Compositions and methods for treating arthritis

Abstract

Disclosed herein are compositions and methods for treating arthritis (e.g., osteoarthritis or rheumatoid arthritis) in a subject. The methods provided herein include intra-articular administration to a subject in need thereof using a composition comprising one or more nucleic acids that collectively encode an interleukin-1 receptor antagonist (IL-1Ra) polypeptide and an FGF18 polypeptide. Adeno-associated virus (AAV) viral genomes and particles carrying such nucleic acids and uses thereof are further described.

Description

[0001] This application claims priority to PCT patent application No. PCT / CN2023 / 123973, filed on October 11, 2023, the entire contents of which are incorporated herein by reference. Electronically submitted sequence list reference

[0002] This application incorporates an XML-formatted sequence list named “098A001WO02_SL”, which was created on October 10, 2024, and is 39,659 bytes in size. Technical Field

[0003] This invention relates to molecular biology and medicine. This document provides information on recombinant viral constructs and their use in gene therapy for the treatment of inflammatory arthritis. background

[0004] Arthritis is a prevalent disease affecting millions of people worldwide. It refers to inflammation and degeneration of the joints, leading to pain, stiffness, and decreased mobility. Two common types of arthritis include osteoarthritis (OA) and rheumatoid arthritis (RA). Osteoarthritis is characterized by cartilage damage and typically affects weight-bearing joints such as the knees, hips, and spine. OA is a leading cause of disability. RA is an autoimmune disease that typically affects the hands, wrists, and feet, causing joint deformities, fatigue, and systemic symptoms such as fever and weight loss. Both OA and RA can significantly impact quality of life. However, currently, few long-term treatments can relieve symptoms or restore joint function.

[0005] Therefore, there is an urgent need for new compositions for treating this disease. The compositions and methods disclosed herein address this need and offer related advantages. Summary of the Invention

[0006] This document provides compositions for treating arthritis comprising (i) an FGF18 peptide or an FGF18 transgene, and (ii) an interleukin-1 receptor antagonist (IL-1Ra) peptide or an IL-1Ra transgene. In some embodiments, the FGF18 peptide is human FGF18 (hFGF18) or a functional variant thereof, or rat FGF18 (rFGF18) or a variant thereof. In some embodiments, the FGF18 peptide has an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO:1. In some embodiments, the IL-1Ra peptide is human IL-1Ra (hIL-1Ra) or a functional variant thereof, or rat IL-1Ra (rIL-1Ra) or a variant thereof. In some embodiments, the IL-1Ra peptide has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO:3.

[0007] In some embodiments, this document provides a composition for treating arthritis comprising an FGF18 transgene and an IL-1Ra transgene. In some embodiments, the nucleotide sequence of the FGF18 transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO:5. In some embodiments, the nucleotide sequence of the IL-1Ra transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO:8. In some embodiments, (1) the FGF18 transgene is codon-optimized, (2) the IL-1Ra transgene is codon-optimized, or both are optimized. In some embodiments, the nucleotide sequence of the FGF18 transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO:6. In some embodiments, the nucleotide sequence of the IL-1Ra transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO:9.

[0008] In some embodiments, the compositions provided herein comprise nucleic acids comprising expression cassettes containing an FGF18 transgene and an IL-1Ra transgene linked by an adapter sequence. In some embodiments, the compositions provided herein comprise nucleic acids comprising an FGF18 expression cassette containing an FGF18 transgene and an IL-1Ra expression cassette containing an IL-1Ra transgene. In some embodiments, the compositions provided herein comprise a first nucleic acid and a second nucleic acid, the first nucleic acid comprising an FGF18 expression cassette containing an FGF18 transgene and the second nucleic acid comprising an IL-1Ra expression cassette containing an IL-1Ra transgene. In some embodiments, the expression cassette comprises a promoter and a polyA sequence. In some embodiments, the expression cassette further comprises an enhancer.

[0009] In some embodiments, one or more nucleic acids of the compositions provided herein are recombinant vectors.

[0010] In some embodiments, one or more nucleic acids of the compositions provided herein are viral genomes. In some embodiments, the nucleic acids are self-complementary.

[0011] In some embodiments, the compositions provided herein comprise one or more recombinant viruses that encapsulate the viral genome. In some embodiments, the one or more recombinant viruses comprise retroviruses, adenoviruses, lentiviruses, or adeno-associated viruses (AAVs). In some embodiments, the one or more recombinant viruses comprise AAVs. In some embodiments, the AAV is a serotype of AAV2, AAV2.5, AAV5, AAV8, or AAV9, or a mixture thereof. In some embodiments, the AAV serotype is AAV5.

[0012] In some embodiments, the viral genome of the AAV provided herein comprises 5' to 3': a first ITR, a first promoter, an IL-1Ra transgene, a first poly A tail, a second promoter, an FGF18 transgene, a second poly A tail, and a second ITR. In some embodiments, the viral genome of the AAV provided herein comprises 5' to 3': a first ITR, a first promoter, an FGF18 transgene, a poly A tail, a second promoter, an IL-1Ra transgene, a second poly A tail, and a second ITR.

[0013] In some embodiments, the viral genome of the AAV provided herein comprises 5' to 3': a first ITR, a promoter, an IL-1Ra transgene, an adapter sequence, an FGF18 transgene, a polyA tail, and a second ITR. In some embodiments, the viral genome of the AAV provided herein comprises 5' to 3': a first ITR, a promoter, an FGF18 transgene, an adapter sequence, an IL-1Ra transgene, a polyA tail, and a second ITR.

[0014] In some embodiments, the compositions provided herein comprise (1) a first AAV viral genome comprising: a first ITR, a first promoter, an IL-1Ra transgene, a first poly A tail, and (2) a second AAV viral genome comprising a second promoter, an IL-1Ra transgene, a second poly A tail, and a second ITR.

[0015] In some embodiments, the compositions provided herein are pharmaceutical compositions further comprising a pharmaceutically acceptable carrier. In some embodiments, the compositions provided herein are formulated for intra-articular injection.

[0016] In some embodiments, this document provides kits comprising unit doses of the compositions disclosed herein.

[0017] In some embodiments, methods for expressing FGF18 peptides and IL-1Ra peptides in cells are provided herein, including contacting the cells with the compositions disclosed herein. In some embodiments, the cells are chondrocytes or synovial cells.

[0018] In some embodiments, this document provides a method for delivering the FGF18 peptide and the IL-1Ra peptide to a region of interest of a subject in need, including introducing the composition disclosed herein into the region. In some embodiments, the region of interest is a joint, synovium, subsynovial layer, joint capsule, tendon, ligament, cartilage, or periarticular muscle.

[0019] In some embodiments, this document provides a method for treating arthritis in a subject in need, comprising administering to the subject a therapeutically effective amount of the composition disclosed herein. In some embodiments, the method disclosed herein includes intra-articular injection.

[0020] In some embodiments of the methods disclosed herein, the subjects are humans.

[0021] In some embodiments, this document provides for the use of the compositions disclosed herein in the treatment of arthritis. In some embodiments, this document provides for the use of the compositions disclosed herein in the preparation of a medicament for the treatment of arthritis. In some embodiments, arthritis includes the hand, knee, hip, shoulder, ankle, elbow, temporomandibular joint, spine, or any combination thereof.

[0022] In some embodiments, the arthritis treated by the compositions disclosed herein is osteoarthritis. In some embodiments, the arthritis treated by the compositions disclosed herein is rheumatoid arthritis. Attached Figure Description

[0023] Figure 1Schematic diagrams of the three expression constructs disclosed in this paper are provided. ITR: inverted terminal repeat; mITR: modified inverted terminal repeat; hIL-1Ra Co: codon-optimized hIL-1Ra transgene; hFGF18-Co: codon-optimized hFGF18 transgene.

[0024] Figure 2 Representative ELISA results are provided, showing the expression of IL-1Ra in synovial fluid after a single injection of AAV-IL-1Ra of various serotypes.

[0025] Figure 3 Representative bipedal balance test results are provided, demonstrating the effect of AAV5-IL-1Ra treatment on weight-bearing of the operated limb in OA model animals. L / R: left / right side, or contralateral / operated side.

[0026] Figure 4 Representative results were provided, demonstrating the effect of AAV5-IL-1Ra treatment on the mechanical pain threshold of the operated limb in OA model animals.

[0027] Figure 5 Representative results were provided, demonstrating the effect of AAV5-IL-1Ra treatment on the comprehensive knee joint injury score in OA model animals.

[0028] Figure 6 Representative ELISA results are provided, showing the expression of IL-1Ra in synovial fluid after a single injection of AAV5-IL-1Ra.

[0029] Figure 7 Representative bipedal balance test results are provided, demonstrating the effect of AAV5-IL-1Ra and / or FGF18 treatment on weight-bearing of the operated limb in OA model animals. L / R: left / right side, or contralateral / operated side.

[0030] Figure 8 Representative results were provided, demonstrating the effect of AAV5-IL-1Ra and / or FGF18 treatment on the mechanical pain threshold of the operated limb in OA model animals.

[0031] Figure 9 Representative results were provided, showing the effects of AAV5-IL-1Ra and / or FGF18 treatment on the comprehensive knee joint injury score in OA model animals.

[0032] Figure 10 Representative OARSI scores are provided, demonstrating the effects of AAV5-IL-1Ra and / or FGF18 treatment on tibial plateau cartilage scores in OA model animals.

[0033] Figure 11Representative Image J results are provided, showing the effects of AAV5-IL-1Ra and / or FGF18 treatment on the tibial plateau cartilage osteophyte score in OA model animals.

[0034] Figure 12A-12B Representative Image J results are provided, showing the expression level of IL-1Ra in synovial fluid after a single injection of AAV5-IL-1Ra and / or FGF18. Figure 12A ) and FGF18 expression level ( Figure 12B ). Invention Details

[0035] Arthritis is a disease that affects the joints in the body. It is characterized by inflammation, pain, stiffness, and limited movement in the affected joints.

[0036] There are different types of arthritis, with osteoarthritis (OA) being the most common. OA is a degenerative joint disease characterized by progressive damage to articular cartilage, subchondral bone sclerosis, osteophyte formation, ligament laxity, weakness of the muscles around the joint, and inflammation of the synovium. Weight-bearing joints, such as the knees, hips, and spine, are commonly affected by osteoarthritis, as are the hands and feet.

[0037] Osteoarthritis is currently incurable and exhibits significant resistance to treatment. Intra-articular delivery of targeted drugs for joint disease is challenging because molecules of various sizes and particles are rapidly cleared from the joint. This makes achieving sustained therapeutic doses of anti-OA drugs within the joint difficult. Furthermore, the size-dependent limitations of the fenestrated endothelium of the synovial capillaries hinder systemic delivery to the joint. Additionally, systemic administration exposes non-target sites to high doses of the treatment, leading to unwanted side effects, especially with repeated administration. In the absence of disease-modifying osteoarthritis drugs (DMOADs), the current standard of care is conservative treatment. Current treatment methods fall into three categories. Non-pharmacological treatments include various strategies such as patient education and self-help, exercise programs, and weight loss. Pharmacological treatments include the use of acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), opioids, and intra-articular injections of corticosteroids or hyaluronic acid; a completely reliable and effective pharmacological intervention is lacking.

[0038] Rheumatoid arthritis (RA) is a complex and heterogeneous disease involving multiple biological pathways that lead to chronic inflammation of the synovium in affected joints. Without timely treatment, RA patients typically face higher morbidity and mortality rates, as well as a decline in quality of life due to loss of daily function and even permanent disability caused by chronic pain, fatigue, and deterioration of articular cartilage and bone. The pathogenesis of RA is difficult to explain, involving multiple exogenous or endogenous antigenic triggers that, combined with a genetic predisposition, collectively induce an autoimmune cascade within the synovial cavity. Interactions among monocytes, macrophages, B cells, T cells, endothelial cells, and fibroblasts result in the eventual release of inflammatory cytokines such as TNF-α, IL-6, and IL-1, which work together to contribute to the symptomatic presentation of RA patients.

[0039] Disease-modifying antirheumatic drugs (DMARDs) are immunosuppressants and immunomodulators used to treat rheumatoid arthritis (RA). Methotrexate is considered the gold standard for RA treatment. Other DMARDs such as sulfasalazine, leflunomide, and hydroxychloroquine are also available. Biologic DMARDs, including TNF inhibitors (such as adalimumab and etanercept) and novel drugs such as JAK inhibitors (such as tofacitinib and baricitinib), have revolutionized RA treatment by targeting specific components of the immune system. While these drugs can control inflammation and slow disease progression, they may have potential side effects, including increased susceptibility to infections, hepatotoxicity, and gastrointestinal complications. Furthermore, some patients do not respond adequately to these drugs or develop resistance over time, necessitating the search for alternative treatment options.

[0040] The present invention is characterized by compositions and methods for the sustained delivery of therapeutic gene products (e.g., FGF18 and IL-1Ra) to target regions (e.g., joints). Furthermore, compositions and methods for treating arthritis (e.g., OA or RA) are also provided herein. The compositions described herein comprise, for example, one or more recombinant vectors (e.g., adeno-associated virus (AAV) vectors) encoding hFGF18 and hIL-1Ra. Without being mechanistically limited, the compositions described herein can simultaneously address pathological problems and improve symptoms associated with arthritis (e.g., OA or RA) by effectively stimulating the expression of hFGF18 and hIL-1Ra, particularly promoting cartilage repair and reducing joint pain and inflammation. As detailed below, for illustrative purposes, a single intra-articular injection of an AAV containing the hFGF18 and hIL-1Ra transgene allows for sustained expression of these two proteins and achieves a synergistic therapeutic effect, demonstrating the significant therapeutic potential of the compositions described herein.

[0041] Before further describing this disclosure, it should be understood that this disclosure is not limited to the specific embodiments described herein, and it should also be understood that the terminology used herein is for describing specific embodiments and is not intended to be limiting.

[0042] 6.1 Definition Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the terms and techniques described herein relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well-known and commonly used in the art.

[0043] The term “one” or “an entity” refers to one or more of that entity; for example, “antibody” is understood to represent one or more antibodies.

[0044] The term “and / or” as used herein should be considered as a specific disclosure of each of the two specified features or components, whether or not the other is included. Therefore, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, A (alone) and B (alone). Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0045] As used herein, the term “about” is used to indicate a value that includes variations in the inherent error of the device, the methods used to determine the value, or the subjects of study. The word “about” includes the exact figures listed. In some embodiments, “about” means within ±10% of a given value or range. In some embodiments, “about” means a variation of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by “about”. In some embodiments, “about” means a variation of ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by “about”.

[0046] As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally present in a particular organism (e.g., a human) or in a specific location within an organism (e.g., an organ, tissue, or cell, such as a human cell).

[0047] "Isolated" polypeptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions refer to substances that exist in a form not found in nature. Isolated polypeptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions include those that have been purified to the point that they no longer occur in nature. In some embodiments, the so-called "isolated" polypeptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are highly purified.

[0048] The terms “peptide,” “polypeptide,” and “protein,” used interchangeably herein, refer to a polymer of amino acids of any length, which may be linear or branched. It may include non-natural or modified amino acids and may be interrupted by non-amino acid components. Peptides, polypeptides, polypeptide chains, polypeptide chains, or proteins may also be modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

[0049] As used herein, the term "variant," when in relation to a protein or polypeptide ("reference protein" or "reference polypeptide") having specific sequence characteristics, refers to a different protein or polypeptide compared to the reference protein or polypeptide that has one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions. Changes in the amino acid sequence can be amino acid substitutions. These changes can be conserved amino acid substitutions. Functional fragments or functional variants of the protein or polypeptide retain the fundamental structural and functional properties of the reference protein or polypeptide.

[0050] The terms “polynucleotide” and “nucleic acid”, used interchangeably in this document, refer to polymers of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerases.

[0051] The terms “identical,” “percentage of identity,” and their grammatical equivalents, when used herein to describe two or more nucleotides or polypeptides, refer to the fact that when two or more identical sequences or subsequences are compared and aligned (with gaps introduced where necessary) to achieve maximum correspondence, they are identical or have a specified percentage of identical nucleotide or amino acid residues. Conserved amino acid substitutions are not considered part of sequence identity. Percentage identity can be measured by sequence comparison software or algorithms or by visual inspection. Various algorithms and software available for obtaining amino acid or nucleotide sequence alignments are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG WisconsinPackage, and variants thereof. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum full-length alignment of the compared sequences. For example, the percentage sequence identity value can be generated using the sequence comparison computer program BLAST.

[0052] In some embodiments, the two polynucleotides or polypeptides provided herein are substantially identical, meaning that when performing maximum correspondence comparisons and alignments using sequence comparison algorithms or visual inspection, they have at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% nucleotide or amino acid residue identity, and in some embodiments, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide or amino acid residue identity. In some embodiments, identity exists in regions of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, or at least about 60-80 residues, or any integer value in between. In some embodiments, identity exists in regions longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical across the full length of the compared sequences, such as the coding region of a target protein or antibody. In some embodiments, identity exists in a region of a nucleotide sequence having a length of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, or at least about 60-80 bases, or any integer value between these lengths. In some embodiments, identity exists in a region longer than 60-80 bases, for example, at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical in length across the entire length of the compared sequences, such as nucleotide sequences encoding a target protein.

[0053] Those skilled in the art will understand that, as used herein, uridine ribonucleotides in RNA molecules are considered equivalent to thymidine ribonucleotides in DNA molecules in order to determine the percentage of sequence identity. Therefore, if the RNA equivalent and the DNA polynucleotide are different from each other only by the substitution of uridine ribonucleotides in the RNA equivalent for thymidine ribonucleotides in the DNA polynucleotide, then the RNA equivalent and the DNA polynucleotide can be considered to have 100% sequence identity.

[0054] As used herein, the term "gene" refers to a region of DNA that encodes a protein. Genes may include regulatory regions and protein-coding regions. In some embodiments, a gene comprises two or more introns and three or more exons, wherein each intron forms an intermediate sequence between two exons.

[0055] As used herein, the term "encoding" and its syntactic equivalents refer to the inherent properties of a specific nucleotide sequence in a polynucleotide or nucleic acid (such as a gene, cDNA, or mRNA) that serves as a template in biological processes for the synthesis of other polymers and macromolecules having specific nucleotide sequences (such as rRNA, tRNA, and mRNA) or specific amino acid sequences and the resulting biological characteristics. Thus, if the mRNA corresponding to a gene is transcribed and translated to produce a protein, then that gene encodes that protein. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences in degenerate form that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0056] As used herein, the term “RNA equivalent” for a gene refers to an RNA polynucleotide corresponding to the DNA polynucleotide encoding that gene, such as an RNA transcript obtained by transcribing the DNA polynucleotide containing that gene. Exemplary RNA equivalents include synthetically produced mRNA transcripts, for example, by solid-phase nucleic acid synthesis techniques known in the art and / or described herein, as well as by recombinant nucleic acid preparation methods.

[0057] As used herein, the term "intron" refers to a region within the coding region of a gene whose nucleotide sequence has not been translated into the amino acid sequence of the corresponding protein. The term intron also refers to the corresponding region of RNA transcribed from a gene. In some embodiments, for example, a gene may contain at least two introns, each forming an intermediate sequence between two exons. Introns are transcribed into precursor mRNA but are removed during processing and are not included in the mature mRNA.

[0058] As used in this article, the term "codon" refers to any set of three consecutive nucleotide bases in a given messenger RNA molecule or DNA coding strand that designates a specific amino acid or a signal to initiate or stop translation. The term codon also refers to a triplet of bases in a DNA strand.

[0059] As used herein, “codon optimization” refers to the process of modifying a nucleic acid sequence based on the principle that the frequency of synonymous codons (e.g., codons encoding the same amino acid) in encoding DNA varies across different species. This codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Sequences modified in this way are referred to herein as “codon-optimized.” This process can be performed on any sequence described in this specification to enhance expression or stability. Codon optimization can be performed in ways known in the art, such as those described in U.S. Patent Nos. 7,561,972, 7,561,973, and 7,888,112, the entire contents of which are incorporated herein by reference. For example, according to known methods, sequences around translation start sites can be converted into consensus Kozak sequences. See Kozak et al. , Nucleic Acids Res. 15 (20): 8125-8148, the entire contents of which are incorporated herein by reference.

[0060] As used herein, the term “mutation” and its grammatical equivalents refer to a change in the nucleotide sequence of a gene or the polypeptide sequence of a protein. Mutations in genes or proteins can occur naturally, for example, due to errors during DNA replication, DNA repair, radiation, and exposure to carcinogens, or can be induced by the administration of a transgene expressing a mutated gene. Mutations can be caused by single or multiple nucleotide insertions, deletions, or substitutions.

[0061] As used herein, the terms “conservative mutation,” “conservative substitution,” and “conservative amino acid substitution” refer to the replacement of one or more amino acids with another one or more different amino acids that have similar physicochemical properties (such as polarity, electrostatic charge, and steric volume). Conservative amino acid families are generally considered to include, for example, (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Therefore, a conservative mutation or substitution refers to the replacement of a member of the same amino acid family with an amino acid (e.g., replacing Thr with Ser or Arg with Lys).

[0062] As used herein, the term "vector" refers to a nucleic acid, such as DNA or RNA, that can serve as a tool for delivering a target gene into a host cell (e.g., mammalian cells, such as human cells), for example, for replication and / or expression. Exemplary vectors used in conjunction with the compositions and methods described herein are plasmids, DNA vectors, RNA vectors, viral particles, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994 / 11026, the disclosure of which is incorporated herein by reference. The expression vectors described herein comprise polynucleotide sequences and additional sequence elements, for example, for protein expression and / or integration of these polynucleotide sequences into the mammalian cell genome. Some vectors that can be used to express the transgenes described herein include plasmids containing regulatory sequences, such as promoter and enhancer regions, which guide gene transcription. Other useful vectors for expressing transgenes comprise polynucleotide sequences that can increase the rate of these genes or improve the stability or nuclear translocation of the mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signaling sites that guide efficient transcription of the gene carried on the expression vector. The expression vectors described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, or notracin.

[0063] As used herein, the term "operably linked" refers to a first molecule linked to a second molecule, wherein the molecules are arranged such that the first molecule influences the second molecule. These two molecules may or may not be part of a single, continuous molecule, and may or may not be adjacent. For example, if a promoter regulates the transcription of a target polynucleotide molecule that can be transcribed in a cell, then the promoter is operably linked to the transcribed polynucleotide molecule. Furthermore, if two parts of a transcriptional regulatory element are linked together such that the transcriptional activation function of one part is not adversely affected by the presence of the other part, then they are operably linked to each other. Two transcriptional regulatory elements can be operably linked via an adapter nucleic acid (e.g., an intermediate non-coding nucleic acid), or they can be operably linked to each other without the presence of an intermediate nucleotide.

[0064] When two or more polynucleotides are co-expressed, these two polynucleotides can be inserted into, for example, a single expression vector or a separate expression vector. For single-vector expression, the encoding polynucleotide can be operatively linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of the polynucleotide into the host cell can be confirmed using methods well known in the art. Those skilled in the art will understand that the polynucleotide expresses the desired product in sufficient quantities, and further understand that expression levels can be optimized using methods well known in the art to obtain sufficient expression product.

[0065] As used herein and understood in the art, an "expression cassette" is a unique and continuous component of vector DNA, comprising regulatory sequences that control the expression of the nucleotide sequences that the expression cassette may carry. Regulatory sequences include, for example, transcription initiation (promoter) and termination sequences, enhancers, introns, origins of replication, polyadenylation sequences, polypeptide signaling, and chromatin insulating elements. For examples of regulatory sequences, see, for instance, Goeddel, *GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY*, Vol. 185 (Academic Press, San Diego, CA, 1990). Simply put, the expression cassette directs the host cell's machinery to produce RNA and proteins encoded by the nucleotide sequences contained within the expression cassette. Therefore, expression in different organisms or species, such as bacteria, yeast, plants, and mammalian cells, requires different regulatory sequences. The vectors described herein may have one or more expression cassettes. An expression cassette may be "empty" containing a multiple cloning site (MCS) for inserting a nucleotide sequence encoding a transgenic sequence. An expression cassette may be loaded with a transgenic sequence.

[0066] As used herein, the term "cloning site" refers to a nucleic acid sequence containing a restriction site mediated by restriction endonuclease to link nucleic acids via compatible sticky or blunt ends; or a nucleic acid region that serves as a primer binding site for cloning inserted DNA via PCR-mediated homologous and extended "overlapping PCR splicing"; or a recombination site for inserting target nucleic acid via a recombinase-mediated recombination exchange reaction; or a chimeric end for transposon-mediated insertion of target nucleic acid; and other techniques common in the art. As used herein and understood in the art, a "multiple cloning site" or "MCS" refers to a short segment of DNA on a vector containing multiple cloning sites to allow for the insertion of transgenic sequences.

[0067] As used herein, the term "inverted terminal repeat" or "ITR" is a palindromic nucleic acid sequence of approximately 120 to 250 nucleotides in length, capable of forming a hairpin structure. The term "ITR" includes a viral genome replication site that can be recognized and bound by parvovirus proteins (e.g., Rep78 / 68). ITRs can originate from any AAV. An ITR includes a replication protein binding element (RBE) and a terminal resolution sequence (TRS). The term "ITR" includes wild-type ITRs and their variants (e.g., wild-type ITRs can be altered by insertion, deletion, truncation, or missense mutations, as long as the ITR has the function of mediating viral packaging, replication, integration, and / or precursor virus rescue). "5'ITR" refers to a parvovirus ITR located at the 5' boundary of a nucleic acid molecule; the term "3'ITR" refers to a parvovirus ITR located at the 3' boundary of a nucleic acid molecule.

[0068] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent administered to a subject (e.g., a mammal, such as a human) to prevent, treat, or control a specific disease or condition that affects or may affect the subject. As used herein, the term "pharmaceuticalally acceptable" means compounds, materials, compositions, and / or dosage forms suitable for contact with the tissues of a subject (e.g., a mammal, such as a human) without causing excessive toxicity, irritation, allergic reactions, or other problems, and in proportion to a reasonable benefit / risk ratio.

[0069] As used herein, the term "treatment" and its grammatical equivalents relate to a disease or condition or a subject suffering from a disease or condition (e.g., OA), and refer to actions that prevent or mitigate (alleviate) undesirable physiological changes or disease in order to suppress, eliminate, reduce, and / or improve symptoms, the severity of symptoms, and / or the frequency of symptoms associated with the treated disease or condition. Beneficial or desirable clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, stabilization of the disease state (i.e., no worsening), slowing or delaying disease progression, improving or alleviating the disease state, and remission (whether partial or complete), whether these outcomes are detectable or undetectable.

[0070] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that will receive specific treatment. Subjects may be human. Subjects may suffer from specific diseases or conditions.

[0071] As used herein, the term "administration" and its grammatical equivalents refer to the act of delivering a therapeutic agent or pharmaceutical composition into a subject by methods described herein or known in the art. A therapeutic agent can be any compound, such as a genetically modified organism, a vector, a peptide, or a virus. Administering a therapeutic agent or pharmaceutical composition includes prescribing the therapeutic agent or pharmaceutical composition for delivery into a subject. Example forms of administration include oral dosage forms such as tablets, capsules, syrups, and suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP) injections; transdermal dosage forms, including creams, gels, powders, or patches; buccal dosage forms; and inhaled powders, sprays, suspensions, and rectal suppositories.

[0072] As used herein, the terms "effective amount," "therapeutic effective amount," and their grammatical equivalents refer to a dose (alone or as part of a pharmaceutical composition, in a single dose or as part of a series of doses) administered to a subject that produces any detectable positive effect on any symptom, aspect, or characteristic of the subject's disease, disorder, or condition. Therapeutic effective amounts can be determined by measuring the associated physiological effects. The exact amount required varies from subject to subject, depending on the subject's age, weight and general condition, the severity of the condition being treated, the clinician's judgment, etc. In any individual case, a person skilled in the art can determine the appropriate "effective amount" using routine laboratory methods.

[0073] Scope: In this disclosure, various aspects of the invention may be presented in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as an inflexible limitation of the scope of the invention. Therefore, the description of a scope should be considered as having specifically disclosed all possible sub-scopes and the individual numerical values ​​within that scope. For example, a description of a scope such as 1 to 6 should be considered as having specifically disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the scope.

[0074] This document describes exemplary genes and peptides with reference to GenBank numbers, GI numbers, and / or SEQ ID NOs. It should be understood that those skilled in the art can easily identify homologous sequences using reference sequence sources, including but not limited to GenBank (ncbi.nlm.nih.gov / GenBank / ) and EMBL (EMBL.org / ).

[0075] Suitable methods and materials for implementing and / or testing embodiments of this disclosure are described below. These methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar to or equivalent to those described herein may be used. For example, conventional methods well known in the art to which this disclosure pertains are described in various general and more specific references, such as "Sambrook..." et al ., Molecular Cloning: A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory Press, 1989; Sambrook et al ., Molecular Cloning: ALaboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al .,Current Protocols in Molecular Biology, Greene Publishing Associates, 1992(and Supplements to 2000); Ausubel et al "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999."; its entire contents are incorporated herein by reference.

[0076] 6.2 Overview This document provides compositions and methods for delivering recombinant FGF18 peptides and IL-1Ra peptides to a target region (e.g., a joint) in a subject, including but not limited to patients diagnosed with OA or RA. In some embodiments, treatment is performed via gene therapy, for example, by administering a viral vector or other DNA expression construct encoding hFGF18 peptides and hIL-1Ra peptides to the joints of a subject diagnosed with OA or RA (e.g., a human), thereby generating a continuous transduced cell bank that continuously supplies the transgenic products to the target region (e.g., a joint). The transduced cell bank in the target region can continuously deliver the recombinant transgenic products, which can reduce or eliminate the need for repeated treatments.

[0077] In another embodiment, the recombinant FGF18 peptide and IL-1Ra peptide can be generated in cell cultures (e.g., bioreactors) and can be administered systemically or injected into the target area (e.g., a joint). Local gene therapy offers several advantages because systemic delivery can be inefficient due to limitations in penetrating the pore-filled endothelium of synovial capillaries, and unlike the gene therapies disclosed herein, direct delivery of peptides to the target area (e.g., a joint) requires repeated injections, which is not only cumbersome but also carries the risk of infection.

[0078] The therapeutic agents described in this article include FGF18, or fibroblast growth factor 18. FGF18 is an anabolic factor that activates fibroblast growth factor receptor 3 (FGFR3), stimulates chondrocyte proliferation and extracellular matrix (ECM) synthesis, stabilizes the anabolic chondrocyte phenotype, inhibits proteolytic enzyme activity, and increases cartilage thickness, etc. (Xie Y, et al. , Nat Rev Rheumatol. 2020 Oct;16(10):547-564; Jia Li, et al. (2021), Expert Opinion on Investigational Drugs (30:9, 923-930). An exemplary sequence of hFGF18 is shown below (SEQ ID NO:1). The secreted protein hFGF18 contains a 27-amino acid signal sequence that is cleaved during processing. Further information about hFGF18 can be found in the following databases: Uniprot Accession: O76093; HGNC: 3674; MIM: 603726; NCBI GeneID: 8817; RefSeq Protein: NP_003853.1.

[0079] MYSAPSACTCLCLHFLLLCFQVQVLVAEENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIKGKE TEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKRSRRIRPTHPA (SEQ ID NO:1) As used herein, the term "FGF18 polypeptide" refers to the wild-type FGF18 protein or a variant thereof. FGF18 polypeptides can be derived from any organism, such as humans, rats, monkeys, cattle, etc. FGF18 polypeptides can be any subtype of FGF18. A variant of wild-type FGF18 is a different polypeptide that, compared to a reference wild-type FGF18, has one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions. Variants of wild-type FGF18 retain the basic structural and functional properties of the reference wild-type FGF8 (e.g., activation of FGFR3 signaling). Changes in the amino acid sequence can be amino acid substitutions. Changes in the amino acid sequence can be substitutions of conserved amino acids. In some embodiments, a variant of wild-type FGF18 may have a subsequence of wild-type FGF18, which consists of fragments of wild-type FGF18 or multiple wild-type FGF18 fragments linked together. Variations can occur naturally, such as allelic variations or splicing variations. Alternatively, these variants can be obtained through genetic engineering.

[0080] In some embodiments, the FGF18 transgene is delivered to a target region (e.g., a joint) of the subject to form a genetically engineered cell bank for secretion of FGF18 in the target region. In some embodiments, the FGF18 transgene is transduced into chondrocytes or synovial cells. In some embodiments, the subject lacks FGF18 activity. In some embodiments, the FGF18 peptide is hFGF18 (SEQ ID NO:1).

[0081] Interleukin-1 (IL-1) is a pro-inflammatory cytokine that plays a crucial role in promoting inflammation and the development and progression of osteoarthritis. The IL-1-mediated inflammatory response is a complex series of events involving various cell types and molecular pathways. IL-1 exerts its effects by binding to its cell surface receptor, the interleukin-1 receptor (IL-1R). IL-1R is expressed in multiple cell types within the joint and triggers a series of intracellular signaling events, leading to the production of various pro-inflammatory mediators and other cytokines, thereby further maintaining the inflammatory response. IL-1 also stimulates the production of matrix metalloproteinases (MMPs) and proteoglycans, which degrade extracellular matrix components in cartilage, such as collagen and proteoglycans, resulting in loss of joint tissue integrity and exacerbating cartilage destruction. Furthermore, IL-1 induces the production of catabolic factors, promotes chondrocyte apoptosis, inhibits the synthesis of cartilage matrix components, and disrupts the balance between anabolism and catabolism within the joint. One of the hallmark features of arthritis is chronic inflammation and tissue damage caused by an overproduction of IL-1.

[0082] IL-1Ra (Interleukin-1 receptor antagonist) counteracts the effects of IL-1 by acting as a competitive inhibitor of the IL-1 receptor. IL-1Ra also has analgesic effects. IL-1a has at least four isoforms, of which isoform 1 is a secreted protein. An exemplary sequence of IL-1Ra (SEQ ID NO:3) is provided below, which includes a 25-amino acid signal sequence cleaved during processing. Isoforms 2-4 are cytoplasmic proteins. Further information on IL-1Ra can be found in the following databases: Uniprot Accession: P18510; HGNC: 6000; MIM:147679; NCBI GeneID: 3557; RefSeq Protein: NP_000568.1, NP_001305843.1, NP_776213.1, NP_776214.1, NP_776215.1, XP_005263718.1, XP_011509423.1. MEICRGLRSHLITLLLFLFHSETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO:3) As used herein, the term "IL-1Ra polypeptide" refers to the wild-type IL-1Ra protein or a variant thereof. IL-1Ra polypeptides can be derived from any organism, such as humans, rats, monkeys, cattle, etc. IL-1Ra polypeptides can be any subtype of IL-1Ra. A variant of wild-type IL-1Ra refers to a different polypeptide that, compared to a reference wild-type IL-1Ra, has one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions. Wild-type IL-1Ra variants retain the basic structural and functional properties of the reference wild-type IL-1Ra (e.g., inhibition of IL-1R signaling). Changes in the amino acid sequence can be amino acid substitutions. Changes in the amino acid sequence can be conserved amino acid substitutions. In some embodiments, a variant of wild-type IL-1Ra may have a subsequence of wild-type IL-1Ra, which consists of fragments of wild-type IL-1Ra or multiple wild-type IL-1Ra fragments linked together. Variations can occur naturally, such as allelic variations or splicing variations. Alternatively, these variants can be obtained through genetic engineering.

[0083] In some embodiments, delivery of the IL-1Ra transgene to a target region (e.g., a joint) in a subject can create a genetically engineered cell bank to secrete IL-1Ra in the target region. In some embodiments, the IL-1Ra transgene is transduced into chondrocytes or synovial cells. In some embodiments, the subject lacks IL-1Ra activity. In some embodiments, the subject has excessive IL-1 activity. In some embodiments, the IL-1Ra peptide is hIL-1Ra (SEQ ID NO:3).

[0084] In some embodiments, this document describes a method for treating subjects with or at risk of developing arthritis (e.g., OA or RA), comprising administering to a target region (e.g., a joint) a recombinant expression vector encoding one or more integral FGF18 and IL-1Ra peptides, wherein the expression vector expresses the FGF18 and IL-1Ra peptides when used to transduce cells in culture. In some embodiments, the therapeutic transgene is delivered using a recombinant adeno-associated virus vector (rAAV).

[0085] In some alternative embodiments, this document provides methods for treating subjects who have arthritis (e.g., OA or RA) or are at risk of developing arthritis (e.g., OA or RA), including administering therapeutically effective amounts of the FGF18 peptide and the IL-1Ra peptide to a target region (e.g., a joint) of the subject. The FGF18 peptide and the IL-1Ra peptide can be recombinantly produced in vitro using methods described herein or known in the art.

[0086] In some embodiments of the methods described herein, the recombinant expression vector is administered via intra-articular injection. In some embodiments of the methods described herein, the recombinant peptide is administered via intra-articular injection.

[0087] 6.3 Composition This document provides compositions comprising (i) an FGF18 peptide or an FGF18 transgene and (ii) an IL-1Ra peptide or an IL-1Ra transgene. The compositions provided herein can be used, for example, to treat arthritis (e.g., OA or RA). In some embodiments, the composition comprises (i) an FGF18 peptide and (ii) an IL-1Ra peptide. In some embodiments, the composition comprises (i) an FGF18 transgene and (ii) an IL-1Ra transgene, wherein the FGF18 transgene encodes an FGF18 peptide and the IL-1Ra transgene encodes an IL-1Ra peptide.

[0088] In some embodiments, the FGF18 peptide is hFGF18 (SEQ ID NO:1). In some embodiments, the FGF18 peptide is a variant of hFGF18 (SEQ ID NO:1) that retains the basic structural and functional properties of hFGF18 (e.g., activation of FGFR3 signaling). In some embodiments, the FGF18 peptide has at least 85% of its amino acid sequence (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:1. The FGF18 peptide may have at least 88% of its amino acid sequence identical to SEQ ID NO:1. The FGF18 peptide may have at least 90% of its amino acid sequence identical to SEQ ID NO:1. The FGF18 peptide may have at least 92% of its amino acid sequence identical to SEQ ID NO:1. The FGF18 polypeptide may have at least 95% of its amino acid sequence identical to SEQ ID NO:1, or at least 98% identical to SEQ ID NO:1. The FGF18 polypeptide may also have at least 99% of its amino acid sequence identical to SEQ ID NO:1. In some embodiments, compared to hFGF18 (SEQ ID NO:1), the FGF18 polypeptide has one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions. Changes in the amino acid sequence may be amino acid substitutions. Changes in the amino acid sequence may be conserved amino acid substitutions. In some embodiments, the FGF18 polypeptide has a subsequence of hFGF18 (SEQ ID NO:1), which consists of linked fragments of hFGF18 (SEQ ID NO:1) or multiple hFGF18 fragments (SEQ ID NO:1).

[0089] In some embodiments, the FGF18 peptide is rat FGF18 (SEQ ID NO:2). In some embodiments, the FGF18 peptide is a variant of rFGF18 (SEQ ID NO:2) that retains the basic structural and functional properties of rFGF18 (e.g., activation of FGFR3 signaling). In some embodiments, the FGF18 peptide has at least 85% of its amino acid sequence (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:2. The FGF18 peptide may have at least 88% of its amino acid sequence identical to SEQ ID NO:2. The FGF18 peptide may have at least 90% of its amino acid sequence identical to SEQ ID NO:2. The FGF18 peptide may have at least 92% of its amino acid sequence identical to SEQ ID NO:2. The FGF18 peptide may have at least 95% of its amino acid sequence identical to SEQ ID NO:2. The FGF18 polypeptide may have at least 98% of its amino acid sequence identical to SEQ ID NO:2. The FGF18 polypeptide may have at least 99% of its amino acid sequence identical to SEQ ID NO:2. In some embodiments, compared to rFGF18 (SEQ ID NO:2), the FGF18 polypeptide has one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions. Changes in the amino acid sequence may be amino acid substitutions. Changes in the amino acid sequence may be conserved amino acid substitutions. In some embodiments, the FGF18 polypeptide has a subsequence of rFGF18 (SEQ ID NO:2), which consists of linked rFGF18 (SEQ ID NO:2) fragments or multiple fragments of rFGF18 (SEQ ID NO:2).

[0090] In some embodiments, the IL-1Ra peptide is hIL-1Ra (SEQ ID NO:3). In some embodiments, the IL-1Ra peptide is a variant of hIL-1Ra (SEQ ID NO:3) that retains the basic structure and functional properties of hIL-1Ra (e.g., inhibition of IL-1R signaling). In some embodiments, the IL-1Ra peptide has at least 85% of its amino acid sequence (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:3. The IL-1Ra peptide may have at least 88% of its amino acid sequence identical to SEQ ID NO:3. The IL-1Ra peptide may have at least 90% of its amino acid sequence identical to SEQ ID NO:3. The IL-1Ra peptide may have at least 92% of its amino acid sequence identical to SEQ ID NO:3. The IL-1Ra peptide may have at least 95% of its amino acid sequence identical to SEQ ID NO:3. The IL-1Ra polypeptide may have at least 98% of its amino acid sequence identical to SEQ ID NO:3. The IL-1Ra polypeptide may have at least 99% of its amino acid sequence identical to SEQ ID NO:3. In some embodiments, compared to hIL-1Ra (SEQ ID NO:3), the IL-1Ra polypeptide has one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions. Changes in the amino acid sequence may be amino acid substitutions. Changes in the amino acid sequence may be conserved amino acid substitutions. In some embodiments, the IL-1Ra polypeptide has a subsequence of hIL-1Ra (SEQ ID NO:3), which consists of linked hIL-1Ra (SEQ ID NO:3) fragments or multiple fragments of hIL-1Ra (SEQ ID NO:3).

[0091] In some embodiments, the IL-1Ra peptide is rat IL-1Ra (SEQ ID NO:4). In some embodiments, the IL-1Ra peptide is a variant of rIL-1Ra (SEQ ID NO:4) that retains the basic structural and functional properties of rIL-1Ra (e.g., inhibition of IL-1R signaling). In some embodiments, the IL-1Ra peptide has at least 85% of its amino acid sequence (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:4. The IL-1Ra peptide may have at least 90% of its amino acid sequence identical to SEQ ID NO:4. The IL-1Ra peptide may have at least 92% of its amino acid sequence identical to SEQ ID NO:4. The IL-1Ra peptide may have at least 95% of its amino acid sequence identical to SEQ ID NO:4. The IL-1Ra peptide may have at least 98% of its amino acid sequence identical to SEQ ID NO:4. The IL-1Ra polypeptide may have at least 99% of its amino acid sequence identical to SEQ ID NO:4. In some embodiments, compared to rIL-1Ra (SEQ ID NO:4), the IL-1Ra polypeptide has one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions. Changes in the amino acid sequence may be amino acid substitutions. Changes in the amino acid sequence may be conserved amino acid substitutions. In some embodiments, the IL-1Ra polypeptide has a subsequence of rIL-1Ra (SEQ ID NO:4), which consists of linked rIL-1Ra (SEQ ID NO:4) fragments or multiple rIL-1Ra (SEQ ID NO:4) fragments.

[0092] The expression and activity of FGF18 variants or IL-1Ra variants can be tested in vitro, in cell culture, or in experimental animals using routine assays to ensure that the mutations do not impair their therapeutic function. Preferred amino acid substitutions, deletions, or additions should be those that maintain or increase protein activity, stability, or half-life, specifically as determined by routine in vitro experiments, cell culture, or animal models of arthritis (e.g., OA or RA). Any assay method disclosed herein or known in the art can be used.

[0093] 6.3.1 Genetically Modified Organisms The compositions used herein can be used for gene therapy. In some embodiments, the compositions comprise (i) an FGF18 transgene and (ii) an IL-1Ra transgene. In some embodiments, (1) the FGF18 transgene is codon-optimized, (2) the IL-1Ra transgene is codon-optimized, or both.

[0094] As used herein, the term "FGF18 transgene" refers to a region of DNA or its RNA equivalent encoding an FGF18 polypeptide. The FGF18 polypeptide can be any FGF18 polypeptide disclosed herein. In some embodiments, the FGF18 polypeptide is hFGF18 (e.g., SEQ ID NO:1). In some embodiments, the FGF18 polypeptide is rFGF18 (e.g., SEQ ID NO:2). In addition to the FGF18 coding region, the FGF18 transgene may also include a regulatory region. For example, in some embodiments, the FGF18 transgene may also include introns.

[0095] In some embodiments, the FGF18 transgene encodes hFGF18 and has at least 85% of its nucleotide sequence (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, co-or 99%) identical to SEQ ID NO:5. For example, in some embodiments, the FGF18 transgene has at least 86% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgene may have at least 87% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgene may have at least 88% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgene may have at least 89% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgene may have at least 90% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgene may have at least 91% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgenic gene can have at least 92% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgenic gene can have at least 93% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgenic gene can have at least 94% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgenic gene can have at least 95% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgenic gene can have at least 96% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgenic gene can have at least 97% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgenic gene can have at least 98% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:5. The FGF18 transgenic gene can have at least 99% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:5.

[0096] In some embodiments, the FGF18 transgene encodes rFGF18 and has at least 85% of its nucleotide sequence (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, co-or 99%) identical to SEQ ID NO:7. For example, in some embodiments, the FGF18 transgene has at least 86% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgene may have at least 87% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgene may have at least 88% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgene may have at least 89% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgene may have at least 90% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgene may have at least 91% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgenic gene can have at least 92% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgenic gene can have at least 93% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgenic gene can have at least 94% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgenic gene can have at least 95% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgenic gene can have at least 96% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgenic gene can have at least 97% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgenic gene can have at least 98% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:7. The FGF18 transgenic gene can have at least 99% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:7.

[0097] As used herein, the term "IL-1Ra transgene" refers to a region of DNA or its RNA equivalent encoding an IL-1Ra polypeptide. The IL-1Ra polypeptide can be any IL-1Ra polypeptide disclosed herein. In some embodiments, the IL-1Ra polypeptide is hIL-1Ra (e.g., SEQ ID NO:3). In some embodiments, the IL-1Ra polypeptide is rIL-1Ra (e.g., SEQ ID NO:4). In addition to the IL-1Ra coding region, the IL-1Ra transgene may also include a regulatory region. For example, in some embodiments, the IL-1Ra gene may also include introns.

[0098] In some embodiments, the IL-1Ra transgene encodes hIL-1Ra and has at least 85% of its nucleotide sequence (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, co-or 99%) identical to SEQ ID NO:8. For example, in some embodiments, the IL-1Ra transgene has at least 86% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 87% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 88% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 89% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 90% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 91% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 92% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 93% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 94% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 95% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 96% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 97% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 98% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:8. The IL-1Ra transgene may have at least 99% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:8.

[0099] In some embodiments, the IL-1Ra transgene encodes rIL-1Ra and has at least 85% of its nucleotide sequence (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, co-or 99%) identical to SEQ ID NO:10. For example, in some embodiments, the IL-1Ra transgene has at least 86% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 87% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 88% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 89% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 90% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 91% of its nucleotide sequence identical to the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 92% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 93% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 94% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 95% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 96% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 97% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 98% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:10. The IL-1Ra transgene may have at least 99% nucleotide sequence identity with the nucleic acid sequence of SEQ ID NO:10.

[0100] FGF18 and / or IL-1Ra transgenes can be optimized to achieve, for example, enhanced protein expression in specific cell types. Any codon optimization technique known to those skilled in the art can be employed. (See, for example, Quax) et al. ,2015, Mol Cell59:149-161). In some embodiments, the FGF18 transgene is codon-optimized and has the nucleotide sequence of SEQ ID NO:6. In some embodiments, the FGF18 transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% identical nucleotide sequence to SEQ ID NO:6. In some embodiments, the IL-1Ra transgene is codon-optimized and has the nucleotide sequence of SEQ ID NO:9.

[0101] Table 1: Exemplary transgenic sequences

[0102] 6.3.2 Expression Boxes and Constructs In some embodiments, the compositions provided herein comprise an FGF18 transgene and an IL-1Ra transgene. The FGF18 transgene and the IL-1Ra transgene can be expressed separately in two expression cassettes. The FGF18 transgene and the IL-1Ra transgene can also be expressed in a single expression cassette. In some embodiments, the compositions provided herein comprise an FGF18 expression cassette containing an FGF18 transgene and an IL-1Ra expression cassette containing an IL-1Ra transgene. In some embodiments, the compositions provided herein comprise an expression cassette containing both the FGF18 transgene and the IL-1Ra transgene. The FGF18 and IL-1Ra transgenes can be linked by an adapter sequence.

[0103] The adapter sequence can be a polynucleotide sequence encoding a cleavable adapter. The cleavable adapter can be a 2A polypeptide. As known in the art, a 2A polypeptide encodes a self-cleaving short polypeptide (approximately 20 amino acids), providing a mechanism for subsequent separation of equimolarly generated target polypeptides. Exemplary self-cleaving 2A polypeptides include P2A, E2A, F2A, and T2A.

[0104] In some embodiments, the adapter sequence is a nucleotide sequence including an internal ribosomal entry site (IRES) for initiating translation in the middle of a nucleotide sequence (e.g., an mRNA sequence). et al , 2011. PLoS One 6(4): e18556; the entire contents of which are incorporated herein by reference), IRES can be used, for example, to regulate the expression of one or more transgenes. In some embodiments, the encoded linker includes a cleavage site of a cathepsin, matrix metalloproteinase, or legume aspartate endonuclease, as described in, for example, WO2008052322, the entire contents of which are incorporated herein by reference.

[0105] Therefore, in some embodiments, this document provides nucleic acids that integrally comprise the FGF18 transgene and the IL-1Ra transgene. Nucleic acids may include DNA, RNA, or a combination of DNA and RNA. In some embodiments, the nucleic acids provided herein are single-stranded. In some embodiments, the nucleic acids provided herein are double-stranded.

[0106] In some embodiments, this document provides a nucleic acid comprising an expression cassette containing an FGF18 transgene, an adapter sequence, and an IL-1Ra transgene. In some embodiments, this document provides a nucleic acid comprising an FGF18 expression cassette containing an FGF18 transgene and an IL-1Ra expression cassette containing an IL-1Ra transgene. The FGF18 expression cassette and the IL-1Ra expression cassette may be present in cis or trans configurations. In some embodiments, the compositions provided herein comprise a nucleic acid that simultaneously comprises an FGF18 expression cassette and an IL-1Ra expression cassette. In some embodiments, the compositions provided herein comprise a first nucleic acid comprising an FGF18 expression cassette and a second nucleic acid comprising an IL-1Ra expression cassette.

[0107] In some embodiments, the expression cassette includes components that regulate gene delivery or gene expression (e.g., "expression control elements"). Expression control elements include, for example, transcriptional regulatory elements. As known in the art, transcriptional regulatory elements are nucleic acids that at least partially control the transcription of a target gene. Transcriptional regulatory elements may include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) to control or assist in controlling gene transcription.

[0108] In some embodiments, the expression cassettes provided herein contain components that regulate gene expression. In some embodiments, the expression cassettes provided herein contain one or more promoters. As understood in the art, a "promoter" is a nucleic acid that initiates the transcription of a gene into messenger RNA, such transcription being initiated by an RNA polymerase binding to or near the promoter. Examples of other transcriptional regulatory elements can be found, for example, in Goeddel's *Gene Expression Technology: Methods in Enzymology*, Volume 185 (Academic Press, San Diego, CA, 1990). In some embodiments, the promoter is a constitutive promoter. In alternative embodiments, the promoter is an inducible promoter. In a preferred embodiment, a strong constitutive promoter that provides sustained transgene expression is used.

[0109] In some embodiments, the promoter includes a TATA box. In some embodiments, the promoter comprises one or more elements. In some embodiments, one or more promoter elements may be inverted or movable relative to each other. In some embodiments, the elements of the promoter are positioned to act synergistically. In some embodiments, the elements of the promoter are positioned to act independently. In some embodiments, the expression cassette provided herein comprises one or more promoters selected from the CB promoter, ubiquitin c (UBC) promoter, cytomegalovirus (CMV) promoter, SV40 early promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, β-glucuronidase (GUSB) promoter, chicken beta-actin (CBA) promoter, mouse insulin promoter, CAG promoter, RPE65 promoter, and opsin promoter. In some embodiments, the expression cassette provided herein comprises one or more tissue-specific promoters.

[0110] In some embodiments, the viral genome contains two promoters. As a non-limiting example, the promoters are an EF1α promoter and a CMV promoter.

[0111] In some embodiments, the expression cassettes provided herein include one or more regulatory elements in addition to a promoter. In some embodiments, the expression cassettes provided herein include an enhancer. In some embodiments, the expression cassettes provided herein include an intron or a chimeric intron (e.g., a chicken β-actin intron). In some embodiments, the expression cassettes provided herein include a polyadenylated sequence (e.g., a rabbit β-globin polyadenylate signal). In some embodiments, the expression cassettes provided herein include one or more long terminal repeat (LTR) promoters selected from AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTRs.

[0112] For illustrative purposes, in some embodiments, the expression cassette provided herein includes: a promoter sequence, a coding sequence of the target gene (FGF18 and / or IL-1Ra transgene), an untranslated region, and a termination sequence.

[0113] 6.3.3 Carrier The methods provided herein are used with vectors or other expression constructs that encode, integrally, FGF18 polypeptides, such as hFGF18 (SEQ ID NO:1), and IL-1Ra polypeptides, such as hIL-1Ra (SEQ ID NO:3). Therefore, in some embodiments, the vectors provided herein integrally contain both the FGF18 transgene and the IL-1Ra transgene. The vector may contain DNA, RNA, or a combination of DNA and RNA. In some embodiments, the vectors provided herein are single-stranded. In some embodiments, the vectors provided herein are double-stranded.

[0114] In some embodiments, this document provides vectors comprising one or more expression cassettes disclosed herein, which collectively comprise an FGF18 transgene and an IL-1Ra transgene. In some embodiments, the vectors provided herein comprise expression cassettes containing both transgenes. For example, the vectors provided herein may comprise an expression cassette containing an FGF18 transgene, an adapter sequence, and an IL-1Ra transgene. The adapter sequence may encode a cleavable adapter, such as a 2A peptide. In some embodiments, the vectors provided herein comprise an FGF18 expression cassette containing an FGF18 transgene and an IL-1Ra expression cassette containing an IL-1Ra transgene. The FGF18 expression cassette and the IL-1Ra expression cassette may be present in cis or trans configurations. In some embodiments, the compositions provided herein comprise a vector that simultaneously comprises an FGF18 expression cassette and an IL-1Ra expression cassette. In some embodiments, the compositions provided herein comprise a first vector containing an FGF18 expression cassette and a second vector containing an IL-1Ra expression cassette.

[0115] The vectors and other expression constructs provided herein include any suitable methods for delivering FGF18 and IL-1Ra transgenes to a target region (e.g., a joint). Transgene delivery methods include viral vectors, liposomes, other lipid-containing complexes, other macromolecular complexes, synthetically modified mRNA, unmodified mRNA, small molecules, non-biologically active molecules (e.g., gold particles), polymeric molecules (e.g., dendritic macromolecules), naked DNA, plasmids, bacteriophages, transposons, granules, or free organisms. In some embodiments, viral vectors may be used to deliver transgenes to a target region. In some embodiments, the viral vectors provided herein are recombinant viral vectors. In some embodiments, the viral vectors provided herein are modified to be non-replicating in humans.

[0116] Viral vectors that can be used in the methods described herein include adenoviruses, AAVs, lentiviruses, helper-dependent adenoviruses, herpes simplex viruses, poxviruses, Japanese Sendai virus (HVJ), alpha viruses, vaccinia viruses, and retroviral vectors. In some embodiments, the viral vector is a hybridization vector, for example, an AAV vector placed within a "helpless" adenovirus vector. In some embodiments, the viral vectors provided herein comprise a viral capsid from a first virus and a viral envelope protein from a second virus.

[0117] In some embodiments, the viral vector used in the methods described herein is an adenovirus-based viral vector. The recombinant adenovirus may be a first-generation vector with an E1 deletion, and may or may not have an E3 deletion, with the expression cassette inserted into either deleted region. The recombinant adenovirus may be a second-generation vector containing complete or partial deletions of the E2 and E4 regions. The adenovirus-dependent transgene retains only the adenovirus inverted terminal repeat and the packaging signal (phi). The transgene is inserted between the packaging signal and the 3' ITR, with or without a filler sequence, to maintain the artificial genome close to the wild-type size (approximately 36 kb). Exemplary protocols for producing adenovirus can be found in Alba. et al. , 2005, Gene Therapy The entire contents of 12:S18-S27 are incorporated into this paper by reference.

[0118] In some embodiments, the viral vector used in the methods described herein is a lentiviral viral vector. Four plasmids are used to construct this construct: a plasmid containing the Gag / pol sequence, a vector containing the Rev sequence, a vector containing an envelope protein (i.e., VSV-G), and a cis plasmid containing packaging elements and transgenic genes. To produce the lentiviral vector, these four plasmids are co-transfected into cells (i.e., HEK293-based cells), wherein polyethyleneimine or calcium phosphate can be used as a transfection agent, etc. The lentivirus can then be harvested from the supernatant. Exemplary protocols for producing lentiviral vectors can be developed using Lesch. et al. , 2011, Gene Therapy 18:531-538, and Ausubel et al. , 2012, Bioprocess Int The contents of both were found in 10(2):32-43 and are incorporated into this paper by reference.

[0119] In some embodiments, the viral vectors provided herein are herpes simplex virus-based viral vectors. In some embodiments, the viral vectors provided herein are MLV-based viral vectors. In some embodiments, the viral vectors provided herein are human immunodeficiency virus (HIV)-based vectors. In some embodiments, the viral vectors provided herein are lentivirus-based viral vectors. In some embodiments, the viral vectors provided herein are alpha virus-based viral vectors. Alpha virus vectors include semliki forest virus (SFV) and sindbisvirus (SIN). In some embodiments, the alpha virus vectors provided herein are recombinant, replication-defective alpha viruses.

[0120] In some embodiments, the vectors provided herein contain components that influence cell binding or targeting. In some embodiments, the vectors provided herein contain components that influence the localization of polynucleotides (e.g., transgenes) taken up into cells. In some embodiments, the vectors provided herein include components that can be used as detection or screening tags for purposes such as detecting or screening cells that have taken up polynucleotides.

[0121] The selection tag may include a gene sequence or a protein or polypeptide encoded by a gene sequence expressed in the host cell, which allows for the identification, selection, and / or purification of host cells from a cell population that may or may not express the selection tag. In some embodiments, the selection tag provides resistance to enable the host cell to survive the selection process, which would otherwise kill the host cell, for example, by treatment with an antibiotic. In some embodiments, the antibiotic selection tag may include one or more antibiotic resistance factors, including but not limited to neomycin resistance (e.g., neo), hygromycin resistance, kanamycin resistance, and / or puromycin resistance.

[0122] In some embodiments, the vectors provided herein may contain screening tags, including but not limited to β-lactamase, luciferase, β-galactosidase, or any other reporter gene as understood in the art, including cell surface markers such as CD4 or truncated nerve growth factor (NGFR) (for GFP, see WO 96 / 23810; Heim et al. , Current Biology 2:178-182 (1996); Heim et al ., Proc. Natl. Acad. Sci. USA (1995); or Heim et al. , Science373:663-664 (1995); for β-lactamases, see WO96 / 30540, the entire contents of which are incorporated herein by reference. In some embodiments, the screening tag may include a fluorescent protein. The fluorescent protein described herein may include any fluorescent marker, including but not limited to green, yellow, and / or red fluorescent proteins (GFP, YFP, and / or RFP). In some embodiments, the payload construct encoding the screening tag may include a human influenza hemagglutinin (HA) tag.

[0123] 6.3.4 AAV In some embodiments, the compositions provided herein comprise one or more recombinant AAV vectors that collectively deliver the FGF18 transgene and the IL-1Ra transgene to the target region.

[0124] As understood in the art, "adeno-associated virus" or "AAV" is a small, non-enveloped virus belonging to the Parvoviridae family. It is a single-stranded DNA virus with a genome length of approximately 4.7 kilobases. AAV is characterized by its ability to establish latent infection within host cells. The AAV genome typically contains two open reading frames (ORFs), encoding replication-related proteins (Rep) and capsid structural proteins (Cap). Flanking the ORF are two inverted terminal repeats (ITRs), which are the starting points for viral genome replication. The wild-type AAV genome contains nucleotide sequences from two ORFs: one encoding four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by the Rep gene), and the other encoding three capsid or structural proteins (VP1, VP2, VP3, encoded by the capsid gene or Cap gene). The Rep proteins are important for replication and packaging, while the capsid proteins assemble to form the protein coat of the AAV, or AAV capsid. Alternative splicing and variable start codons and promoters result in the generation of four non-structural Rep proteins from a single ORF. Different Rep proteins are derived, and three capsid proteins are generated from a single open reading frame. VP1 is the full-length capsid sequence, while VP2 and VP3 are shorter components within the entire capsid sequence. The three capsid proteins assemble together to form the AAV capsid protein. While not wishing to be bound by theory, the AAV capsid protein typically contains a VP1:VP2:VP3 molar ratio of 1:1:10. As used herein, “AAV serotype” is primarily defined by the AAV capsid. In some cases, the ITR is also specifically described by the AAV serotype (e.g., AAV2 / 9).

[0125] As used herein, the terms “recombinant AAV” or “rAAV” refer to a modified or engineered version of the naturally occurring AAV.

[0126] As used herein, the terms “AAV vector” or “AAV particle” include the capsid and viral genome. In some embodiments, the viral genome contains one or more polynucleotides or transgenic polynucleotide regions encoding peptides such as FGF18 and / or IL-1Ra peptides.

[0127] AAV typically requires a helper (e.g., adenovirus) to carry out productive infection within a cell. Without this helper function, AAV viral particles essentially just enter the host cell but do not integrate into the cell's genome.

[0128] Due to certain unique characteristics, AAV vectors have been investigated for gene therapy delivery. Non-limiting examples of these characteristics include (i) the ability to infect both dividing and non-dividing cells; (ii) a broad host infection range, including human cells; (iii) wild-type AAVs are not associated with any disease and do not replicate in infected cells; and (iv) the non-integration of the host chromosome, thus reducing the likelihood of long-term genetic alterations. Furthermore, AAV vector infection has little effect on altering cellular gene expression patterns (Stilwell and Samulski). et al. , Biotechniques , 2003, 34, 148; the entire contents of which are incorporated herein by reference.

[0129] Typically, AAV vectors used to deliver the FGF18 peptide and / or IL-1Ra peptide can be replication-deficient recombinant viral vectors because they lack sequences within the viral genome encoding the functional Rep and Cap proteins. In some cases, defective AAV vectors may lack most or all of the coding sequences, essentially containing only one or two AAV ITR sequences and a payload sequence. In some embodiments, the viral genome contains the FGF18 transgene. In some embodiments, the viral genome contains the IL-1Ra transgene. In some embodiments, the viral genome contains both the FGF18 transgene and the IL-1Ra transgene.

[0130] In some embodiments, this document provides a viral genome (e.g., an AAV viral genome) comprising one or more expression cassettes disclosed herein, the expression cassettes collectively comprising an FGF18 transgene and an IL-1Ra transgene. In some embodiments, the viral genome provided herein comprises an expression cassette containing two transgenes. For example, the viral genome provided herein may comprise an expression cassette containing an FGF18 transgene, an adapter sequence, and an IL-1Ra transgene. The adapter sequence may encode a cleavable adapter, such as a 2A peptide. In some embodiments, the viral genome provided herein comprises an FGF18 expression cassette containing an FGF18 transgene and an IL-1Ra expression cassette containing an IL-1Ra transgene. The FGF18 expression cassette and the IL-1Ra expression cassette may be present in cis or trans. In some embodiments, the compositions provided herein comprise a viral genome that simultaneously contains an FGF18 expression cassette and an IL-1Ra expression cassette. In some embodiments, the compositions provided herein comprise a first viral genome containing an FGF18 expression cassette and a second viral genome containing an IL-1Ra expression cassette.

[0131] The viral genome of the AAV particles disclosed herein can be single-stranded or double-stranded. The size of the vector genome can be small, medium, large, or maximum size. In some embodiments, the AAV vector used in this disclosure is a single-stranded vector (ssAAV). In some embodiments, the AAV vector can be a self-complementary AAV vector (scAAVs), for example, see US7,465,583. The scAAV vector contains two DNA strands that, after annealing, form double-stranded DNA. By skipping the synthesis of the second strand, scAAVs can be rapidly expressed in cells. In some embodiments, the AAV vector used in this disclosure is scAAV.

[0132] Methods for producing and / or modifying AAV vectors (e.g., pseudo-AAV vectors) are disclosed in this art (International Patent Publication Nos. WO200028004; WO200123001; WO2004112727; WO2005005610 and WO 2005072364, the entire contents of which are incorporated herein by reference).

[0133] In some embodiments, the AAV particles provided herein contain the viral genome of this disclosure. In some embodiments, the AAV particles disclosed herein can be introduced into mammalian cells.

[0134] In some embodiments, the AAV vector can be modified to improve delivery efficiency. Such modified AAV vectors of this disclosure can be efficiently packaged and used to successfully infect target cells with high frequency and minimal toxicity.

[0135] 6.3.4.1 AAV serotype The AAV particles disclosed herein may comprise or be derived from any natural or recombinant AAV serotype. According to this disclosure, the AAV particles may utilize or be serotype-based, or comprise polypeptides selected from any of the following serotypes: AAV1, AAV2, AAV2.5, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV13, and any other AAV now known or hereafter discovered. See, for example, Fields. et al. Virology, 4th ed. Lippincott-Raven Publishers, Philadelphia, 1996; Other AAV serotypes and clades have recently been discovered. See, for example, Gao et al. J. Virol. 78:6381 (2004); Moris et al. Virol. 33:375 (2004).

[0136] In addition, AAV particles can also utilize or be based on serotypes, or include peptides selected from the following serotypes: VOY101, VOY201, AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS,AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP,AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP,AAVPHP.S / G2A12, AAVG2A15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S,AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b,AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12,AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2,AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61,AAV2-4 / rh.50, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9,AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55,AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11,AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41,AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV33.12 / hu.17,AAV33.4 / hu.15, AAV33.8 / hu.16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58.2 / hu.25,AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8,AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3,AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69,AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1 / hu.1, AAVH-5 / hu.3,AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5,AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVcy.5R3. AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5,AAVhu.6,AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16,AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24,AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32,AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42,AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46,AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51,AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60,AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19,AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13,AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21,AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33,AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39,AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49,AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58,AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74,AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV,bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16,AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4,AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1,AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13,AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAVShuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAVShuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAVSM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62,AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53,AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10,Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAVCBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAVCBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3,AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9,AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7,AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAVCKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1,AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6,AAV CLg-F7, AAVCLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV CLv-18, AAV Clv-9, AAV Clv CLv-2, AAVCLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3,AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-DAV-1, AAV CLv-1, AAV-CLv-1 AAVCLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-MAV, AAV-CLv-AAV7 CLv-M8,AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAVCLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CLv-1, AAV 1-AVCSp, AAV AVCSp CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAVCSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8. CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4,AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14,AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4,AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 and their variants.

[0137] The genomic sequences of various serotypes of AAV, as well as the sequences of the native ITR, Rep protein, and capsid subunit, are known in the art. These sequences can be found in the literature or in public databases such as GenBank. For example, see NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, AY631966, AX753250. EU285562, NC_001358, NC_001540, AF513851, AF513852, and AY530579; the disclosures of which are incorporated herein by reference for their use in the study of AAV nucleic acid and amino acid sequences. See also, for example, Bantel-Schaal et al. J. Virol. 73:939 (1999);Chiorini et al. J. Virol. 71:6823 (1997); Chiorini et al. J. Virol. 73:1309(1999); Gao et al. P roc. Nat. Acad. Sci. USA 99:11854 (2002); Moris et al. Virol . 33:375 (2004); Muramatsu et al. Virol 221:208 (1996); Ruffing et al. J. Gen. Virol. 75:3385 (1994); Rutledge et al. J. Virol. 72:309 (1998);Schmidt et al. J. Virol. 82:8911 (2008); Shade et al. J. Virol. 58:921(1986); Srivastava et al. J. Virol. 45:555 (1983); Xiao et al. J. Virol. 73:3994 (1999); Pulicherla et al. , Molecular Therapy , 19(6):1070-1078 (2011); US6,156,303; US20030138772; US20150159173; US 7,198,951; US9,475,845; US20140359799; US9,233,131; US20150376607; US9,163,261; US20150376240; US20160017295; US20150238550; US20150315612; US9,238,800; US9,193,769; US7,427,396; US9,624,274; S20150159173; US20160017005; US8,734,809; WO 00 / 28061,WO 99 / 61601, WO 98 / 11244; WO1998011244; WO2014144229; WO2005033321;WO2015168666; WO2015121501; WO2015038958; WO2016049230; WO2016065001;WO2017100671; WO2017058892; the contents of these publications are incorporated herein by reference for the purpose of studying AAV nucleic acid and amino acid sequences.

[0138] In some embodiments, AAV serotypes AAV2, AAV2.5, AAV5, AAV8, or AAV9, or mixtures thereof, can be used to deliver FGF18 and / or IL-1Ra transgenes to the target region. In some embodiments, AAV2.5 can be used. In some embodiments, AAV5 can be used. In some embodiments, AAV8 can be used. In some embodiments, AAV9 can be used.

[0139] 6.3.4.2 AAV Virus Genome In some embodiments, the AAV particles of this disclosure are used as expression vectors, comprising a viral genome containing FGF18 and IL-1Ra transgenes. In some embodiments, the AAV particles, such as the AAV particles described herein for vectorized delivery of FGF18 and / or IL-1Ra peptides, comprise a viral genome, such as an AAV viral genome. In some embodiments, for example, the AAV viral genome comprises an inverted terminal repeat (ITR) region, an enhancer, a promoter, an intron region, a Kozak sequence, an exon region, nucleic acid encoding transgenes (FGF18 and / or IL-1Ra), a polyA signaling region, or a combination thereof.

[0140] Reverse end repetition (ITR) In some embodiments, the viral genome includes at least one ITR region. The AAV particle of this disclosure includes a viral genome containing at least one ITR region and a transgenic region. In some embodiments, the viral genome has two ITRs. These two ITRs are located at the 5' and 3' ends of the transgenic region. In some embodiments, the ITR serves as an origin of replication, which includes a recognition site for replication. In some embodiments, the ITR includes a sequence region that may be complementary and symmetrically arranged. In some embodiments, the ITR incorporated into the viral genome described herein may consist of a naturally occurring polynucleotide sequence or a recombinant-derived polynucleotide sequence.

[0141] The ITR can be derived from the same serotype as the capsid, which can be selected from any known serotype. The serotype of the ITR may be different from that of the capsid. In some embodiments, the AAV particle has more than one ITR. In one non-limiting example, the AAV particle has a viral genome comprising two ITRs. In some embodiments, the ITRs have the same serotype as each other. In another embodiment, the ITRs have different serotypes. Non-limiting examples include zero, one, or two ITRs having the same serotype as the capsid. In some embodiments, both ITRs of the viral genome of the AAV particle are AAV2 ITRs.

[0142] Independently, the length of each ITR can be from about 100 to about 150 nucleotides. In some embodiments, the length of the ITR is 100-180 nucleotides, for example, about 100-115, about 100-120, about 100-130, about 100-140, about 100-150, about 100-160, about 110-120, about 110-130, about 110-140, about 110-150, about 110-160, about 110-170, about 110-180, about 120-130, about 120-140, about 120... -150, about 120-160, about 120-170, about 120-180, about 130-140, about 130-150, about 130-160, about 130-170, about 130-180, about 140-150, about 140-160, about 140-170, about 140-180, about 150-160, about 150-170, about 150-180, about 160-170, about 160-180, or about 170-180 nucleotides. In some embodiments, the ITR is about 120-140 nucleotides long, for example, about 130 nucleotides long. In some embodiments, the ITR is 140-142 nucleotides long, for example, 141 nucleotides long. In some embodiments, the ITR comprises 125-135 nucleotides in length, for example, 130 nucleotides in length. Non-limiting examples of ITR length are 102, 130, 140, 141, 142, 145 nucleotides in length, and those having at least 95% identity with it.

[0143] In some embodiments, the ITR comprises the nucleotide sequence of any one of SEQ ID NO:11-15, or substantially the same nucleotide sequence (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical). In some embodiments, the ITR comprises nucleotide sequences having one, two, or three but no more than four modifications (e.g., substitutions) relative to SEQ ID NO:11-15. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO:11, or a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:11. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO:12, or a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:12. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO:13, or a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:13. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO:14, or a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:14. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO:15, or a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:15.

[0144] Promoters and expression enhancers In some embodiments, the viral genome includes an expression cassette disclosed herein, which contains at least one element to enhance transgene targeting specificity and expression. See, for example, Powell. et al. , Discov Med. , 2015, 19(102):49-57; the entire contents of which are incorporated herein by reference. Non-limiting examples of elements that enhance transgenic targeting specificity and expression include promoters, polyadenylation (PolyA) signaling sequences, enhancers, and introns.

[0145] In some embodiments, the viral genome contains a promoter sufficient for expression, for example, for expressing the FGF18 transgene and / or the IL-1Ra transgene in target cells. In some embodiments, a promoter is considered effective when it drives transgene expression of the viral genome of the AAV particle. In some embodiments, the promoter drives the expression of the FGF18 transgene and / or the IL-1Ra transgene in the target tissue for a period of time. Promoter-driven expression can last for hours, days, weeks, months, or years. The promoter can be naturally occurring or non-natural. Non-limiting examples of promoters include viral promoters, plant promoters, and mammalian promoters. In some embodiments, the promoter can be a human promoter. In some embodiments, the promoter can be truncated.

[0146] In some embodiments, the viral genome contains promoters that can be expressed in one or more (e.g., multiple) cells and / or tissues, such as universally expressed promoters. In some embodiments, promoters that drive or promote expression in most mammalian tissues include, but are not limited to, the human elongation factor 1α-subunit (EF1α) promoter, the cytomegalovirus (CMV) immediate early enhancer and / or promoter, the chicken β-actin (CBA) promoter and its derivatives, the CAG promoter, the CB6 promoter, etc., the β-glucuronidase (GUSB) promoter, and the ubiquitin C (UBC) promoter. In some embodiments, the promoter may be a combination of two or more components of the same or different initiating or parental promoters, such as, but not limited to, the CMV and CBA (or CB promoter) promoters.

[0147] In some embodiments, the promoter may be less than 1kb. The promoter length can be 50-100, 50-200, 50-300, 50-400, 100-200, 100-300, 100-400, 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800. Or between 700 and 800 nucleotides.

[0148] In some embodiments, the promoter is a generally expressed promoter, such as Yu. et al. , ( Molecular Pain 2011, 7:63), Soderblom et al. ( E. Neuro 2015), Gill et al. , ( Gene Therapy 2001, Vol. 8, 1539-1546), and Husain et al. ( Gene Therapy As described in (2009), each of which is incorporated herein by reference in its entirety. In some embodiments, the promoter is not cell-specific.

[0149] In some embodiments, the promoter is a ubiquitin c (UBC) promoter. The UBC promoter can be 300-350 nucleotides in size. In some embodiments, the promoter is a β-glucuronidase (GUSB) promoter. The GUSB promoter can be 350-400 nucleotides in size. In some embodiments, the promoter is a chicken β-actin (CBA) promoter or a functional variant thereof. In some embodiments, the promoter is a CB6 promoter or a functional variant thereof. In some embodiments, the promoter is a minimal CB promoter or a functional variant thereof. In some embodiments, the promoter is a CBA promoter or a functional variant thereof. In some embodiments, the promoter is a minimal CBA promoter or a functional variant thereof. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter or a functional variant thereof. In some embodiments, the promoter is a CAG promoter or a functional variant thereof. In some embodiments, the promoter is an EF1α promoter or a functional variant thereof. In some embodiments, the promoter is an RNA pol type III promoter. As a non-limiting example, the RNA pol type III promoter is the U6 promoter. As a non-limiting example, the RNA pol type III promoter is the H1 promoter.

[0150] In some embodiments, the promoter is a CB promoter or a functional variant thereof.

[0151] In some embodiments, the viral genome contains two promoters. As a non-limiting example, the promoters are the EF1α promoter and the CMV promoter.

[0152] In some embodiments, the promoter comprises the nucleotide sequence of any one of SEQ ID NO:16-18, or a nucleotide sequence substantially identical to SEQ ID NO:16-18 (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical). In some embodiments, the promoter comprises a nucleotide sequence having one, two, or three but no more than four modifications (e.g., substitutions) relative to SEQ ID NO:16-18. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:16, or a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:16. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:17, or a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:17. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:18, or the same nucleotide sequence as SEQ ID NO:18 by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%.

[0153] In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO:19, or a nucleotide sequence substantially identical to SEQ ID NO:19 (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical). In some embodiments, the promoter comprises a nucleotide sequence having one, two, or three, but no more than four modifications (e.g., substitutions) relative to SEQ ID NO:19.

[0154] Introns In some embodiments, the vector genome contains at least one intron or a fragment or derivative thereof. In some embodiments, the at least one intron may enhance the expression of the FGF18 and / or IL-1Ra peptides described herein. Non-limiting examples of introns include MVM (67-97 bps), F.IX truncated intron 1 (300 bps), β-globin SD / immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor / immunoglobulin splice acceptor (500 bps), SV40 late splice donor / splicing acceptor (19S / 16S) (180 bps), and hybrid adenovirus splice donor / IgG splice donor acceptor (230 bps).

[0155] In some embodiments, the length of an intron can be 100-500 nucleotides. The length of an intron can be 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nucleotides. The length of introns can be between 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400-500 nucleotides.

[0156] In some embodiments, the intron comprises the nucleotide sequence of SEQ ID NO:20, or a nucleotide sequence substantially identical to SEQ ID NO:20 (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical). In some embodiments, the promoter comprises a nucleotide sequence having one, two, or three, but no more than four modifications (e.g., substitutions) relative to SEQ ID NO:20.

[0157] Non-translated area (UTR) In some embodiments, the wild-type UTR of a gene is transcribed but not translated. Typically, the 5′ UTR begins at the transcription start site and terminates at the start codon, while the 3′ UTR begins immediately after the stop codon and continues until the transcription termination signal.

[0158] Features commonly found in genes highly expressed in specific target organs can be engineered into the UTR to enhance stability and protein production. In some embodiments, the viral genome encoding the transgene described herein includes the Kozak sequence. While not wishing to be bound by theory, the wild-type 5'UTR includes features that play a role in translation initiation. The Kozak sequence is well known to be involved in the ribosomal initiation of translation of many genes and is typically contained in the 5'UTR. The Kozak sequence has a consensus sequence of CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three sites upstream of the start codon (ATG), followed by another "G".

[0159] While not wishing to be bound by theory, it is known that the wild-type 3'UTR contains a region containing both adenosine and guanosine. These AU-rich features are particularly prevalent in genes with high replacement rates. The introduction, removal, or modification of AU-rich elements (AREs) in the 3'UTR can be used to regulate the stability of polynucleotides. When engineering specific polynucleotides, such as payload regions of a viral genome, one or more copies of AREs can be introduced to make the polynucleotide less stable, thereby reducing translation and the production of the resulting protein. Similarly, AREs can be identified, removed, or mutated to improve intracellular stability, thereby increasing the translation and production of the resulting protein.

[0160] In some embodiments, the 3'UTR of the viral genome may include an oligomeric (dT) sequence used as a template for adding a poly-A tail.

[0161] Any UTR of any gene known in the art can be incorporated into the viral genome of an AAV particle. These UTRs, or portions thereof, may be oriented in the same direction as the selected gene, or their orientation or position may be altered. In some embodiments, the UTRs used in the viral genome of the AAV particle may be inverted, shortened, lengthened, or made using one or more other 5' UTRs or 3' UTRs known in the art. In some embodiments, the viral genome of the AAV particle contains at least one artificial UTR that is not a variant of a wild-type UTR. In some embodiments, the viral genome of the AAV particle contains UTRs selected from a family of transcripts sharing common functions, structures, characteristics, or properties.

[0162] Polyadenylated (polyA) sequence In some embodiments, the viral genome of the AAV particle of this disclosure contains at least one polyA sequence. The viral genome of the AAV particle may contain a polyA sequence between the 3' end of the transgene coding sequence and the 5' end of the 3' UTR. In some embodiments, the polyA signaling region is located relative to the 3' end of the nucleic acid containing the transgene.

[0163] In some embodiments, the polyA signal region is approximately 100-600 nucleotides in length, for example, approximately 100-500 nucleotides, approximately 100-400 nucleotides, approximately 100-300 nucleotides, approximately 100-200 nucleotides, approximately 200-600 nucleotides, approximately 200-500 nucleotides, approximately 200-400 nucleosides, approximately 200-300 nucleotides, approximately 300-600 nucleotides, approximately 300-500 nucleotides, approximately 300-400 nucleotides, approximately 400-600 nucleotides, approximately 400-500 nucleotides, or approximately 500-600 nucleotides. In some embodiments, the polyA signal region is approximately 100-150 nucleotides in length, for example, approximately 127 nucleotides. In some embodiments, the polyA signal region is approximately 450-500 nucleotides in length, for example, approximately 477 nucleotides. In some embodiments, the polyA signal region comprises the nucleotide sequence of SEQ ID NO:21, or a nucleotide sequence substantially identical to SEQ ID NO:21 (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical). In some embodiments, the polyA signal region comprises a nucleotide sequence having one, two, or three, but no more than four modifications (e.g., substitutions) relative to SEQ ID NO:21. In some embodiments, the polyA signal region comprises the nucleotide sequence of SEQ ID NO:22, or a nucleotide sequence substantially identical to SEQ ID NO:22 (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical). In some embodiments, the polyA signal region comprises a nucleotide sequence having one, two, or three, but no more than four modifications (e.g., substitutions) relative to SEQ ID NO:22.

[0164] Fill sequence In some embodiments, the viral genome includes one or more filler sequences. The filler sequences can be wild-type sequences or engineered sequences. The filler sequences can be variants of the wild-type sequences. In some embodiments, the viral genome includes one or more filler sequences to make the length of the viral genome the optimal size for packaging. For illustrative purposes, in some embodiments, the viral genome is a single-stranded (SS) viral genome and includes one or more filler sequences, which, independently or together, have a length of about 0.1 kb to 3.8 kb; in some embodiments, the viral genome is a self-complementary (SC) viral genome and includes one or more filler sequences to make the length of the viral genome about 2.3 kb.

[0165] In some embodiments, the viral genome includes one or more filler sequences between one or more regions of the viral genome. In some embodiments, the filler region may precede regions such as, but not limited to, transgenic regions, ITR regions, promoter regions, intron regions, enhancer regions, polyadenylation signal sequence regions, and / or exon regions. In some embodiments, the filler region may follow regions such as, but not limited to, payload regions, ITR regions, promoter regions, intron regions, enhancer regions, polyadenylation signal sequence regions, and / or exon regions.

[0166] The filler sequence can be located at the 3' end of the 5' ITR sequence. The filler sequence can be located at the 5' end of the promoter sequence. The filler sequence can be located at the 3' end of the polyadenylation signal sequence. The filler sequence can be located at the 5' end of the 3' ITR sequence. The filler sequence can be located between two intron sequences. The filler sequence can be located within an intron sequence.

[0167] The filler sequence can be located after the 5′ ITR. The filler sequence can be located after the promoter region. The filler sequence can be located after the transgenic region. The filler sequence can be located after the intron region. The filler sequence can be located after the enhancer region. The filler sequence can be located after the polyadenylation signal sequence region. The filler sequence can be located before the promoter region. The filler sequence can be located before the transgenic region. The filler sequence can be located before the intron region. The filler sequence can be located before the enhancer region. The filler sequence can be located before the polyadenylation signal sequence region. The filler sequence can be located before the 3′ ITR.

[0168] Table 2: Exemplary sequence regions of the viral genome.

[0169] 6.3.4.3 Exemplary Viral Genome In some embodiments, the AAV viral genome comprises 5' to 3': a first ITR (e.g., SEQ ID NO: 11, 12, 13, 14, or 15), a first promoter (e.g., SEQ ID NO: 16, 17, or 18), an IL-1Ra transgene (e.g., SEQ ID NO: 8, 9, or 10), a polyA tail (e.g., SEQ ID NO: 21 or 22), a second promoter (e.g., SEQ ID NO: 16, 17, or 18), an FGF18 transgene (e.g., SEQ ID NO: 5, 6, or 7), a second polyA tail (e.g., SEQ ID NO: 21 or 22), and a second ITR (e.g., SEQ ID NO: 11, 12, 13, 14, or 15). In some embodiments, the AAV viral genome further comprises an enhancer located between the 5' ITR and the promoter (e.g., SEQ ID NO: 19). In some embodiments, the AAV viral genome further comprises an intron located between the promoter and the transgene (e.g., SEQ ID NO: 20).

[0170] In some embodiments, the AAV viral genome comprises 5' to 3': a first ITR (e.g., SEQ ID NO: 11, 12, 13, 14, or 15), a first promoter (e.g., SEQ ID NO: 16, 17, or 18), an FGF18 transgene (e.g., SEQ ID NO: 5, 6, or 7), a polyA tail (e.g., SEQ ID NO: 21 or 22), a second promoter (e.g., SEQ ID NO: 16, 17, or 18), an IL-1Ra transgene (e.g., SEQ ID NO: 8, 9, or 10), a second polyA tail (e.g., SEQ ID NO: 21 or 22), and a second ITR (e.g., SEQ ID NO: 11, 12, 13, 14, or 15). In some embodiments, the AAV viral genome further comprises an enhancer located between the 5' ITR and the promoter (e.g., SEQ ID NO: 19). In some embodiments, the AAV viral genome further comprises an intron located between the promoter and the transgene (e.g., SEQ ID NO: 20).

[0171] In some embodiments, the AAV viral genome or other expression vector provided herein comprises 5' to 3': a first ITR (e.g., SEQ ID NO: 11, 12, 13, 14, or 15), a promoter (e.g., SEQ ID NO: 16, 17, or 18), an IL-1Ra transgene (e.g., SEQ ID NO: 8, 9, or 10), an adapter sequence (e.g., SEQ ID NO: 23, 24, 25, or 26), an FGF18 transgene, a polyA tail (e.g., SEQ ID NO: 21 or 22), and a second ITR (e.g., SEQ ID NO: 11, 12, 13, 14, or 15). In some embodiments, the AAV viral genome further comprises an enhancer located between the 5' ITR and the promoter (e.g., SEQ ID NO: 19). In some embodiments, the AAV viral genome further comprises an intron located between the promoter and the transgene (e.g., SEQ ID NO: 20).

[0172] In some embodiments, the AAV includes 5' to 3': a first ITR (e.g., SEQ ID NO: 11, 12, 13, 14, or 15), a promoter (e.g., SEQ ID NO: 16, 17, or 18), an FGF18 transgene (e.g., SEQ ID NO: 5, 6, or 7), an adapter sequence (e.g., SEQ ID NO: 23, 24, 25, or 26), an IL-1Ra transgene (e.g., SEQ ID NO: 8, 9, or 10), a polyA tail (e.g., SEQ ID NO: 21 or 22), and a second ITR (e.g., SEQ ID NO: 11, 12, 13, 14, or 15). In some embodiments, the AAV viral genome further includes an enhancer located between the 5' ITR and the promoter (e.g., SEQ ID NO: 19). In some embodiments, the AAV viral genome further includes an intron located between the promoter and the transgene (e.g., SEQ ID NO: 20).

[0173] In some embodiments, the compositions provided herein comprise (1) a first AAV, wherein the viral genome comprises: a first ITR (e.g., SEQ ID NO: 11, 12, 13, 14, or 15), a first promoter (e.g., SEQ ID NO: 16, 17, or 18), an FGF18 transgene (e.g., SEQ ID NO: 5, 6, or 7), a polyA tail (e.g., SEQ ID NO: 21 or 22), and (2) a second AAV, wherein the viral genome comprises: a second promoter (e.g., SEQ ID NO: 16, 17, or 18), an IL-1Ra transgene (e.g., SEQ ID NO: 8, 9, or 10), a second polyA tail (e.g., SEQ ID NO: 21 or 22), and a second ITR (e.g., SEQ ID NO: 11, 12, 13, 14, or 15). In some embodiments, the first AAV viral genome further comprises an enhancer (e.g., SEQ ID NO: 19) located between the 5'IT2R and the promoter. In some embodiments, the first AAV viral genome further includes an intron located between the promoter and the transgene (e.g., SEQ ID NO:20). In some embodiments, the second AAV viral genome further includes an enhancer located between the 5'ITR and the promoter (e.g., SEQ ID NO:19). In some embodiments, the second AAV viral genome further includes an intron located between the promoter and the transgene (e.g., SEQ ID NO:20).

[0174] Table 3 below provides an example viral genome.

[0175] Table 3: Exemplary Viral Genome Configurations

[0176] This document explicitly considers all permutations and combinations of different elements of the viral genome (e.g., ITR, promoter, enhancer, intron, polyA, etc.), including those disclosed herein or known in the art. The specific constructs in Table 4 are for illustrative purposes. Those skilled in the art will understand that various specific elements of the exemplary viral genome (e.g., ITR, promoter, enhancer, intron, polyA, etc.) can be replaced by another sequence with similar function disclosed herein or known in the art.

[0177] Table 4: Exemplary Viral Genome Constructs

[0178] As shown in Table 4, the example above illustrates the AAV viral genome, which contains a 5' to 3' mITR (e.g., SEQ ID NO: 11), an enhancer (e.g., SEQ ID NO: 19), a CB promoter (e.g., SEQ ID NO: 17), an intron (e.g., SEQ ID NO: 20), hIL-1Ra Co (e.g., SEQ ID NO: 9), a poly A (e.g., SEQ ID NO: 21), and an ITR (e.g., SEQ ID NO: 12). In some embodiments, the AAV viral genome provided herein contains a 5' to 3' mITR (e.g., SEQ ID NO: 11), an enhancer (e.g., SEQ ID NO: 19), a CB promoter (e.g., SEQ ID NO: 17), an intron (e.g., SEQ ID NO: 20), rIL-1Ra (e.g., SEQ ID NO: 10), a poly A (e.g., SEQ ID NO: 21), and an ITR (e.g., SEQ ID NO: 12). In some embodiments, the AAV viral genome provided herein comprises a 5' to 3' mITR (e.g., SEQ ID NO: 11), an enhancer (e.g., SEQ ID NO: 19), a CB promoter (e.g., SEQ ID NO: 17), an intron (e.g., SEQ ID NO: 20), hFGF18 Co (e.g., SEQ ID NO: 6), a poly A (e.g., SEQ ID NO: 21), and an ITR (e.g., SEQ ID NO: 12). In some embodiments, the AAV viral genome provided herein comprises a 5' to 3' mITR (e.g., SEQ ID NO: 11), an enhancer (e.g., SEQ ID NO: 19), a CB promoter (e.g., SEQ ID NO: 17), an intron (e.g., SEQ ID NO: 20), rFGF18 (e.g., SEQ ID NO: 7), a poly A (e.g., SEQ ID NO: 21), and an ITR (e.g., SEQ ID NO: 12).In some embodiments, the AAV viral genome provided herein contains a 5' to 3' ITR (e.g., SEQ ID NO:13), an enhancer (e.g., SEQ ID NO:19), a CB promoter (e.g., SEQ ID NO:17), an intron (e.g., SEQ ID NO:20), hIL-1Ra Co (e.g., SEQ ID NO:9), a poly A (e.g., SEQ ID NO:21), an EF promoter (e.g., SEQ ID NO:18), an intron (e.g., SEQ ID NO:20), hFGF18 Co (e.g., SEQ ID NO:6), a poly A (e.g., SEQ ID NO:21), and an ITR (e.g., SEQ ID NO:12).

[0179] 6.4 Preparation method AAV or other expression vectors used to express the FGF18 peptide and / or IL-1Ra peptide disclosed herein can be prepared using any method known in the art. AAV or other expression vectors used in the gene therapy methods disclosed herein can be produced using any method known in the art.

[0180] General virus production process Cells used for producing AAV (e.g., rAAV) particles may, in some embodiments, include mammalian cells (e.g., HEK293 cells) and / or insect cells (e.g., Sf9 cells). In various embodiments, AAV production includes processes and methods for producing AAV particles and vectors that can contact target cells to deliver transgenes, such as FGF18 and / or IL-1Ra. In some embodiments, the viral vector is an AAV vector, such as a recombinant AAV vector. In some embodiments, the viral particle is an AAV particle, such as a recombinant AAV particle.

[0181] In some embodiments, this document discloses a vector comprising the viral genome of this disclosure. In some embodiments, this document discloses a cell comprising the viral genome of this disclosure. In some embodiments, the cell is a bacterial cell, a mammalian cell (e.g., HEK293 cell), or an insect cell (e.g., Sf9 cell).

[0182] In some embodiments, this document discloses a method for preparing a viral genome. The method includes providing a nucleic acid encoding the viral genome described herein and a backbone region suitable for replication of the viral genome in a cell (e.g., a bacterial cell) (e.g., where the backbone region contains one or both of a bacterial origin of replication and a selection tag), and cutting the viral genome from the backbone region, for example by cleaving nucleic acid molecules upstream and downstream of the viral genome. In some embodiments, the viral genome includes a promoter operatively linked to a nucleic acid containing a transgene (e.g., the FGF18 transgene or IL-1Ra transgene described herein), and the viral genome is incorporated into generated AAV particles in the cell. In some embodiments, the cell is a bacterial cell, a mammalian cell (e.g., HEK293 cell), or an insect cell (e.g., Sf9 cell).

[0183] In some embodiments, this document discloses a method for preparing recombinant AAV particles of the present disclosure, the method comprising (i) providing a host cell containing the viral genome described herein, and incubating the host cell under conditions suitable for encapsulating the viral genome in a capsid protein, thereby preparing recombinant AAV particles. In some embodiments, the method comprises introducing a first nucleic acid containing the viral genome into the cell prior to step (i). In some embodiments, the host cell contains a second nucleic acid encoding a capsid protein. In some embodiments, the second nucleic acid is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule. In some embodiments, the host cell is a bacterial cell, a mammalian cell (e.g., HEK293 cell)², or an insect cell (e.g., Sf9 cell).

[0184] In various embodiments, this document provides methods for producing AAV particles or vectors by: (a) contacting virus-producing cells with one or more viral expression constructs encoding at least one AAV capsid protein and one or more expression constructs encoding transgenes, polynucleotides encoding proteins, and regulatory nucleic acids; (b) culturing virus-producing cells under conditions capable of producing at least one AAV particle or vector; and (c) isolating AAV particles or vectors from the production process.

[0185] In these methods, the viral expression construct can encode at least one structural protein and / or at least one non-structural protein. The structural protein can include any natural or wild-type capsid protein VP1, VP2, and / or VP3, or chimeric proteins thereof. The non-structural protein can include any natural or wild-type Rep78, Rep68, Rep52, and / or Rep40 protein, or chimeric proteins thereof.

[0186] In some embodiments, contact occurs via transient transfection, viral transduction, and / or electroporation.

[0187] In some embodiments, the virus-producing cells are selected from mammalian cells and insect cells. In some embodiments, the insect cells include fall armyworm cells. In some embodiments, the insect cells include Sf9 insect cells. In some embodiments, the insect cells include Sf21 insect cells.

[0188] AAV particles and expression vectors prepared according to the methods described herein are also provided.

[0189] In various embodiments, the AAV particles of this disclosure can be formulated into pharmaceutical compositions containing one or more acceptable excipients.

[0190] In some embodiments, AAV particles can be produced by contacting virus-producing cells (e.g., insect or mammalian cells) with at least one viral expression construct encoding at least one capsid protein and at least one transgenic expression construct. The virus-producing cells can be contacted via transient transfection, viral transduction, and / or electroporation. The virus-producing cells can be cultured under conditions that generate, isolate (e.g., using temperature-induced lysis, mechanical lysis, and / or chemical lysis), and / or purify (e.g., by filtration, chromatography, and / or immunoaffinity purification) at least one AAV particle or vector.

[0191] In some embodiments, AAV particles are produced in insect cells (e.g., fall armyworm (Sf9) cells) using the methods described herein. As a non-limiting example, insect cells can be treated with viral transduction, which may include baculovirus transduction.

[0192] In some embodiments, AAV particles are produced in mammalian cells (e.g., HEK293 cells) using the methods described herein. As a non-limiting example, mammalian cells can be treated with viral transduction, which may include transient transfection with multiple plasmids (e.g., transient transfection with three plasmids).

[0193] In some embodiments, the method of this disclosure includes producing viral particles in viral production cells using a viral production system comprising at least one viral expression construct and at least one transgenic expression construct. The at least one viral expression construct and the at least one transgenic expression construct can be introduced into the viral production cells via co-transfection (e.g., double transfection, triple transfection). Transfection is performed using standard molecular biology techniques known and routinely performed by those skilled in the art. The viral production cells provide the cellular mechanisms required for expressing the proteins and other biological materials needed to produce AAV particles, including the Rep protein for replicating the payload construct and the Cap protein for assembling the capsid encapsulating the replicated payload construct. The produced AAV particles are extracted from the viral production cells and can be processed into pharmaceutical formulations for drug delivery.

[0194] In various embodiments, once administered, the AAV particles disclosed herein can contact and enter target cells (e.g., enter endosomes) without being constrained by theoretical limitations. The AAV particles (e.g., AAV particles released from endosomes) can then contact the nucleus of the target cell to deliver a transgenic construct. The transgenic construct can be delivered into the nucleus of the target cell, where the transgene can be expressed.

[0195] In some embodiments, the production process of viral particles utilizes seed cultures of viral production cells containing one or more baculoviruses (e.g., baculoviral expression vectors (BEVs) that have been transfected with viral expression constructs and transgenic expression constructs, or baculovirus-infected insect cells (BIICs)).

[0196] In some embodiments, large-scale production of AAV particles utilizes a bioreactor. Without being constrained by theory, the use of a bioreactor allows for the precise measurement and / or control of variables supporting the growth and activity of virus-producing cells, such as mass, temperature, mixing conditions (impeller speed or wave oscillation), CO2 concentration, O2 concentration, gas injection rate and volume, gas coverage rate and volume, pH, viable cell density (VCD), cell viability, cell diameter, and / or optical density (OD). In some embodiments, the bioreactor is used for batch production, where the entire culture is harvested at experimentally determined time points for AAV particle purification. In some embodiments, the bioreactor is used for continuous production, where a portion of the culture is harvested at experimentally determined time points for AAV particle purification, while the remaining culture in the bioreactor is renewed by adding new culture medium components.

[0197] In various embodiments, AAV virus particles can be extracted from virus-producing cells through processes including cell lysis, clarification, sterilization, and purification. Cell lysis includes any process that disrupts the structure of virus-producing cells to release AAV particles. In some embodiments, cell lysis may include thermal shock, chemical, or mechanical lysis methods. Clarification may include the crude purification of a mixture of lysed cells, culture medium components, and AAV particles. In some embodiments, clarification includes centrifugation and / or filtration, including but not limited to depth filtration, tangential flow filtration, and / or hollow fiber filtration.

[0198] In various embodiments, the final product of virus production is purified AAV particles comprising two components: (1) a transgenic expression construct (e.g., a recombinant AAV vector genome construct) and (2) a viral capsid.

[0199] In some embodiments, the virus production system or method of this disclosure includes the step of producing baculovirus-infected insect cells (BIICs) using viral production cells (VPCs) and plasmid constructs. In some embodiments, the virus production system or method of this disclosure includes the step of producing AAV particles using viral production cells (VPCs) and baculovirus-infected insect cells (BIICs).

[0200] Viral expression construct In various embodiments, the virus production system of this disclosure includes one or more viral expression constructs that can be transfected / transduced into virus production cells. In some embodiments, the viral expression construct or transgenic expression construct of this disclosure may be a baculovirus, also known as a baculovirus plasmid or recombinant baculovirus genome. In some embodiments, the viral expression construct includes a protein-coding nucleotide sequence and at least one expression control sequence for expression in virus production cells. In some embodiments, the viral expression construct includes a protein-coding nucleotide sequence operatively linked to at least one expression control sequence for expression in virus production cells. In some embodiments, the viral expression construct comprises a parvovirus gene controlled by one or more promoters. The parvovirus gene may include a nucleotide sequence encoding a non-structural AAV replication protein, such as the Rep gene encoding Rep52, Rep40, Rep68, or Rep78 proteins. The parvovirus gene may include a nucleotide sequence encoding a structural AAV protein, such as the Cap gene encoding VP1, VP2, and VP3 proteins.

[0201] The viral expression constructs disclosed herein may include any compound or formulation, whether biological or chemical, to facilitate the transformation, transfection, or transduction of nucleic acids into cells. Exemplary biological viral expression constructs include plasmids, linear nucleic acid molecules, and recombinant viruses (including baculoviruses). Exemplary chemical vectors include lipid complexes. Based on the present disclosure, the viral expression constructs are used to integrate nucleic acid sequences into viral replication cells. O'Reilly, due to its involvement with viral expression constructs and their uses, et al. , Baculovirus expression vectors: a laboratory manual. Oxford University Press, 1994; Maniatis et al., eds. Molecular Cloning. CSH Laboratory, NY, NY (1982); and Philiport and Scluber, eds. Liposomes as tools in Basic Research and Industry. CRC Press, Ann Arbor, Mich. (1995); The contents of each of the above are incorporated herein by reference in their entirety.

[0202] In some embodiments, the viral expression construct is an AAV expression construct that includes one or more nucleotide sequences encoding a non-structural AAV replication protein, a structural AAV capsid protein, or a combination thereof.

[0203] In some embodiments, the viral expression construct of this disclosure may be a plasmid vector. In some embodiments, the viral expression construct of this disclosure may be a baculovirus construct.

[0204] This invention is not limited by the number of viral expression constructs used to produce AAV particles or viral vectors. In some embodiments, according to this disclosure, AAV particles can be produced in virus-producing cells using one, two, three, four, five, six, or more viral expression constructs. In some embodiments of this disclosure, the viral expression constructs can be used to produce AAV particles in insect cells. In some embodiments, the wild-type AAV sequence of the capsid and / or rep gene can be modified, for example, to improve the properties of the viral particles, such as increasing infectivity or specificity, or increasing production yield.

[0205] In some embodiments, the VP coding region encodes one or more AAV capsid proteins of a specific AAV serotype. The VP coding regions for AAV serotypes may be the same or different. In some embodiments, the VP coding region may be codon-optimized. In some embodiments, the VP coding region or nucleotide sequence may be codon-optimized for mammalian cells. In some embodiments, the VP coding region or nucleotide sequence may be codon-optimized for insect cells. In some embodiments, the VP coding region or nucleotide sequence may be codon-optimized for fall armyworm cells. In some embodiments, the VP coding region or nucleotide sequence may be codon-optimized for Sf9 or Sf21 cell lines.

[0206] This disclosure describes the process and method for producing AAV particles or viral vectors that are contacted with target cells to deliver a transgenic expression construct, such as a recombinant AAV particle or viral construct containing target gene nucleotides. The virus production cells can be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells (e.g., insect cells, yeast cells, and mammalian cells).

[0207] mammalian cells In some embodiments, the AAV particles of this disclosure can be produced in virus-producing cells comprising mammalian cells. Virus-producing cells include mammalian cells such as A549, WEH1, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293, HEK293T (293T), Saos, C2C12, L cells, HT1080, Huh7, HepG2, C127, 3T3, CHO, HeLa cells, KB cells, BHK, and primary fibroblasts, hepatocytes, and myoblasts derived from mammals. Virus-producing cells may include cells derived from any mammalian species, including but not limited to humans, monkeys, mice, rats, rabbits, and hamsters, or cell types including but not limited to fibroblasts, hepatocytes, tumor cells, transformed cell lines, etc.

[0208] AAV virus production cells commonly used for producing recombinant AAV particles include, but are not limited to, U.S. Patent Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, 6,428,988, and 5,688,676; other mammalian cells described in U.S. Patent Application 2002 / 0081721 and International Patent Publications WO 00 / 47757, WO 00 / 24916, and WO 96 / 17947, the contents of which are incorporated herein by reference, provided that they do not conflict with this disclosure.

[0209] In some embodiments, AAV virus production cells are trans-complementary packaging cell lines that can provide functionality removed from replication-defective helper viruses, such as HEK293 cells or other Ea trans-complementary cells. In some embodiments, packaging cell line 293-10-3 (ATCC accession number PTA-2361) can be used to produce AAV particles, as described in U.S. Patent No. 6,281,010, the entire contents of which relate to the 293-10-3 packaging cell line and its uses are incorporated herein by reference. In some embodiments of this disclosure, cell lines for trans-complementary adenovirus vector-deficient E1 can be used to produce AAV particles, such as the HeLA cell line, which encodes adenovirus Ela and adenovirus E1b under the control of the phosphoglycerate kinase (PGK) promoter, as described in U.S. Patent No. 6,365,394, the entire contents of which relate to the HeLa cell line and its uses are incorporated herein by reference.

[0210] In some embodiments, AAV particles are generated in mammalian cells using a multiplasmid transient transfection method (such as triplasmid transient transfection). In some embodiments, the multiplasmid transient transfection method includes transfecting three different constructs: (i) a transgenic expression construct, (ii) a Rep / Cap construct (parvovirus Rep and parvovirus Cap), and (iii) a helper construct. In some embodiments, the method of producing three components of AAV particles through triple transfection can be used to produce small batches of virus for assays including transduction efficiency, target tissue (tropism) assessment, and stability. In some embodiments, the method of producing three components of AAV particles through triple transfection can be used to produce large quantities of material for clinical or commercial applications.

[0211] Insect cells In some embodiments, the AAV particles or viral vectors of this disclosure can be produced in virus-producing cells comprising insect cells. Growth conditions of cultured insect cells and the production of heterologous products in cultured insect cells are well known in the art, see U.S. Patent No. 6,204,059, the entire contents of which are incorporated herein by reference, relating to the growth and use of insect cells in virus production.

[0212] According to this disclosure, any insect cell that allows parvovirus replication and can be maintained in culture may be used. AAV virus production cells commonly used for producing recombinant AAV particles include, but are not limited to, fall armyworm Sf9 or Sf21 cell lines, Drosophila cell lines, or mosquito cell lines such as those derived from Aedes albopictus. Methods for expressing heterologous proteins using insect cells, methods for introducing nucleic acids (such as vectors, e.g., insect cell-compatible vectors) into insect cells, and methods for culturing and maintaining such cells are well documented. See, for example, Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al. , BaculovirusExpression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al. , J. Vir. 63:3822-8 (1989); Kajigaya et al. , Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al ., J. Vir. 66:6922-30 (1992); Kimbauer et al. , Vir. 219:37-44 (1996); Zhao et al. , Vir. 272:382-93 (2000); andSamulski et al. U.S. Patent No. 6,204,059, the contents of which relate to the use of insect cells in virus production, are incorporated herein by reference in their entirety.

[0213] Baculovirus Production System In some embodiments, the methods of this disclosure may include producing AAV particles or viral vectors using a baculovirus system, wherein the baculovirus system uses viral expression constructs and transgenic expression constructs. In some embodiments, the baculovirus system includes baculovirus expression vectors (BEVs) and / or baculovirus-infected insect cells (BIICs). In some embodiments, the viral expression constructs or transgenic expression constructs of this disclosure may be baculoviruses, also known as baculovirus plasmids or recombinant baculovirus genomes. In some embodiments, the viral expression constructs or transgenic expression constructs of this disclosure may be polynucleotides incorporated into baculovirus plasmids via homologous recombination (transposon donor / recipient system) using standard molecular biology techniques known and practiced by those skilled in the art. Transfection of a separate viral replication cell population yields two or more sets of baculoviruses (BEVs) (e.g., two or three sets), wherein one or more sets of baculoviruses may include viral expression constructs (expression BEVs), and one or more sets may include payload constructs (payload BEVs). Baculoviruses can be used to infect virus-producing cells to produce AAV particles or viral vectors.

[0214] In some embodiments, the method includes transfecting a single viral replication cell population to generate a single set of baculoviruses (BEVs), which includes a viral expression construct and a payload construct. These baculoviruses can be used to infect virus-producing cells to produce AAV particles or viral vectors. In some embodiments, the BEVs are produced using a baculovirus transfection agent such as Promega FuGENE® HD, WFI water, or ThermoFisher Cellfectin® II reagent. In some embodiments, the BEVs are produced and amplified in virus-producing cells, such as insect cells.

[0215] In some embodiments, the method utilizes seed cultures of virus-producing cells containing one or more BEVs, including baculovirus-infected insect cells (BIICs). Seed BIICs have been transfected / transduced / infected with expression BEVs, including viral expression constructs, and transgenic expression BEVs, including transgenic expression constructs. BEVs used to generate AAV particles in insect cells (including, but not limited to, fall armyworm (Sf9) cells) can provide high-titer viral vector products.

[0216] In some embodiments, genetically stable baculoviruses can serve as a source of one or more components for producing AAV particles in invertebrate cells. In some embodiments, defective baculovirus expression vectors exist in a free state in insect cells. In these embodiments, the corresponding baculovirus vectors are engineered with replication control elements, including but not limited to promoters, enhancers, and / or replication elements that regulate the cell cycle. In some embodiments, stable virus-producing cells that allow baculovirus infection are genetically engineered to stably integrate at least one copy of the elements required for AAV replication and vector production, including but not limited to the entire AAV genome, Rep and Cap genes, Rep gene, Cap gene, a separate transcript cassette for each Rep protein, a separate transcript cassette for each VP protein, AAP (assemblyactivation protein), or at least one of the following: baculovirus helper genes having a natural or non-natural promoter.

[0217] In some embodiments, the AAV particles disclosed herein can be produced in insect cells (e.g., Sf9 cells).

[0218] In some embodiments, the AAV particles of this disclosure can be prepared using triple transfection.

[0219] In some embodiments, the AAV particles of this disclosure can be produced in mammalian cells.

[0220] In some embodiments, the AAV particles of this disclosure can be produced by triple transfection in mammalian cells.

[0221] In some embodiments, the AAV particles of this disclosure can be prepared by triple transfection in HEK293 cells.

[0222] The AAV viral genome encoding the FGF18 peptide and / or IL-1Ra peptide described herein can be used in human diseases, veterinary applications, and various in vivo and in vitro environments. The AAV particles disclosed herein can be used in the medical field to treat, prevent, alleviate, or improve arthritis, such as OA or RA. In some embodiments, the AAV particles disclosed herein are used for intervention and / or treatment of inflammatory joint diseases.

[0223] The various embodiments disclosed herein provide a pharmaceutical composition comprising the AAV particles described herein and pharmaceutically acceptable excipients.

[0224] The various embodiments disclosed herein provide a method for treating a subject in need, including administering a therapeutically effective amount of the pharmaceutical composition described herein to the subject.

[0225] In some embodiments of this method, a subject receives treatment to address a disease or disorder caused by a deficiency in the quantity or function of FGF18 and / or IL-1Ra gene products. In one aspect of this method, pathological features of FGF18-related disorders are alleviated and / or the progression of FGF18-related disorders is terminated, slowed, improved, or reversed. In one aspect of this method, pathological features of IL-1Ra-related diseases are alleviated and / or the progression of IL-1Ra-related diseases is stopped, slowed, improved, or reversed.

[0226] The various embodiments disclosed herein describe a method for increasing the levels of FGF18 peptide and IL-1Ra peptide in a target region (e.g., a joint), comprising injecting the subject with an effective amount of the pharmaceutical composition described herein.

[0227] This article also describes compositions, methods, processes, kits, and apparatus for designing, preparing, manufacturing, and / or formulating AAV particles.

[0228] This disclosure also provides methods for administering and / or delivering vectors and viral particles (e.g., AAV particles) to treat or improve arthritis (e.g., OA or RA). These results are achieved by using the methods and compositions described herein.

[0229] 6.5 Pharmaceutical Compositions This disclosure also provides pharmaceutical compositions for delivering the FGF18 and / or IL-1Ra peptides described herein to subjects, including human subjects. In some embodiments, the composition comprises any AAV particle or AAV viral genome described herein. In some embodiments, the pharmaceutical compositions disclosed herein comprise any AAV genome or AAV particle disclosed herein, along with one or more pharmaceutically acceptable carriers.

[0230] Although the descriptions of pharmaceutical compositions provided herein (e.g., AAV particles or viral genomes containing an FGF18 expression cassette and / or an IL-1Ra expression cassette to be delivered) are primarily directed at pharmaceutical compositions suitable for human administration, those skilled in the art will understand that such compositions are generally suitable for any other animal, such as non-human animals, e.g., non-human mammals. It is well known that pharmaceutical compositions suitable for human administration are modified to make them suitable for administration to a variety of animals, and such modifications can generally be designed and / or performed by a skilled veterinary pharmacologist simply through routine experiments (if necessary). The intended recipients of the pharmaceutical compositions include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0231] In some embodiments, the composition is applied to humans.

[0232] The pharmaceutical compositions disclosed herein can be prepared, packaged, and / or sold in bulk, in single unit doses, and / or in multiple single unit doses. As used herein, a “unit dose” means an independent dose of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be given to a subject and / or a convenient fractionation of that dose, such as half or one-third of that dose. Examples of unit dose forms include ampoules, vials, pre-filled syringes, or cartridges.

[0233] The pharmaceutical compositions described herein can be prepared by any method known or subsequently developed in the field of pharmacology. Generally, such preparation methods involve the steps of combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if necessary and / or required, dividing, shaping, and / or packaging the product into the desired single or multiple dosage units.

[0234] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any other components in the pharmaceutical compositions according to this disclosure will vary depending on the identity, size, and / or condition of the subject being treated and the route of administration of the composition.

[0235] The AAV particles disclosed herein can be formulated using one or more excipients to: (1) increase stability; (2) promote cell transfection or transduction; (3) allow sustained or delayed release; (4) alter biodistribution (e.g., target viral particles to specific tissues or cell types); (5) increase the translation of encoded proteins in vivo; (6) alter the release profile of encoded proteins in vivo and / or (7) allow tunable expression of the payload.

[0236] The pharmaceutical compositions disclosed herein may include, but are not limited to, saline, lipids, liposomes, liposome nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for transplantation into subjects), nanoparticle mimics, and combinations thereof. Furthermore, the viral vectors disclosed herein may be formulated using self-assembled nucleic acid nanoparticles.

[0237] The pharmaceutical compositions disclosed herein may include one or more excipients, each excipient being present in amounts that collectively increase the stability of AAV particles, increase cellular transfection or transduction of viral particles, increase the expression of proteins encoded by viral particles, and / or alter the release characteristics of proteins encoded by AAV particles. In some embodiments, the purity of the pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the excipient is approved for human and veterinary use.

[0238] As used herein, excipients include, but are not limited to, any and all solvents, dispersion media, diluents or other liquid carriers, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, etc., as long as they are suitable for the desired specific dosage form. Various excipients used to formulate pharmaceutical compositions and techniques for preparing such compositions are known in the art (see: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; the entire contents of which are incorporated herein by reference). Conventional excipient media may be considered within the scope of this disclosure unless any conventional excipient media may be incompatible with the substance or its derivatives, for example, may produce any adverse biological effects or otherwise interact with any other component of the pharmaceutical composition in a harmful manner.

[0239] The pharmaceutical compositions of AAV particles disclosed herein may include cationic or anionic compounds. In some embodiments, the formulation includes metal cations, such as, but not limited to, Zn. 2+ Ca 2+ Cu 2+ Mg 2+ Or a combination thereof. In some embodiments, the pharmaceutical composition may include a polymer or polynucleotide complexed with a metal cation (see, for example, U.S. Patent Nos. 6,265,389 and 6,555,525, the entire contents of which are incorporated herein by reference).

[0240] In some embodiments, the pharmaceutical compositions provided herein are liquid compositions. In some embodiments, the pharmaceutical compositions are frozen compositions. In some embodiments, the pharmaceutical compositions are lyophilized compositions or reconstituted lyophilized compositions. In some embodiments, the pharmaceutical compositions provided herein can be formulated into various dosage forms for administration, such as intra-articular administration.

[0241] 6.6 Methods and Applications The compositions provided herein (e.g., the AAV particles disclosed herein) can be introduced into cells in vitro or in vivo to express FGF18 and / or IL-1Ra transgenes. In some embodiments, the present invention provides a method for delivering any of the above-described AAV particles to cells or tissues, comprising contacting cells or tissues with AAV particles, or binding cells or tissues to any of the compositions described herein, the compositions comprising a pharmaceutical composition containing AAV particles. The method for delivering AAV particles to cells or tissues can be performed in vitro, ex vivo, or in vivo.

[0242] In some embodiments, this disclosure provides methods for administration and / or delivery of vectors and viral particles (e.g., AA particles) encoding the FGF18 and / or IL-1Ra peptides disclosed herein for the prevention, treatment, or improvement of a disease or condition. In some embodiments, this disclosure provides a method for delivering any of the above-described AAV particles or compositions to a subject, including mammals, and the composition comprising a pharmaceutical composition. In some embodiments, systemic delivery is required. In some embodiments, the methods provided herein include intravenous injection of the above-described AAV particles or pharmaceutical compositions. In some embodiments, systemic delivery is required. In some embodiments, the methods provided herein include subcutaneous administration of the above-described AAV particles or pharmaceutical compositions. In some embodiments, local delivery to a target area is required. In some embodiments, the compositions provided herein can be delivered to certain target areas (e.g., joints). For example, in some embodiments, the compositions can be delivered to cells in the joint (e.g., chondrocytes, synovial cells, such as type A, type B, etc.) by direct intra-articular injection. This document provides methods for administering and / or delivering the AAV particles and AAV vector genomes described herein to these target areas.

[0243] In some embodiments, AAV particles can be delivered to the subject via a multi-site administration route. For example, the subject may receive AAV particles at 2, 3, 4, 5, or more than 5 sites.

[0244] In some embodiments, the composition may be applied to a joint, synovium, subsynovial membrane, joint capsule, tendon, ligament, cartilage, or periarticular muscle of a subject in need. The composition may be applied to a joint. The composition may be applied to the synovium or subsynovial membrane. The composition may be applied to the joint capsule. The composition may be applied to a tendon. The composition may be applied to a ligament. The composition may be applied to cartilage. The composition may be applied to periarticular muscles. The subject may have osteoarthritis (OA). The subject may be at risk of developing OA. The subject may have recurrent osteoarthritis (RA). The subject may be at risk of developing RA. The subject may be a human. The subject may be a non-human animal. The subject may be a non-human mammal, such as a dog or cat.

[0245] In some embodiments, the subject treated with the methods disclosed herein is a person who has or is expected to have osteoarthritis (OA). The specific type of OA can vary, and in some cases, OA is selected from OA of the hand, knee, hip, shoulder, ankle, elbow, temporomandibular joint, and spine, and combinations thereof. In some cases, OA is knee OA. In some cases, OA is hip OA. In some cases, OA is hand OA. In some cases, OA is spinal OA, such as facet joints or intervertebral disc joints. In some cases, OA is early OA, moderate OA, or severe OA.

[0246] In some cases, a patient has been diagnosed with OA, and in some situations, the method includes diagnosing the patient with OA. One or more protocols can be used to assess the patient's OA. In some cases, the assessment of OA can be at least partially performed using imaging protocols. Various imaging protocols can be used when assessing OA. In some cases, radiological (i.e., X-ray) protocols are used.

[0247] For example, structural progression of osteoarthritis (OA) can be assessed on planar radiographs by measuring joint space width (JSW) and / or joint space narrowing (JSN) over a period of time. (Altman) et al. : Osteoarthritis Cartilage (1996, 4:217-243.) OA progression is associated with accelerated cartilage degeneration, which can lead to joint nucleation syndrome (JSN), painful joint destruction, and functional impairment. OA disease progression can be measured on the Kellgren-Lawrence grading scale (“KL scale”) to measure the incidence and severity of OA in human subjects. Grade 0 indicates that the subject's joints are normal. Grade 1 indicates that the human subject has suspected joint space narrowing and may have osteophytes. Grade 2 indicates that the human subject has definite osteophytes and significant joint space narrowing. Grade 3 indicates that the human subject has moderate multiple osteophytes, significant joint space narrowing, some sclerosis, and may have bone contour deformities. Grade 4 indicates that the human subject has large osteophytes, significant joint space narrowing, severe sclerosis, and significant bone contour deformities. In some cases, patients with OA have a Kellgren-Lawrence score of 2, 3, or 4.

[0248] In some cases, osteoarthritis (OA) is assessed using magnetic resonance imaging (MRI), for example, to identify bone marrow damage. Bone marrow damage (or edema) is associated with knee osteoarthritis pain (Felson's disease). et al ., Ann Intern Med. 2001; 134(7):541-9) and disease progression (Felson) et al. , Ann Intern Med (2003 Sep. 2; 139(5 Pt 1):330-6) is closely related. See also Stevens’ U.S. Patent 6,564,083, which describes a method for identifying a patient with joint pain’s susceptibility to developing progressive OA or joint space loss by determining the presence or absence of bone marrow edema around or in the joint. See also U.S. Patent Publication 20170202520, the disclosure of which is incorporated herein by reference.

[0249] In some cases, OA is assessed using subjective protocols, such as the pain reporting protocol. In others, pain assessment scales, such as the Visual Analog Scale (VAS), are used. Other subjective methods that can be used to assess osteoarthritis include, but are not limited to: the Western Ontario and MacMaster Universities Osteoarthritis Index (WOMAC) pain questionnaire; the Knee Injury and Osteoarthritis Outcome Score (KOOS); the McGill Pain Questionnaire; the 36-Item Short Form Health Survey (SF-36); and so on.

[0250] In some embodiments, OA has been evaluated, at least in part, using a biomarker protocol. This evaluation may include measuring, observing, or detecting the presence or activity of a biomarker. In various embodiments, a biomarker is a molecule that indicates the presence or degree of pain in an individual. The evaluation may include measuring, observing, or detecting changes in the concentration or activity of the biomarker over a period of time.

[0251] In some embodiments, the subjects treated with the methods disclosed herein are individuals who have or are expected to have RA. In some cases, the subjects have been diagnosed with RA, where in some instances, the method includes diagnosing the patient with RA. Two commonly used diagnostic criteria for RA include the American College of Rheumatology (ACR) 1987 Revised Criteria (“1987 ACR Revised Criteria”) or the American College of Rheumatology / European League Against Rheumatism (ACR / EULAR) 2010 Criteria (“2010 ACR / EULAR Criteria”).

[0252] According to the 1987 ACR criteria, RA can be diagnosed if at least four of the following seven conditions are present:

[0253] According to the 2010 ACR / EULAR criteria, using the following scoring system, the total diagnostic score for RA must be at least 6 points:

[0254] RF: rheumatoid factor; C-reactive protein; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; ACPA: ACPA is a group of antibodies, mainly including anti-nuclear perinuclear factor (APF), anti-keratin antibody (AKA), and anti-cyclic citrullinated peptide antibody (CCP).

[0255] "-" indicates negative, "+" indicates positive, and "++" indicates strong positive. The total score of these items should be at least 6 points to meet the diagnostic criteria for RA.

[0256] The compositions disclosed herein (e.g., AAV particles) can be administered via any route that produces a therapeutic effect. For example, in some embodiments, the compositions (e.g., AAV particles) can be administered subcutaneously, intravenously, intramuscularly, intraperitoneally, intracavernosal, intracartilaginous, intratendinous, or intra-articularly. In some embodiments, the compositions disclosed herein (e.g., AAV particles) can be administered via intra-articular injection.

[0257] The compositions disclosed herein (e.g., AAV particles) can be administered intra-articularly via any convenient method, such as delivery by positioning the distal end of a needle within the target joint, for example, positioning the distal end of the needle at a synovial location such that medication 9 is delivered from the distal end of the needle to the target joint. In some embodiments, injection can be performed under ultrasound guidance. In certain cases, ultrasound guidance can be used to confirm the needle's position within the target joint. Alternatively, a needle-free injection protocol can be employed, for example, when the target joint does not include a fluid space suitable for needle delivery, such as when the target joint is an intervertebral disc. As mentioned above, in some cases, the target joint is a joint of the hand, knee, hip, shoulder, ankle, elbow, temporomandibular joint, or spine, and in some cases, a knee or spinal joint.

[0258] If necessary, the joint can be irrigated (i.e., thoroughly flushed) before intra-articular administration. Irrigation removes floating particles and loose matter from the joint by flushing or irrigating the joint and subsequently aspirating or removing the fluid. This irrigation procedure may be beneficial in relieving pain. In some cases, flushing lesion fluid containing irritants (byproducts of osteoarthritis) is also therapeutic. In some cases, irrigation can be used to remove neutralizing antibodies against gene therapy vectors from the joint before administration. As a non-limiting example, the presence of neutralizing antibodies in the blood and / or synovial fluid of the patient's target joint can be assessed. If neutralizing antibodies are detected above a specific threshold, such as 1:5, 1:10, 1:20, 1:40, 1:60, 1:80, 1:100, 1:120, 1:160, 1:240, or 1:320, irrigation is performed before intra-articular administration. Any convenient irrigation method and system can be used. Examples of possible irrigation methods and systems include, but are not limited to, those described in U.S. Patent Nos. 6,808,505, 6,419,654, and 7,811,321, the disclosure of which is incorporated herein by reference.

[0259] The methods disclosed herein are not limited to the diseases (e.g., osteoarthritis), routes of administration (e.g., intra-articular injection), target locations (e.g., joints), or cell types (e.g., chondrocytes, synovial cells). For example, in some embodiments, other cell types that can be transduced may include mesenchymal stem cells.

[0260] AAV transduces the vector into cells and expresses FGF18 and / or IL-1Ra peptides. In some embodiments, the expressed peptides can improve symptoms associated with various diseases, such as osteoarthritis. In some embodiments, the methods disclosed herein can alleviate or improve one or more symptoms of osteoarthritis. In some embodiments, the methods provided herein can (a) prevent OA from occurring in individuals who may be susceptible to the disease but have not yet been diagnosed with it; (b) inhibit OA, i.e., block its progression; or (c) alleviate OA, i.e., lead to OA remission. In some embodiments, the methods provided herein can (a) prevent RA from occurring in individuals who may be susceptible to the disease but have not yet been diagnosed with it; (b) inhibit RA, i.e., block its progression; or (c) relieve RA, i.e., lead to RA remission. Treatment may result in a variety of different physical manifestations, such as reduced perceived pain, modulated joint structures, etc. In some embodiments, treatment of ongoing OA or RA stabilizes or alleviates adverse clinical symptoms in patients. Symptoms of OA that can be alleviated or improved using the methods disclosed herein include pain or aches, joint stiffness, reduced range of motion, swelling, and inflammation. The methods disclosed in this article can relieve or improve RA symptoms, including joint pain or joint swelling.

[0261] The treatment methods disclosed herein can be used before the affected tissue completely loses function. In some embodiments, the treatment methods disclosed herein can be applied before, during, and in some cases after the symptom stage of the disease.

[0262] In some embodiments, the introduction of the composition (e.g., AAV) is performed once. In some embodiments, the introduction of the composition (e.g., AAV) is performed twice, for example, a first time and a second time after the first time. In some embodiments, the introduction of the composition is performed more than twice, for example, three, four, five, etc. The second introduction of the composition may be performed at a time point after the time of the first execution of the method, for example, 3 months later, 4 months later, 5 months later, 6 months later, 7 months later, 8 months later, 9 months later, 10 months later, 11 months later, 1 year later, more than 1 year later, etc.

[0263] In some embodiments, the methods provided herein reduce osteoarthritis (OA) in at least one metric or criterion selected from: the Western Ontario and McMaster Universities Arthritis Index (WOMAC), the International Knee Documentation Committee (KOOS) score, the Whole-Organ Magnetic Imaging Score (IKDC) score, the Whole-Organ Magnetic Imaging Score (WORMS), the Intermittent and Constant Osteoarthritis Pain (ICOAP) score, the 11-point Numeric Rating Score (NRS) score, and individual assessments (e.g., questionnaires or overall patient assessments). In various embodiments, observations or assessments are performed over a period of time selected from hours, days, weeks, and months. In various embodiments, observations or assessments determine that the method does not have an adverse effect on the individual. In various embodiments of the method, observation or evaluation determines that the method has at least one of the characteristics selected from the group consisting of: effective, therapeutic, safe, and producing beneficial biochemical and / or effects in an individual. In one embodiment, the method reduces OA and / or modulates the indicator by at least about 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more.

[0264] The methods in various embodiments also include observing or detecting a reduction in pain indicators. For example, the pain condition is associated with knee OA or erosive hand OA. In various embodiments of the method, the pain is nociceptive pain associated with OA. For example, the pain is mechanical nociceptive pain associated with osteoarthritis.

[0265] In some cases, embodiments of this method result in sustained improvement in one or more symptoms of OA, such as pain. In some cases, the method can alleviate pain, for example, as determined using one or more of the assessment protocols described above, such as the visual analog scale.

[0266] In some cases, this method can further alter human joint structure, such as reducing tissue degeneration and / or reducing joint space narrowing. Therefore, these methods may at least partially restore human joint structure, where restoration means at least partial restoration to the healthy joint structure of a non-osteoarthritis patient. In some cases, this method can protect the joint structure of the human body, for example, stabilizing the joint structure without further alteration. In this case, alteration or protection can be determined by imaging protocols, such as the aforementioned radiographic and / or magnetic resonance imaging protocols.

[0267] In some embodiments, the methods described herein effectively treat subjects with RA. In some embodiments, the treatment methods described herein improve the functional status of the treated subjects. The functional status of RA patients can be assessed using the Disability Index of the Health Assessment Questionnaire (HAQ-DI). An improvement of ≥0.22 or 0.30 units in the HAQ score is considered clinically significant. In some embodiments, the treatment methods described herein significantly improve the functional status of the treated subjects.

[0268] The Disease Activity Score (DAS) is commonly used to assess disease activity in patients with rheumatoid arthritis (RA). DAS assessment can help monitor disease progression, evaluate treatment effectiveness, and guide treatment decisions for RA patients. DAS28 stands for Disease Activity Score for 28 Joints, which quantitatively measures disease activity by considering various clinical and laboratory parameters. The DAS28 calculation involves the following components: Tender Joint Count (TEN28), Swollen Joint Count (SW28), Acute Phase Reactants (APR), and Global Health (GH).

[0269] EULAR classifies the disease activity of RA into the following categories using the resulting DAS28 score: remission: DAS28 < 2.6; low disease activity: 2.6 ≤ DAS28 ≤ 3.2; moderate disease activity: 3.2 < DAS28 ≤ 5.1; high disease activity: DAS28 > 5.1. As used interchangeably herein, "clinical response" and "DAS response" of RA mean that the treatment of the subject has achieved a good response or a moderate response according to the following EULAR criteria.

[0270] In some embodiments, the treatment methods described herein can result in a clinical response of RA or a DAS response of RA. In some embodiments, the treatment methods described herein can induce clinical remission of RA. In some embodiments, the treatment methods described herein can maintain clinical remission of RA. In some embodiments, the treatment methods described herein can induce and maintain clinical remission of RA.

[0271] ACR response is also commonly used to evaluate the efficacy of RA treatment. Specifically, ACR20, ACR50, and ACR70 are defined based on the percentage improvement in RA symptoms (20%, 50%, and 70% respectively), including tender joint count (TJC), swollen joint count (SJC), patient pain assessment (VAS), patient's overall assessment of disease activity (VAS), doctor's overall assessment of disease activity (VAS), assessment of patient's physical function using the Health Assessment Questionnaire - Disability Index (HAQ - DI), and ARP level (ESR or CRP). In some embodiments, the methods provided herein achieve ACR20 in a subject. In some embodiments, the methods provided herein achieve ACR50 in a subject. In some embodiments, the methods provided herein achieve ACR70 in a subject.

[0272] In some cases, this method essentially does not produce a cell-mediated immune response, i.e., the immune response that occurs when sensitized T cells attack a foreign antigen and secrete lymphokines, triggering a humoral immune response in the body. In other cases, this method may result in a cell-mediated immune response. In these cases, the cell-mediated immune response includes a CD8+ cytotoxic T cell response. If desired, the cell-mediated immune response can be assessed by immunoassays (e.g., the ELISpot assay). In some cases, humans essentially do not produce antibodies against the encoding FGF18 / IL-1Ra. In other embodiments, humans essentially do not produce antibodies against vectors including the FGF18 / IL-1Ra encoding sequence (such as the vector described above).

[0273] In some embodiments, the composition (e.g., the AAV disclosed herein) is administered in combination with a secondary therapy. In some embodiments, the secondary therapy includes a medication for treating arthritis (e.g., OA or RA) or any other appropriate therapy for treating symptoms of the disease. Any convenient treatment may be used. Examples of other OA therapies that may be employed include, but are not limited to: acetaminophen therapy, NSAID therapy, opioid therapy, glucocorticoid therapy, hyaluronic acid therapy, stem cell therapy, autologous blood product therapy (such as platelet-rich plasma therapy), and combinations thereof. In some cases, this may be a steroid, such as triamcinolone acetonide, triamcinolone acetate, including its extended-release form, such as Zilretta®. Examples of other RA therapies that may be used include, but are not limited to: conventional DMARDs (e.g., MTX, leflunomide, hydroxychloroquine, and sulfasalazine), biological DMARDs (e.g., abatacept, adalimumab, anastropeptide, cetuzumab, etanercept, golimab, infliximab, rituximab, thaliximab, and tocilizumab), as well as synthetic DMARDs (e.g., baricitinib, tofacitinib, and utpatinib), NSAIDs (e.g., ibuprofen and naproxen), and steroids (e.g., prednisone).

[0274] 6.7 Reagent Kits and Devices In some embodiments, this disclosure provides various kits for convenient and / or efficient implementation of the methods of this disclosure. Typically, the kits will include sufficient quantities and / or amounts of composition to allow a user to administer multiple treatments and / or conduct multiple experiments on a subject.

[0275] Any composition, vector, construct, or peptide of this disclosure may be included in the kit. In some embodiments, the kit may also include reagents and / or instructions for generating and / or synthesizing the compounds and / or compositions of this disclosure. In some embodiments, the kit may also include one or more buffers. In some embodiments, the kit of this disclosure may include components for preparing protein or nucleic acid arrays or libraries, and therefore may include, for example, a solid support.

[0276] In some embodiments, kit components may be packaged in an aqueous medium or in lyophilized form. The kit's container device typically includes at least one vial, test tube, flask, bottle, syringe, or other container device in which the composition can be placed and appropriately aliquoted. If multiple kit components are present (labeled reagents and labels may be packaged together), the kit may also include second, third, or other additional containers for individually holding the additional components. In some embodiments, the kit may also include a second container device for containing sterile, pharmaceutically acceptable buffers and / or other diluents. In some embodiments, one or more vials may contain various combinations of ingredients. The kits of this disclosure may also typically include a device for containing the compounds and / or compositions of this disclosure, such as proteins, nucleic acids, and any other closed reagent containers intended for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are retained.

[0277] In some embodiments, the kit components are provided in the form of one and / or more liquid solutions. In some embodiments, the liquid solution is an aqueous solution, particularly a sterile aqueous solution. In some embodiments, the kit components may be provided in the form of a dry powder. When reagents and / or components are provided in dry powder form, these powders can be reconstituted by adding an appropriate volume of solvent. In some embodiments, it is conceivable that the solvent may also be provided in another container device.

[0278] In some embodiments, the kit may include instructions for using the kit components and any other reagents not included in the kit. The instructions may include possible variations.

[0279] 6.8 Testing The detection methods disclosed herein and known in the art can be used in the embodiments of this disclosure.

[0280] 6.8.1 Measuring gene expression Many methods are known in the art for measuring the expression of FGF18 and / or IL-1Ra transgenes from expression constructs, nucleic acids, vectors, and viruses (e.g., AAV).

[0281] The expression level of a gene expressed in a single cell or cell type (e.g., cells belonging to a specific tissue) can be determined by assessing the concentration or relative abundance of RNA transcripts (e.g., mRNA) derived from the transcription of the target gene. Alternatively, or as an alternative, gene expression can be determined by assessing the concentration or relative abundance of proteins produced by the transcription and translation of the target gene. Protein concentration can also be assessed using functional analyses, such as enzyme analysis or gene transcription analysis, if the target gene encodes an enzyme or transcription regulator, respectively. The following sections describe exemplary techniques that can be used to measure and sort gene expression levels in target cells, cell types, or cell populations, for example, at the level of a single cell or cell population. Gene expression in a sample can be analyzed using a variety of methods, many of which are known in the art and will be understood by those skilled in the art, including but not limited to nucleic acid sequencing, microarray analysis, proteomics, in situ hybridization (e.g., fluorescence in situ hybridization (FISH)), amplification-based detection, in situ hybridization, fluorescence activated cell sorting (FACS), northern analysis of mRNA, and / or PCR analysis.

[0282] (i) Nucleic acid testing Nucleic acid-based datasets suitable for analyzing target cell-specific gene expression can take the form of gene expression profiles that represent the identity and degree of gene expression in target cells, and can be used to determine the ordering of gene expression levels in target cells, cell types, or cell populations. Such expression profiles may include whole transcriptome sequencing data (e.g., RNA-Seq data), mRNAs, non-coding RNAs, or any other nucleic acid sequences that can be expressed from genomic DNA. Other nucleic acid datasets suitable for the methods described herein may include expression data collected through imaging-based techniques (e.g., Northern or Southern blots known in the art). Northern blot analysis is a well-known and routine technique in the art; see, for example, Molecular Cloning, a Laboratory Manual, second edition, 1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainview, NY 11803-2500, the disclosure of which is incorporated herein by reference. Protocols for assessing gene and gene product status are available, for example, see Ausubel et al.The contents of these publications are found in Curr Protoc Mol Biol, eds., 1995, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting), and 18 (PCR Analysis), and are incorporated herein by reference.

[0283] Gene expression profiles analyzed using the methods described herein may include, for example, microarray data or nucleic acid sequencing data generated by sequencing methods known in the art (e.g., Sanger sequencing and next-generation sequencing methods, also known as high-throughput sequencing or deep sequencing). Exemplary next-generation sequencing technologies include, but are not limited to, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing platforms. Other sequencing methods known in the art may also be used. For example, RNA-Seq (e.g., see Mortazavi) can be used. et al. , Nat. Methods 5:621-628 (2008), the entire contents of which are incorporated herein by reference. RNA-Seq is a reliable technique known in the art for monitoring expression by directly sequencing RNA molecules in a sample. Briefly, this method may involve fragmenting RNA to fragments of average length 200 nucleotides, converting them into cDNA via random initiation, and synthesizing them into double-stranded cDNA (e.g., using Agilent Technologies' Just cDNA Double-Stranded cDNA Synthesis Kit). Then, by adding sequence adapters (e.g., from Illumina O / Solexa) to each library, the resulting 50-100 nucleotide reads can be matched to the genome.

[0284] Gene expression levels can be determined using microarray-based platforms (e.g., single nucleotide polymorphism (SNP) arrays) because microarray technology offers high resolution. Detailed information on various microarray methods can be found in the literature. For example, see U.S. Patent No. 6232068 and Pollack. et al. , Nat. Genet.23:41-46 (1999), the disclosure of each of which is incorporated herein by reference in its entirety. Using nucleic acid microarrays, mRNA samples are reverse transcribed and labeled to produce cDNA. An example of a microarray processor is the Affymetrix GENECHIP® system, which is commercially available and consists of an array made of oligonucleotides synthesized directly on a glass surface. Other systems can be used, as is known to those skilled in the art. Amplification-based assays can also be used to measure the expression levels of one or more biomarkers (e.g., genes). In such assays, the nucleic acid sequence of the gene acts as a template in an amplification reaction (e.g., PCR, such as qPCR). In quantitative amplification, the amount of amplified product is proportional to the amount of template in the original sample. Based on the principles described herein, the gene expression level corresponding to the specific probe used can be determined by comparison with an appropriate control. Real-time qPCR methods using TaqMan probes are well known in the art. See, for example, Gibson. et al. , Genome Res. 6:995-1001 (1996) and inHeid et al. , Genome Res. 6:986-994 (1996) provides a detailed protocol for real-time qPCR, all of which are incorporated herein by reference. The gene expression levels described herein can be determined using RT-PCR. Probes used for PCR can be labeled with detectable markers, such as radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes.

[0285] (ii) Protein detection Gene expression can also be determined by measuring the concentration or relative abundance of the corresponding protein product encoded by the target gene. Protein levels can be assessed using standard detection techniques known in the art. Examples of protein expression analyses suitable for the methods described herein include, but are not limited to, proteomics methods, immunohistochemistry and / or Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, enzyme-linked immunofiltration assays (ELIFA), mass spectrometry, mass spectrometry immunoassays, and biochemical enzyme activity assays. In particular, proteomics methods can be used to generate large-scale multiplex protein expression datasets. Proteomics methods can utilize mass spectrometry to detect and quantify peptides (e.g., proteins) and / or utilize peptide microarrays with capture reagents (e.g., antibodies) specific to a group of target proteins to identify and determine the expression levels of proteins expressed in samples (e.g., single-cell samples or multi-cell populations).

[0286] Exemplary peptide microarrays have multiple peptides bound to a substrate, and the binding of each bound oligonucleotide, peptide, or protein to each of the bound peptides can be detected individually. Alternatively, the peptide microarray may include a variety of binding agents, including but not limited to monoclonal antibodies, polyclonal antibodies, phage display binding agents, yeast two-hybrid binding agents, and aptamers, capable of specifically detecting the binding of specific oligonucleotides, peptides, or proteins. Examples of peptide arrays can be found in U.S. Patents 6,268,210, 5,766,960, and 5,143,854, the disclosure of each of which is incorporated herein by reference.

[0287] Mass spectrometry (MS) can be used in conjunction with the methods described herein to identify and characterize gene expression profiles of single-cell or multi-cell populations. Any MS method known in the art can be used to identify, detect, and / or measure one or more target peptides, such as LC-MS, ESI-MS, ESI-MS / MS, MALDI-TOF-MS, MALDI / TOF / TOF-MS, tandem mass spectrometry, etc. Mass spectrometers typically include ion sources and optical components, mass analyzers, and data processing electronics. Mass spectrometers include scanning and ion beam mass spectrometers, such as time-of-flight (TOF) and quadruple (Q) mass spectrometers, as well as trap mass spectrometers, such as ion trap (IT), orbital trap, and Fourier transform ion cyclotron resonance (FT-ICR), all of which can be used in the methods described herein. Detailed information on various mass spectrometry methods can be found in the literature. For example, see Yates. et al. , Annu. Rev. Biomed. Eng. 11:49-79 (2009), the contents of which are hereby incorporated by reference.

[0288] 6.8.2 Animal Model of Oascoparia Various methods exist in the prior art for determining the therapeutic activity of AAV and the compositions disclosed herein for expressing FGF18 and / or IL-1Ra transgenes. For example, some animals used in this art are provided below.

[0289] Rodent models: Surgical induction: Surgical models, such as medial meniscus destabilization (DMM) instability in mice or anterior cruciate ligament transection (ACLT) in rats, involve surgical instability of the joint to induce OA-like changes. These models mimic post-traumatic OA and have been widely used in OA research (Glasson). et al (2007), Osteoarthritis and Cartilage , 15(9), 1061-1069.; Little et al. (2010) Osteoarthritis and Cartilage , 18(Suppl3), S80-S92.). Please see the Experiments section below for more details.

[0290] Chemical induction model: Intra-articular injection of compounds such as monosodium iodoacetate (MIA) or papain can induce OA-like symptoms in rodents. MIA, in particular, is known to cause chondrocyte death and cartilage damage (Janusz...). et al (2001). Osteoarthritis and Cartilage , 9(4), 316-325;Guzman et al (2003) Toxicologic Pathology , 31(6), 619-624.).

[0291] Monkey models: Rhesus macaques and cynomolgus monkeys are used in OA research due to their anatomical and genetic similarities to humans. These models are particularly useful for testing therapies (Pelletier). et al (2013) Arthritis & Rheumatism , 65(10), 2690-2703.).

[0292] Canine models: Dogs, especially those susceptible to OA, have been used to study the progression of naturally occurring OA. Canine models can provide insights into the long-term effects of OA and potential treatments (Vandeweerd). et al (2012) Journal of Veterinary Pharmacology and Therapeutics , 35(Suppl 1), 168-181.).

[0293] Zebrafish model: Zebrafish are used as a unique model for studying osteoarthritis (OA) due to their transparent embryos and ability to visualize cartilage development and degeneration in real time (Kague). et al (2012) Development , 139(1), 180-185.).

[0294] 6.9 Exemplary Implementation Scheme Implementation Scheme 1: A composition for treating arthritis comprising (i) an FGF18 peptide or an FGF18 transgene, and (ii) an interleukin-1 receptor antagonist (IL-1Ra) peptide or an IL-1Ra transgene.

[0295] Implementation Scheme 2: The composition of Implementation Scheme 1, wherein the FGF18 polypeptide is human FGF18 (hFGF18) or a functional variant thereof, or rat FGF18 (rFGF18) or a variant thereof.

[0296] Implementation Scheme 3: The composition of Implementation Scheme 1, wherein the FGF18 polypeptide has at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO:1.

[0297] Implementation Scheme 4: A composition of any one of Implementation Schemes 1 to 3, wherein the IL-1Ra polypeptide is human IL-1Ra (hIL-1Ra) or a functional variant thereof, or rat IL-1Ra (rIL-1Ra) or a variant thereof.

[0298] Implementation Scheme 5: The composition of Implementation Scheme 4, wherein the IL-1Ra polypeptide has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO:3.

[0299] Implementation Scheme 6: A composition of any one of Implementation Schemes 1 to 5, comprising the FGF18 transgene and the IL-1Ra transgene.

[0300] Implementation Scheme 7: The composition of Implementation Scheme 6, wherein the FGF18 transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identical nucleotide sequence to SEQ ID NO:5.

[0301] Implementation Scheme 8. The composition of Implementation Scheme 6 or 7, wherein the IL-1Ra transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identical nucleotide sequence to SEQ ID NO:8.

[0302] Implementation Scheme 9: The composition of Implementation Scheme 6, wherein (1) the FGF18 transgene is codon-optimized, (2) the IL-1Ra transgene is codon-optimized, or both.

[0303] Implementation Scheme 10: The composition of Implementation Scheme 9, wherein the FGF18 transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% of the same nucleotide sequence as SEQ ID NO:6.

[0304] Implementation Scheme 11: The composition of Implementation Scheme 9 or 10, wherein the IL-1Ra transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identical nucleotide sequence to SEQ ID NO:9.

[0305] Implementation Scheme 12: A composition of any one of Implementation Schemes 6 to 11, comprising a nucleic acid, the nucleic acid comprising an expression cassette, the expression cassette comprising an FGF18 transgene and an IL-1Ra transgene linked by an adapter sequence.

[0306] Implementation Scheme 13: A composition of any one of Implementation Schemes 6 to 11, comprising nucleic acids, wherein the nucleic acids comprise an FGF18 expression cassette and an IL-1Ra expression cassette, wherein the FGF18 expression cassette comprises an FGF18 transgene, and the IL-1Ra expression cassette comprises an IL-1Ra transgene.

[0307] Implementation Scheme 14: A composition of any one of Implementation Schemes 6 to 11, comprising a first nucleic acid and a second nucleic acid, the first nucleic acid comprising an FGF18 expression cassette containing an FGF18 transgene, the FGF18 expression cassette containing an FGF18 transgene, and the second nucleic acid comprising an IL-1Ra expression cassette containing an IL-1Ra transgene.

[0308] Implementation Scheme 15: A composition of any one of Implementation Schemes 12 to 14, wherein the expression cassette comprises a promoter and a polyA sequence.

[0309] Implementation Scheme 16: The composition of Implementation Scheme 15, wherein the expression cassette further includes an enhancer.

[0310] Implementation Scheme 17: A composition of any one of Implementation Schemes 12 to 16, wherein one or more nucleic acids are recombinant vectors.

[0311] Implementation Scheme 18: A composition of any one of Implementation Schemes 12 to 16, wherein one or more nucleic acids are viral genomes.

[0312] Implementation Scheme 19: The composition of Implementation Scheme 18, wherein the nucleic acids are self-complementary.

[0313] Implementation Scheme 20: The composition of Implementation Scheme 18 or 19, comprising one or more recombinant viruses that encapsulate the viral genome.

[0314] Implementation Scheme 21: The composition of Implementation Scheme 20, wherein the one or more recombinant viruses include retroviruses, adenoviruses, lentiviruses, or adeno-associated viruses (AAVs).

[0315] Implementation Scheme 22: The composition of Implementation Scheme 21, wherein the one or more recombinant viruses comprise AAV.

[0316] Implementation Scheme 23: The composition of Implementation Scheme 22, wherein the AAV is a serotype of AAV2, AAV2.5, AAV5, AAV8 or AAV9, or a mixture thereof.

[0317] Implementation Scheme 24: The composition of Implementation Scheme 23, wherein AAV is serotype 5.

[0318] Implementation Scheme 25: A composition of any one of Implementation Schemes 22 to 24, wherein the viral genome of AAV comprises 5' to 3': a first ITR, a first promoter, an IL-1Ra transgene, a first poly A tail, a second promoter, an FGF18 transgene, a second poly A tail, and a second ITR.

[0319] Implementation Scheme 26: A composition of any one of Implementation Schemes 22 to 24, wherein the viral genome of AAV comprises 5' to 3': a first ITR, a first promoter, an FGF18 transgene, a poly A tail, a second promoter, an IL-1Ra transgene, a second poly A tail, and a second ITR.

[0320] Implementation Scheme 27: A composition of any one of Implementation Schemes 22 to 24, wherein the viral genome of AAV comprises 5' to 3': a first ITR, a promoter, an IL-1Ra transgene, an adapter sequence, an FGF18 transgene, a poly A tail, and a second ITR.

[0321] Implementation Scheme 28: A composition of any one of Implementation Schemes 22 to 24, wherein the viral genome of AAV comprises 5' to 3': a first ITR, a promoter, an FGF18 transgene, an adapter sequence, an IL-1Ra transgene, a poly A tail, and a second ITR.

[0322] Implementation Scheme 29: A composition of any one of Implementation Schemes 22 to 24, comprising (1) a first AAV virus genome, the first AAV virus genome comprising: a first ITR, a first promoter, an IL-1Ra transgene, a poly A tail, and (2) a second AAV virus genome, the second AAV virus genome comprising a second promoter, an IL-1Ra transgene, a second poly A tail, and a second ITR.

[0323] Implementation Scheme 30: A composition of any one of Implementation Schemes 1 to 29, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0324] Implementation Scheme 31: The composition of Implementation Scheme 30, wherein the composition is formulated for intra-articular injection.

[0325] Implementation Scheme 32: A kit comprising a unit dose of the composition of any one of Implementation Schemes 1 to 31.

[0326] Implementation Scheme 33: A method for expressing FGF18 peptide and IL-1Ra peptide in cells, comprising contacting the cells with a composition of any one of Implementation Schemes 12 to 31.

[0327] Implementation Scheme 34: The method of Implementation Scheme 33, wherein the cells are chondrocytes or synovial cells.

[0328] Implementation Scheme 35: A method of delivering FGF18 peptide and IL-1Ra peptide to a target region of a subject in need, comprising introducing a composition of any one of Implementation Schemes 12 to 31 into the region.

[0329] Implementation Scheme 36: The method of Implementation Scheme 35, wherein the target area is a joint, synovium, subsynovium, joint capsule, tendon, ligament, cartilage or periarticular muscle.

[0330] Implementation Scheme 37: A method for treating arthritis in a subject in need, comprising administering to the subject a therapeutically effective amount of the composition of any one of Implementation Schemes 1 to 31.

[0331] Implementation Scheme 38: The method of Implementation Scheme 37, including intra-articular injection.

[0332] Implementation Scheme 39: The method according to any one of Implementation Schemes 35 to 38, wherein the subject is a human being.

[0333] Implementation Scheme 40: Use of the composition of any one of Implementation Schemes 1 to 31 in the treatment of arthritis.

[0334] Implementation Scheme 41: Use of the composition of any one of Implementation Schemes 1 to 31 in the preparation of a medicament for treating arthritis.

[0335] Implementation Scheme 42: The method or use of any one of Implementation Schemes 37 to 41, wherein arthritis refers to arthritis of the hand, knee, hip, shoulder, ankle, elbow, temporomandibular joint, spine or any combination thereof.

[0336] Implementation Scheme 43: The method or use of Implementation Scheme 42, wherein the arthritis is osteoarthritis.

[0337] Implementation Scheme 44: The method or use of Implementation Scheme 42, wherein the arthritis is rheumatoid arthritis.

[0338] 6.10 Example The examples provided below are for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the invention should in no way be construed as limited to the examples below, but rather as encompassing any and all variations that become apparent from the examples provided herein.

[0339] In summary, the results of the study described below indicate that FGF18 / IL-1Ra molecular constructs of different AAV serotypes effectively express FGF18 / IL-1Ra-peptides in the joint cavity of mice, alleviating OA-induced pain and reducing articular cartilage damage.

[0340] 6.10.1 Example 1: Preparation of transgenic expression constructs The following transgenic expression constructs were prepared for packaging into AAV (Av). Figure 1 ): (1) Self-complementary AAV vector containing IL-1Ra expression cassette (AAV-IL-1Ra) (2) Self-complementary AAV vector containing FGF18 expression cassette (AAV-FGF18) (3) A single-stranded AAV vector containing cis-arranged IL-1Ra expression cassettes and FGF18 expression cassettes (AAV-FGF18 / IL-1Ra). Note: (1) The IL-1Ra expression cassette contains the codon-optimized hIL-1Ra transgene (SEQ ID NO:6); (2) The FGF18 expression cassette contains the codon-optimized hFGF18 transgene (SEQ ID NO:9).

[0341] 6.10.2 Example 2: AAVs express IL-1Ra in the joint cavity Methods: Eighty-eight 6-8 week old male C57BL / 6J mice were randomly divided into five groups: vector control group, AAV5-IL-1Ra group, AAV8-IL-1Ra group, AAV9-IL-1Ra group, and AAV2.5-IL-1Ra group. Eight mice were placed in the vector control group, and 20 mice were placed in each of the other groups. AAV solution (or buffer solution of the control group) was injected into the right knee cavity of the mice (25 μL / mouse; 5e10vg / mouse). Synovial fluid was collected from the right knee of each group of mice at 2, 4, 8, and 12 weeks after the single intra-articular injection, and the concentration of IL-1Ra in the synovial fluid was determined by ELISA.

[0342] Results and conclusions: Figure 2 As shown, after a single intra-articular injection of AAV serotypes, IL-1Ra was continuously expressed in the joint cavity up to 12 weeks post-injection. The expression efficiency of different AAV serotypes was AAV5~AAV2.5>AAV8>AAV9. AAV5-IL-1Ra was selected for further research.

[0343] 6.10.3 Example 3: AAV5-IL-1Ra reduces OA-induced pain and alleviates articular cartilage damage. Methods: The efficacy of AAV5-IL-1Ra in treating osteoarthritis (OA) was determined in rats. Medial meniscal tear (MMT) was performed on male rats to establish an OA model. Rats were randomly divided into four groups: sham-operated group, model control group, AAV group, and AAV-Co group. The sham-operated group consisted of 6 rats, and the other groups each contained 18 rats. Fifty-four model rats were randomly assigned to their bipedal weight-bearing ratios one week post-surgery (D0). One week post-surgery, all rats received a single intra-articular injection (50 μL / rat). The AAV group received AAV5-IL-1Ra WT (wild-type; SEQ ID NO:5), the AAV-Co group received AAV5-IL-1Ra-Co (codon-optimized IL-1Ra; SEQ ID ID:6) at a dose of 1e11-vg / rat, and the sham-operated group and model control group received buffer injections. The day of administration was designated D1. Before injection and at D15, D36, and D58 post-injection, surviving rats in each group underwent bipedal balance tests and mechanical pain threshold tests to assess pain relief. At D15, D36, and D58 post-injection, synovial fluid was collected from the right knee of the rats to observe the gross anatomy of the joint, and the knee joint was removed for fixation and sectioning to assess cartilage damage.

[0344] Results and conclusions: Bipedal weight As shown in Table 5 and Figure 3As shown, at D0, the bipedal weight-bearing ratio in the model control group (1.36±0.08) was significantly higher than that in the sham surgery group (1.00±0.00), indicating that the OA model had been successfully established. The ratios in the two treatment groups and the model group were comparable at D0, indicating that the severity of OA was similar in all groups before treatment.

[0345] Table 5. Effect of AAV5-IL-1Ra treatment on weight-bearing of the operated limb in OA model animals (contralateral / operated side ratio)

[0346] Note: During the study, bipedal balance tests were performed on each group of animals. All individual data are expressed as mean ± standard deviation (SD). There were statistically significant differences between the different treatment groups and the sham-operated group. #### P<0.0001 (unlabeled). # P<0.05 ## P<0.01 ### P<0.001 and #### P<0.0001 represents the statistical analysis results between different treatment groups and the model control group.

[0347] As shown in the figure, compared with the model control group, the contralateral / operational side ratios in both treatment groups were statistically significantly reduced at D15 and D36, indicating that the treatment groups experienced pain relief.

[0348] Mechanical pain threshold As shown in Table 6 and Figure 4 As shown, at D0, the mechanical pain threshold of the operated limb in the model control group (40.21±5.70g) was statistically significantly lower than that in the sham surgery group (58.96±6.00g), indicating that the OA model had been successfully established. The mechanical pain thresholds of all model groups were comparable, indicating that the severity of OA was the same in these groups before treatment.

[0349] Table 6. Effects of AAV5-IL-1Ra treatment on the mechanical pain threshold of the surgical limb in OA model animals.

[0350] Note: During the study, the mechanical pain threshold of the operated limb was measured in animals in each treatment group. All individual data are expressed as mean ± standard deviation (SD). There were statistically significant differences between the different treatment groups and the normal control group (****P<0.05, unmarked). # P<0.05 ## P<0.01 ###P<0.001 and #### P<0.0001 represents the statistical analysis results between different treatment groups and the model control group.

[0351] At day 15, the mechanical pain threshold of the operated limb in the model control group was statistically significantly lower than that in both treatment groups, indicating that AAV-IL-1Ra treatment in both groups effectively relieved pain caused by OA. At days 36 or 58, there were no significant differences between the model groups.

[0352] Overall knee injury (Pelletier score) As shown in Table 7 and Figure 5 As shown, during the study, the comprehensive score of surgical knee joint injury in the model control group was significantly higher than that in the sham surgery group, indicating that the OA model has been successfully established.

[0353] Table 7. Effects of AAV5-IL-1Ra treatment on overall injury score (Pelletier score) in OA model animals

[0354] As shown in the figure, during the study period, the total score of the surgical knee joint in the model control group was significantly higher than that in the sham surgery group, indicating that the osteoarthritis model had been successfully established. At 2, 5, and 8 weeks post-injection, the total scores of the surgical knee joint in both treatment groups were lower than those in the model control group, indicating an effective reduction in OA-induced damage. The AAV-Co group showed better results compared to the AAV group.

[0355] Express like Figure 6 As shown, human IL-1Ra expression was detected in both treatment groups at 2, 5, and 8 weeks post-drug administration, with expression gradually decreasing over time. At each time point, comparable IL-1Ra expression was detected in both the AAV and AAV-Co groups. Human IL-1Ra was not detected in the synovial fluid of either the sham-operated group or the model control group.

[0356] 6.10.4 Example 4: AAV5-IL-1Ra / FGF18 treatment synergistically reduces pain caused by OA and alleviates articular cartilage damage. Methods: The efficacy of AAV5-IL-1Ra / FGF18 in treating osteoarthritis (OA) was measured in rats. Medial meniscal tear (MMT) was performed on male rats to establish an OA model. Rats were randomly divided into 7 groups: sham-operated group, model control group, AAV group 1, AAV group 2, AAV group 3, AAV group 4, and AAV group 6. The sham-operated group consisted of 6 rats, and the other groups each contained 12 rats. The 72 model rats were randomly assigned to their respective groups based on their bipedal weight-bearing ratio one week post-surgery (D0). One week post-surgery, all rats received a single intra-articular injection (50 μL / rat). AAV groups 1-5 received AAV5-hIL-1Ra, AAV5-rIL-1Ra, AAV5-hFGF18, AAV5-rFGF18, and AAV5-hIL-12Ra-hFGF18, respectively, at a dose of 1e11 vg / rat. The sham-operated group and the model control group received buffer injections. The sequence characteristics of these AAVs are shown in Table 4 above. The administration date was designated D1. Before injection and at D15, D36, D57, and D85 post-injection, bipedal balance tests and mechanical pain threshold tests were performed on surviving rats in each group to assess pain relief. At D36 and D85 post-injection, synovial fluid was collected from the right knee of each rat group to observe the gross anatomy of the joint. The knee joint was removed, fixed, and sectioned to assess cartilage damage. Furthermore, at 5 and 12 weeks post-injection, knee joint specimens were collected, stained with Safranin-Fix-Green, and subjected to systematic histopathological analysis, including microscopic observation, OARSI scoring, measurement of cartilage degeneration (width and area), and measurement of cartilage osteophyte height.

[0357] Results and conclusions: Bipedal weight As shown in Table 8 and Figure 7 As shown, at D0, the bipedal weight-bearing ratio in the model control group (1.33±0.04) was significantly higher than that in the sham surgery group (1.00±0.00), indicating that the OA model had been successfully established. At D0, the bipedal weight-bearing ratios were comparable between all treatment groups and the control model group, indicating that the severity of OA was the same in all groups before treatment.

[0358] Table 8. Effects of AAV5-IL-1Ra / FGF18 treatment on weight-bearing of the operated limb in OA model animals (ratio of contralateral to operated side)

[0359] Note: During the study, bipedal balance tests were performed on animals in each treatment group. All individual data are expressed as mean ± standard deviation (SD). There were no statistically significant differences between the different treatment groups and the sham-operated group. #### P<0.0001 (unlabeled). #P<0.05 ## P<0.01 ### P<0.001 and ### P<0.0001 represents the statistical analysis results between different treatment groups and the model control group.

[0360] As shown in the figure, during the study period, compared with the model control group, the contralateral / operational side ratios in all five treatment groups were statistically significantly reduced, indicating pain relief in the treatment groups. Furthermore, at D15, D36, and D57, the bipedal weight-bearing ratio in AAV group 5 was lower than that in AAV groups 1 and 3, indicating a synergistic effect of FGF18 / IL-1Ra dual treatment.

[0361] Mechanical pain As shown in Table 9 and Figure 8 As shown, throughout the study period, the mechanical pain threshold of the operated limb in the model control group was statistically significantly lower than that in the sham surgery group, indicating that the OA model was successfully established. At D0, the mechanical pain thresholds of all model groups were comparable, indicating that these groups had similar OA severity before treatment.

[0362] Table 9. Effects of AAV5-IL-1Ra / FGF18 treatment on the mechanical pain threshold of the surgical limb in OA model animals.

[0363] Note: Electromechanical pain tests were performed on animals in each experimental group during the study. All data are expressed as mean ± standard deviation (SD). There were statistically significant differences between the experimental groups and the normal control group (****P<0.0001, unmarked). # P<0.05 ## P<0.01 ### P<0.001 and #### P<0.0001 represents the statistical analysis results between different experimental groups and the model control group.

[0364] During the study, the mechanical pain threshold of the operated limb in AAV treatment groups 1–5 was consistently higher than that in the model control group, indicating that all treatments—AAV5-hIL-1Ra, AAV5-rIL-1Ra, AAV5-hFGF18, AAV5-rFGF18, and AAV5-hIL-12Ra-hFGF18—effectively reduced OA-induced pain. Compared to other treatment groups, AAV group 1 (AAV5-hIL-1Ra) and AAV group 2 (AAV5-rIL-1Ra) showed greater improvement.

[0365] Overall knee injury (Pelletier score) As shown in Table 10 and Figure 9 As shown, during the study period, the total injury score of the surgical knee joint in the model control group was significantly higher than that in the sham surgery group, indicating that the OA model has been successfully established.

[0366] Table 10. Effects of AAV5-IL-1Ra / FGF18 treatment on total injury score (Pelletier score) in OA model animals

[0367] Note: During the experiment, the overall injury score of each experimental group of animals was calculated, and all individual data are expressed as mean ± standard deviation (SD). # P<0.05 ## P<0.01 ### P<0.001 and #### P<0.0001 represents the statistical analysis results between different experimental groups and the model control group.

[0368] At 5 and 12 weeks post-injection, the total scores in all treatment groups were lower than those in the model control group, indicating that all treatments effectively reduced OA-induced damage. Furthermore, at 12 weeks post-injection, AAV group 3 (AAV5-hFGF18) showed greater improvement compared to other treatment groups.

[0369] Cartilage degeneration (OARSI criteria) To assess tibial plateau cartilage degeneration, knee joint tissues were stained with Safranin-Fix-Green and evaluated according to the criteria of the Osteoarthritis Research Society International (OARSI). See Table 11 and... Figure 10 As shown, during the study period, the OARSI score of the model control group was consistently higher than that of the sham-operated group, with cartilage degeneration mainly concentrated in zones 1 and 2, especially zone 2. The OARSI scores of AAV treatment groups 1-5 were lower than those of the model control group, indicating that the treatment improved OA-induced cartilage degeneration. AAV group 3 (AAV5-hFGF18) and AAV group 4 (AAV5-rFGF18) showed greater effects than the other groups.

[0370] Table 11. Scoring of tibial plateau cartilage degeneration in animals (unit: points)

[0371] Note: All individual data are expressed as mean ± standard deviation (SD).

[0372] Cartilage degeneration (percentage of degeneration width / area) Tables 12 and 13 show the degeneration width and area of ​​the most severely damaged tibial plateau cartilage in knee joint tissue, measured using ImageJ software with safranin-fast green staining. Throughout the study, the degeneration width and area of ​​the cartilage in the model control group were consistently higher than those in the AAV treatment groups 1–5, indicating that this treatment improved OA-induced cartilage degeneration. AAV groups 3 (AAV5-hFGF18) and 4 (AAV5-rFGF18) showed better results than the other groups.

[0373] Table 12. Changes in the width and area of ​​tibial plateau cartilage degeneration in animals 5 weeks after drug administration

[0374] Note: All individual data are expressed as mean ± standard deviation (SD). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 represent statistical analysis results between different experimental groups and sham surgery groups. #P<0.05, ##P<0.01, ##P<0.001 and ###P<0.0001 represent statistical analysis results between different experimental groups and model control groups.

[0375] Table 13. Changes in the width and area of ​​tibial plateau cartilage degeneration in animals 12 weeks after drug administration

[0376] Note: All individual data are expressed as mean ± standard deviation (SD). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 represent statistical analysis results between different experimental groups and sham surgery groups. #P<0.05, ##P<0.01, ##P<0.001 and ###P<0.0001 represent statistical analysis results between different experimental groups and model control groups.

[0377] Cartilage osteophyte score Table 14 and Figure 11 The image shows the tibial plateau cartilage height of the knee joint tissue stained with Safranin-Fix Green and measured using ImageJ software. At 5 weeks post-injection, the osteophyte score in the model control group was higher than that in AAV treatment groups 1 and 2. At 12 weeks post-injection, the osteophyte score in the model control group was higher than that in all AAV treatment groups 1–5, indicating that treatment reduced osteophyte formation. AAV groups 3 (AAV5-hFGF18) and 4 (AAV5-rFGF18) showed superior effects compared to the other groups.

[0378] Table 14. Effects of AAV5-IL-1Ra / FGF18 treatment on tibial plateau cartilage osteophyte score in OA model animals (unit, points)

[0379] Note: All individual data are expressed as mean ± standard deviation (SD). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 represent statistical analysis results between different experimental groups and sham surgery groups. #P<0.05, ##P<0.01, ###P<0.001 and ####P<0.0001 represent statistical analysis results between different experimental groups and model control groups.

[0380] Express like Figure 12A As shown, human IL-1Ra expression was detected in AAV group 1 (AAV5-hIL-1Ra) and AAV group 5 (AAV5-hIL-1Ra-hFGF18) at 5 and 12 weeks post-injection, while rat IL-1Ra expression was detected in AAV group 2 (AAV5-rIL-1Ra), with a gradual decrease over time. Human IL-1Ra was not detected in the synovial fluid of the sham-operated group, model control group, AAV group 2 (AAV5-rIL-1Ra), AAV group 3 (AAV5-hTFGF18), or AAV group 4 (AAV3-rFGF18).

[0381] like Figure 12B As shown, human hFGF18 expression was detected in both AAV group 3 (AAV5-hFGF18) and AAV group 5 (AAV5-h-IL-1Ra-hFGF18) at 5 and 12 weeks post-injection, and its expression gradually decreased over time. Human hFGF18 was not detected in the synovial fluid of the sham-operated group, model control group, AAV group 1 (AAV5-hIL-1Ra), AAV group 2 (AAV5-rIL-1Ra), or AAV group 4 (AAV5-rFGF18).

[0382] Although the invention has been described in detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. Unless the context otherwise indicates, the various features described herein may be used in any combination. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0383] Therefore, the foregoing has only illustrated the principles of the invention. It should be understood that those skilled in the art will be able to devise various arrangements, which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language cited herein are primarily intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to the further development of the art, and should be interpreted as not being limited to these specifically cited examples and conditions. Moreover, all statements herein recounting the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to cover their structural and functional equivalents. Furthermore, such equivalents include both currently known equivalents and those developed in the future, i.e., any element developed that performs the same function, regardless of its structure. Furthermore, nothing disclosed herein is intended to be donated to the public, whether or not it is expressly referenced in the claims.

[0384] All publications and patents referenced in this specification are incorporated herein by reference, just as each individual publication or patent is specifically and individually indicated to be incorporated herein by reference, and are incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials of those publications. Reference to any publication is for disclosure prior to the application date and should not be construed as an admission that the invention is not entitled to prior publication by virtue of prior invention.

Claims

1. A composition for treating arthritis comprising (i) an FGF18 peptide or an FGF18 transgene, and (ii) an interleukin-1 receptor antagonist (IL-1Ra) peptide or an IL-1Ra transgene.

2. The composition according to claim 1, wherein the FGF18 polypeptide is human FGF18 (hFGF18) or a functional variant thereof, or rat FGF18 (rFGF18) or a functional variant thereof.

3. The composition according to claim 1, wherein the FGF18 polypeptide has at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO:

1.

4. The composition according to any one of claims 1 to 3, wherein the IL-1Ra polypeptide is human IL-1Ra (hIL-1Ra) or a functional variant thereof, or rat IL-1Ra (rIL-1Ra) or a functional variant thereof.

5. The composition according to claim 4, wherein the IL-1Ra polypeptide has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO:

3.

6. The composition according to any one of claims 1 to 5, comprising FGF18 transgene and IL-1Ra transgene.

7. The composition according to claim 6, wherein the FGF18 transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% of the same nucleotide sequence as SEQ ID NO:

5.

8. The composition according to claim 6 or 7, wherein the IL-1Ra transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identical nucleotide sequence to SEQ ID NO:

8.

9. The composition according to claim 6, wherein (1) the FGF18 transgene is codon-optimized, (2) the IL-1Ra transgene is codon-optimized, or both are codon-optimized.

10. The composition according to claim 9, wherein the FGF18 transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% of the same nucleotide sequence as SEQ ID NO:

6.

11. The composition according to claim 9 or 10, wherein the IL-1Ra transgene has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% of the same nucleotide sequence as SEQ ID NO:

9.

12. The composition according to any one of claims 6-11, comprising nucleic acid, the nucleic acid comprising an expression cassette, the expression cassette comprising an FGF18 transgene and an IL-1Ra transgene linked by an adapter sequence.

13. The composition according to any one of claims 6-11, comprising nucleic acids, said nucleic acids comprising an FGF18 expression cassette and an IL-1Ra expression cassette, said FGF18 expression cassette comprising an FGF18 transgene and said IL-1Ra expression cassette comprising an IL-1Ra transgene.

14. The composition according to any one of claims 6-11, comprising a first nucleic acid and a second nucleic acid, the first nucleic acid comprising an FGF18 expression cassette containing an FGF18 transgene, the second nucleic acid comprising an IL-1Ra expression cassette containing an IL-1Ra transgene.

15. The composition according to any one of claims 12-14, wherein the expression cassette comprises a promoter and a polyA sequence.

16. The composition of claim 15, wherein the expression cassette further comprises an enhancer.

17. The composition according to any one of claims 12-16, wherein the one or more nucleic acids are recombinant vectors.

18. The composition according to any one of claims 12-16, wherein one or more nucleic acids are viral genomes.

19. The composition according to claim 18, wherein the nucleic acid is self-complementary.

20. The composition of claim 18 or 19, comprising one or more recombinant viruses that encapsulate the viral genome.

21. The composition of claim 20, wherein the one or more recombinant viruses comprise retroviruses, adenoviruses, lentiviruses, or adeno-associated viruses (AAVs).

22. The composition of claim 21, wherein the one or more recombinant viruses comprise AAV.

23. The composition of claim 22, wherein the AAV is a serotype of AAV2, AAV2.5, AAV5, AAV8, or AAV9, or a mixture thereof.

24. The composition of claim 23, wherein the AAV is AAV5.

25. The composition according to any one of claims 22-24, wherein the viral genome of the AAV comprises 5' to 3': a first ITR, a first promoter, an IL-1Ra transgene, a first poly A tail, a second promoter, an FGF18 transgene, a second poly A tail, and a second ITR.

26. The composition according to any one of claims 22-24, wherein the viral genome of the AAV comprises 5' to 3': a first ITR, a first promoter, an FGF18 transgene, a first poly A tail, a second promoter, an IL-1Ra transgene, a second poly A tail, and a second ITR.

27. The composition according to any one of claims 22-24, wherein the viral genome of the AAV comprises 5' to 3': a first ITR, a promoter, an IL-1Ra transgene, an adapter sequence, an FGF18 transgene, a poly A tail, and a second ITR.

28. The composition according to any one of claims 22-24, wherein the viral genome of the AAV comprises 5' to 3': a first ITR, a promoter, an FGF18 transgene, an adapter sequence, an IL-1Ra transgene, a poly A tail, and a second ITR.

29. The composition according to any one of claims 22-24, comprising (1) a first AAV viral genome comprising: a first ITR, a first promoter, an IL-1Ra transgene, a first poly A tail, and (2) a second AAV viral genome comprising a second promoter, an IL-1Ra transgene, a second poly A tail, and a second ITR.

30. The composition according to any one of claims 1-29, further comprising a pharmaceutical composition of a pharmaceutically acceptable carrier.

31. The composition of claim 30, wherein the composition is formulated for intra-articular injection.

32. A kit comprising a unit dose of the composition according to any one of claims 1-31.

33. A method for expressing FGF18 peptide and IL-1Ra peptide in cells, comprising contacting the cells with the composition of any one of claims 12-31.

34. The method of claim 33, wherein the cell is a chondrocyte or a synovial cell.

35. A method of delivering FGF18 peptide and IL-1Ra peptide to a target region of a subject in need, comprising introducing the composition of any one of claims 12-31 into the region.

36. The method of claim 35, wherein the target region is a joint, synovium, subsynovium, joint capsule, tendon, ligament, cartilage, or periarticular muscle.

37. A method of treating arthritis in a subject in need, comprising administering to the subject a therapeutically effective amount of the composition according to any one of claims 1-31.

38. The method of claim 37, comprising intra-articular injection.

39. The method according to any one of claims 35-37, wherein the subject is a human.

40. Use of the composition according to any one of claims 1-31 in the treatment of arthritis.

41. Use of the composition according to any one of claims 1-31 in the preparation of a medicament for treating arthritis.

42. The method or use according to any one of claims 37-41, wherein the arthritis refers to the hand, knee, hip, shoulder, ankle, elbow, temporomandibular joint, spine, or any combination thereof.

43. The method or use according to claim 42, wherein the arthritis is osteoarthritis.

44. The method or use according to claim 42, wherein the arthritis is rheumatoid arthritis.

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