Means and methods for the treatment of musculoskeletal diseases

Abstract

The invention relates to the treatment of musculoskeletal diseases, particularly joint inflammation disorders such as rheumatoid arthritis, spondyloarthritis, psoriatic arthritis, tendinitis and osteoarthritis involving the overexpression of the transcription factor BHLHE40 in the synovial fibroblasts of an affected mammal. The invention provides viral and non-viral vectors comprising BHLHE40 for the use to treat musculoskeletal diseases, particularly joint inflammation disorders.

Description

[0001] DiEI / synovfibros / 874

[0002] MEANS AND METHODS FOR THE TREATMENT OF MUSCULOSKELETAL DISEASES

[0003] Field of the invention

[0004] The invention relates to the field of musculoskeletal disorders, particularly joint inflammation disorders such as rheumatoid arthritis, spondyloarthritis, psoriatic arthritis and osteoarthritis. More specifically the invention relates to the treatment of musculoskeletal disorders, particularly joint inflammation disorders involving the overexpression of the transcription factor BHLHE40 in synovial fibroblasts of an affected mammal.

[0005] Introduction to the invention

[0006] Musculoskeletal disorders (MSDs) are conditions that affect the muscles, bones, joints, tendons, ligaments, and related tissues of the body. MSDs are complex and multifactorial diseases. One example is osteoarthritis (OA) which affects over 500 million people globally (1). As the most prevalent form of arthritis, OA significantly impacts the quality of life of afflicted individuals and imposes a substantial socio-economic healthcare burden (2, 3). The knee is the most common joint afflicted by OA, with patients experiencing chronic joint pain and stiffness, culminating in a progressive decline in mobility (4, 5). To measure the severity of knee (K)OA joint degeneration, radiographs are graded using the Kellgren-Lawrence (KL) system, with higher grades associated with more advanced disease (6). The field has acknowledged the importance of pre-radiographic, or "early OA", as a critical timepoint in which effective disease modifying therapeutics / treatments could be administered (7). In this study, we refer KL I as early-stage and KL 111 / IV as advanced-stage radiographic KOA. OA pathogenesis involves degradation of the articular cartilage, subchondral bone remodeling, and synovial fibrosis (9). Extracellular matrix (ECM)-rich synovium lines the joint capsule and functions to support joint health through the production of synovial fluid (required for lubrication), transport of nutrients into the joint, and removal of debris and waste (10). The synovium is made up of lining and sub-lining layers that are comprised of several different cell types including fibroblasts, macrophages, lymphocytes, and mast cells, among others (11). The most abundant cell type found in normal synovium is the fibroblast-like synoviocyte (referred to as fibroblasts throughout this invention). These cells secrete lubricating molecules, provide plasma-derived nutrients for joint tissues, and produce ECM components to maintain tissue architecture (12-14). OA synovium presents with severe structural changes including inflammation, hyperplasia of the lining layer, and increased ECM deposition (fibrosis) (15, 16). These histological changes are reflected by cellular and molecular changes evident even before cartilage degeneration becomes visible, which include fibroblasts proliferation, angiogenesis, mononuclear cell infiltration, and production of pro-inflammatory cytokines (17). Although some studies have investigated fibroblast populations associatedDiEI / synovfibros / 874

[0007] with lining and sublining layers in advanced stages of OA (18, 19), fibroblast subtypes in early and advanced stages of KOA, and their contribution to synovial fibrosis, are not well characterized.

[0008] To examine synovial fibroblasts in KOA, in the present invention we took a step-wise approach utilizing bulk RNA sequencing, single-nuclei RNA sequencing (snRNA-seq), flow cytometry, and advanced bioinformatic analyses, coupled with transgenic and surgical OA mouse models. We first created a catalogue of cells in the synovium, focusing on distinct fibroblast sub-populations present in early and advanced stages of radiographic KOA.

[0009] We have identified the transcription factor BHLHE40 as a negative regulator of fibrosis in synovial fibroblasts. We conclude that the presence of BHLHE40 in fibroblasts protects against arthritis. In the prior art opposite findings are disclosed by Zhang Y et al (2022) Cell & Bioscience 12, 70 who point at BHLHE40 as a causal protein for promoting arthritis, our own previous published conference abstract in Gracey, E etal (2022) Annals of Rheumatic Dis. Vol 81, issue Suppl. 1 and a granted Korean patent (KR10-2275313B1) which claims the use of antisense oligonucleotides against BHLHE40 for the treatment of osteoarthritis. Our recent findings were made possible by examining the expression of BHLHE40 specifically in fibroblasts through in vitro culture and conditional knockout mice. Our results differ from previous publications due to careful studying of the cell specific actions of BHLHE40. We observed that in macrophages, the other dominant cell type in the synovium, BHLHE40 is a negative regulator of the anti-inflammatory response. Therefore, the presence of BHLHE40 in macrophages would promote arthritis. In addition, we recently phenotyped a statistically more relevant number of full KO mice of BHLHE40 and we reached the conclusion that these mice had a worsening of the pathological bone formation despite a protection against soft tissue swelling. We therefore conclude that the target cell population for overexpression of BHLHE40 in the synovium are synovial fibroblasts which overexpression protects against synovial fibrosis and leads to a reduced joint damage and joint pain. Thus the present invention provides viral and non-viral vectors comprising the nucleotide sequence of BHLHE40 for treatment of musculoskeletal disorders, particularly for treatment of joint diseases.

[0010] Figures

[0011] Figure 1: Structural and transcriptomic shifts in the synovium correlate with knee OA progression. A) Schematic outlining the workflow of human and mouse experiments to identify cellular populations of synovial tissue. B) Masson's Trichrome staining of KLI (left) and KLIV (right) graded radiographic knee OA synovium. C) Volcano plot showing the Iog2 fold change (FC) of differentially-expressed genes (DEGs) in advanced stage samples compared to early samples. Genes with a log2FC > 0.5, q<0.05 are upregulated while genes with a log2FC < -0.5, q<0.05 are downregulated. D) Principal component analysis of gene expression profiles of early- and advanced-stage samples from bulk RNA sequencing.DiEI / synovfibros / 874

[0012] Figure 2: SnRNA-seq reveals major cell types and fibroblast subtypes in early- and advanced- stage knee OA synovium. A) Uniform manifold approximation and projection (UMAP) of all clusters identified in human synovium (left) through snRNA-seq. Proportion of cell types found in each sample (n=9) shown as a stacked bar graph (right). B) Dot plot showing the expression of marker genes used to annotate the clusters from KOA human synovium. C) All fibroblast subclusters identified in KLI and KLIII / IV graded radiographic knee OA synovium represented as a UMAP. D) Heatmap displaying gene expression differences between KLI and KLIII / IV dominant fibroblast subclusters. E) Density and violin plots of top two DEGs from fibroblast subclusters. F) Percent contribution of fibroblast subcultures to KLI & KLIII / IV stages. G) Individual patient UMAPs of fibroblast subclusters.

[0013] Figure 3: Fibroblasts putatively contribute to emerging populations and have unique communication patterns. A) Trajectory analysis showing changes in gene expression in fibroblast subclusters across pseudotime. B) Pseudotemporal changes in gene expression patterns across fibroblast subclusters. C) Jitter plots showing gene expression changes of top two DEGs across pseudotime in fibroblast subclusters. D) River plot showing outgoing communication pattern of secreting cells where outgoing pattens reveal how the sender cells coordinate with each other as well as how they coordinate with certain signaling pathways to drive communication (left). Dot plot showing contribution of outgoing communication of pathways in different Fibroblast sub-clusters (right).

[0014] Figure 4: DPP4 and ITGB8 define early- and advanced stage fibroblast subsets.. An independent cohort was generated to explore synovial fibroblast endotype transition with OA progression. A) Schematic outlining the workflow of human synovial tissue used in flow cytometry experiments. B) Gating strategy used to identify PDPN+ fibroblasts. C) FloSOM illustration of PDPN+ fibroblast subclusters, segregated by lining (CD55+) and sublining (CD90+). D) FloSOM illustration showing separate healthy control, early OA and advanced OA data. E) Percent positive ITGB8+ and DPP4+ fibroblasts depicted by OA grade. Multiple unpaired t-tests were performed and corrected for multiple testing. F) IHC staining of both DPP4+ and ITGB8+ cells in KLI- and KLI I l / l V-graded radiographic KOA human synovial tissue and positive cell count scoring. Positive cell counts were statistically analyzed using an unpaired, nonparametric, Mann Whitney test. Data in (E) and (F) are presented as mean ± standard deviation. Not significant (n.s.), *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

[0015] Figure 5: 1 tgb8+ fibroblasts emerge in synovium of a surgically-induced mouse model of KOA. A) Workflow showing mouse synovial tissue isolation, snRNA sequencing followed by bioinformatics analyses. B) All mouse synovial fibroblast subclusters identified in naive (non-surgical control; n=4), 2 week (n=3) and 10 week (n=4) post-DMM surgery represented in a UMAP. C) Individual fibroblast UMAPs of each mouse synovial sample analyzed. D) Stacked bar graph illustrating proportions of fibroblast subclusters in eachDiEI / synovfibros / 874

[0016] mouse subject. E) Percent positive nuclei for Dpp4 and Itgb8 in mouse synovium in control, 2 week and 10-week conditions. Data presented as mean ± standard deviation. Performed one-way ANOVA and corrected for multiple comparisons by controlling the false discovery rate using the two-stage step-up method of Benjamini, Krieger and Yekutieli. Not significant (n.s.), *, P < 0.05, **, P < 0.01, and ***, P < 0.001. F) Violin plots showing analogous human clusters 0 & 1 found in mouse clusters and density plots showing expression of ITGB8+ and DPP4+ fibroblasts in mouse snRNA sequencing data. G) Expression of human markers; ITGB8 and DPP4, on mouse fibroblast clusters. H) Heatmap illustrating the expression of top human DEGs from ITGB8+ and DPP4+ fibroblasts on mouse clusters 1 and 4. I) Dot plot showing expression of top human DEGs from each fibroblast subcluster onto mouse fibroblast clusters.

[0017] Figure 6: BHLHE40 is a key transcription factor regulating synovial fibroblast activation. A) Pathway-gene-TF interaction network showing putative transcription factors regulating early- and advanced-stage ECM genes and the pathways the genes are enriched for. B) Expression of advanced and early ECM genes across KLI (early) and KLIII / IV (advanced) predominant clusters. C) Volcano plot of matrisome-annotated genes up and downregulated in advanced- versus early-stage bulk RNA sequencing. Genes with a log2FC > 0.5, q<0.05 are upregulated while genes with a log2FC < -0.5, q<0.05 are downregulated. D) Heatmap illustrating matrisome genes upregulated in advanced- versus early-stage tissue. E) Expression of BHLHE40 / Bhlhe40 across all fibroblast subclusters in human and mouse. F) Schematic of in in vitro siRNA knockdown of transcription factor in advanced-stage fibroblasts. G) Graph showing relative gene expression of siRNA knockdown of BHLHE40 in advanced-stage fibroblasts (n=5) in vitro. Significance was determined using a two-tailed paired T-Tests, *, P < 0.05 was considered statistically significant. H & I) Heatmap and bar graph showing Log2FC of ECM and fibrotic genes significantly modulated by BHLHE40 siRNA knockdown. J) a-SMA and rhodamine phalloidin staining of BHLHE40 siRNA treated advanced-stage fibroblasts. Data presented as mean ± standard deviation, *, P < 0.05. Rhodamine Phalloidin = RP.

[0018] Figure 7: Fibroblast expression of Bhlhe40 negatively regulates synovial fibrosis in surgically-induced murine KOA. A) Schematic showing joint collection of WT and CKO mice being subjected to imaging and histology. B) Genotyping of Bhlhe40 CKO mice in ere- and cre+ mice. Bar graph showing knockdown of Bhlhe40 in fibroblasts and macrophages from synovium of WT and CKO mice. Multiple unpaired t-tests was performed and corrected for multiple testing. C) Masson's trichrome staining shown at 4 and 20X images of WT sham, WT DMM, CKO sham and CKO DMM mice synovium. D) Synovitis scoring of WT and CKO mice in both sham and DMM conditions. E) Safranin O fast green staining of the cartilage shown at 20x and 40X images. F) OARSI cartilage scoring illustrating cartilage degeneration in both WT and CKO mice under sham and DMM conditions. G) aSMA and Ki67 IHC staining of mouse synovium. H) Positive cell count scoring for aSMA and Ki67 IHC. Synovitis, OARSI and IHC positive cell count scoring wereDiEI / synovfibros / 874

[0019] statistically analyzed using a two-way ANOVA and corrected for multiple comparisons by controlling the false discovery rate using the two-stage step-up method of Benjamini, Krieger and Yekutieli. Data in (B), (D), (F) and (H) are presented as mean ± standard deviation. P values are shown as follows: not significant (n.s.) difference, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

[0020] Figure 8: Overexpression of BHLHE40 attenuates TGF-p-induced fibroblast activation. A) Western blot (left) showing lentivirus transduction of 50 MOI or 100 MOI results similar BHLHE40 expression after 2 days in advanced-stage fibroblasts from KOA synovium. B) Fibroblast cultures (right) were transduced with control or BHLH40 lentivirus (50 MOI) for 24 hours and subsequently treated with or without TGF-P (lOng / ml) for an additional 24 hours. Cells were immunostained to detect aSMA, and stained with rhodamine phalloidin to detect stress fibers and imaged by fluorescence microscopy. C) Schematic diagram depicting key findings of this study. Multiple cell types found in synovium of early and advanced-stage synovium, of which fibroblasts were the predominant cell type. DPP4+ fibroblasts were found to be the dominating fibroblast subtype in the synovium of early-stage KOA synovium while ITGB8+ fibroblasts were found to be the predominant subtype in the advanced-stage tissue. BHLHE40, a crucial transcription factor in ITGB8+ fibroblasts, was identified to regulate ECM expression and fibrosis. In vitro and in vivo knockout / knockdown of BHLHE40 was found to be associated with increased synovial fibrosis and excessive ECM deposition.

[0021] Figure 9: Overexpression of murine Bhlhe40 in mouse joints. A. Bhlhe40 adenovirus vector construct. B. Schematic of mouse joint collections subjected to sham and DMM surgeries and injected with Bhlhe40 and control AAV's. C. I VIS (Xenogen) live imaging of DMM mice injected with Bhlhe40 AAV. D. Masson's Trichrome staining at 4X and 20X images and (E) synovitis scoring of Bhlhe40 AAV and control AAV mice in both sham and DMM conditions. Data is presented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 and ****p < 0.0001.

[0022] Figure 10: A. Safranin O Fast Green stain of cartilage from mouse knee joints. B. OARSI cartilage scoring of Bhlhe40 AAV and control AAV mice in both sham and DMM conditions. Data is presented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

[0023] Detailed description of the invention

[0024] The present invention is described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are nonlimiting.DiEI / synovfibros / 874

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0026] In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0027] In referring to genes or proteins herein, no distinction is made in the annotation. Thus, whereas for example the human BHLHE40 gene would be referred to as the BHLHE40 gene, the mRNA as BHLHE40 mRNA, and the protein as BHLHE40, such distinction is not, or not always, made hereinabove or hereinafter.

[0028] It is to be understood that although particular embodiments, specific configurations as well as materials and / or molecules, have been discussed herein for methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.DiEI / synovfibros / 874

[0029] The term "treatment" refers to both therapeutic treatment and prophylactic / preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventative measures).

[0030] In the context of the present invention, the terms "protein" or "polypeptide" are to be understood as encompassing any oligopeptide comprising at least two amino acids covalently bonded to one another. The term "a protein" or "a polypeptide," as used herein, should be interpreted broadly to include the entirety or a portion of the referenced protein or polypeptide, as well as orthologous sequences from different mammals.

[0031] The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met, also indicated in one-letter code herein) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. A "substitution", or "mutation", or "variant" as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. It is understood that the skilled person can identify orthologous mammalian sequences of the human BHLHE40 nucleotide sequence, by searching databases such as NCBI-Genbank, EMBL Bl ENA, , Ensemble, KEGG and the like for example.

[0032] Synovial fibrosis is a common feature across musculoskeletal diseases such as for example arthritic diseases, such as but not limited to, osteoarthritis, spondyloarthritis, psoriatic arthritis, tendinitis and rheumatoid arthritis. Synovial fibrosis involves remodeling of the synovium, the layer of tissue that produces lubricants for the synovial joint and removes debris in the joint. Fibroblasts are producers of extracellular matrix. During arthritis, they change their phenotype to produce excess matrix, resulting in fibrosis. No disease modifying therapy is available for synovial joint diseases. Fibroblasts can respond to cytokines such as TNF and IL6. Biologies targeting these cytokines have only a clinical effect in about 60% of RA or SpA patients, but do not work at all in osteoarthritis.DiEI / synovfibros / 874

[0033] In the present invention we have found that the transcription factor BHLHE40 is a negative regulator of fibrosis in synovial fibroblasts. Therefore, the presence of BHLHE40 in fibroblasts protects against arthritis, thus the present invention is based on the finding that overexpression of BHLHE40 in fibroblasts is beneficial to prevent and / or to treat joint diseases, particularly joint diseases with a component of synovial fibrosis. This finding was made possible by examining the presence of BHLHE40 specifically in fibroblasts through in vitro culture and conditional knockout mice.

[0034] Therefore in a first embodiment the invention provides a polynucleotide sequence encoding the transcriptional repressor BHLHE40 for use to treat mammals suffering from musculoskeletal disorders. In another embodiment the invention provides a polynucleotide sequence encoding the transcriptional repressor BHLHE40 for use to treat mammals suffering from inflammatory joint diseases.

[0035] In a particular embodiment the invention provides a polynucleotide sequence encoding the transcriptional repressor BHLHE40 for use to treat mammals suffering from synovial fibrosis.

[0036] In another particular the invention provides a polynucleotide sequence encoding the transcriptional repressor BHLHE40 for use to treat mammals suffering from inflammatory joint diseases wherein a component (or hallmark of, or the underlying condition) of synovial fibrosis is present.

[0037] The term "musculoskeletal diseases or disorders" refers to conditions are conditions that affect the muscles, bones, joints, tendons, ligaments, and related tissues of the body. They can arise from both acute injuries and chronic overuse, often manifesting as pain, inflammation, stiffness, and reduced mobility. A non-limiting list of musculoskeletal disorders comprises rheumatoid arthritis, carpal tunnel syndrome, tendinitis and bursitis. The synovium is a specialized connective tissue that lines the inner surfaces of joint capsules, tendon sheaths, and bursae. It plays a critical role in joint function by producing synovial fluid, which lubricates and nourishes the joint.

[0038] The term "synovial fibrosis" refers to a pathological condition where excessive scar tissue (fibrosis) develops in the synovial membrane, the soft tissue lining the joints, tendons, and bursae. This process results from chronic inflammation, trauma, or prolonged joint stress, leading to thickening, stiffness, and reduced mobility in the affected joint. Typical causes of synovial fibrosis are i) chronic inflammation -which is common in rheumatoid arthritis (RA) or other inflammatory joint diseases, ii) trauma or injury because joint injuries or repeated microtrauma can trigger fibrosis, iii) post-surgical complications which can occur after joint surgeries, such as total knee replacement (TKA) or arthroscopy, iv) osteoarthritis such as knee osteoarthritis (KOA) wherein long-term joint wear and tear can lead to fibrosis and v) infections which can cause septic arthritis or other infections can cause synovial tissue damage and fibrosis. Symptoms of synovial fibrosis are recognized by joint stiffness and reduced rangeDiEI / synovfibros / 874

[0039] of motion, pain and discomfort, especially with movement, swelling and thickening of the affected synovial membrane and crepitus (grating sensation) in severe cases. Synovial fibrosis can be diagnosed by imaging wherein for example MRI or ultrasound can detect thickened synovium and fibrosis, synovial biopsy can confirm excessive fibrotic tissue and arthroscopy wherein a direct visualization of the joint can also assess fibrosis severity.

[0040] The wording "inflammatory joint diseases' generally refers to conditions that cause chronic inflammation in the joints, leading to pain, swelling, stiffness, and, in some cases, synovial fibrosis. These diseases can involve the immune system attacking the joints or other inflammatory triggers that damage joint structures. Examples of Inflammatory Joint Diseases are rheumatoid arthritis (RA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), reactive arthritis which develops after an infection (e.g. gastrointestinal or sexually transmitted infection), gout caused by uric acid crystal buildup in the joints, leading to acute inflammation, pseudogout caused by calcium pyrophosphate crystals, which also trigger inflammation, juvenile idiopathic arthritis (JIA), lupus arthritis (Systemic Lupus Erythematosus - SLE), infectious (septic) arthritis.

[0041] The polynucleotide sequence for use as defined in claim 1 which encodes the BHLHE40 polypeptide depicted in SEQ ID NO: 2.

[0042] BHLHE40 (Basic Helix-Loop-Helix Family Member E40) is a transcription factor involved in various cellular processes, including circadian rhythm regulation, immune response, and cancer progression. BHLHE40 acts as a transcriptional repressor or activator, depending on the target gene. The name BHLHE40 is also known in the literature by various synonyms, including Basic Helix-Loop-Helix Family Member E40, SHARP2, DECI, CAST5, BHLHB2, and STRA13. In the instant invention we use the name BHLHE40. The polynucleotide sequence of BHLHE40 in the instant invention also encompasses homologous nucleotide sequences from mammals such as humans, chimps, bonobos and veterinary animals such as livestock and pets including but not limited to horses, cows, dogs, cats, rabbits, sheep, goats. Thus the present invention also includes veterinary applications.

[0043] The mRNA sequence of the human BHLHE40 is depicted in SEQ ID NO: 1.

[0044] SEQ ID NO: 1 - Homo sapiens mRNA for BHLHE40

[0045] 5'-GGACACCGGGCCATGCACGCCCCCAACTGAAGCTGCATCTCAAAGCCGAAGATTCCAGCA GCCCAGGGGATTTCAAAGAGCTCAGACTCAGAGGAACATCTGCGGAGAGACCCCCGAAGC CCTCTCCAGGGCAGTCCTCATCCAGACGCTCCGCTAGTGCAGACAGGAGCGCGCAGTGGC CCCGGCTCGCCGCGCCATGGAGCGGATCCCCAGCGCGCAACCACCCCCCGCCTGCCTGCCDiEI / synovfibros / 874 CAAAGCACCGGGACTGGAGCACGGAGACCTACCAGGGATGTACCCTGCCCACATGTACCA AGTGTACAAGTCAAGACGGGGAATAAAGCGGAGCGAGGACAGCAAGGAGACCTACAAATT GCCGCACCGGCTCATCGAGAAAAAGAGACGTGACCGGATTAACGAGTGCATCGCCCAGCT GAAGGATCTCCTACCCGAACATCTCAAACTTACAACTTTGGGTCACTTGGAAAAAGCAGT GGTTCTTGAACTTACCTTGAAGCATGTGAAAGCACTAACAAACCTAATTGATCAGCAGCA GCAGAAAATCATTGCCCTGCAGAGTGGTTTACAAGCTGGTGAGCTGTCAGGGAGAAATGT CGAAACAGGTCAAGAGATGTTCTGCTCAGGTTTCCAGACATGTGCCCGGGAGGTGCTTCA GTATCTGGCCAAGCACGAGAACACTCGGGACCTGAAGTCTTCGCAGCTTGTCACCCACCT CCACCGGGTGGTCTCGGAGCTGCTGCAGGGTGGTACCTCCAGGAAGCCATCAGACCCAGC TCCCAAAGTGATGGACTTCAAGGAAAAACCCAGCTCTCCGGCCAAAGGTTCGGAAGGTCC TGGGAAAAACTGCGTGCCAGTCATCCAGCGGACTTTCGCTCACTCGAGTGGGGAGCAGAG CGGCAGCGACACGGACACAGACAGTGGCTATGGAGGAGAATCGGAGAAGGGCGACTTGCG CAGTGAGCAGCCGTGCTTCAAAAGTGACCACGGACGCAGGTTCACGATGGGAGAAAGGAT CGGCGCAATTAAGCAAGAGTCCGAAGAACCCCCCACAAAAAAGAACCGGATGCAGCTTTC GGATGATGAAGGCCATTTCACTAGCAGTGACCTGATCAGCTCCCCGTTCCTGGGCCCACA CCCACACCAGCCTCCTTTCTGCCTGCCCTTCTACCTGATCCCACCTTCAGCGACTGCCTA CCTGCCCATGCTGGAGAAGTGCTGGTATCCCACCTCAGTGCCAGTGCTATACCCAGGCCT CAACGCCTCTGCCGCAGCCCTCTCTAGCTTCATGAACCCAGACAAGATCTCGGCTCCCTT GCTCATGCCCCAGAGACTCCCTTCTCCCTTGCCAGCTCATCCGTCCGTCGACTCTTCTGT CTTGCTCCAAGCTCTGAAGCCAATCCCCCCTTTAAACTTAGAAACCAAAGACTAAACTCT CTAGGGGATCCTGCTGCTTTGCTTTCCTTCCTCGCTACTTCCTAAAAAGCAACAAAAAAG TTTTTGTGAATGCTGCAAGATTGTTGCATTGTGTATACTGAGATAATCTGAGGCATGGAG AGCAGATTCAGGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTGCGTGTGCGTGCAC ATGTGTGCCTGCGTGTTGGTATAGGACTTTAAAGCTCCTTTTGGCATAGGGAAGTCACGA AGGATTGCTTGACATCAGGAGACTTGGGGGGGATTGTAGCAGACGTCTGGGCTTTTCCCCDiEI / synovfibros / 874

[0046] ACCCAGAGAATAGCCCCCTTCGATACACATCAGCTGGATTTTCAAAAGCTTCAAAGTCTT GGTCTGTGAGTCACTCTTCAGTTTGGGAGCTGGGTCTGTGGCTTTGATCAGAAGGTACTT TCAAAAGAGGGCTTTCCAGGGCTCAGCTCCCAACCAGCTGTTAGGACCCCACCCTTTTGC CTTTATTGTCGACGTGACTCACCAGACGTCGGGGAGAGAGAGCAGTCAGACCGAGCTTTC TGCTAACATGGGGAGGTAGCAGGCACTGGCATAGCACGGTAGTGGTTTGGGGAGGTTTCC GCAGGTCTGCTCCCCACCCCTGCCTCGGAAGAATAAAGAGAATGTAGTTCCCTACTCAGG CTTTCGTAGTGATTAGCTTACTAAGGAACTGAAAATGGGCCCCTTGTACAAGCTGAGCTG CCCCGGAGGGAGGGAGGAGTTCCCTGGGCTTCTGGCACCTGTTTCTAGGCCTAACCATTA GTACTTACTGTGCAGGGAACCAAACCAAGGTCTGAGAAATGCGGACACCCCGAGCGAGCA CCCCAAAGTGCACAAAGCTGAGTAAAAAGCTGCCCCCTTCAAACAGAACTAGACTCAGTT TTCAATTCCATCCTAAAACTCCTTTTAACCAAGCTTAGCTTCTCAAAGGCCTAACCAAGC CTTGGCACCGCCAGATCCTTTCTGTAGGCTAATTCCTCTTGCCCAACGGCATATGGAGTG TCCTTATTGCTAAAAAGGATTCCGTCTCCTTCAAAGAAGTTTTATTTTTGGTCCAGAGTA CTTGTTTTCCCGATGTGTCCAGCCAGCTCCGCAGCAGCTTTTCAAGATGCACTATGCCTG ATTGCTGATCGTGTTTTAACTTTTTCTTTTCCTGTTTTTATTTTGGTATTAAGTCGTTGC CTTTATTTGTAAAGCTGTTATAAATATATATTATATAAATATATTAAAAAGGAAAATGTT TCAGATGTTTATTTGTATAATTACTTGATTCACACAGTGAGAAAAAATGAATGTATTCCT GTTTTTGAAGAGAAGAATAATTTTTTTTTCTCTAGGGAGAGGTACAGTGTTTATATTTTG GAGCCTTCCTGAAGGTGTAAAATTGTAAATATTTTTATCTATGAGTAAATGTTAAGTAGT TGTTTTAAAATACTTAATAAAATAATTCTTTTCCTGTGGAAG-3'

[0047] The polypeptide sequence of the human BHLHE40 is depicted in SEQ. ID NO: 2:

[0048] Polypeptide sequence of Homo sapiens BHLHE40 (from the amino-terminal to carboxy-terminal) NH2- MERIPSAQPPPACLPKAPGLEHGDLPGMYPAHMYQVYKSRRGIKRSEDSKETYKLPHRLIEKKRRDRINECIAQLKDLLPDiEI / synovfibros / 874

[0049] EHLKLTTLGHLEKAVVLELTLKHVKALTNLIDQQQQKIIALQSGLQAGELSGRNVETGQEMFCSGFQTCAREVLQYLAKH ENTRDLKSSQLVTHLHRVVSELLQGGTSRKPSDPAPKVMDFKEKPSSPAKGSEGPGKNCVPVIQRTFAHSSGEQSGSDT DTDSGYGGESEKGDLRSEQPCFKSDHGRRFTMGERIGAIKQESEEPPTKKNRMQLSDDEGHFTSSDLISSPFLGPHPH QPPFCLPFYLIPPSATAYLPMLEKCWYPTSVPVLYPGLNASAAALSSFMNPDKISAPLLMPQRLPSPLPAHPSVDSSVLLQ ALKPIPPLNLETKD-COOH

[0050] In a particular embodiment the polynucleotide encoding the BHLHE40 protein is a chemically modified polynucleotide sequence wherein the modification is at least one chemically modified nucleobase, sugar, backbone, or any combination thereof.

[0051] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a chemically modified nucleobase, for example, a chemically modified uracil, e.g., pseudouracil, Nl-methylpseudouracil, 5-methoxyuracil, or the like.

[0052] In certain aspects of the invention, when the modified uracil base is connected to a ribose sugar, as it is in polynucleotides, the resulting modified nucleoside or nucleotide is referred to as modified uridine. In some embodiments, uracil in the polynucleotide is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, at least 95%, at least 99%, or about 100% modified uracil. In one embodiment, uracil in the polynucleotide is at least 95% modified uracil. In another embodiment, uracil in the polynucleotide is 100% modified uracil. In embodiments where uracil in the polynucleotide is at least 95% modified uracil, overall uracil content can be adjusted such that an mRNA provides suitable protein expression levels while inducing little to no immune response.

[0053] In further embodiments, the ORF of the mRNA encoding a BHLHE40 polypeptide having modified uracil and adjusted uracil content has increased Cytosine (C), Guanine (G), or Guanine / Cytosine (G / C) content (absolute or relative). In some embodiments, the overall increase in C, G, or G / C content (absolute or relative) of the ORF is at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% relative to the G / C content (absolute or relative) of the wildtype ORF.

[0054] In further embodiments, alternative lower frequency codons are employed. At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, atDiEI / synovfibros / 874

[0055] least about 90%, at least about 95%, at least about 99%, or 100% of the codons in the BHLHE40 polypeptide encoding ORF of the modified uracil-comprising mRNA are substituted with alternative codons, each alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set. The ORF also has adjusted uracil content, as described above. In some embodiments, at least one codon in the ORF of the mRNA encoding the BHLHE40 polypeptide is substituted with an alternative codon having a codon frequency lower than the codon In some embodiments, the mRNA of the present invention induces a detectably lower immune response (e.g., innate or acquired) relative to the immune response induced by a corresponding wild-type mRNA under the same conditions. In other embodiments, the mRNA of the present disclosure induces a detectably lower immune response (e.g., innate or acquired) relative to the immune response induced by an mRNA that encodes for a BHLHE40 polypeptide but does not comprise modified uracil under the same conditions, or relative to the immune response induced by an mRNA that encodes for a BHLHE40 polypeptide and that comprises modified uracil but that does not have adjusted uracil content under the same conditions.

[0056] The disclosure includes modified polynucleotides comprising a polynucleotide described herein (e.g., a polynucleotide, e.g. mRNA, comprising a nucleotide sequence encoding a BHLHE40 polypeptide). The modified polynucleotides can be chemically modified and / or structurally modified. When the polynucleotides of the present invention are chemically and / or structurally modified the polynucleotides can be referred to as "modified polynucleotides."

[0057] The present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as mRNA polynucleotides) encoding a BHLHE40 polypeptide. A "nucleoside" refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase"). A "nucleotide" refers to a nucleoside including a phosphate group. Modified nucleotides can be synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides. The modified polynucleotides disclosed herein can comprise various distinct modifications. In some embodiments, the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide, introduced to a cell can exhibit one or more desirable properties, e.g., improved proteinDiEI / synovfibros / 874

[0058] expression, reduced immunogenicity, or reduced degradation in the cell, as compared to an unmodified polynucleotide.

[0059] In yet another particular embodiment the polynucleotide encoding the BHLHE40 protein is comprised in a viral vector.

[0060] In a particular embodiment the viral vector is a gene therapy vector.

[0061] In another particular embodiment said gene therapy viral vector comprises a lentivirus, an adenovirus, a herpes simplex virus or an adeno-associated virus.

[0062] In another particular embodiment the polynucleotide encoding the BHLHE40 protein is comprised in a non-viral vector.

[0063] In a particular embodiment said non-viral vector is comprises a liposome, a nanoparticle, or a lipid nanoparticle.

[0064] Gene therapy vectors

[0065] As used herein "vector", or "gene therapy vector", used interchangeably herein, refers to gene therapy delivery vehicles, or carriers, that deliver therapeutic genes to cells. A gene therapy vector is any vector suitable for use in gene therapy, e.g., any vector suitable for the therapeutic delivery of nucleic acid polymers (encoding a polypeptide or a variant thereof) into target cells (e.g. synovial fibroblasts) of a patient. In some embodiments, the gene therapy vector delivers the nucleic acid encoding a therapeutic protein or therapeutic fusion protein to a cell where the therapeutic protein or fusion is expressed and secreted from the cell. The vector may be of any type, for example it may be a plasmid vector or a minicircle DNA. Typically, the vector is a viral vector. These include both genetically disabled viruses such as adenovirus and nonviral vectors such as liposomes. The viral vector may for example be derived from an adeno-associated virus (AAV), a retrovirus, a lentivirus, a herpes simplex virus, or an adenovirus. AAV derived vectors. The vector may comprise an AAV genome or a derivative thereof.

[0066] In some embodiments, the viral vector is an AAV9 vector. In some embodiments, the expression cassette of the viral vector is flanked by AAV2 inverted terminal repeats (ITRs). ITRs used in alternative embodiments of the disclosed vectors include, but are not limited to, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the viral vector is an AAV2 / 9 vector. The notation AAV2 / 9 refers to an AAV vector have the ITRs of AAV2 and the capsid of AAV9. Other embodiments of the disclosure include without limitation AAV2 / 9, AAV5 / 9, AAVrh74, AAV2 / rh74, AAV5 / 9, and AAV5 / rh74 vectors. Other ITRs known in the art may be used. Exemplary ITRs (and other AAV components) useful in the vectors of the present disclosure include, without limitation, those described in U.S. Pat. No.DiEI / synovfibros / 874

[0067] 6,936,466B2, U.S. Pat. No. 9,169,494B2, US20050220766A1, US20190022249A1, and U.S. Pat. No.

[0068] 7,282,199B2, which are each incorporated by reference herein in their entireties.

[0069] In some embodiments, the vector is a retroviral vector, or more specifically, a lentiviral vector. As used herein, the term "retrovirus" or "retroviral" refers an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Retrovirus vectors are a common tool for gene delivery (Miller, 2000, Nature. 357: 455-460). Once the virus is integrated into the host genome, it is referred to as a "provirus." The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoded by the virus.

[0070] Illustrative retroviruses (family Retroviridae) include, but are not limited to: (1) genus gammaretrovirus, such as, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), and feline leukemia virus (FLV), (2) genus spumavirus, such as, simian foamy virus, (3) genus lentivirus, such as, human immunodeficiency virus-1 and simian immunodeficiency virus.

[0071] As used herein, the term "lentiviral" or "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2; visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (i.e., HIV cis-acting sequence elements) are preferred.

[0072] Retroviral vectors, and more particularly, lentiviral vectors, may be used in practicing the present invention. Accordingly, the term "retroviral vector," as used herein is meant to include "lentiviral vector"; and the term "retrovirus" as used herein is meant to include "lentivirus."

[0073] The term viral vector may refer either to a vector or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors contain structural and / or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term "lentiviral vector" refers to a viral vector containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus. The term "hybrid" refers to a vector, LTR or other nucleic acid containing both retroviral, e.g., lentiviral, sequences and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a vector orDiEI / synovfibros / 874

[0074] transfer plasmid comprising retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration and / or packaging.

[0075] In particular embodiments, the terms "lentiviral vector" and "lentiviral expression vector" may be used to refer to lentiviral transfer plasmids and / or infectious lentiviral particles. Where reference is made herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of these elements are present in RNA form in the lentiviral particles of the invention and are present in DNA form in the DNA plasmids of the invention.

[0076] Lipid nanoparticles (LNP)

[0077] In another specific embodiment the invention provides a lipid nanoparticle (LNP) comprising a polynucleotide or a polypeptide of the invention. Lipid-based nanoparticles are very small spherical particles composed of lipids. A lipid nanoparticle is typically spherical with an average diameter between 10 and 1000 nanometers. LNPs are made up of phospholipids, cholesterol, ionizable lipids, and polyethylene glycol-derived lipids (PEGylated lipids). The ionizable cationic lipids bind to mRNA, PEGylated lipids stabilize LNPs, and phospholipids and cholesterol give LNPs their structure. Because of rapid clearance by the immune system of the positively charged lipid, neutral ionizable amino lipids were developed. Solid lipid nanoparticles (SLNs) possess a solid lipid core matrix that solubilizes lipophilic molecules. Surfactants (emulsifiers) stabilize the lipid core. The emulsifier used depends on administration routes, and is more limited for parenteral administrations. The term "lipid" refers to a broader class of molecules, and includes triglycerides, diglycerides, monoglycerides, fatty acids, steroids (e.g. cholesterol), and waxes (e.g. cetyl palmitate). All classes of emulsifiers (with respect to charge and molecular weight) have been used to stabilize the lipid dispersion. It has been found that the combination of emulsifiers might prevent particle agglomeration more efficiently.

[0078] Pharmaceutical compositions

[0079] This invention also relates to pharmaceutical compositions containing a polynucleotides or polypeptides of the present invention for use to treat musculoskeletal diseases, particularly diseases affecting the joints. These compositions can be utilized to achieve the desired pharmacological effect by administration to a mammal, such as a patient in need thereof. A patient, for the purpose of this invention, is a mammal, including a human, in need of treatment for the particular condition or disease. Therefore, the present invention includes pharmaceutical compositions that are comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound, or salt thereof, of the present invention for use to treat musculoskeletal diseases, particularly joint diseases, particularly joint diseases which display synovial fibrosis. A pharmaceutically acceptable carrier isDiEI / synovfibros / 874

[0080] preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. A pharmaceutically effective amount of compound is preferably that amount which produces a result or exerts an influence on the particular condition being treated. The polynucleotides and polypeptides of the present invention can be administered with pharmaceutically-acceptable carriers well known in the art using any effective conventional dosage unit forms, including immediate, slow and timed release preparations, orally, parenterally, topically, nasally, ophthalmically, intra-articularly, intra-joint, optically, sublingually, rectally, vaginally, intrathecally, intracerobroventricully and the like.

[0081] Dose and administration:

[0082] Based upon standard laboratory techniques known to evaluate compounds useful for the treatment of joint diseases, by standard toxicity tests and by standard pharmacological assays for the determination of treatment of the conditions identified above in mammals, and by comparison of these results with the results of known medicaments that are used to treat these conditions, the effective dosage of the compounds of this invention can readily be determined for treatment of the indications cited herein. The amount of the active ingredient to be administered in the treatment can vary widely according to such considerations as the particular compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated.

[0083] The total amount of the active ingredient to be administered will generally range from about 0.001 mg / kg to about 200 mg / kg body weight per day, and preferably from about 0.01 mg / kg to about 50 mg / kg body weight per day. Clinically useful dosing schedules will range from one to three times a day dosing to once every four weeks dosing. In addition, "drug holidays" in which a patient is not dosed with a drug for a certain period of time, may be beneficial to the overall balance between pharmacological effect and tolerability. A unit dosage may contain from about 0.5 mg to about 1500 mg of active ingredient, and can be administered one or more times per day or less than once a day. The average daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg / kg of total body weight. The average daily rectal dosage regimen will preferably be from 0.01 to 200 mg / kg of total body weight. The average daily topical dosage regimen will preferably be from 0.1 to 200 mg administered between one to four times daily. The transdermal concentration will preferably be that required to maintain a daily dose of from 0.01 to 200 mg / kg. The average daily inhalation dosage regimen will preferably be fromDiEI / synovfibros / 874

[0084] 0.01 to 100 mg / kg of total body weight. The average daily oral dosage regimen will preferably be from 0.01 to 100 mg / kg of total body weight.

[0085] It is evident for the skilled artisan that the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific compound employed, the age and general condition of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like. The desired mode of treatment and number of doses of a compound of the present invention or a pharmaceutically acceptable salt or ester or composition thereof can be ascertained by those skilled in the art using conventional treatment tests.

[0086] Examples

[0087] 1.Distinct transcriptomic profiles of early- and advanced-stage KOA synovium

[0088] To begin exploring synovial pathologies during early and advanced stages of KOA, we first acquired synovial tissue samples from patients with KL I radiographic KOA (early-stage, undergoing arthroscopic surgery) and from patients with KL 111 / IV radiographic KOA [advanced-stage, undergoing total knee arthroplasty (TKA)] from a single center (Schroeder Arthritis Institute, Toronto Western Hospital, University Health Network, Toronto, ON, Canada). Harvested tissues were processed for histological investigation, or flash frozen for subsequent sequencing studies (Fig. 1A). Discernible structural differences were observed in the synovium of early and advanced stages of radiographic KOA using the Masson's Trichrome staining. Compared to early-stage synovia, enhanced deposition of ECM and thickening of the synovial lining layer in advanced-stage tissues was notable (Fig. IB).

[0089] To evaluate transcriptomic differences between early- and advanced-stage synovia, we subjected synovial tissue from early (KL I; n=6) and advanced stages (KL 111 / IV; n=8) of radiographic KOA to bulk RNA sequencing, identifying a total of 4,515 upregulated genes and 4,389 downregulated genes in advanced-compared to early-stage tissue (Fig. 1C). Principal component analysis confirmed separation of samples based on disease stage, with PCI explaining 91% of the variance (Fig. ID). These findings prompted us to perform a more granular investigation to determine which cell types may be driving synovial transcriptomic profiles during early and advanced stages of KOA.

[0090] 2.Cellular composition of early- and advanced-stage KOA synovium

[0091] We next subjected nine KOA synovium samples (five KL I and four KL 111 / IV) to snRNA-seq. After quality control and filtering, 25,285 nuclei were analyzed. Unsupervised clustering identified nine distinct cell types using canonical cell type markers (Fig. 2A and B). Identified cell types, listed from most to least abundant, were fibroblasts, macrophages, endothelial cells, adipocytes, lymphocytes, mural cells,DiEI / synovfibros / 874

[0092] dendritic cells, proliferating cells and mast cells. While the proportions of the cell types identified did have some variation between patients, the largest shift in proportion was observed between disease stages (Fig. 2A). In all subjects, fibroblasts were the predominant cell type identified, comprising approximately 50% of the sequenced nuclei in each synovial tissue sample.

[0093] 3. Nine

[0094]

[0095] distinct fibroblast su

[0096]

[0097] identified in human KOA

[0098]

[0099] Since fibroblasts were the predominant cell type identified in human KOA synovium, our subsequent analyses focused on this cell type. Unbiased clustering analysis was performed on nuclei determined to be fibroblasts, revealing nine transcriptionally distinct fibroblast subtypes based on differentially-expressed gene (DEG) profiles (Fig. 2C&D). These subtypes were defined by unique DEG lists consisting of a maximum of 235 genes (subcluster 0), to a minimum of 5 genes (subcluster 7). Of note, subcluster 8 had 0 DEGs. The top 5 genes associated with fibroblast subclusters 0 to 7, based on decreasing Log2FC (q<0.05, Log2FC>0.5, min.pct>0.25) included subcluster 0: PRG4, ITGB8, SEMA5A, CRTAC1, CLIC5; subcluster 1: DOCK4, PXDNL, KCNB2, FHOD3, KANK1; subcluster 2: ROBO2, DHRS3, AFF3, HMCN1, FBLN1; subcluster 3: KAZN, GLIS3, PTGFR, CXCL12, RUNX1; subcluster 4: LRRTM4, COL15A1, LAMA2, ABCA10, BMPER; subcluster 5: TEX41, STEAP2, EFNA5; subcluster 6: CNTN4, NRP1, PAK3, ELN, MBNL1; subcluster 7: MTSS1, TRIM22, SYNPO2, KLF7, MGLL (Fig. 2E).

[0100] We next compared our identified fibroblast clusters to characterizations from previously published studies. Fibroblasts from advanced-stage OA synovium have been categorized into two distinct populations: synovial sublining fibroblasts (SSF) and synovial intimal fibroblasts (SIF) (20). In our study, cluster 0 fibroblasts closely resembled the SIF (lining) population, while cluster 1 fibroblasts were more similar to the SSF (sublining) population. Comparing to the AMP-RA dataset of rheumatoid arthritis and late stage OA fibroblasts (21), early-stage OA fibroblasts from our study (clusters 1, 2, 4, and 6) resembled the SC-F1 (CD34+) population, while advanced-stage OA fibroblasts from our study (clusters 0, 3, and 5) resembled the SC-F4 (CD55+) population. These comparisons indicate that the fibroblasts identified in our study align with those from previously published studies.

[0101]

[0102] fibroblasts from early to advanced KOA

[0103] We next investigated whether there was disproportionate contribution of nuclei from early- or advanced-stage tissues to each identified fibroblast cluster. Interestingly, we determined that early-stage tissues contributed proportionally more nuclei to fibroblast clusters 1, 2, 4 and 6, while advanced-stage tissues contributed proportionally more nuclei to clusters 0, 3 and 5 (Fig. 2F, G). Subclusters 7 and 8 showed no significant differences in proportion of nuclei contributed by early or advanced-stage tissues. Subcluster 0, with the highest proportion of nuclei derived from advanced-stage KOA synovial tissues, was theDiEI / synovfibros / 874

[0104] predominant fibroblast subtype in advanced-stage tissue. In contrast, subcluster 1, with the highest proportion of nuclei contributed from early-stage tissues, was the predominant fibroblast subtype from early-stage KOA synovial tissues.

[0105] We next used trajectory analysis to determine if fibroblasts from early-stage tissue had the potential to differentiate to those found in advanced-stage tissue. Trajectory analysis suggested that advanced-stage fibroblast subtypes, namely clusters 0, 3 and 5, may be derived from early-stage fibroblast populations (Fig. 3A), through intermediate populations, via progressive changes in gene expression (Fig. 3A&B). This data suggests an endophenotypic shift in fibroblast subtypes in synovium from early to advanced stages of KOA. Of note, key genes contributing to the determined trajectory included DOCK4, PXDNL, CNTN4, ROBO2, LRRTM4, DHRS3 and COL15A1, which were also highly expressed in early-stage predominant clusters 1, 2, 4 and 6, and KAZN, TEX41, GLIS3, ITGB8, and PRG4, which were also highly expressed in advanced-stage predominant clusters 0, 3 and 5 (Fig. 3C).

[0106] We further investigated putative cell-cell interaction patterns between major cell types and subtypes using CellChat v2.0(22). Comparing the top four major cell types (fibroblasts, macrophages, endothelial cells, adipocytes), signaling within fibroblasts exhibited the highest interaction strength, prompting us to further investigate interactions among fibroblast subtypes. Within fibroblast subtypes, the strongest interaction strengths were observed directed towards fibroblast cluster 0. To further define signaling towards fibroblast cluster 0 from other subtypes, ligand-receptor cell signaling interactions and signaling pathways were investigated with fibroblast cluster 0 set as the "receiver," expressing receptors, and all other fibroblast subtypes set as "senders," expressing ligands. A number of communication patterns to fibroblast cluster 0 from other fibroblast subtypes were identified, with the highest proportion of signals being received by fibroblast subcluster 0. We also found that the various fibroblast subtypes had multiple signaling patterns composed of various pathways. Interestingly fibroblast subtypes 0 and 5 both followed pattern 2, which associated with TGF-P, FN1, PDGF and other outgoing communication pathways. In contrast, fibroblast subtypes 1, 3, 4 and 6 were associated with distinct communication patterns linked to various unique outgoing communication pathways including NOTCH, VCAM, and non-canonical (nc)WNT. (Fig. 3D).

[0107] Overall, fibroblast subcluster 0 was found to be predominant in the advanced stages of the disease while subcluster 1 was predominant in the synovium of early-stage KOA. Furthermore, we determined that synovial fibroblasts likely undergo an endotypic shift from early to advanced stages of KOA.DiEI / synovfibros / 874

[0108] 5.Flow cytometry confirms distinct fibroblast sub-populations in KOA

[0109] To independently confirm the presence of major fibroblast clusters observed by snRNA-seq, an independent cohort of early- and advanced-stage KOA patients was generated at a second center (Ghent, Belgium). This cohort also included anatomically matched healthy synovium obtained from post-mortem donors with no history of musculoskeletal disease. Suprapatellar pouch synovial tissue was formalin fixed for histology and cryopreserved for flow cytometry (Fig. 4A).

[0110] Cryopreserved synovial tissue was thawed and enzymatically digested. Consistent with previous reports of synovial hypertrophy in OA (1), a progressive increase in cell yield was observed from healthy tissue through to advanced-stage KOA synovium. A 19-marker flow cytometry panel was assembled to examine subsets of fibroblasts identified by snRNA-seq. First, pan-MHCI (HLA-ABC) and a fixable viability marker were used to separate live cells from debris, following which, lineage markers were used to gate out immune cells (CD45+), endothelial cells (CD31+) and mural cells (CD146+) before using PDPN to identify fibroblasts (Fig. 4B). As expected, immune cells and fibroblasts were the dominant cell types identified in the synovium. Of note, we found a moderate increase in immune cells with progressive KOA; noteworthy given that OA is often used as "non-inflammatory" control tissue for inflammatory arthritis studies (21).

[0111] A clear shift in broad KOA fibroblast populations was observed when using conventional markers for subsets and activation. CD90 and CD55 are commonly used to identify sub lining and lining fibroblast respectively (23). Using these markers, we found an increase in the frequency of lining fibroblasts in advanced- compared to early-stage KOA synovium, consistent with lining layer hyperplasia formation observed by histology (Fig. IB) (1). Further, fibroblast activation protein (FAP) and CD63, additional fibroblast activation markers, were elevated with KOA compared to healthy controls, but were only moderately increased with stage of KOA.

[0112] FlowSOM was next used to unbiasedly cluster fibroblasts in our flow cytometry data using markers identified by snRNA-seq (Fig. 4C). By examining the intensity of markers on each cluster, we identified 11 metaclusters. Many of the metaclusters appeared to match those in our snRNA-seq analyses based on expression profiles of key markers including DPP4, CD34, ITGB8 and PTGFR. Two notable metaclusters included DPP4+CD34+ fibroblasts, which endotypically resembled snRNA-seq cluster 1 fibroblasts, and ITGB8+ fibroblasts, which resembled snRNA-seq cluster 0 fibroblasts. Thus, cluster 0, the major predominant fibroblast population in advanced-stage KOA synovium, will now be referred to as ITGB8+ fibroblasts, and cluster 1, the predominant fibroblast population in early-stage KOA synovial tissue, will now be referred to as DPP4+ fibroblasts.DiEI / synovfibros / 874

[0113] We found a gradient in the distribution of fibroblasts across metaclusters from healthy to early- and advanced-stage KOA, with ITGB8+CD55+ fibroblast clusters coming primarily from advanced-stage KOA synovium (Fig. 4D). Examination of these clusters on a per subject basis revealed a significant reduction in the frequency of DPP4+ fibroblasts and an increase in ITGB8+ fibroblasts with KOA progression (Fig.

[0114] 4E). Immunohistochemistry (IHC) further showed that ITGB8+ cells were more frequent in advanced-stage tissue (Fig. 4F). Conversely, DPP4+ cells were more frequent in early-stage tissues compared to advanced-stage tissue.

[0115] Overall, this data confirms the endotypic shift in fibroblasts from early- to advanced-stages of the disease, and demonstrates more subtle phenotypic changes from healthy to the early stage. Consistent with the findings from our snRNA-seq data, flow cytometry further validated DPP4+ fibroblasts as the major fibroblast subcluster found in the synovium during early-stage KOA whereas ITGB8+ fibroblasts are the major fibroblast subcluster found in synovium of advanced-stage KOA.

[0116] 6. fibroblasts

[0117]

[0118] KOA in a mouse model

[0119] Having observed the same fibroblast endotypes with different techniques in two independent human OA cohorts, we next sought to determine if similar early- and advanced-stage predominant fibroblast populations were present in murine KOA. Destabilization of the medial meniscus (DMM) surgery was performed in mice and synovia was collected at two (n=3) or ten weeks (n=4) post-surgery. Naive mouse synovia (non-surgical) was collected as a control (n=4). Collected tissue was subjected to snRNA-seq (Fig.

[0120] 5A). A total of 19,304 nuclei were analyzed after filtering and subjected to unsupervised clustering, which resolved a total of ten distinct cell types utilizing canonical cell surface markers. Further clustering analysis of fibroblasts identified eight transcriptionally distinct fibroblast subclusters (Fig. 5B). A change in the proportion of the fibroblast subtypes was identified as the model progressed from early (two weeks) to advanced stages (10 weeks) (Fig. 5C, D). Higher proportions of Dpp4+ nuclei were present in synovia from control and week 2 post-surgery mice, whilst higher proportions of Itgb8+ nuclei were found in tissues from both week 2 and week 10 post-surgical mice compared to controls (Fig. 5E). These findings emphasize that transcriptomically distinct fibroblast subsets are also present at various stages of murine KOA, akin to our findings in synovia from human KOA.

[0121] We next sought to identify if analogous mouse fibroblast subtypes resembled human fibroblast subtypes identified by snRNA-seq analysis of human synovia. Modules of DEGs from human fibroblast subpopulations were assessed in each mouse fibroblast subtype, illustrating that human ITGB8+ fibroblasts (cluster 0) were most similar to mouse cluster 1 fibroblasts, while human DPP4+ fibroblasts (cluster 1) were most similar to mouse cluster 4 fibroblasts (Fig. 5F). Itgb8 exhibited highest expression in mouse fibroblast cluster 1, with Col22al, Fnl, and Ccn2, also highly expressed in both mouse fibroblastDiEI / synovfibros / 874

[0122] cluster 1 and human ITGB8+ population (cluster 0). In contrast, Dpp4 was predominantly expressed in mouse fibroblast cluster 4, with Fbnl, Flrt2 and Ltbpl also highly expressed in both mouse fibroblast cluster 4 and the human DPP4+ fibroblast population (cluster 1) (Fig. 5G&H). Additionally, top DEGs from each human fibroblast cluster were scored against each mouse cluster, further confirming similarities between human ITGB8+ and DPP4+ fibroblasts and mouse fibroblast clusters 1 and 4, respectively (Fig.

[0123] 51). These findings support similarities between key human and mouse fibroblast subtypes during progressive stages of KOA.

[0124] 7.DPP4+ and ITGB8+ fibroblasts are associated with ECM-related pathways

[0125] Following investigation of fibroblast subpopulations in two independent human cohorts, and in a KOA mouse model, we next investigated putative functional roles of major human fibroblast clusters. Pathway enrichment analysis was performed on DEGs from human fibroblast subclusters 0-6 using gene ontology and pathDIP v4 (24). Comprehensive pathway analysis identified many pathways enriched in each fibroblast subset. Interestingly both predominant human fibroblast subpopulations (ITGB8+ and DPP4+) shared common pathways including: extracellular matrix organization, extracellular structure organization, neuron projection guidance, axon guidance, axonogenesis, positive regulation of neuron projection development and regulation of cell morphogenesis. It is established that excessive fibroblast proliferation and ECM deposition can contribute to the development of synovial fibrosis (25, 26). Given that ITGB8+ and DPP4+ fibroblasts were both associated with ECM-related pathways, we directed our subsequent studies to investigate these two major subpopulations.

[0126] 8. Identification of BHLHE40 as an upstream transcriptional regulator linked to ECM pathways

[0127] A total of 48 ECM-related DEGs from ITGB8+ (24 genes) and DPP4+ (24 genes) fibroblast populations were enriched with ECM-related pathways (Fig. 6A&B). These genes were then subjected to Catrin (27) analysis to identify putative enriched upstream transcription factors regulating these genes (Fig. 6A). Catrin is an integrated database used to predict regulatory transcription factors (TFs) of query genes (https: / / ophid.utoronto.ca / Catrin / ). Catrin analysis identified that ECM pathway-related DEGs from ITGB8+ fibroblasts were putatively regulated by 31 transcription factors, while ECM pathway-related DEGs from DPP4+ fibroblasts were putatively regulated by 39 transcription factors.

[0128] To investigate differences in ECM-related transcriptomes in early- and advanced-stage KOA synovia, we also assessed DEGs in our bulk-RNAseq analysis for matrisome (ECM-associated genes) annotations. Out of a total of 1027 matrisome genes, 731 were collectively expressed in early and advanced stage KOA synovium. Of the 731 expressed genes, 50.69% (n=372) were differentially-expressed in advanced- versusDiEI / synovfibros / 874

[0129] early-stage KOA synovium, with 37.48% (n=274) upregulated in advanced-stage tissue and 13.41% (n=98) upregulated in early-stage tissue (Fig. 6C, & D).

[0130] We next focused on the advanced-stage KOA synovium, particularly ITGB8+ fibroblasts, due to high ECM gene expression and evident synovial fibrosis observed in tissues from this disease stage. Further investigation of the ECM-regulatory transcription factors from our network analysis found that BHLHE40 was the only TF that putatively regulated at least 80% (20 / 24) of the ECM gene list in ITGB8+ cells, and its expression was significantly higher between ITGB8+ and DPP4+ fibroblasts in humans(Fig. 6E). BHLHE40 (DEC1 / STRA13) is a broadly expressed transcription factor with repressive functions (28). It was initially identified for its minor role in circadian rhythms (29), and more recently was found to regulate immune cell function (30, 31). Since our data show that BHLHE40 is a crucial upstream transcriptional regulator linked to ECM pathways, this prompted us to further evaluate its role in fibroblast activation and synovial fibrosis.

[0131] 9.BHLHE40 negatively regulates ECM expression and fibroblast activation in vitro

[0132] To begin investigating the link between BHLHE40 and the ECM transcriptome, fibroblast activation and fibrosis, we targeted BHLHE40 in cultures of fibroblasts obtained from synovium of advanced-stage KOA. Using siRNA knockdown, we reduced BHLHE40 expression by an average of 66.4% (KLIII / IV; n=5) (Fig. 6F, G). RNA collected from BHLHE40 siRNA- and control siRNA-treated cultures was subjected to analysis using a custom-made NanoString panel comprising of 767 (fibrosis-related genes and ECM genes related to BHLH40). Knockdown of BHLHE40 in fibroblasts led to a notable increase in the expression of 41 fibrosis-related genes and a significant decrease in expression of only 5 genes (Fig. 6H, I). Next, we investigated the effects of BHLHE40 silencing on fibroblast activation. Activated fibroblasts express a-smooth muscle actin (aSMA), a marker of fibroblast activation (32), and induce stress fiber formation (14). Cultured fibroblasts with siRNA-targeted BHLHE40 knockdown were stained for aSMA and rhodamine phalloidin. BHLHE40 knockdown revealed fibroblast activation associated with enhanced stress fiber formation, cell spreading and increased aSMA expression (Fig. 6J). These data suggest that BHLHE40 may play a role in inhibiting fibroblast activation and ECM gene expression, consistent with its proposed function as a transcriptional repressor.

[0133] 10.BHLHE40 conditional knockout mice exhibit severe synovial fibrosis

[0134] To further investigate the influence of BHLHE40 on ECM regulation and synovial fibrosis in vivo, we generated a conditional knock out (CKO) mouse. Here, Bhlhe40 was deleted by Cre-mediated recombination in cells expressing Col6al (33), which include synovial fibroblasts (Fig. 7A). CKO mouse knee fibroblasts showed a significant reduction in Bhlhe40ger\e expression compared to those from wild-DiEI / synovfibros / 874

[0135] type (WT) mice, without affecting expression in synovial macrophages (Fig. 7B), confirming selectivity of targeted deletion of Bhlhe40 in knee synovial fibroblasts in vivo. 12-week-old WT and CKO mice were subjected to sham or DMM surgery to evaluate the contribution of mouse fibroblast-intrinsic Bhlhe40to KOA pathogenesis. Interestingly, even in the absence of surgery, sham CKO mice exhibited significant development of synovial fibrosis, evidenced by increased thickening of the synovial lining and matrix deposition, as compared to sham WT mice. Furthermore, DMM CKO mice exhibited even more pronounced synovial fibrosis in comparison to DMM WT, Sham WT and Sham CKO mice (Fig. 7C), with DMM CKO mice having the highest synovial fibrosis scores (Fig. 7D). Cartilage integrity was also evaluated in safranin O-stained sections using OARSI grading criteria for mice (34) to assess differences between WT and CKO mice subjected to sham or DMM surgery. Prominent medial cartilage degeneration was present in WT and CKO mice subjected to DMM surgery, as compared to sham-surgical animals (Fig. 7E); however, no significant differences in OARSI scores of the tibial plateau (TP) or femoral condyle (FC) were observed in sham or DMM groups between genotypes (Fig. 7F).

[0136] Since we observed enhanced synovial thickening and fibrosis in CKO mice compared to WT mice, we further analyzed changes in protein expression of aSMA (activated fibroblasts), and Ki67, a marker of proliferation (35). IHC scoring of aSMA-stained sham mouse synovia showed significant increases in the number of aSMA-positive cells in the CKO mice compared to WT mice, however no significant differences were observed between DMM WT and DMM CKO mice (Fig. 7G, H). Ki67 scoring illustrated a significantly increased number of positive cells in Sham CKO mice compared to Sham WT mice. DMM CKO mouse synovia also had significantly higher Ki67 scores compared to DMM WT mouse synovia (Fig. 7H). Overall, these results show that loss of Bhlhe40 leads to severe synovial fibrosis associated with increased fibroblast activation and proliferation in vivo.

[0137] 11.BHLHE40 ion suppresses TGF-B-induced fibroblast activation

[0138] Since BHLHE40 CKO mice developed severe synovial fibrosis, and BHLHE40 knockdown in vitro resulted in enhanced aSMA expression, fibroblast activation and stress fiber formation, we sought to overexpress BHLHE40 in KOA fibroblasts using a lentivirus approach to evaluate its effects on fibroblast activation. We first tested our lentivirus transduction efficiency, and observed that 50MOI was sufficient for transduction and target protein expression (Figure 8A). We next used four experimental conditions to evaluate the effect of BHLHE40 on fibroblast activation: control lentivirus; control lentivirus + TGF-P; BHLHE40 lentivirus; and BHLHE40 lentivirus + TGF-p. TGF-P is a known inducer of fibroblast activation and was therefore used in this investigation's). As expected, fibroblast activation, stress fiber formation, and cellular aggregation were increased in cultures treated with control lentivirus + TGF-p. In contrast, BHLHE40 transduction alone did not result in significant morphological or phenotypic changes inDiEI / synovfibros / 874

[0139] fibroblasts compared to the control lentivirus-treated cultures alone. Notably, fibroblasts transduced with BHLHE40 lentivirus and treated with TGF-P exhibited a marked reduction in the activated phenotype, with reduced aSMA expression, stress fibers and a more spindle-shaped morphology, compared to cultures transduced with control lentivirus and treated with TGF-p. These findings show that overexpression of BHLHE40 suppresses fibroblast activation under fibrotic conditions and further supports its role as a crucial mediator of fibroblast activation.

[0140] 12.Surgical induction of osteoarthritis and intra-articular injection of a recombinant lentivirus

[0141] We generated four different recombinant lentiviral viruses which express the following:

[0142] 1. human BHLHE40 and eGFP

[0143] 2. eGFP alone, as a control for construct 1,

[0144] 3. mouse BHLHE40 and nano-Luc

[0145] 4. nano-luc alone as a control for construct 3.

[0146] For the mouse studies, we use virus constructs 3. and 4.

[0147] Lentiviral stocks containing lentivirus capable of transducing cells to express BHLHE40, in addition to control vectors are intra-articularly injected into C57BL / 6 mice. Prior to injection, animals are subjected to the destabilization of the medial meniscus (DMM) surgery, or Sham surgery. For the DMM surgery, an 8 mm skin incision centered 3 mm medially to the patella is performed. The subcutaneous tissues is dissected to the joint capsule. A medial arthrotomy is performed and the patella is then luxated laterally. The cranial pole of the medial meniscus is visualized; the cranial medial menisco-tibial ligament identified and transacted at its insertion to the meniscus. The arthrotomy is closed followed by closure of subcutaneous tissue and skin closure. Sham surgery consists of the same procedures with the exception of the cranial medial menisco-tibial ligament transaction. Two and 4 weeks-post DMM or sham surgery, either BHLHE40 (construct 3) or control lentivirus (construct 4) is injected into the surgical joints at a concentration of 5.6x10slentiviral particles in up to 7ul PBS per joint using a Hamilton syringe. Joints are collected up to 10 weeks post-surgery.

[0148] Joint Histology and Disease Scoring

[0149] Mouse joint tissue is collected and fixed with 10% neutral buffered formalin, transferred into 70% ethanol, and decalcified, embedded, and processed. Five-micron sections are be stained with Masson's trichrome (HT10516, Sigma Aldrich) (26367-04, Electron Microscopy Sciences) for analysis of synovial fibrosis and Safranin O (S2255-100G, Signma Aldrich) / Fast Green staining (2353-45-9, Bio Basic Canada)DiEI / synovfibros / 874

[0150] (26367-02, Electron Microscopy Sciences) for analysis of cartilage integrity. Both synovial fibrosis and cartilage integrity are evaluated by synovitis / OARSI scoring described for mouse (see PMID: 20864019).

[0151] 13. intra-articular injection of a recombinant lentivirus in a RA model

[0152] The collagen-induced arthritis model is outlined in PMID: 17546023. In brief mice are immunized at the base of the tail with type II collagen in complete freund adjuvant, a repeat immunization is given 3 weeks after the first injection. Joint swelling occurs following the second injection and joint swelling is monitored visually. After the second injection either the recombinant lentivirus comprising BHLHE40 (construct 3 from examplel2) or control lentivirus (construct 4 from example 12) is injected into the surgical joints at a concentration of 109AAV virus particles in up to 8ul PBS per joint using a Hamilton syringe. Joints are collected up to 10 weeks post-surgery.

[0153] At the endpoint, arthritis is assessed by histopathology. Synovial fibrosis (thickening of the lining layer and infiltration of the sublining) is monitored.

[0154] 14. intra-articular injection of a recombinant AAV2 virus in an osteoarthritis model

[0155] Mice were subjected to destabilization of the medial meniscus (DMM) or SHAM surgeries and received intraarticular injections of either Bhlhe40 or control AAV2 particles (lxlO9particles / joint) (see materials and methods for the production of recombinant AAV2) beginning two weeks post-surgery with subsequent injections administered every four days for ten weeks (Fig 9B). The four experimental groups included: control AAV sham (n=9), control AAV DMM (n=10), Bhlhe40 AAV sham (n=9) and Bhlhe40 AAV DMM (n=10). DMM mice injected with Bhlhe40 AAV2 particles were administered Nano-Gio Fluorofurimazine In Vivo Substrate (Promega) according to manufacturer's directions and subjected to MS (Xenogen) live Imaging 3 weeks post-first injection to detect NanoLuc and confirm adenoviral transduction activity (Fig 9C). Mouse Knee joints were collected 10 weeks post-surgery and assessed for synovitis and cartilage degeneration. Mouse joints were processed for histopathological analysis and stained with Masson's Trichrome and Safranin O-Fast Green stain. Synovitis scoring was performed on mouse synovial tissue and cartilage degeneration was evaluated using the OARSI scoring system on both tibial plateaus and femoral condyles. Masson's Trichrome staining of mouse synovium revealed a significant reduction in synovial hyperplasia and fibrosis in DMM mice injected with Bhlhe40 AAV in comparison to Control AAV DMM mice, suggesting that Bhlhe40 overexpression attenuates OA synovial pathology in mouse model of KOA (Fig 9 D, E). In contrast, quantification of cartilage degeneration by OARSI scoring revealed no significant differences in cartilage integrity between groups (Figure 10 A and B).DiEI / synovfibros / 874

[0156] Materials and methods

[0157] 1. & OA Patient Recruitment

[0158] Patients were recruited from the Schroeder Arthritis Institute, Toronto Western Hospital, University Health Network using Research Ethics Board (REB)-approved protocols and under informed written consent (early samples 16-5969-AE, advanced samples 07-0383-BE). All patients met the American College of Rheumatology's definition of knee OA(52). This study ensures compliance with all relevant ethical regulations. For single-nuclei RNA sequencing and bulk RNA sequencing, OA human knee synovial tissue samples were retrieved from patients exhibiting KL I radiographic knee OA undergoing arthroscopic surgery and from with KL 111 / IV undergoing total knee arthroplasty. Synovial tissue samples were processed for histology and stored in liquid nitrogen for sequencing. For our independent second cohort, KOA synovium was obtained from patients during total knee arthroplasty, routine knee arthroscopy or cadavers for healthy controls. All patients provided informed consent to participate in this study, under study number 2019 / 1922-BC-06496 (Commissie voor Medische Ethiek, UZ Gent).

[0159] 2. Human ium Hi:

[0160]

[0161] Harvested human knee OA synovial tissues were isolated and fixed in 10% neutral buffered formalin for at least 48 hours at 4°C. The tissues were embedded in paraffin and serial sections (4pm) were stained with Masson's trichrome (Sigma-Aldrich) and, as per manufacturers guidelines, after the sections were de-paraffinized and rehydrated.

[0162] 3. Bulk RNA sequencing

[0163] For bulk RNA sequencing analysis, n=6 KL I and n=8 KL 111 / IV radiographic knee OA synovial tissues were used. RNA was isolated from frozen synovia (~50 mg) using the Qiagen RNeasy Mini kit (Qiagen, 74104). Quality of total RNA isolated was assessed using an RNA Nano chip on an Agilent Bioanalyzer (Agilent, Santa Clara, CA, USA). Samples were fluorometrically quantified using a Qubit RNA BR assay (ThermoFisher) on a Denovix DS-11 spectrophotometer (Denovix, Wilmington, DE, USA). For each sample, 200 ng RNA was used to prepare sequencing libraries using the TruSeq Stranded Total RNA with RiboZero, as per manufacturer's recommendations (Illumina, San Diego, CA, USA), as previously described(53). Library quality was assessed on a high-sensitivity DNA chip on the Bioanalyzer (Agilent). Sequencing libraries were volumetrically pooled and sequenced on an Illumina NextSeq 550 sequencer for 150 paired end read-cycles at the Schroeder Arthritis Institute (Krembil, Toronto, ON, Canada). Raw sequenced reads were assessed for quality with Cutadapt (v3.0)(54), used to maintain minimum read length of 25 bp post trimming of adapters along with trimming of N's. Splice-aware alignment of reads using a Hierarchical Graph FM index (HGFM) method was performed using HISAT2DiEI / synovfibros / 874

[0164] software (v2.2.1)(55) against human reference genome (vGRCh38). To populate the abundance of transcripts based on the reference genome and transcriptome, StringTie (v2.1.4)(56) was run to generate outputs as table format files.

[0165] Gene expression read counts were analyzed to identify DEGs using Bioconductor package DESeq2 (version 1.36.0)(57). Lowly expressed genes, where less than 2 samples had a counts less than or equal to 10, were filtered out. Genes with adjusted P value <0.05 and absolute Iog2 fold change >0.5 were considered to have significant altered expression. Unsupervised clustering was used to generate the heatmap using heatmap.2 function.

[0166] 4.Single-Nuclei RNA

[0167]

[0168] Single-nuclei RNA sequencing was performed at the Princess Margaret Genomics Centre, UHN. OA synovial tissue was disaggregated into single-nuclei suspension, as described (58). To prepare for mechanical and enzymatic disaggregation, 30-50 mg of synovial tissue were cut into l-2mm3using a cold razor on dry ice. The tissue was covered with lysis buffer (IM sucrose, IM CaCL, IM Mg(Ac)j, IM Tris-HCI, Triton X-100, 0.5M EDTA pH 8, RNase Inhibitor (40U / pl), H2O) on ice and was mechanically disaggregated. A Kimble Dounce tissue grinder (Sigma-Aldrich) was used to homogenize the tissue and the supernatant was collected in lysis buffer. The solution was centrifuged three times at 800g for 10 minutes and washed with 2 ml wash buffer (IX PBS, 10% BSA, RNase Inhibitor (40U / pl), with the final resuspension using 1 ml wash buffer. The solution was filtered using a 40pm Flowmi® cell strainer (Sigma-Aldrich) and transferred into a 1.5 ml LoBind tube on ice. Single-nuclei suspension was assessed for nuclei quantity and viability with DAPI (Sigma-Aldrich) staining. FAC's was used to sort out the nuclei and was centrifuged at 800g for 10 minutes. The pellet was resuspended in wash buffer to adjust the final concentration to 1000 nuclei / pl. The lOx Genomics Chromium system was used to construct a lOx barcoded library. During the reaction, each 10X barcoded bead (v3.1) was encapsulated with one nucleus by portioning oil into one droplet. The reaction mixture containing the nuclei, the barcoded beads and partitioning oil were loaded separately on a 10X chip and a collection of gel beads in emulsions were generated. This was further processed by performing the reverse transcription (RT) reaction to form cDNA that shares a lOx Barcode with all cDNA from its individual nuclei of origin, followed by preparation of the lOx barcoded sequencing library, which was subjected to Illumina sequencing. The sequenced data was processed using Cell Ranger pipeline (v 6.1.2) by lOx Genomics (https: / / www.10xgenomics.com). Sequencing reads were aligned to human reference genome (vGRCh38) and mouse reference genome (vGRCm38) for mouse and human samples respectively, followed by filtering and correction of cell barcodes and Unique Molecular Identifiers (UMIs). Reads associated with retained barcodes were quantified and used to build gene count matrix.DiEI / synovfibros / 874

[0169] 5. Single-Nuclei RNA

[0170] Single-nuclei RNA sequencing of human and mouse dataset follows the standard procedures of filtering, normalization, dimensionality reduction and clustering which was performed using an R package Seurat (v.4.1.0)(59). A total of 51,527 nuclei were sequenced from human tissue and 28,188 were sequenced from mouse tissue. All genes not detected in at least three nuclei and all nuclei expressing less than 200 genes were excluded from analyses. In addition, nuclei with high mitochondrial content and potential doublets or multiplets were filtered out from downstream analyses. DoubletFinder was used for the detection of doublets (60). Early- (KL I) and advanced-stage (KL 111 / IV) OA samples were merged for subsequent clustering and visualization. Batch correction between samples was performed using the Harmony R package (vl.0)(61).

[0171] Data was normalized using the log normalization method and highly variable genes were selected using the variance stabilization (vst) method. PCA was then performed on the highly variable genes and the number of significant PCs to include for clustering was determined based on the elbow plot of standard deviations of PCs. A graph-based clustering method was implemented by calculating k-nearest neighbours, followed by Louvian modularity optimization to cluster cells. Non-linear dimensionality reduction and visualization was performed using UMAP (Uniform Manifold Approximation and Projection). Clusters were annotated based on canonical markers and differential gene expression testing was used to determine transcriptomic signatures for each cluster using the Wilcoxon Rank Sum test. Subclustering of fibroblast clusters was performed in a similar manner. A total of 533 mural cell nuclei were identified only in early samples and were excluded from the human dataset prior to cluster analysis, as they were considered contaminants from surrounding tissue.

[0172] Similarly, all mouse synovium samples from naive, 2 week and 10 week post-surgery samples were merged for clustering and visualization. The mouse fibroblast clusters were scored against human fibroblast clusters using 'AddModuleScore' function in Seurat.

[0173] 6. Flow Cytometry

[0174] Using synovia from our independent second cohort (UZ Gent), synovium was obtained from patients during total knee arthroplasty, routine knee arthroscopy or from cadaveric subjects.

[0175] All patients provided informed consent to participate in this study, under study number 2019 / 1922-BC-06496 (Commissie voor Medische Ethiek, UZ Gent). Knee synovium was dissected from adjacent joint capsule / fat tissue and sectioned into lOOmg pieces for cryopreservation using cryostor following published protocols (https: / / pubmed.ncbi.nlm.nih.gov / 17951672 / ). Cryopreserved synovium samples were thawed, minced and immediately digested at 37C with 0.5mg / ml collagenase VIII and O.lmg / mlDiEI / synovfibros / 874

[0176] DNAse in phenol red free RPML Cells from the digested synovium were counted with trypan blue. The synovium single cell suspensions were stained in 96 well microplates using the following protocol: Cells were first stained with a fixable viability stain. After washing, the cells were treated with Fc receptor blocking reagent, followed by staining for surface antigens. The cells were fixed with PFA prior to acquisition on the BD Symphony. Two panels were designed with 15-25 channels each, depending on antibody and fluorochrome availability. Basic lineage markers were included to positively identify cells (eg, fibroblasts were identified as CD31-CD235a-CD45-HLA-ABC+ cells, with CD90 and CD55 defining sublining and lining subsets, respectively). Specific markers identified by snRNA-seq were used to identify cell populations of interest identified in early vs. advanced stage OA synovium (e.g. ITGB8 for advanced-staged OA fibroblasts). Data was analyzed with FlowJo.

[0177] 7.DMM Surgery

[0178] The right knees of 12-week-old C57BL / 6 [The Jackson Laboratory (RRID: IMSR_JAX:000664)] mice were subjected to destabilization of the medial meniscus surgery with synovium collected at 2 weeks (n=3) or 10 weeks (n=4) post-surgery (62). Synovia from naive mice (n=4) were also collected for analysis. Extracted synovia were subjected to snRNA-seq, as described above. Of note, collections include some contaminating tissue including patella, ligament, fat and muscle, as previously described (63).

[0179] 8.Pathway and Transcription factor Analyses

[0180] The list of genes from each cluster linked to early- or advanced-stage fibroblasts were used to perform pathway and gene ontology enrichment analysis. Pathway enrichment analysis was performed using pathDIP 4 (http: / / ophid.utoronto.ca / pathDIP, ref: PMID: 31733064) API in R 4.0.3, excluding KEGG, OntoCancro, ACSN2, RB-Pathways and WikiPathways as sources. Only pathways with adjusted P-value <0.01 were further considered. Gene Ontology Biological Processes enrichment analysis was performed using clusterProfiler v3.16.1(64) n R and retaining terms with adjusted P-value < 0.01. ECM related genes from DEG lists of cluster 0 and cluster 1 that were associated with identified pathways of cluster 0 and 1 were extracted. Catrin database v 1.0.6.2. b(27) was used to identify transcription factors (TF) targeting the ECM genes for each cluster. Hypergeometric test to identify upstream regulators was performed in R, and TFs with adjusted P-value <0.01 were retained. Furthermore, we only considered TFs that targeted early- or advanced-stage genes. A network of pathway - gene - TF interactions was built using NAViGaTOR v3.0.16 (65). Interactions among genes were retrieved using HD v2020-ll (66). The network was then reduced to show only terms related to extracellular matrix processes.DiEI / synovfibros / 874

[0181] 9.Fibroblast- specific BHLHE40 Conditional Knockout Mice

[0182] Bhlhe4C / fmice were generously gifted by Dr. Brian Edelson (Washington University) (31). Col6al-cre / + mice (B6.Cg-Tg(Col6al-cre)lGkl / Flmg), were purchased from the European Mutant Mouse Archive repository as sperm and re-derived in-house. Fibroblast-specific Bhlhe40 knockout mice were generated by crossing Bhlhe4Of / fmice to Col6al-cre / + mice to generate Col6al-cre / +; Bhlhe4Of / fmice. Cre-; Bhlhe4Of / fmice were used as controls. Right knee joints from WT or Bhlhe40 CKO mice were subjected to sham or DMM surgeries (WT sham, n=9; CKO sham, =10; WT DMM, n=13; CKO DMM, n=ll) and collected 10 weeks post-surgery. Genotyping was assessed by standard PCR and confirmed by sorting synovial fibroblasts and macrophages for qPCR. To sort synovial fibroblasts and macrophages for confirmation of cell-specific Bhlhe40 deletion, mouse knee synovium was dissected as above and subjected to enzymatic digestion using 1 mg / ml collagenase IV, 0.75 mg / ml collagenase VIII and 0.1 mg / ml DNAse. Cells were stained with CD31-APC.Cy7, F / 480-APC, CD45-PB, Ly6G-FITC, CDllb-BV785, PDPN-PECy7 and 7AAD. Live fibroblasts (CD31-CD45-PDPN+) and macrophages (CD31-CD45+Ly6G-CDllb+F4 / 80+) were sorted using the ARIA III (BD). RNA was extracted from cells using RNease micro kit (Qiagen) before qPCRfor the genes of interest. Eefla (F CGTCAGAACGCAGGTGTTG, R TCGATGGTTCGCTTGTCGAT) and Rpll3a (F AAGCAGGTACTTCTGGGCCG, R CCTCGGGAGGGGTTGGTATT) were used as housekeepers for Bhlhe40 (F CGTTGAAGCACGTGAAAGCA, R TCCCGACAAATCACCAGCTT).

[0183] lO.Statistical Analysis

[0184] The proportions of human fibroblast subtypes in early- versus advanced-stage synovia were statistically analyzed using two-tailed t-tests with correction for false detection rate (FDR). P-value <0.05 is considered statistically significant. Data are expressed as mean ± standard deviation (SD). Default statistical tests from Seurat, Monocle3, and CellChat R packages were used for snRNA-seq data analysis. These packages include FDR testing to account for multiple comparisons in large datasets. OARSI, synovitis scores and IHC positive cell count for CKO mice (Ki67 & a-SMA) were statistically analyzed using a two-way ANOVA and corrected for multiple comparisons by controlling the false discovery rate using the two-stage step-up method of Benjamini, Krieger and Yekutieli. Adjusted P-values <0.05 were considered statistically significant. Finally, IHC positive cell count scoring for ITGB8 and DPP4 were analyzed using an unpaired, nonparametric Mann Whitney test, with P-values < 0.05 being considered as statistically significant.

[0185] ll.Generation of recombinant lentivirus

[0186] The full-length mouse or human BHLHE40 gene tagged with eGFP (human) or nano-Luc (nLuc, mouse) using T2A peptide, and control eGFP or nano-luc transgenes were individually cloned into the pRS-EFla-DiEI / synovfibros / 874

[0187] MCS-WPRE lentiviral vector (Tailored Genes Inc, Ontario, Canada) using In-Fusion HD cloning kit (638946, In-Fusion® Snap Assembly Value Bundle Takara Bio USA Inc, San Jose, CA, USA). The source sequence for huBHLHE40-T2A was amplified from a pAAV[Exp]- EFlA>hBHLHE40[NM_003670.3](ns) / HA:T2A:Nluc:WPRE3 vector (VB240712-1285nmb; Vector Builder, Chicago, IL). The source sequence for mBHLHE40-T2A-nLuc was amplified from pAAV[Exp]-EFlA>mBHLHE40[NM_003670.3](ns) / HA:T2A:Nluc:WPRE3 (VB240108-1168msu; Vector Builder). Lentivirus vectors (LVV) were produced by transfection of HEK293T cells with pRS-EFla-hBHLHE40:HA-T2A-eGFP-WPRE / pRS-EFla-eGFP-WPRE / mBHLHE40:HA-T2A-nLuc-WPRE / pRS-EFla-nLuc-WPRE pDNAs and with packaging plasmids pLenti gag-pol, pRSV Rev, and pHCMV- VSV-G (Tailored Genes Inc, Ontario, Canada). The supernatant containing the LVVs was collected at 48 h post-transfection, filtered using 0.45-micron filters and concentrated / purified using Amicon ultrafiltration (EMD Millipore corporation, MA, USA). Infectious titer was determined by transducing human osteosarcoma cells with serial dilution of LVV, genomic DNA isolation from transduced cells, and determining vector copy number in transduced cells by digital PCR using primers / probe specific to WPRE sequence.

[0188] 12. Generation of recombinant AAV2 particle production

[0189] Recombinant adeno-associated virus serotype 2 (AAV2) vectors were constructed and produced by VectorBuilder Inc. The murine Bhlhe40 sequence (NM_011498.4) was cloned into the pAAV expression vector under the control of the constitutive EFla promoter (pAAV[Exp]-EFla>mBhlhe40; VectorBuilder ID: VB240108-1168msu) (see Fig 9A). Downstream of the Bhlhe40 sequence, the vector construct included an HA tag, a T2A self-cleaving peptide, and a NanoLuc luciferase reporter, following aWPRE3 element to enhance transcript stability. The Bhlhe40 transgene construct was flanked by AAV2 inverted terminal repeats. Replication deficient AAV2 particles were subsequently generated. Control vectors do not contain the transgene or HA tag (i.e transgene empty) but all other indicated sequences.DiEI / synovfibros / 874

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Claims

DiEI / synovfibros / 874Claims1. A polynucleotide sequence encoding the transcriptional repressor BHLHE40 for use to treat mammals suffering from musculoskeletal diseases2. The polynucleotide sequence for use as defined in claim 1 which encodes the BHLHE40 polypeptide depicted in SEQ. ID NO: 2.

3. The polynucleotide for use according to claims 1 or 2 which is a chemically modified polynucleotide sequence wherein the modification is at least one chemically modified nucleobase, sugar, backbone, or any combination thereof.

4. The polynucleotide sequence for use according to claims 1, 2 or 3 wherein said musculoskeletal diseases comprise osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, bursitis and tendinitis.

5. The polynucleotide sequence for use as defined in claims 1 or 2 which is comprised in a viral vector.

6. The polynucleotide sequence for use according to claim 5 wherein said viral vector comprises a lentivirus, an adenovirus, a herpes simplex virus or an adeno-associated virus.

7. The polynucleotide sequence for use as defined in claims 1, 2 or 3 which is comprised in a non- viral vector.

8. The polynucleotide sequence for use according to claim 7 wherein said non-viral vector comprises a liposome, a nanoparticle, or a lipid nanoparticle.

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