LP1-GLA at different doses of 5E+11vg / kg, 1.5E+11vg / kg, and 5E+10vg / kg (approximately 20g / mouse), respectively. Each dosing group contained six animals, including three male mice and three female mice. Two groups of control animals were used in the experiment, including wild-type mice and blank model mice (Gla- / -). Animals in all groups were sacrificed 7 days after intravenous tail administration, and serum, liver tissues, heart tissues, and kidney tissues were collected using known techniques. Tissues were homogenized as described above, and protein concentrations were measured by BCA assay. GLA activity in serum and tissues was measured by substrate fluorescence method.

[0078] The results showed that in serum, GLA enzyme activity gradually increased with increasing virus titer, and even at the lowest titer (i.e., 5E+10vg / kg), the level of GLA enzyme activity was higher than that of control wild-type animals. In liver, the level of GLA enzyme activity also increased with increasing virus titer, and at the lowest titer (i.e., 5E+10vg / kg), the level of GLA enzyme activity was equivalent to that of control wild-type animals. In kidney, the level of GLA enzyme activity was very low at the lowest and intermediate virus titers, but at the highest virus titer (i.e., 5E+11vg / kg), the level of GLA enzyme activity was significantly higher than that of control wild-type animals. In heart, the level of GLA enzyme activity increased with increasing virus titer, and at the intermediate virus titer of 1.5E+11vg / kg, the level of GLA enzyme activity was equivalent to that of control wild-type animals. As a result, as shown in Figures 14A to 14D, when the virus dose was less than 1.5E+11 vg / kg, the number of viruses that reached the kidney after systemic administration was relatively small, and after administration at this dose, the level of GLA enzyme activity in the kidney was lower than the level of enzyme activity in wild-type mice.

[0079] Renal damage was a hallmark of Fabry disease and was primarily caused by the accumulation of globotriaosylceramide (Gb3). The ultrastructure of mouse renal parenchyma was evaluated by electron microscopy. In untreated Fabry model mice, podocytes formed foot process fusion, Gb3 accumulated, and permeated the slits to form multivesicular bodies that disintegrated, forming slit diaphragm complexes. These changes may progress to proteinuria and glomerulosclerosis. In model mice administered a high dose (i.e., 5E+11vg / kg) of ssAAV2 / X-LP1-GLA, we observed that lipid accumulation was reduced throughout the renal parenchyma and that kidney structure returned to normal (data not shown). The ultrastructure of the kidneys of treated model mice showed a decrease in the number of lysosomes, a decrease in lysosome size, or less densely packed lysosomes, indicating that administration of rAAV at a dose of 5E+11vg / kg was able to reduce Gb3 accumulation and prevent re-accumulation of Gb3 in mice.

[0080] Example 5.2: AAV2 / X for the treatment of Hepatitis B In vitro analysis of suppression of HBV HBeAg, HBsAg, and HBV DNA by scAAV2 / X-H1-shRNA-intron2 or scAAV2 / 8-H1-shRNA-intron2 scAAV2 / X-H1-shRNA-intron2 and scAAV2 / 8-H1-shRNA-intron2 were infected into HepG2.2.15 cells at increasing multiplicity of infection (MOI). Hepatitis B virus e antigen diagnostic kit (Beijing Wantai Biopharmaceuticals Ltd.Co.), Hepatitis B virus surface antigen diagnostic kit (Beijing Wantai Biopharmaceuticals Ltd.Co.), and Hepatitis B virus nucleic acid quantitative detection kit (QIAGEN) were respectively employed to detect the levels of HBV HBeAg, HBV HBsAg, and HBV DNA in the samples. In the experiment, 1 μg / mL lamivudine (LAM) was used as a positive control. scAAV2 / X-H1-NC-intron2 (X-NC) and scAAV2 / 8-H1-NC-intron2 (8-NC) served as negative controls. The NC sequence was a sequence unrelated to HBV and did not downregulate HBV when expressed as shRNA. The NC sequence was also unrelated to the human genome and could not produce an RNAi effect on human gene expression when expressed as shRNA. Samples were taken daily for 9 days to detect the levels of HBeAg, HbsAg, and HBV DNA.

[0081] The results showed that HBsAg levels began to decrease from day 1 after infection and reached the lowest level on day 3. Such low levels were maintained until day 9. When the MOI of scAAV2 / X-H1-shRNA-intron2 was greater than 6E+2, the suppression of HBsAg levels was strongest. When the MOI of scAAV2 / X-H1-shRNA-intron2 was 6E+2, the expression level of HBsAg tended to approach 0. When the MOI of scAAV2 / 8-H1-shRNA-intron2 was greater than 2E+4, the suppression of HBsAg was strongest. When the MOI of scAAV2 / 8-H1-shRNA-intron2 was 2E+4, the expression level of HBsAg tended to approach 0. The results showed that the MOI of scAAV2 / 8-H1-shRNA-intron2 used was 30 times that of scAAV2 / X-H1-shRNA-intron2 used when the two AAVs achieved the same inhibitory effect on the expression of HBsAg. In comparison, lamivudine did not show significant inhibition of HBeAg. The results are shown in Figures 15A-15B and Tables 10 and 11.

[0082] [Table 10]

[0083] [Table 11]

[0084] The HBeAg expression level also began to decrease on day 1 after infection. Meanwhile, scAAV2 / X-H1-shRNA-intron2 showed the strongest suppression of HBeAg on day 2, and scAAV2 / 8-H1-shRNA-intron2 showed the strongest suppression of HBeAg on day 3. When the MOI of scAAV2 / X-H1-shRNA-intron2 was 2E+4, the HBeAg level tended to approach 0 and was maintained at a low level from day 3 to day 9. When the MOI of scAAV2 / 8-H1-shRNA-intron2 was 5E+5, the HBeAg level tended to approach 0 and was maintained at a low level from day 4 to day 9. The results also showed that when the two AAV serotypes achieved similar inhibitory effects on HBeAg expression levels, the MOI of scAAV2 / 8-H1-shRNA-intron2 used was 25 times that of scAAV2 / X-H1-NC-intron2 used. In comparison, lamivudine did not show significant inhibition of HBeAg. The results are shown in Figures 16A-16B and Tables 12 and 13.

[0085] [Table 12]

[0086] [Table 13]

[0087] HBV DNA results showed that the DNA levels of cells infected with scAAV2 / X-H1-shRNA-intron2 or scAAV2 / 8-H1-shRNA-intron2 began to decrease from day 1 postinfection and reached the lowest level at day 5. In cells infected with either of the two rAAV serotypes, the HBV DNA was maintained at low levels from day 6 to day 9. The lowest HBV DNA levels were observed when the MOI of scAAV2 / X-H1-shRNA-intron2 was greater than 2E+3. An MOI of scAAV2 / X-H1-shRNA-intron2 of 2E+3 led to a trend of HBV DNA levels approaching 0. The lowest HBV DNA levels were observed when the MOI of scAAV2 / 8-H1-shRNA-intron2 was greater than 2E+5. The MOI of scAAV2 / 8-H1-shRNA-intron2 of 2E+5 resulted in HBV DNA tending to approach 0. The results also showed that the MOI of scAAV2 / 8-H1-shRNA-intron2 used was 100-fold higher than that of scAAV2 / X-H1-shRNA-intron2 used when the two AAVs achieved similar inhibitory effects on HBV DNA levels. The results are shown in Figures 17A-17B and Tables 14 and 15.

[0088] [Table 14]

[0089] [Table 15]

[0090] In vivo analysis of shRNA efficacy using self-complementary AAV2 / 8 and AAV2 / X scAAV2 / X-H1-shRNA-intron2 and scAAV2 / 8-H1-shRNA-intron2 were administered to HBV transgenic mice (Beijing Vitalstar Biotechnology Co., Ltd, B6-Tg HBV / Vst; C57BL / 6-HBV) by intravenous injection, respectively. Lamivudine and entecavir were used as controls. The experimental groupings are shown in Table 16. Blood samples were collected, centrifuged, and assayed for HBsAg, HBeAg, and HBV DNA levels on days 0, 7, 14, 21, 28, 35, 56, 84, 112, 140, 168, 196, 224, and 252, respectively.

[0091] [Table 16]

[0092] As a result, the suppression of HBsAg was higher in animals administered scAAV2 / X-H1-shRNA-intron2 or scAAV2 / 8-H1-shRNA-intron2 than in the control group (Table 17). Furthermore, the reduction of HBsAg in animals administered scAAV2 / X-H1-shRNA-intron2 was 5-6 times higher than that in animals administered scAAV2 / 8-H1-shRNA-intron2 (Table 17).

[0093] Furthermore, as shown in Table 18, although scAAV2 / 8-H1-shRNA-intron2 and lamivudine had similar effects on HBV DNA levels, HBV DNA suppression was 70-fold higher in animals treated with scAAV2 / X-H1-shRNA-intron2 than in animals treated with scAAV2 / 8-H1-shRNA-intron2.

[0094] In animals administered scAAV2 / 8-H1-shRNA-intron2, HBeAg levels were below 1 at most time points, and HBeAg was undetectable in more than 50% of animals (hence, no statistics or analysis was performed on these data). scAAV2 / X-H1-shRNA-intron2 resulted in reduced HBeAg levels starting from day 7 after administration. For example, on day 56, administration of scAAV2 / X-H1-shRNA-intron2 resulted in reduced HBeAg levels to 17.4±3.6 PEIU / mL (as shown in Table 19), whereas in control animals administered DPBS, HBeAg levels were unchanged (129.0 PEIU / mL, compared to 133.9 PEIU / mL on day 0). Administration of scAAV2 / X-H1-shRNA-intron2 also resulted in a 96%, 68% and 91.6% reduction in HBsAg, HBeAg and HBV DNA levels in the liver compared to controls at the end of the experiment (Table 20).

[0095] [Table 17]

[0096] [Table 18]

[0097] [Table 19]

[0098] [Table 20]

[0099] Sequence information [Table 21] [Table 22]

Table 23

Table 24

Table 25

Table 26

Table 27

Table 28

Table 29

Table 30

Table 31

Table 32

Table 33

Claims

1. A recombinant adeno-associated virus (rAAV), comprising: (a) an adeno-associated virus (AAV) capsid protein having the amino acid sequence set forth in SEQ ID NO:1; and (b) an expression cassette comprising a polynucleotide sequence encoding a therapeutic agent useful in gene therapy of liver disease; An rAAV, wherein the therapeutic agent encoded by the polynucleotide sequence is an shRNA that targets alpha-galactosidase A (GLA) or the genome of Hepatitis B virus (HBV).

2. The rAAV of claim 1, wherein the expression cassette further comprises a promoter located upstream of the polynucleotide sequence and / or a human non-coding sequence located downstream of the polynucleotide sequence.

3. The rAAV of claim 2, wherein the promoter is an RNA polymerase II promoter or an RNA polymerase III promoter.

4. The rAAV of claim 2 or 3, wherein the promoter is an LP1 promoter, an ApoE / hAAT promoter, a DC172 promoter, a DC190 promoter, an ApoA-I promoter, a TBG promoter, an LSP1 promoter, a 7SK promoter, an H1 promoter, a U6 promoter, or an HD-IFN promoter.

5. The promoter is (i) the LP1 or DC172 promoter for the polynucleotide sequence encoding GLA; or (ii) an H1 promoter for a polynucleotide sequence encoding an shRNA; be, The rAAV according to any one of claims 2 to 4.

6. The rAAV of any one of claims 1 to 5, wherein the expression cassette further comprises a first AAV inverted terminal repeat (ITRs) and a second AAV ITR.

7. 7. The rAAV of claim 6, wherein the first and / or second ITRs are derived from any serotype of AAV selected from the group consisting of clades A-F, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and any hybrid / chimeric forms thereof.

8. The rAAV of claim 6 or 7, wherein the first and / or second ITRs are derived from the AAV2 serotype.

9. (i) GLA comprises the sequence shown in SEQ ID NO:2; or (ii) the polynucleotide sequence encoding the shRNA comprises the sequence shown in SEQ ID NO:3; The rAAV according to any one of claims 1 to 8.

10. 10. The rAAV of any one of claims 2 to 9, wherein the human non-coding filling sequence is a human factor IX intron sequence, a human cosmid C346 sequence, a HPRT-intron sequence, or a combination thereof.

11. The rAAV of claim 10, wherein the human non-coding filling sequence is an HPRT-intron sequence comprising the sequence shown in SEQ ID NO:

4.

12. The rAAV of claim 1, wherein the expression cassette comprises the sequence shown in SEQ ID NO:

5.

13. The rAAV of claim 1 , wherein the expression cassette comprises the sequence shown in SEQ ID NO: 6 or 7.

14. The rAAV of claim 12 or 13, wherein the expression cassette further comprises the sequence shown in SEQ ID NO:

8.

15. A composition comprising an rAAV according to any one of claims 1 to 14, and a pharma- ceutically acceptable excipient and / or diluent.

16. A composition for use in the treatment of liver disease in a patient, the composition comprising an rAAV according to any one of claims 1 to 14.

17. 17. The composition for use according to claim 16, wherein the liver disease is Fabry disease or Hepatitis B.

18. 18. The composition for use according to claim 16 or 17, wherein the rAAV or the composition is suitable for intravenous administration or intravenous injection.

19. 19. A composition for use according to any one of claims 16-18, further comprising a second therapeutic agent.

20. Administration of the rAAV or the composition (i) increased levels of GLA expression in liver tissue compared to a corresponding rAAV containing an AAV2 / 8 serotype capsid protein; or (ii) enhanced inhibition of Hepatitis B surface antigen (HBsAg), Hepatitis B E antigen (HBeAg) or HBV DNA compared to the corresponding rAAV comprising the AAV2 / 8 serotype capsid protein; A composition for use according to any one of claims 16 to 19.

21. The composition for use according to any one of claims 16 to 20, wherein the therapeutically effective amount of the rAAV comprises between 1x106VG and 1x1018VG.

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