NZ767106B2 - A modified raav capsid protein for gene therapy - Google Patents

Abstract

The invention relates to recombinant adeno-associated virus (rAAV) virions for gene therapy, wherein the rAAV virions comprise a novel capsid protein. In particular,the invention relates to the use of such virions in gene therapy for the treatment of an arthritic disease, such as for example rheumatoid arthritis, or symptoms thereof, preferably by intraarticular administration.

Description

A modified rAAV capsid protein for gene therapy Field of the invention The ion relates to the field of recombinant adeno-associated virus (rAAV) based gene therapy, in particular to the use of a mutant capsid rAAV in the treatment or prevention of an arthritic disease.

Background of the invention Recombinant adeno-associated virus (rAAV) vectors have trated excellent safety 1O and efficacy profiles for the delivery of genes in humans in vivo. Therefore, rAAV s have been extensively used for in vivo gene y and have been shown safe and effective in pre-clinical models as well as in clinical trials. rAAV vectors have been successful in a number of gene therapy clinical trials for a range of diseases including haemophilia B, haemophilia A, cystic fibrosis, alpha- 1 rypsin deficiency, spinal muscular atrophy (SMA), Parkinson disease, Duchenne muscular dystrophy and Leber’s congenital amaurosis (Selot et 51]., Current Pharmaceutical Biotechnology, 2013, 14, 1072-1082). Alipogene tiparvovec (Glybera®, uniQure) has been granted marketing authorization in Europe as a gene therapy for the treatment of lipoprotein lipase ency .

Subsequently, gene therapy drug approval was granted for herpes-virus based gene laherparepvec for the treatment of skin cancer (T-Vec, lmlygic®, Amgen) and for ex vivo stem cell retroviral-based gene therapy Strimvelis for the treatment of ADA-SCID (GSK). rAAV vector-based gene therapy has also been applied in toid arthritis (RA), which is a chronic inflammatory e that s ~1% of the population. The pathology of RA extends throughout the synovial joint. The localized nature of the joint makes in vivo gene y very attractive. Therapies providing anti-inflammatory ns aimed at shifting the balance in RA towards an anti-inflammatory state have been applied.

Much work has focused on the development of AAV capsid proteins with desired properties.

Such properties can include higher uction efficiency, tissue/organ tropism, de-targeting of non-desired tissues/organs, or avoidance of pre-existing lizing antibodies.

There is, however, 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 y vectors in arthritic disease and, more precisely, to improve the efficiency of ring genetic material to the targeted tissue, such as the synovial joint or specific cell types within the synovial joint, preferably to fibroblast-like synoviocytes (FLS).

Summary of the invention In a first aspect, the present invention relates to a recombinant adeno-associated virus (rAAV) virion comprising a modified capsid protein for use in treating or preventing an arthritic disease or for use in treating or preventing symptoms associated with an arthritic disease, wherein the modified capsid protein comprises in the C-terminal part of the protein an amino acid sequence Z, residues of which are exposed on the surface of the capsid protein. Preferably, amino acid sequence Z: a. comprises or consists of a sequence of amino acid residues of the formula I: x—G—(x)3—R—(x)3—y—A—Q—A—A wherein x represents a single amino acid residue and wherein y represents 0, 1 or 2 amino acid residues; and b. is present at a location corresponding to a position 100 — 200, preferably 120 — 180, more preferably 130 — 170, more preferably 140 — 160 amino acid residues from the C terminus of a wild-type AAV capsid protein.

Preferably, amino acid residues of the formula I are exposed on the surface of the capsid protein.

In a preferred embodiment, sequence Z is comprised in the ed capsid protein at a location represented by the formula II: EEEIXXXXPVATEXXGXXXXNXQy — Z — (x)nLPGMVWQXRDVYLQGPIWAKIPHTDG wherein Z, x and y are as defined above; and wherein n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In a preferred embodiment, the present ion relates to an rAAV virion comprising a modified capsid protein for use in treating or preventing an arthritic disease or for use in treating or preventing ms associated with an tic disease, wherein the capsid protein comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 70% sequence identity with an amino acid sequence having SEQ ID NO: 1 and wherein 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% ce identity with an amino acid sequence having SEQ ID NO: 2 and n 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 ce having at least 70% sequence ty with an 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 ty with SEQ ID NO: 9; iv) an amino acid sequence having at least 70% sequence ty with an amino acid sequence having SEQ ID NO: 4 and wherein amino acids 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% ce identity with an amino acid sequence having SEQ ID NO: 5 and wherein 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 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 ce having at least 70% sequence identity with an amino acid sequence having SEQ ID NO: 7 and wherein amino acids at positions 587 — 601 of SEQ ID NO: 7 have at least 80% sequence identity with SEQ ID 40 NO: 12; wherein the modified capsid protein provides for an at least two-fold increase in expression in comparison 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 has the same serotype as the modified capsid protein.

In a preferred embodiment, the ed capsid protein provides for an at least two-fold increase in expression in human FLS cells in comparison to the unmodified capsid protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 13 — 19, when tested under the same ions, wherein preferably the unmodified capsid protein has the amino acid sequence having SEQ ID NO: 19 or has the same serotype as the modified capsid protein. 1O Alternatively, or in combination with any one of the preceding ments, in a preferred embodiment of the present invention, the capsid protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 — 7.

Alternatively, or in combination with any one of the preceding embodiments, in a red 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 comprises a nucleotide sequence encoding a gene product of interest. Even more preferably, the nucleotide sequence encoding a gene t of interest is located between two AAV ITR sequences.

In a preferred embodiment, the gene product of interest treats, prevents or sses symptoms associated with an tic disease, wherein ably the gene product of interest is selected from the group consisting ofinterleukins, immune-modulators, antibodies, shRNA, miRNA, guide RNA, , growth factors, proteases, tidases/nucleosidases, peptides, protease inhibitors, inhibitors, enzymes and combinations thereof, and wherein more preferably the gene product of interest is at least one of CD39, CD73 and IFN-B.

Alternatively, or in combination with any one of the preceding embodiments, in a preferred ment of the present invention, the rAAV virion comprises at least one of: (i) a polynucleotide comprising a sequence encoding at least one guide RNA; wherein the or each guide RNA is substantially mentary to a target polynucleotide sequence(s) in a genome; and (ii) a polynucleotide comprising a sequence encoding a nuclease; wherein the nuclease forms a ribonuclease complex with the guide RNA, and n the clease complex makes site- ic double-stranded DNA breaks (DSDB) in the genome.

In another aspect, the present invention relates to an rAAV composition for use in treating or ting an tic disease or for use in treating or ting symptoms associated with an arthritic disease, wherein the rAAV composition comprises an rAAV virion according to the invention and a pharmaceutically acceptable carrier. In an embodiment, the rAAV composition further comprises an empty capsid in a ratio of empty capsid to rAAV virion of at least 1:1, at least 5:1 or at least 10:1.

In another aspect, the present invention s to an rAAV composition and an immunosuppressant for use in treating or preventing an arthritic disease or for use in treating or ting symptoms associated with an arthritic disease, wherein the rAAV composition is an rAAV 40 composition according to the invention and preferably wherein the treatment or prevention comprises the stration of the rAAV composition and the administration of the immunosuppressant to an individual.

Alternatively, or in combination with any one of the preceding embodiments, in a preferred ment of the present invention, the arthritic disease is selected from the group consisting of rheumatoid arthritis (RA), juvenile toid tis, rthritis (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 one of the preceding embodiments, in a preferred embodiment of the present invention, the rAAV virion or the rAAV composition is administered ically and / or y. In a red embodiment, at least one of the rAAV composition and the immunosuppressant is administered locally. Preferably, the local administration is intraarticular administration.

In a further aspect, the present invention relates to a method for treating, preventing, or suppressing symptoms associated with an arthritic disease, wherein the method comprises the step of intraarticular administration of a medicament comprising an effective amount of an rAAV virion or an rAAV composition according to the invention.

Detailed description of the invention The inventors have discovered that a recombinant adeno-associated virus (rAAV) virion comprising a modified capsid protein is surprisingly efficient at transducing cells, and in particular efficient at transducing cells of the synovial joint. As last-like synoviocytes (FLS) typically are the primary target cell in the joint in the treatment of arthritic diseases, such as for example rheumatoid arthritis, the aim of the present invention is to provide for capsid proteins which are improved in one or more of the following properties: i) higher expression levels in the synovial tissue, in particular in FLS; ii) improved synovial tissue m, in particular ed tropism for FLS; and/or iii) improved de-targeting to non-desired tissues/organs upon rAAV administration as compared to capsid proteins known in the art. In particular, these ties of the rAAV virion comprising a modified capsid protein of the invention are improved as compared to unmodified capsid proteins, preferably the ype capsid protein of the same serotype as the modified capsid protein and/or AAV5 capsid proteins. It has been previously established that AAV5 capsid gives rise to the highest FLS sion levels when ed with other AAV serotypes (Adriaansen et al. (2005) Ann Rheum Dis 64:1677-1684; Apparailly et al. (2005) Hum. Gene Ther. 16:426-434).

As it is the capsid that confers the tissue/cell tropism properties, the modified capsids described in this invention have the property of enhanced FLS transduction potential preferably when compared with fied AAV5. In particular, it is preferred that the capsid proteins of the t ion provide higher expression levels in synovial tissue, in particular in FLS, preferably upon intraarticular administration, as compared to unmodified capsid proteins (that is, the same capsid protein without the modification that is to be tested, preferably of the same serotype as the ed capsid ns), preferably wild-type unmodified capsid proteins (preferably of the same serotype 40 as the modified capsid proteins), more preferably unmodified AAV5 or thAV5 capsid proteins.

Hence, in a first aspect, the invention ns to an rAAV virion sing a modified capsid protein. The rAAV virion as defined herein is particularly useful for use in gene therapy.

As used herein, "gene y" is the insertion of nucleic acid ces (such as for example a transgene (also ed to as a nucleotide sequence encoding a gene product of interest) as defined herein below) into an individual's cells and/or tissues to treat or prevent a disease or er or to treat or prevent the ms of a disease or disorder.

AAV can infect both dividing and quiescent cells and infection occurs by interaction of the capsid proteins with a cell-membrane receptor, followed by endocytosis of the AAV virion. AAV s to the genus Dependovirus, which in turn belongs to the subfamily of the Parvovirinae, also 1O referred to as parvoviruses, which are capable of infecting vertebrates. Parvovirinae belong to a family of small DNA animal viruses, i.e. the Parvoviridae family. As may be deduced from the name of their genus, members of the Dependovirus are unique in that they usually require coinfection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture. The genus Dependovirus es AAV, which normally infects humans, and d viruses that infect other warm-blooded animals (6.9., bovine, canine, equine, and ovine adeno-associated viruses). Further information on parvoviruses and other s of the Parvoviridae is described in Kenneth l.

Berns, "Parvoviridae: The s and Their Replication," Chapter 69 in Fields Virology (3d Ed. 1996). For convenience, the t invention is further exemplified and described herein by reference to AAV. It is however understood that the invention is not d to AAV but may equally be applied to other parvoviruses.

The c organization of all known AAV serotypes is very r. The genome of AAV is a linear, -stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. lnverted terminal repeats (lTRs) flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP1, -2 and -3) form the capsid or protein shell with the help of the assembly-activating protein (AAP) (for some serotypes), which is encoded in an alternative open reading frame overlapping with that of VP2NP3. The terminal nucleotides are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. The size of the terminal nucleotides is serotype-dependent. For example, in the case of AAV2, of the terminal 145 nt, 125 nt are self-complementary and the remaining 20 nt remain single-stranded. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wild-type AAV (thAV) infection in mammalian cells the Rep proteins (i.e. Rep78 and Rep52) are expressed from mRNAs transcribed by the p5 promoter and the p19 promoter, respectively. Both Rep proteins have a function in the replication of the viral genome. A splicing event in the Rep ORF results in the expression of actually four Rep ns (i.e. Rep78, Rep68, Rep52 and Rep40). However, it has been shown that the Rep78 and Rep52 proteins, encoded by the unspliced mRNAs, in mammalian cells are sufficient for AAV vector production. Production of thAV or rAAV in mammalian cells moreover relies on a combination of alternate usage of two splice acceptor sites and the suboptimal utilization of an ACG initiation codon for VP2, which ensures proper expression of all three capsid proteins in an approximate 1:1 :10 ratio (VP1:VP2:VP3).

An "rAAV virion" (also referred to as an "rAAV vector" or an "rAAV transgene vector" herein) as used herein means an AAV capsid comprising a non-native nucleic acid sequence. Such a sequence in rAAV is generally flanked by ITR sequences, preferably from thAV, and preferably encodes a gene product of st, such as for example a transgene or homology arms. Said differently, an rAAV virion means an rAAV genome, comprising (i) a nucleotide sequence encoding a gene product ofinterest and (ii) at least one AAV ITR sequence, encapsidated by capsid ns.

An rAAV genome may have one or preferably all thAV genes deleted, but may still comprise 1O functional ITR nucleic acid sequences. Preferably, the rAAV virion does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV. Thus, an rAAV virion is guished from a thV virion, since all or a part of the viral genome has been replaced with a nucleotide sequence encoding a gene product of interest, which is a non-native nucleic acid with respect to the AAV nucleic acid sequence as further defined herein.

In a preferred embodiment, an rAAV virion comprising a modified capsid protein of the invention is for use in ng or preventing an arthritic disease or for use in treating or ting symptoms ated with an arthritic e. The medical use (6.9. gene therapy for treatment or tion of (symptoms associated with) arthritic e) herein described is formulated as an rAAV virion according to the ion for use as a medicament for prevention or treatment of the disease(s) and/or disorder(s) defined herein, but could equally be formulated as (i) a method of tion or treatment of the disease(s) and/or disorder(s) defined herein or symptoms thereof comprising administering a sufficient or an effective amount of an rAAV virion according to the invention to a subject in need thereof, as (ii) an rAAV virion according to the invention for use in the preparation of a ment to prevent or treat the disease(s) and/or disorder(s) defined herein, or as (iii) use of an rAAV virion according to the invention for the prevention or treatment of the disease(s) and/or disorder(s) defined herein. Such medical uses are all envisaged by the present invention. Preferably, the modified capsid protein comprises in the C-terminal part of the protein an amino acid sequence Z, es of which are exposed on the e of the capsid protein.

As used herein, the terms "treat", "treatment", or "treating" refer to application or administration of an rAAV virion of the invention to a subject who has an arthritic disease, n the object is to cure, partially or tely reverse, alleviate, ameliorate, inhibit, delay, suppress, slow down or stop the progression or severity of an arthritic disease, or of the symptoms associated with the arthritic disease. The term "treating" includes reducing or ating at least one adverse effect or symptom of the arthritic disease. Treatment is generally "effective" if one or more ms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the arthritic disease is reduced or . That is, "treatment" includes notjust the improvement of ms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of 40 disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and ion (whether partial or total), whether detectable or undetectable. The term "treatment" of an arthritic disease also includes providing relief from the symptoms or side-effects of the arthritic disease ding palliative treatment). As used herein, the term nt", "prevention", or "preventative" (also referred to as prophylactic) refer to application or administration of an rAAV virion according to the invention to a subject who has a predisposition toward an arthritic disease, with the e to delay or prevent onset of, alleviate, ameliorate, relieve, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a future arthritic e. Thus, an rAAV virion according to the invention may be administered to a subject who does not exhibit signs of an arthritic disease and/or 1O to a subject who exhibits only early signs of an arthritic disease, preferably for the purpose of decreasing the risk of developing pathology associated with the arthritic disease.

The term "cure" or "curing" as used herein means to completely alleviate one or more, preferably all of the symptoms or features of an tic disease. The term "delay" or "delaying" as used herein means to delay onset of and/or inhibit progression of and/or reduce severity of one or more of the symptoms or features of the arthritic disease.

In a preferred embodiment, the modified capsid protein of the invention provides for an at least two-fold increase in expression in comparison to an unmodified capsid protein, when tested under the same conditions. Preferably, the fied capsid protein, is a capsid protein of the same serotype as the modified capsid protein, but without the modification that is to be tested. More preferably, the fied capsid n is a wild-type (wt) capsid protein of the same pe as the modified capsid protein, wherein the wt capsid protein preferably has an amino acid sequence selected from the group consisting of SEQ ID NO: 13 — 19. Alternatively, it is preferred that the unmodified capsid protein has an amino acid sequence ed from the group consisting of SEQ ID NO: 13 — 19. Most ably, the unmodified capsid n has the amino acid sequence as shown in SEQ ID NO: 19. The preferred unmodified capsid proteins may depend on the tissue that is to be ed by the rAAV virion. For example, an rAAV with AAV5 capsid proteins appears to be a virion of choice for FLS cells (Apparailly et al. (2005) Human Gene Therapy 16(4):426—434; Adriaansen et al. (2005) Ann. Rheum. Dis. 64(12):1677-1684) and therefore — irrespective of the original serotype of the rAAV mutant virions — the rAAV control virion preferably comprises AAV5 capsid proteins, more preferably ype AAV5 (thAV5) capsid protein, more preferably the AAV5 capsid protein has the amino acid sequence shown in SEQ ID NO:19, even more preferably the rAAV control virion is an rAAV5 . An rAAV control virion is an rAAV virion comprising unmodified capsid ns as defined herein instead of modified capsid proteins. In a red embodiment, the rAAV virion (comprising modified capsid proteins) provides for higher expression upon in vitro transduction in Fibroblast Like Synoviocytes from rheumatoid athritis patients (RA- FLS) and/or HEK 293, preferably HEK293T, cells as compared to the same rAAV virion with unmodified capsid proteins as defined herein instead of modified capsid proteins, using a method as described in the es. In other words, apart from the capsid proteins, the rAAV virion and the rAAV control virion preferably are identical. ably, transduction efficiency is detected in an 40 in vitro uction assay: by measuring expression levels of a reporter gene encoded by the transgene, such as GFP, YFP and/or Luciferase. In a preferred embodiment, the test to determine the expression 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) are plated at 2500 cells/well or HEK293T cells (human nic kidney cells) are plated at 40,000 cells/well in a 96-well plate (DMEM-GlutaMAX—l (Gibco, ref. 31966-021), 10% FBS (heat inactivated (Hl) Bovine Serum Gold, Gibco ref. A15-151), 100 pg/ml Penicillin/100 pg/ml omycin (Sigma- Aldrich, ref.P0781; 37°C / 5% C02). After 24 hours, supernatant is removed and ed by medium (DMEM-glutaMAX—l (Gibco, ref. 31966-021), 0.001% pluronic F68 (Sigma, ref. p5559)) containing the rAAV mutant virions or the rAAV control virions — all expressing yellow fluorescent 1O protein (yFP) and/or luciferase under control of a galovirus (CMV) promoter— at a licity of infection (MOI) of 10,000, 20,000 and 100,000. Crude lysates (i.e. non-purified supernatants of cells transfected with all plasmids needed for rAAV production and containing reporter-expressing virions) or purified AAV (preferably based on lodixanol purification or cesium chloride (CsCl) density gradient purification) can be used. Four hours after transduction, medium (DMEM-GlutaMAX—l, 10% FBS 100 U/ml llin, 100 pg/ml streptomycin) ning doxorubicin (Sigma, ref. D1515; final concentration 0.4 pM), FBS (final concentration 1%), is added. After 48h hours (HEK293T) or 4-6 days (RA-FLS), cells are assayed for the tage 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.

A "serotype" is traditionally defined on the basis of a lack of cross-reactivity between antibodies to one virus as compared to another virus. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (6.9., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes). Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus es are discovered and capsid mutants generated, there may or may not be gical ences with any of the currently existing serotypes. Thus, in cases where the new AAV has no serological difference, this new AAV would be a subgroup or variant of the corresponding serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype.

Accordingly, for the sake of convenience and to avoid repetition, the term "serotype" y refers to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are not serologically ct that may be within a up or a variant of a given serotype.

"Transduction" refers to the transfer of a transgene into a recipient host cell by a viral vector.

Transduction of a target cell by an rAAV virion of the invention leads to er of the transgene contained in that rAAV virion into the transduced cell. "Host cell" or "target cell" refers to the cell into which the DNA delivery takes place, such as the ocytes or al cells of an individual, or 40 such as isolated FLS cells from patients or T cells in case of the in vitro transduction assay.

AAV s are able to transduce both dividing and non-dividing cells. In a cell comprising a gene product of interest, such as for example GFP, the gene product of interest has been introduced/transferred/transduced by rAAV "transduction" of the cell. A cell into which the transgene has been introduced is referred to as a "transduced" cell.

The recipient host cell wherein the transgene is transduced preferably is a cell that is affected by the e that is to be treated, such as for example synovial cells, more ically FLS, macrophages, monocytes, neutrophils, osteoblasts, osteoclasts, ocytes, T-lymphocytes, dendritic cells, plasma cells, mast cells, B lymphocytes in case of an arthritic disease. The "synovium" or "synovial tissue" or "synovial cells" as used herein refers to the ar lining covering the non-cartilaginous surfaces of the synovial joints, as further described in Tak (2000, Examination of the synovium and synovial fluid. In: ein GS, Panyani GS, Wollheim FA s. Rheumatoid Arthritis. New York: Oxford Univ. Press, Inc. 55-68) and incorporated herein by reference. The synovium consists of the intimal lining layer (or synovial lining layer) and the synovial sublining (subsynovium), which merges with the joint capsule. The intimal lining layer comprises intimal macrophages (or macrophage-like synoviocytes or type A synoviocytes) and FLS (or type B synoviocytes). "Synovium" may therefore be replaced by, or is synonymous with, "synovial ".

A synovial cell can include any cell present in the um including FLS and macrophage-like synoviocytes. A synoviocyte cell may also be a neutrophil, T, B cells and/or connective tissue cells, which may all be present in the synovium.

"Fibroblast-like synoviocytes" (FLS) are cells of mesenchymal origin that display many characteristics that are in common with fibroblasts, such as expression of specific proteins, such as for example several types of collagens. However, FLS also secrete ns that are normally absent in otherfibroblast lineages, such as for example lubricin. In addition, FLS express molecules that are important for the mediation of cell adhesion, such as cadherin-11, VCAM-1, several integrins and their receptors. ic for FLS is the expression of CD55 and this protein is therefore typically used to fy FLS in the synovium by immunohistochemistry. FLS represent a specialized cell type located inside joints in the synovium, whose cells play a crucial role in the pathogenesis of chronic inflammatory diseases, such as rheumatoid arthritis (RA). The term "rheumatoid synovium" or "rheumatoid synovial cells" or "rheumatoid synovial tissue" refers to the inflamed um of the joints of an individual suffering from RA. The rheumatoid synovium is characterized by intimal lining hyperplasia and by accumulation of FLS, T-cells, plasma cells, hages, B-cells, natural killer cells and dendritic cells in the al sublining. These accumulated cells are comprised in the definition of rheumatoid synovial cells. During the progression of RA, the synovial tissue becomes a place where constant inflammatory processes take place, which can eventually lead to age damage and joint destruction and deformation.

FLS that are t in the um during RA have been reported to y an altered phenotype compared to the FLS present in normal tissues. For example, the FLS in toid synovium lose "contact inhibition", i.e., they lose the property to arrest their growth when more cells come into contact with each other. In addition, they lose the dependency to grow on adhesive surfaces. As a 40 result, the number of FLS in the diseased synovium increases. The inflammation is further enhanced by the production of several pro-inflammatory signaling les, particularly interleukins lL-6 and lL-8, prostanoids and matrix metalloproteinases (MMPs).

Alternatively, or in ation with r embodiment, in a further preferred embodiment of the present invention, an rAAV virion comprising a modified capsid protein according to the invention provides for an at least two-fold increase in sion of the transgene in human FLS in comparison to an rAAV virion comprising an fied capsid protein as defined herein, preferably to the 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 same serotype as the modified capsid n or has the amino acid 1O sequence shown in SEQ ID NO: 19.

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

Also preferred, or in addition to the above, the rAAV virion provides for increased expression of the ene upon in vivo administration to the air pouch synovium (APS) mouse model (adapted from Edwards et al (1981) J Pathol 134: 147-156 as bed in Example 4) as compared to an rAAV control virion, preferably as compared to an rAAV virion comprising thAV5 capsid proteins, provided that the rAAV is othenNise identical (apart from its capsid protein(s)).

Preferably, sion of the transgene is at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30- -fold increased using an rAAV comprising the mutant capsid proteins of the method of the invention. An exemplary method is provided in the es.

Also preferred, or in addition to the above, the rAAV virion comprising a modified capsid protein provides for similar or lower neutralizing antibody (nAb) titers as compared with the same rAAV virion comprising unmodified, preferably wild-type, AAV capsid n of the same serotype.

WtAAV5 capsids are known to have an attractive nAb profile, and therefore, similar or lower nAb titers of rAAV comprising a modified capsid protein ing to the present invention as compared to thAV5 is preferred.

Alternatively, the in vitro uction assay could be performed similarly as described above, but in a cell type/cell line different from FLS, depending on the cell type that is to be targeted, such as for example, in cells selected from the group consisting of primary hepatocytes, liver cell lines, e.g. HuH, HepG2, HepA1-6, heart cells, skeletal muscle cells, lung cells such as the cell line A549, CNS cells, eye cells, gastrointestinal tract cells, bone marrow cells and blood cells, such as the cell line THP-1. This also may require a ent AAV serotype as preferred control, depending on the tropism of the wild-type capsid proteins. In general, a control vector preferably comprises wild-type capsid proteins that naturally target to the tissue of choice. As the skilled person will appreciate, this may also depend on mode of administration: locally or systemically. For example AAV2, which has been the most extensively examined AAV, presents tropism towards skeletal muscle cells, s, vascular smooth muscle cells and hepatocytes; AAV6 presents tropism towards ainNay 40 epithelial cells; AAV7 presents tropism s skeletal muscle cells; AAV8 ts tropism towards hepatocytes; AAV1 and AAV5 present tropism towards vascular endothelial cells. Upon ic administration, AAV 1-3 and 5-9 have tropism towards the liver, with high n levels observed with AAV9, 8, 7, 6, 1 and to a lesser extent 5 and 2; heart is transduced by AAV4, 6, 7, 8 and 9; thoracic expression is seen for AAV4 and 6 relli et al (2008) Molecular Therapy 16(6): 1073-1080).

Without wishing to be bound by any theory, we believe that the increased expression achieved by the rAAV virions sing a modified capsid protein of the invention as compared to rAAV control virions is caused by an improved transduction of the rAAV into the cell, possibly by altered tropism, resulting in (i) an increased number of cells within the cell population being 1O transduced, and/or (ii) an sed level of expression per cell, for example, due to better virion uptake and/or ellular processing..

Another advantage of the rAAV virion with a modified capsid protein according to the ion could preferably be other improvements such as possible avoidance of pre-existing neutralizing antibodies.

In a preferred ment of the t ion, the modified capsid protein comprises an amino acid sequence Z, preferably amino acid ce Z is comprised in the C-terminal part of the protein. Preferably, sequence Z is 12 — 18 amino acid residues in length (herein also referred to as "loop " and as "insert"). In a preferred embodiment, sequence Z is located in the C- terminal part of the capsid protein, ably at a location corresponding to a position at 100 — 200, preferably 120 — 180, more preferably 130 — 170, more preferably 140 — 160, most ably about 150 amino acids from the C-terminus of the wild-type capsid protein, such as for example shown in SEQ ID NO’s: 13 - 19. Residues of amino acid sequence Z are preferably exposed on the surface of the capsid protein, such as for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17 or 18 residues are exposed on the surface of the capsid protein (a so-called "loop"). In a preferred embodiment, the sequence Z is 14 — 18 amino acid residues in length, more ably, 15, 16, 17 or 18 residues in length, most preferably, 15, 17 or 18 amino acid residues in length. Sequence Z may replace some amino acid residues as compared to the unmodified, 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 of the unmodified, more preferably of the wild-type sequence. Apart from the sequence Z/insert, thus in the framework, the capsid protein may comprise further modifications, such as amino acid substitutions (for example conservative amino acid substitutions) or the ork capsid protein may be as the wild-type amino acid sequence. The framework AAV wherein the insert is comprised can be of any serotype, such as for example AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10 or AAVDJ. Preferably, the ork AAV wherein the sequence Z is comprised is selected from the group consisting of AAV1, AAV2, AAV7, AAV9, AAVrh10, AAVDJ, more preferably from the unmodified capsid proteins having an amino acid sequence as shown in any one of SEQ ID NO: 13 — 19. The insert according to the invention is preferably comprised in the C-terminal part of the capsid protein, preferably at a location 40 corresponding to 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 wild-type capsid protein, such as for example shown in SEQ ID NO’s: 13 — 19, wherein the location of the insert is represented by formula II: xxPVATEXXGXXXXNXQy — Z — (x)nLPGMVWQXRDVYLQGPlWAKlPHTDG wherein x represents a single amino acid residue, wherein y represents 0, 1 or 2 amino acid residue(s) (which thus may be absent), and wherein n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 8, 9 or 10 or wherein the location of the insert is represented by a sequence having at 1O least 90, 93, 95, 96, 97, 98 or 99% sequence identity with formula II. Preferably, the last three amino acid residues preceding the inal end of sequence Z of the invention are NLQ, NHQ or NFQ. ably, y represents 0 or 2 amino acid residues. In some cases, y ents 2 amino acid residues, thus two additional amino acid es, preferably two serine residues, may be present between the NXQ motif and the insert of the ion. This is preferably the case where the NXQ motif is NFQ, for example if the AAV capsid is an AAV1 capsid ce as represented in SEQ ID NO:1. The skilled person will be able to determine these motifs and this region where the insert is located, also if it harbors some variations, such as amino acid substitutions or deletions, which are also encompassed in the scope of the present invention.

In a preferred embodiment, based on the alignments shown in Figures 4 and 5, the insert (sequence Z) comprises or consists of a sequence of the formula: X1-G-Q-X2-G-X3-X4-X5-R-X6-X7-X8- X9-X10-X11-X12-X13-X14-X15, wherein X1 is Q or none, X2 is S or R, X3 is N or C, X4 is D, Y or E, X5 is C, V, S or A, X6 is G, S or V, X7 is none, A, V or R, X8 is D, N or E, X9 is C or A, X1o is F or Q, X11 is none, C or A, X12 is none or A, X13 is none or Q, X14 is none or A and X15 is none or A. Alternatively, the insert (sequence Z) comprises or ts of a sequence of the formula: y1-G-Q-y2-G-y3-y4-y5-R-y6- y7-ys-y9-y1o-A-y11-y12-y13, wherein y1 is Q or none, y2 is S or R, y3 is N or C, y4 is D, Y or E, y5 is C, V, S or A, ya is G, S or V, y7 is none or D, ys is none or C, y9 is A, V, R or F, y1o is N, D, E or C, y11 is none or Q, y12 is none or A, y13 is none or A. In yet another alternative, in the most red ment, based on the alignments shown in Figures 6 and 7, the insert (sequence Z) comprises or consists of a sequence of the general a I: y—G—Q—x—G—(x)3—R—(x)3—y—A—Q—A—A wherein x represents a single amino acid residue and wherein y represents 0, 1 or 2 amino acid residues (which thus may be absent). Preferably, (i) if at the N-terminus y represents 0 amino acids, then the other y within formula I represents 0 amino acid residues or (ii) if at the N-terminus y represents 1 amino acid residue, then the other y within formula I represents 2 amino acid es.

More preferably, the insert (sequence Z) comprises or consists of a sequence of the more specific formula: 40 Zo—G—Q-Z1—G-22—23-Z4—R-Z5Z7—Zs-Ze—A—Q—A-A wherein 20 is none or Q, 21 is R or S, 22 is C or N, Z3 is D, E or Y, 24 is C, A, S or V, 25 is G, V or S, Z6 is d or none, 27 is C or none, 28 is F, R, V or A, 29 is C, D, N or E. More preferably, if 20 is none, then also Z6 and 27 both represent none.

More ably, sequence Z/insert ses or consists of an amino acid sequence that has at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, most preferably 100% sequence identity with any one of the amino acid sequences ed from the group ting of SEQ ID NOs: 8 — 12. It is preferred that sequence Z/insert comprises or consists of an amino acid sequence represented by any one of the formulae above and that has at least 80, 85, 87, 90, 91, 92, 93, 94, 1O 95, 96, 97, 98 or 99%, most preferably 100% sequence identity with any one of the amino acid sequences ed from the group consisting of SEQ ID NOs: 8 — 12.

In mammalian cells, expression of the three AAV capsid proteins (VP1, VP2 and VP3) in the correct iometry relies on a combination of alternate usage of two splice acceptor sites and the imal utilization of a ACG initiation codon for VP2 that is not accurately reproduced by insect cells. Correct stoichiometry is ant for infectivity of the AAV particles. For production of the three AAV capsid proteins in insect cells in the correct stoichiometry, it is common in the art to use a construct that is transcribed into a single polycistronic messenger that is able to express all three VP proteins without requiring splicing. In order to achieve this, the VP1 protein could be under control of a suboptimal translation initiation codon instead of ATG. Examples of such a suboptimal translation tion codon are ACG, TTG, CTG and GTG (Urabe et al. (2002) Human Gene Therapy 13: 1935-1943; US 20030148506; US 20040197895; the production of rAAV in insect cells a nucleic acid cassette can be used for sing VP1, VP2 and VP3 proteins, where these proteins are encoded by a nucleic acid sequence comprising overlapping open reading frames (ORFs) as described in European patent No. 891 B1, where a VP expression cassette is disclosed that comprises an intron comprising a promoter prior to the VP2 ACG initiation codon. The modified capsid protein of the invention is defined with t to the n sequence of the VP1 capsid protein. However, since sequence Z/insert is located in the C-terminal part of the VP1 protein, it is included in the invention that also VP2 and VP3 proteins harbor the sequence Z/insert and thus are modified (irrespective of the method of production of rAAV, such as for example in insect cells or in mammalian cells).

Alternatively, or in combination with another embodiment, in a further preferred ment of the present invention, the modified capsid protein according to the invention comprises or consists of an amino acid ce 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%, most preferably having 100% sequence identity with an amino acid sequence having SEQ ID NO: 1 and wherein amino acids at positions 588 — 602 of SEQ ID NO: 1 have at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, most ably having 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%, most preferably having 100% sequence identity with an amino acid sequence having SEQ ID NO: 40 2 and wherein amino acids at positions 585 — 599 of SEQ ID NO: 2 have at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, most preferably having 100% sequence identity with SEQ ID NO: , iii) an amino acid sequence having at least 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, most preferably having 100% sequence identity with an amino acid ce having SEQ ID NO: 3 and wherein amino acids at positions 587 — 601 of SEQ ID NO: 3 have at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, most preferably having 100% sequence identity with SEQ ID NO: 9, iv) an amino acid sequence having at least 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, most preferably having 100% sequence identity with an amino acid sequence having SEQ ID NO: 4 and wherein amino acids at positions 586 — 600 of SEQ ID NO: 4 have at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, most preferably having 100% sequence identity with 1O 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%, most preferably having 100% sequence identity with an amino acid sequence having SEQ ID NO: 5 and n amino acids at positions 588 - 602 of SEQ ID NO: 5 have at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, most preferably having 100% sequence identity with SEQ ID NO: 9, vi) an amino acid sequence having at least 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, most preferably having 100% sequence identity with an amino acid sequence having SEQ ID NO: 6 and wherein 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%, most preferably having 100% sequence identity with SEQ ID NO: 8, and vii) an amino acid sequence having at least 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, most preferably having 100% sequence identity with an amino acid sequence having SEQ ID NO: 7 and wherein amino acids at positions 587 — 601 of SEQ ID NO: 7 have at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, most preferably having 100% sequence identity with SEQ ID NO: 12. Preferably, the framework AAV capsid protein wherein the insert is comprised, has the amino acid sequence of a wild-type AAV capsid, such as for example of AAV5, AAV1, AAV2, AAV7, AAV9, AAVrh10 or AAVDJ or an amino acid ce sing conservative amino acid substitutions. More ably, the framework AAV capsid protein wherein the insert is comprised, has the amino acid sequence of a thV5 capsid or an amino acid sequence comprising conservative amino acid substitutions. nce ty" is herein d as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as 3O determined by comparing the ces. In a preferred embodiment, ce identity is calculated based on the full length of two given SEQ ID NO or on part f. Part thereof preferably means at least 10, 20, 30, 40, 50, 60, 70, 80, 90%, or 100% of both SEQ ID NO. In the art, "identity" also means the degree of sequence dness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.

Unless otherwise indicated herein, identity or similarity with a given SEQ ID NO means identity or similarity based on the full length of said sequence (i.e. over its whole length or as a whole).

"Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its ved amino acid tutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but 40 not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: atics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer is of Sequence Data, Part |, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular y, von Heine, G., Academic Press, 1987; and Sequence is Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and , D., SIAM J. d Math., 48:1073 (1988)).

Preferred methods to determine identity are designed to give the t match between the sequences tested. Methods to determine identity and rity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity 1O n two sequences include 6.9., the GCG program 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 NCBl and other sources (BLAST Manual, Altschul, S. et al., NCBl NLM NIH Bethesda, MD 20894; Altschul, S. et al., J. Mol.

Biol. 215:403-410 (1990)). The well-known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison 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 Length Penalty: 4. Such a program is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).

Preferred parameters for nucleic acid comparison include the following Algorithm: Needleman and , J. Mol. Biol. 48:443-453 ; Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap m from Genetics Computer Group, located in Madison, Wis (www.biology.wustl.edu/gcg/gap). Given above are the default parameters for nucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is 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 containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is alanine, ne, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred vative amino acid substitution groups are: -leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. tutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is 40 conservative. Preferred conservative substitutions for each of the naturally occurring amino acids 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 to Pro; His to Asn or Gln; lie to Leu or Val; Leu to lie or Val; Lys to Arg; Gln or Glu; Met to Leu or lle; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Val to lie or Leu.

Alternatively, or in combination with another embodiment, in a further preferred embodiment of the present invention, the capsid protein comprises 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, still more preferably from the group consisting of SEQ ID NO: 4 and 6, most preferably 1O SEQ ID NO:4.

Functional ITR sequences are necessary for the replication, rescue and packaging of rAAV virions. The ITR sequences may be wild-type sequences or may have at least 80%, 85%, 90%, 95%, or 100% sequence identity with wild-type sequences or may be altered by, for example, insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional. In this context, functionality refers to the ability to ly package the genome into the capsid shell and then allow for expression in the host cell to be transduced or target cell. Typically, the ITRs of the wild-type AAV genome are retained in the rAAV-vector. The ITRs can be cloned from the AAV viral genome or excised from a vector comprising the AAV ITRs. The ITR tide sequences can be either ligated at either end to a transgene as defined herein using standard molecular biology 2O techniques, or the ype AAV sequence n the ITRs can be replaced with the desired nucleotide sequence. The ector preferably comprises at least the nucleotide sequences of the ITR regions of one of the AAV pes, or nucleotide sequences substantially identical o, and at least one nucleotide sequence ng a therapeutic protein (under control of a suitable regulatory element) inserted between the two ITRs. The majority of tly used rAAV- vectors use the ITR ces from AAV serotype 2. Most preferred ITRs present in an rAAV- vector are of the AAV2 serotype. Other preferred ITRs are of the AAV1, AAV3, AAV5 or AAV6 serotype (Grimm et al. (2006) J Virol 80(1):426-439). An rAAV genome can comprise single- stranded or double-stranded (self-complementary) DNA. The single-stranded nucleic acid molecule is either sense or antisense strand, as both polarities are equally e of packaging into AAV capsids. Single-stranded rAAV-vectors may e the wild-type AAV pe 2 (AAV2) ITR sequences, and double-stranded (self-complementary) rAAV-vectors may utilize a modified version of the ITRs. Alternatively, in an ment, a double-stranded vector comprises one ITR, which ITR is from AAV4. The rAAV-vector may further se a marker or reporter gene, such as a gene for example ng an antibiotic resistance gene, a fluorescent protein (6.9., gfp) or a gene encoding a chemically, enzymatically or otherwise detectable and/or selectable product (6.9., [5102, alkaline phosphatase (AP), SEAP, Luc, Neo, Bla, etc.) known in the art.

The rAAV-vector, including any possible combination of AAV serotype capsid and AAV genome ITRs, is produced using methods known in the art, for example using a mammalian rAAV tion system or an insect cell rAAV production system. Methods known in the art are for 40 example described 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 based on Urabe et al (Human Gene Therapy (2002) 13:1935-1943), Kohlbrenner et al (Molecular Therapy (2005) 12(6):1217-1225), International Patent publication WO 2007/046703, International Patent publication WO 2007/148971, International Patent publication publication 6,723,551 B, which are incorporated herein by reference. In short, the methods generally may involve (a) the introduction of the rAAV genome construct into a host cell, (b) the introduction of an 1O AAV helper uct into the host cell, wherein the helper uct comprises the viral functions missing from the wild-type rAAV genome and (c) introducing a helper virus construct into the host cell. All functions for rAAV vector replication and packaging need to be present, to achieve replication and packaging of the rAAV genome into rAAV vectors. The introduction into the host cell can be carried out using rd molecular y techniques and can be simultaneously or sequentially. Finally, the host cells are cultured to produce rAAV vectors which are then purified using standard techniques such as CsCI 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 of more than 1012 particles per ml and high purity (free of 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 generally smaller than 5 ses (kb) in size.

In the context of the present invention a capsid protein shell may be of a different serotype than the rAAV-genome, comprising (i) a nucleotide sequence encoding a gene product of interested and (ii) at least one AAV ITR sequence. An rAAV-genome of the ion may thus be encapsidated by a capsid protein shell of the t invention, i.e. the icosahedral capsid, which comprises capsid proteins (VP1, VP2, and/orVP3) according to the present ion, e.g., mutants of AAV capsid proteins according to the invention, whereas the ITRs sequences contained in that rAAV-vector may be any of the AAV serotypes described above, including for example AAV2 or AAV5. In an ment, the rAAV genome or ITRs present in the rAAV virion are derived from AAV serotype 2 or AAV serotype 5 or AAV serotype 8. The te 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 No. AF085716). The ITR nucleotide sequences of AAV2 and AAV5 are thus readily available to a skilled person. The complete genome of AAV2 is available in NCBI (NCBI Reference Sequence 401.2). They can be either cloned or made by chemical synthesis as known in the art, using for example an oligonucleotide synthesizer as supplied 6.9., by d Biosystems Inc. (Fosters, CA, USA) or by standard lar biology techniques.

Alternatively, or in combination with another embodiment, in a r preferred ment of the present ion, the rAAV vector comprises a tide ce encoding a gene 40 product of interest.

The terms "transgene" or "gene product of interest" are used hangeably herein and refer to a tive 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 ribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (e.g., lacks an expression control element, such as a promoter that drives transcription). A gene product of interest of the invention may comprise at least two nucleotide sequences each being different or encoding different therapeutic molecules. The at least two different nucleotide sequences may be linked by an IRES (internal ribosome entry site) element, providing a bicistronic transcript under control of a single promoter. Suitable IRES elements are described in 6.9., Hsieh et al. (1995, Biochem. Biophys. Res. Commun. 214:910-917). Furthermore, the at least two different nucleotide sequences ng different (therapeutic) polypeptides or proteins may be linked by a viral 2A sequence to allow for efficient expression of both transgenes from a single er. Examples of 2A sequences include those from foot and mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1 (Kim et al., PLoS One (2011) 6(4): e18556). A gene product of interest is preferably ed within the rAAV genome or between lTR sequences. A gene product of interest may also be an sion construct comprising an expression tory element such as a promoter or transcription regulatory sequence operably linked to a coding sequence and a 3’ termination sequence. The gene product of interest can be a functional mutant allele that replaces or ments a defective one. Gene therapy also includes insertion of transgenes that are tory in nature, i.e., that inhibit, decrease or reduce expression, activity or on of an endogenous or exogenous gene or n, such as an undesirable or aberrant (e.g., pathogenic) gene or protein. Such transgenes may be exogenous. An exogenous molecule or sequence is understood to be a molecule or sequence not normally occurring in the cell, tissue and/or individual to be treated. Both ed and congenital diseases are amenable to gene therapy.

"Gene" or "coding sequence" refers to a DNA or RNA region which "encodes" a particular protein. A 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 comprise several ly linked fragments, such as a promoter, a 5’ leader sequence, an intron, a coding sequence and a 3’ non-translated sequence, comprising a polyadenylation site or a signal sequence. A chimeric or recombinant gene is a gene not normally found in , such as a gene in which for example the promoter is not associated in nature with part or all of the transcribed DNA . "Expression of a gene" refers to the process wherein a gene is transcribed into an RNA and/or translated into an active protein.

As used herein, the term "promoter" or "transcription regulatory sequence" refers to a c acid nt that ons to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the ce of a binding site for DNA- 40 dependent RNA polymerase, ription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, sor and tor protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act ly or indirectly to regulate the amount of transcription from the promoter. A itutive" er is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a er that is physiologically or developmentally regulated, 6.9., by the application of a chemical inducer. A preferred inducible promoter is an NF-KB responsive er which is inducible upon inflammation. A "tissue specific" promoter is preferentially active in specific types of tissues or cells. The selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of a DNA segment. Preferred promoter sequences within the rAAV and/or transgene of the invention are promoters which confer expression in cells of the rheumatoid synovium, such as in intimal macrophages and/or in FLS and/or other synovial cells such as, but not limited to, T-cells. Preferred promoters are for example the promoters of genes known to be expressed in synovial cells, such as the CMV promoter, the promoter of the lL-6 gene or the SV40 promoter, or an NF-KB inducible promoter as earlier identified herein and others, as readily determined by a skilled person. Alternatively, a transgene is operably linked to a promoter that allows for efficient systemic sion. Suitable promoter sequences are CMV promoter, CBA (chicken beta-actin), or liver-specific promoters such as human alpha-1 anti-trypsin (hAAT) or TBG (thyroxine-binding globulin). ably, 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 , the term "operably linked" refers to a linkage of polynucleotide (or polypeptide) elements in a onal relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another c acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it s the ription ofthe coding sequence. "Operably linked" means that the DNA sequences being linked are typically contiguous and, where ary to join two protein-encoding regions, contiguous and in reading frame.

A "gene product of interest" can be a "therapeutic polypeptide" or "therapeutic protein" which are 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 human suffers from a disease.

Such therapeutic polypeptide may be selected from, but is not limited to, the group consisting of an enzyme, a co-factor, a cytokine, an antibody, a growth , a hormone and an anti-inflammatory protein. atively, or in combination with another embodiment, in a further preferred embodiment of the present invention, the nucleotide sequence encoding a gene product of interest is located between two AAV ITR sequences. Alternatively said, the nucleotide sequence ng the gene product of interest is flanked by two AAV ITR sequences, i.e., one ITR on either end of the nucleotide sequence encoding the gene product of interest.

Alternatively, or in combination with another embodiment, in a r preferred embodiment of the 40 present invention, the gene product of interest treats, prevents or suppresses symptoms associated with an arthritic disease, wherein preferably the gene product of interest is selected from the group consisting of interleukins, immune-modulators, antibodies, shRNA, miRNA, growth factors, proteases, nucleotidases/nucleosidases, peptides, se inhibitors, inhibitors, enzymes and combinations thereof, and wherein more preferably the gene product of interest is at least one of CD39, CD73 and lFN-B. Examples of these are: interleukin1 (IL-1) inhibitor (e.g. ra, canakinumab, rilonacept), tumor is factor alpha (TNFq) inhibitor (e.g. etanercept, infliximab, adalimumab, certilizumab pegol, golimumab), lL-1 receptor antagonist, soluble lL-1 receptor, lL-17 inhibitor (e.g. secukinumab, brodalumab, ixekizumab, lL-23 inhibitor (ustekinumab, risankizumab, guselkumab, kizumab), T-cell costimulation inhibitor (e.g.abatacept), B cell 1O depleting and inhibiting agents (e.g.rituximab, belimumab, mab, mab), lL-15 tor (e.g. AMG-714), lL-22 inhibitor (e.g. Fezakunimab), inhibitor of GM-CSF (lenzilumab, namilumab) insulin-like growth factor (lGF-1), fibroblast-growth factor (FGF) (e.g. thGF-18/sprifermin), receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor (e.g. mab), complement 5a inhibitor (e.g. C5aR-151), bone morphogenetic protein family member (BMPs), transforming growth factor-beta (TGF-B), growth differentiation factor family , interleukin-18 inhibitor (e.g. nig alfa/ lL-18 binding protein), lL-2 inhibitors (e.g. basiliximab, daclizumab), soluble TNFq (sTNFq) receptor p55 or sTNFq receptor p75, sTNFq ors fused with an lgG, inhibitors of TNFq receptor p55, inhibitors of sTNFq receptor p75, dominant ve lKB-kinase (dn-lKK-B), interleukin-4 (IL-4), interleukin-10 (IL-10) (F8lL10/Dekavil), interleukin-13 (IL-13), interferon beta ), tissue inhibitor of MMP family (TlMPs), plasminogen-activator inhibitor (PAls), serine protease inhibitors ns), ing molecules/transcription factors (e.g. SMAD, 80x9, lkB), extracellular matrix components (e.g. collagen, cartilage eric matrix protein (COMP), proteoglycans, elastin), vasoactive intestinal peptide (VIP), Cluster of Differentiation 39 (CD39) and Cluster of Differentiation 73 (CD73), Superoxide dismutase (SOD), and ations f.

Functional genome editing systems for use in all embodiments of the invention are known to the person skilled in the art and include: ription Activator-Like or Nucleases (TALENs, Gaj et al. (2013) Trends Biotechnol. 31(7):397-405), zinc-finger nucleases (ZFNs, Gaj et al. (2013) supra), meganucleases such as l-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 like CRISPR/Cas (Mali et al. (2013) Nat methods 10(10):957-963; Mali et al. (2013) Nat Biotechnol 31(9):833-838; Cong et al. (2013) Science 339(6121):819-823) and CRISPR/Cpf1 he et al. (2015) Cell 163(3):759-771), triplex-forming molecules, synthetic polyamides and designer zinc- finger proteins (Uil et al. (2003) Nucleic Acids Res 31(21):6064-6078). Functional genome editing systems employ nucleases which create site-specific double-strand breaks at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining or homologous recombination. As a result, targeted mutations are obtained. It is advantageous to replace a defective gene (causing a disease or disorder) with a normal allele at its natural location by any of these methods, because it does not require that the full coding sequences and regulatory 40 sequences are included in the rAAV virion when only a small portion of the gene needs to be altered.

Expression of the partially ed gene is also thought to be more consistent with normal cell biology than full genes that are carried by virions. The preferred gene editing system is CRISPR (comprising /Cpf1 and CRISPR-Cas), because it is quicker and cheaper than other methods. A major advantage is also that CRISPR can be easily repurposed to target ent DNA sequences using the CRISPR single guide RNAs. Thus, alternatively, or in combination with another embodiment, in a further preferred embodiment of the t ion, the rAAV genome comprises at least one of: (i) a polynucleotide comprising a sequence encoding at least one guide RNA (gRNA); n the guide RNA is substantially complementary — ably complementary — to a target polynucleotide sequence(s) in a genome; and (ii) a polynucleotide comprising a sequence encoding a nuclease; wherein the nuclease forms a ribonuclease x with the guide RNA, and wherein the ribonuclease complex makes site-specific double-stranded DNA breaks in the genome.

In a second aspect, the present invention relates to an rAAV composition for use in treating, preventing or suppressing symptoms associated with an arthritic e, wherein the rAAV composition ses an rAAV virion of the invention and a pharmaceutically acceptable carrier, diluant, solubilizer, filler, preservative and/or excipient, preferably a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carrier, diluents, lizer, filler, preservative and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000. Any suitable pharmaceutically acceptable carrier, diluant, solubilizer, filler, preservative and/or excipient can be used in the present compositions (See e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997). red pharmaceutical forms would be in combination with sterile saline, se on, or buffered solution, or other pharmaceutically acceptable sterile fluids. Alternatively, a solid carrier may be used such as, for example, microcarrier beads.

Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to accommodate high drug concentration. The carrier may be a t or sion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the ed particle size in the case of dispersion and by the use of tants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

Prolonged absorption of the injectable itions can be brought about by including in the composition an agent which delays tion, for example, monostearate salts and gelatin. The parvoviral virion may be administered as a bolus or in a controlled release formulation, for example in a composition which includes a slow release r or other rs that will protect the compound against rapid release, including implants and microencapsulated delivery s. 40 Biodegradable, biocompatible polymers may for example be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, thoesters, polylactic acid and polylactic, ycolic copolymers (PLG). As used herein, "pharmaceutically acceptable carrier" or ient" preferably includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are logically compatible.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or sion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical itions of the invention is contemplated. 1O It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as y dosages for ts to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention may be ed by the unique characteristics of the active compound and the particular eutic effect to be ed, and by the limitations inherent in the art of compounding such an active compound for the treatment of a condition in individuals.

Supplementary active compounds can also be incorporated into the pharmaceutical compositions of the invention. Guidance on co-administration of additional therapeutics may for example be found in the Compendium of Pharmaceutical and Specialties (CPS) of the an Pharmacists Association.

In an embodiment, the rAAV composition further comprises empty particles (i.e., capsid- only particles, thus not comprising an rAAV genome). Therefore, alternatively, or in ation with another embodiment, in a further embodiment of the t invention, the rAAV composition of the invention further ses an empty capsid in a ratio of empty capsid to rAAV virion of at least 1:1, more preferably at least 5:1, even more preferably at least 10:1. The rAAV composition can comprise the rAAV virion as defined above and an empty capsid, such as for example defined (2017) Hum. Gene Ther. 28(2):168—178. The empty capsid can be of the same serotype or of a different serotype as compared to the rAAV-transgene vector of the composition of the invention.

Preferably, the empty capsid is of the same serotype as the rAAV virion.Within such rAAV composition, the empty capsid and the capsid of the rAAV virion can comprise a ed capsid protein of the invention, preferably the same type of modified capsid proteins. r, also assed is an rAAV composition wherein the empty capsids have a different serotype or are differently modified capsid proteins as compared to the modified capsid proteins of the rAAV virion. r encompassed is an rAAV composition wherein the empty capsids have a mixture of serotypes, such as, but not limited to, a mixture of AAV2 and AAV5 capsids. The inventors report an increasing effect of transgene expression in joints after intraarticular administration of rAAV- virions admixed with a significant amount of empty capsids. Preferably in the irion and the 40 empty capsid are present within the composition in 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 at least 5:1 (i.e. an amount of empty capsids that is at least 5 times the amount of rAAV-transgene vectors). Preferably, said composition comprises rAAV-virion and empty capsid in a ratio of empty capsid to rAAV-transgene vector of at most 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 at most 1000:1.

Preferably said composition comprises rAAV-virion and empty capsids in a ratio of empty capsid to rAAV-virion of between 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 between 5:1 to 20:1.

Provided herein above is an embodiment wherein the rAAV-virion and the empty capsids 1O are present in a single composition. Also encompassed within the present invention is an alternative embodiment wherein the rAAV-virion and the empty capsids are present in (at least two or more) separate, distinct compositions. In this alternative embodiment, the rAAV-virion and the empty capsids can be administered separately in time (6.9., tially) and/or localization, wherein localization is to be understood as the site of administration. rmore, the irion and the empty capsids can be administered aneously, 6.9., at substantially the same timing, optionally at a separate location.

In a third aspect, the present invention relates to an rAAV composition and an immunosuppressant for use in treating or preventing an tic disease or for use in treating or preventing symptoms associated with an arthritic disease, wherein the rAAV composition is as defined above and wherein the treatment or tion ses the administration of the rAAV composition and the administration of the immunosuppressant to an individual. WO 55437, herein incorporated by reference, discloses an increasing effect of an immunosuppressant on AAV transgene expression when subjects were treated with both suppressants and rAAV- virions. Furthermore, WO 2016/055437 discloses a surprising synergistic effect of the immunosuppressant together with empty vectors on rAAV transgene expression. In one embodiment, the immunosuppressant is applied tely from the rAAV composition, separate meaning separate in location and/or time. In such an embodiment, the immunosuppressant and the rAAV composition may be present in separate and distinct compositions. The immunosuppressant, the rAAV-virion and optionally the empty vectors may even each be t in a te, distinct composition. In another embodiment, the immunosuppressant and the rAAV composition may be t in a single ition. In a further ment, the rAAV-virion and the suppressant are present in a single composition, and preferably this composition is used in treatment together with a separate composition comprising the empty capsid. In an even further embodiment, the immunosuppressant and the empty capsid are present in a single composition, and ably this composition is used in treatment together with a separate composition comprising the rAAV-virion. Therefore, the invention also provides for a composition comprising an empty capsid and an suppressant as defined herein, for a composition sing an rAAV- virion and an immunosuppressant as defined herein, and for a composition comprising an rAAV composition and an immunosuppressant as defined herein.

Preferably, an immunosuppressant for use in the present invention is an innate immune cell inhibitor, preferably a macrophage inhibitor. An innate immune cell is d herein as a neutrophil, hage, monocyte, eosinophil, basophil, or dendritic cell, that has the potential to ipate in the matory response to a foreign nce. An innate immune cell inhibitor is herein defined as an agent that results in a decrease in innate immune cell ty and/or innate immune cell number. A macrophage inhibitor is defined herein as an agent that results in a decrease in macrophage activity and/or macrophage number. A "macrophage" is tood 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 1O inhibitor of the invention, results in a se of at least 1, 2, 5, 10, 15, 20, 25, 30, 35, 45, 55, 65, 75, 85, 95% or preferably of 100% of the number or activity of innate immune cells or macrophages as compared to the initial number or activity of innate immune cells or macrophages before treatment. lnnate immune cell or macrophage activity and/or number can be detected by any suitable assay known by the person skilled in the art, such as, but not limited to, MTT (3-(4,5- dimethylthiazolyl)2,5-diphenyl tetrazolium bromide) colorimetric assay for testing macrophage xic activity in vitro as described by Ferrari et al. (Journal of Immunological Methods, 131 (1990) 165-172), by measurement of ne levels (6.9., CCL2, TNF), by histological and histochemical detection methods, for ce, by CD68 labeling or by in vivo magnetic resonance imaging (MRI) detection of superparamagnetic iron oxide (SPIO) uptake by macrophages, preferably after intravenous administration of SPIO as ed by Yi-Xiang J. Wang (Quant. lmaging Med Surg (2011)1:35—40). The detection can either be in vitro or in vivo. Preferably, in vivo detection is in an animal model, preferably a rat or murine model.

Preferably, the immunosuppressant is a glucocorticoid and/or a bisphosphonate, preferably a liposomal bisphosphonate. Particular non-limiting examples of glucocortocoids are cortisol, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate and eron. Preferably, the immunosuppressant is triamcinolone. ular non-limiting examples of sphonates are etidronate, clodronte, tiludronate, pamidronate, neridronate, onate, alendronate, onate, risedronate and zoledronate. Preferably, the bisphosphonate is a liposome-encapsulated bisphosphonate or liposomal bisphosphonate, preferably liposomal clodronate. Preferably, the glucocorticoid is not dexamethasone. It is to be understood that the matory or macrophage inhibitor of the invention is not d to a glucocorticoids and/or a sphonate. For instance, the inflammatory or macrophage inhibitor of the invention can also be an matory or macrophage-depleting antibody such as an anti-F4/80 antibody. Preferably, such antibody is a human or humanized antibody. Further relevant immunosuppressants to be used in the present invention are cytostatic drugs (e.g. alkylating agents and/or antimetabolites such as methotrexate), drugs that modify the purinergic signaling y (e.g. methotrexate, adenosine analogs, adenosine receptor antagonists or agonists), non-steroidal anti-inflammatory drugs (NSAIDS, e.g. ibuprofen, diclofenac, meloxicam, naproxen, acetylsalicylic acid), biologicals such 40 as TNF blockers (e.g. infliximab, etanercept, adalimumab, certolizumab, golimumab), lL-6 blockers (6.9. tocilizumab), |L-2 blockers (e.g. basiliximab, daclizumab), lL-1B blockers (e.g. anakinra, rilonacept, canakinumab) |L-17 (secukinumab, brodalumab, ixekinumab), anti-lL-12/lL-23 (ustekinumab), a PDE4-inhibitor (apremilast) muromonab, abatacept, and/or rituximab, and/or other compounds such hydroxychloroquine, chloroquine, omide, sulfasalazine, azathioprine, cyclophosphamide, cyclosporine, gold salt, mTOR inhibitors (e.g. rapamycin/sirolimus, everolimus) and penicillamine.

Preferably, the rAAV ition and/or composition comprising empty s and/or the composition comprising the immunosuppressant further ses a pharmaceutically acceptable carrier, diluents, solubilizer, filler, preservative and/or excipient as defined elsewhere herein. 1O Preferably, gene therapy according to the present ion further comprises the administration of an immunosuppressant as defined herein, either present within the rAAV composition, or comprised within a separate, distinct composition, i.e. te and distinct from the rAAV ition. At administration, the rAAV composition and/or empty capsids and/or immunosuppressant of the invention is delivered to an individual, a cell, tissue or organ of said individual, preferably an individual suffering from a condition or disease as defined herein.

Preferably, the rAAV composition and the immunosuppressant are administered simultaneously.

Simultaneous administration is to be understood herein as administration at more or less the same time, preferably no longer separated than 15 min, 30 min, 1 hour, 2 hours, 3 hours, 12 hours or 24 hours in time, preferably no longer separated than 15 min in time. In another embodiment, the rAAV composition and the immunosuppressant are administered tially, wherein ably the immunosuppressant is administered prior to 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 stration of the rAAV composition. In case the rAAV-virions and the empty capsids are t in separate itions, the immunosuppressant may be administered simultaneously or within at least 15 min, 1 hour, 2 hours, 3 hours, 1 day, 2 days or 1 week prior to the empty capsids and the empty capsids in turn are administered simultaneously or within at least min, 1 hour, 2 hours, 3 hours, 1 day, 2 days or 3 days prior to the rAAV-virion.

Within the embodiments defined herein, the immunosuppressant may be administered edly, i.e. prior to and/or simultaneously with the rAAV ition. As indicated herein above, preferably the rAAV composition comprises a significant amount of empty capsids. Furthermore, the invention encompasses the administration of both rAAV-transgene vectors and empty capsids in separate, ct compositions, which may be administered simultaneously or sequentially in a method or use of the invention. lf comprised in separate compositions, the rAAV-transgene vectors and empty capsids are preferably stered simultaneously. In a further embodiment, the empty capsids are administered at most 3 days, 2 days, 1 day, 24 hours, 12 hours, 3 hours, 2 hours, 1 hour, 30 min, 15 min or 5 min, preferably at most 24 hours, prior to rAAV-transgene vector stration. Furthermore, if comprised in separate compositions, the rAAV-transgene s and empty capsids are preferably administered at the same site.

The immunosuppressant dose s on the type of immunosuppressant. Effective 40 dosages are known by the skilled person. A preferred therapeutic effective dosage of triamcinolone is indicated above. A preferred therapeutic effective dosage of mal clodronate is preferably a therapeutic effective dose as known by the skilled person, 6.9. preferably 80-320 mg/dose intraarticular, more preferably 160 mg/dose intraarticular (Barrera et al. 2000, Arthritis & Rheumatism Vol 43(9), p1951-1959).

Generally, a joint disorder is termed an arthropathy, and when involving inflammation of one or more joints the disorder is termed an arthritis. Most joint disorders e arthritis, however, joint damage caused by external physical trauma is typically not termed arthritis. The term "arthritic disease" as used herein, also referred to as "arthritis", is herein defined as a form t disorder that involves inflammation of one or more joints. Currently, it is estimated that there are over a hundred different forms of arthritis. The arthritic disease is herein understood as referring to "joint pain" or "joint disease". In a red ment, the arthritic disease is selected from the group consisting of Adult-onset Still’s disease, ankylosing spondylitis, arthritis, back pain, 's disease, blunt trauma, bursitis, calcium osphate deposition disease (CPPD), carpal tunnel syndrome, chondromalacia a, chronic fatigue syndrome, complex regional pain syndrome, cryopyrin-associated periodic syndromes (CAPS), degenerative disc e, pmental- dysplasia of hip, Ehlers-Danlos, familial rranean fever, fibromyalgia, fifth disease, giant cell arteritis, gout, hemochromatosis, infectious tis, inflammatory arthritis, inflammatory bowel disease, joint replacement, juvenile arthritis, juvenile dermatomyositis (JD), juvenile idiopathic arthritis (JIA), juvenile toid arthritis, juvenile scleroderma, Kawasaki disease, lupus, lupus in children & teens, Lyme disease, mixed connective tissue disease, myositis (inc. polymyositis, dermatomyositis), osteoarthritis (OA), orosis, pagets, palindromic rheumatism, patellofemoral pain syndrome, pediatric rheumatic diseases, pediatric 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, systemicjuvenile idiopathic arthritis, systemic lupus matosus, systemic lupus erythematosus in children & teens, systemic sclerosis, temporal arteritis, tendinitis, vasculitis and Wegener’s granulomatosis. In a further preferred embodiment the tic e is selected from the group consisting of toid arthritis (RA), juvenile rheumatoid arthritis, rthritis (OA), gout, pseudogout, spondyloarthritis (SpA), tic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement and Still’s disease. In a more preferred embodiment, the arthritic disease is a joint disorder that involves inflammation of one or more joints. Preferably, the arthritic disease is selected from the group consisting of rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, gout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, septic arthritis, tis, juvenile idiopathic arthritis and Still’s disease.

Alternatively, or in combination with another ment, in a further preferred embodiment of the present invention, the rAAV virion or the rAAV composition is administered systemically and/or locally. An rAAV ition and/or empty capsids and/or an immunosuppressant of the 40 invention may be directly or indirectly administrated using suitable means known in the art. Methods and uses ofthe invention include delivery and administration of the rAAV ition and/or empty vector and/or immunosuppressant systemically, regionally or locally, or by any route, for example, by injection, infusion, orally (e.g., ingestion or inhalation), or lly (e.g., transdermally).

Exemplary administration and delivery routes include intravenous (iv), intraarticular, intraperitoneal (i.p.), intraarterial, uscular, eral, subcutaneous, leural, topical, dermal, intradermal, transdermal, parenterally, e.g. transmucosal, intracranial, intraspinal, oral (alimentary), mucosal, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, avity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, intralymphatic. Improvements in means for ing an individual or a cell, tissue, organ of said individual with an rAAV composition and/or empty capsids and/or an immunosuppressant of the invention, are anticipated considering the progress that has already been achieved thus far. Such future improvements may of course be incorporated to achieve the mentioned effect of the invention. When administering an rAAV composition and/or empty capsids and/or an immunosuppressant of the invention, it is preferred that such combination and/or composition is dissolved in a solution that is compatible with the ry method. For intravenous, aneous, intramuscular, intrathecal, intraarticular and/or intraventricular administration it is preferred that the solution is a physiological salt solution. In case an immunosuppressant is present within the rAAV composition of the invention, the immunosuppressant is administered at the same site as the rAAV composition, i.e. preferably locally as indicated above. In the embodiment wherein the immunosuppressant is comprised within a separate composition distinct from the rAAV ition, the immunosuppressant may be stered systemically, preferably intramuscularly or enously. The rAAV composition may also be administered locally, preferably at a site of the body comprising substantive numbers of macrophages as defined herein, and the immunosuppressant is administered systemically, preferably intramuscularly or enously. Also encompassed in the invention is an embodiment wherein the immunosuppressant and the rAAV composition, even though present in distinct compositions, are administered at the same site, ably locally, more preferably intraarticularly.

As further indicated herein, administration of such distinct compositions may be either simultaneously or sequentially. In a preferred embodiment of the present invention, at least one of the rAAV composition and the immunosuppressant is administered y. More preferably, the local administration is intraarticular stration. "lntraarticular injection" (also known as "joint injection" or "intraarticular injection") is herein defined as injection or infusion into the joint. lntraarticular injection is typically used for administration of an anti-inflammatory agent into a joint affected by inflammation.

In a further aspect, the present invention relates to an rAAV composition comprising an rAAV virion of the invention and a pharmaceutically acceptable carrier, diluant, lizer, filler, preservative and/or excipient, preferably a pharmaceutically acceptable carrier as defined herein.

In a preferred embodiment, the composition r comprises empty s as herein defined and/or an immunosuppressant as herein defined.

In a further aspect, the present invention relates to a method for treating, preventing, or suppressing symptoms associated with an arthritic disease, wherein the method comprises the step of intraarticular administration of a medicament comprising an effective amount of an rAAV virion as defined in any one of claims 1 — 8 or of an rAAV composition as defined above.

A "therapeutically effective amount" refers to an amount ive, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a nucleic acid, nucleic acid construct, rAAV virion or pharmaceutical composition may vary according to factors such as the disease state, age, sex, and weight of the subject to be treated, and the ability of the nucleic acid, c acid construct, rAAV virion or pharmaceutical composition to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically ive amount is also typically one in which any toxic or detrimental effects of the nucleic acid, nucleic acid construct, rAAV virion or pharmaceutical composition are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the d prophylactic result, such as preventing or inhibiting various conditions. A lactic dose may be used in subjects prior to or at an earlier stage of disease, and a prophylactically effective amount may be more or less than a therapeutically ive amount in some cases. The dosage to be administered may depend to a large extent on the condition and size of the subject being treated as well as the eutic formulation, ncy of treatment and the route of administration.

Regimens for continuing therapy, including dose, formulation, and frequency may be guided by the initial response and clinical judgment.

The term "subject" or "patient" is used interchangeably herein and refers to an animal, including the human species, that is treatable with the compositions and/or rAAV of the present invention. Accordingly, the term "subject" or "patient" includes, but is not limited to, human, non- human primate such as chimpanzees, and other apes and monkey species, or any mammal such as dog, cat, horse, sheep, pig, cow etc. In a red embodiment of the present invention, the subject treated with an rAAV according to the present ion is a , more preferably a human, dog, cat or horse, most preferably a human.

In this document and in its claims, the verb "to comprise" and its conjugations are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The word "approximately" or "about" when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 10% of the value.

All patent and ture references cited in the present ication are hereby orated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Description of the figures The t invention will be discussed in more detail below, with reference to the attached drawings: Figure 1: Screening of capsid serotypes on HEK293T and FLS cells. Crude lysate containing 7 mutant capsid serotypes (plus AAV5) expressing yellow fluorescent protein (YFP) were used to transduce HEK293T cells or 3 different FLS cell lines (each from a different RA patient). 1O After 72 hours 3T) or 6 days (FLS), cells were assayed for percentage of cells expressing YFP by FLOW cytometry. Panel A shows % of HEK 293T cells expressing YFP; Panel B shows the % of YFP-expressing cells in 3 different FLS cell lines; Panel C shows the mean scent intensity (MFI) in HEK293T cells; Panel D shows MFI in 3 different FLS cell lines (all ; Panel E shows MFI in 3 ent FLS cell lines (only positive population). The sample legend is depicted in Table 2.

Figure 2: Capsid mutants show increased luciferase expression vs wt-AAV5 in FLS cells.

Purified AAV (4 mutant serotypes or AAV5) expressing YFP-Luc fusion protein were used to transduce three different FLS cell lines from ent RA patients: BB5498 (FLS 1), BB5540 (FLS 2) and BB7144 (FLS 3) using two MOIs (20000 or 100000 rAAV particles per cell). After 4 days, cells were lysed and luciferase expression was measured. Data is ted as absolute luciferase expression levels (RLU; white bars) or fold increase over AAV5 (black bars). Panel A shows FLS 1 at MOI 20K; Panel B shows FLS 1 at MOI 100K; Panel C shows FLS 2 at MOI 20K; Panel D shows FLS 2 at MOI 100K; Panel E shows FLS 3 at MOI 20K; and Panel F shows FLS 3 at MOI 100K.

Open bars show luciferase (RLU) and filled bars show "fold increase" over AAV5. In a ent experiment, three additional FLS cell lines from RA patients were transduced with AAV (7 mutant serotypes or AAV5) expressing luciferase: BB4308 (FLS 4), BX 1592 (FLS 5), BB4426 (FLS 6) using 2 MOIs (10K or 100K rAAV particles per cell). Panel G shows FLS 4 at MOI 10K; Panel H shows FLS 4 at MOI 100K; Panel I shows FLS 5 at MOI 10K; Panel J shows FLS 5 at MOI 100K; Panel K shows FLS 6 at MOI 10K; and Panel L shows FLS 3 at MOI 100K.

Transduction efficacy of the 7 mutant serotypes or AAV5 (MOI 100K) was also ted in HEK293T cells (Panel M). Open bars show luciferase expression (RLU) and filled bars show "fold increase" over AAV5.

Figure 3A: Capsid mutants exhibit increased gene expression in vivo. Two capsid mutants (AAV9-A2 and AAV7-A6) were compared with thAV5 using the air pouch synovium model.

Luciferase-expressing vector was administered on day 0 following air pouch formation and luciferase expression was measured by live animal imaging (IVIS) on day 3 following transduction.

Data shown is the scence (photon/second/square centimeter m2/steradian) in air pouch in mean+SEM.

Figure 3B: In a second experiment, 5 selected capsid s (AAV1-P4, AAV7-A6, AAV9- 40 A2, AAVrh10-A2, 0-A6) and thAV5 were injected into the knee joints of mice. A luciferase expressing vector was injected on day 0 and expression was measured by live imaging (IVIS) at indicated time points after administration. Data shown is the luminescence (photon/second/square centimeter m2/steradian)(left panel) in mean+SEM. **P<0.05, ***P<0.01, ****P<0.00001 vs. thAV5 at day 14. Figure 30: Fold increase vs. thAV5.

Figure 4: CLUSTAL format alignment by MAFFT FFT-NS-I (V7215). Below the alignment is a key denoting a conserved residue (*);and a non-conservative mutation ( ).

Figure 5: CLUSTAL multiple sequence alignment by MUSCLE (3.8). Below the alignment is a key denoting a conserved residue (*); a conservative mutation (:); a semi-conservative mutation (.); and a non-conservative mutation ( ). 1O Figure 6: CLUSTAL format alignment of inserts P4, A2, A6, P2 and QR-P2 (SEQ ID NOS: 8 — 12) by MAFFT FFT-NS-I (V7215). Below the alignment is a key denoting a ved residue (*); and a non-conservative mutation ( ).

Figure 7: CLUSTAL multiple ce alignment of inserts P4, A2, A6, P2 and QR-P2 (SEQ ID NO’s: 8-12) by MUSCLE (3.8). Below the alignment is a key denoting a ved residue (*); and a nservative mutation ( ).

Sequence listing Table 1 provides an explanation of the sequence references in correlation with the SEQ ID No’s.

Table 1: Explanation of sequence nces SEQ ID NO: serotype Modified linsertlwild-type 1 AAV1 Modified capsid 2 AAV2 Modified capsid 3 AAV7 Modified capsid 4 AAV9 Modified capsid 0 Modified capsid 6 AAVrh10 Modified capsid 7 AAV DJ-QR Modified capsid 8 Insert A2 Insert 9 Insert A6 Insert Insert P2 Insert 11 Insert P4 Insert 12 Insert QR-P2 Insert 13 AAV1 ype capsid 14 AAV2 Wild-type capsid AAV7 Wild-type capsid 16 AAV9 Wild-type capsid 17 AAVrh10 Wild-type capsid 18 AAV DJ-QR tic capsid 19 AAV5 Wild-type capsid Examples Example 1 Initial screening of capsid library 1.1. Materials and methods 96-well plates spotted (and subsequently dried) with crude lysate containing AAV from 91 ent AAV capsid pes were obtained from Dirk Grimm and Kathleen Borner at the University of Heidelberg. Each vector encoded a YFP transgene driven by a CMV promoter. As FLS are the primary target cells in the joint, an AAV capsid mutant library was screened for serotypes that show increased expression in human FLS isolated from joints of rheumatoid arthritis patients (RA-FLS) 1O (as described in van de Sande MG et al., (2011)Ann Rheum Dis 70: 423-427). RA—FLS were plated (2500/well, 37°C / 5% C02) directly onto the spotted plates (DMEM-GlutaMAX—l (Gibco, 966- 021), 10% FBS (heat inactivated (HI) Bovine Serum Gold, Gibco, ref A15—151), 10 mM HEPES (Gibco, ref.15630-056), 50 pg/ml gentamycin (Gibco, ref.15710-049), 100U/ml penicillin/100pg/ml streptomycin (Sigma-Aldrich, ref.P0781) and all wells were visualized for YFP expression by fluorescence microscopy after 6 days. 1.2. Results Transduction efficacy of capsid mutants vs. 5 in FLS from RA patients.

In screening of the 91 capsid mutants, while the overall expression levels were low, the t inventors identified 7 different serotypes that showed higher expression than thAV5: AAV9-A2, AAV7-A6, AAV1-P4, AAVDJ-QR-P2, AAVrh10-A6, AAVrh10-A2 and AAV2-P2 (amino acid sequences SEQ ID NO: 1 — 7; thAV5 SEQ ID NO: 19).

Crude lysates of all 7 vectors were used in an in vitro transduction assay in 3 ent t FLS cell lines and in HEK293T cells le 2).

Table 2: Sample legend for figure 1 Sample Capsid Insert/modified sequence Insert Position SEQ pe in VP1 ID NO: 5 none none - 19 61 AAV1 GQSGNDVRSANAQAA P4 588 — 602 1 33 AAV9 GQRGNYSRGVDAQAA A2 586 — 600 4 34 AAVrh10 GQRGNYSRGVDAQAA A2 588 — 602 6 50 AAV2 QGQSGCDCRGDCFCA P2 585 — 599 2 88 AAV-DJ-QR DCRGDCFCA(QAA) QR-P2 587 — 601 7 43 AAV7 GQRGNEARVREAQAA A6 587 — 601 3 46 AAVrh10 GQRGNEARVREAQAA A6 588 — 602 5 Example 2 Expression of crude lysates of 7 selected mutants 2.1. Materials and methods AAV production Details on the production of the crude AAV lysates can be found in Grosse et al. (J. Virol, 2017, doi: .1128/JV|.01198-17).

Aliquots of crude lysate for each of the selected 7 capsid mutants (plus thAV5 as a l) 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 3T) -5 days (FLS) following transduction. In 1O , HEK293T were seeded in a 96-well plate (Greiner Bio-One, 5180) at 45000 cells per well. RA—FLS were seeded in a 96-well plate at 2500 cells per well. After overnight incubation, the cell supernatants were replaced with 40 pl DMEM-glutaMAX—l (Gibco 31966-021) ning 0.001% pluronic F68 solution (Sigma P5556). The virus lysates were added in duplo, 10 pl per well.

After 4 hours, doxorubicin (final concentration 0.4 pM) (Sigma D1515) in DMEM-glutaMAX—l containing FBS (heat inactivated (HI) Bovine Serum Gold, Gibco, ref 1), final concentration 1%) was added to the wells (50 pl per well). The day after, the medium of FLS was removed and DMEM-glutaMAX—l (10% FBS (heat inactivated (HI) Bovine Serum Gold, Gibco, ref A15-151), 10 mM HEPES (Gibco, ref.15630), 50 pg/ml gentamycin (Gibco ref. 15710-049), 100 U/ml penicillin/100 pg/ml streptomycin -Aldrich, ref. P0781)) was added (200 pl per well). The medium of HEK293T cells was not changed. Three (HEK293T cells) or 6 days (FLS) after transduction cells were trypsinized using 0.5% Trypsin/EDTA (Gibco ref.15400-054) in PBS (Gibco, ref. 10010) and analyzed for YFP expression by FLOW cytometry (FACSCanto ll, BD Biosciences).

Both percentage of expressing cells and mean fluorescence intensity (MFI) for all cells was determined. 2.2. Results Crude lysates of all 7 vectors were used in an in vitro transduction assay in 3 different patient FLS cell lines and in HEK293T cells. Cells were assayed for the percentage of cells expressing YFP by fluorescence microscopy (data not shown) or FLOW try (Figure 1 panels A — E). While there was some variability between cell types, all mutant capsids gave higher expression in both, FLS and HEK293T cells than AAV5-WT (Figure 1). Table 2 provides the sample legend for Figure 1. Based on these results, four capsid mutants were selected for further igation (see example 3).

Example 3 In vitro testing of capsid ts in HEK293T and FLS 3.1 materials and methods 3.1.1 Four of the mutant capsid proteins, AAV9-A2, 6, AAV1-P4, and AAVDJ-QR—P2, were 40 further igated. Purified vector (lodixanol gradient) expressing a YFP-Luciferase fusion protein (to allow for visualization (YFP) as well as quantification by luciferase assay) was generated. Three different primary FLS lines isolated from rheumatoid arthritis patients (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 by rase assay ga rase assay Kit).

In detail, RA-FLS were plated at 2500 cell/well in a 96-well plate (Greiner Bio-One, ref.655207) in medium (DMEM-GlutaMAX (Gibco ref.31966—021), 10% FBS (heat inactivated (HI) Bovine Serum Gold, ref A15-151), 10 mM HEPES (Gibco ref. 15630-056), 50 pg/ml gentamycin (Gibco, ref 15710- 049), 100u/ml penicillin/100pg/ml streptomycin (Sigma-Aldrich Merck ref. . After 48h, 1O medium was removed and virus (in DMEM-Glutamax containing 0.001% ic-68 (Sigma, ref. p5556)) was added at an MOI of 20,000 or 100,000. After 4h, medium containing bicin (Sigma, ref.D1515, final concentration 0.4 pM) and FBS (final concentration 1%) was added. 24h later, medium was ed with DMEM-GIutaMAX, (10% FBS, 10 mM HEPES, 50 pg/ml gentamycin, 100u/ml penicillin, 100pg/ml streptomycin). Four days post-transduction, cells were washed 1x with 100 pl PBS (Gibco, ref. 10010) and luciferase ty was determined using the ONE GloTM luciferase assay system ga, ref.E6110): 100 pl Lysis buffer was added and cells were placed on a shaker for 10’, 900 rpm at RT. Subsequently, 20 pl lysate was transferred to a white 96-well plate, 80 pl substrate (was added for 3’ (dark) and rase activity was determined on a luminometer (1 sec/well, synergy HT, Biotek). 3.1.2. In a similar experiment, three additional FLS cell lines isolated from rheumatoid arthritis ts were transduced with AAV5 and 7 capsid s from a different AAV preparation than described in 3.1.1 (AAV9-A2, AAV1-P4, AAV7-A6, AAVDJ-QR-P2, AAVrh10-A6, AAVrh10-A2, AAV2-P2) containing a luciferase gene (MOI 10,000 and 100,000). The number of empty particles differed between the AAV preparations. To exclude a possible effect on transduction efficacy, empty capsid correction was done by adding AAV5 empty particles to equalize the percentage of empty particles per preparation. 3.1.3. The 7 capsid mutants from the same preparation as described in 3.1.2 were also tested in HEK293T cells. In detail, HEK293T were seeded in a 96-well plate (Greiner Bio-One, ref.655180) at 50000 cells per well. After overnight incubation, the cell supernatants were replaced with DMEM- glutaMAX—I (Gibco 021) containing 0.001% pluronic F68 solution (Sigma P5556). The different vectors were added in duplo, at an MOI of 100,000. In this protocol, empty capsid correction was done as described for 3.1.2. After 4 hours, doxorubicin (final concentration 0.4 pM) (Sigma D1515) in DMEM-gIutaMAX—I-containing FBS (heat inactivated (HI) Bovine Serum Gold, Gibco, ref A15-151 ), final concentration 1%, was added to the wells. Three days after transduction, cells were harvested and gene expression was quantified by luciferase assay (Promega Luciferase assay Kit) on a luminometer (BMG Labtech Fluostar Omega). 3.2. s 3.2.1 In vitro transductions of three different FLS cell lines were performed using 40 recombinant AAV comprising one of the 4 mutant capsids (as well as AAV5 as control, made in the cal manner) following the protocol described in 3.1.1. All 4 serotypes showed increased expression levels when compared with AAV5, ranging from 2-fold to 35-fold increases, depending on the pe and cell line used (Figure 2A—F). 3.2.2 In another series of experiments, in vitro transduction efficacy of 7 mutant capsids (as well as AAV5 control, made in the identical manner) was assessed in 3 FLS cell lines. All 7 serotypes showed sed luciferase expression levels when compared with AAV5, g from 6-fold to 55-fold increases depending on the serotype and cell line used (Figure 2G-L) 3.2.3 A similar experiment was performed in HEK293T cells. Transduction with all 7 serotypes resulted in enhanced luciferase expression compared with thAV5, ranging from 2-fold 1O to 12-fold increases e 2M).

Example 4 In vivo study in the air pouch synovium model 4.1. Materials and methods Animals Female Balb/c mice (8—10 weeks old and weighing 20—25 g; (Harlan, r, the Netherlands)) were housed in individual ventilated cages at the animal facility of the Academic Medical Center, Amsterdam. Food and water were available ad Iibitum. All animal experiments were performed ing to the guidelines of the Animal Research Ethics Committee of the University of Amsterdam.

Air pouch synovium (APS) model Two pes, AAV9-A2 and AAV7-A6, were compared t thV5. The air pouch synovium model was adapted from Edwards et al (1981; J Pathol 134: 147-156). At day 0, 3 ml of air was ed subcutaneously into the dorsal skin of 7-9 week-old female Balb/cOlaHsd mice n) (day 0). Immediately following the ion of the air pouch, 1 ml of air was removed and 1 ml of AAV (2e10 vector genomes/mouse in PBS (Gibco, ref.10010 containing 0.001% pluronic F68 (Sigma, ref.p5556) was added directly into the air pouch. Three days following transduction, gene expression was measured by in vivo animal imaging.

Imaging of luciferase expression Luciferase expression was measured at day 3. It was initially planned to continue monitoring expression for up to 3 months following vector administration, however, a irus infection of the animal facility resulted in the premature termination of all ongoing experiments. D-luciferin potassium-salt substrate (Caliper Life Sciences, Hopkinton, MA, USA) was injected intraperitoneally (150 mg/kg of body weight, in a volume of imately 200 pl). Photon counts were acquired 10 min after substrate administration for 5 min using a cooled charge-coupled device (CCD) camera system (Photon lmager, Biospace Lab, Paris, France) and image processing and signal intensity 40 quantification and analysis were performed using M3 Vision (Biospace Lab). The number of photons emitted per second per square centimeter per ian was calculated as a measure of luciferase activity.

General animal conditions and ethics statement Air pouch formation, vector administration and in vivo imaging were performed under isoflurane anaesthesia (3% isoflurane and oxygen). At the end of the experiments, animals were sacrificed by cardiac puncture under isoflurane hesia, followed by cervical dislocation. The studies were ed and approved by the animal care and use committee of the University of Amsterdam and carried out in strict accordance with the recommendations in the Dutch Law on Animal e 1O (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 med using the air pouch synovium (APS) model, where two serotypes, AAV9-A2 and AAV7-A6, were compared against thAV5. Due to an unfortunate infection in the animal ty that necessitated the premature termination of this study, we were only able to obtain data from a single time point, day 3 post vector stration. At this time point it was clear that the capsid mutants were giving rise to increased gene expression when compared with AAV5, with AAV7-A6 showing ~6—fold increased expression and AAV9-A2 ~22-fold (Figure 3A).

Example 5: In vivo study: intra-articular iniections in healthy animals 5.1. Material and methods Animals Male DBA1/J mice (12 weeks old, Envigo) were housed in individual ventilated cages at the animal facility of the Academic Medical Center, dam. Food and water were ble ad libitum. All animal experiments were performed after approval of the Central Commission Animal ments (CCD) and the Animal Research Ethics Committee of the University of Amsterdam, the Netherlands.

Expression study Five rAAV comprising capsid mutants, i.e., AAV9-A2, AAV1-P4, 6, AAVrh10-A6 and AAVrh10-A2, were compared against thAV5. As capsid load may affect expression (Aalbers CJ et al., Hum Gene Ther 2017;28 (2):168—178), rAAV preparations were corrected for the capsid load by adding thAV5 empty particles. Healthy mice (n=9 per group) received articular injections of AAV vector carrying the luciferase gene in both knees (7.5x109 viral genomes per knee). Gene 40 expression was determined by in vivo g at several time points after vector administration.

Imaging of luciferase expression Luciferase expression was determined at indicated time points (Fig 38). At each time point, D- luciferin potassium-salt substrate (Caliper Life Sciences, Hopkinton, MA, USA) was injected intraperitoneally (150 mg/kg of body weight, in a volume of approximately 200 pl). Photon counts were acquired 15 min after substrate stration for 5 min using a cooled charge-coupled device (CCD) camera system (Photon lmager, Biospace Lab, Paris, France). Image processing and signal ity quantification and analysis were med using M3 Vision (Biospace Lab). The number of photons emitted per second per square centimeter per steradian was calculated as a measure 1O of luciferase activity.

General animal conditions and ethics statement Vector administration and in vivo imaging were performed under isoflurane anaesthesia (4% isoflurane and oxygen). The s were carried out in strict accordance with the recommendations in the Dutch Law on Animal Welfare (Dutch: "Wet op Dierproeven"). s were maintained under pathogen-free conditions in the animal facility of the University of Amsterdam. .2. Results At the first time point, day 3, AAV-mediated expression in the knee is detected in all groups and increases in time (Figure 38). All capsid mutants except AAV1-P4 show increased expression vs. thAV5 with AAV9-A2 showing the highest expression (~5 fold increased vs. thAV5 at day 14) (Figure 3C). Expression levels at day 14 from high to low: AAV9-A2 > AAVrh10-A2 > AAVrh10-A6 > AAV7-A6 > thAV5 > AAV1-P4 . On day 7, AAVrh10-A2, AAV9-A2 and AAVrh10-A6 show significantly increased expression vs. thAV5 (**P<0.05, ***P<0.01, ****P<0.00001 vs. thAV5 at day 14. (Figure 38).

Example 6: Determination of lizing antibody titers against capsid s in human sera 6.1 Material and methods HEK293T cells were plated in DMEM containing 9% FBS, 0.9% penicillin/streptomycin in l clear-bottomed plates. Cells were allowed to rest for 24 hours (at 37°C, 5% C02) before transduction. Human serum s (obtained from the French blood institute) where diluted as follows: neat undiluted serum — 1:4 -1 :16 — 1:64 — 1: 256 — 1:1,024 (neat serum means 1 volume of virus for 1 volume of serum). A pooled mouse plasma sample (from 10 DBA/1 mice, taken 42 days after intra-articular ion of an AAV5-vector) was serially diluted in FBS as follows: 1:10 — 1:50 — 1:250 — 1:6,250 — 1:31,250. A solution of human Intravenous lmmunoglobulin (lVig, Sanquin, lot 15D30H4560A) was serially semi-log diluted from 1:10 down to 1:10,000. Samples and ls were incubated er with the appropriate capsid mutant or thAV5 vector for 30’ i 5 min. at 40 C at an MOI of 2,500 (as determined previously). After 48 i 2 hours, luciferase reagent was added and luminescence emission was measured with the VictorX microplate reader. Transduction inhibition titers were determined as the highest dilution of serum still associated to a detectable neutralizing ty, Le. a neutralizing activity >50%. 6.2 Results As presented in Table 3, 70%-85% of the samples did not contain neutralizing antibodies against thAV5 or the 7 capsid mutants. Most of the samples shared vity t the seven capsid mutants, thus a serum sample having reactivity against the wild type AAV5 capsid also reacted against other capsids. In terms of the level of response, they were also comparable between capsid mutants. The number of samples that did not react (ND = not detected) is ted for each capsid mutant. These data are only given as information as it is very difficult to compare titers with ent vectors. Regarding the pooled mouse serum sample from intra-articular injected joints, it only reacted against the WT AAV5 capsid that was used to immunize the animals, whereas no response was observed against mutant capsids (Table 3). All capsid mutants and WT AAV5 were neutralized by lVlg (titers >100) (data not shown). $5083 E<-oHE><< EEHA oz oz oz oz oz EN E oz E oz oz oz H oz H 8:26 0530200 Hmong; E<-n><< EEHA oz oz oz oz oz EN E oz E oz oz oz oz oz H 9: 02 otoo .o__E_\U_0o_ 2-9/2 EEN oz oz oz oz oz E E oz E oz H oz H oz EH USE UEEQEQE votoooo EH><< EEHA oz oz oz oz oz EN EH oz E oz oz oz E oz E LEE 29.9w EBEEE o>:_won_ N<-oHE><< EN oz oz oz oz oz EN E oz E oz oz oz oz oz E .mema .o>:_mooooow oz oz oz oz oz E EH oz EH oz oz oz oz oz E $50.: mm Hood22 EN mm 9208 voomecoo 9: 9m E H H w N<< EN oz oz oz oz oz E EH oz oz oz oz oz mm 92:- 2998 .>H_>_Hom m><< EN E oz oz oz oz E EH oz E oz oz oz oz oz H comm mc_N__m::mc H N m E m E H w o OH HH NH mH EH EH EH HooEoEHEOEn. o0". "m EEEQBEU 0388 0388 0388 0388 0388 0388 0388 0388 0388 0359- 2993 29:8 29:8 2993 2993 0359. o_o_m-_- m oz oz oz 3 m: oz oz oz oz 3 m: oz oz oz oz wow on oz oz oz : oz 8 oz oz oz oz 9 mm oz oz oz oz E m: oz oz oz oz wow 8 oz oz oz oz : on mom 2 2 2 om "8838:; 29:8 29:8 29:8 2:58 3:53: 8.989.. 8:9: mEmmE The present invention has been described above with reference to a number of exemplary embodiments as shown in the gs. Modifications and alternative implementations of some parts or elements are possible, and are included in the scope of protection as defined in the appended claims

Claims (2)

Claims

1. Use of a recombinant adeno-associated virus (rAAV) virion comprising a ed capsid protein in the manufacture of a medicament for treating or preventing an arthritic disease or for 5 treating or preventing a symptom associated with an arthritic disease, wherein the symptom is joint pain or the mation of one or more tic joints,; wherein the modified capsid protein comprises in the C-terminal part of the protein an amino acid sequence Z, residues of which are exposed on the surface of the capsid protein, wherein the amino acid sequence Z: 10 a. comprises or consists of a sequence of amino acid residues of the formula I: y – G – Q – x – G – (x)3 – R – (x)3 – y – A – Q – A – A wherein x represents a single amino acid e and wherein y represents 0, 15 1 or 2 amino acid es; and b. is present at a location corresponding to a position 100 – 200 amino acid residues from the C terminus of a wild-type AAV capsid protein; and wherein the rAAV virion comprises: i) a nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) 20 sequence and ii) a nucleotide sequence encoding a gene t of interest

2. The use according to claim 1, wherein b. is present at a location corresponding to a position