NFAT1-based therapeutics for joint diseases

Abstract

A composition may have an adeno-associated virus (AAV) vector containing a nucleic acid sequence encoding NFAT1 (Nuclear Factor of Activated T Cells 1) protein for treatment of osteoarthritis and other joint diseases. The AAV vector may be AAV serotype 5, other AAV serotypes, and AAV subtype variants. The nucleic acid sequence encoding NFAT1 may be constitutively active NF ATI or wild-type NF ATI. The nucleic acid sequence encoding NFAT 1 may comprise a hemagglutinin epitope tag. At least one AAV vector may comprise a viral genome concentration of about 2x1010 viral genomes. The NFAT 1 may be human NFAT1. The NFAT1 may be mouse NFAT1. A pharmaceutical composition may comprise the composition and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be suitable for intra-articular or local tissue injection. The pharmaceutically acceptable carrier may be sterile saline.

Description

NF ATI -BASED THERAPEUTICS FOR JOINT DISEASESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. Provisional Application No. 63 / 677,321 filed July 30, 2024, which provisional application is incorporated herein by specific reference in its entirety.SEQUENCE LISTING STATEMENT

[0002] The Sequence Listing submitted as the XML file named K126210108W001, created on July 29, 2025, and having a size of 19,128 bytes, is hereby incorporated by reference in its entirety.BACKGROUNDField:

[0003] The present disclosure relates to compositions and pharmaceuticals for use as treatment for joint pain or inflammation, such as osteoarthritis. More particularly, the compositions can include gene delivery vectors, pharmaceutical compositions, and therapeutic methods.Description of Related Art:

[0004] Osteoarthritis is a chronic, degenerative joint disease characterized by the progressive breakdown of articular cartilage, subchondral bone remodeling, and synovial inflammation, leading to pain, stiffness, and loss of joint function. It represents the most common form of arthritis and is a leading cause of disability worldwide, particularly among aging populations. Current treatment options are primarily palliative, focusing on symptom relief through analgesics, anti-inflammatory drugs, or physical therapy, and ultimately joint replacement surgery in advanced cases. Accordingly, there remains a significant unmet medical need for disease-modifying therapies that can prevent, halt, or reverse the progression of osteoarthritis.

[0005] Gene therapy offers a promising approach to treating osteoarthritis by enabling sustained, localized expression of therapeutic proteins directly within the affected joint. Unlike conventional pharmacologic treatments that require repeated administration and may have systemic side effects, gene therapy can provide long-term therapeutic benefit from a single administration by delivering nucleic acid sequences encoding anti-inflammatory cytokines, growth factors, or other disease-modifying agents. Advances in vector design, including the use of viral vectors such as adeno-associated virus (AAV), have improved the safety, specificity, and efficiency of gene delivery to cells of joint tissues, including chondrocytes and synoviocytes. As such, gene therapy holds significant potential for modifying the disease course of osteoarthritis and other joint diseases by addressing their underlying molecular and cellular mechanisms.

[0006] The AAV is a widely used gene delivery vector due to its favorable safety profile, ability to mediate long-term gene expression in both dividing and non-dividing cells, and low immunogenicity. AAV is a non-pathogenic, replication-deficient parvovirus that can be engineered to carry therapeutic nucleic acid sequences within its capsid and efficiently transduce a range of cell types. Multiple AAV serotypes and engineered capsid variants have been developed to enhance tissue tropism and transduction efficiency, including for musculoskeletal and joint-associated tissues. In the context of osteoarthritis, AAV vectors offer an attractive platform for delivering genes encoding anti-inflammatory or anabolic proteins directly to the synovium or cartilage, thereby achieving sustained, localized therapeutic effects with minimal systemic exposure.SUMMARY

[0007] In some embodiments, a composition may comprise an adeno-associated virus (AAV) vector containing a nucleic acid sequence encoding NFAT1 (Nuclear Factor of Activated T Cells 1) protein. The AAV vector may be AAV serotype 5 or other AAV serotypes and engineered capsid variants with tissue tropism and transduction efficiency for musculoskeletal and joint diseases. The nucleic acid sequence encoding NFAT1 may be constitutively active NFAT1. The nucleic acid sequence encoding NF ATI may comprise a hemagglutinin epitope tag. At least one AAV vector may comprise a viral genome concentration of about 2xlO10viral genomes. The NFAT1 may be human NF ATI. The nucleic acid may include SEQ ID NO: 1 or SEQ ID NO: 2, or at least 80% identity therewith. The NFAT1 may be mouse NFAT1. The nucleic acid may include SEQ ID NO: 3 or SEQ ID NO: 4, or at least 80% identity therewith.

[0008] In some embodiments, a pharmaceutical composition may comprise the composition having the AAV vector and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be suitable for intra- articular injection. The pharmaceutically acceptable carrier may be sterile saline.

[0009] In some embodiments, a method of providing a therapy to a joint may comprise providing the composition having NFAT1 or a nucleic acid encoding for NFAT1. The method may comprise administering the composition to a joint cavity and / or tissues surrounding a joint to provide a therapy to the joint of a subject. The therapy may be a preventative treatment to inhibit onset or progression of a joint disease. The therapy may be a treatment after onset of the joint disease. The joint disease may be selected from the group consisting of osteoarthritis, rheumatoid arthritis, joint contracture, osteochondroma of the joint condyle, inflammatory synovitis, psoriatic arthritis, juvenile idiopathic arthritis, reactive arthritis, ankylosing spondylitis, crystal-induced arthropathies, autoimmune polyarthritis, and combinations thereof. The composition may include NFAT1 protein in a controlled release formulation. The NFAT1 composition may include a nucleic acid encoding for NFAT1. The NF ATI may be human NFAT1. The nucleic acid may include SEQ ID NO: 1 or SEQ ID NO: 2, or at least 80% identity therewith. The joint may be selected from the group consisting of knee, hip, ankle, shoulder, elbow, wrist, spinal facet joint, and other synovial joints. The subject may have NF ATI deficiency in joint tissues. The NFAT1 deficiency may comprise NF ATI expression levels less than 80% (or 70%, or 60%, or 50%) of normal control levels. The subject may have a deleterious mutation in the NFAT1 DNA binding domain. The deleterious mutation may be selected from the group consisting of Q571H and R572G amino acid substitutions. The composition may comprise an adeno-associated virus (AAV) vector containing a nucleic acid sequence encoding NFAT1 (Nuclear Factor of Activated T Cells 1) protein. The AAV vector may be AAV serotype 5 or other AAV serotypes and engineered capsid variants with tissue tropism and transduction efficiency for musculoskeletal and joint diseases. The nucleic acid sequence encoding NF ATI may comprise a hemagglutinin epitope tag. The administrating may be gene delivery.

[0010] In some embodiments, a pharmaceutical composition may comprise the composition having the AAV vector and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be suitable for intra- articular injection. The pharmaceutically acceptable carrier may be sterile saline.

[0011] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0012] The foregoing and following information as well as other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings.

[0013] Figure 1A includes an AAV-Nfatl construct map.

[0014] Figure IB includes images for immunohistochemistry (IHC) that shows no staining in controls (Ctrl, no primary antibody) but displays positive staining of NF AT 1 protein in chondrocytes and synovial cells of Nfatl+ / - mouse knees at 6 and 12 months after intraarticular (IA) injection of AAV-Nfatl (2xlO10vg / joint) when using anti-HA (linked to NFAT1 ) antibodies.

[0015] Figure 1C includes images of NFTA1 IHC and Safranin-O-stained images of Nfatl- / - mouse knees, which show that AAV-Nfatl treated knees have significantly less severe OA, compared to the knees receiving AAV alone, at 6 months after IA injections. N= 4 mice (8 knees) per treatment group. s=synovium, m=meniscus, ACL= anterior cruciate ligament, op= osteophyte, arrowheads point to osteoarthritic cartilage lesions.

[0016] Figure ID includes a graph that compares AAV versus AAV-Nfatl for knee OA grade, where the AAV-Nfatl group is in better condition with a lower OA grade compared to the higher OA grade AAV group.

[0017] Figure 2A includes photomicrographs of mouse knees showing that DMM-induced knee injury receiving intra- articular (IA) AAV control resulted in severe knee OA, while OA changes in the knee joint receiving IA AAV-Nfatl are limited to the superficial layer of the articular cartilage (AC / cartilage). Arrowheads point to osteoarthritic cartilage lesions.

[0018] Figure 2B includes a graph of OA scores for the medial compartment of the knee joint. The average OA score is significantly higher in the AAV group than the AAV-Nfatl group at 26 weeks post-DMM. AC = articular cartilage, s = synovium, m = meniscus. Arrowheads indicate cartilage lesions. N = 4.

[0019] Figure 3 A includes a graph of a linear regression analysis comparing NFAT1 mRNA fold change and average OA grades, including a correlation between averageKellgren and Lawrence (KL) radiographic OA grades (from 3 observers) and NFAT1 mRNA levels in the cartilage of primary OA patients.

[0020] Figure 3B includes a graph of the correlation between average KL radiographic OA grades (from 3 observers) and NFAT1 mRNA levels in the cartilage of secondary OA patients.

[0021] Figure 4A includes a graph of a correlation analysis of cartilage NFAT1 protein levels that are significantly correlated with radiographic OA severity in combined primary OA and PTOA patients (p < 0.0001), N = 82.

[0022] Figure 4B includes a graph showing that blood NF ATI protein levels are not significantly correlated with cartilage NF ATI levels.

[0023] Figure 4C includes a graph showing the synovium NF ATI levels are significantly correlated with cartilage NFAT1 levels (p < 0.00001) in OA patients. N = 67 for both Figures 4B and 4C.

[0024] Figure 5A includes an example AAV-NFAT1 (human) construct map.

[0025] Figure 5B includes images of human articular cartilage and synovium explants in culture.

[0026] Figure 5C includes immunofluorescent images show positive staining of NFAT1 protein expression in human synovial cells and articular chondrocytes released from cultured synovium and AC explants after transduction of AAV-NFAT1. Anti-HA tag (linked to NF ATI transgene) antibody was used for immunocytochemistry.

[0027] Figure 5D includes graphs for cartilage and synovium qPCR analysis, which indicates that AAV-NFAT1 treatment can restore aberrant expression of specific genes in the primary cells at day 14 of culture. N=6. *=p<0.05, **=p<0.01, ***=p<0.001.

[0028] Figure 6A includes an example Col2-CA-Nfatl transgenic construct map.

[0029] Figure 6B includes a gel for RT-PCR showing cartilage-specific expression of CA- Nfatl transgene. AC=articular cartilage; Synov= synovium.

[0030] Figure 7 includes a graph that shows the semiquantitative histopathologic grades of mouse knee OA at 2, 4, and 8 weeks after surgical destabilization of the medial meniscus (DMM). OA severity in each genotype group at each timepoint was determined using the OARSI knee OA histopathology grading system (Osteoarthritis and Cartilage 2010; 18 :S 17-S23). Values are the means and standard deviations of averaged OA grades scored blindly by three individuals. N = 6 mice per genotype group / timepoint; *p < 0.05, **p < 0.01, ***p < 0.001.

[0031] Figure 8 illustrates the alignment of wild and mutated NFAT1 cDNA (A) and protein (B) sequences.DETAILED DESCRIPTION

[0032] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0033] Generally, the present disclosure provides gene therapy compositions and methods for the treatment of osteoarthritis. In various embodiments, nucleic acid sequences encoding therapeutic proteins are delivered to joint tissues using viral or non-viral gene delivery vectors to achieve localized, sustained expression of disease-modifying agents. The disclosed gene therapy approaches are designed to address the underlying molecular and cellular mechanisms of osteoarthritis, such as inflammation, cartilage degradation, and impaired tissue regeneration. In preferred embodiments, adeno-associated virus (AAV) vectors are utilized to deliver therapeutic transgenes to synovial cells, chondrocytes, or other joint-associated cells. By enabling long-term expression of anti-inflammatory cytokines, growth factors, or cartilage-protective proteins, the disclosed compositions and methods offer potential for modifying disease progression, alleviating symptoms, and improving joint function in patients with osteoarthritis.

[0034] Osteoarthritis (OA) is the most common joint disorder among middle-aged and elderly individuals, affecting more than 32.5 million adults in the United States [1-3]. While OA involves multiple joint tissues, degeneration of the articular cartilage (AC) is widely recognized as its primary pathological feature [4-8]. OA is generally categorized into two forms: primary OA, which arises spontaneously or idiopathically, and secondary OA, which results from joint trauma or structural abnormalities. Despite the identification of numerous potential risk factors contributing to OA onset and progression, the underlying disease mechanisms remain incompletely understood. At present, no approvedpharmacologic treatment exists that can effectively stop or reverse OA progression [1, 2, 4, 9].

[0035] NFAT1, also referred to as NFATc2, is a transcription factor within the nuclear factor of activated T cells (NFAT / Nfat) family. Mice deficient in NFAT1 exhibit an exaggerated immune response, increased susceptibility to hapten-induced allergic contact hypersensitivity (CHS), and age-related development of CHS-like skin inflammation [10, 11]. Research has demonstrated that NF ATI directly associates with the promoters of 21 genes responsible for encoding various pro- and anti-inflammatory cytokines, matrixdegrading enzymes, growth factors, and cartilage matrix collagens, among others, thereby regulating their expression in articular cartilage (AC) cells of adult mice

[0012] . Although NFAT1 knockout (Nfatl- / -) mice develop normally in terms of skeletal formation, they later exhibit osteoarthritic (OA) changes accompanied by abnormal gene expression in joint tissues, mirroring primary OA observed in humans [13—15]. Notably, early signs of hip OA emerge in all Nfatl - / - mice between 4 and 6 months of age, with knee O A following at 7 to 8 months. In contrast, Nfatl+ / - mice develop both hip and knee OA after 15 months, whereas only 20% of aged wild-type (WT) mice show knee OA at 18 months, highlighting a “gene dosage” effect of Nfatl on the onset and progression of OA [12-16].

[0036] Human studies have identified that a subset of osteoarthritis (OA) patients (approximately 40%) exhibit a marked reduction in articular cartilage (AC) NFAT1 mRNA expression, defined as less than 50% of the mean NF ATI expression level observed in age- and body mass index (BMI)-matched control subjects. In these patients, elevated AC NFAT1 expression levels were found to correlate inversely with radiographic OA severity, such that higher NF ATI expression was associated with milder OA grades, and lower expression with more severe disease. Additionally, somatic mutations within the NFAT1 DNA-binding domain were detected in AC cells (but not in peripheral blood cells) in approximately 13% of tested OA patients, with potentially deleterious variants identified in 6% of cases. These findings suggest that NFAT1 deficiency or dysfunction may contribute to OA pathogenesis in a subset of patients and that NF ATI may play a protective role in modulating OA progression in humans.

[0037] Described herein are therapeutic agents developed for the treatment of OA in human patients. These agents include: (1) an adeno-associated virus (AAV) vector encoding a mouse Nfatl transgene (AAV-Nfatl), (2) an AAV vector encoding a human NFAT1 transgene (AAV-NFAT1), and (3) a cartilage-specific transgenic construct, Col2-CA-Nfatl, designed to drive overexpression of NFAT1 under the control of a collagen type II promoter in mice. The therapeutic efficacy of these agents has been evaluated in murine models of OA as well as in ex vivo cultured cartilage and synovium explants obtained from human OA patients. A single intra-articular (LA) injection of AAV-NFAT1 into the knee joints of NF ATI -deficient mice, as well as wild-type mice subjected to joint injury, resulted in a significant reduction in OA severity. Furthermore, transduction of human OA cartilage and synovial explants with AAV-NFAT1 led to the correction of aberrant gene expression patterns associated with OA pathogenesis, supporting the potential therapeutic utility of NFATl-based gene therapy approaches in human osteoarthritis.

[0038] In this disclosure, the assays utilized AAV-NFAT1 viral vectors containing eGFP to label the presence of NF ATI protein fused with eGFP in animal and tissue culture studies. For future clinical trial using AAV-NFAT1, AAV-NFAT1 vectors without eGFP can be generated. Accordingly, the inventive AAV-NFAT1 vectors can be prepared with or without eGFP.

[0039] In some embodiments, the vector can be AAV5 in the AAV-NFAT1 viral vectors. However, any other AAV serotypes or subclasses may also be used as those serotypes might also be functional for generating AAV-NFAT1 vectors.

[0040] In some embodiments, the vector can be a viral vector other than AAV. The non- AAV viral vectors are well known in the art. The viral vector may be selected from the group consisting of an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, a retroviral vector, a herpes simplex virus (HSV) vector, a vaccinia virus vector, a baculoviral vector, a foamy virus vector, a human foamy virus vector, a Sendai virus vector, a vesicular stomatitis virus (VSV) vector, a measles virus vector, a human immunodeficiency virus (HlV)-derived vector, a murine leukemia virus (MLV)-derived vector, a simian immunodeficiency virus (SlV)-derived vector, a human T-lymphotropic virus (HTLV)-derived vector, and combinations thereof.

[0041] In some embodiments, the NAFT1 can be provided by therapeutics such as non- viral delivery, biologies, oligonucleotide, nanomaterial-controlled stable release of NFAT1 or an NF ATI stimulator, or the like. The non-viral gene carrier may be selected from the group consisting of a lipid-based carrier, a liposome, a cationic liposome, a lipid nanoparticle, a polymer-based carrier, a dendrimer, a polyethyleneimine (PEI) polymer, a poly(lactic-co-glycolic acid) (PLGA) nanoparticle, a chitosan-based nanoparticle, a cyclodextrin-containing polymer, a poly (beta- amino ester) (PBAE), a poly(ethyleneglycol) (PEG) -modified polymer, a solid lipid nanoparticle, a silica nanoparticle, a carbon nanotube, a graphene oxide nanoparticle, a gold nanoparticle, a calcium phosphate nanoparticle, a cell -penetrating peptide (CPP), a peptide-based nanocarrier, a DNA origami structure, an exosome, a micelle, a nanogel, a magnetic nanoparticle, and combinations thereof.

[0042] THERAPIES

[0043] In some embodiments, a single injection of AAV-NFAT1 attenuates OA progression by regulating the expression of multiple target genes. This novel therapeutic strategy using NFAT1, an upstream transcriptional regulator, as a molecular target appears to be more effective than the previously tested disease-modifying osteoarthritis drug (DMOAD) candidates that target a single downstream catabolic or anabolic molecule. However, a yearly, monthly, or weekly or other time period duration for repeated administrations can be used. This allows the dosing to be administered as needed or desired.

[0044] In some embodiments, the NFAT1 gene can be delivered to provide treatment for individual OA patients with quantitative NF ATI deficiency (significantly reduced NFAT1 expression) and / or qualitative NF ATI deficiency (pathogenic somatic mutations in the NFAT1 DNA binding domain). The treatment described herein can improve the effectiveness of OA therapy in humans.

[0045] The AAV-NAFT1 particles of the present invention may be administered by any delivery route which results in a therapeutically effective outcome. These include, but are not limited to, intra- articular, intracartilaginous (within a cartilage), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), and spinal canal.

[0046] The AAV particles of the present invention may be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for liquid solution or suspension in a liquid solution. The AAV particles may be formulated with any appropriate and pharmaceutically acceptable excipient.

[0047] PHARMACEUTICAL COMPOSITIONS

[0048] Provided herein are compositions (e.g., pharmaceutical compositions) comprising AAV particles. The AAV particles may comprise a viral genome comprising a nucleic acid sequence encoding a protein of interest (e.g., NF ATI).

[0049] According to the present invention the AAV particles may be prepared as pharmaceutical compositions. It will be understood that such compositions necessarily comprise one or more active ingredients and, most often, a pharmaceutically acceptable excipient.

[0050] Relative amounts of the active ingredient (e.g. AAV particle), a pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and / or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w / v) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w / w) active ingredient.

[0051] In some embodiments, the AAV particle pharmaceutical compositions described herein may comprise at least one payload (e.g., nucleic acid encoding NFAT1). As a nonlimiting example, the pharmaceutical compositions may contain an AAV particle with 1, 2, 3, 4 or 5 payloads.

[0052] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated 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, rats, birds including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0053] In some embodiments, compositions are administered to humans, human patients or subjects.

[0054] FORMULATIONS

[0055] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. As used hereinthe term “pharmaceutical composition” refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.

[0056] In general excipient and / or one or more other accessory ingredients. As used herein, the phrase “active ingredient” generally refers either to an AAV particle carrying a payload region encoding the polypeptides of the invention or to the end product encoded by a viral genome of an AAV particle as described herein.

[0057] Formulations of the AAV particles and pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0058] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0059] In one embodiment, the AAV particles of the invention may be formulated in PBS with 0.001% of pluronic acid (F-68) at a pH of about 7.0.

[0060] In some embodiments, the AAV formulations described herein may contain sufficient AAV particles for expression of at least one expressed functional payload. As a non-limiting example, the AAV particles may contain viral genomes encoding 1, 2, 3, 4 or 5 functional payloads.

[0061] In some embodiments, the formulations described herein may contain at least one AAV particle comprising the nucleic acid sequence encoding a protein of interest, such as NAFT1.

[0062] The AAV particles of the invention can be formulated using one or more excipients or diluents to (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release of the payload; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase the translation of encodedprotein; (6) alter the release profile of encoded protein; and / or (7) allow for regulatable expression of the payload of the invention.

[0063] In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0064] Excipients, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

[0065] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and / or combinations thereof.

[0066] In some embodiments, AAV particle formulations may comprise at least one inactive ingredient. As used herein, the term “inactive ingredient” refers to one or more agents that do not contribute to the activity of the active ingredient of the pharmaceutical composition included in formulations. In some embodiments, all, none or some of the inactive ingredients which may be used in the formulations of the present invention may be approved by the US Food and Drug Administration (FDA).

[0067] As used herein, the phrase “pharmaceutically acceptable route of administration” or “route of administration” includes, but is not limited to, oral, sublingual, buccal, nasal, inhalational (pulmonary), rectal, vaginal, topical, transdermal, ocular (ophthalmic), nasal, otic, parenteral, and mucosal routes. In some aspects, parenteral administration of the compounds is provided. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, and the like.

[0068] JOINT DISEASES AND NFAT1 DEFICIENCY

[0069] NFAT1 deficiency may be treated by administering a gene therapy vector comprising a nucleic acid sequence encoding a functional NFAT1 protein or a biologically active variant thereof. In certain embodiments, the gene therapy vector comprises a recombinant adeno-associated virus (rAAV) vector in which a polynucleotide encoding human or murine NFAT1 is operably linked to a regulatory element such as a tissue-specific or constitutive promoter. The rAAV vector may be formulated for local or systemic delivery and is capable of transducing target cells and directing expression of the encoded NFAT1 protein therein. In some embodiments, the rAAV vector is delivered intraarticularly to joint tissues, such as articular cartilage or synovium, to restore NFAT1 activity in situ and mitigate molecular and cellular pathways associated with osteoarthritis or cartilage degeneration. In other embodiments, the vector may be administered systemically or ex vivo to transduce progenitor or stromal cells that can home to affected tissues and supply therapeutic NFAT1 function. The encoded NFAT1 may restore transcriptional regulation of genes involved in cartilage homeostasis, matrix production, inflammation modulation, and chondrocyte survival.

[0070] The gene therapy vector may optionally include post- transcriptional regulatory elements, codon optimization, or signal sequences to enhance expression, stability, or activity of the NFAT1 protein. In some embodiments, the NFAT1 coding sequence is delivered using a self-complementary AAV vector, or an AAV serotype selected for high tropism toward chondrocytes, synovial fibroblasts, or mesenchymal stromal cells. This approach enables durable restoration of NF ATI expression and functional correction of NF ATI deficiency in affected tissues.

[0071] In some embodiments, the treatment can be for joint contractures. NF ATI may be used as a therapeutic agent for the treatment of joint contracture by modulating gene expression pathways involved in fibrosis, inflammation, and extracellular matrix remodeling within joint tissues. In certain embodiments, a nucleic acid encoding NFAT1is delivered to affected joints using a gene therapy vector, such as a recombinant adeno- associated virus (rAAV), to restore or enhance NF ATI activity in cells such as fibroblasts, synoviocytes, or myofibroblasts. Therapeutic expression of NFAT1 may reduce fibrotic tissue deposition, inhibit pro-inflammatory signaling, and promote tissue remodeling, thereby improving joint mobility and reducing stiffness associated with contracture.

[0072] In some embodiments, NF ATI may be used as a therapeutic agent for the treatment of osteochondroma affecting the joint condyle by regulating abnormal chondrocyte proliferation and differentiation associated with ectopic cartilage growth. In certain embodiments, a nucleic acid encoding NFAT1 is delivered to the osteochondroma site using a gene therapy vector, such as a recombinant adeno-associated virus (rAAV), to restore NFAT1 activity in condylar cartilage or periosteal cells. Expression of NF ATI may normalize gene expression pathways involved in skeletal patterning, inhibit aberrant cartilage matrix production, and suppress osteochondroma progression, thereby preserving joint structure and function.

[0073] NF ATI may be used as a therapeutic agent for the treatment of rheumatoid arthritis by modulating immune response pathways and suppressing pro-inflammatory gene expression in joint tissues. In certain embodiments, a nucleic acid encoding NFAT1 is delivered using a gene therapy vector, such as a recombinant adeno-associated virus (rAAV), to transduce synovial fibroblasts, macrophages, or T cells within the affected joint. Therapeutic expression of NF ATI may reduce the production of inflammatory cytokines, inhibit synovial hyperplasia, and limit joint destruction, thereby alleviating inflammation, pain, and structural damage associated with rheumatoid arthritis.

[0074] EXPERIMENTAL

[0075] Methods of NFATl-Based Intraarticular Therapy for Osteoarthritis in Mice.

[0076] NFATl-based intraarticular therapy for spontaneous osteoarthritis in NFATl-deficient mice:

[0077] A gene therapy method was developed to test therapeutic effects of stable overexpression of NF ATI for OA in mice. The gene therapy method includes using adeno- associated virus (AAV) serotype-5 (AAV5) carrying mouse Nfatl transgene to generate AAV-Nfatl vectors. AAV5 was chosen because it has been confirmed to be safe and effective for delivering transgenes into animal and human joint tissues [17,18]. However, it is expected that any AAV subtype and any other viral delivery vector as well as other gene delivery vectors could be used to deliver the gene for NFATl.

[0078] The mouse AAV-Nfatl vector is mainly composed of a plasmid containing wholelength mouse NF ATI cDNA tagged with a hemagglutinin (HA)-epitope (purchased from Addgene, with an MTA), a CMV promoter, and eGFP sequence for labeling purpose (Fig. 1A). The first two batches of mouse AAV5-NFAT1 viral vectors used in the animal study were manufactured by Virovek (Hayward, CA).

[0079] Fig. 1A includes an AAV-NFAT1 construct map. Fig. IB includes immunohistochemistry (IHC) that shows no staining in controls (IHC Ctl, no primary antibody) but displays positive staining of NFAT1 protein in chondrocytes, synovial cells, and ligament cells of Nfatl+I~ mouse knees at 6 and 12 months after IA injections of AAV5- NFAT1 (2x1010vg / joint) when using anti-HA (linked to NFAT1) antibodies. Fig. 1C shows NFTA1 IHC and Safranin-O-stained images of Nfatl- / - mouse knees show that AAV- NFAT1 treated knees have significantly less severe OA, compared to the knees receiving AAV alone, at 6 months after IA injections. S = synovium, m = meniscus, ACL = anterior cruciate ligament, op = osteophyte, arrowheads point to osteoarthritic AC lesions. Fig. ID compares AAV versus AA-NFAT1, which shows AAV group has significantly more knee osteoarthritis.

[0080] The assay tested the efficiency of AAV5-NFAT1 expression with three doses (lxl010vg, 2xl0lovg, and 5xl010vg) and found that 2xl0lovg was sufficient to have stable NFAT1 mRNA (detected by qPCR) and protein overexpression (detected by immunohistochemistry) in mouse knee joint tissues after intra-articular (IA) injections of AAV-NAT1. NFAT1 mRNA levels in mouse joint tissues after a single IA injection was significantly higher in the AAV-NFAT1 (2xlOlovg) group than the AAV control group, consistent with previous reports from others that a single IA injection of AAV-transgenes provided stable expression in rodent knee tissues for up to one year [19-21]. Immunohistochemistry (IHC) analysis using an anti-HA (linked to Nfatl transgene) antibody confirmed NF AT 1 protein expression in AC and synovial cells at 6 and 12 months after a single IA injection of AAV5-NFAT1 (2x1010vg). (Fig. IB). More importantly, the data confirmed that a single IA injection of AAV5-NFAT1 significantly attenuated the severity of knee OA in NFAT1 -deficient mice (Fig. 1C-D).

[0081] NFATl-based intraarticular therapy for posttraumatic osteoarthritis (PTOA) in wild-type mice:

[0082] Intraarticular (IA) delivery of AAV5-NFAT1 into the knee of young adult wildtype (WT) mice at 2 weeks post-DMM (OA initiation phase) can halt or attenuate kneePTOA progression, using age-matched post-DMM WT mice receiving IA AAV alone (placebo) as controls. Mouse knee OA was induced by surgical destabilization of the medial meniscus (DMM)

[0023] , which was performed in the right knee of 10-12 weeks old male mice. The contralateral left knee was not operated on and served as an internal control. Animals with DMM knee injury were euthanized at 8, 16, and 26 weeks after DMM for histopathology, OA grading, and gene expression analyses to determine the severity of knee OA using a semiquantitative mouse knee OA scoring system

[0024] by three observers blinded from the treatment information. Gene expression analysis demonstrated that IA treatment with AAV-NFAT1 significantly down-regulated the expression of many catabolic genes responsible for AC degradation. Histopathological and OA grading analyses indicated that IA therapy with AAV-NFAT1 significantly reduced knee OA severity compared AAV alone group at all time points. The therapeutic outcomes from 26 weeks after DMM knee injury are presented in Figs. 2A-2B.

[0083] Fig. 2A includes photomicrographs of mouse knees showing that DMM-induced knee injury receiving IA AAV control resulted in severe knee OA, while OA changes in the knee joint receiving IA AAV -NF AT 1 are limited to the superficial layer of the articular cartilage (AC / cartilage). Arrowheads indicate articular cartilage lesions, s = synovium, m = meniscus.

[0084] Fig. 2B includes a graph of OA scores for the medial compartment of the knee joint is significantly higher in the AAV group than the AAV-NFAT1 group at 26 weeks post- DMM.

[0085] Discovery of NFAT1 deficiency in articular cartilage (AC):

[0086] It was discovered in human studies that 42% (155 / 372) of the total tested OA patients had a significant reduction of AC NFAT1 mRNA expression (<50% of the mean NFAT1 levels of age- and BMI-matched controls, n=41). Higher AC NF ATI levels correlated with less severe radiographic OA grades, and vice versa in both primary and secondary OA. A semiquantitative radiographic OA grading system published by Kellgren and Lawrence (KL OA Grading System)

[0022] was used to determine the OA grades, as shown in Figs. 3A-3B.

[0087] Figs. 3A-3B include graphs that show the correlation between average KL radiographic OA grades (from 3 observers) and NFAT1 mRNA levels in the AC. Fig. 3 A shows the correlation for a primary OA, and Fig. 3B shows the correlation for a secondary OA.

[0088] Additionally, somatic mutations in the NF ATI DNA binding domain were detected in the AC (not in blood) cells in 13% (12 / 93) of tested OA patients. Software-assisted in silico bioinformatic analysis

[0032] suggested that 1713G>T and 1714C>G are potentially deleterious / pathogenic or disease-causing mutations, which were detected in 6 / 93 patients (6%). The 1713G>T mutation causes Q571H amino acid (AA) substitution and 1714C>Gmutation causes R572G AA substitution (Table 1).Table 1 Footnote: Three distinct bioinformatics tools were used for in silico analysis.PROVEAN (Protein Variation Effect Analyzer) provean.jcvi.org / seq_submit.php), PolyPhen-2 (Polymorphism Phenotyping v2) (genetics.bwh.harvard.edu / pph2 / ), and MutPred2 (mutpred.mutdb.org / index.html) to predict functional effects of the mutations identified in this study. The cutoff values of PROVEAN, PolyPhen-2 and MutPre2 were set at -2.5, 0.5 and 0.5, respectively. Amino acid substitutions that exceed the cut off values were considered deleterious or pathogenic.

[0089] Further analysis confirmed that a significant NF ATI reduction and / or potentially pathogenic mutation in the NF ATI binding domain was identified in 45% of the examined patients with primary OA and in 40% of the patients with PTOA. This is because 4 of the 13 patients with NFAT1 mutations had both a significant NFAT1 reduction (quantitative NF ATI deficiency) and potentially pathogenic NFAT1 mutation (qualitative NFAT1 deficiency).

[0090] Reduced NF ATI protein levels are correlated with more severe radiographic O grades:

[0091] To examine if NF ATI deficiency is detectable at the protein level and is correlated with radiographic OA severity in OA patients, NF ATI protein levels were measured by enzyme-linked immunosorbent assay (ELISA) in 82 human AC and synovium samples harvested from arthroplasty (67 cases) and arthroscopic surgeries (15 cases). The results revealed that a significant NF ATI reduction (<0.50-fold of the mean NFAT levels of age-and B Mi-matched controls, n=20) in AC was identified in 47% of 53 patients with primary OA and in 38% of 29 patients with PTOA.

[0092] It is noteworthy that OA also occurred in a subset of patients whose NF ATI levels were within or above the normal control range, suggesting OA is a multifactorial disease in humans. Importantly, statistical analysis confirmed that lower NFAT1 protein levels in AC significantly correlated with more severe radiographic OA, while higher NFAT1 protein levels significantly correlated with less severe radiographic OA (Fig. 4A). The data suggest that NF ATI may play a protective role against OA progression, in both primary and secondary OA, though other risk factors may also play a role in the etiopathogenesis of OA.

[0093] Synovium NFAT1 levels highly significantly correlates with cartilage NFAT1 levels:

[0094] It was confirmed that synovium NF ATI levels (by ELISA) are significantly correlated with AC (cartilage) NF ATI levels in OA patients (p<0.00001), while blood NFAT1 protein levels are not significantly correlated with cartilage NF ATI levels (Figs 4B-4C). This finding is very important to future clinical research or new drug clinical trials using an NFAT1 level in joint tissues as a biomarker. Clinically, it would be much easier to obtain a large amount of the synovial tissue than the cartilage tissue, especially from patients with early OA through arthroscopic surgeries.

[0095] AAV-NFAT1 Treatment Corrects Aberrant Expression of Specific Genes in Cultured OA Cartilage and Synovium.

[0096] To test therapeutic effects of stable NFAT1 overexpression on human OA joint tissues, an adeno-associated virus (AAV) serotype-5 (AAV5) carrying human NFAT1 trans gene to generate AAV5-NFAT1 vectors was used. AAV5 was chosen as it has been confirmed to be safe and effective for delivering transgenes into animal and human joint tissues [17,18]. The human AAV-NFAT1 vector is composed of a plasmid containing whole-length human NFAT1 cDNA (purchased from Addgene, Catalog #74050, with an MTA), a hemagglutinin (HA)-epitope tag, a CMV promoter, and eGFP sequence for labeling purpose (Fig. 5A). The first two batches of human AAV-NFAT1 viral vectors used in our tissue culture study were manufactured by Virovek (Hayward, CA). Notably, the mouse version may be AAV-Nfatl, which is referred to herein as AAV-NFAT1.

[0097] The AAV-NFAT1 treatment was tested to show that it attenuates OA-like cellular dysfunction of cultured cartilage and synovium explants with quantitative NFAT1deficiency. Articular cartilage (AC) and synovium tissues were harvested from the hip and knee joints of patients with relatively early OA (KL grades 1-2)

[0022] . Full thickness AC explants were cut into ~10 mm2in size and synovium explants were cut into 50 mg per culture. Tissue explants were cultured in DMEM / F12 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator with 5% CO at 37°C. To increase transduction efficiency of AAC-NFAT1, the explants were pretreated with 20 p M of Etoposide (Sigma #E1383, Saint Louis, MO) for AC and 10 M of Etoposide for synovium overnight (about 16 hours) incubations as previously described [25,26]. Next day, cultures were washed twice with DMEM, AAV- NFAT1 vectors (4 x 109vg / ml for synovium and 6xl09vg / ml for AC cultures) were added for transduction, and the cultures were maintained in DMEM / F12 containing 10% FBS (synovium) or DMEM containing 5% FBS and 1 M dexamethasone (AC). Cultured tissues and cells released from tissue samples were harvested for transduction efficiency testing, protein assay, as well as gene expression analysis following RNA isolation and reverse transcription (RT) at various times depending on specific study goals.

[0098] It was found that AAV can efficiently deliver the NFAT 1 transgene into human AC and synovium. Explants in tissue culture conditions after Etoposide treatment (Fig. 5B). Immunofluorescence staining using an anti-HA antibody confirmed NFAT 1 expression in AC and synovial cells released from or within the cultured AC and synovium explants (Fig. 5C). AAV5-NFAT1 expression significantly corrected aberrant expression of specific genes known to be involved in OA pathogenesis in cultured OA cartilage and synovium (Fig. 5D).

[0099] Fig. 5A includes an example AAV-NFAT1 (human) construct map. Fig. 5(B) includes images of human articular cartilage and synovium explants in culture. Fig. 5C includes immunofluorescent images show positive staining of NF ATI protein expression in human synovial cells and articular chondrocytes released from cultured synovium and AC explants 21 days after transduction of AAV-NFAT1 . An anti- HA tag (linked to NFAT1 transgene) antibody was used for immunocytochemistry. Fig. 5D includes a graph showing data of relative fold change for AAV versus AAV-NFAT1 in cartilage or synovium. The qPCR analysis indicates that AAV-NFAT1 can correct aberrant expression of specific genes in OA cartilage and synovium explants at day 14 of culture. N = 8. *=p<0.05,**=p<0.01, ***=p<0.001.

[0100] Also, it was found that cartilage-specific NFAT1 overexpression attenuates OA progression in mice.

[0101] To examine if NFAT1 overexpression in cartilage can attenuate AC aging or OA in animals, a novel plasmid construct was prepared for generation of cartilage specific NFAT1 -overexpressing transgenic mice using the following methods: Constitutively active (CA)-NFATl cDNA tagged with an hemagglutinin (HA)-epitope in the pENTR vector (purchased from Addgene, with an MTA - Attachment 1) was inserted into the pCol2al construct (gift from Dr. Benoit de Crombrugghe) containing Col2al promoter driving cartilage-specific expression

[0027] , which generated a Col2al-CA-NFAT 1 cDNA construct. The Notl restriction site in CA-NFAT1 cDNA is eliminated by site- directed mutation using a QuickChange Site Directed Mutagenesis Kit (Stratagen). This mutation allows us to remove the plasmid backbone in the final construct (Fig. 6A). Fig. 6A shows a map of the Col2-C A-NFAT 1 transgenic construct.

[0102] The Col2al -CA-NFAT1 transgenic mice (cNfatlTg mice) were generated by pronuclear injection of the Col2al-CA-NFAT1 cDNA construct into fertilized eggs isolated from FVB female mice. Three founder mice were identified. Cartilage-specific expression of the transgene was confirmed by RT-PCR analysis (Fig. 6B). Continued genotype and cartilage phenotype monitoring of cNATITg mice indicated that the CA- Nfatl transgene remained specifically and efficiently expressed in cartilage cells after 10 generations.

[0103] Quantitative laser scanning cytometry (LSC) using NFAT1 antibodies showed that the levels of nuclear NFAT1 protein in the AC cells of the cNfatlTg+ / ‘ mice were 4.78+1.05-fold at 6 months, 4.12+0.61-fold at 12 months, and 3.95+0.65-fold at 18 months, compared to the cNfatlTg7' littermates (equivalent to WT mice).

[0104] Fig. 6B includes an image of a gel from RT-PCR showing cartilage-specific expression of Col2-CA-NFAT1 transgene. AC = articular cartilage; Synov = synovium.

[0105] To test if NF ATI plays a protective role against posttraumatic osteoarthritis (PTOA) progression, knee PTOA was created through surgical destabilization of the medial meniscus (DMM)

[0023] in 10-12 weeks old male Nfatl knockout (Nfatl- / -), Nfatl+ / -, WT, and cNfatlTg mice. Animals were euthanized at 2, 4, and 8 weeks after surgery. The knee joints were harvested and processed for histochemical and OA grading analyses to assess the severity of knee OA. Cartilage and synovium samples harvested from the medial compartment of the knee joints were processed for gene expression analyses by quantitativereal-time RT-PCR (qPCR). The results demonstrated that aberrant expression of mRNAs for specific matrix-degrading enzymes, pro-inflammatory cytokines, and major collagens in AC also displayed dose-related changes in both AC and synovium at each time point, namely, most severe changes in the Nfatl- / - and least remarkable changes in the cNfatlTg knees. Structural OA changes such as cartilage lesions, chondro-osteophyte formation and subchondral bone thickening were significantly more apparent in the medial compartment than the lateral compartment of the knee joints in all four genotype groups. Knee OA severity in each genotype group at each timepoint was determined using the OARSI mouse knee OA histological grading system

[0024] . The average grades from three independent observers showed that OA changes in the knee (femorotibial) joints were most severe in Nfatl- / - knees and least severe in cNfatlTg knees at 2, 4, and 8 weeks after DMM (Fig. 7). The data suggest that NFAT1 plays a protective role against PTOA progression

[0106] Fig. 7 includes a graph of data showing the knee OA score after DMM for the test agents. Semiquantitative histopathologic grades of mouse knee OA at 2, 4, and 8 weeks after DMM joint injury. OA severity in each genotype group at each timepoint was determined using the OARSI knee OA histopathology grading system

[0024] . Values are the means and standard deviations of averaged OA grades scored blindly by three individuals. N = 8 mice per genotype group / timepoint; *p < 0.05, **p < 0.01, ***p < 0.001.

[0107] SEQUENCES

[0108] Generally, embodiments of the invention include the nucleic acids that encode for NAFT1. The nucleic acid encoding NFAT1 may be present in any suitable form for gene therapy, including but not limited to DNA, RNA, or combinations thereof. In some embodiments, the nucleic acid comprises a coding sequence encoding a full-length human or murine NF ATI protein, an isoform or variant thereof, or a biologically active fragment. The nucleic acid may be in the form of a plasmid, linear or circular DNA, messenger RNA (mRNA), or self-amplifying RNA. The nucleic acid may be codon-optimized for expression in a target host cell and may optionally include regulatory elements such as a promoter, enhancer, polyadenylation signal, untranslated regions (UTRs), and / or introns. The nucleic acid may be incorporated into a viral vector (e.g., AAV, lentivirus) or formulated with a non-viral delivery vehicle (e.g., lipid nanoparticle, polymer complex, or exosome). In certain embodiments, the nucleic acid may be chemically modified to enhance stability, reduce immunogenicity, or improve cellular uptake. The nucleic acid may beadministered directly to a subject or used to transduce or transfect cells ex vivo prior to administration.

[0109] In some embodiments, the NAFT1 can include a human NFAT1 nucleic acid coding sequence (SEQ ID NO: 1).SEQ ID NO: 1 atgaacgcccccgagcggcagccccaacccgacggcggggacgccccaggccacgagcctgggggcagcccccaagac gagcttgacttctccatcctcttcgactatgagtatttgaatccgaacgaagaagagccgaatgcacataaggtcgccagcccacc ctccggacccgcataccccgatgatgtcctggactatggcctcaagccatacagcccccttgctagtctctctggcgagcccccc ggccgattcggagagccggatagggtagggccgcagaagtttctgagcgcggccaagccagcaggggcctcgggcctgag ccctcggatcgagatcactccgtcccacgaactgatccaggcagtggggcccctccgcatgagagacgcgggcctcctggtg gagcagccgcccctggccggggtggccgccagcccgaggttcaccctgcccgtgcccggcttcgagggctaccgcgagcc gctttgcttgagccccgctagcagcggctcctctgccagcttcatttctgacaccttctccccctacacctcgccctgcgtctcgcc caataacggcgggcccgacgacctgtgtccgcagtttcaaaacatccctgctcattattcccccagaacctcgccaataatgtcac ctcgaaccagcctcgccgaggacagctgcctgggccgccactcgcccgtgccccgtccggcctcccgctcctcatcgcctggt gccaagcggaggcattcgtgcgccgaggccttggttgccctgccgcccggagcctcaccccagcgctcccggagcccctcgc cgcagccctcatctcacgtggcaccccaggaccacggctccccggctgggtacccccctgtggctggctctgccgtgatcatg gatgccctgaacagcctcgccacggactcgccttgtgggatcccccccaagatgtggaagaccagccctgacccctcgccggt gtctgccgccccatccaaggccggcctgcctcgccacatctacccggccgtggagttcctggggccctgcgagcagggcgag aggagaaactcggctccagaatccatcctgctggttccgcccacttggcccaagccgctggtgcctgccattcccatctgcagca tcccagtgactgcatccctccctccacttgagtggccgctgtccagtcagtcaggctcttacgagctgcggatcgaggtgcagcc caagccacatcaccgggcccactatgagacagaaggcagccgaggggctgtcaaagctccaactggaggccaccctgtggtt cagctccatggctacatggaaaacaagcctctgggacttcagatcttcattgggacagctgatgagcggatccttaagccgcacg ccttctaccaggtgcaccgaatcacggggaaaactgtcaccaccaccagctatgagaagatagtgggcaacaccaaagtcctg gagatacccttggagcccaaaaacaacatgagggcaaccatcgactgtgcggggatcttgaagcttagaaacgccgacattga gctgcggaaaggcgagacggacattggaagaaagaacacgcgggtgagactggttttccgagttcacatcccagagtccagtg gcagaatcgtctctttacagactgcatctaaccccatcgagtgctcccagcgatctgctcacgagctgcccatggttgaaagacaa gacacagacagctgcctggtctatggcggccagcaaatgatcctcacggggcagaactttacatccgagtccaaagttgtgttta ctgagaagaccacagatggacagcaaatttgggagatggaagccacggtggataaggacaagagccagcccaacatgcttttt gttgagatccctgaatatcggaacaagcatatccgcacacctgtaaaagtgaacttctacgtcatcaatgggaagagaaaacgaa gtcagcctcagcactttacctaccacccagtcccagccatcaagacggagcccacggatgaatatgaccccactctgatctgca gccccacccatggaggcctggggagccagccttactacccccagcacccgatggtggccgagtccccctcctgcctcgtggc caccatggctccctgccagcagttccgcacggggctctcatcccctgacgcccgctaccagcaacagaacccagcggccgtac tctaccagcggagcaagagcctgagccccagcctgctgggctatcagcagccggccctcatggccgccccgctgtcccttgcggacgctcaccgctctgtgctggtgcacgccggctcccagggccagagctcagccctgctccacccctctccgaccaaccagca ggcctcgcctgtgatccactactcacccaccaaccagcagctgcgctgcggaagccaccaggagttccagcacatcatgtactg cgagaatttcgcaccaggcaccaccagacctggcccgcccccggtcagtcaaggtcagaggctgagcccgggttcctacccc acagtcattcagcagcagaatgccacgagccaaagagccgccaaaaacggacccccggtcagtgaccaaaaggaagtattac ctgcgggggtgaccattaaacaggagcagaacttggaccagacctacttggatgatgagctgatagacacacaccttagctgga tacaaaacatattatga

[0110] In some embodiments, the NAFT1 can include a Human NF ATI coding sequence with 3 HA tags and GFP (SEQ ID NO: 2).SEQ ID NO: 2ATGATCTTTTACCCATACGATGTTCCTGACTATGCGGGCTATCCCTATGACGT CCCGGACTATGCAGGATCCTATCCATATGACGTTCCAGATTACGCTatgaacgcccc cgagcggcagccccaacccgacggcggggacgccccaggccacgagcctgggggcagcccccaagacgagcttgacttct ccatcctcttcgactatgagtatttgaatccgaacgaagaagagccgaatgcacataaggtcgccagcccaccctccggacccg cataccccgatgatgtcctggactatggcctcaagccatacagcccccttgctagtctctctggcgagccccccggccgattcgg agagccggatagggtagggccgcagaagtttctgagcgcggccaagccagcaggggcctcgggcctgagccctcggatcga gatcactccgtcccacgaactgatccaggcagtggggcccctccgcatgagagacgcgggcctcctggtggagcagccgccc ctggccggggtggccgccagcccgaggttcaccctgcccgtgcccggcttcgagggctaccgcgagccgctttgcttgagcc ccgctagcagcggctcctctgccagcttcatttctgacaccttctccccctacacctcgccctgcgtctcgcccaataacggcggg cccgacgacctgtgtccgcagtttcaaaacatccctgctcattattcccccagaacctcgccaataatgtcacctcgaaccagcct cgccgaggacagctgcctgggccgccactcgcccgtgccccgtccggcctcccgctcctcatcgcctggtgcccaggattctc ccaagcggaggcattcgtgcgccgaggccttggttgccctgccgcccggagcctcaccccagcgctcccggagcccctcgcc gcagccctcatctcacgtggcaccccaggaccacggctccccggctgggtacccccctgtggctggctctgccgtgatcatgg atgccctgaacagcctcgccacggactcgccttgtgggatcccccccaagatgtggaagaccagccctgacccctcgccggtg tctgccgccccatccaaggccggcctgcctcgccacatctacccggccgtggagttcctggggccctgcgagcagggcgaga ggagaaactcggctccagaatccatcctgctggttccgcccacttggcccaagccgctggtgcctgccattcccatctgcagcat cccagtgactgcatccctccctccacttgagtggccgctgtccagtcagtcaggctcttacgagctgcggatcgaggtgcagccc aagccacatcaccgggcccactatgagacagaaggcagccgaggggctgtcaaagctccaactggaggccaccctgtggttc agctccatggctacatggaaaacaagcctctgggacttcagatcttcattgggacagctgatgagCGGATCcttaagccgca cgccttctaccaggtgcaccgaatcacggggaaaactgtcaccaccaccagctatgagaagatagtgggcaacaccaaagtcc tggagatCcccttggagcccaaaaacaacatgagggcaaccatcgactgtgcggggatcttgaagcttagaaacgccgacatt gagctgcggaaaggcgagACGgacattggaagaaagaacacgcgggtgagactggttttccgagttcacatcccagagtcc agtggcagaatcgtctctttacagactgcatctaaccccatcgagtgctcccagcgatctgctcacgagctgcccatggttgaaag acaagacacagacagctgcctggtctatggcggccagcaaatgatcctcacggggcagaactttacatccgagtccaaagttgtgtttactgagaagaccacagatggacagcaaatttgggagatggaagccacggtggataaggacaagagccagcccaacatg ctttttgttgagatccctgaatatcggaacaagcatatccgcacacctgtaaaagtgaacttctacgtcatcaatgggaagagaaaa cgaagtcagcctcagcactttacctaccacccagtcccagccatcaagacggagcccacggatgaatatgaccccactctgatct gcagccccacccatggaggcctggggagccagccttactacccccagcacccgatggtggccgagtccccctcctgcctcgt ggccaccatggctccctgccagcagttccgcacggggctctcatcccctgacgcccgctaccagcaacagaacccagcggcc gtactctaccagcggagcaagagcctgagccccagcctgctgggctatcagcagccggccctcatggccgccccgctgtccct tgcggacgctcaccgctctgtgctggtgcacgccggctcccagggccagagctcagccctgctccacccctctccgaccaacc agcaggcctcgcctgtgatccactactcacccaccaaccagcagctgcgctgcggaagccaccaggagttccagcacatcatg tactgcgagaatttcgcaccaggcaccaccagacctggcccgcccccggtcagtcaaggtcagaggctgagcccgggttccta ccccacagtcattcagcagcagaatgccacgagccaaagagccgccaaaaacggacccccggtcagtgaccaaaaggaagt attacctgcgggggtgaccattaaacaggagcagaacttggaccagacctacttggatgatgttaatgaaattatcaggaaggag ttttcaggacctcctgccagaaatcagacgTCTGGATCCggagagggcagaggaagtctgctaacatgcggtgacgtc gaggagaatcctggcccaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggc gacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatct gcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctacccc gaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgac ggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttca aggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaa gaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaac acccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacg agaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa

[0111] The first all capital region is the HA tag. The first lowercase region is the human NF ATI coding sequence. The next all capital region is a connection sequence, the next lower case region includes T2A and GFP.

[0112] In some embodiments, the NAFT1 can include mouse NFAT1 nucleic acid coding sequence (SEQ ID NO: 3).SEQ ID NO: 3 atggacgtcccggagccgcagcccgaccccgatggcggggacggccccggccacgagcccgggggcagtccccaagacg agctggacttttccatcctcttcgattatgactatctgaaccctatcgaagaagaaccgatcgcacataaggccatcagctcaccct ccggactcgcatacccggatgatgtcctggactatggcctcaagccatgcaacccccttgccagtctctctggcgagccccctgg ccggttcggagagccggatagtatagggttccagaactttctgagcccggtcaagccagcaggggcttcgggcccgagccctc ggatcgagatcactccatcccacgaactgatgcaggcagggggggccctccgtgggagagacgccggcctgtcccccgagcagccggccctggccctggccggcgtggccgccagcccgaggttcacactgcccgtgcccggctacgagggctaccgcgagc cgctttgcttgagccccgctagcagcggctcctctgccagcttcatttctgacaccttctccccctacacctcgccctgcgtctcacc caataacgccgggcccgacgacctgtgtccccagtttcaaaacatccctgctcattattcccccagaacctctccaataatgtcac ctcgaaccagcctcgccgaggacagctgcctgggccgacactcgcccgtgccccgtccggcatcccgctcctcctcacccggt gccaagcggaggcattcgtgcgcagaggctttggttgctcctctgcccgcagcctcaccccagcgctcccggagcccctcgcc acagccctcgcctcacgtggcactgcaggacgacagcatccccgctgggtacccccccacggccggctctgctgttctcatgg atgccctcaacaccctggccaccgactcgccctgcgggatcccctccaagatatggaagaccagtcctgacccgacgcctgtgt ccaccgctccgtccaaggctggcctggcccgccacatctaccctactgtggagttcctggggccatgtgagcaggaggagagg aggaattccgctccagagtccatcctgctggtaccacctacttggcccaagcagttggtgccggccattcccatctgcagcatccc tgtgactgcatccctcccaccactcgagtggccactctccaatcagtcgggctcctatgagctacggattgaggtccaacccaag ccccatcaccgggcccactatgagacggagggcagccgtggcgctgtcaaagccccaacaggaggacaccctgtggtgcag ctccacggctacatggagaacaagcctctggggcttcagatcttcattgggacagcagatgagaggatccttaagccgcacgcc ttctaccaagtacacaggatcactgggaaaacggtcaccaccacgagctatgagaagatcgtaggcaacaccaaggtcctgga gatccccctggagccaaagaacaacatgagagccaccatcgactgtgcaggcatcctgaagctccgaaacgctgacatcgag ctgcggaagggcgagacggacatcggcaggaagaacacgcgtgtgcgcctggtgttccgcgtgcacgtcccagagcccagt gggcgcatcgtctccctgcaggctgcgtccaaccccatcgagtgctctcagcgctctgcccacgagctgcccatggtggagag acaagacatggacagctgcctggtctacgggggccagcagatgatcctcacgggccagaacttcacagcggagtccaaggtt gtgttcatggagaagactacagatgggcagcagatttgggagatggaagctacggtggataaagacaagagccagcctaacat gctttttgttgagatccccgagtatcggaacaagcacatccgcgtgcccgtgaaagtcaacttctacgtcatcaacggaaagagg aaacgaagtcagccacagcactttacctaccacccagtccctgccatcaagacagagcccagcgatgagtatgaaccatctttga tctgcagccccgcccatggaggcctggggagccagccatattacccacagcacccaatgctggccgagtccccctcctgcctt gtggctaccatggccccctgccaacagttccgctcggggctctcatcccccgatgctcgctaccaacagcagagccccgcagct gccctctaccagagaagcaagagcctgagtcccggcctgctgggctaccagcagccgtccctcctggcagcacccttgggtct ggctgatgcccaccgctctgtgctggtgcatgctggttctcaggggcaggggcagggctccaccctgccacacacatcctcgg ccagccagcaggcctcacccgtgatccactactcacccaccaaccagcagcttcgcggtgggggtcaccaggagttccagcat atcatgtactgtgaaaacttcggccccagctctgccaggcctggcccgcctcccatcaaccaaggtcagaggctgagcccggg cgcctaccccacagtcatccaacaacagactgccccgagccaaagagctgccaaaaacggacccagtgaccagaaggaagc tctgcccacgggagtgaccgtcaaacaggaacagaacctggaccagacctacctggatgacgttaatgaaatcatcaggaagg agttttcaggacctccctcccgaaatcagacctag

[0113] In some embodiments, the NAFT1 can include mouse NFAT1 coding sequence with 3 HA tags and GFP(SEQ ID NO: 4).SEQ ID NO: 4ATGATCTTTTACCCATACGATGTTCCTGACTATGCGGGCTATCCCTATGACGTCCCGGACTATGCAGGATCCTATCCATATGACGTTCCAGATTACGCTGCTCAGTGCGGCCGCGACGTCCCGGAGCCGCAGCCCGACCCCGATGGCGGGGACGGCCCCGGCCACGAGCCCGGGGGCAGTCCCCAAGACGAGCTGGACTTTTCCATCCTCTTCGATTATGACTATCTGAACCCTATCGAAGAAGAACCGATCGCACATAAGGCCATCAGCTCACCCTCCGGACTCGCATACCCGGATGATGTCCTGGACTATGGCCTCAAGCCATGCAACCCCCTTGCCAGTCTCTCTGGCGAGCCCCCTGGCCGGTTCGGAGAGCCGGATAGTATAGGGTTCCAGAACTTTCTGAGCCCGGTCAAGCCAGCAGGGGCTTCGGGCCCGAGCCCTCGGATCGAGATCACTCCATCCCACGAACTGATGCAGGCAGGGGGGGCCCTCCGTGGGAGAGACGCCGGCCTGTCCCCCGAGCAGCCGGCCCTGGCCCTGGCCGGCGTGGCCGCCAGCCCGAGGTTCACACTGCCCGTGCCCGGCTACGAGGGCTACCGCGAGCCGCTTTGCTTGAGCCCCGCTGCAGCAGGCGCAGCAGCCGCATTCATTTCTGACACCTTCTCCCCCTACACCTCGCCCTGCGTCTCACCCAATAACGCCGGGCCCGACGACCTGTGTCCCCAGTTTCAAAACATCCCTGCTCATTATGCCCCCCGGACCGCTCCAATAATGGCACCTCGAACCAGCCTCGCCGAGGACAGCTGCCTGGGCCGACACTCGCCCGTGCCCCGTCCGGCATCCCGCTCCTCCTCACCCGGTGCCAAGCGGAGGCATTCGTGCGCAGAGGCTTTGGTTGCTCCTCTGCCCGCAGCCGCACCCCAGCGCGCCCGGAGCCCCGCGCCACAGCCCGCGCCTCACGTGGCACCGCAGGACGACAGCATCCCCGCTGGGTACCCCCCCACGGCCGGCTCTGCTGTTCTCATGGATGCCCTCAACACCCTGGCCACCGACTCGCCCTGCGGGATCCCCTCCAAGATATGGAAGACCAGTCCTGACCCGACGCCTGTGTCCACCGCTCCGTCCAAGGCTGGCCTGGCCCGCCACATCTACCCTACTGTGGAGTTCCTGGGGCCATGTGAGCAGGGTGACTGCATCCCTCCCACCACTCGAGTGGCCACTCTCCAATCAGTCGGGCTCCTATGAGCTACGGATTGAGGTCCAACCCAAGCCCCATCACCGGGCCCACTATGAGACGGAGGGCAGCCGTGGCGCTGTCAAAGCCCCAACAGGAGGACACCCTGTGGTGCAGCTCCACGGCTACATGGAGAACAAGCCTCTGGGGCTTCAGATCTTCATTGGGACAGCAGATGAGAGGATCCTTAAGCCGCACGCCTTCTACCAAGTACACAGGATCACTGGGAAAACGGTCACCACCACGAGCTATGAGAAGATCGTAGGCAACACCAAGGTCCTGGAGATCCCCCTGGAGCCAAAGAACAACATGAGAGCCACCATCGACTGTGCAGGCATCCTGAAGCTCCGAAACGCTGACATCGAGCTGCGGAAGGGCGAGACGGACATCGGCAGGAAGAACACGCGTGTGCGCCTGGTGTTCCGCGTGCACGTCCCAGAGCCCAGTGGGCGCATCGTCTCCCTGCAGGCTGCGTCCAACCCCATCGAGTGCTCTCAGCGCTCTGCCCACGAGCTGCCCATGGTGGAGAGACAAGACATGGACAG CTGCCTGGTCTACGGGGGCCAGCAGATGATCCTCACGGGCCAGAACTTCACA GCGGAGTCCAAGGTTGTGTTCATGGAGAAGACTACAGATGGGCAGCAGATTT GGGAGATGGAAGCTACGGTGGATAAAGACAAGAGCCAGCCTAACATGCTTTT TGTTGAGATCCCCGAGTATCGGAACAAGCACATCCGCGTGCCCGTGAAAGTC AACTTCTACGTCATCAACGGAAAGAGGAAACGAAGTCAGCCACAGCACTTTA CCTACCACCCAGTCCCTGCCATCAAGACAGAGCCCAGCGATGAGTATGAACC ATCTTTGATCTGCAGCCCCGCCCATGGAGGCCTGGGGAGCCAGCCATATTACC CACAGCACCCAATGCTGGCCGAGTCCCCCTCCTGCCTTGTGGCTACCATGGCC CCCTGCCAACAGTTCCGCTCGGGGCTCTCATCCCCCGATGCTCGCTACCAACA GCAGAGCCCCGCAGCTGCCCTCTACCAGAGAAGCAAGAGCCTGAGTCCCGGC CTGCTGGGCTACCAGCAGCCGTCCCTCCTGGCAGCACCCTTGGGTCTGGCTGA TGCCCACCGCTCTGTGCTGGTGCATGCTGGTTCTCAGGGGCAGGGGCAGGGCT CCACCCTCCGACACACATCCTCGGCCAGCCAGCAGGCCTCACCCGTGATCCAC TACTCACCCACCAACCAGCAGCTTCGCGGTGGGGGTCACCAGGAGTTCCAGC ATATCATGTACTGTGAAAACTTCGGCCCCAGCTCTGCCAGGCCTGGCCCGCCT CCCATCAACCAAGGTCAGAGGCTGAGCCCGGGCGCCTACCCCACAGTCATCC AACAACAGACTGCCCCGAGCCAAAGAGCTGCCAAAAACGGACCCAGTGACC AGAAGGAAGCTCTGCCCACGGGAGTGACCGTCAAACAGGAACAGAACCTGG ACCAGACCTACCTGGATGACGTTAATGAAATCATCAGGAAGGAGTTTTCAGG ACCTCCCTCCCGAAATCAGACCTCTGGATCCggagagggcagaggaagtctgctaacatgcggtg acgtcgaggagaatcctggcccaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctgga cggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagtt catctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgcta ccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaagga cgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcga cttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagc agaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagca gaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagacccc aacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggatggacgagctgtacaagtaa

[0114] The first all capital region is the HA tag. The first lowercase region is the human NFAT1 coding sequence. The next all capital region is a connection sequence, the next lower case region includes T2A and GFP.

[0115] The following protein is the full amino acid sequence of human NFAT1 (NFATC2) — UniProt ID: Q13469, canonical isoform, 925 amino acids (SEQ ID NO: 5).SEQ ID NO: 5MNAPERQPQPDGGDAPGHEPGGSPQDELDFSILFDYEYLNPNEEEPNAHKVASPP SGPAYPDDVLDYGLKPYSPLASLSGEPPGRFGEPDRVGPQKFLSAAKPAGASGLS PRIEITPSHELIQAVGPLRMRDAGLLVEQPPLAGVAASPRFTLPVPGFEGYREPLCL SPASSGSSASFISDTFSPYTSPCVSPNNGGPDDLCPQFQNIPAHYSPRTSPIMSPRTS LAEDSCLGRHSPVPRPASRSSSPGAKRRHSCAEALVALPPGASPQRSRSPSPQPSS HVAPQDHGSPAGYPPVAGSAVIMDALNSLATDSPCGIPPKMWKTSPDPSPVSAAPSKAGLPRHIYPAVEFLGPCEQGERRNSAPESILLVPPTWPKPLVPAIPICSIPVTASL PPLEWPLSSQSGSYELRIEVQPKPHHRAHYETEGSRGAVKAPTGGHPVVQLHGY MENKPLGLQIFIGTADERILKPHAFYQVHRITGKTVTTTSYEKIVGNTKVLEIPLEP KNNMRATIDCAGILKLRNADIELRKGETDIGRKNTRVRLVFRVHIPESSGRIVSLQ TASNPIECSQRSAHELPMVERQDTDSCLVYGGQQMILTGQNFTSESKVVFTEKTT DGQQIWEMEATVDKDKSQPNMLFVEIPEYRNKHIRTPVKVNFYVINGKRKRSQPQHFTYHPVPAIKTEPTDEYDPTLICSPTHGGLGSQPYYPQHPMVAESPSCLVATM APCQQFRTGLSSPDARYQQQNPAAVLYQRSKSLSPSLLGYQQPALMAAPLSLAD AHRSVLVHAGSQGQSSALLHPSPTNQQASPVIHYSPTNQQLRCGSHQEFQHIMYC ENFAPGTTRPGPPPVSQGQRLSPGSYPTVIQQQNATSQRAAKNGPPVSDQKEVLP AGVTIKQEQNLDQTYLDDVNEIIRKEFSGPPARNQT

[0116] NFAT1 and NFATC2 are the same protein. NFAT1 is a common name for the transcription factor, while NFATC2 is its official gene symbol according to UniProt and NCBI. Both terms refer to the same protein, Nuclear factor of activated T-cells, cytoplasmic 2. This protein is a member of the NFAT family of transcription factors.

[0117] The alignment of wild and mutated NFAT1 cDNA (A) and protein (B) sequences is presented in Fig. 8.

[0118] As used herein, a nucleic acid encoding NFAT1 may include any polynucleotide that encodes a protein having functional activity comparable to a reference NFAT1 sequence, including variants and homologs exhibiting a specified degree of sequence identity. In certain embodiments, the nucleic acid has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to a reference nucleotide sequence encoding human NF ATI, such as SEQ ID NO: 1, 2, 3, or 4. Percent identity may be calculated overthe full length of the coding sequence or over a functionally relevant region thereof using a standard alignment algorithm (e.g., BLAST or Clustal). Such variants may include silent substitutions, conservative amino acid changes, or codon optimizations, provided they retain the ability to express a functionally active NFAT1 protein. The nucleic acid may further comprise regulatory elements such as promoters, enhancers, untranslated regions, and polyadenylation signals, which may or may not share identity with corresponding sequences in the native gene.

[0119] ADENO-ASSOCIATED VIRUS

[0120] Adeno-associated virus (AAV) is a non-enveloped virus comprising a capsid structure encapsulating a single- stranded DNA genome approximately 4.7 kilobases in length. The AAV genome encodes multiple functional proteins, including four replication (Rep) proteins, three capsid (Cap) proteins, and an assembly-activating protein (AAP). The genome is flanked by two inverted terminal repeats (ITRs), which form secondary structures that serve as self-priming elements for replication. Upon entry into a host cell, the single-stranded DNA genome is converted into a double- stranded replicative form by host cellular DNA polymerases. The large Rep proteins (e.g., Rep78 and Rep68) recognize and bind to the ITRs, introducing site-specific nicks and using helicase activity to unwind the terminal structures, enabling replication by host polymerases. This process facilitates the synthesis of a new DNA strand and displacement of the original strand through a mechanism referred to as single-strand displacement, thereby producing both single-stranded genomes and double- stranded replicative intermediates.

[0121] In the absence of helper virus functions, AAV is replication-deficient. However, when co-infected with helper viruses such as adenovirus or herpesvirus, AAV can replicate efficiently. Helper virus elements enhance AAV replication by promoting AAV transcription, augmenting replication processivity, inhibiting cellular DNA damage responses that suppress AAV replication, and enhancing translation of AAV-encoded proteins. For example, certain viral proteins degrade host DNA repair complexes that otherwise recognize AAV ITRs as aberrant DNA structures, while non-coding RNAs from helper viruses facilitate efficient translation of AAV mRNAs.

[0122] For productive AAV packaging, single-stranded AAV genomes must be encapsidated into pre-assembled capsids. The AAV capsid is composed of three structurally related viral proteins, VP1, VP2, and VP3, which are expressed from the cap gene and assemble in a specific stoichiometric ratio. VP3 constitutes the primary structuralframework, while VP1 contains a unique N-terminal domain with phospholipase activity required for infectivity. VP2 is non-essential for assembly or infection, but may contribute to overall capsid architecture. The surface-exposed loops of the VP proteins, particularly VP3, influence receptor binding and serotype specificity. The AAP protein, encoded in an alternative reading frame within the cap gene, mediates nucleolar localization of capsid proteins and promotes their assembly into intact capsids. During encapsidation, the small Rep proteins (e.g., Rep52 and Rep40) utilize their helicase activity to translocate the newly synthesized AAV genomes into the preformed capsids, thereby completing the production of infectious viral particles.

[0123] As the use of recombinant adeno-associated virus (rAAV) in preclinical and clinical applications continues to expand, the development of efficient and scalable production methods for rAAV has become increasingly critical. Among the commonly utilized approaches is a plasmid-based transient transfection system employing either adherent or suspension cultures of HEK293 cells, which inherently express adenoviral El gene products. In this method, rAAV particles are produced without the need for coinfection with wild-type helper viruses. Typically, three plasmids are co-transfected into the producer cells: a first plasmid comprising a gene of interest flanked by inverted terminal repeats (ITRs); a second plasmid encoding the AAV rep and cap genes; and a third plasmid encoding one or more adenoviral helper functions, such as E2A, E4, and VA RNA. Alternatively, rAAV production may be achieved using recombinant helper viruses, such as modified herpes simplex virus or Baculovirus vectors, which deliver AAV components and helper functions into the production cells via viral infection. While these systems can facilitate rAAV production, they present various challenges. In the transfection-based approach, low co-transfection efficiency can result in a limited proportion of cells receiving all necessary genetic components, necessitating the use of excessive plasmid quantities. In virus-based systems, there is a risk of contamination with replication-competent helper viruses, such as adenovirus or recombinant herpesvirus, which may copurify with the rAAV during downstream processing. These limitations underscore the need for improved systems and methods for the safe, efficient, and scalable production of rAAV vectors.

[0124] To facilitate large-scale production of recombinant adeno-associated virus (rAAV), various approaches have been explored to generate stable cell lines that incorporate essential genetic elements required for rAAV production, thereby avoiding the need for transient transfection or viral infection. In some strategies, stable producer celllines were engineered by integrating both the rAAV vector genome and the rep and cap genes into human cell lines, enabling rAAV production upon introduction of helper functions. In related approaches, packaging cell lines were developed with stable integration of only the rep and cap genes. These packaging lines could be combined with a hybrid adenovirus vector carrying the rAAV genome in place of a deleted adenoviral region, allowing delivery of both the rAAV vector genome and helper functions necessary for replication and packaging. However, certain cell types, such as HEK293, exhibit challenges due to constitutive expression of the El A gene, which can activate AAV promoters and cause cytotoxic effects when rep proteins are expressed. To address this, regulatory systems were implemented to minimize basal expression of the rep genes, such as transcriptional switches controlling all four rep isoforms in packaging cell lines. Further improvements included development of inducible cell lines containing integrated elements for E1A / E1B, the rAAV vector genome, rep and cap genes, and additional helper elements such as E2, E4, and VA RNA. While such systems enabled rAAV production without exogenous adenoviral infection, they exhibited limited stability over extended cell culture, likely due to leaky expression of cytotoxic adenoviral helper genes. These efforts underscore the complexity of creating a stable, high-yielding, and self-contained rAAV production platform suitable for large-scale manufacturing.

[0125] The AAV cap and rep sequences may be independently selected from different AAV parental sequences and be introduced into the host cell in a suitable manner known to one in the art. In certain embodiments, the rAAV particles have an AAV capsid gene from an AAV serotype comprising AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV- 6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13. In certain embodiments, the rAAV particles have an AAV capsid gene from an AAV serotype selected from the group consisting of AAV2, 3, 8, 9 and 10.

[0126] In certain embodiments, the rAAV particles comprise an AAV rep gene from an AAV serotype comprising AAV-1 , AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13. In certain embodiments, the rAAV particles have an AAV rep gene from an AAV serotype selected from the group consisting of AAV2, 3, 8, 9 and 10

[0127] DEFINITIONS

[0128] Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, includingall numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0129] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements-or, as appropriate, equivalents thereof— and that other elements can be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0130] As used herein, the term “helper virus” or “contaminating helper virus” refers to a virus used when producing copies of a helper virus -dependent viral vector, such as adeno- associated virus, which does not have the ability to replicate on its own. The helper virus is used to co-infect cells alongside the viral vector and provides the necessary proteins for replication of the genome of the viral vector. The term encompasses intact viral particles, empty capsids, viral DNA and the like. Helper viruses commonly used to produce rAAV particles include adenovirus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, and vaccinia virus.

[0131] Helper viruses include Adenovirus (AV), and herpes simplex virus (HSV), and systems exist for producing AAV in insect cells using baculovirus. Helper viruses include any virus capable of creating an allowing AAV replication. AV is a nonenveloped nuclear DNA virus with a double-stranded DNA genome of approximately 36 kb. AV is capable of rescuing latent AAV provirus in a cell, by providing Ela, Elb55K, E2a, E4orf6, and VA genes, allowing AAV replication and encapsidation. HSV is a family of viruses that have a relatively large double-stranded linear DNA genome encapsidated in an icosahedral capsid, which is wrapped in a lipid bilayer envelope. HSV are infectious and highly transmissible. The following HSV-1 replication proteins were identified as necessary for AAV replication: the helicase / primase complex (UL5, UL8, and UL52) and the DNA binding protein ICP8 encoded by the UL29 gene, with other proteins enhancing the helper function.

[0132] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0133] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. A “tissuespecific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0134] A “protelomerase” target sequence is any DNA sequence whose presence in a DNA template allows for its conversion into a closed linear DNA by the enzymatic activity of protelomerase. In other words, the protelomerase target sequence is required for the cleavage and religation of double stranded DNA by protelomerase to form covalently closed linear DNA. Typically, a protelomerase target sequence comprises any perfect palindromic sequence i.e. any double-stranded DNA sequence having two-fold rotational symmetry, also described herein as a perfect inverted repeat. The length of the perfect inverted repeat differs depending on the specific organism. In Borrelia burgdorferi, the perfect inverted repeat is 14 base pairs in length. In various mesophilic bacteriophages, the perfect inverted repeat is 22 base pairs or greater in length. Also, in some cases, e.g. E. coli N15, the central perfect inverted palindrome is flanked by inverted repeat sequences, i.e. forming part of a larger imperfect inverted palindrome.

[0135] As used herein, the terms “recombinant AAV (rAAV) vector” or “gene delivery vector” refer to a virus particle that functions as a nucleic acid delivery vehicle, and which comprises the vector genome (e.g., viral DNA [vDNA]) packaged within an AAV capsid. Alternatively, in some contexts, the term “vector” may be used to refer to the vector genome / vDNA alone.

[0136] A “rAAV vector genome” or “rAAV genome” is an AAV genome (i.e., vDNA) that comprises one or more heterologous nucleotide sequences. rAAV vectors generally require only the 145 base terminal repeat(s) (TR(s)) in cis to generate virus. Typically, the rAAV vector genome will only retain the minimal TR sequence(s) so as to maximize the size of the transgene that can be efficiently packaged by the vector. The structural and non- structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell). The rAAV vector genome comprises at least one TR sequence (e.g., AAV TR sequence, synthetic, or other parvovirus TR sequence), optionally two TRs (e.g., two AAV TRs), which typically will be at the 5' and 3' ends of the heterologous nucleotide sequence(s), but need not be contiguous thereto. The TRs can be the same or different from each other.

[0137] An “AAV terminal repeat” or “AAV TR” may be from any AAV, including but not limited to serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, A AV-8, A AV-9, AAV- 10, AAV-11 , AAV-12, AAV- 13, or any other AAV now known or later discovered. The AAV terminal repeats need not have a wild-type terminal repeat sequence (e.g., a wild-type sequence may be altered by insertion, deletion, truncation or missense mutations), as long as at least one of the terminal repeat mediates the desired functions, a functional TR, e.g., replication, virus packaging, integration, and / or provirus rescue, and the like. One of skill in the art understands to choose a Rep protein that is functional for replication of the functional TR.

[0138] A “transgene” is used herein to conveniently refer to a polynucleotide or a nucleic acid that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene that encodes a polypeptide or protein. Suitable trans genes, for example, for use in gene therapy are well known to those of skill in the art. For example, the vectors described herein can deliver transgenes and uses that include, but are not limited to, those described in U.S. Pat. Nos. 6,547,099; 6,506,559; and 4,766,072; Published U.S. Application No. 20020006664; 20030153519; 20030139363; and published PCT applications of WO 01 / 68836 and WO 03 / 010180, and e.g. miRNAs and other transgenes of WO2017 / 152149; each of which are hereby incorporated herein by reference in their entirety.

[0139] The term “tropism” as used herein refers to preferential entry of the virus into certain cells or tissues, optionally followed by expression (e.g., transcription and,optionally, translation) of a sequence(s) carried by the viral genome in the cell, e.g., for a recombinant virus, expression of a heterologous nucleic acid(s) of interest.

[0140] The term “variant,” when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, “allelic,” “splice,” “species,” or “polymorphic” variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention are variants of wild type gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

[0141] One skilled in the art will appreciate that, for this and other processes and methods disclosed herein, the functions performed in the processes and methods may be implemented in differing order. Furthermore, the outlined steps and operations are only provided as examples, and some of the steps and operations may be optional, combined into fewer steps and operations, or expanded into additional steps and operations without detracting from the essence of the disclosed embodiments.

[0142] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0143] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0144] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art wouldunderstand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0145] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0146] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0147] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0148] All references recited herein are incorporated herein by specific reference in their entirety.References:1. CDC: Center for Disease Control and Prevention. Arthritis / Types / Osteoarthritis. cdc.gov / arthri tis / osteoarthritis / ?CDC_AAref_Val= cdc.gov / arthritis / types / osteoarthritis.htm2. Buckwaiter JA, Martin JA. Osteoarthritis. Advanced drug delivery reviews. 2006 May 20;58(2):150-67. pub 2006 / 03 / 15.3. Lawrence RC, Felson DT, Helmick CG, Arnold LM, Choi H, Deyo RA, Gabriel S, Hirsch R, Hochberg MC, Hunder GG, Jordan JM, Katz JN, Kremers HM, Wolfe F. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis and rheumatism. 2008 Jan;58(l):26-35. pub 2008 / 01 / 01.4. Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis and rheumatism. 2012 Jun;64(6): 1697-707. pub 2012 / 03 / 07.5. Lotz M, Loeser RF. Effects of aging on articular cartilage homeostasis. Bone. 2012 Aug;51(2):241-8. Epub 2012 / 04 / 11.6. Scanzello CR, Goldring SR. The role of synovitis in osteoarthritis pathogenesis.Bone. 2012 Aug;51(2):249-57. Epub 2012 / 03 / 06.7. Alliston T, Hernandez CJ, Findlay DM, Felson DT, Kennedy OD. Bone marrow lesions in osteoarthritis: What lies beneath. Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 2018 Jul;36(7): 1818-25. Epub 2017 / 12 / 22.8. Zhu S, et al. X. Subchondral bone osteoclasts induce sensory innervation and osteoarthritis pain. The Journal of clinical investigation. 2019 Mar 1; 129(3): 1076-93. Epub 2018 / 12 / 12.9. Le Graverand-Gastineau MP. Disease modifying osteoarthritis drugs: facing development challenges and choosing molecular targets. Curr Drug Targets 2010; 11:528-35.10. Xanthoudakis S, Viola J, Shaw K, Luo C, Wallace J, Bozza P, Luk D, Curran T, Rao A. An enhanced immune response in mice lacking the transcription 5 factor NF ATI . Science. 1996; 272(5263): 892-5.11. Kwon HK, Kim GC, Hwang JS, Kim Y, Chae CS, Nam JH, Jun CD, Rudra D, Surh CD, Im SH. Transcription factor NFAT 1 controls allergic contact hypersensitivity through regulation of activation induced cell death program. Sci Rep. 2016; 6: 19453.12. Zhang M, Lu Q, Budden T, Wang J. NFAT1 protects articular cartilage against osteoarthritic degradation by directly regulating transcription of specific anabolic and catabolic genes. Bone & joint research. 2019; 8(2):90-100. Epub 2019 / 03 / 28.13. Wang J, Gardner BM, Lu Q, Rodova M, Woodbury BG, Yost JG, Roby KF, Pinson DM, Tawfik O, Anderson HC. Transcription factor Nfatl deficiency causes osteoarthritis through dysfunction of adult articular chondrocytes. J Pathol. 2009; 219(2): 163-72. Epub 2009 / 06 / 16.14. Rodova M, Lu Q, Li Y, Woodbury BG, Crist JD, Gardner BM, Yost JG, Zhong XB, Anderson HC, Wang J. Nfatl regulates adult articular chondrocyte function through its age-dependent expression mediated by epigenetic histone methylation. Journal of bone and mineral research 2011; 26(8): 1974-86. Epub 2011 / 04 / 01.15. Zhang M, Lu Q, Egan B, Zhong XB, Brandt K, Wang J. Epigenetically mediated spontaneous reduction of NFAT1 expression causes imbalanced metabolic activities of articular chondrocytes in aged mice. Osteoarthritis and cartilage 2016; 24(7): 1274-83. Epub 2016 / 02 / 24.16. Wang J, Lu Q, Mackay MJ, Liu X, Feng Y, Burton DC, Asher MA. Spontaneous Facet Joint Osteoarthritis in NF ATI-Mutant Mice: Age-Dependent Histopathologic Characteristics and Molecular Mechanisms. The Journal of bone and joint surgery American volume. 2022; 104(10):928-40. Epub 20220215.17. Ortved KF, Begum L, Mohammed HO, Nixon AJ. Implantation of rAAV5-IGF-I transduced autologous chondrocytes improves cartilage repair in full-thickness defects in the equine model. Molecular therapy : the journal of the American Society of Gene Therapy. 2015 Feb;23(2):363-73. Epub 20141014.18. Evans CH, Ghivizzani SC, Robbins PD. Osteoarthritis gene therapy in 2022. Curr Opin Rheumatol. 2023; 35(l):37-43. Epub 20221109.19. Takahashi H, Kato K, Miyake K, Hirai Y, Yoshino S, Shimada T. Adenoassociated virus vector-mediated anti-angiogenic gene therapy for collagen-induced arthritis in mice. Clinical and experimental rheumatology. 2005; 23(4).20. Payne KA, Lee HH, Haleem AM, Martins C, Yuan Z, Qiao C, Xiao X, Chu CR.Single intra-articular injection of adeno-associated virus results in stable and controllable in vivo transgene expression in normal rat knees. Osteoarthritis and cartilage 2011; 19(8): 1058-65. pub 2011 / 05 / 17.21. Wang G, [vans CH, Benson JM, Hutt JA, Seagrave J, Wilder JA, Grieger JC, Samulski RJ, Terse PS. Safety and biodistribution assessment of scrAAV2.5IL-lRa administered via intra- articular injection in a mono-iodoacetate45 induced osteoarthritis rat model. Mol Ther Methods Clin Dev. 2016;3: 15052. pub20160113.22. Kellgren JH, Lawrence JS. Radiological assessment of osteo-arthrosis. 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Claims

CLAIMS1. A composition comprising an adeno-associated virus (AAV) vector containing a nucleic acid sequence encoding Nuclear Factor of Activated T Cells 1 (NFAT1) protein.

2. The composition of claim 1, wherein the AAV vector is selected from AAV serotype 5, other AAV serotypes, recombinant AAV, or engineered AAV capsid variants.

3. The composition of claim 1, wherein the nucleic acid sequence encoding NFAT1 is constitutively active NF ATI or wild-type NFAT1 without any functional mutations or variations.

4. The composition of claim 1, wherein the nucleic acid sequence encoding NFAT1 comprises a hemagglutinin epitope tag.

5. The composition of claim 1, wherein at least one AAV vector comprises a viral genome concentration of about 2xlO10viral genomes.

6. The composition of claim 1, wherein the NFAT1 is human NFAT1.

7. The composition of claim 6, wherein the nucleic acid includes SEQ ID NO: 1 or SEQ ID NO: 2, or at least 80% identity therewith.

8. The composition of claim 1, wherein the NFAT1 is mouse NFAT1.

9. The composition of claim 8, wherein the nucleic acid includes SEQ ID NO: 3 or SEQ ID NO: 4, or at least 80% identity therewith.

10. The composition of claim 1, wherein the AAV vector is AAV serotype 5.

11. A pharmaceutical composition comprising: the composition of claim 1 ; and a pharmaceutically acceptable carrier.

12. The method of claim 11, wherein the pharmaceutically acceptable carrier is suitable for intra- articular injection or local tissue injection.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutically acceptable carrier is sterile saline.

14. A method of providing a therapy to a joint, comprising: providing the composition having Nuclear Factor of Activated T Cells 1 (NAFT1) or a nucleic acid encoding for NAFT1; and administering the composition to a joint to provide a therapy to the joint of a subject.

15. The method of claim 1 , wherein the therapy is: a preventative treatment to inhibit onset of a joint disease; or a treatment after onset of the joint disease to attenuate, halt, or reverse the progression of a joint disease.

16. The method of claim 15, wherein the joint disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, joint contracture, osteochondroma of the joint condyle, inflammatory synovitis, psoriatic arthritis juvenile idiopathic arthritis, reactive arthritis, ankylosing spondylitis, crystal-induced arthropathies, autoimmune polyarthritis, and combinations thereof.

17. The method of claim 14, wherein the composition includes NAFT1 protein in a controlled release formulation.

18. The method of claim 14, wherein the NAFT1 composition includes a nucleic acid encoding for NAFT1.

19. The method of claim 18, wherein the NFAT1 is human NFAT1.

20. The method of claim 19, wherein the nucleic acid includes SEQ ID NO: 1 or SEQ ID NO: 2, or at least 80% identity therewith.

21. The method of claim 14, wherein the joint is selected from the group consisting of knee, hip, ankle, shoulder, elbow, wrist, spinal facet joint, and other synovial joints.

22. The method of claim 14, wherein the subject has NF ATI deficiency in joint tissues.

23. The method of claim 22, wherein the NF ATI deficiency comprises NFAT1 expression levels less than 80% of normal control le vels.

24. The method of claim 14, wherein the subject has a deleterious mutation in the NF ATI DNA binding domain.

25. The method of claim 24, wherein the deleterious mutation is selected from the group consisting of Q571H and R572G amino acid substitutions.

26. The method of claim 14, wherein the composition comprises an adeno- associated virus (AAV) vector containing a nucleic acid sequence encoding NF ATI protein.

27. The method of claim 26, wherein the AAV vector is selected from AAV serotype 5, other AAV serotypes, recombinant AAV, or engineered AAV capsid variants.

28. The method of claim 26, wherein the nucleic acid sequence encoding NFAT1 comprises a hemagglutinin epitope tag.

29. The method of claim 16, wherein the administrating is gene delivery.

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