Mutated adeno-associated virus capsid proteins, AAV particles containing the same, and liver-directed AAV vector gene therapy

Abstract

In a first aspect, the present invention relates to a mutated adeno-associated virus (AAV) capsid protein or fragment thereof, optionally having an insert composed of an oligopeptide with additional amino acids representing linker sequences at both ends of the oligopeptide. These mutated AAV capsid proteins are more efficient in targeting liver tissue, hepatocytes, liver cells, liver cell lines, or hepatocellular carcinoma (HCC) with partially higher specificity. Furthermore, mutated AAV particles containing the mutated AAV capsid proteins of the present invention are provided. Additionally, nucleic acids encoding the mutated AAV capsid proteins of the present invention are identified along with corresponding nucleic acid vectors, particularly plasmids. Additionally, host cells containing the nucleic acid vectors or nucleic acid molecules of the present invention are also provided. Furthermore, the use of the AAV particles, nucleic acids, or nucleic acid vectors of the present invention in the manufacture of AAV particles or in the manufacture of pharmaceuticals for gene therapy is described. Furthermore, the described proteins, particle molecules, and nucleic acid vectors for use in targeting hepatocytes and / or HCC are described. In particular, the compositions are disclosed for use in treating diseases involving liver cells or for treating HCC, in particular for use in gene therapy, e.g., for use in transferring a gene of interest into liver cells, liver tissue, or HCC.

Description

[Technical Field]

[0001] In a first aspect, the present invention relates to mutated adeno-associated virus (AAV) capsid proteins or fragments thereof having inserts composed of oligopeptides, optionally with additional amino acids representing linker sequences at both ends of the oligopeptide. These mutated AAV capsid proteins are more efficient at transferring genetic material to liver tissue, hepatocytes, liver cells, and hepatocyte cell lines or hepatocellular carcinoma (HCC), in part with improved affinity for these cell types. Furthermore, mutated AAV particles (either homogeneous or hybrid) containing the mutated AAV capsid proteins of the present invention are provided. Additionally, nucleic acids encoding the mutated AAV capsid proteins of the present invention are identified together with corresponding nucleic acid vectors, particularly plasmids. Additionally, host cells containing the nucleic acid vectors or nucleic acid molecules of the present invention are also provided. Furthermore, the use of AAV particles, nucleic acids, or nucleic acid vectors of the present invention in the manufacture of pharmaceuticals for gene therapy is also described. Furthermore, the described proteins, particle molecules, and nucleic acid vectors for use in targeting hepatocytes and / or HCC are also described. In particular, the components are disclosed for use in treating diseases involving liver cells or for treating HCC, and in particular, the components are disclosed for use in gene therapy, e.g., for use in transferring a gene of interest to liver cells, liver tissue, or HCC. [Background technology]

[0002] The liver is an essential organ with complex metabolic, immunological, and hemostatic functions. It is affected by a wide range of genetic disorders and chronic metabolic conditions (see Marcellin, P. and Kutala, BK, Liver International, 2018, 38 (S1), 2-6). Consequently, the liver has become a prime target for the development of gene therapy strategies, particularly using AAV vectors, as summarized in Baruteau, J., Journal of Inherited Metabolic Disease, 2017, 497-517.

[0003] As recently reviewed, promising results have been obtained, for example, in patients with hemophilia. See Pierce, GF and Iorio, A., Hamophilia, 1018, 24 (Suppl 6), 60-67. However, high vector doses are still required to compensate for the low efficacy of AAV vectors in human patients. While efficacy is sufficient to achieve moderate physiological levels of blood coagulation factors necessary to convert severe hemophilia phenotypes to milder forms, more effective AAV vectors for other liver diseases are urgently needed. In addition, patients develop neutralizing capsid antibodies upon the first AAV vector administration, as described by Fitzpatrick et al., Molecular Therapy: Methods & Clinical Development. Elsevier Ltd., 9 (June), 119-129. This renders re-administration of AAV vectors using the same or cross-reactive AAV capsids ineffective in improving transgene expression levels. Therefore, AAV vectors with novel capsid properties for efficient liver-directed gene transfer are of utmost importance to improve current and even future gene therapy approaches.

[0004] In addition to the diseases mentioned above, the liver is prone to the development of cancer, particularly hepatocellular carcinoma (HCC), or to become a "home" for metastases originating from tumors other than HCC. Globally, HCC is reported to be the third leading cause of cancer-related death. It is a severe cancer disease with a poor prognosis in advanced stages of HCC. To date, potentially curative treatments have been limited to surgical procedures such as tumor resection, orthotopic liver transplantation, or percutaneous radiofrequency ablation. See Waghray, A. et al., WJH, 2015, 7(8), 1020–1029. The majority of patients are diagnosed at an advanced stage of HCC, for which current treatment options are highly inadequate. Therefore, there is a strong need for novel therapeutic approaches, including AAV vector-based cancer gene therapy. AAV vectors can be used to deliver suicide genes, tumor suppressor genes, or transgenes for immunotherapy to tumor sites, as discussed, for example, in Dhungel, B. et al., 2017, 7544. To provide efficient and safe delivery of transgenes for cancer gene therapy, it is necessary to engineer novel AAV vectors that are directed to HCC target tissues and capable of efficient therapeutic transgene expression.

[0005] AAV vectors are based on adeno-associated viruses (AAVs), a nonpathogenic genus of the Parvoviridae family and the Dependoparvovirus genus. AAVs consist of a single-stranded DNA genome of approximately 4.7 kB packaged in a non-enveloped icosahedral protein capsid. It is a small (25 nm diameter) virus composed of 60 monomers that form the icosahedral capsid. These monomers are composed of viral capsid proteins (VPs) VP1, VP2, and VP3 in a 1:1:10 ratio. The 4.5 kB single-stranded coding DNA genome (plus inverted terminal repeats (ITRs)) is bounded by two genes (rep and cap) that encode nonstructural (Rep protein and assembly activator protein) and structural proteins (VP1, VP2, and VP3). The AAV genome is flanked by ITRs, which serve as packaging and replication signals. The biological characteristics of AAV infection are primarily determined by the interaction of the AAV capsid with the host cell. Specifically, the capsid interacts with host cell surface receptors that mediate virion processing as well as cell entry and intracellular trafficking. AAV has not been associated with any disease, and controversy exists as to whether it may even be tumor-protective. AAV is unique in its dependence on the presence of a helper virus-like adenovirus for progeny production. Furthermore, AAV can transduce mitotic and postmitotic tissues, demonstrating broad tissue tropism. Indeed, for in vivo applications, a major drawback of AAV vectors, particularly AAV serotype 2-based vectors, is their broad tropism. Because AAV vectors can transduce diverse cell types, transduction efficiency in distinct target organs is low, requiring the administration of higher vector doses to achieve therapeutic target cell transduction levels.

[0006] However, AAV, particularly AAV2, has gained immense popularity not only as a vector for gene therapy but also as a platform for vaccine development and as a tool in preclinical research. AAV vectors are considered the gene delivery system of choice for in vivo gene therapy. The advantageous features of AAV include its nonpathogenicity, high stability, ability to transduce both dividing and nondividing cells, long-term gene expression in postmitotic or slowly proliferating cells, and low immunogenicity. Additionally, AAV vectors lack native genomic integration activity and are therefore defined as a nonintegrating vector system. This is a major advantage over retroviral / lentiviral vectors, as it significantly reduces the risk of insertional mutagenesis. Furthermore, due to their stability, vectors can be produced to high titers and purity.

[0007] To date, 13 different natural human and non-human primate AAV serotypes and over 100 natural variants have been isolated. Most serotypes differ in terms of tropism and epitopes recognized by the immune system and have been vectorized as alternatives to the prototypic AAV vector, AAV serotype 2. For vectorization, the viral genome is replaced with a transgene cassette. The use of serotypes is simplified by pseudopackaging technology, which allows packaging of an AAV vector genome flanked by ITR2 into a non-AAV2 capsid. This allows the production of AAV vectors that differ in capsid but deliver the same vector genome. AAV vector capsids and genomes can be modified and optimized for efficient and directed gene delivery in vitro and in vivo.

[0008] Pseudotyping (pseudopackaging) is a representative example of a strategy for altering the tropism of AAV vector systems. However, it cannot redirect tropism to distinct cell types. In addition, its effectiveness in increasing the efficiency of AAV vector-mediated cell transduction is limited. Therefore, alternative strategies, simply referred to as capsid engineering techniques, have been developed to adapt host-AAV interactions, including the first step of transducing cells. As a result, nonpermissive cell types can be made susceptible to AAV transduction, and transduction efficiency can be improved in permissive cells. In addition, it is possible to redirect the in vivo tropism of viral vectors to defined cell surface structures (Buning, H. et al., Current Opinion in Pharmacology, 2015, 24, 94-104).

[0009] In genetic capsid engineering approaches focused on cell surface targeting, peptide ligands are inserted into suitable positions on the capsid. These positions must be exposed on the capsid surface to affect the infectivity of target cells. To date, peptide insertion for affinity modification has primarily been achieved by the following techniques: (i) fusion to the N-terminus of VP2 in the case of AAV2, or (ii) insertion of a peptide at the tip of variable region (VR)-VIII (insertion at amino acid position 587 and insertion at position 588), or insertion in VR-IV (insertion at amino acid position 453), as described in Buning et al. (see above); and Buning and Svrivastava, Mol Ther Methods Clin Dev, 2019, 12, 248-265. For example, EP2158211B1 identifies the insertion of structural proteins 4 to 13 amino acids long that represent ligands mediating cell targeting. Additionally, approaches have been taken to replace specific amino acids present in the capsid protein.

[0010] Because suitable peptide ligands expected to mediate target cell transduction are unknown for many therapeutically interesting target cells or tissues, high-throughput screening of AAV peptide display libraries has been described. For example, libraries are disclosed in Muller, OJ et al., Nature Biotechnology, 2003, 21(9), 1040-1046 and Perabo, L. et al., Molecular Therapy, 2003, 8(1), 151-157. The AAV peptide display library established by Perabo et al. consists of more than four E6 capsid variants. The library was generated by inserting random 7-mer peptides into amino acid position 587 (VP1 numbering) of the AAV2 capsid protein. The random peptides have linker sequences at the ends to facilitate optimal display. This library is described, for example, in EP1456383B1 and also in EP2363487A2. Selection of this library on target cells has been used to identify AAV capsid variants with novel and improved transduction properties.

[0011] US2002 / 0192823A1 describes AAV capsid variants and methods for deriving said AAV capsid variants from shuffled AAV capsid libraries based on AAV2, AAV8, and AAV9.

[0012] Further strategies include attaching foreign molecules to the outer surface of the vector, which can mediate specific interactions with cellular receptors expressed on the membrane of target cell types.Chemical modification of amino acids present on the surface or exterior of the capsid surface has been discussed.These modifications include, for example, the attachment of biotin to allow the coupling of additional components via streptavidin.Furthermore, the introduction of non-natural moieties has been disclosed.For example, WO2012 / 149160A2 describes a strategy for modifying viral capsid proteins based on the introduction of non-natural amino acid moieties, which ultimately allows the introduction of additional groups. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] US2002 / 0192823A1 [Patent Document 2] WO2012 / 149160A2 [Non-patent literature]

[0014] [Non-Patent Document 1] Marcellin, P. and Kutala, B.K., Liver International, 2018, 38(S1), 2–6 [Non-patent document 2] Baruteau, J., Journal of Inherited Metabolic Disease, 2017, 497~517 [Non-patent document 3] Pierce, G.F. and Iorio, A., Hamophilia, 1018, 24(Suppl 6), 60–67 [Non-patent document 4] Fitzpatrick et al., Molecular Therapy: Methods & Clinical Development. Elsevier Ltd., 9(June), 119–129 [Non-patent document 5] Waghray, A. et al., WJH, 2015, 7(8), 1020~1029 [Non-patent document 6] Dhungel, B. et al., 2017, 7544 [Non-Patent Document 7] Buning, H. et al., Current Opinion in Pharmacology, 2015, 24, 94~104 [Non-patent document 8] Buning and Svrivastava, Mol Ther Methods Clin Dev, 2019, 12, 248–265 [Non-Patent Document 9] Muller, OJ et al., Nature Biotechnology, 2003, 21 (9), 1040-1046 [Non-Patent Document 10] Perabo, L. et al., Molecular Therapy, 2003, 8(1), 151~157 Summary of the Invention [Problem to be solved by the invention]

[0015] As mentioned above, the main problem with AAV vector systems is the in vivo tropism as well as the transduction efficiency of specific tissue types, thus necessitating the use of high vector doses. [Means for solving the problem]

[0016] Summary of the Invention Consequently, it is an object of the present invention to provide AAV vectors and capsid proteins that overcome the problems described in the art, namely, broad tropism and low transduction efficiency.

[0017] In a first aspect, the present invention provides a mutated adeno-associated virus (AAV) capsid protein or fragment thereof, wherein an insert is inserted after at least one of the amino acids having amino acid numbers 139, 161, 261, 381, 447, 453, 459, 534, 570, 573, 584, 585, 586, 587, 588, 589 of SEQ ID NO: 2, which correspond to the capsid protein of AAV2 or the homologous capsid proteins of other AAV serotypes, i.e., AAV1, AAV3b to AAV11, and bovine AAV, and the insert is inserted after an amino acid sequence of at least 4 amino acids and at most 30 amino acids. The insert comprises an oligonucleotide, for example an oligonucleotide consisting of 5 to 12 amino acids, particularly 7 amino acids, wherein the insert may optionally contain additional amino acids representing linker sequences at both sites of the oligopeptide, which linker sequences, if present, have a size of 1 to 3 amino acids independently of each other; and the mutated AAV capsid protein or a fragment thereof or a homolog thereof relates to an AAV capsid protein or a fragment thereof that is more efficient in targeting liver tissue, hepatocytes, liver cells and liver cell lines or hepatocellular carcinoma (HCC) with higher specificity.

[0018] In a second aspect, the present invention relates to a mutated AAV particle comprising an AAV capsid protein or a fragment thereof according to the present invention. Furthermore, the present invention relates to a nucleic acid encoding an AAV viral capsid protein or a fragment thereof according to the present invention. Additionally, nucleic acid vectors, in particular plasmids, comprising the nucleic acid molecules according to the present invention, as well as host cells containing said nucleic acid vectors or said nucleic acids according to the present invention are disclosed.

[0019] Furthermore, the AAV particles of the present invention, the nucleic acid molecules of the present invention, or the nucleic acid vectors of the present invention are used in the manufacture of AAV particles or in the manufacture of medicines for gene therapy. Furthermore, the components described herein, including mutated AAV capsid proteins or fragments or homologs thereof, as well as mutated AAV particles, nucleic acid molecules, or nucleic acid vectors, are suitable for use in targeting hepatocytes and / or HCC and / or improving the transduction efficiency of AAV vectors in hepatocytes and / or HCC. Furthermore, these components are suitable for use in transferring a gene of interest into hepatocytes or HCC, particularly for use in gene therapy.

[0020] That is, the inventors have identified new variants of mutated AAV capsid proteins or fragments thereof that are more efficient in transducing liver tissue, hepatocytes, liver cells and liver cell lines, and HCC with higher specificity and / or efficiency, respectively. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows transgene expression of rAAV vectors in Balb / c mice over (A) 28 days and (B) at day 28. Animals were given 4.8E11 particles of the indicated AAV vector via tail vein injection. Seven, 15, and 28 days later, mice were analyzed for vector transduction. Specifically, animals were given 1.5 mg of D-luciferin via tail vein injection and placed in the IVIS® instrument. Five minutes later, images were taken with a 5-minute exposure. Luminance represents the luminescence signal intensity induced by firefly luciferase, measured as photons / second / cm² / steradian [p / s / cm² / sr]. Statistics: Error bars indicate SD. Two-way general ANOVA and Tukey's multiple comparison test were performed. *p<0.05; **p<0.01; ***p<0.001; ****p<0.00001. [Figure 2]Biodistribution of rAAV vector genomes determined by qPCR quantification. Total DNA was isolated from animals from the in vivo experiments described above to determine vector genome content in the liver relative to the major off-target tissues (LIV = liver; SPL = spleen; LNG = lung; HRT = heart) (Figure 1). The indicated DNA samples were analyzed by relative qPCR quantification using firefly luciferase as the target and mHPRT as the reference gene. Biodistribution is shown as the relative transgene content for each different tissue sample. Target gene content was defined as the cTarget / Reference ratio and represents the target / reference ratio calibrated by a plate-by-plate calibrator. Error bars indicate SD. The mean relative vector genome content of rAAV8 in liver tissue was set as 1. Statistics: Error bars indicate SD. The significance shown is relative to rAAV2. One-way ANOVA and Tukey's multiple comparison test: **p<0.01; ****p<0.0001. [Figure 3] Transduction of primary human and mouse hepatocytes with rMLIV1 and rMLIV3 compared with the parental rAAV2 and hepatotropic rAAV8 controls. Primary human (A) or mouse (B) hepatocytes were seeded and then transduced at a ratio of 10,000 vector particles to cells (GOI). 72 hours after transduction, cells were lysed and a luciferase assay was performed to determine the level of transduction as the mean luciferase activity. Luminescence signal was expressed in relative luminescence units (RLU). Mean RLU was normalized to protein content measured by Bradford assay. Statistics: Error bars indicate SD. One-way ANOVA and Tukey's multiple comparison test: *p ≤ 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001. [Figure 4]HCC transduction efficiency of novel AAV2-based capsid variants (rHCCM1, rHCCM2, and rHCCM3) compared with the parental rAAV2wt control. A) Representative images of in vivo and in situ imaging of luciferase activity / luminescence signal 3 days after vector injection into TGFα / c-myc HCC mice. B) Quantification of in situ luciferase activity / luminescence of tumor nodules; animals received 1.5E11 particles of the indicated vector via tail vein injection. Three days after injection, animals were prepared for in vivo imaging. Five minutes after D-luciferin injection (1 mg per animal), a series of images was taken with an IVIS® system with a 3-minute exposure time. One hour after the first measurement, animals received a second injection of D-luciferin. Five minutes later, animals were sacrificed, and the livers were isolated and then imaged with a 3-minute exposure time. Counts represent the luminescence signal intensity induced by firefly luciferase, measured as relative light units (RLU). Signal overlay images (A) were generated using a color scale ranging from 50 to 1200 counts. 100 RLU was defined as the background level. The dashed rectangle marks the background (BKG) ROI on the animal skin, and the RLU values ​​represent the background-corrected signal. The circle represents the ROI marking the tumor nodule. The dashed circle marks the background (BKG) ROI on a non-tissue area. The ROI (BKG) tag indicates the BKG-corrected total RLU value measured within the ROI, and the BKG tag indicates the total RLU value measured within the BKG ROI. [Figure 5] Transduction of human hepatoma and human liver cell lines with the indicated novel capsid variants compared to the parental rAAV2wt control. A) HepG2, B) Huh7, and C) Pop10 were transduced at a ratio of 1000 particles to cells (GOI). 24 hours after transduction, cells were lysed and luciferase assays were performed. Luciferase activity as a luminescent signal is expressed in relative luminescence units (RLU). The mean RLU of rAAV2 was set as 1. Statistics: One-way ANOVA and Tukey's multiple comparison test: *p≦0.05, **p<0.01; ***p<0.001; ****p<0.0001. [Figure 6] Transduction of human hepatoma cell lines with the indicated capsid variants compared to the parental rAAV2wt control. HepG2 were transduced with 1000 GOIs and harvested 24 hours post-transduction. Cells were lysed, and luciferase assays were performed to detect luciferase activity as a luminescent signal expressed in relative light units (RLU). The mean RLU of rAAV2 (rAAV2wt control) was set as 1. Statistics: One-way ANOVA and Tukey's multiple comparison test: *p≦0.05, **p<0.01; ***p<0.001; ****p<0.0001. [Figure 7A] VP1 amino acid sequence alignment of AAV serotypes AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, and bovine AAV. I-587 and I-453 represent preferred homologous insertion sites after amino acid positions 587 and 453 (AAV2 VP1 numbering) (bovine, consensus). [Figure 7B] VP1 amino acid sequence alignment of AAV serotypes AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, and bovine AAV. I-587 and I-453 represent preferred homologous insertion sites after amino acid positions 587 and 453 (AAV2 VP1 numbering) (bovine, consensus). [Figure 7C] VP1 amino acid sequence alignment of AAV serotypes AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, and bovine AAV. I-587 and I-453 represent preferred homologous insertion sites after amino acid positions 587 and 453 (AAV2 VP1 numbering) (bovine, consensus). [Figure 7D]VP1 amino acid sequence alignment of AAV serotypes AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, and bovine AAV. I-587 and I-453 represent preferred homologous insertion sites after amino acid positions 587 and 453 (AAV2 VP1 numbering) (bovine, consensus). DETAILED DESCRIPTION OF THE INVENTION

[0022] The present inventors have identified new mutant AAV capsid proteins or fragments thereof that are more efficient and, in part, more specific in targeting liver tissue, hepatocytes, liver cells and liver cell lines, or hepatocellular carcinoma (HCC).

[0023] The vector particle, including the capsid protein, is characterized by an insert inserted after at least one of the amino acids 139, 161, 261, 381, 447, 453, 459, 534, 570, 573, 584, 585, 586, 587, 588, and 589 of SEQ ID NO: 2, which correspond to the capsid protein of AAV or the homologous capsid proteins of other AAV serotypes, i.e., AAV1, AAV3 to AAV11, and bovine AAV, where the insert comprises an oligopeptide of at least 4 amino acids and up to 30 amino acids, for example, an oligopeptide of 5 to 12 amino acids, particularly 7 amino acids. The insert may optionally contain additional amino acids at both positions of the oligopeptide representing linker sequences, which, if present, independently have a size of 1 to 3 amino acids.

[0024] The term "fragment thereof" as used herein refers to a protein or polypeptide derived from a mutated adeno-associated virus capsid protein. Typically, the size of a fragment is at least 50%, e.g., at least 70%, 80%, 90%, e.g., 95%, of the size or length of the reference sequence.

[0025] The term "mutated AAV capsid protein" means a capsid protein containing an insert consisting of amino acids at discrete positions identified herein with reference to the sequence of SEQ ID NO: 2, which corresponds to the capsid protein VP1 of AAV2.

[0026] The term "homologous capsid protein" refers to the homologous capsid protein VP1 of other AAV serotypes, namely, AAV1 (SEQ ID NO: 1), AAV3 (SEQ ID NO: 3), AAV4 (SEQ ID NO: 4), AAV5 (SEQ ID NO: 5), AAV6 (SEQ ID NO: 6), AAV7 (SEQ ID NO: 7), AAV8 (SEQ ID NO: 8), AAV9 (SEQ ID NO: 9), AAV10 (SEQ ID NO: 10), AAV11 (SEQ ID NO: 11), and bovine AAV (SEQ ID NO: 12). That is, homologous capsid proteins of serotypes derived from other AAV serotypes. An overview is provided in Vance, MA et al., DOI: 10.5772 / 61988. Serotype alignments are provided in Figures 7A-D. That is, homologous insertion sites in other AAV serotypes corresponding to the insertion site in AAV2 can be easily determined by those skilled in the art.

[0027] The term "external surface of the capsid" refers to the outside of the capsid, where the insert is present to allow interaction with an interaction partner. The term "external surface of the capsid" is used synonymously with the term "exterior of the capsid."

[0028] The term "linker" refers to an amino acid sequence optionally present in the mutated AAV capsid protein of the present invention, which is a linking element between the oligopeptide present in the insert of the mutated AAV capsid protein and the wild-type amino acid sequence of the VP1 protein.

[0029] The term "particle" or "AAV particle" refers to either a DNA-free or DNA-containing capsid. The term "vector particle" or "vector" refers to a DNA-containing capsid. The term "AAV" refers to an adeno-associated virus or its derivatives, such as recombinant AAV vector particles, and the term "AAV wild-type particle" refers to a naturally occurring adeno-associated virus capsid that is DNA-free or DNA-containing. The term "recombinant AAV" or "recombinant vector particle" refers to an AAV in which the viral genome has been replaced with a vector genome, i.e., foreign DNA to be introduced into cells, and is also referred to as "rAAV." The term "AAV" or each of recombinant AAV vector particles and AAV wild-type particles includes at least 13 different serotypes known in the art. Human serotype 2 AAV is also referred to as AAV2, AAV2 particles, or AAV2 vector particles.

[0030] The term "targeting" refers to genetic modification of the AAV capsid to improve specificity to and / or transduction efficiency in a target tissue or cell. The term "targeting molecule" refers to a molecule that enables targeting of the AAV particles described herein to a target cell.

[0031] The term "target cell" or "target tissue," as used herein, refers to a specific cell type or tissue that represents a target defined by the targeting molecule present in the mutated AAV capsid protein or fragment thereof of the present invention, i.e., the target cell or tissue is liver tissue, hepatocytes, liver cells and liver cell lines, or hepatocellular carcinoma.

[0032] The present inventors have recognized that inserting an insert into a distinct location of the capsid protein allows for providing capsid proteins such that the mutated adeno-associated virus particles are more efficient in targeting liver tissue, hepatocytes, liver cells and liver cell lines or hepatocellular carcinoma with partially higher specificity.

[0033] In one embodiment, the mutated AAV capsid protein, fragment thereof or homolog thereof is a mutated AAV capsid protein derived from AAV types 1, 2, 3b, 4, 5, 6, 7, 8, 9, 10, 11 and bovine AAV. In one embodiment, the mutated AAV capsid protein is derived from VP1 of serotype AAV2.

[0034] In a further embodiment of the invention, the mutated AAV capsid protein, fragment thereof or homolog thereof is a protein in which an insert is located at a position after at least one of amino acids 261, 453, 534, 570, 573, 587 and 588. In a preferred embodiment, the positions at which the insert is introduced are after positions 453 and 587, respectively. That is, the insertion is made between positions 453 and 454 of SEQ ID NO:2 and / or at positions 587 and 588 of SEQ ID NO:2. In one embodiment, the insertion is located between positions 587 and 588 of SEQ ID NO:2.

[0035] The sequence of SEQ ID NO:2 corresponds to the coding sequence of the VP1 protein of AAV2. In another embodiment, the mutated AAV capsid protein, fragment thereof, or homolog thereof according to the invention is a mutated AAV capsid protein, fragment thereof, or homolog thereof comprising at least one additional mutation selected from a point mutation, an internal or terminal deletion, a second insertion, and a mutation.

[0036] For example, the additional mutations may be mutations already described in the art, or may be mutations described in EP17193742.8, which has not been disclosed.

[0037] The mutated AAV capsid proteins of the present invention were obtained by in vivo screening of the library in two different orthotopic HCC mouse models after preliminary screening for heparin-binding variants. This characteristic correlates with nonspecific binding to a wide variety of cell types expressing heparan sulfate proteoglycans (HSPGs), the primary cell surface receptor for AAV2. One HCC mouse model was generated by transplanting a mouse hepatoma cell line (Hepa129) into the livers of C3H mice (Schmitz et al., J. Hepatol. 2004, pp. 787-797). An additional mouse model represents a transgenic mouse model overexpressing TGF-α and c-myc, controlled by ZnCl2- and albumin-inducible promoters, respectively (Haupenthal J. et al., Neoplasia, 2012, 14(5), pp. 410-419).

[0038] The mutated AAV capsid proteins disclosed herein, which have insertions at predetermined positions, are characterized by presenting the introduced insert from the capsid on the outer surface of the capsid when the capsid is assembled into an AAV particle. Specifically, the inventors aimed to improve the AAV vector system based on the mutated AAV capsid proteins of the present invention in terms of the efficiency and selectivity of gene transfer into hepatocytes and their malignant counterparts, hepatocellular carcinoma cells, in vivo. The superior transduction efficiency of hepatocytes and HCC was achieved through a higher cell entry rate and more efficient vector genome accessibility for transcription. The variants described herein transduced hepatocytes and HCC more efficiently than wild-type AAV2 or other known mutated capsid strains. Additionally, the mutated AAV capsid proteins of the present invention are also discussed here for their more efficient intracellular processing compared to wild-type AAV2, and have been found to be superior in terms of expression efficiency, i.e., the number of transcripts per vector copy.

[0039] That is, the displayed insert, present on the outer surface of the capsid when it is assembled into an AAV particle, increases the efficiency of transfection, resulting in lower doses required to achieve therapeutic target cell transduction levels in hepatocytes and HCC, respectively.

[0040] In one embodiment of the present invention, the mutated AAV capsid protein, fragment thereof or homolog thereof of the present invention is a capsid protein or protein fragment or homolog thereof in which a linker sequence in the amino acid sequence contains or consists of the amino acids G, S and / or A. In a preferred embodiment, the linker consists of at least one of the amino acids A or S, and in particular has amino acids AAA at the N-terminus and AA at the C-terminus, or ASA at the N-terminus and AA at the C-terminus, at the ends of the amino acid sequence of the oligopeptide inserted at the positions identified herein.

[0041] In one embodiment, the insert does not contain any stop codons. That is, the mutated AAV capsid proteins of the present invention contain an insert followed by a wild-type amino acid after the amino acid into which the insert is introduced. In addition, the nucleic acid molecules of the present invention do not encode a stop codon that would terminate translation of the encoded peptide.

[0042] In one embodiment, the linker, particularly the linker described above, is an oligopeptide of at least 5 amino acids and up to 12 amino acids, such as 7 amino acids, as described herein.

[0043] In one embodiment, the mutated AAV capsid protein, fragment, or homolog thereof is a capsid protein, protein fragment, or homolog thereof having a size of 7 amino acids, wherein the oligonucleotide has a sequence according to any one of SEQ ID NOs: 13 to 275. In another embodiment, the mutated AAV capsid protein, fragment, or homolog thereof contains an insert having a sequence according to any one of SEQ ID NOs: 276 to 538. The sequence according to any one of SEQ ID NOs: 13 to 275 is an amino acid sequence representing an insert without any linker group, while the insert according to any one of SEQ ID NOs: 276 to 538 represents the amino acid sequence of a preferred insert to be present as an insert in a mutated AAV capsid protein according to the present invention containing linker sequences at both positions of the peptide sequence set forth in SEQ ID NOs: 13 to 275.

[0044] Thus, an embodiment of the invention is a protein having an insertion between positions 587 and 588 of SEQ ID NO: 2 with an oligonucleotide of 7 amino acids and a linker sequence of 1 to 3 amino acids on either side. Preferably, the sequence is any one of SEQ ID NOs: 13 to 21 and 276 to 284.

[0045] In another aspect of the present invention, a mutated adeno-associated virus particle, also referred to as a vector particle, is provided, which comprises the mutated adeno-associated virus capsid protein of the present invention, or a fragment thereof, or a homolog thereof.The AAV particle is more efficient in targeting liver tissue, hepatocytes, liver cells and liver cell lines or hepatocellular carcinoma, in part with higher specificity.That is, the efficiency of transduction of target cells or target tissues ex vivo or in vivo, as well as the applicability of AAV as a delivery tool, are increased by increasing the ability of the mutated AAV particle to transduce cells with higher specificity and / or efficiency.

[0046] For example, this mutated AAV particle is useful in the production of AAV particles in the manufacture of pharmaceuticals for gene therapy. For example, in the case of pharmaceuticals for gene therapy, the AAV particles of the present invention have a functional nucleic acid fragment or a nucleic acid fragment of a molecule of interest. The molecule of interest includes a nucleic acid fragment encoding the molecule of interest. Targeting liver tissue, hepatocytes, liver cells and liver cell lines, or HCC, with more efficient and partially higher specificity in targeting target cells or target tissues, allows for increased efficacy of vaccination strategies in addition to gene therapy to prevent or treat malignant tumors in the mentioned targeted cells and target tissues.

[0047] In particular, the mutated AAV particles of the present invention are suitable for use in targeting hepatocytes and / or HCC. The mutated AAV particles of the present invention are particularly useful in the transfer of a gene of interest into hepatocytes or HCC, particularly for use in gene therapy.

[0048] That is, the mutated AAV particles of the present invention are useful for expressing therapeutically effective transgenes. For example, metabolic factors such as enzymes may be expressed to overcome defects such as genetic defects. For example, in the case of HCC, expression of tumor suppressor genes, suicide genes, pro-inflammatory factors, etc. may be envisioned.

[0049] Furthermore, the mutated AAV particles are suitable for inducing immune tolerance to the transgene or antigen introduced into cells. This can occur in target cells and target tissues, particularly in the liver. Tolerance is useful for treating autoimmune diseases.

[0050] In a further aspect, the present invention relates to a nucleic acid molecule encoding a mutated AAV capsid protein according to the present invention, or a fragment thereof, or a homolog thereof. That is, the nucleic acid molecule according to the present invention is a nucleic acid molecule encoding an AAV capsid protein or a fragment thereof as defined herein, for example, an AAV capsid protein or a fragment thereof containing an oligopeptide according to any one of SEQ ID NOs: 13 to 275, or an insert according to any one of SEQ ID NOs: 276 to 538. Of course, codon optimization can be performed depending on the host cell.

[0051] A further aspect relates to a nucleic acid vector, particularly a plasmid, containing the nucleic acid molecule of the present invention. Those skilled in the art are familiar with suitable plasmids and vectors. In particular, the plasmid or vector is selected based on the host cell to be transfected. Furthermore, the nucleic acid vector may contain additional genetic AAV helper elements, such as the rep open reading frame.

[0052] In a further aspect, there is provided a host cell containing a nucleic acid vector according to the invention or a nucleic acid molecule according to the invention. The host cell may contain additional helper plasmids or nucleic acid molecules, for example containing the adenoviral helper genes E4, E2A, and VA, for recombinant AAV particle production. In one embodiment of the invention, the host cell is a producer for the production of AAV particles according to the invention.

[0053] In a further aspect, the present invention relates to a composition as described herein comprising a mutated AAV particle according to the present invention, or a nucleic acid molecule according to the present invention, or a nucleic acid vector according to the present invention, or a suitable transgene DNA component. The transgene DNA component can therefore be a known transgene DNA or a DNA encoding a suitable peptide or protein. In another embodiment, a composition comprising a mutated AAV particle according to the present invention is provided, for example, the composition is a pharmaceutical composition for use in transducing liver tissue, hepatocytes, liver cells and liver cell lines, or hepatocellular carcinoma. Furthermore, the present invention relates to the use of an AAV particle according to the present invention, a nucleic acid molecule according to the present invention, or a nucleic acid vector according to the present invention in the manufacture of an AAV particle or in the manufacture of a medicament for gene therapy.

[0054] The mutated AAV capsid proteins of the present invention, fragments or homologs thereof, the mutated AAV particles of the present invention, the nucleic acid molecules of the present invention, or the nucleic acid vectors of the present invention are particularly useful in targeting hepatocytes and / or HCC, as demonstrated herein.

[0055] Another aspect of the present invention relates to a mutated AAV capsid protein or a fragment thereof or a homolog thereof according to the present invention, a mutated AAV particle according to the present invention, a nucleic acid molecule according to the present invention, or a nucleic acid vector according to the present invention for use in treating diseases involving liver cells or for treating HCC: bleeding disorders such as hemophilia A, hemophilia B, von Willebrand disease, metabolic disorders such as alpha 1 antitrypsin deficiency, familial hypercholesterolemia, ornithine transcarbamylase deficiency, Crigler-Najjar syndrome, acute intermittent porphyria, glycogen storage disease type 1a, liver cancers such as hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma, autoimmune disorders such as multiple sclerosis, latent autoimmune diabetes, induction of tolerance to allogeneic transplants of liver, kidney, heart, stem cells, etc., lipoprotein lipase deficiency, diabetes, and ornithine transcarbamylase deficiency, and alternative liver-directed gene therapy.

[0056] That is, the components described herein, including a mutated AAV capsid protein, a fragment thereof or a homolog thereof, a mutated AAV particle, a nucleic acid molecule, or a nucleic acid vector, are suitable for use in a method of treating an individual in need thereof. Accordingly, another aspect relates to a method of treating an individual in need thereof, comprising administering to the individual a mutated AAV particle of the present invention, or a nucleic acid molecule encoding a mutated AAV viral capsid protein, a fragment thereof, or a homolog thereof, and / or a nucleic acid vector of the present invention.

[0057] Furthermore, administration can be carried out by known means: in particular, administration can be carried out by the intravenous route or by injection into the (hepatic) portal vein, or intrahepatically, or intratumorally, respectively.

[0058] In particular, the therapeutic treatment method of the present invention is a method for the therapeutic treatment of diseases involving hepatocytes or for the treatment of HCC, including bleeding disorders such as hemophilia A, hemophilia B, and von Willebrand's disease, metabolic disorders such as alpha-1 antitrypsin deficiency, familial hypercholesterolemia, ornithine transcarbamylase deficiency, Crigler-Najjar syndrome, acute intermittent porphyria, and glycogen storage disease type 1a, liver cancers such as hepatocellular carcinoma, hepatoblastoma, and cholangiocarcinoma, autoimmune disorders such as multiple sclerosis, latent autoimmune diabetes, induction of tolerance to allogeneic transplants such as liver, kidney, heart, and stem cell transplants, and liver-directed gene therapy for lipoprotein lipase deficiency, diabetes, and ornithine transcarbamylase deficiency.

[0059] In one embodiment of the invention, the method of prophylactic or therapeutic treatment is a gene therapy comprising the step of administering an AAV particle according to the invention, or an AAV particle comprising at least one of the mutated AAV capsid proteins according to the invention or fragments thereof or homologs thereof, a nucleic acid molecule according to the invention, and / or a nucleic acid vector according to the invention, for example in the form of a pharmaceutical composition. The pharmaceutical composition or medicament may contain other suitable ingredients, such as diluents, excipients or carriers.

[0060] Furthermore, methods are disclosed for the transfer of a gene of interest into hepatocytes or HCC ex vivo or in vivo, particularly by gene therapy, namely liver-directed AAV gene therapy.

[0061] The present invention is further illustrated by, but not limited to, examples. [Example]

[0062] Characterization of MLIV capsid variants Prior to AAV peptide display library selection, the library was precleaned from HSPG-binding capsid variants by heparin affinity column purification. In the library, the insert is located between positions 587 and 588 of SEQ ID NO: 2. Capsid variants accumulated in liver tissue after in vivo AAV peptide display library selection in two different mouse models were analyzed using MLIV, respectively. * The two libraries were named MLIV1 (Hepa129 transplanted) and MLIV2 (TGF-α / c-myc transgenic). Ninety-two candidate variants were identified for the transplanted mouse model and 84 for the transgenic mouse model. The most abundant variants in each of the two libraries were designated MLIV1 (MLIV2). *These vectors, named MLIV1 (SEQ ID NO: 13) and MLIV3 (MLIV) (SEQ ID NO: 14), were characterized in detail. For in vivo evaluation, MLIV1 and MLIV3 were produced as recombinant (r)AAV vectors expressing a luciferase transgene cassette. Transgene expression was controlled by the ubiquitously expressed CMV promoter. Specifically, 4.8E11 rAAV vector particles were administered to healthy mice via tail vein injection. As a control, and for comparison, mice were given rAAV2 and rAAV8 with native serotype capsids delivering the same vector genome. Seven, 14, and 28 days after injection, mice were injected with D-luciferin (1.5 mg per animal), and luciferase activity in various body regions was monitored by in vivo imaging using a camera to detect luminescent signals. Significant luciferase activity was detected in the upper abdominal and liver regions of the animals in all mouse cohorts. The novel capsid variants rMLIV1 and rMLIV3 demonstrated a significant improvement (up to 26-fold) in mouse liver transduction efficiency compared to rAAV2 (Figure 1, A and B). The mouse liver transduction efficiency of the new capsid variants was similar to that of the control vector (rAAV8) currently used in liver-directed human clinical gene therapy trials. Regarding the biodistribution of rMLIV1 and rMLIV3, as determined by qPCR analysis of the vector genome, a clear affinity for the mouse liver was observed. Specifically, the amount of vector genome detected in the liver was up to 51-fold higher than in the spleen, up to 18-fold higher than in the lungs, and 3-fold higher than in the heart for the novel capsid variants (Figure 2). In particular, the observed detargeting from the mouse spleen, one of the major off-target tissues of AAV2 (in mouse off-target tissues: rAAV2 genomic content > rMLIV capsid variant genomic content (182-fold in spleen; up to 24-fold in lung, 7-fold in heart)) (Figure 2) is an important novel feature of the variants of the present invention for in vivo gene therapy.

[0063] The non-human primate serotype rAAV8 is known to exhibit high transduction efficiency in mouse liver tissue. However, this does not correlate with the transduction efficiency achieved in human liver. In comparison, human serotype rAAV2 vectors are more efficiently taken up and processed in human hepatocytes. To investigate the efficiency of the newly developed capsid variants in human liver tissue, primary human hepatocytes were transduced with rMLIV1 and rMLIV3 (as well as controls rAAV2 and rAAV8). In addition, the same vectors were assayed in primary mouse hepatocytes (GOI of 1E4 rAAV vector particles per cell, 72 h). To quantify luciferase expression, luciferase activity in cell lysates was measured by adding luciferin followed by luminescence signal detection (Promega luciferase assay kit). Here, rAAV2 and the novel capsid variants rMLIV1 and rMLIV3 clearly outperformed rAAV8 used in human clinical trials (Nathwani 2014 doi: 10.1056 / NEJMoa 1407309; Pierce 2018, doi:10.1111 / hae.13489) (Fig. 3, A and B).

[0064] Due to their strong liver tropism across species and high liver transduction efficiency, MLIV1 and MLIV3 represent promising new developments for in vivo gene therapy. Furthermore, the significantly higher efficiency observed after ex vivo transduction in primary human hepatocytes compared with rAAV8 has now been confirmed in humanized mice, providing an excellent foundation for evolving these novel vectors into tools for liver-directed human clinical gene therapy trials.

[0065] Characterization of HCCM capsid variants Prior to AAV peptide display library selection, the library was precleaned from HSPG-binding capsid variants by heparin affinity column purification. The capsid variants that accumulated most abundantly in HCC tissues after in vivo AAV peptide display library selection in two different mouse models were identified as HCCM. * (selected in a Hepa129 transplant mouse model) and HCCM (selected in a TGF-α / c-myc transgenic mouse model). * For the selection pathway, the AAV peptide display library was pre-selected in vitro in target cells, Hepa129, prior to in vivo selection. We identified 103 candidate variants in a transplanted mouse model and 89 candidate variants in a transgenic mouse model. These capsid variants were further scored for enrichment in HCC (target) tissue compared to liver (major off-target) tissue. The best-scoring capsid variants were ranked for enrichment in HCC compared to all analyzed off-target tissues (liver, heart, spleen, skeletal muscle, kidney, and pancreas). The best-scoring capsid variants were identified as HCCM1 (SEQ ID NO: 15), HCCM2 (SEQ ID NO: 16), and HCCM3 (SEQ ID NO: 17) (HCCM library), as well as HCCM. * 1 (SEQ ID NO: 18), HCCM * 2 (SEQ ID NO: 19), HCCM * 3 (SEQ ID NO: 20), and HCCM * 4 (SEQ ID NO: 21) (HCCM * These were named the rAAV library. For characterization, they were produced as rAAV vectors expressing a luciferase reporter transgene cassette. Transgene expression was controlled by the SFFV promoter.

[0066] HCCM1, HCCM2, and HCCM3 were administered to TGF-α / c-myc mice via tail vein injection (1.5E11 rAAV vector particles per animal). For comparison, the parent AAV (a naturally occurring serotype rAAV2 vector delivering the same vector genome) was administered. Three days after injection, the mice were injected with D-luciferin (1 mg per animal), and 5 minutes later, luciferase activity in different regions of the body was measured by in vivo imaging analysis using a luminescence signal detection camera (3-minute exposure). In addition, tumor-bearing livers were isolated from all animals 5 minutes after repeated administration of luciferin, and luminescence signals were determined in situ (3-minute exposure). The mouse cohort treated exclusively with the novel rHCCM capsid variant demonstrated significant luciferase activity in the tumors, whereas in the rAAV2 cohort, the luminescence signal remained at low background levels (100 RLU) (Figure 4, A and B). The novel capsid variants rHCCM1, rHCCM2, and rHCCM3 demonstrated clear improvements in HCC transduction efficiency compared to controls.

[0067] rAAV2 is highly efficient in transducing human hepatoma and hepatic cell lines (1000 AAV vector particles of GOI per cell, 24 hours). * To estimate the efficiency of the capsid variants, several human hepatoma / liver cell lines were tested with the newly developed capsid variants rHCCM1, rHCCM2, rHCCM3, and rHCCM. * 1. rHCCM * 2. rHCCM * 3, and rHCCM * 4, as well as the parental control (rAAV2). The rAAV vector expressed Renilla luciferase under the control of the CMV promoter. Transgene expression levels were quantified by luciferase assay (Promega Renilla luciferase assay kit). The novel capsid variants rHCCM1, rHCCM2, and rHCCM3 (Figure 5, A-B) and rHCCM* 2. rHCCM * 3, and rHCCM * 4 (Fig. 6 ) demonstrated transduction efficiencies comparable to or superior to rAAV2 in these human hepatoma cell lines (Fig. 5, A and B ).

[0068] To date, HCC treatment itself, and therefore AAV-based gene therapy targeting HCC, has been limited by low efficacy. The novel HCCM capsid variant exhibited significantly improved transduction of HCC nodules after intravenous injection and is therefore a promising candidate for novel treatment strategies employing AAV vector systems. [1] A mutated adeno-associated virus (AAV) capsid protein or fragment thereof, the insert is inserted after at least one amino acid having amino acid numbers 139, 161, 261, 381, 447, 453, 459, 534, 570, 573, 584, 585, 586, 587, 588, 589 of SEQ ID NO: 2, which correspond to the capsid protein of AAV type 2 or the homologous capsid proteins of other AAV serotypes, i.e., AAV1, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and bovine AAV; the insert comprises an oligopeptide of at least 4 amino acids and at most 30 amino acids, for example an oligopeptide of 5 to 12 amino acids, particularly 7 amino acids; The insert may optionally have additional amino acids representing linker sequences at both sites of the oligopeptide, the linker sequences, if present, having a size of 1 to 3 amino acids independently from each other; The mutated AAV capsid protein or a fragment thereof or a homolog thereof is more efficient in targeting liver tissue, hepatocytes, liver cells and liver cell lines, or hepatocellular carcinoma (HCC) with higher specificity, the mutated AAV capsid protein or a fragment thereof. [2] The mutated AAV capsid protein, fragment thereof or homolog thereof according to [1], wherein the AAV is type 1, 2, 3b, 5, 6, 7, 8, 9, 10, 4, 11, or bovine. [3] A mutated AAV capsid protein, fragment thereof or homolog thereof according to [1], wherein the insert is located at at least one position following amino acids 261, 453, 534, 570, 573, 587 and 588, more preferably 453 and 587, and particularly preferably 587, of SEQ ID NO: 2. [4] A mutated AAV capsid protein, a fragment thereof, or a homolog thereof described in any one of [1] to [3], wherein the insert is inserted after amino acid 587 of SEQ ID NO: 2 and has a 7 amino acid oligonucleotide and linker sequences of 1 to 3 amino acids on both sides. [5] A mutated AAV capsid protein, a fragment thereof, or a homolog thereof according to any one of [1] to [4], wherein the insert is a sequence of SEQ ID NOs: 13 to 21 and 276 to 284. [6] A mutated AAV capsid protein, a fragment thereof or a homolog thereof described in any one of [1] to [5], which comprises at least one further mutation selected from a point mutation, an internal or terminal deletion, a second insertion and a substitution. [7] When the linker sequence is an amino acid sequence containing or consisting of the amino acids G, S and / or A, the linker particularly consists of at least one of the amino acids A or S, and particularly has an AAA amino acid at the N-terminus and an AA amino acid at the C-terminus, or an ASA at the N-terminus and an AA amino acid at the C-terminus, at the end of an oligopeptide of at least 4 and at most 30 amino acids, for example at least 5 and at most 12 amino acids, for example 7 amino acids. A mutated AAV capsid protein, fragment thereof, or homolog thereof described in any one of [1] to [6]. [8] A mutated AAV capsid protein, a fragment thereof, or a homolog thereof described in any one of [1] to [7], wherein the oligopeptide having a size of 7 amino acids is an array set forth in any one of SEQ ID NOs: 13 to 275. [9] A mutated AAV capsid protein, a fragment thereof, or a homolog thereof according to any one of [1] to [8], wherein the insert is any one of SEQ ID NOs: 276 to 538.

[10] A mutated adeno-associated virus (AAV) particle, comprising the mutated AAV capsid protein, a fragment thereof, or a homolog thereof described in any one of [1] to [9].

[11] A nucleic acid molecule encoding a mutated AAV capsid protein, a fragment thereof, or a homolog thereof according to any one of [1] to [9].

[12] A nucleic acid vector, particularly a plasmid, comprising the nucleic acid molecule described in

[11] .

[13] A host cell containing the nucleic acid vector according to

[12] or the nucleic acid molecule according to

[11] .

[14] Use of an AAV particle according to

[10] , or a nucleic acid molecule according to

[11] , or a nucleic acid vector according to

[12] in the manufacture of an AAV particle or in the manufacture of a medicament for gene therapy.

[15] A mutated AAV capsid protein, fragment or homolog thereof described in any one of [1] to [9], a mutated AAV particle described in [6], a nucleic acid molecule described in [4], a nucleic acid vector described in

[12] , or a nucleic acid vector described in

[13] for use in transducing hepatocytes and / or HCC in vivo or ex vivo.

[16]

[0033] The mutated AAV capsid protein, fragment or homolog thereof according to any one of [1] to [9], the mutated AAV particle according to

[10] , the nucleic acid molecule according to

[11] , or the nucleic acid vector according to

[12] for use in treating diseases related to liver cells: bleeding disorders such as hemophilia A, hemophilia B, von Willebrand's disease, metabolic disorders such as alpha-1 antitrypsin deficiency, familial hypercholesterolemia, ornithine transcarbamylase deficiency, Crigler-Najjar syndrome, acute intermittent porphyria, glycogen storage disease type 1a, liver cancers such as hepatocellular carcinoma, hepatoblastoma, and cholangiocarcinoma, autoimmune disorders such as multiple sclerosis, latent autoimmune diabetes, induction of tolerance to allogeneic transplants of liver, kidney, heart, stem cells, etc., lipoprotein lipase deficiency, diabetes, and ornithine transcarbamylase deficiency, and alternative liver-directed gene therapy; or treating HCC.

[17] A mutated AAV capsid protein, fragment or homolog thereof described in any one of [1] to [9], a mutated AAV particle described in

[10] , a nucleic acid molecule described in

[11] , a nucleic acid vector described in

[12] , or a nucleic acid vector described in

[13] for use in transferring a gene of interest into hepatocytes or HCC, particularly for use in gene therapy.

Claims

1. A mutated adeno-associated virus (AAV) capsid protein, a fragment thereof or a homolog thereof, an insert after amino acid number 587 of SEQ ID NO: 2, which corresponds to the capsid protein AAV2, or homologous capsid proteins AAV1, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and bovine AAV; the insert consists of a sequence selected from SEQ ID NOs: 13-21 and 276-284; The mutated AAV capsid protein, or a fragment thereof, or a homolog thereof, which specifically and efficiently targets liver tissue, hepatocytes, liver cells and liver cell lines, or hepatocellular carcinoma (HCC), is described above.

2. 2. The mutated AAV capsid protein, fragment thereof or homolog thereof of claim 1, comprising at least one further mutation selected from a point mutation, an internal or terminal deletion, a second insertion and a substitution.

3. 1. A mutated adeno-associated virus (AAV) particle comprising:

3. An AAV particle comprising a mutated AAV capsid protein, a fragment thereof or a homolog thereof according to claim 1 or 2.

4. A nucleic acid molecule encoding the mutated AAV capsid protein of claim 1 or 2, a fragment thereof or a homolog thereof.

5. A nucleic acid vector comprising the nucleic acid molecule of claim 4.

6. The nucleic acid vector of claim 5 which is a plasmid.

7. A host cell containing the nucleic acid vector of claim 5 or 6 or the nucleic acid molecule of claim 4.

8. Use of an AAV particle according to claim 3, a nucleic acid molecule according to claim 4, a nucleic acid vector according to claim 5 or 6 or a host cell according to claim 7 in the manufacture of AAV particles or in the manufacture of a medicament for gene therapy.

9. A mutated AAV capsid protein, fragment or homolog thereof described in claim 1 or 2, a mutated AAV particle described in claim 3, a nucleic acid molecule described in claim 4, a nucleic acid vector described in claim 5 or 6 or a host cell described in claim 7 for use in transducing hepatocytes and / or HCC in vivo or ex vivo.

10. A mutated AAV capsid protein, fragment or homolog thereof described in claim 1 or 2, a mutated AAV particle described in claim 3, a nucleic acid molecule described in claim 4, a nucleic acid vector described in claim 5 or 6 or a host cell described in claim 7 for use in the treatment of a disease associated with liver cells.

11. A mutated AAV capsid protein, fragment or homolog thereof described in claim 1 or 2, a mutated AAV particle described in claim 3, a nucleic acid molecule described in claim 4, a nucleic acid vector described in claim 5 or 6 or a host cell described in claim 7 for use in liver-directed gene therapy for the treatment of bleeding disorders, metabolic disorders, liver cancer, autoimmune disorders, for inducing tolerance to or replacing allogeneic transplants.

12. A mutated AAV capsid protein, fragment or homolog thereof described in claim 1 or 2, a mutated AAV particle described in claim 3, a nucleic acid molecule described in claim 4, a nucleic acid vector described in claim 5 or 6 or a host cell described in claim 7 for use in transferring a gene of interest into hepatocytes or HCC.

13. A mutated AAV capsid protein, fragment or homolog thereof described in claim 1 or 2, a mutated AAV particle described in claim 3, a nucleic acid molecule described in claim 4, a nucleic acid vector described in claim 5 or 6 or a host cell described in claim 7 for use in transferring a gene of interest into hepatocytes or HCC and for use in gene therapy.

14. A mutated AAV capsid protein, a fragment or a homologue thereof according to claim 1 or 2, The mutated AAV particle of claim 3. The nucleic acid molecule of claim 4. A nucleic acid vector according to claim 5 or 6, or The host cell of claim 7 .

10. A pharmaceutical composition comprising:

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