Treatment of osteoarthritis

Abstract

The present invention is applicable, at least in part, to gene therapy treatment of diseases associated with cartilage loss, such as osteoarthritis, including administration of FGF-18 gene therapy to the intra-articular space of a joint to promote cartilage thickening.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of 35 U.S.C. § 119(e). Provisional Application No. 63 / 404,294, filed September 7, 2022, is entitled "TREATMENT FOR OSTEOARTHRITIS," the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (R087270002WO00-SEQ-KVC.xml; size: 29,526 bytes; and creation date: August 31, 2023) are incorporated herein by reference in their entirety.

[0003] FIELD OF THE INVENTION The present invention relates, at least in part, to gene therapy treatments for diseases associated with cartilage loss, such as osteoarthritis, including administration of FGF-18 gene therapy to the intra-articular space of a joint to promote cartilage thickening. [Background technology]

[0004] Summary of the Invention Growth factors (e.g., fibroblast growth factor 18 (FGF-18)) are proteins that regulate many biological functions, including cell proliferation, migration, survival, differentiation, and tissue deposition, turnover, and maintenance. Growth factor levels in tissues generally decline with age. This decline is thought to be due, at least in part, to reduced gene expression mediated by one of many mechanisms of genetic silencing, a general decrease in cell density (hypothesized as a positive feedback loop with declining growth factor levels), decreased translation efficiency and effectiveness, and / or an increase in the proportion of senescent cells. Cell density within tissues correlates with the composition and physicochemical properties of the tissue. At least in part, the decline in growth factor concentrations is associated with disease, tissue atrophy, and tissue degeneration. Osteoarthritis is a disease primarily of aging and progressive cartilage loss, leading to debilitating pain and loss of function. This process occurs in humans, nonhuman primates, and other animals, such as horses, dogs, cats, and pigs. Although some genetic factors have been hypothesized as contributing factors, age is by far the strongest predictor of osteoarthritis prevalence and progression.

[0005] Currently, there is no approved treatment that can stop or reverse cartilage loss in osteoarthritis, but recent clinical studies have demonstrated that exogenous FGF-18 protein can promote and increase cartilage thickness in a dose-dependent manner compared to placebo.For example, intra-articular FGF-18 protein injection has been demonstrated to reduce the rate of cartilage loss and, in some treatment regimens, increase cartilage thickness compared to placebo in controlled randomized clinical trials.Thus, protein injections as a means of supplementing, replacing, or replacing growth factors have previously demonstrated efficacy in reversing disease progression, but the treatment paradigm remains challenging, requiring up to 12 injections per year for bilateral treatment of knee osteoarthritis, with a treatment regimen of up to three injections per week every six months.

[0006] Provided herein are compositions and methods for treating cartilage disorders in a subject using gene therapy comprising a nucleic acid encoding a fibroblast growth factor 18 (FGF-18) polypeptide. As used herein, a "cartilage disorder" refers to a condition in which cartilage thickening, growth, regeneration, and / or repair is beneficial, and / or in which there is cartilage loss, degeneration, and / or damage. Cartilage disorders include, but are not limited to, degenerative diseases of the cartilage and meniscus, meniscal tears, focal cartilage lesions, and osteoarthritis (e.g., secondary osteoarthritis). The subject may be a mammal. The subject may be a human, dog, cat, cow, sheep, goat, horse, or other animal. The nucleic acid may be under the control of an optimal promoter and / or regulatory sequence. The nucleic acid may also encode a secretory signal. In some embodiments, the gene therapy is administered by local or intra-articular administration. In some embodiments, the gene therapy is in an optimal dosage range.

[0007] It is also recognized that it may be beneficial to administer gene therapy locally and in a manner such that the dose correlates with the amount of target tissue, e.g., at the time of administration. Joint surface area has been commonly characterized in a wide range of animals. As an example, the total articular surface area of ​​the knee cartilage plate is 102-163 cm in adult humans. 2 range, with a mean of 121 and a standard deviation of 14.1 cm 2 It has been demonstrated that the patella has an average articular surface area of ​​12 cm 2 The total volume of knee cartilage in an average adult human is estimated to be approximately 23.3 cm 3 Dhollander et al., using 3D MRI analysis, found that the average knee cartilage volume of male and female beagles weighing 7.2 to 17.1 kg was 319.7 to 647.3 mm. 3It has been demonstrated that the thickness of cartilage in rabbits, sheep, dogs, goats, and horses is in the range of 0.3 mm, 0.4-0.5 mm, 0.6-1.3 mm, 0.7-1.5 mm, and 1.5-2.0 mm, respectively. Therefore, by determining the cartilage volume, thickness, or surface area and appropriately adjusting the dose of gene therapy, optimal dosages can be provided for regenerative gene therapy aimed at halting or reversing cartilage loss, such as in osteoarthritis. Any one of the compositions and methods provided herein may be or can result in the administration of such optimal doses. Also provided herein are methods for determining such optimal doses, as well as compositions for gene therapy at such optimal doses.

[0008] Furthermore, clinical data demonstrates that when treatment is stopped, cartilage gain is reversed and cartilage loss resumes.Therefore, in order to reduce the progressive cartilage loss of osteoarthritis and prevent osteoarthritis-related complications, optimal and / or more durable treatment approaches are needed.The compositions and methods provided herein can be such optimal and / or durable treatments.

[0009] Aspects of the present disclosure relate to genetic constructs that include the following: a nucleic acid encoding an FGF-18 polypeptide, a promoter, and, optionally, regulatory elements (such as post-translational regulatory elements) (e.g., from the WPRE sequence family, encoding a polyA signal, an enhancer, a translation termination sequence, and a sequence that promotes binding of one or more DNA-binding proteins).

[0010] In some embodiments of any one of the compositions or methods provided herein, the FGF-18 polypeptide is mammalian, such as a human or non-human primate. In some embodiments of any one of the compositions or methods provided herein, the FGF-18 polypeptide is human, canine, feline, bovine, ovine, caprine, or equine FGF-18 polypeptide. In some embodiments of any one of the compositions or methods provided herein, the FGF-18 polypeptide is encoded by the sequence of AF075292, AB007422, AF211188, BT019570, BTo19571, CH471062, BC006245, AY358811, NM_003862.2, or a portion thereof. In some embodiments of any one of the compositions or methods provided herein, the FGF-18 polypeptide is encoded by at least a portion of the sequence of gene ID 8817 from HGNC:3674.

[0011] In some embodiments of any one of the compositions or methods provided herein, the nucleic acid encodes another protein or a portion thereof. In some embodiments of any one of the compositions or methods provided herein, the nucleic acid further encodes at least one auxiliary and / or regulatory sequence that facilitates expression. In some embodiments of any one of the compositions or methods provided herein, the nucleic acid further encodes at least one intron from another gene sequence (e.g., human).

[0012] In some embodiments of any one of the compositions or methods provided herein, the promoter is a CMV promoter, a CMV promoter with an MVM1 intron, a CAG promoter, an EF1α promoter, a UBC promoter, a CBh promoter, an MSCV promoter, an hPGK promoter, an SFFV promoter, or an SV40 promoter. In some embodiments of any one of the compositions or methods provided herein, the promoter is a constitutive promoter (e.g., a mammalian promoter). In some embodiments of any one of the compositions or methods provided herein, the promoter drives expression in at least one cell type that is at least transiently present in the joint or tissue surrounding the joint. In some embodiments of any one of the compositions or methods provided herein, the promoter is an inducible promoter. In some embodiments of any one of the compositions or methods provided herein, the inducible promoter up- or down-regulates expression in response to an external or internal stimulus (e.g., inflammation, heat, light, stress, or administration of steroids, tetracycline, antibiotics, rapamycin, ganciclovir, acyclovir), or is induced by up- or down-regulated heat, ROS, NOS, or cytokine release. In some embodiments of any one of the compositions or methods provided herein, the promoter is a circadian rhythmic or cycling promoter (e.g., one that changes its activity level by at least 2%, 5%, or 10% over a period of hours to months, including weeks), in response to cortisol levels, menstrual cycle, circadian cycle, exercise level, etc. In some embodiments of any one of the compositions or methods provided herein, the promoter is a tissue-specific promoter. In some embodiments of any one of the compositions or methods provided herein, the promoter is a chondrocyte-specific promoter. In some embodiments of any one of the compositions or methods provided herein, the promoter is a synoviocyte-specific promoter.

[0013] In some embodiments of any one of the compositions or methods provided herein, the regulatory sequence element is one or more of Argc1, Col2a1, Col6a1, Col10a1, Col11a2, Matn1, Gdf5, IL1B, and Prx1. In some embodiments of any one of the compositions or methods provided herein, the regulatory element is one or more of Adam12, α-SMA, Col1a1, Col1a2, FGF18, FGF10, FGF-2, FoxD1, Fsp1, FoxJ1, Gli1, PDGFa, PDGFb, PDFR-α, PDGFR-β, Twist2, and TCF4. In some embodiments of any one of the compositions or methods provided herein, the regulatory element is an intron, a portion of an intron, a post-translational regulatory element, an enhancer, a repressor, a genetic sequence that can form a multidimensional structure with a portion of the genome or gene construct itself, or a gene regulatory element in general.

[0014] Aspects of the present disclosure relate to compositions comprising any of the genetic constructs described herein, wherein the nucleic acid is contained in a delivery vector. In some embodiments of any one of the compositions or methods provided herein, the delivery vector is a polyelectrolyte complex or polypeptide, a virus (e.g., an adeno-associated virus (e.g., AAV2), an adenovirus, a lentivirus, a herpes simplex virus, a poxvirus, a measles virus, an alphavirus, or a mimivirus), a polymeric carrier, or a lipid carrier, optionally conjugated to a ligand (e.g., to increase selectivity and / or specificity and / or target a tissue or cell type) (e.g., at least a portion of an Fc fragment, at least a portion of a cytokine, at least a portion of a growth factor, at least a portion of a growth factor receptor, at least a portion of a molecule that increases the residence time of a growth factor, or at least a portion of a molecule that increases binding affinity to a receptor).

[0015] In some embodiments of any one of the compositions or methods provided herein, the viral carrier is from a virus with a synthetic or hybrid capsid. In some embodiments of any one of the compositions or methods provided herein, the viral carrier is from a virus with a natural or synthetic capsid, and is optionally bound to a ligand. In some embodiments of any one of the compositions or methods provided herein, the viral carrier is composed of multiple virus types. In some embodiments of any one of the compositions or methods provided herein, at least 5% of the capsids of the viral carrier are complete capsids.

[0016] In some embodiments of any one of the compositions or methods provided herein, the delivery vector is a polymer, optionally linked to a ligand. In some embodiments of any one of the compositions or methods provided herein, the delivery vector is a polyelectrolyte complex comprising at least one polymer, optionally linked to a ligand. In some embodiments of any one of the compositions or methods provided herein, the polymer is a cationic polymer, an anionic polymer, and / or a non-ionic polymer.

[0017] In some embodiments of any one of the compositions or methods provided herein, the delivery vector comprises chitosan, polyethyleneimine, or a polypeptide with an overall positive charge. In some embodiments of any one of the compositions or methods provided herein, the ligand targets any one of the tissues or cell types described herein. In some embodiments of any one of the compositions or methods provided herein, the delivery vector is a lipid nanoparticle or liposome, optionally conjugated to a ligand. In some embodiments of any one of the compositions or methods provided herein, the lipid carrier comprises up to 60% cholesterol by molar ratio. In some embodiments of any one of the compositions or methods provided herein, the lipid carrier comprises up to 80% cationic or ionizable lipid by molar ratio. In some embodiments of any one of the compositions or methods provided herein, the lipid carrier comprises a glyceride, polyglyceryl and / or polyoxyglyceride. In some embodiments of any one of the compositions or methods provided herein, the lipid carrier comprises an oil / water nanoemulsion or an oil / water microemulsion. In some embodiments of any one of the compositions or methods provided herein, the lipid carrier is a nanocapsule, a self-nanoemulsifying or self-microemulsifying system, a micelle, a lipid-polymer hybrid, or comprises a biopolymer or biomimetic.

[0018] In some embodiments of any one of the compositions or methods provided herein, the ligand comprises a peptide, protein, polysaccharide, small molecule, or a combination thereof (e.g., for targeting, increasing uptake, or increasing in vivo residence time). Aspects of the present disclosure relate to a method of administering any one of the gene constructs or compositions described herein to a subject in need thereof. In some embodiments of any one of the methods provided herein, the subject has or is at risk of cartilage damage, cartilage loss, and / or is in need of cartilage regeneration. In some embodiments of any one of the methods provided herein, the subject has or is at risk of osteoarthritis. In some embodiments of any one of the methods provided herein, the subject has meniscus tear.

[0019] In some embodiments of any one of the methods provided herein, the genetic construct or composition is administered locally or intra-articularly. In some embodiments of any one of the methods provided herein, the genetic construct or composition is administered to the meniscus of the joint. In some embodiments of any one of the methods provided herein, the subject is a human subject. In some embodiments of any one of the methods provided herein, the subject is a horse, dog, or cat.

[0020] In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is a dose calculated or adjusted using the subject's average joint size (e.g., average joint surface area or average joint volume), average articular cartilage volume, body weight, age, and / or disease state, or representative values ​​of the subject, compared to another subject, such as of a different species. In another aspect, a composition is provided that comprises (or is capable of providing) one or more of any one of the doses provided herein.

[0021] In some embodiments of any one of the methods provided herein, a metric representative of the subject is determined for the subject, and optionally, the method further comprises determining the subject's average joint size (e.g., average joint surface area or average joint volume), average articular cartilage volume, body weight, age, and / or disease state. In some embodiments of any one of the methods provided herein, the metric representative of the subject is of another subject representative of the subject, e.g., a healthy subject or another subject of the same species, and optionally, the method further comprises determining the other subject's average joint size (e.g., average joint surface area or average joint volume), average articular cartilage volume, body weight, age, and / or disease state.

[0022] In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to body weight. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to age. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to body weight and age. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to a disease state. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using the average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to the disease state and body weight. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using the average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to the disease state and age. In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is calculated or adjusted or made using the average expression ratio (e.g., based on the promoter and / or regulatory element of the construct) (such as the average promoter ratio and / or regulatory element ratio).

[0023] In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is 2x10 9 ~1x10 11In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 1x10 genome copies / joint (rat) or an equivalent amount as defined herein, e.g., a human, horse, dog, or cat equivalent amount. 10 ~6x10 11 In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 5x10 genome copies / joint (human) or an equivalent amount as defined herein, e.g., an equine, canine, or feline equivalent amount. 11 ~3x10 13 In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 5x10 genome copies / joint (human) or an equivalent amount as defined herein, e.g., an equine, canine, or feline equivalent amount. 11 ~3.5x10 12 In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 1x10 genome copies / joint (dog) or an equivalent amount as defined herein, e.g., a human, horse, or cat equivalent amount. 10 ~7x10 10 In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 1x10 genome copies / joint (dog) or an equivalent amount as defined herein, e.g., a human, horse, or cat equivalent amount. 12 ~8.5x10 13 In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 3x10 genome copies / joint (horse) or an equivalent amount as defined herein, e.g., a human, dog, or cat equivalent amount. 10 ~2x10 12 In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 1x10 genome copies / joint (horse) or an equivalent amount as defined herein, e.g., a human, dog, or cat equivalent amount. 8 ~6.5x10 9In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is 5x10 genome copies / joint / kg (human) or an equivalent amount as defined herein, e.g., an equine, canine, or feline equivalent amount. 9 ~3.5x10 11 In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is 2x10 genome copies / joint / kg (human) or an equivalent amount as defined herein, e.g., an equine, canine, or feline equivalent amount. 4 Genome copies / joint / kg ~6.3x10 13 In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is 3.8x10 genome copies / kg or an equivalent amount as defined herein. 4 Genome copies / joint / kg ~4.7x10 13 In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is 4.5x10 genome copies / kg (human), or an equivalent amount as defined herein. 5 Genome copies / joint / kg ~6.3x10 13 In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is 3.8x10 genome copies / kg (horse), or an equivalent amount as defined herein. 4 Genome copies / joint / kg ~1.2x10 13 In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is 2x10 genome copies / kg dog, or an equivalent amount as defined herein. 4 Genome copies / joint / kg ~4.3x10 12 In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is 5x10 genome copies / kg of cat, or an equivalent amount as defined herein. 7 Genome copies / knee, hip, or shoulder joint ~2x10 14In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 5x10 genome copies / knee joint, hip or shoulder joint (human), or an equivalent amount as defined herein. 7 Genome copies / knee, hip, or shoulder joint ~5x10 13 In some embodiments of any one of the compositions or methods provided herein, the dose of the genetic construct is 1x10 genome copies / knee, hip, or shoulder joint (horse), or an equivalent amount as defined herein. 5 Genome copies per knee, hip, or shoulder joint ~1x10 12 In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 5x10 genome copies / knee joint, hip joint, or shoulder joint (dog), or an equivalent amount as defined herein. 4 Genome copies / knee, hip, or shoulder joint ~7x10 11 In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 4x10 genome copies / knee, hip, or shoulder joint (cat), or an equivalent amount as defined herein. 7 Genome copies / knee, hip, or shoulder joint ~2x10 14 In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 5x10 genome copies / knee joint, hip joint, or shoulder joint, or an equivalent amount as defined herein. 8 Genome copies per knee, hip, or shoulder joint ~1x10 13 In some embodiments of any one of the compositions or methods provided herein, the dose of the gene construct is 5x10 genome copies / knee joint, hip joint, or shoulder joint, or an equivalent amount as defined herein. 8 Genome copies / knee, hip, or shoulder joint ~8x10 12 genome copies / knee joint, hip or shoulder joint, or an equivalent amount as defined herein.

[0024] Aspects of the present disclosure relate to methods that include determining or adjusting the dose based on the subject's average joint size (e.g., average joint surface area or average joint volume), average joint cartilage volume, body weight, age, and / or disease state, or using representative values ​​for the subject, compared to another subject, such as a different species.

[0025] In some embodiments of any one of the methods provided herein, a metric representative of the subject is determined for the subject, and optionally, the method further comprises determining the subject's average joint size (e.g., average joint surface area or average joint volume), average articular cartilage volume, body weight, age, and / or disease state. In some embodiments of any one of the methods provided herein, the metric representative of the subject is of another subject representative of the subject, e.g., a healthy subject or another subject of the same species, and optionally, the method further comprises determining the other subject's average joint size (e.g., average joint surface area or average joint volume), average articular cartilage volume, body weight, age, and / or disease state.

[0026] In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to body weight. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to age. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to body weight and age. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to a disease state. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using the average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to the disease state and body weight. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using the average joint size (e.g., average joint surface area or average joint volume) or average joint cartilage volume in relation to the disease state and age. In some embodiments of any one of the compositions or methods provided herein, the dose is calculated or adjusted or made using the average expression ratio (e.g., based on the promoters and / or regulatory elements of the construct) (such as the average promoter ratio and / or regulatory element ratio).

[0027] Aspects of the present disclosure relate to compositions comprising any of the genetic constructs provided herein in any one of the doses provided herein, and a pharmaceutically acceptable carrier. Aspects of the present disclosure relate to a genetic construct as described in any one of the Examples herein. [Brief explanation of the drawings]

[0028] The accompanying drawings are not intended to be drawn to scale. The drawings are for illustrative purposes only and are not required to enable disclosure. For clarity, not every component is labeled in every drawing. In the drawings:

[0029] [Figure 1] Figure 1 shows the cytocompatibility and transfection efficiency of AAV2 vectors with primary human chondrocytes and synoviocytes (cytocompatibility: pTime>0.05, pDose>0.05; transfection efficiency: pTime<0.05, pDose<0.05; ANOVA). [Figure 2] FIG. 2 shows the proliferation of human chondrocytes stimulated by rhFGF18 protein (left) and AAV2-FGF18 (right) (protein: pDose<0.05, pTime<0.05, ANOVA; AAV: PDose<0.05, ANOVA).

[0030] [Figure 3] Figure 3 shows the effects of AAV2-FGF18 gene therapy and rhFGF18 protein on chondrocytes in transwell culture with synoviocytes compared with the AAV2-GFP negative control (ANOVA, p<0.05; Tukey's pairwise comparison groups indicated by letter codes, pcrit=0.05). [Figure 4] Figure 4 shows the set of genes up- and down-regulated by chondrocytes treated with AAV2-FGF18 compared to the complete set of genes up- and down-regulated by chondrocytes treated with rhFGF18 protein. *ESM1 was up-regulated by AAV2-GFP control over PBS, although by less than 3-fold.

[0031] [Figure 5]Figure 5 shows the effects of AAV2-FGF18 gene therapy (left) and rhFGF18 protein treatment (right) on gene expression in human primary chondrocytes compared to PBS treatment (* indicates statistical significance using RESeq2, pcrit=0.01). [Figure 6] Figure 6 shows, from left to right: 1) AAV2-nLuc bioluminescence reporter analysis, 4 months after the first dose, 2) AAV2-FGF18 active group hFGF18 antibody immunohistochemical staining, 3) AAV2-GFP control hFGF18 antibody immunohistochemical staining, 4) AAV2-GFP control hFGF18 antibody immunohistochemical staining without primary antibody (Ab) as a negative control.

[0032] [Figure 7] Figure 7 shows Safranin-O stained sagittal tissue sections of rat knee joints injected with AAV2-GFP (top), AAV2-FGF18 (middle), and rhFGF18 protein (bottom), indicating the anatomical locations of cartilage thickness measurements (semitransparent white boxes). The tibia locations were selected as six evenly spaced rectangles of equal width, while the meniscus apex locations were obtained as a single width at the thickest point of the apex.

[0033] [Figure 8] Figure 8 shows the mean body weight-normalized cartilage thickness by anatomical location after administration of the rhFGF18 protein positive control, three doses of AAV2-FGF18 (Dose 1 = 2x109 vg / joint, Dose 2 = 1x1010 vg / joint, Dose 3 = 1x1011 vg / joint), and the AAV2-GFP negative control. All groups within anatomical location reached statistical significance versus the AAV2-GFP negative control (ANOVA, p<0.05; Tukey's pairwise comparison groups indicated by letter codes, p=0.05).

[0034] [Figure 9]Figure 9 shows joint diameters at the 1-month and 2-month study time points compared to baseline with no injections; AAV2-FGF18 single injection, AAV2-GFP control single injection, and rhFGF18 protein biweekly injections (ANOVA, p<0.05; Tukey's pairwise comparison groups indicated by letter codes, p=0.05; dashed red lines indicate 99% confidence intervals of the mean of non-operated controls). [Figure 10] FIG. 10 shows gene expression changes following administration of rhFGF18 (left) and AAV2-FGF18 (right) compared to the PBS negative control.

[0035] [Figure 11] Figure 11 shows the hyaline cartilage- and fibrocartilage-related pathways affected by AAV2-FGF18 administration, as determined by RNA-Seq analysis. Direct hyaline cartilage-promoting effects observed in RNA-Seq analysis: lubricin (PRG4) and collagen 2 (COL2A1). Indirect hyaline cartilage-promoting effects: ADAMTS1, 5, 15, and matrix metalloproteinase 2 (MMP2). Direct fibrocartilage-inhibiting effects: collagen 1 (COL1A1) and lysine 6-oxidase (LOX). Indirect fibrocartilage-inhibiting effects: PTX3 and IGF1. [Figure 12] FIG. 12 shows cartilage thickness normalized by body weight by anatomical location.

[0036] Detailed Description of the Invention Provided herein are compositions and methods for preventing, reducing, or reversing cartilage loss, such as in osteoarthritic joints. More specifically, provided herein are compositions and methods for preventing, reducing, or reversing cartilage loss, such as in osteoarthritic joints, by administering a nucleic acid encoding a human growth factor, such as human FGF (e.g., FGF-18) or a functional portion thereof.

[0037] In one embodiment, the compositions provided herein are administered via intra-articular injection to deliver a nucleic acid encoding an FGF-18 polypeptide. As used herein, "FGF-18 polypeptide" includes any FGF-18 polypeptide that exhibits one or more functions as a full-length FGF-18 protein from any species, such as humans, dogs, cats, cows, sheep, goats, horses, or other animals. FGF-18 polypeptides also include full-length FGF-18 proteins from any species, as well as functional portions or fragments of such full-length FGF-18 proteins. Thus, FGF-18 polypeptides also include non-human primate FGF-18 proteins, full-length or functional fragments or portions thereof. FGF-18 polypeptides also include mammalian or non-mammalian homologs, paralogs, or orthologs, mammalian or non-mammalian functional analogs, and the like.

[0038] Thus, as used herein, "FGF-18 gene" refers to a sequence encoding an FGF-18 polypeptide. Thus, an FGF-18 gene can encode a full-length FGF-18 protein from any species or a functional portion thereof. Examples of FGF-18 polypeptides containing functional portions include, but are not limited to, the following: AEENVDFRIH VENQTRARDD VSRKQLRLYQ LYSRTSGKHI QVLGRRISAR GEDGDKYAQL LVETDTFGSQ VRIKGKETEF YLCMNRKGKL VGKPDGTSKE CVFIEKVLEN NYTALMSAKY SGWYVGFTKK GRPRKGPKTR ENQQDVHFMK RYPKGQPELQ KPFKYTTVTK RSR (SEQ ID NO: 7), amino acids 28-207 of uniprot.org / uniprotkb / O76093 / entry#sequences, and Sprifermin (amino acids 28-196).

[0039] The sequence encoding the FGF-18 polypeptide is preferably under the control of a promoter, such as a constitutive, inducible, tissue-specific, or cycling promoter, and optionally also under the control of regulatory sequences. In one embodiment, the nucleic acid is at least partially encased in a viral capsid, lipid-based carrier, or polymer-based carrier capable of delivering the nucleic acid to the interior of a nuclear cell, with or without secondary modifications. The sequences provided herein may be RNA, DNA, or a hybrid of RNA and DNA, or chemically modified sequences based on RNA, DNA, or RNA and DNA.

[0040] In one embodiment, the FGF-18 gene and / or any other coding or non-coding sequences (e.g., other auxiliary non-coding RNA sequences or other coding sequences) delivered in cis or trans can be under the control of a constitutive promoter. As used herein, a "constitutive promoter" drives expression in a tissue-, cell-type-, or cell-stage-independent manner without significant fluctuations in expression. In one embodiment, the constitutive promoter can be a CMV promoter with or without hybrid elements, such as the MVM1 intron, CAG promoter, EF1-α promoter, UBC promoter, CBh promoter, MSCV promoter, hPGK promoter, SFFV promoter, SV40 promoter, etc., or generally any constitutive promoter capable of driving expression in at least one cell type present at least transiently within a joint or surrounding tissue such as the joint capsule, or a combination of one or more of the foregoing promoters or their functional elements.

[0041] In another embodiment, the FGF-18 gene and / or any other coding or non-coding sequences (e.g., other auxiliary non-coding RNA sequences or other coding sequences) delivered in cis or trans may be driven by an inducible promoter. Some examples of inducible promoters include LexA, AlcaA, araBAD, PtxA, SPLs, GAL7, TRE, etc., or more generally, steroid-inducible promoters, tetracycline-inducible promoters, rapamycin-inducible promoters, ganciclovir-inducible promoters, acyclovir-inducible promoters, temperature-inducible promoters, stress-inducible promoters, promoters induced by increased oxidative status, or promoters induced by upregulation of one or more of ROS, NOS, cytokines, or another exogenously administered molecule, or a combination or one or more functional elements of the aforementioned inducible promoters.

[0042] In another embodiment, it may be beneficial to drive expression at alternating levels periodically without using external stimuli. For example, the FGF-18 gene and / or any other coding or non-coding sequence (e.g., other auxiliary non-coding RNA sequences or other coding sequences) delivered in cis or trans can be driven through the use of a cycling promoter. Examples of cycling promoters include promoters that change their expression regulation level in response to some natural or semi-natural cycle of an organism or its surroundings (e.g., circadian cycle, weekly cycle, lunar cycle, menstrual cycle, cortisol synthesis cycle, diurnal cycle, rest-activity cycle, or exercise level). Generally, it may be beneficial for such cycling promoters to change their activity by at least 2%, at least 5% or more, or in some cases, such as circadian cycle, cortisol response, diurnal cycle, menstrual cycle, or exercise, at least 10% of the average or other activity level (e.g., compared to the minimum or maximum or opposite cyclic activity). Some specific examples of cycling promoters include, without limitation, one or more of the CLOCK promoter, BMAL1 promoter, PER promoter, Cry promoter, NFIL3 promoter, DEC promoter, or PPAR-γ promoter, or elements thereof.

[0043] In yet another embodiment, the FGF-18 gene and / or any other coding or non-coding sequence (e.g., other auxiliary non-coding RNA sequences or other coding sequences) delivered in cis or trans can be driven by a tissue-specific promoter that exhibits at least a preferential expression pattern in cells that are at least temporarily resident in the joint, joint capsule, or surrounding tissue. Examples of such promoters include promoters that exhibit preferentially increased expression in chondrocytes, chondroblasts, synoviocytes, synoviblasts, fibroblast-like synoviocytes, or cells of the chondrocyte or synoviocyte lineage. In one embodiment, the promoter can promote gene expression in connective tissue cells or resident immune cells of the joint, joint capsule, or surrounding tissue. Some specific examples of tissue-specific promoters include one or more of the promoters or enhancers of Argc1, Col2a1, Col6a1, Col10a1, Col11a2, Matn1, Gdf5, IL1B, Prx1, Adam12, α-SMA, Col1a1, Col1a2, FGF18, FGF10, FGF-2, FoxD1, Fsp1, FoxJ1, Gli1, PDGFa, PDGFb, PDFR-α, PDGFR-β, Twist2, or TCF4, or functional fragments thereof.

[0044] In one embodiment, the FGF-18 gene encodes the entire coding sequence of the FGF-18 protein, or the cDNA sequence of the FGF-18 protein, or a functional portion thereof. The FGF-18 protein may be a human FGF-18 protein. In one embodiment, at least a portion of the following gene sequences or a combination thereof is encoded: AF075292, AB007422, AF211188, BT019570, BTo19571, CH471062, BC006245, AY358811, or NM_003862.2. The FGF-18 gene may be a codon-optimized version of any one of the sequences provided herein. As an example, the FGF-18 polypeptide may be encoded by at least a portion of gene ID 8817 from HGNC:3674. The FGF-18 polypeptide provided herein may also be encoded by a codon-optimized version of any one of the sequences provided herein, or other sequences known to those skilled in the art.

[0045] In one embodiment, the FGF-18 polypeptide can be fused with another protein or a fragment of another protein, such as a fragment crystallizable region of an antibody, to facilitate optimal retention time and alternative clearance mechanisms. Thus, a genetic construct can encode a fusion protein that combines the FGF-18 polypeptide provided herein with at least one functional element of another protein. In one embodiment, the fusion partner can be at least a portion of an Fc fragment of an antibody, at least a portion of a cytokine, at least a portion of a growth factor, at least a portion of a growth factor receptor, at least a portion of a molecule that increases the retention time of a growth factor, or at least a portion of a molecule that increases the binding affinity to a receptor.

[0046] In one embodiment, the nucleic acid encodes at least one auxiliary or regulatory sequence that facilitates expression of the FGF-18 gene and FGF-18 polypeptide. Such regulatory sequences may include one or more introns from the FGF-18 gene or other human genes, post-translational regulatory elements such as WPRE or oPRE (e.g., OPRE, WPREmut6, or WPREmut1), genetic sequences encoding poly(A) signals, transcription initiation complex binding sequences, protein binding sequences (which bind DNA-binding proteins such as TATA-binding protein, GATA1, zinc finger proteins, helicases, nickases, or nucleases, single-strand binding proteins, transcription initiation complex proteins, or other proteins that can interact with DNA), enhancer sequences, distal and proximal enhancer elements, insulating sequences, Kozak sequences, termination signals, internal ribosome entry sites, or other genetic sequences that affect transcription, replication, translation, insertion into the genome, recombination, persistence, expression levels, nuclear transport, or entry into cells or specific cellular compartments.

[0047] The therapeutic construct may be delivered locally, such as to an osteoarthritic or pre-arthritic joint, by local or intra-articular injection. In one embodiment, the therapeutic construct is delivered to cells in at least a portion of the joint. The cells may be any one of the cells provided herein. In another embodiment, the therapeutic construct is in a formulation that facilitates delivery of the FGF-18 gene and / or other sequences to cells in at least a portion of the joint. Such cells may be any of the cells provided herein.

[0048] Therapeutic formulations may contain a delivery vector for delivering the FGF-18 gene and / or other sequences to cells, target tissues, or desired cellular compartments. In one embodiment, the gene construct can be delivered by a viral, lipid-based, polymer-based, or hybrid carrier. Some specific viral vectors that can be used include one or more of adeno-associated virus, adenovirus, lentivirus, herpes simplex virus, poxvirus, measles virus, alphavirus, mimivirus, or other enveloped or non-enveloped viruses or their functional elements. In another embodiment, the viral carrier is a viral vector engineered as a synthetic recombinant viral vector, or a viral vector chemically modified after capsid assembly. The viral vector can be synthetic, semi-synthetic, engineered, or contain a hybrid protein or complete hybrid capsid. Preparations of viral capsids may contain empty or full capsids, or capsids containing different sequences, or may be composed of different viral strains, species, families, or genera, and may be at least 5% filled and contain the genetic construct of interest, or may be up to 100% filled and contain the genetic construct of interest.

[0049] In another embodiment, viral carriers, natural or synthetic capsids, lipid nanoparticles (LNPs), liposomes, polymeric carriers, or other non-viral carriers can be conjugated to one or more ligands to enhance the functionality, selectivity, specificity, residence time, or other physical or chemical properties of the capsid or capsid cargo. As used herein, "couple" or "coupled" or "coupling" (and the like) means chemically associating one entity (e.g., a ligand) with another entity. In some embodiments, the coupling is covalent, meaning that the coupling occurs in the context of a covalent bond between the two entities. In non-covalent embodiments, the non-covalent coupling is mediated by non-covalent interactions, including, but not limited to, charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and / or combinations thereof. In one embodiment, encapsulation is a form of coupling. In another embodiment, coupling is by conjugation via a direct linkage, such as covalent conjugation.

[0050] In another embodiment, a genetic construct encoding an FGF-18 gene or other sequence may be incorporated into a polymeric carrier, such as a solid polymeric carrier, a polyelectrolyte complex comprising at least one polymer and a nucleic acid, or a plurality of polymers and nucleic acids. The polymeric carrier may be composed at least in part of a cationic polymer, an anionic polymer, an amphiphilic polymer, a nonionic polymer, or a polymer capable of changing its overall charge from positive to negative, neutral to positive, or neutral to negative depending on the physiological pH range or on changes in pH between the original formulation and physiological conditions. In one embodiment, the polymeric carrier may be composed at least in part of chitosan, polyethyleneimine, or a polypeptide having an overall positive charge, or a combination of one or more of the foregoing polymers.

[0051] In one embodiment, the gene construct encoding the FGF-18 gene and / or other sequences is formulated into at least one type of lipid nanoparticle, liposome, or lipid emulsion, with or without the use of a ligand to enhance targeting, improve bioavailability, increase residence time, or promote more optimal clearance. In one embodiment, the lipid carrier can contain up to 60% cholesterol by molar ratio, or up to 80% cationic or ionizable lipid by molar ratio. In another embodiment, the lipid carrier can be at least partially composed of glycerides, polyglyceryl, or polyoxyglycerides, or generally, can be an oil / water nanoemulsion, oil / water microemulsion, nanocapsule, or self-nanoemulsifying or self-microemulsifying system. In another embodiment, the lipid carrier is a micelle, a lipid-polymer hybrid, or at least partially composed of biopolymers or biomimetics. In one embodiment, the lipid carrier contains a peptide, protein, polysaccharide, small molecule, or combination thereof for targeting, increasing uptake, increasing nuclear delivery of a DNA payload, or increasing in vivo residence time, or for specifically and generally altering one or more physical, chemical, or biological properties of the lipid carrier, or for altering one or more components of the formulation as a whole.

[0052] In one embodiment, the dosage is calculated based on the surface area of ​​the joint, which can be estimated (for example, from an MRI image scan). Alternatively, the dosage can be estimated from the volume of the articular cartilage, either nominal (defined by a healthy state) or at the time of treatment. Alternatively, the dosage can also be estimated from correlates of the surface area or volume of the articular cartilage, such as age, disease state, weight, or species being treated.

[0053] In one embodiment, the therapeutic construct is administered as a function of joint surface area. For example, the therapeutic construct is administered at a dose of 1.2x10 2 Genome copies / cm of joint surface area 2 ~6.1x10 11 Genome copies / cm of joint surface area 2(or a correlate thereof) is administered. As another example, a therapeutic construct may be administered such that 4.5x10 3 Genome copies / cm of joint surface area 2 ~2.3x10 10 Genome copies / cm of joint surface area 2 (or a correlate) may be administered.

[0054] Alternatively, the therapeutic construct may be administered at a dose calculated using the average joint size of a given species relative to body weight, or the average joint size of a given species relative to age, or the average joint size of a given species relative to body weight and age. In one embodiment, the therapeutic construct is administered at a dose of 2x10 for humans. 4 Genome copies / kg ~ 6.3 x 10 13 at a dose of genome copies / kg, or 3.8x10 4 Genome copies / kg ~ 4.7x10 13 In horses, 4.5x10 genome copies / kg joint surface area correlates. 5 Genome copies / kg ~ 6.3 x 10 13 via genome copies / kg joint surface area correlate, or 3.8x10 for dogs 4 Genome copies / kg ~ 1.2 x 10 13 via genome copies / kg joint surface area correlates, or 2x10 for cats 4 Genome copies / kg ~ 4.3 x 10 12 Administered via genome copies / kg joint surface area correlates. It should be understood that the genetic constructs provided herein can be administered to humans or other mammals as appropriate.

[0055] In one embodiment, the therapeutic construct is administered as a function of joint surface area to a large or small joint, which may be generally defined as a knee, hip, shoulder, or elbow, or a joint approximately the size of a knee, hip, shoulder, or elbow, or a joint in an extremity, respectively. In one embodiment, the dose is 5x10 for humans. 7 Genome copies / knee, hip, or shoulder joint ~2x1014 5x10 for horses via genome copies / joint surface area correlation for knee, hip, or shoulder joints 7 Genome copies / knee, hip, or shoulder joint ~5x10 13 Through the genome copies / joint surface area correlation of the knee, hip, or shoulder joint, the dog field is 1x10 5 Genome copies per knee, hip, or shoulder joint ~1x10 12 5x10 for cats via genome copies / joint surface area correlation for knee, hip, or shoulder joints 4 Genome copies / knee, hip, or shoulder joint ~7x10 11 via genome copies / joint surface area correlation for knee, hip, or shoulder joints, or 4x10 7 Genome copies / knee, hip, or shoulder joint ~2x10 14 In another embodiment, the therapeutic construct dose is administered via a genome copy / joint surface area correlation of knee, hip, or shoulder. 8 Genome copies per knee, hip, or shoulder joint ~1x10 13 Genome copies / knee, hip, or shoulder, or 5x10 8 Genome copies / knee, hip, or shoulder joint ~8x10 12 Administration is via a genome copy / knee, hip or shoulder joint volume or surface area correlation.

[0056] Any one of the methods provided herein may include one or more steps of determining a dose based on the concepts provided herein. In one aspect, a method is provided that includes one or more steps of determining a dose based on the concepts provided herein. Any one of the methods provided herein may include administering a genetic construct or a composition comprising a genetic construct at a dose provided herein or at a dose determined by a method provided herein. Any one of the compositions provided herein may include a genetic construct at a dose provided herein or at a dose determined by a method provided herein.

[0057] In one embodiment, the dose can be adjusted based on age, sex, and disease severity by utilizing the average multiple of cartilage thickness, volume, or surface area for each species, age, sex, and disease severity. Any one of the methods provided herein can include such an adjustment step. Any one of the methods provided herein can include administering a gene construct or a composition comprising the gene construct at a dose provided herein or at a dose determined by a method including an adjustment step. Any one of the compositions provided herein can include a gene construct at a dose provided herein or at a dose determined by a method including an adjustment step.

[0058] The compositions according to the present invention can contain pharmaceutically acceptable excipients, such as preservatives, buffers, saline, or phosphate-buffered saline. "Pharmaceutically acceptable excipients" or "pharmaceutically acceptable carriers" refer to pharmacologically inactive materials used to formulate compositions with pharmacologically active materials. Pharmaceutically acceptable excipients include various materials known in the art, including, but not limited to, sugars (e.g., glucose, lactose), preservatives such as antibacterial agents, reconstitution aids, coloring agents, saline (e.g., phosphate-buffered saline), and buffers. The compositions can be prepared using conventional pharmaceutical manufacturing and compounding techniques to achieve useful dosage forms. In one embodiment, the composition is suspended in sterile saline for injection together with a preservative.

[0059] The compositions provided herein may include inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonic acid, acetic acid, or citric acid) and pH adjusters (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citric acid or acetic acid, amino acids and their salts), antioxidants (e.g., ascorbic acid, α-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), Solution and / or freeze / lyophilization stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmolality adjusters (e.g., salts or sugars), antimicrobial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity adjusters (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose), and cosolvents (e.g., glycerol, polyethylene glycol, ethanol).

[0060] The composition can be prepared using conventional pharmaceutical manufacturing and compounding techniques to achieve a useful dosage form.Suitable techniques for implementing the present invention can be found in Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed. Edited by ME Auten, 2001, Churchill Livingstone.The composition of the present invention can be prepared by any suitable method, and it should be understood that the present invention is not limited to the composition that can be prepared using the method described herein.The selection of an appropriate manufacturing method may require attention to the desired functionality.

[0061] In some embodiments, the composition is produced under aseptic conditions or terminally sterilized.This ensures that the resulting composition is sterile and non-infectious, thus improving safety compared to non-sterile compositions.This provides a valuable safety measure, especially when the subject receiving the composition has immunodeficiency, is suffering from infectious diseases, and / or is susceptible to infection.In some embodiments, the composition can be lyophilized and stored for a long period of time without losing activity in suspension or as lyophilized powder depending on the formulation strategy.The compositions referred to herein can be produced and prepared for administration using conventional methods.

[0062] The compositions or genetic construct(s) provided herein may be delivered via a drug delivery device, such as, without limitation, a pre-filled syringe, or may be stored in an ampoule, vial, form-fill-seal, or blow-fill-seal container for subsequent administration via a secondary appropriate means. Kits containing any one of the compositions or genetic construct(s) provided herein are also provided.

[0063] It should be understood that within the scope of the present invention, the formulations, materials, genetic constructs, sequences, biological and chemical compositions, or methods of use may be varied by those skilled in the art to the extent that they perform the desired functions defined herein. Various parts, components, or features may be used in combination, with or without modification by those skilled in the art, to achieve the desired functions defined herein.

[0064] Furthermore, all individual features and methods of use described herein, and each and every combination of two or more of such features and methods of use, are included within the scope of the present invention, provided that the features and methods of use in such combinations are not mutually inconsistent. It is understood that particular moieties or combinations of such moieties can be modified by one skilled in the art while still achieving the objectives of the present invention. Finally, it is understood that the specific ranges provided in the present invention are not limiting but are for illustrative purposes only, and that values ​​outside the specified ranges may be used to achieve the objectives of the present invention without altering the principles of the proposed mechanism. [Example]

[0065] Example 1 An AAV2 vector encoding the hFGF18 gene without codon optimization, driven by the cartilage-specific promoter Col2a1 and regulated by the WPRE post-translational response element. Thus, in one embodiment, a therapeutic agent can be an AAV2 vector encoding the hFGF18 gene without codon optimization, driven by the cartilage-specific promoter Col2a1, and optionally regulated by the WPRE post-translational response element. This therapeutic agent can be delivered intra-articularly. Transduction of chondrocytes can occur, providing autocrine and paracrine cues for cartilage repair. The cartilage-specific promoter Col2a1 can serve as a means to increase the specificity of treatments administered locally to the joint.

[0066] [ka] [ka] [ka]

[0067] Example 2 A construct encoding a CMV promoter-driven, codon-optimized hFGF-18 with an oPRE post-translational response element, AAV delivery. Thus, in another embodiment, the therapeutic agent can be an AAV vector encoding a CMV promoter and codon-optimized hFGF-18. The therapeutic agent can be delivered intra-articularly, providing local transduction efficiency within the joint and promoting high levels of expression relative to the number of viral particles administered.

[0068] [ka] [ka] [ka]

[0069] Example 3 A tetracycline-inducible promoter activation construct delivers hFGF18 with a WPRE post-translational response element. During periods of increased pain or after cartilage loss, it may be desirable to induce or activate periodic expression. In one embodiment, the therapeutic agent comprises a tetracycline-inducible promoter that regulates the expression of hFGF-18. This therapeutic agent can be delivered intra-articularly, allowing for temporal control of local expression within the joint.

[0070] [ka] [ka]

[0071] Example 4 A self-complementary AAV vector that delivers codon-optimized hFGF18 under the control of a tissue-specific promoter and contains an SV40 late polyA sequence. In another embodiment, the therapeutic agent comprises a self-complementary AAV vector that delivers codon-optimized hFGF18 under the control of a tissue-specific promoter and optionally contains an SV40 late polyA sequence.The therapeutic agent can be delivered intra-articularly.This therapeutic agent can be used to optimize the local expression level of FGF-18 in the joint relative to the number of viral particles delivered, and the strong polyA signal stops expression at the end of the coding sequence and stabilizes messenger RNA.

[0072] [ka] [ka]

[0073] Example 5 An AAV vector delivering dgFGF18 without codon optimization under the control of the CBh promoter and containing the oPRE post-translational regulatory element.

[0074] [ka] [ka] [ka]

[0075] Example 6 An AAV vector that delivers ctFGF18 without codon optimization under the control of the CAG promoter and contains the WPRE post-translational regulatory element.

[0076] [ka] [ka] [ka] [ka]

[0077] Example 7 An AAV vector that delivers hrFGF18 without codon optimization under the control of the CAG promoter and contains the WPRE post-translational regulatory element.

[0078] Example 8 We compared the effects of rhFGF18 protein and AAV2-delivered hFGF18 gene therapy on cartilage anabolism. The chondrogenic properties of AAV2-FGF18 were compared in vitro with full-length, fully glycosylated rhFGF18 protein and Escherichia coli-expressed rhFGF18 protein analogs compared with PBS and AAV2-GFP negative controls. Gene expression analysis was performed using RNA-seq on primary human chondrocytes from middle-aged adults. In vivo cartilage anabolism was assessed by measuring cartilage thickness in the tibial plateau and the anterior horn of the medial meniscus in Sprague-Dawley rats after administration of increasing doses of AAV2-FGF18, rhFGF18 protein analogs, and AAV2-GFP5 control, confirming long-term gene expression using AAV2-nLuc.

[0079] AAV2-FGF18, AAV2-GFP, and AAV2-nLuc were produced using triple transfection in suspension cell cultures of HEK293 cells, followed by downstream purification by clarification, ion exchange chromatography, and UF / DF followed by controlled-temperature freezing at -80°C. Viral vector preparations were diluted to the target dose 30 minutes before injection using 20 μL of room temperature 1x PBS solution. The in vivo dose was 2x10 per joint. 9 ~1x10 11The viral genomes ranged from 10 to 1000 vg / cell, and were administered via 20 μL intra-articular injection (Sprague-Dawley rats) using a 30G needle. In vitro doses ranged from MOI 10 to MOI 1000 vg / cell. Viral vector preparations were diluted in 1x PBS, 1x PBS with 1% albumin solution, or 0.5% sucrose in USP WFI solution. Best practices for blinding and randomization were applied to all experiments whenever possible. Raw data were analyzed for normality using the Anderson-Darling normality test. Normally distributed data were analyzed using ANOVA, followed by Tukey's post hoc test for multifactorial experiments, or Student's t-test for one-factor, multilevel, or two-factor, single-level studies. Data are presented as mean ± standard deviation. A p-critical value (p-crit) of <0.05 was considered significant; however, for RNA-Seq analysis, a p-crit of <0.01 was used to align with RNA-Seq best practices. All statistical analyses were performed using Minitab version 21.1.

[0080] result AAV2-delivered hFGF18 represents a promising strategy for hyaline articular cartilage repair by promoting anatomically relevant extracellular matrix production, chondrocyte proliferation, and increased articular and meniscal cartilage thickness in vivo. FGF18 delivered as a protein or via an AAV2 vector exhibits chondrocyte anabolic activity by promoting chondrocyte proliferation and upregulation of hyaline cartilage-related genes such as COL2A1 and HAS2, while downregulating fibrocartilage-associated COL1A1. This activity translates into statistically significant increases in in vivo cartilage thickness in the tibial plateau and meniscal tip regions after a single intra-articular injection of AAV2-FGF18 and a regimen of six 2-weekly injections of rhFGF18 protein compared with AAV2-GFP controls. Furthermore, a single injection of AAV2-delivered hFGF18 offers a potential safety advantage over multiple protein injections, as evidenced by reduced joint swelling during the treatment period.

[0081] To evaluate the cytocompatibility of AAV2 vectors, primary human chondrocytes and primary human synoviocytes were treated with increasing doses of AAV2 encoding a green fluorescent protein (GFP) reporter transgene, ranging from an MOI of 1,000 to an MOI of 500,000 (Figure 1). Despite the high multiplicity of infection assessed, there was no statistical significance between the test groups (doses) and test times (hours) (two-way ANOVA, p crit Dose-dependent cytotoxicity was not observed in either chondrocytes or synoviocytes in vitro, as evidenced by a β-reactive protein (MOI) of 0.05. Both synoviocytes and chondrocytes appeared to be highly permissive to AAV2-mediated transduction, as evidenced by a rapid increase in the number of GFP-positive cells between 24 and 168 hours. Chondrocytes appear to be more amenable to AAV2 transduction in vitro, as chondrocytes reached 97% GFP+ cells at an MOI of 50,000, whereas synoviocyte cultures only reached 58% GFP+ cells at the same MOI.

[0082] Because the rhFGF18 protein analog evaluated in clinical trials was bacterially expressed in Escherichia coli (15), we tested the ability of eukaryotically expressed hFGF18 to promote proliferation in a dose-dependent manner compared to bacterial rhFGF18 analog. Both E. coli-expressed rhFGF18 protein analog (Bac) and HEK293-expressed rhFGF18 protein (HEK) demonstrated dose-dependent proliferation, with no statistical difference between the two test groups, but statistical significance for the dose factor (Figure 2). A similar dose-response curve was observed for chondrocytes treated with AAV2-FGF18 at a multiplicity of infection (MOI) of 100 and 1,000. The 1,000 MOI increased cell numbers by 84% compared to the pre-treatment time point (t = 0), comparable to the 10,000 ng / mL dose of bacterially expressed rhFGF18 analog (85%).

[0083] The effect of hFGF18 paracrine signaling from AAV2-FGF18-transduced synoviocytes was evaluated in transwell cultures with primary human chondrocytes (Figure 3). The number of proliferating chondrocytes increased by 92–135% between 48 and 72 hours after exposure to synoviocytes treated with AAV2-FGF18 and rhFGF18 protein, whereas only a 6% increase was observed with the AAV2-GFP negative control administered at an MOI of 500,000.

[0084] In addition to the chondrogenic effects of hFGF18 protein and AAV2-FGF18 gene therapy, we compared the gene expression profiles of chondrocytes exposed to a high dose of rhFGF18 protein (1,000 ng / mL) and a subproliferative dose (MOI 10) of AAV2-FGF18. This experimental design allowed us to identify the full set of genes upregulated by rhFGF18 protein and compare them with the most significantly upregulated subset in the AAV2-FGF18 gene therapy treatment group. Results showed that 94% of genes upregulated in AAV2-FGF18-treated chondrocytes at a 3-fold cutoff, or all but two genes (TGM2 and SERPINA1), were also upregulated by rhFGF18. Similarly, all genes whose expression was downregulated by 100% by AAV2-FGF18 gene therapy at a 3-fold cutoff, were also downregulated by chondrocytes treated with rhFGF18 (Figure 4).

[0085] Analysis of genes specifically associated with the hyaline cartilage phenotype, including HAS2 (20), ACAN (21), COL2A1 (22), and PRG4 (23), showed that chondrocytes treated with AAV2-FGF18 upregulated all genes tested (only ACAN upregulated p crit = 0.01, which did not achieve statistical significance), whereas chondrocytes treated with rhFGF18 protein only upregulated HAS2, COL2A1, and PRG4 (PRG4 was p crit(P < 0.01, which did not achieve statistical significance) (Figure 5). All fibrocartilage extracellular matrix-related genes (COL1A1 (24), ADAMTS15 (25), LOX (26)) were down-regulated in the AAV2-FGF18-treated group and the rhFGF18 protein-treated group (ADAMTS15 was down-regulated in the protein-treated group). crit (Statistical significance was not achieved at p < 0.01.) Interestingly, AAV2-FGF18-treated chondrocytes also upregulated the SOX9 chondrocyte differentiation marker (27), whereas a similar upregulation was not observed in the rhFGF18 protein-treated group.

[0086] Gene expression was monitored in vivo over a 4-month period using a nanoluciferase reporter gene under the control of a CMV promoter delivered via an AAV2 vector (Figure 6). After intra-articular administration of furimazine substrate, a strong bioluminescent signal was detected locally within the joint. Signal intensity appeared most consistent when measurements were taken 12 minutes after substrate injection using a 20 μL intra-articular injection volume. Furthermore, hFGF18 expression was confirmed at the final test time point using a 1:100 dilution of anti-hFGF18 antibody in the AAV2-FGF18 active group compared with the AAV2-GFP control and the AAV2-GFP negative control without primary antibody (all transgenes under the control of the same CMV promoter).

[0087] Analysis of sagittal sections of the knee joints demonstrated a qualitative increase in cartilage thickness at the tibial surface and meniscal tip (white area of ​​the anterior horn of the medial meniscus) in the AAV2-FGF18 and rhFGF18-treated groups compared with the AAV2-GFP negative control. No observable effects on subchondral bone, bone marrow content or morphology, or the intermediate portion of the meniscus were observed in any of the histological sections (Figure 7). Finally, no signs of inflammation, degenerative processes within the cartilage or underlying bone, or abnormal growths were observed in the examined areas.

[0088] After normalization by animal weight, quantitative and statistically significant increases in cartilage thickness were observed at the distal end of the tibia and meniscus (Figure 8). Normalized tibial cartilage thickness for AAV2-FGF18 gene therapy ranged from 0.72 ± 0.15 μm / g (mean ± standard deviation) to 0.67 ± 0.18 μm / g; normalized tibial cartilage thickness for the rhFGF18 protein-treated group was 0.70 ± 0.15 μm / g, respectively. Control AAV2-GFP-treated joints demonstrated a mean cartilage thickness of 0.64 ± 0.17 μg / g at 2 months. Statistically significant differences in cartilage thickness were observed in the protein-treated groups and the highest gene therapy dose in the tibia compared to negative controls known to lack chondrogenic or cartilage-degenerating properties (28-30) (one-way ANOVA followed by Tukey's pairwise comparison post-hoc test, p < 0.05). crit = 0.05). Within the meniscus, the top two gene therapy doses combined with the protein treatment achieved statistically significant differences versus the negative control, whereas the protein did not achieve statistically significant differences versus the negative control using Tukey's pairwise comparison post-hoc test. A dose-response curve was observed in the tibia, with dose 2 of AAV2-FGF18 appearing to work best in the meniscal tip cartilage.

[0089] Finally, increased joint swelling was observed after repeated administration of FGF18 over the 2-month study period. Swelling was observed at both the 1-month and 2-month time points and partially resolved toward the end of the study (Figure 9). At 1 month, the diameter of joints treated with multiple-injection rhFGF18 protein increased by 22.8% compared to the no-injection baseline control. The 1-month and 2-month time points for the protein-treated group were statistically different from all other test groups except for AAV2-GFP at 2 months (ANOVA, Tukey's pairwise comparison, p crit =0.05).

[0090] Consideration Osteoarthritis shares many characteristics of classic aging diseases, with age-related tissue cellularity loss and structurally important ECM deposition resulting in a progressive degenerative phenotype ultimately necessitating surgical intervention (4, 31). Although inflammatory cytokines have been hypothesized to play both leading and contributing roles (32, 33), none of the anti-inflammatory therapies investigated to date have been able to demonstrate disease improvement in controlled randomized clinical trials. Repeated administration of Sprifermin, a cartilage-anabolic rhFGF18 protein analog, has been demonstrated to increase cartilage thickness compared with placebo controls (15). However, the protein injection approach is a multi-dose regimen requiring up to 12 injections per year for the treatment of bilateral osteoarthritis and may need to be continued indefinitely to prevent reversal of cartilage gain (15). To overcome the delivery challenges associated with rhFGF18 protein therapy, hFGF18 gene therapy using an AAV2 delivery vector has been developed.

[0091] The results demonstrate the excellent transfection efficiency and cytocompatibility of AAV2 vectors for primary human synoviocytes and chondrocytes, cell types suggested as primary targets for stable transgene expression in joints (35). Chondrocytes appear more amenable to AAV2-mediated transduction, as evidenced by the high percentage of GFP-positive cells at significantly lower doses. After 168 hours of culture, more than 90% of chondrocytes were assessed as GFP+ by quantitative fluorescence microscopy at an MOI of 1,000, whereas only approximately 80% of GFP+ synoviocytes were observed over the same period at an MOI of 500,000 AAV2-GFP / cell (Figure 1). Despite these differences in transduction efficiency, both cell lines expressed significant amounts of GFP protein and showed no statistically significant reduction in cell numbers at any of the doses evaluated, confirming the lack of dose-dependent cytotoxicity of the delivered vector.

[0092] In parallel, significantly lower doses of AAV2-FGF18 (MOI 100 and MOI 1,000) were able to induce chondrocyte proliferation effects of a similar magnitude to those observed at protein doses previously evaluated in vitro to aid in in vivo and subsequent clinical dose selection (36) (Figure 2). This effect may be due to the previously suggested autocrine activity of FGF18 protein (37). Because transduction of synoviocytes may be predominant in vivo despite the low transduction potential in vitro, the paracrine effect was further evaluated using a transwell coculture assay. AAV2-FGF18-transduced synoviocytes were cultured with primary human synoviocytes in transwell plates, confirming the ability of AAV2-FGF18 to mediate chondrocyte proliferation in a paracrine manner at doses comparable to 1 μg / mL of rhFGF18 protein. These results support the potential of AAV2-FGF18 gene therapy to promote chondrogenic effects after intra-articular delivery, regardless of the precise biodistribution within the joint, as long as at least a portion of the resident cells proximal to the joint capsule are transduced.

[0093] We next evaluated the ability of AAV2-FGF18 gene therapy to promote the upregulation of hyaline cartilage-related genes while downregulating fibrocartilage-related genes in culture. Hyaline cartilage is the natural form of cartilage within joints, but the presence of fibrocartilage after surgical local defect repair procedures (e.g., microfractures) has previously been suggested to reduce the durability of the repair (38). Treatment of primary human chondrocytes with AAV2-FGF18 and rhFGF18 proteins upregulated HAS2 and COL2A1 compared to a PBS baseline, but only AAV2-FGF18 was able to achieve a statistically significant increase in PRG4. HAS2 is an essential component for HA synthesis, particularly involved in the production of high molecular weight hyaluronan, which is abundant in hyaline cartilage (20). Meanwhile, COL2A1 and PRG4 are secreted proteins that are essential components of hyaline cartilage (22, 23) and have structural and anti-adhesion-specific roles (39). Therefore, the upregulation of these hyaline cartilage-related genes further supported the cartilage anabolic activity of hFGF18.

[0094] Conversely, COL1A1 and LOX were downregulated in the protein and AAV2-FGF18-treated groups, but only the AAV2-FGF18-treated group achieved a statistically significant reduction in ADAMTS15. COL1A1 is a fibrocartilage matrix component and is upregulated in abnormal cartilage repair after microfractures (40). Similarly, copper enzymes of the lysyl oxidase family (LOX) have been suggested to play a role in collagen cross-linking, and modulation of LOX has been observed to promote cartilage regeneration (26). Similarly, ADAMTS15, a key metalloproteinase responsible for catabolic activity, has been associated with pathological osteoarthritis (41), and modulation of ADAMTS15 has been suggested as a potentially viable approach to prevent progressive cartilage degeneration (42).

[0095] Interestingly, Sox9, a chondrocyte differentiation-related transcription factor, was upregulated only by AAV2-FGF18 and not by rhFGF18 protein treatment, although the degree of upregulation was relatively small compared to baseline, achieving only a 1.3-fold increase. Many other genes were similarly up- and down-regulated, including ESM1, NTSR1, MMP1, and ANGPTL7, COPG2IT1, and PTX3 (Figure 10), all of which have previously been linked to skeletal development and maintenance (43-48). These findings further suggest similarities in activity between rhFGF18 protein and AAV2-FGF18 gene therapy, despite the different protein delivery vehicles. Conversely, the differential upregulation of TGM2, a calcium-dependent acyltransferase observed to be involved in cartilage homeostasis ( 49 ), and SERPINA1, previously linked to chondrogenesis and chondrocyte differentiation ( 50 ), by AAV2-FGF18 but not rhFGF18 protein requires further investigation using pathway analysis and mechanistic studies to elucidate whether the differences are due to the degree of hFGF18 accumulation in the cytoplasm, delivery, or pathways activated by the AAV2 capsid.

[0096] Overall, the results of gene expression analysis suggest that the mechanism of hFGF18 activity consists of two complementary anabolic components that promote chondrocyte proliferation and hyaline cartilage extracellular matrix production through multiple direct (lubricin / PRG4, collagen 2 / COL2A1) and indirect (ADAMTS1, 5, 15, and MMP2) pathways (Figure ​(Figure11)) (51-53). In parallel, FGF18 appears to suppress the expression of fibrocartilage-related genes through the direct downregulation of COL1A1 and LOX, and the indirect downregulation of pro-fibrotic genes, including PTX3 and IGF1 (54,55).

[0097] The safety of AAV2 delivery vectors was demonstrated in up to 1x10 per joint via intra-articular injection. 11 This was confirmed by administering a dose of 1000 mg / kg. No abnormal growths or tumors were observed in the cartilage, meniscus, subchondral bone, or proximal bone marrow during the study period. Furthermore, neither the AAV2-GFP control nor the AAV2-FGF18 gene therapy treatment groups showed clearly observable cartilage or bone degenerative processes, inflammatory infiltrates, or other qualitative attributes of tissue degradation or cellular inflammation, consistent with previous studies using viral vector-delivered gene therapy (18,34,37).

[0098] Statistically significant increases in articular and meniscal cartilage thickness were observed after delivery of the hFGF18 transgene and rhFGF18 protein analog (37). Both the rhFGF18 protein injection and AAV-hFGF18 treatment groups demonstrated statistically significant increases in tibial cartilage thickness compared to the AAV2-GFP negative control, reaching increases of 9.4% and 12.5%, respectively. The highest doses of the AAV2-FGF18 and protein treatment groups achieved statistical significance over the tibial negative control. Previous studies using fewer animals and without normalization for body weight did not observe statistically significant increases in cartilage thickness in healthy rodent joints treated with rhFGF18 protein; however, this study was limited by sample size and did not normalize for body weight, which has previously been demonstrated to be associated with cartilage thickness (16). Furthermore, early studies using adenoviral vector-delivered hFGF18 observed increased local tissue thickness due to chondrocyte proliferation, upregulated extracellular matrix production, and increased cartilage accumulation (37). The overall increase in cartilage thickness was relatively small, involving approximately 28–37 μm of additional cartilage (compared to a control baseline of 298 μm). Because hyaline cartilage thinning has been demonstrated to occur as a function of age and osteoarthritis progression (8–10), these increases may be sufficient to delay the onset of symptomatic osteoarthritis or slow age-related cartilage erosion over time. Interestingly, the increase in cartilage thickness was most pronounced at the meniscus tip, where administration of a medium dose of AAV2-FGF18 resulted in a 30% increase in white cartilage area compared to AAV2-GFP controls. In the protein-treated group, meniscus tip thickness increased by 18%, but statistical significance was not achieved, likely due to sample size and effect size. This increase in cartilage correlates with meniscal section, which is known to be composed of a more hyaline cartilage phenotype with increased concentrations of type II collagen.(56,57) Cartilage regeneration procedures focused on articular cartilage are currently undergoing late-stage clinical evaluation, but the application of these procedures to meniscal tissue repair has not previously been reported.Concurrently, no joint swelling was observed after a single injection of AAV2-GFP or AAV2-FGF18, but the protein-treated groups showed a significant increase in joint diameter compared with baseline controls without injection, peaking at 1 month and partially resolving by 2 months. Whether swelling is a common phenomenon in human joints is unclear, and although rhFGF18 protein injection treatment appears safe in placebo-controlled randomized human trials (15), further evaluation of the effects of swelling associated with multiple injections, focusing on the aspect ratio of the joint to the injection needle and injection frequency, is warranted.

[0099] In conclusion, hFGF18 gene therapy exhibits many mechanistic similarities with the activity of rhFGF18 protein analogs, which are currently being developed for the treatment of osteoarthritis and have been demonstrated to promote increased articular cartilage thickness in vivo (15,16). The ability to increase chondrocyte proliferation, upregulate several hyaline cartilage extracellular matrix-related genes while downregulating fibrocartilage-related genes, and promote increased cartilage thickness in rat knee joints supports the potential for hFGF18 gene therapy to be developed for disease model efficacy testing in rodent models of osteoarthritis and focal cartilage damage repair. Finally, the observed effects of rhFGF18 protein and hFGF18 gene therapy on the meniscal tip support the potential for hFGF18 to promote regenerative repair of meniscal tears.

[0100] References [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0101] Example 9 Three doses of gene therapy treatment using the CMV promoter were tested in rats (Figure 12). Doses capable of increasing cartilage thickness compared to the negative control (PBS) included 2x10 9 , 1x10 10 and 1x10 11 The gene construct was also found to be superior to administered protein in meniscus repair at doses including 1 x 10 vg / joint. 10 The dose of 1x10 vg / joint showed the highest effect on the meniscus. 11 The vg / joint dose showed the greatest effect in the tibia (articular cartilage).

[0102] Any one of the genetic constructs or compositions provided herein can be present or administered in an amount that is delivered in any one of the doses provided herein. Thus, any one of the methods provided herein includes administering a genetic construct or composition provided herein, and any one of the doses provided herein is provided to a subject. The dose can be determined based on the dose administered to a similar test subject, for example, a test subject that is similar in terms of any one or more of species, age, weight, sex, and disease state. In another embodiment, the dose can be determined based on the dose administered to a dissimilar test subject, for example, a test subject that is dissimilar in terms of any one or more of species, age, weight, sex, and disease state, but adjusted. The adjustment can be based on the average joint surface area, average joint volume, and average volume of articular cartilage between the subject (or expected subject) and the test subject. Any one of the methods provided herein can include one or more steps for determining the dose and / or adjusting the dose as described above.

[0103] For example, the average joint volumes for rats and humans are listed as follows: [Table 2]

[0104] Therefore, a human dose range equivalent to that used in rats (using the lowest to highest ratio, the highest to lowest ratio, and the mean joint volume, respectively) can be determined. Illustrative exemplary values ​​are shown below (per knee joint): [Table 3]

[0105] Similarly, dosages (low and high) for other species can be determined. Illustrative exemplary values ​​are also provided below. [Table 4]

[0106] Generally, higher doses are used for more advanced disease, such as KL3 and KL4 osteoarthritis, while lower doses are generally used for less advanced disease, such as KL1 and KL2 osteoarthritis, or may be used for disease prevention. Any one of the methods provided herein can be used for the treatment of advanced disease or progressive cartilage loss, preferably at higher doses, for example, at any one of the high doses provided herein or calculated herein or that can be calculated herein. Any one of the methods provided herein can be used for the treatment of less advanced disease or disease prevention, preferably at lower doses, for example, at any one of the low doses provided herein or calculated herein or that can be calculated herein. Any one of the methods provided herein can include one or more steps for determining or adjusting the dose accordingly. Such determined or adjusted doses are also provided herein, and the dose of any one of the methods or compositions provided herein can be such a dose.

[0107] Furthermore, a statistically significant correlation has been observed between animal weight and cartilage thickness.The heavier the animal weight, the smaller the cartilage at the tip of the meniscus, so in any one of the methods provided herein, dosage can be determined by body weight.Examples include: [Table 5]

[0108] [Table 6]

[0109] As an example, for humans, the higher dose range may be: [Table 7]

[0110] As another example, for humans, a lower dose range may be: [Table 8]

[0111] Correlated doses can also be determined (e.g., for horses, dogs, or cats) according to the principles provided herein, and such doses are also provided herein. Any one of the methods provided herein can include one or more steps for determining or adjusting the dose accordingly. Such determined or adjusted doses are also provided herein, and the dose of any one of the methods or compositions provided herein can be such doses.

[0112] Finally, the dosage may be adjusted based on the promoter used. The CMV promoter is an example, and the dosage may be determined based on such a promoter. When a different promoter is used, the dosage can be determined and / or adjusted based on the relative dosage ratio depending on the type of promoter (e.g., journals.plos.org / plosone / article?id=10.1371 / journal.pone.0010611 and nature.com / articles / gt20093).

[0113] Examples of ratios are provided below: [Table 9]

[0114] Therefore, any one of the doses provided herein may be adjusted based on the type of promoter of the gene construct. Any one of the methods provided herein may include one or more steps for determining or adjusting the dose accordingly. Such determined or adjusted doses are also provided herein, and the dose of any one of the methods or compositions provided herein may be such doses.

[0115] As another example, an exemplary vector may include a post-translational regulatory element (e.g., a WPRE regulatory element such as OPRE, WPREmut6, or WPREmut1). Such regulatory elements can reduce the overall dose range, for example, by 25%, 20%, and 10%, respectively. Thus, the dose can also be adjusted based on the regulatory elements of the gene construct. Any one of the methods provided herein can include one or more steps for determining or adjusting the dose accordingly. Such determined or adjusted doses are also provided herein, and the dose of any one of the methods or compositions provided herein can be such doses.

[0116] Example 10 Exemplary doses (average, high and low) are provided. Any one of these doses or equivalent doses described herein can be applied to any one of the compositions or methods provided herein. As can be understood, the doses provided herein also represent ranges from low dose to high dose. Any one of these dose ranges or equivalent dose ranges described herein can be applied to any one of the compositions or methods provided herein.

[0117] [Table 10]

Claims

1. A genetic construct comprising a nucleic acid encoding an FGF-18 polypeptide, a promoter, and optionally, regulatory elements (such as post-translational regulatory elements) (for example, from the WPRE sequence family, encoding a polyA signal, an enhancer, a translation termination sequence, and a sequence that promotes binding of one or more DNA-binding proteins).

2. 2. The genetic construct of claim 1, wherein the FGF-18 polypeptide is mammalian, such as a human or non-human primate.

3. 3. The genetic construct of claim 2, wherein the FGF-18 polypeptide is a human, canine, feline, bovine, ovine, caprine, or equine FGF-18 polypeptide.

4. 2. The genetic construct of claim 1, wherein the FGF-18 polypeptide is encoded by the sequence of AF075292, AB007422, AF211188, BT019570, BTo19571, CH471062, BC006245, AY358811, NM_003862.2 or a portion thereof.

5. 2. The genetic construct of claim 1, wherein the FGF-18 polypeptide is encoded by at least a portion of the sequence of gene ID 8817 from HGNC:3674.

6. 10. A genetic construct according to any one of the preceding claims, wherein the nucleic acid encodes another protein or part thereof.

7. 10. A genetic construct according to any one of the preceding claims, wherein the nucleic acid further encodes at least one auxiliary and / or regulatory sequence that facilitates expression.

8. 10. A genetic construct according to any one of the preceding claims, wherein the nucleic acid further encodes at least one intron from another genetic sequence (e.g., human).

9. 10. The genetic construct of any one of the preceding claims, wherein the promoter is a CMV promoter, a CMV promoter with an MVM1 intron, a CAG promoter, an EF1α promoter, a UBC promoter, a CBh promoter, an MSCV promoter, an hPGK promoter, an SFFV promoter, or an SV40 promoter.

10. 10. A genetic construct according to any one of the preceding claims, wherein the promoter is a constitutive promoter (e.g., a mammalian promoter).

11. 10. A genetic construct according to any one of the preceding claims, wherein the promoter drives expression in at least one cell type that is at least transiently present within the joint or tissue surrounding the joint.

12. 10. A genetic construct according to any one of the preceding claims, wherein the promoter is an inducible promoter.

13. 13. The genetic construct of claim 12, wherein the inducible promoter up- or down-regulates expression in response to external or internal stimuli (e.g., inflammation, heat, light, stress, or administration of steroids, tetracycline, antibiotics, rapamycin, ganciclovir, acyclovir), or is induced by up- or down-regulated heat, ROS, NOS, or cytokine release.

14. 10. The genetic construct of any one of the preceding claims, wherein the promoter is a circadian rhythmic or cycling promoter (e.g., one that changes its activity level by at least 2%, 5%, or 10% over periods such as hours to months, including weeks), in response to cortisol levels, menstrual cycle, circadian cycle, exercise level, etc.

15. 10. The genetic construct of any one of the preceding claims, wherein the promoter is a tissue-specific promoter.

16. 10. A genetic construct according to any one of the preceding claims, wherein the promoter is a chondrocyte-specific promoter.

17. 10. The genetic construct of any one of the preceding claims, wherein the promoter is a synovial cell-specific promoter.

18. 10. A genetic construct according to any one of the preceding claims, wherein the regulatory sequence elements are one or more of Argc1, Col2a1, Col6a1, Col10a1, Col11a2, Matn1, Gdf5, IL1B, and Prx1.

19. 10. The genetic construct of any one of the preceding claims, wherein the regulatory element is one or more of Adam12, α-SMA, Col1a1, Col1a2, FGF18, FGF10, FGF-2, FoxD1, Fsp1, FoxJ1, Gli1, PDGFa, PDGFb, PDFR-α, PDGFR-β, Twist2, and TCF4.

20. A genetic construct according to any one of the preceding claims, wherein the regulatory element is an intron, part of an intron, a post-translational regulatory element, an enhancer, a repressor, a genetic sequence capable of forming a multidimensional structure with part of the genome or the genetic construct itself, or a genetic regulatory element in general.

21. 10. A composition comprising the genetic construct of any one of the preceding claims, wherein the nucleic acid is contained in a delivery vector.

22. 22. The composition of claim 21, wherein the delivery vector is a polyelectrolyte complex or polypeptide, a virus (e.g., an adeno-associated virus (e.g., AAV2), an adenovirus, a lentivirus, a herpes simplex virus, a poxvirus, a measles virus, an alphavirus, or a mimivirus), a polymeric carrier, or a lipid carrier, optionally conjugated to a ligand (e.g., to increase selectivity and / or specificity and / or target a tissue or cell type) (e.g., to at least a portion of an Fc fragment, at least a portion of a cytokine, at least a portion of a growth factor, at least a portion of a growth factor receptor, at least a portion of a molecule that increases the residence time of a growth factor, or at least a portion of a molecule that increases binding affinity to a receptor).

23. 23. The composition of claim 21 or 22, wherein the viral carrier is from a virus with a synthetic or hybrid capsid.

24. The composition of any one of claims 21 to 23, wherein the viral carrier is from a virus with a natural or synthetic capsid, optionally conjugated to a ligand.

25. The composition of any one of claims 21 to 24, wherein the viral carrier is composed of multiple virus types.

26. 26. The composition of any one of claims 21 to 25, wherein at least 5% of the capsids of the viral carrier are complete capsids.

27. 23. The composition of claim 21 or 22, wherein the delivery vector is a polymer, optionally linked to a ligand.

28. 23. The composition of claim 21 or 22, wherein the delivery vector is a polyelectrolyte complex comprising at least one polymer, optionally linked to a ligand.

29. 29. The composition of any one of claims 21, 22, 27 or 28, wherein the polymer is a cationic polymer, an anionic polymer and / or a non-ionic polymer.

30. 30. The composition of any one of claims 21, 22 or 27 to 29, wherein the delivery vector comprises chitosan, polyethyleneimine, or a polypeptide with an overall positive charge.

31. 31. The composition of any one of claims 21 to 30, wherein the ligand targets any one of the tissues or cell types described herein.

32. 32. The composition of claim 21, 22 or 31, wherein the delivery vector is a lipid nanoparticle or liposome, optionally conjugated to a ligand.

33. 33. The composition of claim 21, 22, 31 or 32, wherein the lipid carrier comprises up to 60% cholesterol by molar ratio.

34. 33. The composition of claim 21, 22, 31 or 32, wherein the lipid carrier comprises up to 80% by molar ratio of cationic or ionizable lipids.

35. 33. The composition of claim 21, 22, 31 or 32, wherein the lipid carrier comprises a glyceride, polyglyceryl and / or polyoxyglyceride.

36. 33. The composition of claim 21, 22, 31 or 32, wherein the lipid carrier comprises an oil / water nanoemulsion or an oil / water microemulsion.

37. 33. The composition of claim 21, 22, 31 or 32, wherein the lipid carrier is a nanocapsule, a self-nanoemulsifying or self-microemulsifying system, a micelle, a lipid-polymer hybrid, or comprises a biopolymer or biomimetic.

38. 38. The composition of any one of claims 21 to 37, wherein the ligand comprises a peptide, protein, polysaccharide, small molecule, or combination thereof (e.g., for targeting, increasing uptake, or increasing in vivo residence time).

39. A method comprising administering any one of the genetic constructs or compositions of any one of claims 1 to 38 to a subject in need thereof.

40. 40. The method of claim 39, wherein the subject has or is at risk of having cartilage damage, cartilage loss, and / or is in need of cartilage regeneration.

41. 41. The method of claim 39 or 40, wherein the subject has or is at risk for osteoarthritis.

42. 41. The method of claim 39 or 40, wherein the subject has a meniscus tear.

43. 42. The method of any one of claims 39 to 41, wherein the genetic construct or composition is administered locally or intra-articularly.

44. 43. The method of any one of claims 39 to 42, wherein the genetic construct or composition is administered to the meniscus of the joint.

45. 44. The method of any one of claims 39 to 43, wherein the subject is a human subject.

46. 44. The method of any one of claims 39 to 43, wherein the subject is a horse, dog, or cat.

47. 46. ​​The method of any one of claims 39 to 45, wherein the dose of the genetic construct is calculated or adjusted using the subject's average joint size (e.g., average joint surface area or average joint volume), average articular cartilage volume, body weight, age and / or disease state, or representative values ​​for the subject, compared to another subject, such as of a different species.

48. 48. The method of claim 47, wherein a subject's representative metric is determined for the subject, and optionally, the method further comprises determining the subject's average joint size (e.g., average joint surface area or average joint volume), average articular cartilage volume, body weight, age, and / or disease status.

49. 48. The method of claim 47, wherein the subject's metric representative values ​​are those of another subject representative of the subject, such as a healthy subject or another subject of the same species, and optionally the method further comprises determining the average joint size (e.g., average joint surface area or average joint volume), average articular cartilage volume, body weight, age and / or disease status of the other subject.

50. 50. The method of any one of claims 47 to 49, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in relation to body weight.

51. 50. The method of any one of claims 47 to 49, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in relation to age.

52. 50. The method of any one of claims 47 to 49, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in relation to body weight and age.

53. 50. The method of any one of claims 47 to 49, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in the context of the disease state.

54. 50. The method of any one of claims 47 to 49, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in relation to the disease state and body weight.

55. 50. The method of any one of claims 47 to 49, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in relation to disease state and age.

56. 56. The method of any one of claims 39 to 55, wherein the dosage of the genetic construct is calculated or adjusted or is a dose calculated or adjusted using an average expression ratio (e.g. based on the promoters and / or regulatory elements of the construct) (such as an average promoter ratio and / or regulatory element ratio).

57. The dose of the gene construct is 2x10 9 ~1x10 11 57. The method of any one of claims 39 to 56, wherein the amount is genome copies / joint (rat) or an equivalent amount as defined herein, such as a human, horse, dog or cat equivalent amount.

58. The dose of gene construct is 1x10 10 ~6x10 11 57. The method of any one of claims 39 to 56, wherein the genome copies / joint (human) or an equivalent amount as defined herein, such as an equine, canine or feline equivalent amount.

59. The dose of the gene construct is 5x10 11 ~3x10 13 57. The method of any one of claims 39 to 56, wherein the genome copies / joint (human) or an equivalent amount as defined herein, such as an equine, canine or feline equivalent amount.

60. The dose of the gene construct is 5x10 11 ~3.5x10 12 57. The method of any one of claims 39 to 56, wherein the genome copies / joint (dog) or an equivalent amount as defined herein, such as a human, horse or cat equivalent amount.

61. The dose of gene construct is 1x10 10 ~7x10 10 57. The method of any one of claims 39 to 56, wherein the genome copies / joint (dog) or an equivalent amount as defined herein, such as a human, horse or cat equivalent amount.

62. The dose of gene construct is 1x10 12 ~8.5x10 13 57. The method of any one of claims 39 to 56, wherein the genome copies / joint (horse) or an equivalent amount as defined herein, such as a human, dog or cat equivalent amount.

63. The dose of the gene construct is 3x10 10 ~2x10 12 57. The method of any one of claims 39 to 56, wherein the genome copies / joint (horse) or an equivalent amount as defined herein, such as a human, dog or cat equivalent amount.

64. The dose of gene construct is 1x10 8 ~6.5x10 9 57. The method of any one of claims 39 to 56, wherein the amount is genome copies / joint / kg (human) or an equivalent amount as defined herein, such as an equine, canine or feline equivalent amount.

65. The dose of the gene construct is 5x10 9 ~3.5x10 11 57. The method of any one of claims 39 to 56, wherein the amount is genome copies / joint / kg (human) or an equivalent amount as defined herein, such as an equine, canine or feline equivalent amount.

66. The dose of the gene construct is 2x10 4 Genome copies / joint / kg ~ 6.3 x 10 13 57. The method of any one of claims 39 to 56, wherein the amount is in genome copies / kg, or an equivalent amount as defined herein.

67. The dose of the gene construct was 3.8x10 4 Genome copies / joint / kg ~ 4.7x10 13 57. The method of any one of claims 39 to 56, wherein the amount is in genome copies / kg human, or an equivalent amount as defined herein.

68. The dose of the gene construct was 4.5x10 5 Genome copies / joint / kg ~ 6.3 x 10 13 57. The method of any one of claims 39 to 56, wherein the amount is genome copies / kg of horse, or an equivalent amount as defined herein.

69. The dose of the gene construct was 3.8x10 4 Genome copies / joint / kg ~ 1.2 x 10 13 57. The method of any one of claims 39 to 56, wherein the amount is genome copies / kg dog, or an equivalent amount as defined herein.

70. The dose of the gene construct is 2x10 4 Genome copies / joint / kg ~ 4.3 x 10 12 57. The method of any one of claims 39 to 56, wherein the amount is genome copies / kg of cat, or an equivalent amount as defined herein.

71. The dose of the gene construct is 5x10 7 Genome copies / knee, hip, or shoulder joint ~2x10 14 57. The method of any one of claims 39 to 56, wherein the genome copies per knee, hip or shoulder joint (human) or an equivalent amount as defined herein.

72. The dose of the gene construct is 5x10 7 Genome copies / knee, hip, or shoulder joint ~5x10 13 57. The method of any one of claims 39 to 56, wherein the genome copies per knee, hip or shoulder joint (horse) or an equivalent amount as defined herein.

73. The dose of gene construct is 1x10 5 Genome copies per knee, hip, or shoulder joint: 1x10 12 57. The method of any one of claims 39 to 56, wherein the genome copies per knee, hip or shoulder joint (dog) or an equivalent amount as defined herein.

74. The dose of the gene construct is 5x10 4 Genome copies / knee, hip, or shoulder joint ~7x10 11 57. The method of any one of claims 39 to 56, wherein the genome copies per knee, hip or shoulder joint (cat) or an equivalent amount as defined herein.

75. The dose of the gene construct is 4x10 7 Genome copies / knee, hip, or shoulder joint ~2x10 14 57. The method of any one of claims 39 to 56, wherein the genome copies per knee joint, hip joint or shoulder joint, or an equivalent amount as defined herein.

76. The dose of the gene construct is 5x10 8 Genome copies per knee, hip, or shoulder joint: 1x10 13 57. The method of any one of claims 39 to 56, wherein the genome copies per knee joint, hip joint or shoulder joint, or an equivalent amount as defined herein.

77. The dose of the gene construct is 5x10 8 Genome copies / knee, hip, or shoulder joint ~8x10 12 57. The method of any one of claims 39 to 56, wherein the genome copies per knee joint, hip joint or shoulder joint, or an equivalent amount as defined herein.

78. determining or adjusting the dose based on the subject's average joint size (e.g., average joint surface area or average joint volume), average joint cartilage volume, body weight, age and / or disease state compared to another subject, such as a different species, or using representative values ​​for the subject.

79. 79. The method of claim 78, wherein a subject's representative metric is determined for the subject, and optionally, the method further comprises determining the subject's average joint size (e.g., average joint surface area or average joint volume), average articular cartilage volume, body weight, age, and / or disease status.

80. 79. The method of claim 78, wherein the subject's metric representative values ​​are those of another subject representative of the subject, such as a healthy subject or another subject of the same species, and optionally the method further comprises determining the average joint size (e.g., average joint surface area or average joint volume), average articular cartilage volume, body weight, age and / or disease status of the other subject.

81. 81. The method of any one of claims 78 to 80, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in relation to body weight.

82. 81. The method of any one of claims 78 to 80, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in relation to age.

83. 81. The method of any one of claims 78 to 80, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in relation to body weight and age.

84. 81. The method of any one of claims 78 to 80, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in the context of the disease state.

85. 81. The method of any one of claims 78 to 80, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in relation to the disease state and body weight.

86. 81. The method of any one of claims 78 to 80, wherein the dose is or has been calculated or adjusted using average joint size (e.g. average joint surface area or average joint volume) or average joint cartilage volume in relation to disease state and age.

87. 87. The method of any one of claims 78 to 86, wherein the dosage is or has been calculated or adjusted using an average expression ratio (e.g. based on the promoters and / or regulatory elements of the construct) (such as an average promoter ratio and / or regulatory element ratio).

88. The method of any one of claims 39 to 77, further comprising the steps of the method of any one of claims 78 to 86.

89. A composition comprising any of the genetic constructs provided herein in any one dose provided herein and a pharmaceutically acceptable carrier.

90. A genetic construct according to any one of the Examples.

Images