EA045763B1 - MODIFIED RAAV CAPSIDS PROTEIN FOR GENE THERAPY - Google Patents

Description

Field of technology

This invention relates to the field of recombinant adeno-associated virus (rAAV) gene therapy, in particular to the use of capsid mutant rAAV in the treatment or prevention of arthritis.

BACKGROUND OF THE INVENTION

Recombinant adeno-associated virus (rAAV) vectors have demonstrated excellent safety and efficacy profiles for gene delivery to humans in vivo. Therefore, rAAV vectors have been widely used for in vivo gene therapy and have been shown to be safe and effective in preclinical models as well as in clinical trials. rAAV vectors have been successful in a number of clinical gene therapy trials for a number of diseases, including hemophilia B, hemophilia A, cystic fibrosis, alpha-1 anti-trypsin deficiency, spinal muscular atrophy (SMA), Parkinson's disease, Duchenne muscular dystrophy and Leber congenital amaurosis ( Selot et al., Current Pharmaceutical Biotechnology, 2013, 14, 1072-1082). Alipogen tiparvovec (Glybera®, uniQure) has received European medical approval as a gene therapy for the treatment of lipoprotein lipase deficiency (LPLD). Subsequent drug approvals were made for the herpes virus-based gene therapy drug talimogene laherparepvec for the treatment of skin cancer (T-Vec, Imlygic®, Amgen) and for ex vivo gene therapy based on the retroviral stem cells Strimvelis for the treatment of ADA-SCID (GSK).

rAAV vector-based gene therapy is also used for rheumatoid arthritis (RA), which is a chronic inflammatory disease affecting ~1% of the population. The pathology of RA spreads throughout the synovial joint. The localized nature of the joint makes in vivo gene therapy very attractive. Treatments that provide anti-inflammatory proteins have been used to shift the balance in RA toward an anti-inflammatory state.

Much effort has been focused on developing AAV capsid proteins with desired properties. Such properties may include higher transduction efficiency, tissue/organ tropism, avoidance of targeting of undesired tissues/organs, or avoidance of pre-existing neutralizing antibodies.

However, there is still a need in the art to further improve rAAV gene therapy vectors. In particular, there is a need to improve the use of rAAV gene therapy vectors in arthritis and, more specifically, to improve the efficiency of delivery of genetic material to target tissue, such as the synovial junction or specific cell types at the synovial junction, preferably fibroblast-like synoviocytes (FLS).

The essence of the invention

In a first aspect, the present invention provides a recombinant adeno-associated virus (rAAV) virion comprising a modified capsid protein for use in the treatment or prevention of arthritis, or in the treatment or prevention of symptoms associated with arthritis, wherein the modified capsid protein comprises at the C-terminal portion of the protein amino acid sequence Z, the residues of which are located on the surface of the capsid protein. Preferably, the amino acid sequence of Z is:

a) contains or consists of a sequence of amino acid residues of formula I:

yGQxG - (x) ₃ - R - (x) ₃ - y- A- Q- A- A where x represents one amino acid residue, and where y represents 0, 1 or 2 amino acid residues; and b) is present at the region corresponding to position 100-200, preferably 120-180, more preferably 130-170, more preferably 140-160 amino acid residues from the C-terminus of the wild-type AAV capsid protein.

Preferably, the amino acid residues of formula I are located on the surface of the capsid protein. In a preferred embodiment, the Z sequence is contained in the modified capsid protein at the site represented by formula II:

EEEIxxxxPVATExxGxxxxNxQy - Z (x) _n LPGMVWQxRDVYLQGPIWAKIPHTDG where Z, x and y have the meanings defined above; and where n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In a preferred embodiment, the present invention relates to a rAAV virion comprising a modified capsid protein for use in the treatment or prevention of arthritis or in the treatment or prevention of symptoms associated with arthritis, wherein the capsid protein comprises an amino acid sequence selected from the group consisting of: i) an amino acid a sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 1, and wherein the amino acids at positions 588-602 of SEQ ID NO: 1 have at least 80% sequence identity with SEQ ID NO: 11, ii) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 2, and wherein the amino acids at positions

- 1 045763

585-599 SEQ ID NO: 2 have at least 80% sequence identity with SEQ ID NO: 10, iii) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 3, and wherein amino acids at positions 587-601 of SEQ ID NO: 3 have at least 80% sequence identity with SEQ ID NO: 9, iv) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 4, and wherein the amino acids at positions 586-600 of SEQ ID NO: 4 have at least 80% sequence identity to SEQ ID NO: 8, v) an amino acid sequence having at least 70% sequence identity to the amino acid sequence having SEQ ID NO: : 5, and wherein the amino acids at positions 588-602 of SEQ ID NO: 5 have at least 80% sequence identity with SEQ ID NO: 9, vi) an amino acid sequence having at least 70% sequence identity with an amino acid sequence having SEQ ID NO: 6, and wherein the amino acids at positions 588-602 of SEQ ID NO: 6 have at least 80% sequence identity with SEQ ID NO: 8, and vii) an amino acid sequence having at least 70% sequence identity with amino acid sequence having SEQ ID NO: 7, and wherein the amino acids at positions 587-601 of SEQ ID NO: 7 have at least 80% sequence identity with SEQ ID NO: 12, wherein the modified capsid protein provides at least a twofold increase expression, preferably in human FLS cells, compared to an unmodified capsid protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-19, when tested under the same conditions, wherein preferably the unmodified capsid protein has the amino acid sequence of SEQ ID NO: 19 or has the same serotype as the modified capsid protein.

In a preferred embodiment, the modified capsid protein provides at least a two-fold increase in expression in human FLS cells, compared to an unmodified capsid protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 13-19, when tested under the same conditions, wherein preferably the unmodified capsid protein has the amino acid sequence having SEQ ID NO: 19 or is the same serotype as the modified capsid protein.

Alternatively or in combination with any of the previous embodiments, in a preferred embodiment of the present invention, the capsid protein contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-7.

Alternatively, or in combination with any of the preceding embodiments, in a preferred embodiment of the present invention, the rAAV virion comprises a nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) sequence. Preferably, the virion further contains a nucleotide sequence encoding a gene product of interest. Even more preferably, the nucleotide sequence encoding the gene product of interest is located between two AAV ITR sequences.

In a preferred embodiment, the gene product of interest treats, prevents or suppresses symptoms associated with arthritis, wherein preferably the gene product of interest is selected from the group consisting of interleukins, immunomodulators, antibodies, shRNA, microRNA, guide RNA, lncRNA, growth factors, protease, nucleotidase/nucleosidase, peptides, protease inhibitors, inhibitors, enzymes and combinations thereof, and more preferably the gene product of interest is at least one of CD39, CD73 and IFN-β.

Alternatively, or in combination with any of the preceding embodiments, in a preferred embodiment of the present invention, the rAAV virion contains at least one of: (i) a polynucleotide containing a sequence encoding at least one guide RNA; wherein said or each guide RNA is substantially complementary to the target sequence (polynucleotides) in the genome; and (ii) a polynucleotide containing a sequence encoding a nuclease; in this case, the nuclease forms a ribonuclease complex with guide RNA, and at the same time, the ribonuclease complex causes site-specific double-strand breaks (DSDB) of DNA in the genome.

In another aspect, the present invention provides a rAAV composition for use in the treatment or prevention of arthritis or for the treatment or prevention of symptoms associated with arthritis, the rAAV composition comprising the rAAV virion of the invention and a pharmaceutically acceptable carrier. In one embodiment, the rAAV composition further comprises an empty capsid in an empty capsid to rAAV virion ratio of at least 1:1, at least 5:1, or at least 10:1.

In another aspect, the present invention provides a rAAV composition and an immunosuppressant for use in the treatment or prevention of arthritis or for the treatment or prevention of symptoms associated with arthritis, wherein the rAAV composition is the rAAV composition of the invention, and preferably wherein treatment or prophylaxis involves administering the rAAV composition and administering an immunosuppressant to the individual.

Alternatively or in combination with any of the preceding embodiments, in a preferred embodiment of the present invention, the arthritis is selected from the group consisting of rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement and Still's disease.

Alternatively, or in combination with any of the preceding embodiments, in a preferred embodiment of the present invention, the rAAV virion or composition is administered systemically and/or locally. In a preferred embodiment, at least one of the rAAV composition and an immunosuppressant is administered locally. Preferably, local administration is intra-articular administration.

In a further aspect, the present invention provides a method of treating, preventing, or suppressing symptoms associated with arthritis, comprising the step of intra-articular administration of a medicament containing an effective amount of a rAAV virion or a rAAV composition of the invention.

Detailed Description of the Invention

The inventors have discovered that a recombinant adeno-associated virus (rAAV) virion containing a modified capsid protein is surprisingly effective in cell transduction and is particularly effective in transducing synovial junction cells. Since fibroblast-like synoviocytes (FLS) are typically the primary target cell in the joint in the treatment of arthritis-related diseases, such as, for example, rheumatoid arthritis, the purpose of this invention is to provide capsid proteins that improve one or more of the following properties: i) higher levels of expression in synovial tissue, particularly in FLS; ii) improved tropism for synovial tissue, in particular improved tropism for FLS; and/or iii) improved elimination of targeting of unwanted tissues/organs upon rAAV administration compared to capsid proteins known in the art. In particular, these properties of the rAAV virion containing the modified capsid protein of the invention are improved compared to unmodified capsid proteins, preferably a wild-type capsid protein of the same serotype as the modified capsid protein and/or AAV5 capsid proteins. The AAV5 capsid has previously been shown to induce the highest levels of FLS expression compared to other AAV serotypes (Adriaansen et al. (2005) Ann Rheum Dis 64:1677-1684; Apparaily et al. (2005) Hum. Gene Ther. 16:426 -434). Since it is the capsid that imparts tissue/cell tropism, the modified capsids described herein have the property of increased FLS transduction potential, preferably over unmodified AAV5. In particular, it is preferred that the capsid proteins of the present invention provide higher levels of expression in synovial tissue, in particular in FLS, preferably when administered intra-articularly, compared to unmodified capsid proteins (i.e. the same capsid protein without the modification that must be tested, preferably the same serotype as the modified capsid proteins), preferably unmodified wild-type capsid proteins (preferably the same serotype as the modified capsid proteins), more preferably unmodified AAV5 or wtAAV5 capsid proteins.

Therefore, in a first aspect, the present invention provides an rAAV virion containing a modified capsid protein. The rAAV virion, as defined herein, is particularly useful for use in gene therapy.

As used herein, gene therapy is the insertion of nucleic acid sequences (such as, for example, a transgene (also referred to as a nucleotide sequence encoding a gene product of interest), as defined herein below) into cells and/or tissues of an individual for treatment or preventing a disease or disorder or treating or preventing a symptom of a disease or disorder.

AAV can infect both dividing and quiescent cells, and infection occurs through the interaction of capsid proteins with a cell membrane receptor, followed by endocytosis of the AAV virion. AAV belongs to the genus Dependovirus, which in turn belongs to the subfamily Parvovirinae, also called parvoviruses, which are capable of infecting vertebrates. Parvovirinae belongs to a family of small animal DNA viruses, that is, the family Parvoviridae. As their genus name suggests, members of Dependovirus are unique in that they typically require co-infection with a helper virus, such as an adenovirus or herpes virus, for productive infection in cell culture. The genus Dependovirus includes AAV, which commonly infects humans, and related viruses that infect other warm-blooded animals (eg, adeno-associated viruses of cattle, dogs, horses, and sheep). Additional information on parvoviruses and other members of the Parvoviridae is described in Kenneth I. Berns, Parvoviridae: The Viruses and Their Replication, Chapter 69 in Fields Virology

- 3 045763 (3d Ed. 1996). For convenience, the present invention is further illustrated and described herein with reference to AAV. However, it should be understood that the invention is not limited to AAV, but may equally apply to other parvoviruses.

The genomic organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule less than 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank unique coding nucleotide sequences for nonstructural replication proteins (Rep) and structural proteins (VP). The VP proteins (VP1, -2, and -3) form the capsid or shell protein by assembly-activating protein (AAP) (for some serotypes), which is encoded in an alternative open reading frame overlapping that of VP2/VP3. The terminal nucleotides are self-complementary and are organized so that an energetically stable intramolecular duplex can be formed, forming a T-shaped hairpin. The size of the terminal nucleotides depends on the serotype. For example, in the case of AAV2, the terminal 145 nt 125 nt are self-complementary and the remaining 20 nt remain single-stranded. These hairpin structures function as the origin of viral DNA replication by serving as primers for the cellular DNA polymerase complex. Following wild-type AAV (wtAAV) infection in mammalian cells, Rep proteins (i.e., Rep78 and Rep52) are expressed from mRNA transcribed by the p5 promoter and the p19 promoter, respectively. Both Rep proteins perform specific functions during viral genome replication. The splicing event at the Rep ORF results in the expression of essentially four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, the proteins Rep78 and Rep52, encoded by unspliced mRNAs, have been shown to be sufficient in mammalian cells to produce the AAV vector. Production of wtAAV or rAAV in mammalian cells further depends on a combination of alternative use of two splice acceptor sites and suboptimal use of the ACG start codon for VP2, which ensures proper expression of all three capsid proteins in an approximately 1:1:10 ratio (VP1:VP2 : VP3).

An rAAV virion (also referred to herein as an rAAV vector or rAAV transgenic vector), as used herein, refers to an AAV capsid containing a non-native nucleic acid sequence. Such a sequence in rAAV is typically flanked by ITR sequences, preferably from wtAAV, and preferably encodes a gene product of interest, such as, for example, a transgene or homologous arms. In other words, an rAAV virion means a rAAV genome containing (i) a nucleotide sequence encoding a gene product of interest, and (ii) at least one AAV ITR sequence encapsidated by capsid proteins. The rAAV genome may have one or preferably all wtAAV genes, but may still contain functional ITR nucleotide sequences. Preferably, the rAAV virion does not contain any nucleotide sequences encoding viral proteins, such as the AAV rep (replication) or cap (capsid) genes. Thus, the rAAV virion differs from the wtAAV virion because all or part of the viral genome is replaced by a nucleotide sequence encoding a gene product of interest that is a non-native nucleic acid to the AAV nucleotide sequence as further defined herein.

In a preferred embodiment, the rAAV virion containing the modified capsid protein of the invention is for use in the treatment or prevention of arthritis or for the treatment or prevention of symptoms associated with arthritis. The medical use (e.g., gene therapy for the treatment or prevention of (symptoms associated with) arthritis) described herein is provided by the rAAV virion of the invention for use as a medicament for the prevention or treatment of specified disease(s) and/or disorder(s) defined herein, but can equally be formulated as (i) a method of preventing or treating the disease(s) and/or disorder(s) defined herein or symptoms thereof, comprising administering a sufficient or effective amount of a rAAV virion according to the invention to a subject in need thereof in the form of (ii) an rAAV virion according to the invention for use in the manufacture of a medicament for the prevention or treatment of the disease(s) and/or disorder(s) defined herein, or in as (iii) the use of the rAAV virion in accordance with the invention for the prevention or treatment of the disease(s) and/or disorder(s) defined herein. Such medical applications are all contemplated by the present invention. Preferably, the modified capsid protein contains in the C-terminal part of the protein the amino acid sequence Z, the residues of which are located on the surface of the capsid protein.

As used herein, the terms treat, treatment, or treatment process refer to the use or administration of a rAAV virion of the invention to a subject suffering from arthritis, the purpose being to cure, partially or completely reverse, attenuate, improve, inhibit, delay, suppress, slow, or stop the progression or the severity of arthritis or symptoms associated with arthritis. The term treatment includes reducing or ameliorating at least one adverse effect or symptom of arthritis. Treatment is usually effective if one

- 4 045763 or more symptoms or clinical markers decrease. Alternatively, treatment is effective if the progression of the arthritis disease is reduced or stopped. That is, treatment involves not only improving symptoms or markers, but also stopping or at least slowing the progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desirable clinical outcomes include, but are not limited to, relief of one or more symptom(s), reduction in the severity of the disease, stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, improvement or temporary alleviation of the disease state, and remission (partial or complete), detectable or undetectable. The term treatment of arthritis also includes relief of symptoms or side effects of arthritis (including palliative treatment). As used herein, the term prevent, prevent, or preventive (also referred to as prophylactic) refers to the use or administration of an rAAV virion of the invention to a subject who is predisposed to arthritis in order to delay or prevent the onset, attenuate, improve, alleviate, inhibit the progression, reduce the severity of severity and/or reduce the frequency of one or more symptoms or signs of future arthritis. Thus, the rAAV virion of the invention can be administered to a subject who has no signs of arthritis and/or a subject who is showing only early signs of arthritis, preferably for the purpose of reducing the risk of developing pathology associated with arthritis.

As used herein, the term cure or cure means complete relief of one or more, preferably all, symptoms or signs of arthritis. As used herein, the term delay or delay means delaying the onset and/or inhibiting the progression and/or reducing the severity of one or more symptoms or signs of an arthritic disease.

In a preferred embodiment, the modified capsid protein of the invention provides at least a twofold increase in expression compared to the unmodified capsid protein when tested under the same conditions. Preferably, the unmodified capsid protein is a capsid protein of the same serotype as the modified capsid protein, but without the modification to be tested. More preferably, the unmodified capsid protein is a wild-type (wt) capsid protein of the same serotype as the modified capsid protein, wherein the wt capsid protein preferably has an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-19. Alternatively, it is preferred that the unmodified capsid protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-19. Most preferably, the unmodified capsid protein has the amino acid sequence set forth in SEQ ID NO: 19. The preferred unmodified capsid proteins may depend on the tissue to which the rAAV virion will target. For example, rAAV with AAV5 capsid proteins is the preferred virion for FLS cells (Apparilly et al. (2005) Human Gene Therapy 16(4):426-434; Adriaansen et al. (2005) Ann. Rheum. Dis. 64(12) : 1677-1684) and therefore - regardless of the original serotype of the mutant rAAV virions - the control rAAV virion preferably contains AAV5 capsid proteins, more preferably wild-type AAV5 capsid protein (wtAAV5), more preferably the AAV5 capsid protein has the amino acid sequence presented in SEQ ID NO: 19, even more preferably, the rAAV control virion is an rAAV5 virion. The rAAV control virion is an rAAV virion containing unmodified capsid proteins as defined herein instead of modified capsid proteins. In a preferred embodiment, the rAAV virion (containing modified capsid proteins) provides higher expression when transduced in vitro in fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA-FLS) and/or HEK 293, preferably HEK293T cells, compared to the rAAV virion with unmodified capsid proteins as defined herein, instead of modified capsid proteins using the method described in the examples. In other words, other than the capsid proteins, the rAAV virion and the control Raav virion are preferably identical. Preferably, transduction efficiency is determined in an in vitro transduction assay: by measuring the expression levels of a reporter gene encoded by the transgene, such as GFP, YFP and/or luciferase. In a preferred embodiment, the expression test is an in vitro transduction assay as described in Example 2/3. Briefly, RA-FLS (isolated as described in van de Sande MG et al., (2011) Ann Rheum Dis 70: 423-427) is seeded at 2500 cells/well or HEK293T cells (human embryonic kidney cells) are seeded at 40,000 cells /well in a 96-well plate (DMEM-GlutaMAX-I (Gibco, ref. 31966-021), 10% FBS (heat inactivated (HI) bovine serum Gold, Gibco cat. A15-151), 100 μg/ml penicillin/ 100 µg/ml streptomycin (Sigma-Aldrich, cat. P0781; 37°C/5% CO ₂ ) After 24 hours, the supernatant was removed and replaced with medium (DMEM-glutaMAX-I (Gibco, cat. 31966-021), 0.001% pluronic F68 (Sigma, cat. p5559)), containing mutant rAAV virions or control rAAV virions - all of which express yellow fluorescent protein (yFP) and/or luciferase under the control of the cytomegalovirus promoter

- 5 045763 (CMV), at multiplicity of infection (MOI) of 10,000, 20,000 and 100,000. Crude lysates (i.e., crude supernatants of cells transfected with all plasmids required for rAAV production and containing reporter-expressing virions) or purified AAV can be used. (preferably based on iodixanol purification or cesium chloride (CsCl) density gradient purification). Four hours after transduction, medium (DMEM-GlutaMAX-I, 10% FBS 100 U/ml penicillin, 100 μg/ml streptomycin) containing doxorubicin (Sigma, cat. D1515; final concentration 0.4 μM), FBS (final concentration 1%). After 48 hours (HEK293T) or 4–6 days (RA-FLS), cells were analyzed for the percentage of cells expressing YFP or luciferase by fluorescence microscopy or flow cytometry. Preferably, the in vitro transduction assay is performed multiple times with FLS isolated from different patients, such as, for example, FLS isolated from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more patients.

Serotype is traditionally determined based on the lack of cross-reactivity between antibodies to one virus compared to another virus. Such differences in cross-reactivity are typically due to differences in capsid protein/antigenic determinant sequences (eg, due to sequence differences between AAV serotypes VP1, VP2 and/or VP3). By traditional definition, a serotype means that the virus of interest has been tested for neutralizing activity using sera specific for all existing and characterized serotypes, and no antibodies have been detected that neutralize the virus of interest. As naturally occurring viral isolates are further discovered and capsid mutants are generated, serological differences may or may not exist with any of the currently extant serotypes. Thus, in cases where a new AAV is not serologically distinct, the new AAV will be a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine whether they are of a different serotype according to the traditional definition of serotype. Accordingly, for convenience and to avoid repetition, the term serotype broadly refers to both serologically distinct viruses (e.g., AAV) and viruses (e.g., AAV) that are not serologically distinct, which may be found in a subgroup or variant of a given serotype .

Transduction refers to the transfer of a transgene into a recipient host cell using a viral vector. Transduction of a target cell with the rAAV virion of the invention results in the transfer of the transgene contained in the rAAV virion into the transduced cell. Host cell or target cells refers to the cell into which the DNA is delivered, such as synoviocytes or synovial cells of an individual or such as isolated FLS cells from patients or HEK293T cells in the case of an in vitro transduction assay. AAV vectors are capable of converting both dividing and non-dividing cells. In a cell containing a gene product of interest, such as, for example, GFP, the gene product of interest has been introduced/transferred/transformed by rAAV transduction of the cell. The cell into which the transgene has been introduced is called a transduced cell.

The recipient host cell in which the transgene is transduced is preferably a cell affected by the disease being treated, such as, for example, synovial cells, more particularly FLS, macrophages, monocytes, neutrophils, osteoblasts, osteoclasts, chondrocytes, T lymphocytes, dendritic cells, plasma cells, mast cells, B lymphocytes in arthritis. The term synovial, synovial tissue, or synovial cells as used herein refers to the cellular lining covering the noncartilaginous surfaces of synovial joints, as further described in Tak (2000, Examination of the synovium and synovial fluid. In: Firestein GS, Panyani GS, Wollheim FA editors. Rheumatoid Arthritis. New York: Oxford Univ. Press, Inc. 55-68) and is incorporated herein by reference. The synovium consists of a layer of inner membrane (or synovial lining layer) and a synovial membrane (subsynovium), which merges with the joint capsule. The tunica intima layer contains intimate macrophages (or macrophage-like synoviocytes or type A synoviocytes) and FLS (or type B synoviocytes). Therefore, synovium can be replaced or synonymous with synovial tissue. A synovial cell may include any cell present in the synovium, including FLS and macrophage-like synoviocytes. The synoviocyte cell may also be a neutrophil, T cell, B cell, and/or connective tissue cell, which may all be present in the synovium.

Fibroblast-like synoviocytes (FLS) are cells of mesenchymal origin that share many characteristics with fibroblasts, such as the expression of specific proteins, such as several types of collagens. However, FLS also secrete proteins that are typically absent from other fibroblast lineages, such as lubricin. In addition, FLS express molecules that are important for mediating cell adhesion, such as cadherin-11, VCAM-1, several integrins and their receptors. Specific to FLS is the expression of CD55, and therefore this protein is commonly used to identify FLS in the synovium using immunohistochemistry. FLS are a specialized cell type located

- 6 045763 located inside joints in the synovium, the cells of which play a decisive role in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis (RA). The term rheumatoid synovial or rheumatoid synovial cells or rheumatoid synovial tissue refers to the inflamed synovial joint of the joints of a person suffering from RA. Rheumatoid synovium is characterized by intimal mucosal hyperplasia and accumulation of FLS, T cells, plasma cells, macrophages, B cells, natural killer cells, and dendritic cells in the synovial sublination. These accumulated cells are included in the definition of rheumatoid synovial cells. As RA progresses, synovial tissue becomes the site of ongoing inflammatory processes that can ultimately lead to cartilage damage and joint destruction and deformity. FLS that are present in synovium during RA have been reported to exhibit an altered phenotype compared to FLS present in normal tissues. For example, FLS in rheumatoid synovium lose contact inhibition, meaning they lose the ability to stop growing when more cells come into contact with each other. In addition, they lose the ability to grow on adhesive surfaces. As a result, the number of FLS in the diseased synovium increases. Inflammation is further enhanced by the production of several proinflammatory signaling molecules, particularly the interleukins IL-6 and IL-8, prostanoids, and matrix metalloproteinases (MMPs).

Alternatively, or in combination with another embodiment, in a further preferred embodiment of the present invention, a rAAV virion containing a modified capsid protein of the invention provides at least a twofold increase in transgene expression in human FLS compared to a rAAV virion containing an unmodified capsid protein, as defined in herein, preferably with an unmodified capsid protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-19, when tested under the same conditions, wherein preferably the unmodified capsid protein is of the same serotype as the modified capsid protein, or has the amino acid sequence set forth in SEQ ID NO: 19.

More preferably, the rAAV virion of the invention results in increased levels of transgene expression when transduced in vitro as described above by at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- 10- , 15-, 20-, 25-, 30-, 35-, 40-, 50-fold increase in expression levels in human FLS cells compared to control rAAV virion.

Also preferably, or in addition to the above, the rAAV virion provides increased transgene expression when administered in vivo in the mouse air sac (APS) model, (adapted from Edwards et al. (1981) J Pathol 134: 147-156 as described in the example 4) compared to a control rAAV virion, preferably compared to a rAAV virion containing wtAAV5 capsid proteins, provided that the rAAV is otherwise identical (except for its capsid protein(s)). Preferably, the transgene expression has at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35- fold magnification using rAAV containing mutant capsid proteins from the method of the invention. An illustrative method is provided in the examples.

Also preferably, or in addition to the foregoing, a rAAV virion containing a modified capsid protein provides similar or lower titers of neutralizing antibody (nAb) compared to the same rAAV virion containing an unmodified, preferably wild-type, AAV capsid protein of the same serotype. WtAAV5 capsids are known to have an attractive nAb profile, and therefore similar or lower nAb titers for rAAV containing the modified capsid protein of the present invention are preferred compared to wtAAV5.

Alternatively, an in vitro transduction assay can be performed in a manner similar to that described above, but in a cell type/cell line other than FLS, depending on the cell type to be affected, for example, in cells selected from the group consisting of primary hepatocytes, liver cell lines such as HuH, HepG2, HepA1-6, heart cells, skeletal muscle cells, lung cells such as the A549 cell line, CNS cells, eye cells, gastrointestinal cells, bone marrow cells and blood cells , such as the THP-1 cell line. This may also require a different AAV serotype as a preferential control depending on the tropism of the wild-type capsid proteins. Typically, the control vector preferably contains wild-type capsid proteins that are naturally targeted to the tissue of choice. As one skilled in the art will appreciate, this may also depend on the route of administration: local or systemic. For example, AAV2, which has been the most widely studied AAV, shows tropism for skeletal muscle cells, neurons, vascular smooth muscle cells, and hepatocytes; AAV6 shows tropism for airway epithelial cells; AAV7 exhibits tropism for skeletal muscle cells; AAV8 exhibits tropism for hepatocytes; AAV1 and AAV5 show tropism for vascular endothelial cells. When administered systemically, AAVs 1-3 and 5-9 have a tropism for the liver, with high protein levels observed with AAV9, 8, 7, 6, 1 and to a lesser extent 5 and 2; the heart is transduced by AAV4, 6, 7, 8, and 9; thoracic expression is observed for AAV4 and 6 (Zincarelli et al. (2008) Molecular Therapy 16(6): 1073-1080).

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Without wishing to be bound by any theory, we believe that the increased expression achieved by rAAV virions containing the modified capsid protein of the invention compared to rAAV control virions is due to improved rAAV transduction into the cell, possibly altered tropism leading to (i ) an increase in the number of cells in a population of cells and/or (ii) an increased level of expression per cell, for example, due to better virion uptake and/or intracellular processing.

Another advantage of the rAAV virion with a modified capsid protein in accordance with the invention may advantageously be other improvements, such as the possible avoidance of pre-existing neutralizing antibodies.

In a preferred embodiment of the present invention, the modified capsid protein contains an amino acid sequence Z, preferably the amino acid sequence Z is contained in the C-terminal portion of the protein. Preferably, the Z sequence is 12-18 amino acid residues in length (also referred to herein as the loop and insert region). In a preferred embodiment, the Z sequence is located at the C-terminal portion of the capsid protein, preferably at the region corresponding to position 100-200, preferably 120-180, more preferably 130-170, more preferably 140-160, most preferably about 150 amino acids with C- end of the wild-type capsid protein, as, for example, shown in SEQ ID NO: 13-19. The amino acid sequence residues Z are preferably located on the surface of the capsid protein such that, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. 16, 17 or 18 residues are located on the surface of the capsid protein (the so-called loop). In a preferred embodiment, the Z sequence is 14-18 amino acid residues in length, more preferably 15, 16, 17 or 18 amino acid residues, most preferably 15, 17 or 18 amino acid residues in length. The Z sequence may replace certain amino acid residues compared to unmodified ones, such as the wild-type capsid protein sequence. Preferably, the insert replaces 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues, more preferably 6 or 7 amino acid residues of the same sequence but without the insert, preferably unmodified, more preferably from the wild type sequence. In addition to the Z/insertion sequence, the framework capsid protein may thus contain additional modifications such as amino acid substitutions (eg, conservative amino acid substitutions), or the framework capsid protein may be a wild-type amino acid sequence. The AAV framework into which the insert is included can be of any serotype, such as, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrMO, or AAVDJ. Preferably, the AAV framework containing the Z sequence is selected from the group consisting of AAV1, AAV2, AAV7, AAV9, AAVrMO, AAVDJ, more preferably from unmodified capsid proteins having an amino acid sequence as shown in any of SEQ ID NO: 13 -19. The insert of the invention is preferably contained in the C-terminal portion of the capsid protein, preferably at a region corresponding to amino acid residues 100-200, preferably 120-180, more preferably 130-170, more preferably 140-160, most preferably approximately 150 amino acid residues from the C-terminus of the capsid protein. wild type protein, as for example shown in SEQ ID NO: 13-19, where the location of the insert is represented by Formula II:

EEEIxxxxPVATExxGxxxxNxQy - Z(x) _n LPGMVWQxRDVYLQGPIWAKIPHTDG where x represents one amino acid residue, where y represents 0, 1 or 2 amino acid residue(s) (which may thus be missing), and where n represents 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 8, 9 or 10, or where the insertion location is represented by a sequence having at least 90, 93, 95, 96, 97, 98 or 99% sequence identity with formula II. Preferably, the last three amino acid residues preceding the N-terminus of the Z sequence of the invention are NLQ, NHQ or NFQ. Preferably, y represents 0 or 2 amino acid residues. In some cases, y represents 2 amino acid residues, so two additional amino acid residues, preferably two serine residues, may be present between the NxQ motif and the insert of this invention. This is preferably the case where the NxQ motif is an NFQ, for example if the AAV capsid is the AAV1 capsid sequence as set out in SEQ ID NO: 1. One skilled in the art will be able to identify these motifs and this region in which the insert is located, also if it contains some variations, such as amino acid substitutions or deletions, which are also included within the scope of this invention.

In a preferred embodiment, based on the alignments shown in FIGS. 4 and 5, the insert (sequence Z) contains or consists of a sequence of the formula: xi-GQx ₂ -Gx ₃ x ₄ -x ₅ -Rx ₆ -x ₇ -x ₈ -xx ₁₀ -x ₁₁ -x- ₁₂ -x- ₁₃ -x- ₁₄ -x- ₁₅ where x1 is Q or none, x ₂ is S or R, x ₃ is N or C, x ₄ is D, Y or E, x ₅ is C, V, S or A, x ₆ is G, S or V, x ₇ is missing or is A, V or R, x ₈

- 8 045763 is D, N or E, x ₉ is C or A, x ₁₀ is F or Q, x11 is missing or is C or A, x ₁₂ is missing or is A, x ₁₃ is missing or is Q, x ₁₄ is missing or is A and x ₁₅ is missing or is A. Alternatively, the insertion (sequence Z) contains or consists of the sequence of the formula: y ₁ -GQy ₂ -Gy ₃ -y ₄ -y ₅ -Ry ₆ - y ₇ -y ₈ -y ₉ -y ₁₀ -Ay ₁₁ -y ₁₂ -y ₁₃ where y1 is Q or none, y ₂ is S or R, y ₃ is N or C, y ₄ is D , Y or E, y ₅ is C, V, S or A, y ₆ is G, S or V, ₇ is absent or is D, ₈ is absent or is C, y ₉ is A, V , R or F, y ₁₀ is N, D, E or C, y11 is absent or is Q, y ₁₂ is absent or is A, y ₁₃ is absent or is A. In yet another alternative, in the most preferred embodiment ,based on the alignments shown in FIG. 6 and 7, the insert (sequence Z) contains or consists of a sequence of general formula I:

yGQxG- (x) ₃ - R - (x) ₃ -yAQAA where x represents one amino acid residue and where y represents 0, 1 or 2 amino acid residues (which may therefore be absent). Preferably, (i) if the N-terminal y represents 0 amino acid residues, then the other y in Formula I represents 0 amino acid residues, or (ii) if the N-terminal y represents 1 amino acid residue, then the other y in Formula I represents 2 amino acid residues remainder. More preferably, the insert (sequence Z) contains or consists of a sequence of the more specific formula:

zo - G - Q - ζ _ή - G - z ₂ - z ₃ - z ₄ - R - z ₅ - z ₆ - z ₇ - z ₈ - z ₉ - A - Q - A - A where z ₀ is absent or represents is Q, z1 is R or S, z ₂ is C or N, z ₃ is D, E or Y, z ₄ is C, A, S or V, z ₅ is G, V or S, z ₆ is d or absent, z ₇ is C or absent, z ₈ is F, R, V or A, z ₉ is C, D, N or E. More preferably, if z ₀ is absent, then also z ₆ or z ₇ are both missing.

More preferably, the Z/insert sequence contains or consists of an amino acid sequence that is at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, most preferably 100% sequence identity with any of the amino acid sequences selected from the group consisting of SEQ ID NO: 8-12. Preferably, the Z/insert sequence contains or consists of an amino acid sequence represented by any of the above sequences and having at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 %, most preferably 100% sequence identity with any of the amino acid sequences selected from the group consisting of SEQ ID NO: 8-12.

In mammalian cells, expression of the three AAV capsid proteins (VP1, VP2, and VP3) in the correct stoichiometry depends on a combination of alternative use of two splice acceptor sites and suboptimal use of the ACG start codon for VP2, which is not accurately replicated in insect cells. Correct stoichiometry is important for the infectivity of AAV particles. To produce the three AAV capsid proteins in insect cells with the correct stoichiometry, the art typically uses a construct that is transcribed into a single polykistronic messenger that is capable of expressing all three VP proteins without the need for splicing. To achieve this, the VP1 protein may be under the control of a suboptimal translation initiation codon instead of ATG. Examples of such a suboptimal translation initiation codon are ACG, TTG, CTG and GTG (Urabe et al. (2002) Human Gene Therapy 13: 1935-1943; US 20030148506; US 20040197895; WO 2007/046703). Alternatively, when producing rAAV in insect cells, a nucleic acid cassette can be used to express the VP1, VP2 and VP3 proteins, where these proteins are encoded by a nucleic acid sequence containing overlapping open reading frames (ORFs), as described in European Patent No. 2061891 B1, where disclosed a VP expression cassette that contains an intron containing a promoter upstream of the ACG VP2 initiation codon. The modified capsid protein of the invention is defined in relation to the protein sequence of the VP1 capsid protein. However, since the Z/insert sequence is located in the C-terminal part of the VP1 protein, it is included in the invention that also the VP2 and VP3 proteins carry the Z/insertion sequence and are thus modified (regardless of the method of preparation of rAAV, such as, for example, in insect cells or mammalian cells).

Alternatively or in combination with another embodiment, in a further preferred embodiment of the present invention, the modified capsid protein of the invention contains or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 70, 75, 80, 85 , 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably having 100% sequence identity with the amino acid sequence having SEQ ID NO: 1 and wherein the amino acids are at positions 588-602 SEQ ID NO: 1 have at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably 100% sequence identity with SEQ ID NO: 11 , ii) an amino acid sequence having at least 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more

- 9 045763 preferably having 100% sequence identity with the amino acid sequence having SEQ ID NO: 2 and wherein the amino acids at positions 585-599 of SEQ ID NO: 2 are at least 80, 85, 87, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99%, more preferably having 100% sequence identity to SEQ ID NO: 10, iii) an amino acid sequence having at least 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably having 100% sequence identity with the amino acid sequence having SEQ ID NO: 3 and wherein the amino acids at positions 587-601 are SEQ ID NO: 3 have at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably 100% sequence identity with SEQ ID NO: 9, iv) amino acid sequence having at least 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably having 100% sequence identity with the amino acid sequence having SEQ ID NO: 4 and wherein the amino acids at positions 586 to 600 of SEQ ID NO: 4 are at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably having 100% sequence identity with SEQ ID NO: 8, v) an amino acid sequence having at least 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably, having 100% sequence identity with the amino acid sequence having SEQ ID NO: 5 and wherein the amino acids at positions 588-602 of SEQ ID NO: 5 are at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably having 100% sequence identity with SEQ ID NO: 9, vi) amino acid sequence having at least 70, 75, 80, 85, 87, 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably having 100% sequence identity with the amino acid sequence having SEQ ID NO: 6 and wherein the amino acids at positions 588-602 of SEQ ID NO: 6 have at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably 100% sequence identity with SEQ ID NO: 8 and vii) amino acid sequence having at least 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably having 100% sequence identity with an amino acid sequence having SEQ ID NO: 7 and wherein the amino acids at positions 587-601 of SEQ ID NO: 7 are at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, more preferably having 100% sequence identity to SEQ ID NO: 12. Preferably, the AAV framework capsid protein that contains the insert has the amino acid sequence of a wild-type AAV capsid, such as, for example, AAV5, AAV1, AAV2, AAV7, AAV9, AAVrh10 or AAVDJ , or an amino acid sequence containing conservative amino acid substitutions. More preferably, the AAV framework capsid protein that contains the insert has the amino acid sequence of a wtAAV5 capsid or an amino acid sequence containing conservative amino acid substitutions.

Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences determined by sequence comparison. In a preferred embodiment, sequence identity is calculated based on the total length of the two SEQ ID NOs or portions thereof. Parts thereof preferably represent at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of both SEQ ID NOs. In the art, identity also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the correspondence between strings of such sequences. Unless otherwise indicated herein, identity or similarity to a given SEQ ID NO means identity or similarity based on the full length of the specified sequence (that is, along its entire length or as a whole).

The similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substituents of one polypeptide with the sequence of a second polypeptide. Identity and similarity can be easily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed. ., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G. , Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math ., 48:1073 (1988)).

Preferred methods for determining identity have been developed to identify maximum similarity between the sequences under study. These methods of determining identity and similarity are programmed into publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, the GCG software package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990)). The BLAST X program is publicly available from the National Center for Biotechnology Information and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S. et al., J. Mol. Biol. 215: 403-410 (1990)). To determine identity can also be applied generally

- 10 045763 well-known Smith-Waterman algorithm.

Preferred parameters for comparing polypeptide sequences include the following algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); gap penalty: 12 and gap extension penalty: 4. Such a program is publicly available as the Ogap program from Genetics Computer Group, located in Madison, Wisconsin. The above options are the default options for amino acid comparisons (along with no penalty for end gaps).

Preferred parameters for comparing nucleic acids include the following algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); comparison matrix: matches = + 10, mismatch = 0; gap penalty: 50; penalty for gap extension: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wisconsin (www.biology.wustl.edu/gcg/gap). The above are the default parameters for nucleic acid comparisons.

Optionally, when determining the degree of amino acid similarity, one skilled in the art may also take into account so-called conservative amino acid substitutions, as will be appreciated by one skilled in the art. Conservative amino acid substitutions refer to the interchangeability of residues that have similar side chains. For example, the group of amino acids having aliphatic side chains are glycine, alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; the group of amino acids having basic side chains is lysine, arginine and histidine, and the group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. Substitutive variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences is deleted and another residue is inserted in its place. Preferably, the amino acid substitution is conservative. Preferred conservative substitutions for each naturally occurring amino acid are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly on Pro; His to Asn or Gln; Ile to Leu or Val; Leu on Ile or Val; Lys to Arg; Gln or Glu; Met on Leu or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe and Val to Ile or Leu.

Alternatively or in combination with another embodiment, in a further preferred embodiment of the present invention, the capsid protein contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-7, more preferably from the group consisting of SEQ ID NO: 1 , 2, 3, 4, 6 and 7, even more preferably from the group consisting of SEQ ID NO: 3, 4 and 6, even more preferably from the group consisting of SEQ ID NO: 4 and 6 most preferably SEQ ID NO: 4.

Functional ITR sequences are required for replication, release, and packaging of rAAV virions. ITR sequences may be wild-type sequences or may have sequence identity equal to at least 80, 85, 90, 95, or 100% with wild-type sequences or may be altered, for example, by insertion, mutation, deletion, or nucleotide substitution, while they remain functional. In this context, functionality refers to the ability to directly package a genome into a capsid shell and then allow expression in a host or target cell. Typically, the ITRs of the wild-type AAV genome are retained in the rAAV vector. ITRs can be cloned from the AAV viral genome or excised from a vector containing the AAV ITR. The ITR nucleotide sequences can either be ligated at either end to the transgene as defined herein using standard molecular biology techniques, or the wild-type AAV sequence between the ITRs can be replaced with the desired nucleotide sequence. The rAAV vector preferably contains at least nucleotide sequences of the ITR regions of one of the AAV serotypes, or nucleotide sequences substantially identical thereto, and at least one nucleotide sequence encoding a therapeutic protein (under the control of a suitable regulatory element) inserted between the two ITRs. Most rAAV vectors currently in use use ITR sequences from the AAV2 serotype. The most preferred ITRs present in the rAAV vector are of the AAV2 serotype. Other preferred ITRs are serotype AAV1, AAV3, AAV5 or AAV6 (Grimm et al. (2006) J Virol 80(1):426-439). The rAAV genome may contain single-stranded or double-stranded (self-complementary) DNA. A single-stranded nucleic acid molecule is either a sense or an antisense strand, since both polarities are equally capable of packaging into AAV capsids. Single-stranded rAAV vectors can use wild-type AAV serotype 2 (AAV2) ITR sequences, and double-stranded (self-complementary) rAAV vectors can use a modified version of the ITR. Alternatively,

- 11 045763 in an embodiment, the double-stranded vector contains one ITR, which is an ITR from AAV4. The rAAV vector may further comprise a marker or reporter gene, such as, for example, a gene encoding an antibiotic resistance gene, a fluorescent protein (e.g., gfp), or a gene encoding a chemically, enzymatically, or otherwise detectable and/or selectable product (e.g., lacZ , alkaline phosphatase (AP), SEAP, Luc, Neo, Bla, etc.) known in the art.

The rAAV vector, including any possible combination of an AAV serotype capsid and an AAV genome ITR, is produced using methods known in the art, for example, using a mammalian rAAV production system or an insect cell rAAV production system. Methods known in the art are described, for example, in Pan et al. (J. of Virology (1999) 73: 3410-3417), Clark et al. (Human Gene Therapy (1999) 10: 1031-1039), Wang et al. (Methods Mol. Biol. (2011) 807: 361-404), Grimm (Methods (2002) 28(2): 146-157), and the insect cell system are based on Urabe et al. (Human Gene Therapy (2002) 13:1935-1943), Kohlbrenner et al. (Molecular Therapy (2005) 12(6): 12171225), International Patent Publication WO 2007/046703, International Patent Publication WO 2007/148971, International Patent Publication WO 2009/014445, International Patent Publication WO 2009/104964, International Patent Publication WO 2009/154452, international patent publication WO 2011/112089, international patent publication WO 2013/036118, US patent no. 6723551 B, which are incorporated herein by reference. Briefly, the methods may generally include (a) introducing an rAAV genome construct into a host cell, (b) introducing an AAV helper construct into the host cell, wherein the helper construct contains viral functions not present in the wild-type rAAV genome, and (c) ) introduction of a helper virus construct into a host cell. All functions for rAAV vector replication and packaging must be present to allow replication and packaging of the rAAV genome into rAAV vectors. Introduction into a host cell can be accomplished using standard molecular biology techniques and can be done simultaneously or sequentially. Finally, host cells are cultured to produce rAAV vectors, which are then purified using standard methods such as CsCl gradients (Xiao et al. 1996, J. Virol. 70: 8098-8108) or iodixanol purification. The purified rAAV vector is then typically ready for use in the methods. High titers greater than 10 ¹² particles per ml and high purity (no detectable helper and wild-type viruses) can be achieved (see, for example, Clark et al. supra and Flotte et al. 1995, Gene Ther. 2: 29- 37). The total size of the transgene inserted into the rAAV vector between the ITR regions is typically less than 5 kilobases (kb).

In the context of the present invention, the capsid protein shell may be of a different serotype than the rAAV genome, comprising (i) a nucleotide sequence encoding the gene product of interest, and (ii) at least one AAV ITR sequence. Thus, the rAAV genome of the present invention can be encapsidated by a capsid protein shell of the present invention, i.e. an icosahedral capsid that contains capsid proteins (VP1, VP2 and/or VP3) according to the present invention, for example, mutant AAV capsid proteins according to the invention, while the ITR sequences contained in this rAAV vector may represent any of the AAV serotypes described above , including, for example, AAV2 or AAV5. In one embodiment, the rAAV genome or ITRs present in the rAAV virion are derived from AAV serotype 2, AAV serotype 5, or AAV serotype 8. The complete genome of AAV5 and other AAV serotypes has been sequenced (Chiorini et al. 1999, J. of Virology Vol. 73, No. 2, p1309-1319), and the nucleotide sequence of AAV5 is available in GenBank (accession number AF085716). Thus, the ITR nucleotide sequences of AAV2 and AAV5 are readily available to those skilled in the art. The complete genome of AAV2 is available from NCBI (NCBI reference sequence NC_001401.2). They can be cloned or prepared by chemical synthesis known in the art, using, for example, an oligonucleotide synthesizer available, for example, from Applied Biosystems Inc. (Fosters, California, USA) or using standard molecular biology techniques.

Alternatively, or in combination with another embodiment, in a further preferred embodiment of the present invention, the rAAV vector contains a nucleotide sequence encoding a gene product of interest.

The terms transgene or gene product of interest are used interchangeably herein and refer to a non-native nucleic acid with respect to the AAV nucleic acid sequence. They are used to refer to a polynucleotide that can be introduced into a cell or organism. Gene products of interest include any polynucleotide, such as a gene that encodes a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (eg, lacking an expression control element, such as a promoter, that drives transcription). The gene product of interest according to the invention may contain at least two nucleotide sequences, each of which is different or encodes different therapeutic molecules. At least two different nucleotide sequences can be linked by an IRES (internal ribosome entry site) element, providing a bicistronic transcript under the control of one

- 12 045763rd promoter. Suitable IRES elements are described, for example, in Hsieh et al. (1995, Biochem. Biophys. Res. Commun. 214:910-917). In addition, at least two different nucleotide sequences encoding different (therapeutic) polypeptides or proteins can be linked by the viral 2A sequence to ensure efficient expression of both transgenes from the same promoter. Examples of 2A sequences include those of foot-and-mouth disease virus, equine rhinitis virus A, Tesa Asigna virus, and porcine teschovirus-1 (Kim et al., PLoS One (2011) 6(4): e18556). The gene product of interest is preferably inserted into the rAAV genome or between ITR sequences. The gene product of interest may also be an expression construct comprising an expression regulatory element, such as a promoter or transcription regulatory sequence, operably linked to a coding sequence and a 3' terminal sequence. The gene product of interest may be a functional mutant allele that replaces or complements the defective allele. Gene therapy also includes the insertion of transgenes that are inhibitory in nature, that is, that inhibit, reduce, or reduce the expression, activity, or function of an endogenous or exogenous gene or protein, such as an unwanted or aberrant (eg, pathogenic) gene or protein. Such transgenes may be exogenous. By exogenous molecule or sequence is meant a molecule or sequence not normally found in the cell, tissue and/or individual being treated. Both acquired and congenital diseases are amenable to gene therapy.

A gene or coding sequence refers to a region of DNA or RNA that codes for a specific protein. The coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide when placed under the control of an appropriate regulatory region, such as a promoter. A gene may contain several functionally linked fragments, such as a promoter, a 5' leader sequence, an intron, a coding sequence, and a 3' untranslated sequence containing a polyadenylation site or signal sequence. A chimeric or recombinant gene is a gene not normally found in nature, such as a gene in which, for example, a promoter is not naturally associated with part or all of the region of transcribed DNA. Gene expression refers to the process in which a gene is transcribed into RNA and/or translated into an active protein.

As used herein, the term promoter or transcription regulatory sequence refers to a nucleic acid fragment that controls the transcription of one or more coding sequences and is located upstream of the transcription initiation site in the coding sequence relative to the direction of transcription and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, without limitation, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to one of ordinary skill in the art to directly or indirectly act to regulate the amount of transcription from a promoter. A constitutive promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An inducible promoter is a promoter that is physiologically or developmentally regulated, for example, by the use of a chemical inducer. A preferred inducible promoter is an NF-κB-responsive promoter that is inducible under inflammation. A tissue-specific promoter is preferentially active in certain types of tissues or cells. The selection of an appropriate promoter sequence generally depends on the host cell chosen to express the DNA segment. Preferred promoter sequences within the rAAV and/or transgene of the invention are promoters that allow expression in rheumatoid synovium cells, such as in vitro macrophages and/or FLS and/or other synovial cells, such as, but not limited to, T cells. Preferred promoters are, for example, promoters of genes known to be expressed in synovial cells, such as the CMV promoter, the IL-6 gene promoter or the SV40 promoter or the NF-κB inducible promoter as previously identified herein, and others. which can be easily determined by a qualified specialist. Alternatively, the transgene is operably linked to a promoter that allows efficient systemic expression. Suitable promoter sequences are the CMV promoter, CBA (chicken beta actin) or liver specific promoters such as human alpha 1 antitrypsin (hAAT) or TBG (thyroxine binding globulin). Preferably, the promoter within the rAAV and/or transgene is not a steroid-inducible promoter. More preferably, the promoter within the rAAV and/or transgene is not a dexamethasone-inducible promoter.

As used herein, the term operably linked refers to the linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is operably linked when it is in a functional relationship with another nucleic acid sequence. For example, a transcription regulatory sequence is operably linked to a coding sequence if it affects transcription of the coding sequence. Functionally linked means that the DNA sequences being joined are typically contiguous and, if necessary, join two protein-coding regions adjacent and in frame.

The gene product of interest may be a therapeutic polypeptide or therapeutic protein, which is to be understood herein as a polypeptide or protein that can have a beneficial effect on an individual, preferably said individual is a human, more preferably said individual is suffering from a disease. Such a therapeutic polypeptide may be selected from, without limitation, the group consisting of an enzyme, a cofactor, a cytokine, an antibody, a growth factor, a hormone, and an anti-inflammatory protein.

Alternatively, or in combination with another embodiment, in a further preferred embodiment of the present invention, the nucleotide sequence encoding the gene product of interest is located between two AAV ITR sequences. Alternatively, the nucleotide sequence encoding the gene product of interest is said to be flanked by two AAV ITR sequences, that is, one ITR at either end of the nucleotide sequence encoding the gene product of interest.

Alternatively, or in combination with another embodiment, in a further preferred embodiment of the present invention, the gene product of interest treats, prevents or suppresses symptoms associated with arthritis, wherein preferably the gene product of interest is selected from the group consisting of interleukins, immunomodulators, antibodies , shRNA, microRNA, growth factors, protease, nucleotidase/nucleosidase, peptides, protease inhibitors, inhibitors, enzymes and combinations thereof, and more preferably the gene product of interest is at least one of CD39, CD73 and IFN-β. Examples of these are: interleukin 1 (IL-1) inhibitor (eg anakinra, canakinumab, rilonacept), tumor necrosis factor alpha (TNFa) inhibitor (eg etanercept, infliximab, adalimumab, certilizumab, pegol, golimumab), IL-1 receptor antagonist , soluble IL-1 receptor, IL-17 inhibitor (e.g. secukinumab, brodalumab, ikekizumab), IL-12/IL-23 inhibitor (ustekinumab, risankizumab, guselkumab, tildrakizumab), T-cell costimulation inhibitor (e.g. abatacept), B -cell depleting and inhibitory agents (eg, rituximab, belimumab, ianalumab, tabalumab), IL-15 inhibitor (eg, AMG-714), IL-22 inhibitor (eg, fezacunimab), GM-CSF inhibitor (lenzilumab, namilumab) insulin-like growth factor (IGF-1), fibroblast growth factor (FGF) (eg, rhFGF-18/sprifermin), receptor activator of nuclear factor kappa B ligand (RANKL) inhibitor (eg, denosumab), complement 5a inhibitor (eg, C5aR151) , bone morphogenetic protein (BMP) family member, transforming growth factor-beta (TGF-β), growth differentiation factor (GDF) family, interleukin-18 inhibitor (eg Tadekinig alpha/IL-18 binding protein), IL-2 inhibitors ( eg basiliximab, daclizumab), soluble TNFa receptor (sTNFa) p55 or sTNFa receptor p75, sTNFa receptors fused to IgG, TNFa p55 receptor inhibitors, sTNFa p75 receptor inhibitors, dominant negative IkB kinase (dnIKK-β), interleukin-4 (IL-4), interleukin-10 (IL-10) (F8IL10/decavil), interleukin-13 (IL-13), interferon beta (IFN-β), tissue inhibitor of the MMP family (TIMP), plasminogen activator inhibitor (PAI ), serine protease inhibitors (serpins), signaling molecules/transcription factors (e.g., SMAD, Sox9, IkB), extracellular matrix components (e.g., collagen, cartilage oligomeric matrix protein (COMP), proteoglycans, elastin), vasoactive intestinal peptide (VIP) ), cluster of differentiation 39 (CD39) and cluster of differentiation 73 (CD73), superoxide dismutase (SOD) and combinations thereof.

Functional genome editing systems for use in all embodiments of the invention are known to those skilled in the art and include: transcription activator-like effector nucleases (TALEN, Gaj et al. (2013) Trends Biotechnol. 31(7):397405), nucleases zinc fingers (ZFN, Gaj et al. (2013) above), meganucleases such as I-Scel (Arnould et al. (2007) J Mol Biol 371(1):49–65; Takeuchi et al. (2011) PNAS USA 108(32): 13077-13082), RNA-guided endonuclease systems such as CRISPR/Cas (Mali et al. (2013) Nat methods 10(10):957963; Mali et al. (2013) Nat Biotechnol 31(9) :833-838; Cong et al. (2013) Science 339(6121):819-823) and CRISPR/Cpf1 (Zetsche et al. (2015) Cell 163(3):759-771), triplex-forming molecules, synthetic polyamides and engineered zinc finger proteins (Uil et al. (2003) Nucleic Acids Res 31(21):6064-6078). Functional genome editing systems use nucleases that create site-specific double-strand breaks at desired locations in the genome. Induced double-strand breaks are repaired by nonhomologous end joining or homologous recombination. The result is targeted mutations. It is advantageous to replace a defective gene (causing a disease or disorder) with a normal allele in its natural location by any of these methods, since it is not required that complete coding sequences and regulatory sequences be included in the rAAV virion when only a small portion of the gene needs to be

- 14 045763 to be changed. It is also believed that the expression of the partially replaced gene is more consistent with normal cell biology than the full genes carried by the virions. The preferred gene editing system is CRISPR (including CRISPR/Cpfl and CRISPR-Cas) because it is faster and cheaper than other methods. A major advantage is also that CRISPR can be easily reconfigured to target different DNA sequences using single CRISPR guide RNAs. Thus, alternatively or in combination with another embodiment, in another preferred embodiment of the present invention, the rAAV genome comprises at least one of: (i) a polynucleotide comprising a sequence encoding at least one guide RNA (gRNA); wherein the guide RNA is essentially complementary, preferably complementary, to the target sequence (polynucleotides) in the genome; and (ii) a polynucleotide containing a sequence encoding a nuclease; in this case, the nuclease forms a ribonuclease complex with guide RNA, and the ribonuclease complex causes site-specific double-stranded DNA breaks in the genome.

In a second aspect, the present invention provides rAAV compositions for use in the treatment, prevention or suppression of symptoms associated with arthritis, wherein the rAAV composition comprises an rAAV virion of the invention and a pharmaceutically acceptable carrier, diluent, solubilizer, excipient, preservative and/or excipient, preferably pharmaceutically acceptable carrier. Such pharmaceutically acceptable carrier, diluents, solubilizer, excipient, preservative and/or excipient, for example, can be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000. Any suitable pharmaceutically acceptable carrier, diluent, solubilizer, excipient, preservative and/or excipient can be used in these compositions (see, for example, Remington: The Science and Practice of Pharmacy, Alfonso R Gennaro (Editor) Mack Publishing Company, April 1997). Preferred pharmaceutical forms will be in combination with sterile saline, dextrose or buffered saline or other pharmaceutically acceptable sterile liquids. Alternatively, a solid carrier may be used, such as, for example, microcarrier beads.

Pharmaceutical compositions are generally sterile and stable under conditions of manufacture and storage. Pharmaceutical compositions may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to accommodate high drug concentrations. The carrier may be a solvent or dispersion containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Proper flow can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. The parvovirus virion can be administered as a bolus or as a controlled release formulation, for example, in a composition that contains a sustained release polymer or other vehicles that will protect the compound from rapid release, including implants and microencapsulated delivery systems. For example, biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyesters, polylactic acid and polylactic-polyglycol copolymers (PLG) can be used. As used herein, the term pharmaceutically acceptable carrier or excipient preferably includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for ready-to-use preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional carrier or agent is incompatible with the active compound, it is contemplated for use in the pharmaceutical compositions of the invention.

It may be advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specification of the unit dosage forms of the invention may be dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as limitations inherent in the state of the art for preparing such

- 15 045763 active compound for the treatment of a condition in individuals.

Additional active compounds may also be included in the pharmaceutical compositions of the invention. Guidance on the concomitant use of additional drugs can be found, for example, in the Compendium of Pharmaceutical and Specialty Products (CPS) of the Canadian Pharmacists Association.

In one embodiment, the rAAV composition further comprises empty particles (ie, capsid-only particles, thus not containing the rAAV genome). Therefore, alternatively or in combination with another embodiment, in a further embodiment of the present invention, the rAAV composition of the invention further comprises an empty capsid with an empty capsid to rAAV virion ratio of at least 1:1, more preferably at least 5:1, even more preferably at least 10:1. The rAAV composition may contain the rAAV virion as defined above and an empty capsid, such as, for example, defined in WO 2016/055437, which is incorporated herein by reference, and as described in Aalbers et al. (2017) Hum. Gene Ther. 28(2): 168-178. The empty capsid may be the same serotype or a different serotype compared to the rAAV transgenic vector composition of the invention. Preferably, the empty capsid is of the same serotype as the rAAV virion. In such a rAAV composition, the empty capsid and the rAAV virion capsid may contain a modified capsid protein of the invention, preferably the same type of modified capsid proteins. However, also included is a rAAV composition in which the empty capsids are of a different serotype or are differently modified capsid proteins compared to the modified rAAV virion capsid proteins. Also covered is an rAAV composition in which the empty capsids are of a mixture of serotypes, such as, but not limited to, a mixture of AAV2 and AAV5 capsids. We report an enhanced effect of transgene expression in joints following intra-articular administration of rAAV virions mixed with a significant amount of empty capsids. Preferably, the rAAV virion and empty capsid are present within the composition at a ratio of empty capsid to rAAV virion of at least 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 15:1, 20 :1, 50:1, 100:1 or 1000:1, preferably equal to at least 5:1 (ie, the number of empty capsids that is at least 5 times the number of rAAV transgenic vectors). Preferably, said composition contains an rAAV virion and an empty capsid in a ratio of empty capsid to rAAV transgenic vector of not more than 10000:1, 5000:1, 4000:1, 3000:1, 2000:1, 1000:1, 500:1, 400: 1, 300:1, 200:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1 or 5:1, preferably no more than 1000:1. Preferably, said composition contains the rAAV virion and empty capsids in an empty capsid-rAAV virion ratio of 1:1 to 100:1, 2:1 to 100:1, 5:1 to 100:1, 1:1 to 20:1, 2:1 to 20:1, or preferably 5:1 to 20:1.

Proposed above herein is an embodiment in which the rAAV virion and empty capsids are present in a single composition. Also included in the present invention is an alternative embodiment wherein the rAAV virion and empty capsids are present in (at least two or more) separate separated compositions. In this alternative embodiment, the rAAV virion and empty capsids may be administered separately in time (eg, sequentially) and/or location, where by location is meant the site of administration. In addition, the rAAV virion and empty capsids can be administered simultaneously, for example, at substantially the same time, not necessarily at a separate location.

In a third aspect, the present invention provides compositions of rAAV and an immunosuppressant for use in the treatment or prevention of arthritis or for use in the treatment or prevention of symptoms associated with arthritis, wherein the rAAV composition is as defined above, and wherein the treatment or prevention comprises administering rAAV compositions and administering an immunosuppressant to the individual. WO 2016/055437, incorporated herein by reference, discloses the potentiating effect of an immunosuppressant on AAV transgene expression when subjects were treated with both immunosuppressive drugs and rAAV virions. In addition, WO 2016/055437 discloses the surprising synergistic effect of an immunosuppressant along with empty vectors on rAAV transgene expression. In one embodiment, the immunosuppressant is administered separately from the rAAV composition, which means separate at location and/or time. In such an embodiment, the immunosuppressant and the rAAV composition may be present in separate and distinct compositions. The immunosuppressant, rAAV virion, and optionally empty vectors may even be present in each individual composition. In another embodiment, the immunosuppressant and the rAAV composition may be present in the same composition. In a further embodiment, the rAAV virion and the immunosuppressant are present in the same composition, and preferably the composition is used for treatment in conjunction with a separate composition containing the empty capsid. In yet another embodiment, the immunosuppressant and the empty capsid are present in the same composition, and preferably the composition is used for treatment in conjunction with a separate composition containing the rAAV virion. Therefore, the present invention also provides a composition comprising an empty capsid and an immunosuppressant as defined herein, a composition comprising an rAAV virion and an immunosuppressant as defined herein, and a composition comprising a composition of rAAV and

- 16 045763 immunosuppressant as defined herein.

Preferably, the immunosuppressant for use in the present invention is an innate immune cell inhibitor, preferably a macrophage inhibitor. An innate immune cell is defined herein as a neutrophil, macrophage, monocyte, eosinophil, basophil, or dendritic cell that has the potential to participate in an inflammatory response to a foreign substance. An innate immune cell inhibitor is defined herein as an agent that results in a decrease in the activity of innate immune cells and/or the number of innate immune cells. A macrophage inhibitor is defined herein as an agent that results in a decrease in macrophage activity and/or macrophage number. A macrophage is defined herein as an innate immune cell that engulfs and digests cellular debris, foreign substances, microbes, and cancer cells in a process called phagocytosis. Preferably, the innate immune cell or macrophage inhibitor of the invention results in a reduction of at least 1, 2, 5, 10, 15, 20, 25, 30, 35, 45, 55, 65, 75, 85, 95%, or preferably 100% the number or activity of innate immune cells or macrophages compared with the baseline number or activity of innate immune cells or macrophages before treatment. The activity and/or number of innate immune cells or macrophages can be detected using any suitable assay known to one skilled in the art, such as, without limitation, MTT (3-(4,5-dimethylthiazol-2-yl)2,5- diphenyl tetrazolium bromide) colorimetric assay to determine the cytotoxic activity of macrophages in vitro as described in Ferrari et al. (Journal of Immunological Methods, 131 (1990) 165-172), by measuring cytokine levels (eg, CCL2, TNF), by histological and histochemical detection methods, for example, by CD68 labeling or by in vivo magnetic resonance imaging detection ( MRI) of superparamagnetic iron oxide (SPIO) uptake by macrophages, preferably after intravenous administration of SPIO, as reviewed by Yi-Xiang J. Wang (Quant. Imaging Med Surg (2011)1:35-40). Detection may be in vitro or in vivo. Preferably, the in vivo determination is carried out in an animal model, preferably a rat or mouse model.

Preferably the immunosuppressant is a glucocorticoid and/or a bisphosphonate, preferably a liposomal bisphosphonate. Specific non-limiting examples of glucocortocoids include cortisol, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate, and aldosterone. Preferably the immunosuppressant is triamcinolone. Specific non-limiting examples of bisphosphonates include etidronate, clodronate, tiludronate, pamidronate, neridronate, oladronate, alendronate, ibandronate, risedronate, and zoledronate. Preferably the bisphosphonate is a liposome-encapsulated bisphosphonate or a liposomal bisphosphonate, preferably a liposomal clodronate. Preferably the glucocorticoid is not dexamethasone. It should be understood that the inflammatory or macrophage inhibitor of the invention is not limited to glucocorticoids and/or bisphosphonate. For example, the inflammatory or macrophage inhibitor of the invention may also be an inflammatory or macrophage-depleting antibody, such as an anti-F4/80 antibody. Preferably, such antibody is a human or humanized antibody. Other relevant immunosuppressants for use in this invention are cytotoxic drugs (eg, alkylating agents and/or antimetabolites such as methotrexate), drugs that modify the purinergic signaling pathway (eg, methotrexate, adenosine analogues, adenosine receptor antagonists or agonists), non-steroidal anti-inflammatory drugs drugs (NSAIDs, eg, ibuprofen, diclofenac, meloxicam, naproxen, acetylsalicylic acid), biologics, such as TNF blockers (eg, infliximab, etanercept, adalimumab, certolizumab, golimumab), IL-6 blockers (eg, tocilizumab), blockers IL-2 (eg, basiliximab, daclizumab), IL-1e blockers (eg, anakinra, rilonacept, canakinumab) IL-17 (secukinumab, brodalumab, ikekinumab), anti-IL-12/IL-23 (ustekinumab), PDE4- inhibitor (apremilast) muromonab, abatacept and/or rituximab, and/or other compounds such as hydroxychloroquine, chloroquine, leflunomide, sulfasalazine, azathioprine, cyclophosphamide, cyclosporine, sodium tetrachloroauric acid, mTOR inhibitors (eg, rapamycin/sirolimus, everolimus) and penicillamine.

Preferably, the rAAV composition and/or the empty capsid-containing composition and/or the immunosuppressant-containing composition further comprises a pharmaceutically acceptable carrier, diluents, solubilizer, excipient, preservative and/or excipient, as defined elsewhere herein.

Preferably, the gene therapy of the present invention further comprises administering an immunosuppressive agent, as defined herein, either present in the rAAV composition or included in a separate and distinct composition, that is, separate and distinct from the rAAV composition. Upon administration, the rAAV composition and/or empty capsids and/or immunosuppressive agent of the invention are delivered to an individual, cell, tissue or organ of said individual, preferably an individual suffering from a condition or disease as defined herein. Prefer

- 17 045763 In particular, the rAAV composition and the immunosuppressant are administered simultaneously. By simultaneous administration as used herein is meant administration at more or less the same time, preferably no more than 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 12 hours or 24 hours apart, preferably separated by no more than 15 minutes. In another embodiment, the rAAV composition and the immunosuppressant are administered sequentially, with the immunosuppressant preferably administered before the rAAV composition. Preferably, the immunosuppressant is administered at least 1 hour, 3 hours, 12 hours, 24 hours, 2 days, 4 days or 1 week before administration of the rAAV composition. In the case where rAAV virions and empty capsids are present in separate compositions, the immunosuppressant can be administered simultaneously or over a period of at least 15 minutes, 1 hour, 2 hours, 3 hours, 1 day, 2 days or 1 week before the empty capsid, and the empty capsids are in turn administered simultaneously or within at least 15 minutes, 1 hour, 2 hours, 3 hours, 1 day, 2 days, or 3 days prior to administration of the rAAV virion.

In embodiments defined herein, the immunosuppressant can be administered repeatedly, that is, before and/or simultaneously with the rAAV composition. As stated above, it is preferred that the rAAV composition contains a significant amount of empty capsids. The invention further covers the administration of both rAAV transgenic vectors and empty capsids in separate, separate compositions that can be administered simultaneously or sequentially in a method or application of the invention.

If included in separate formulations, the rAAV transgenic vectors and empty capsids are preferably administered simultaneously. In a further embodiment, empty capsids are administered in no more than 3 days, 2 days, 1 day, 24 hours, 12 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes or 5 minutes, preferably in no more than 24 hours before administration of the rAAV transgenic vector. In addition, when included in separate compositions, the rAAV transgenic vectors and empty capsids are preferably administered at the same site.

The dose of the immunosuppressant depends on the type of immunosuppressant. Effective dosages are known to one skilled in the art. The preferred therapeutically effective dosage of triamcinolone is indicated above. The preferred therapeutically effective dose of liposomal clodronate is preferably a therapeutically effective dose known to one skilled in the art, for example, preferably 80-320 mg/dose intra-articular, more preferably 160 mg/dose intra-articular (Barrera et al. 2000, Arthritis & Rheumatism Vol 43( 9), p1951-1959).

Typically, joint disease is called arthropathy, and when one or more joints are inflamed, the disorder is called arthritis. Most joint diseases are associated with arthritis, but joint damage caused by external physical trauma is not usually called arthritis. As used herein, the term arthritic disease, also referred to as arthritis, is defined herein as a form of joint disorder that involves inflammation of one or more joints. Currently, it is estimated that there are more than one hundred different forms of arthritis. Arthritis as used herein refers to joint pain or joint disease. In a preferred embodiment, the arthritic disease is selected from the group consisting of adult Still's disease, ankylosing spondylitis, arthritis, back pain, Behçet's disease, blunt trauma, bursitis, calcium pyrophosphate deposition disease (CPPD), carpal tunnel syndrome, chondromalacia patellar syndrome, chronic fatigue, reflex sympathetic dystrophy, cryopyrin-associated periodic syndromes (CAPS), degenerative disc disease, hip dysplasia, Ehlers-Danlos disease, familial Mediterranean fever, fibromyalgia, erythema infectiosum, giant cell arthritis, gout, hemochromatosis, infectious arthritis, inflammatory arthritis , inflammatory bowel disease, endoprosthetics, juvenile arthritis, juvenile dermatomyositis (JD), juvenile idiopathic arthritis (JIA), juvenile rheumatoid arthritis, juvenile scleroderma, Kawasaki disease, lupus, lupus in children and adolescents, Lyme disease, mixed connective tissue disease, myositis (incl. polymyositis, dermatomyositis), osteoarthritis (OA), osteoporosis, Paget's disease, palindromic rheumatism, patellofemoral pain syndrome, childhood rheumatic disease, pediatric systemic lupus erythematosus (SLE), polymyalgia rheumatica, pseudogout, psoriatic arthritis, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, rheumatic fever, rheumatism, rheumatoid arthritis, scleroderma, septic arthritis, Sjogren's disease, spinal stenosis, spondyloarthritis, Still's disease, systemic juvenile idiopathic arthritis, systemic lupus erythematosus, systemic lupus erythematosus in children and adolescents, systemic sclerosis, temporal arteritis, tendinitis, vasculitis and Wegener's granulomatosis. In a further preferred embodiment, the arthritic disease is selected from the group consisting of rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, arthroplasty and Still's disease. In a more preferred embodiment, the arthritic disease is a joint disease that involves inflammation of one or more joints. Preferably, the arthritic disease is selected from the group consisting of rheumatoid arthritis

- 18 045763 (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis and Still's disease.

Alternatively, or in combination with another embodiment, in a further preferred embodiment of the present invention, the rAAV virion or rAAV composition is administered systemically and/or locally. The rAAV composition and/or empty capsids and/or immunosuppressive agent of the invention can be administered directly or indirectly using suitable means known in the art. The methods and uses of the invention include delivery and administration of an rAAV and/or empty vector and/or immunosuppressant composition systemically, regionally or locally or by any route, for example, by injection, infusion, orally (for example, by ingestion or inhalation) or topically (for example, transdermal ). Exemplary routes of administration and delivery include intravenous (IV), intraarticular, intraperitoneal (IP), intraarterial, intramuscular, parenteral, subcutaneous, intrapleural, topical, cutaneous, intradermal, transdermal, parenteral, e.g., transmucosal, intracranial, intraspinal, oral (alimentary), mucosal, respiratory, intranasal, by intubation, intrapulmonary, by intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavitary, iontophoretic, intraocular, ophthalmic, ophthalmic, intraglandular, intraorgan, intralymphatic. Improvements in the means of providing an individual or cell, tissue, organ of said individual with the rAAV composition and/or empty capsids and/or immunosuppressant of the invention are expected, taking into account the progress that has already been made to date. To achieve the mentioned effect of the invention, such future improvements may of course be included. When administering the rAAV composition and/or empty capsids and/or immunosuppressive agent of the invention, it is preferred that such combination and/or composition be dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal, intraarticular and/or intraventricular administration, it is preferred that the solution is physiological saline. In the case where an immunosuppressant is present in the rAAV composition of the invention, the immunosuppressant is administered at the same site as the rAAV composition, that is, preferably locally, as indicated above. In an embodiment where the immunosuppressant is contained in a separate composition other than the rAAV composition, the immunosuppressant can be administered systemically, preferably intramuscularly or intravenously. The rAAV composition can also be administered topically, preferably to an area of the body containing a significant number of macrophages, as defined herein, and the immunosuppressant is administered systemically, preferably intramuscularly or intravenously. Also included in the present invention is an embodiment wherein the immunosuppressant and the rAAV composition, even if present in different compositions, are administered at the same site, preferably locally, more preferably intra-articularly. As further stated herein, the administration of such separate compositions can be carried out either simultaneously or sequentially. In a preferred embodiment of the present invention, at least one of the rAAV composition and an immunosuppressant is administered locally. More preferably, local administration is intra-articular administration. An intra-articular injection (also known as a joint injection or intra-articular injection) is defined herein as an injection or infusion into a joint. An intra-articular injection is usually used to administer an anti-inflammatory drug to an inflamed joint.

In a further aspect, the present invention provides a rAAV composition comprising a rAAV virion of the invention and a pharmaceutically acceptable carrier, diluent, solubilizer, excipient, preservative and/or excipient, preferably a pharmaceutically acceptable carrier as defined herein. In a preferred embodiment, the composition further comprises empty capsids, as defined herein, and/or an immunosuppressant, as defined herein.

In a further aspect, the present invention provides a method of treating, preventing or suppressing symptoms associated with arthritis, comprising the step of intra-articular administration of a medicament containing an effective amount of a rAAV virion as defined in any one of claims 1 to 8 or a rAAV composition as defined above .

A therapeutically effective amount refers to an amount effective in dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the nucleic acid, nucleic acid construct, rAAV virion, or pharmaceutical composition may vary depending on factors such as the disease state, age, sex, and weight of the subject being treated, and the ability of the nucleic acid, nucleic acid construct, rAAV virion, or pharmaceutical composition. compositions to evoke the desired response in the subject. Dosage regimens may be adjusted to ensure optimal therapeutic response. A therapeutically effective amount is also generally one in which any toxic or harmful effects of the nucleic acid, nucleic acid construct, rAAV virion or pharmaceutical composition are outweighed by the therapeutically beneficial effects

- 19 045763 effects. A prophylactically effective amount refers to an amount effective at dosages and for periods of time necessary to achieve the desired prophylactic result, such as preventing or inhibiting various conditions. A prophylactic dose may be used in subjects before or at an earlier stage of the disease, and in some cases, the prophylactically effective amount may be greater or less than the therapeutically effective amount. The dosage administered may depend largely on the condition and size of the subject being treated, as well as the therapeutic composition, frequency of treatment, and route of administration. Continued therapy regimens, including dose, formulation, and frequency, may be based on initial response and clinical assessment.

The terms subject or patient are used interchangeably herein and refer to an animal, including the human species, that is amenable to treatment with the compositions and/or rAAVs of this invention. Accordingly, the term subject or patient includes, without limitation, a human, a non-human primate such as chimpanzees and other species of apes and apes, or any mammal such as a dog, cat, horse, sheep, pig, cow, etc. d. In a preferred embodiment of the present invention, the subject being treated with rAAV according to the present invention is a mammal, more preferably a human, a dog, cat or horse, most preferably a human.

In this document and in its claims, the verb contain and its derivatives are used in their non-limiting sense to mean that elements following the word are included, but elements not specifically mentioned are not excluded. In addition, a reference to an element specified in the singular does not exclude the possibility of the presence of more than one element unless the context clearly implies the presence of one and only one element. Thus, a singular noun usually means at least one.

The word approximately or about when used in conjunction with a numerical value (about 10, about 10) preferably means that the value may be a specified value equal to 10 or more or less than 10% of the value.

All patent and literature references cited herein are incorporated herein by reference in their entirety.

The following examples are illustrative only and are in no way intended to limit the scope of the present invention.

Description of the figures

This invention will be discussed in more detail below with reference to the accompanying drawings:

fig. 1: capsid serotype screening on HEK293T and FLS cells. A crude lysate containing 7 mutant capsid serotypes (plus AAV5) expressing yellow fluorescent protein (YFP) was used to transduce HEK293T cells or 3 different FLS cell lines (each from a different RA patient). After 72 hours (HEK293T) or 6 days (FLS), cells were analyzed for the percentage of cells expressing YFP by flow cytometry. Panel A shows the % of HEK293T cells expressing YFP; panel B shows the % of cells expressing YFP in 3 different FLS cell lines; panel C shows the mean fluorescence intensity (MFI) in HEK293T cells; panel D shows MFI in 3 different FLS cell lines (all cells); Panel E shows MFI in 3 different FLS cell lines (positive population only). The symbols of the sample are presented in table. 2.

Fig. 2: Capsid mutants show increased luciferase expression compared to wt-AAV5 in FLS cells. Purified AAV (4 mutant serotypes or AAV5) expressing the YFP-Luc fusion protein was used to transduce three different FLS cell lines from different RA patients: BB5498 (FLS 1), BB5540 (FLS 2) and BB7144 (FLS 3) using two MOI (20,000 or 100,000 rAAV particles per cell). After 4 days, the cells were lysed and luciferase expression was measured. Data are presented as absolute luciferase expression levels (RLU; white bars) or fold increases relative to AAV5 (black bars). Panel A shows FLS 1 at MOI 20,000; Panel B shows FLS 1 at MOI 100,000; Panel C shows FLS 2 at MOI 20,000; Panel D shows FLS 2 at MOI 100,000; Panel E shows FLS 3 at MOI 20,000; and panel F shows FLS 3 at an MOI of 100,000. Open bars represent luciferase (RLU) and filled bars represent fold increase over AAV5. In another experiment, three additional FLS cell lines from RA patients were transduced with AAV (7 mutant serotypes or AAV5) expressing luciferase: BB4308 (FLS 4), BB 1592 (FLS 5), BB4426 (FLS 6) using 2 MOI (10,000 or 100,000 RAAV particles per cell). Panel G shows FLS 4 at MOI 10,000; Panel H shows FLS 4 at MOI 100,000; Panel I shows FLS 5 at MOI 10,000; Panel J shows FLS 5 at MOI 100,000; Panel K shows FLS 6 at MOI 10,000; and panel L shows FLS 3 at MOI 100,000.

Transduction efficiency of 7 mutant serotypes or AAV5 (MOI 100,000) was also assessed in HEK293T cells (Panel M). Open bars represent luciferase (RLU) expression, and

- 20 045763 filled bars represent fold increases over AAV5.

Figure 3A: Capsid mutants show increased gene expression in vivo. Two capsid mutants (AAV9-A2 and AAV7-A6) were compared to wtAAV5 using the air sac synovium model. The luciferase expression vector was administered at day 0 after air sac formation, and luciferase expression was measured by live animal imaging (MS) at day 3 post-transduction. Data shown are luminescence (photon/second/square centimeter/steradian) in the air sac as mean+SEM.

Fig. 3B: In the second experiment, 5 selected capsid mutants (AAV1-P4, AAV7-A6, AAV9A2, AAVrh10-A2, AAVrh10-A6) and wtAAV5 were injected into the knee joints of mice. The luciferase expression vector was administered on day 0, and expression was measured by real-time imaging (MS) at the indicated time points after administration. Data shown are luminescence (photon/second/square centimeter/steradian) (left panel) as mean+SEM. **P<0.05, ***P<0.01, ****P<0.00001 against wtAAV5 on day 14. Fig. 3C: fold increase compared to wtAAV5.

Fig. 4: CLUSTAL format alignment using MAFFTFFT-NS-I (v7.215). Below the alignment is a key indicating a conserved residue (*) and a non-conservative mutation ().

Fig. 5: CLUSTAL alignment of multiple sequences using MUSCLE (3.8). Below the alignment is a key indicating a conserved residue (*); conservative mutation (:); semi-conservative mutation (.) and non-conservative mutation ().

Fig. 6: CLUSTAL format alignment of inserts P4, A2, A6, P2 and QR-P2 (SEQ ID NO: 8-12) using MAFFT FFT-NS-I (v7.215). Below the alignment is a key indicating a conserved residue (*) and a non-conservative mutation ().

Fig. 7: CLUSTAL alignment of several insertion sequences P4, A2, A6, P2 and QR-P2 (SEQ ID NO: 8-12) using MUSCLE (3.8). Below the alignment is a key indicating a conserved residue (*) and a non-conservative mutation ().

List of sequences

In table 1 provides an explanation of sequence references in correlation with SEQ ID numbers.

Table 1

Sequence references explained

SEQ ID NO: serotype Modified capsid/insert/wild type 1 AAV1 Modified capsid 2 AAV2 Modified capsid 3 AAV7 Modified capsid 4 AAV9 Modified capsid 5 AAVrhIO Modified capsid 6 AAVrhIO Modified capsid 7 AAV DJ-QR Modified capsid 8 Box A2 Insert 9 Insert Ab Insert 10 Insert P2 Insert eleven Insert P4 Insert 12 QR-P2 insert Insert 13 AAV1 Wild type capsid 14 AAV2 Wild type capsid 15 AAV7 Wild type capsid 16 AAV9 Wild type capsid 17 AAVrhIO Wild type capsid 18 AAV DJ-QR Synthetic capsid 19 AAV5 Wild type capsid

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Examples

Example 1.

Initial screening of capsid library.

1.1. Materials and methods.

96-well plates unevenly coated (and subsequently dried) with crude lysate containing AAV from 91 different AAV capsid serotypes were obtained from Dirk Grimm and Kathleen Borner at the University of Heidelberg. Each vector encoded a YFP transgene driven by a CMV promoter. Because FLS are the primary target cells in the joint, a library of AAV capsid mutants was screened for serotypes that show increased expression in human FLS isolated from the joints of patients with rheumatoid arthritis (RA-FLS) (as described in van de Sande MG et al., (2011) Ann Rheum Dis 70: 423-427). RAFLS were seeded (2500/well, 37°C/5% CO2) directly into unevenly coated plates (DMEM-GlutaMAX-I (Gibco, cat. 31966-021), 10% FBS (heat inactivated (HI) bovine serum Gold, Gibco, cat. A15-151), 10 mM HEPES (Gibco, cat. 15630-056), 50 μg/ml gentamicin (Gibco, cat. 15710-049), 100 U/ml penicillin/100 μg/ml streptomycin (Sigma -Aldrich, cat. P0781) and all wells were visualized for YFP expression by fluorescence microscopy after 6 days.

1.2. Results.

Transduction efficiency of capsid mutants against WT-AAV5 (wild type) in FLS from RA patients.

In a screen of 91 capsid mutants, although overall expression levels were low, we identified 7 different serotypes that showed higher expression than wtAAV5: AAV9-A2, AAV7-A6, AAV1-P4, AAVDJ-QR-P2, AAVrh10- A6, AAVrh10-A2 and AAV2P2 (amino acid sequences SEQ ID NO: 1-7; wtAAV5 SEQ ID NO: 19).

Crude lysates from all 7 vectors were used in an in vitro transduction assay in 3 different patient FLS cell lines and in HEK293T cells (Example 2).

table 2

Sample symbols for Figs. 1

Sample Capsid serotype Inserted/modified nth sequence Insert Position in VP1 SEQ ID NO: 5 5 absent absent - 19 61 AAV1 GQSGNDVRSANAQAA P4 588 - 602 1 33 AAV9 GQRGNYSRGVDAQAA A2 586 - 600 4 34 AAVrhIO GQRGNYSRGVDAQAA A2 588 - 602 6 50 AAV2 QGQSGDCRGDCFCA (QAA) P2 585 - 599 2 88 AAV-DJ- QR QGQRGCDCRGDCFCA(QAA) QR-P2 587 - 601 7 43 AAV7 GQRGNEARVREAQAA Ab 587 - 601 3 46 AAVrhIO GQRGNEARVREAQAA Ab 588 - 602 5

Example 2.

Expression of crude lysates of the 7 selected mutants. 2.1. Materials and methods. Receiving AAV.

Detailed information on the preparation of crude AAV lysates can be found in Grosse et al. (J. Virol, 2017, doi: 10.1128/JVI.01198-17).

Aliquots of the crude lysate for each of the 7 selected capsid mutants (plus wtAAV5 as a control) were used to transduce cells (HEK293T or 3 different FLS cell lines isolated from RA patients) and YFP expression was measured by flow cytometry 3 (HEK293T) - 5 days (FLS) after transduction. In detail, HEK293T was seeded in a 96-well plate (Greiner Bio-One, cat. 655180) at 45,000 cells per well. RA-FLS were seeded in a 96-well plate at 2500 cells per well. After overnight incubation, cell supernatants were replaced with 40 μl of DMEM-glutaMAX-I (Gibco 31966-021) containing 0.001% Pluronic F68 (Sigma P5556). Viral lysates were added in duplo, 10 μl per well. After 4 hours, doxorubicin (final concentration 0.4 μM) (Sigma D1515) in DMEM-glutaMAX-I containing FBS (heat inactivated (HI) bovine serum Gold, Gibco, cat. A15-151), final concentration 1%), was added to the wells (50 μl per well). The next day, the FLS medium was removed and DMEMglutaMAX-I (10% FBS (heat inactivated (HI) Gold bovine serum, Gibco, cat. A15) was added

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151), 10 mM HEPES (Gibco, cat. 15630), 50 μg/ml gentamicin (Gibco cat. 15710-049), 100 U/ml penicillin/100 μg/ml streptomycin (Sigma-Aldrich, cat. P0781)) ( 200 µl per well). The medium of HEK293T cells was not changed. Three days (HEK293T cells) or 6 days (FLS) after transduction, cells were trypsinized using 0.5% trypsin/EDTA (Gibco cat. 15400-054) in PBS (Gibco cat. 10010) and analyzed for YFP expression using flow cytometry FLOW (FACSCanto II, BD Biosciences). Both the percentage of expressing cells and the mean fluorescence intensity (MFI) of all cells were determined.

2.2. Results.

Crude lysates from all 7 vectors were used in an in vitro transduction assay in 3 different patient FLS cell lines and in HEK293T cells. Cells were analyzed for the percentage of cells expressing YFP by fluorescence microscopy (data not shown) or flow cytometry (Fig. 1, panels A–E). Although some variability was observed between cell types, all mutant capsids provided higher expression in FLS and HEK293T cells than AAV5-WT (Fig. 1). In table 2 shows the legend for FIG. 1. Based on these results, four capsid mutants were selected for further study (see example 3).

Example 3.

In vitro testing of capsid variants in HEK293T and FLS.

3.1. Materials and methods.

3.1.1. Four mutant capsid proteins AAV9-A2, AAV7-A6, AAV1-P4 and AAVDJ-QR-P2 were further investigated. A purified vector (iodixanol gradient) expressing a YFP-luciferase fusion protein was prepared (to allow visualization of (YFP) as well as quantification by luciferase assay). Three different primary FLS lines isolated from patients with rheumatoid arthritis (as described in van de Sande MG et al., (2011) Ann Rheum Dis 70: 423-427) were transduced with each serotype at 2 vector doses (MOI 20,000 or 100,000) and after 4 days, cells were harvested and gene expression was quantified using a luciferase assay (Promega Luciferase assay Kit).

In detail, RA-FLS were seeded at 2500 cells/well in a 96-well plate (Greiner BioOne, cat. 655207) in DMEM-GlutaMAX (Gibco cat. 31966-021), 10% FBS (heat inactivated (HI) bovine serum Gold, cat. A15-151), 10 mM HEPES (Gibco cat. 15630-056), 50 μg/ml gentamicin (Gibco, cat. 15710-049), 100 U/ml penicillin/100 μg/ml streptomycin (Sigma -Aldrich Merck cat. P0781) After 48 hours, the medium was removed and virus was added (in DMEM-Glutamax containing 0.001% Pluronic-68 (Sigma, cat. P5556)) at an MOI of 20,000 or 100,000. After 4 hours, medium containing doxorubicin was added (Sigma, cat. D1515, final concentration 0.4 µM) and FBS (final concentration 1%).

After 24 hours, the medium was replaced with DMEM-GlutaMAX (10% FBS, 10 mM HEPES, 50 μg/ml gentamicin, 100 μg/ml penicillin, 100 μg/ml streptomycin). Four days after transduction, the cells were washed once with 100 μl of PBS (Gibco, cat. 10010) and luciferase activity was determined using the ONE Glo™ luciferase assay system (Promega, cat. E6110): 100 μl of lysis buffer was added and the cells were placed on a shaker for 10 minutes, 900 rpm at room temperature. Then 20 μl of lysate was transferred to a white 96-well plate, 80 μl of substrate was added over 3 minutes (darkness) and luciferase activity was determined using a luminometer (1 sec/well, Synergy HT, Biotek).

3.1.2. In a similar experiment, three additional FLS cell lines isolated from patients with rheumatoid arthritis were transduced with AAV5 mutants and 7 capsids from an AAV preparation other than that described in 3.1.1 (AAV9-A2, AAV1-P4, AAV7-A6, AAVDJ-QR -P2, AAVrh10-A6, AAVrh10-A2, AAV2-P2) containing the luciferase gene (MOI 10,000 and 100,000). The number of empty particles varied between AAV preparations. To exclude a possible effect on transduction efficiency, empty capsid correction was performed by adding empty AAV5 particles to equalize the percentage of empty particles per preparation.

3.1.3. 7 capsid mutants from the same preparation described in 3.1.2 were also tested in HEK293T cells. In detail, HEK293T was seeded in a 96-well plate (Greiner Bio-One, cat. 655180) at 50,000 cells per well. After overnight incubation, cell supernatants were replaced with DMEM-glutaMAX-I (Gibco 31966-021) containing 0.001% Pluronic F68 (Sigma P5556). Various vectors were added in duplo, at an MOI of 100,000. In this protocol, empty capsid correction was performed as described for 3.1.2. After 4 hours, doxorubicin (final concentration 0.4 μM) (Sigma D1515) in DMEM-glutaMAX-I-containing FBS (heat inactivated (HI) Gold bovine serum, Gibco, cat. A15-151), final concentration 1%, was added into the holes. Three days after transduction, cells were harvested and gene expression was quantified using a luciferase assay (Promega Luciferase assay Kit) on a luminometer (BMG Labtech Fluostar Omega).

3.2. Results.

3.2.1. In vitro transduction from three different FLS cell lines was performed using recombinant AAV containing one of the 4 mutant capsids (as well as AAV5 as a control made in an identical manner) according to the protocol described in 3.1.1. All 4 serotypes are pro-

- 23 045763 showed increased expression levels compared to AAV5, ranging from 2-fold to

35-fold magnification, depending on the serotype and cell line used (Fig. 2A-F).

3.2.2. In another series of experiments, the in vitro transduction efficiency of 7 capsid mutants (as well as an AAV5 control produced in an identical manner) was assessed in 3 FLS cell lines. All 7 serotypes showed increased levels of luciferase expression compared to AAV5, ranging from 6-fold to 55-fold increase depending on the serotype and cell line used (Fig. 2G-L).

3.2.3. A similar experiment was performed on HEK293T cells. Transduction with all 7 serotypes resulted in increased luciferase expression compared with wtAAV5, ranging from a 2-fold to a 12-fold increase (Fig. 2M).

Example 4.

In vivo study in the air sac synovium model.

4.1. Materials and methods.

Animals.

Female Balb/c mice (8–10 weeks old and weighing 20–25 g; (Harlan, Boxmeer, The Netherlands)) were housed in individual ventilated cages in the animal facility of the Academic Medical Center in Amsterdam. Food and water were available ad libitum. All animal experiments were carried out in accordance with the guidelines of the Animal Research Ethics Committee of the University of Amsterdam.

Air sac synovium (APS) model.

Two serotypes, AAV9-A2 and AAV7-A6, were compared with wtAAV5. The air sac synovium model was adapted from the work of Edwards et al. (1981; J Pathol 134: 147-156). On day 0, 3 ml of air was injected subcutaneously on the back of female Balb/cOlaHsd mice 7-9 weeks old (Harlan) (day 0). Immediately after the formation of the air sac, 1 ml of air was removed and 1 ml of AAV (2e10 vector genomes/mouse in PBS (Gibco, cat. 10010, containing 0.001% Pluronic F68 (Sigma, cat. p5556)) was added directly to the air sac. Three days later following transduction, gene expression was measured using in vivo animal imaging.

Visualization of luciferase expression.

Luciferase expression was measured on day 3. It was initially planned to continue monitoring expression for up to 3 months after vector administration, however, parvovirus infection of the animal subject led to the premature termination of all ongoing experiments. Potassium salt substrate D-luciferin (Caliper Life Sciences, Hopkinton, Massachusetts, USA) was administered intraperitoneally (150 mg/kg body weight, in a volume of approximately 200 μl). Photon counts were taken 10 min after substrate injection for 5 min using a cooled charge-coupled device (CCD) camera system (Photon Imager, Biospace Lab, Paris, France), and processing and signal intensity quantification and analysis were performed using M3 Vision (Biospace Lab). The number of photons emitted per second per square centimeter per steradian was calculated as a measure of luciferase activity.

General animal welfare conditions and ethics.

Air sac formation, vector injection, and in vivo imaging were performed under isoflurane anesthesia (3% isoflurane and oxygen). At the end of the experiments, the animals were sacrificed by cardiac puncture under isoflurane anesthesia, followed by cervical dislocation. The studies were reviewed and approved by the Animal Care and Use Committee of the University of Amsterdam and were carried out in strict accordance with the guidelines of the Dutch Animal Welfare Act (Dutch: Wet op Dierproeven). Animals were maintained under pathogen-free conditions in the animal facility of the University of Amsterdam.

4.2. Results.

Based on these promising results, a preliminary in vivo study was performed using an air sac (APS) model in which two serotypes, AAV9A2 and AAV7-A6, were compared with wtAAV5. Due to an unsuccessful infection in the animal facility that required premature termination of this study, we were only able to obtain data at one time point, 3 days after vector administration. At this time point, it was clear that the capsid mutants caused an increase in gene expression relative to AAV5, with AAV7-A6 showing an ∼6-fold increase in expression and AAV9-A2 ∼22-fold ( Fig. 3A ).

Example 5.

In vivo study: intra-articular injections in healthy animals.

5.1. Materials and methods.

Animals.

Male DBA1/J mice (12 weeks old, Envigo) were housed in individual ventilated cages in the animal facility of the Academic Medical Center in Amsterdam. Food and water were available ad libitum. All animal experiments were carried out after approval by the Central Commission on Animal Experimentation (CCD) and the Animal Research Ethics Committee of the University of Amsterdam, the Netherlands.

Expression studies.

Five rAAVs containing capsid mutants, i.e., AAV9-A2, AAV1-P4, AAV7-A6, AAVrh10-A6, and AAVrh10-A2, were compared with wtAAV5. Because capsid load can influence expression (Aalbers CJ et al., Hum Gene Ther 2017;28(2):168-178), rAAV preparations were adjusted for capsid load by adding wtAAV5 empty particles. Healthy mice (n=9 per group) received intra-articular injections of an AAV vector carrying the luciferase gene in both knees (7.5 HHH10 ⁹ viral genomes per knee). Gene expression was determined by in vivo imaging at multiple time points after vector administration.

Visualization of luciferase expression.

Luciferase expression was determined at the indicated time points (Fig. 3B). At each time point, D-luciferin potassium salt substrate (Caliper Life Sciences, Hopkinton, MA, USA) was administered intraperitoneally (150 mg/kg body weight, in a volume of approximately 200 μl). Photon counts were taken 15 min after substrate injection for 5 min using a refrigerated charge-coupled device (CCD) camera system (Photon Imager, Biospace Lab, Paris, France). Image processing, quantification, and signal intensity analysis were performed using M3 Vision (Biospace Lab). The number of photons emitted per second per square centimeter per steradian was calculated as a measure of luciferase activity.

General animal welfare conditions and ethics.

Vector administration and in vivo imaging were performed under isoflurane anesthesia (4% isoflurane and oxygen). The studies were carried out in strict accordance with the recommendations of the Dutch Animal Welfare Act (Dutch: Wet op Dierproeven). Animals were maintained under pathogen-free conditions in the animal facility of the University of Amsterdam.

5.2. Results.

At the first time point, on the third day, AAV-mediated expression in the knee was detected in all groups and increased over time (Fig. 3B). All capsid mutants except AAV1-P4 showed increased expression compared to wtAAV5, with AAV9-A2 showing the highest expression (∼5-fold increase over wtAAV5 at day 14) ( Fig. 3C ). Expression levels on day 14 from high to low: AAV9-A2 > AAVrh10-A2 > AAVrh10A6 > AAV7-A6 > wtAAV5 > AAV1-P4. On day 7, AAVrh10-A2, AAV9-A2 and AAVrh10-A6 showed significantly increased expression compared to wtAAV5 (**P<0.05, ***P<0.01, ****P<0.00001 by compared with wtAAV5 at day 14 (Fig. 3B).

Example 6.

Determination of titers of neutralizing antibodies against capsid mutants in human sera.

6.1. Materials and methods.

HEK293T cells were seeded in DMEM containing 9% FBS, 0.9% penicillin/streptomycin in 96-well clear-bottom plates. Cells were left undisturbed for 24 hours (at 37°C, 5% CO ₂ ) before transduction. Human serum samples (obtained from the French Blood Institute) were diluted as follows: pure undiluted serum -1:4 -1:16 - 1:64 - 1:256 - 1:1024 (pure serum means 1 volume of virus per 1 volume of serum). A pooled mouse plasma sample (from 10 DBA/1 mice, collected 42 days after intra-articular injection of AAV5 vector) was serially diluted in FBS as follows: 1:10 - 1:50 - 1:250 - 1:6250 - 1:31250. Human intravenous immunoglobulin solution (IVig, Sanquin, lot 15D30H4560A) was serially diluted semilogarithmically from 1:10 to 1:10,000. Samples and controls were incubated together with the corresponding capsid mutant or wtAAV5 vector for 30 ± 5 min. at 35-38°C at an MOI of 2500 (as previously determined). After 48±2 hours, luciferase reagent was added and luminescence emission was measured using a VictorX microplate reader. Transduction inhibition titers were defined as the highest serum dilution still associated with detectable neutralizing activity, i.e. >50% neutralizing activity.

6.2. Results.

As presented in table. 3, 70-85% of samples did not contain neutralizing antibodies against wtAAV5 or 7 capsid mutants. Most samples had overall reactivity against the seven capsid mutants, so a serum sample that was reactive against the wild-type AAV5 capsid was also reactive against the other capsids. The level of response was also comparable between capsid mutants. The number of samples that did not react (NO = not detected) is indicated for each capsid mutant. These data are provided for informational purposes only as it is very difficult to compare titers with different vectors. As for the pooled mouse serum sample from intra-articularly injected joints, it responded only to the WT AAV5 capsid that was used to immunize the animals, whereas no response was observed to the mutant capsids (Table 3). All capsid mutants and WT AAV5 were neutralized by IVIg (titers >100) (data not shown).

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Table 3

For each serum sample, as well as for the pooled mouse plasma, the inhibitory titer that corresponds to the highest dilution still associated with detectable neutralizing activity is reported. Titers >8 are considered seropositive. Positive signals are shown in bold/italics. BUT = not detected. ________________________________

AAV5 AAV9A2 AAV-DJ-QR- AAVrhlO- AAV1-P4 AAV2-P2 AAV7-A6 AAVrh10-A6 P2 A2 sample 1 256 256 256 256 >1024 256 >1024 >1024 sample 2 4 BUT BUT BUT BUT BUT BUT BUT sample 3 BUT BUT BUT BUT But BUT But But sample 4 BUT But But But But But But But sample 5 But But But But But But But But sample 6 But But But But But But But But sample 7 64 64 64 256 256 64 256 256 sample 8 16 16 16 64 16 64 64 64 sample 9 BUT BUT BUT BUT BUT BUT BUT BUT sample 10 4 4 16 4 64 64 64 64 sample 11 BUT BUT But BUT BUT BUT BUT BUT sample 12 BUT BUT But BUT But 1 But But sample 13 But But But But But But But But sample 14 But 1 But But 4 1 But 1 sample 15 But But But But BUT But But But sample 16 1 1 4 4 4 16 1 1 sample 17 But But But BUT BUT But But But sample 18 But But But But BUT But But But sample 19 But But But But 4 But But But sample 20 4 256 64 4 BUT 256 16 16 % negative 85 80 75 85 80 70 75 75 s samples mouse plasma 256 BUT BUT BUT BUT BUT BUT BUT

The present invention has been described above with reference to a number of illustrative embodiments, as shown in the drawings. Modifications and alternative implementations of some parts or elements are possible and are included within the scope of protection as defined by the appended claims.

Claims (31)

1. The use of a recombinant adeno-associated virus (rAAV) virion containing a modified capsid protein for the treatment or prevention of arthritis, or for the treatment or prevention of symptoms associated with arthritis, wherein the modified capsid protein contains in the terminal part of the protein the amino acid sequence Z, the residues of which are located on surface of the capsid protein, and in which the amino acid sequence Z is: a) contains or consists of a sequence of amino acid residues of formula I: where x represents one amino acid residue, and where y represents 0, 1 or 2 amino acid residues; And b) is present in the region corresponding to the position of 100-200 amino acid residues from the end of the wild-type AAV capsid protein. 2. Use according to claim 1, wherein the amino acid sequence Z is present at the region corresponding to position 120-180, preferably 130-170, more preferably 140-160 amino acid residues from the C-terminus of the wild-type AAV capsid protein. 3. Use according to any of the preceding claims, wherein the Z sequence is contained in the modified capsid protein in the region represented by formula II: EEEIxxxxPVATExxGxxxxNxQy - Z (x)nLPGMVWQxRDVYLQGPIWAKIPHTDG c) where Z, x and y have the meanings defined in paragraph 1; And d) where n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. 4. Use according to claim 1 or 2, wherein the capsid protein contains an amino acid sequence selected from the group consisting of: - 26 045763 i) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 1, and wherein the amino acids at positions 588-602 of SEQ ID NO: 1 have at least 80% sequence identity with SEQ ID NO: 11, ii) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 2, and wherein the amino acids at positions 585-599 of SEQ ID NO: 2 have at least 80% sequence identity to SEQ ID NO: 10, iii) an amino acid sequence having at least 70% sequence identity to the amino acid sequence having SEQ ID NO: 3, and wherein the amino acids at positions 587-601 of SEQ ID NO: 3 have at least at least 80% sequence identity to SEQ ID NO: 9, iv) an amino acid sequence having at least 70% sequence identity to the amino acid sequence having SEQ ID NO: 4, and wherein the amino acids are at positions 586-600 of SEQ ID NO: 4 have at least 80% sequence identity with SEQ ID NO: 8, v) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 5, and wherein the amino acids at positions 588-602 of SEQ ID NO: 5 have at least 80% sequence identity with SEQ ID NO: : 9, vi) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 6, and wherein the amino acids at positions 588-602 of SEQ ID NO: 6 have at least 80% sequence identity with SEQ ID NO: 8, and vii) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 7, and wherein the amino acids at positions 587-601 of SEQ ID NO: 7 have at least 80 % sequence identity to SEQ ID NO: 12, wherein the modified capsid protein provides at least a twofold increase in expression, preferably in human FLS cells, compared to an unmodified capsid protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 13-19, when tested under the same conditions, preferably the unmodified capsid protein having the amino acid sequence of SEQ ID NO: 19 or the same serotype as the modified capsid protein. 5. Use according to any one of claims 1, 2 and 4, wherein the capsid protein contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-7. 6. Use according to any of the preceding claims, wherein the rAAV virion contains: i) a nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) sequence, and ii) a nucleotide sequence encoding a gene product of interest, wherein preferably the nucleotide sequence encoding the gene product of interest is located between two AAV ITR sequences. 7. The use of claim 6, wherein the gene product of interest treats, prevents or suppresses symptoms associated with arthritis, more preferably the gene product of interest is selected from the group consisting of interleukins, immunomodulators, antibodies, shRNA, microRNA, guide RNA, growth factors, protease, nucleotidase/nucleosidase, peptides, protease inhibitors, inhibitors, enzymes and combinations thereof, even more preferably the gene product of interest is at least one of CD39, CD73 and IFN-β. 8. The use of claim 6, wherein the gene product of interest is a tumor necrosis factor alpha (TNFa) inhibitor. 9. Use according to claim 8, wherein the TNFa inhibitor is selected from the group consisting of the following drugs: etanercept, infliximab, adalimumab, certilizumab pegol, golimumab. 10. Use according to claim 9, wherein the TNFa inhibitor is etanercept. 11. Use according to any of the preceding claims, wherein the rAAV virion contains at least one of the following: (i) a polynucleotide containing a sequence encoding at least one guide RNA; wherein said or each guide RNA is substantially complementary to the target polynucleotide sequence(s) in the genome; and (ii) a polynucleotide containing a sequence encoding a nuclease; in this case, the nuclease forms a ribonuclease complex with guide RNA, and at the same time, the ribonuclease complex carries out site-specific double-strand DNA breaks (DSDBs) in the genome. - 27 045763 12. Use as claimed in any one of the preceding claims, wherein the arthritis is selected from the group consisting of rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, septic arthritis , arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement and Still's disease. 13. Use of a recombinant adeno-associated virus (rAAV) virion containing a modified capsid protein as a drug for the treatment or prevention of arthritis, or for the treatment or prevention of symptoms associated with arthritis, wherein the modified capsid protein contains an amino acid sequence in the C-terminal part of the protein Z, the residues of which are located on the surface of the capsid protein, and in which the amino acid sequence of Z is: a) contains or consists of a sequence of amino acid residues of formula I: where x represents one amino acid residue, and where y represents 0, 1 or 2 amino acid residues; and b) is present in the region corresponding to the position of 100-200 amino acid residues from the end of the wild-type AAV capsid protein. 14. Use according to claim 13, wherein the amino acid sequence Z is present at the region corresponding to position 120-180, preferably 130-170, more preferably 140-160 amino acid residues from the C-terminus of the wild-type AAV capsid protein. 15. Use according to claim 13 or 14, wherein the sequence Z is contained in the modified capsid protein in the region represented by formula II: EEEIxxxxPVATExxGxxxxNxQy - Z (x) _n LPGMVWQxRDVYLQGPIWAKIPHTDG c) where Z, x and y have the meanings defined in paragraph 1; And d) where n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. 16. Use according to any one of claims 14-15, wherein the capsid protein contains an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 1, and wherein the amino acids at positions 588-602 of SEQ ID NO: 1 have at least 80% sequence identity with SEQ ID NO: : 11, ii) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 2, and wherein the amino acids at positions 585-599 of SEQ ID NO: 2 have at least 80% sequence identity with SEQ ID NO: 10, iii) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 3, and wherein the amino acids at positions 587-601 of SEQ ID NO: 3 have at least 80% sequence identity to SEQ ID NO: 9, iv) an amino acid sequence having at least 70% sequence identity to the amino acid sequence having SEQ ID NO: 4, and wherein the amino acids at positions 586-600 of SEQ ID NO: 4 have at least at least 80% sequence identity with SEQ ID NO: 8, v) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 5, and wherein the amino acids at positions 588-602 of SEQ ID NO: 5 have at least 80% sequence identity with SEQ ID NO: : 9, vi) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 6, and wherein the amino acids at positions 588-602 of SEQ ID NO: 6 have at least 80% sequence identity with SEQ ID NO: 8, and vii) an amino acid sequence having at least 70% sequence identity with the amino acid sequence having SEQ ID NO: 7, and wherein the amino acids at positions 587-601 of SEQ ID NO: 7 have at least 80 % sequence identity to SEQ ID NO: 12, wherein the modified capsid protein provides at least a twofold increase in expression in human cells compared to an unmodified capsid protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 13-19, when tested under the same conditions, preferably the unmodified capsid protein having the amino acid sequence of SEQ ID NO: 19 or the same serotype as the modified capsid protein. 17. Use according to any one of claims 13-15 or 17, wherein the capsid protein contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-7. 18. Use according to any one of claims 13-17, in which the rAAV virion contains: i) a nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) sequence; and ii) a nucleotide sequence encoding a gene product of interest, wherein preferably the nucleotide sequence encoding the gene product of interest is located between two AAV ITR sequences. 19. The use of claim 18, wherein the gene product of interest treats, prevents or suppresses symptoms associated with arthritis, more preferably the gene product of interest is selected from the group consisting of interleukins, immunomodulators, antibodies, shRNA, microRNA, guide RNA, growth factors, protease, nucleotidase/nucleosidase, peptides, protease inhibitors, inhibitors, enzymes and combinations thereof, even more preferably the gene product of interest is at least one of CD39, CD73 and IFN-β. 20. The use of claim 18, wherein the gene product of interest is a tumor necrosis factor alpha (TNFa) inhibitor. 21. Use according to claim 20, wherein the TNFa inhibitor is selected from the group consisting of the following drugs: etanercept, infliximab, adalimumab, certilizumab pegol, golimumab. 22. Use according to claim 21, wherein the TNFa inhibitor is etanercept. 23. Use according to any one of claims 13 to 22, wherein the rAAV virion contains at least one of the following: (i) a polynucleotide containing a sequence encoding at least one guide RNA; wherein said or each guide RNA is substantially complementary to the target polynucleotide sequence(s) in the genome; and (ii) a polynucleotide containing a sequence encoding a nuclease; in this case, the nuclease forms a ribonuclease complex with guide RNA, and at the same time, the ribonuclease complex carries out site-specific double-strand DNA breaks (DSDBs) in the genome. 24. Use according to any one of claims 13 to 23, wherein the arthritis is selected from the group consisting of rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis , septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement and Still's disease. 25. Use according to any one of claims 13 to 24, wherein the medicinal product further comprises a pharmaceutically acceptable carrier. 26. Use according to claim 25, wherein the medicinal product further comprises an empty capsid in a ratio of empty capsid to rAAV virion of at least 1:1. 27. Use according to any one of claims 13 to 26, wherein the drug is used in combination with an immunosuppressant. 28. Use according to any one of claims 13 to 27, wherein the treatment or prophylaxis comprises administering a medicament to the individual and administering an immunosuppressant. 29. Use according to any one of claims 13-28, in which the drug is administered systemically and/or locally. 30. Use according to any one of claims 13 to 28, wherein at least one of the drug and the immunosuppressant is administered topically. 31. Use according to claim 29 or 30, wherein the local administration is intra-articular administration.